**Pharmacokinetic of Drugs, Effect of Compound Interactions on Cytochrome P450 Activity**

[41] Siriwardena AN, Qureshi MZ, Dyas JV, Middleton H, Orner R. Magic bullets for insomnia? Patients' use and experiences of newer (Z drugs) versus older (benzodiazepine) hypnotics for sleep problems in primary care. The British Journal of General Practice.

[42] Zosel A, Osterberg EC, Mycyk MB. Zolpidem misuse with other medications or alcohol frequently results in intensive care unit admission. American Journal of Therapeutics.

[43] Tietz E, Rosenbger H. Autoradiographic localization of benzodiazepine receptor downregulation. The Journal of Pharmacology and Experimental Therapeutics. 1986;

[44] Busto U, Sellers EM. Pharmacologic aspects of benzodiazepine tolerance and depen-

[45] Liebrenz M, Schneider M, Buadze A, Gehring M-T, Dube A, Caflisch C. High-dose benzodiazepine dependence: A qualitative study of patients' perceptions on initiation,

[46] Ashton H. Benzodiazepines: How They Work & How to Withdraw. Available from:

[47] Ashton H. The Treatment of Benzodiazepine Dependence. Available from: https://benzo.

[48] Clinical Guidelines for Withdrawal Management and Treatment of Drug Dependence in Closed Settings. WHO Guidelines Approved by the Guidelines Review Committee.

[49] World Benzodiazepine Awareness Day. Available from: http://w-bad.org. [Accessed:

dence. Journal of Substance Abuse Treatment. 1991;**8**:29-33

reasons for use, and obtainment. PLoS One. 2015;**10**:e0142057

org.uk/ashtbd.htm [Accessed: February 23, 2018]

Geneva: World Health Organization; 2009

February 25, 2018]

https://benzo.org.uk/manual/index.htm [Accessed: February 23, 2018]

2008;**58**:417-422

90 Medicinal Chemistry

2011;**18**:305-308

**236**:284-292

**Chapter 6**

**Provisional chapter**

**Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-**

**Clinical Pharmacokinetics of Clavulanic Acid, a Novel** 

**β-Lactamase Isolated from** *Streptomyces clavuligerus*

DOI: 10.5772/intechopen.79409

**Lactamase Isolated from** *Streptomyces clavuligerus* **and**

The clavulanic acid derived by fermentation of *Streptomyces clavuligerus* and possessed the capability to inactivate a broad range of β-lactamase enzymes. A complex physicochemical process involves the binding of clavulanic acid to β-lactamases in which clavulanic acid itself deplete irreversibly along with β-lactamase enzyme rendering amoxicillin spared which otherwise would hydrolyze by an enzyme. It is therefore termed as 'suicide 'inhibitor for β-lactamases. We discussed here pharmacokinetic parameters and identified factors responsible for the variability of absorption of clavulanic acid. The results based on individual plasma concentration-time curve of amoxicillin and clavulanic acid in an open, randomized, two-way crossover study involving 10 healthy male subjects

**Keywords:** clinical, clavulanic acid, pharmacokinetics, variable absorption, AUC total,

Clavulanic acid derived by fermentation of *Streptomyces clavuligerus* and possessed the capability to inactivate a broad range of β-lactamase enzymes. The molecular formula of clavulanate potassium is C₈H₈KNO₅ with the molecular weight of 237.25. Chemically it is potassium

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Anab Fatima, Mohammad Jiyad Shaikh, Hina Zahid,

Hina Zahid, Ishart Younus, Sheikh Abdul Khaliq and

Ishart Younus, Sheikh Abdul Khaliq and

Anab Fatima, Mohammad Jiyad Shaikh,

http://dx.doi.org/10.5772/intechopen.79409

Additional information is available at the end of the chapter

administered with two amoxiclav formulations.

pharmacokinetics, β-lactamase

Additional information is available at the end of the chapter

**Its Variability**

**and Its Variability**

Farah Khalid

**Abstract**

**1. Introduction**

Farah Khalid

#### **Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-Lactamase Isolated from** *Streptomyces clavuligerus* **and Its Variability Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-Lactamase Isolated from** *Streptomyces clavuligerus* **and Its Variability**

DOI: 10.5772/intechopen.79409

Anab Fatima, Mohammad Jiyad Shaikh, Hina Zahid, Ishart Younus, Sheikh Abdul Khaliq and Farah Khalid Anab Fatima, Mohammad Jiyad Shaikh, Hina Zahid, Ishart Younus, Sheikh Abdul Khaliq and Farah Khalid

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79409

#### **Abstract**

The clavulanic acid derived by fermentation of *Streptomyces clavuligerus* and possessed the capability to inactivate a broad range of β-lactamase enzymes. A complex physicochemical process involves the binding of clavulanic acid to β-lactamases in which clavulanic acid itself deplete irreversibly along with β-lactamase enzyme rendering amoxicillin spared which otherwise would hydrolyze by an enzyme. It is therefore termed as 'suicide 'inhibitor for β-lactamases. We discussed here pharmacokinetic parameters and identified factors responsible for the variability of absorption of clavulanic acid. The results based on individual plasma concentration-time curve of amoxicillin and clavulanic acid in an open, randomized, two-way crossover study involving 10 healthy male subjects administered with two amoxiclav formulations.

**Keywords:** clinical, clavulanic acid, pharmacokinetics, variable absorption, AUC total, pharmacokinetics, β-lactamase

#### **1. Introduction**

Clavulanic acid derived by fermentation of *Streptomyces clavuligerus* and possessed the capability to inactivate a broad range of β-lactamase enzymes. The molecular formula of clavulanate potassium is C₈H₈KNO₅ with the molecular weight of 237.25. Chemically it is potassium

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**2.1. Content assay for co-amoxiclav tablets by HPLC**

and 100 to 101.4% for clavulanic acid.

**2.2. Pharmacokinetic evaluation of co-amoxiclav tablet**

**Table 1.** Validation parameters of amoxicillin and clavulanic acid in mobile phase.

The content assay for co-amoxiclav tablets was carried out by validated HPLC method. The method validation was carried out according to USP guidelines. For good and accurate resolution and reproducibility of the presented method various suitability considerations including tailing factor, retention time, resolution, RSD% of retention time and peak areas were determined and were found within acceptable range. The method was found to be specific for the determination of particular analyte. Specificity was determined by injecting the analytical placebo(containing all excipient of tablet except amoxicillin and clavulanic acid). The interference by these excipients were determined by evaluating mixture of all excipient(placebo),standard solutions and commercial pharmaceutical preparations con-

Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-Lactamase Isolated from *Streptomyces clavuligerus*...

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95

tained amoxicillin and clavulanic acid within the same chromatographic condition.

The linearity of HPLC method was determined for amoxicillin and clavulanic acid. Ten dilutions of amoxicillin and eight dilutions clavulanic acid of different concentrations were prepared in mobile phase then sample size of 20 μl of each concentration was injected into HPLC. The detector response was measured at 235 nm and the calibration plots(concentration versus peak area) were obtained using the linear regression method. The linearity data showed linearity over a concentration range of 0.03–31.25 μg/ml for amoxicillin and 0.24–15.6 μg/ml for clavulanic acid. Repeatable and intermediate precision of method was determined. During the same day with the same experimental condition repeatability of four determination (n = 4) at the same concentration was calculated. The results of assay were compared and evaluated on three different days by different analyst for intermediate precision. The precision values for amoxicillin and clavulanic acid were found to be 0.91 and 0.35% for intraday and 0.89 and 0.34% for inter-day respectively. The recovery studies were carried out for the assurance of reliability and accuracy of the proposed method. A known quantity of the drug added with preanalyzed sample and then reanalyzed by the proposed method. The recovery studies for amoxicillin and clavulanic acid were performed at three different concentrations corresponded to 80, 100 and 120% of active ingredients. For each concentration mean %recovery were from 99.7 to 101.4 for amoxicillin

Small variation in the method parameters was created to measure its reliability during routine usage and the robustness. Not any significant effect on the method performance was observed by changing the flow rate of mobile phase, column temperature and ratio of organic content in mobile phase indicated that the test method was robust for all variable conditions (**Table 1**).

In Pakistan Co-amoxiclav tablet of 375 mg marketed by a multinational company Code #1 Pakistan and a local company Code #2 (**Table 2**). Physico-chemical and potency determination

Limit of Detection {LOD(μg/ml)} 0.015/0.0037 0.12/0.06

**Medium Parameters Amoxicillin Clavulanic acid** In mobile phase/plasma Limit of Quantification {LOQ(μg/ml)} 0.030/0.0075 0.243/0.12

**Figure 1.** Structure of clavulanate potassium.

(Z)-(2R,5R)-3-(2-hydroxyethylidine)-7-oxo-4-1-azabicyclo[3.2.0]-heptane-2-carboxylate, represented as in **Figure 1**. Mainly it combines with amoxicillin to broaden its antibacterial spectrum [1]. A complex physicochemical process involve in binding of clavulanic acid to β-lactamases in which clavulanic acid itself deplete irreversibly along with β-lactamase enzyme rendering amoxicillin spared which otherwise would hydrolyzed by enzyme. It is therefore termed as 'suicide 'inhibitor for β-lactamases. A very low plasma concentration of clavulanic acid is required for this target action. After oral administration the pharmacokinetic parameters of both components i.e. amoxicillin and clavulanic acid were similar and they did not affect pharmacokinetic parameters of each other [2–6]. This could be one of reason for their antimicrobial combination. In combination clavulanic acid and amoxicillin used in different composition i.e. 250/125, 500/125 and 850/125 respectively. In this amount of amoxicillin varies but that of clavulanic acid remain constant i.e. 125 mg. It would suggest that there is no significant amount required for clavulanic acid to inhibit β lactamase enzymes [3]. We discussed here pharmacokinetic parameters and identified factors responsible for variability of absorption of clavulanic acid and compared it with previous reported data. The results based on individual plasma concentration-time curve of amoxicillin and clavulanic acid in an open, randomized, two-way crossover study involving 12 healthy male subjects administered with two amoxiclav formulations.

#### **2. Parameter which is associated to show variability of absorption**

Mainly Ct (concentration at time t) is responsible for clinical effects of any drug. When C<sup>t</sup> is higher the AUC (Area under Curve) and Cp (plasma concentration) will also show higher values. Thus they all are co-related to show any clinical effect but question arises that which pharmacokinetic parameter or any other factor is most likely to show variability in absorption of any drug which would ultimately affect its clinical effect. Clavulanic acid along with amoxicillin is well absorbed from stomach after oral administration without having any impact of fasting and fed state on the pharmacokinetics of amoxicillin. While relative bioavailability of clavulanic acid becomes reduced when administered after 30 and 150 min of high fat breakfast. The logic behind reduced bioavailability of clavulanic acid after ingestion with the meal was due to prolong residence time of clavulanic acid in GI due to intragastric tablet deposition in the proximal stomach. The half-life of clavulanic acid is 1.0 h and 25–40% of it is excreted unchanged in urine following first 6 h of administration. Clavulanic acid is difficult to extract out from plasma as it has been bound approximately 25% to human serum and therefore required double extraction procedure to observe by liquid chromatography. After absorption clavulanic acid is well distributed in body tissues [7].

#### **2.1. Content assay for co-amoxiclav tablets by HPLC**

(Z)-(2R,5R)-3-(2-hydroxyethylidine)-7-oxo-4-1-azabicyclo[3.2.0]-heptane-2-carboxylate, represented as in **Figure 1**. Mainly it combines with amoxicillin to broaden its antibacterial spectrum [1]. A complex physicochemical process involve in binding of clavulanic acid to β-lactamases in which clavulanic acid itself deplete irreversibly along with β-lactamase enzyme rendering amoxicillin spared which otherwise would hydrolyzed by enzyme. It is therefore termed as 'suicide 'inhibitor for β-lactamases. A very low plasma concentration of clavulanic acid is required for this target action. After oral administration the pharmacokinetic parameters of both components i.e. amoxicillin and clavulanic acid were similar and they did not affect pharmacokinetic parameters of each other [2–6]. This could be one of reason for their antimicrobial combination. In combination clavulanic acid and amoxicillin used in different composition i.e. 250/125, 500/125 and 850/125 respectively. In this amount of amoxicillin varies but that of clavulanic acid remain constant i.e. 125 mg. It would suggest that there is no significant amount required for clavulanic acid to inhibit β lactamase enzymes [3]. We discussed here pharmacokinetic parameters and identified factors responsible for variability of absorption of clavulanic acid and compared it with previous reported data. The results based on individual plasma concentration-time curve of amoxicillin and clavulanic acid in an open, randomized, two-way crossover study involving 12 healthy male subjects

**2. Parameter which is associated to show variability of absorption**

absorption clavulanic acid is well distributed in body tissues [7].

(concentration at time t) is responsible for clinical effects of any drug. When C<sup>t</sup>

higher the AUC (Area under Curve) and Cp (plasma concentration) will also show higher values. Thus they all are co-related to show any clinical effect but question arises that which pharmacokinetic parameter or any other factor is most likely to show variability in absorption of any drug which would ultimately affect its clinical effect. Clavulanic acid along with amoxicillin is well absorbed from stomach after oral administration without having any impact of fasting and fed state on the pharmacokinetics of amoxicillin. While relative bioavailability of clavulanic acid becomes reduced when administered after 30 and 150 min of high fat breakfast. The logic behind reduced bioavailability of clavulanic acid after ingestion with the meal was due to prolong residence time of clavulanic acid in GI due to intragastric tablet deposition in the proximal stomach. The half-life of clavulanic acid is 1.0 h and 25–40% of it is excreted unchanged in urine following first 6 h of administration. Clavulanic acid is difficult to extract out from plasma as it has been bound approximately 25% to human serum and therefore required double extraction procedure to observe by liquid chromatography. After

is

administered with two amoxiclav formulations.

**Figure 1.** Structure of clavulanate potassium.

94 Medicinal Chemistry

Mainly Ct

The content assay for co-amoxiclav tablets was carried out by validated HPLC method. The method validation was carried out according to USP guidelines. For good and accurate resolution and reproducibility of the presented method various suitability considerations including tailing factor, retention time, resolution, RSD% of retention time and peak areas were determined and were found within acceptable range. The method was found to be specific for the determination of particular analyte. Specificity was determined by injecting the analytical placebo(containing all excipient of tablet except amoxicillin and clavulanic acid). The interference by these excipients were determined by evaluating mixture of all excipient(placebo),standard solutions and commercial pharmaceutical preparations contained amoxicillin and clavulanic acid within the same chromatographic condition.

The linearity of HPLC method was determined for amoxicillin and clavulanic acid. Ten dilutions of amoxicillin and eight dilutions clavulanic acid of different concentrations were prepared in mobile phase then sample size of 20 μl of each concentration was injected into HPLC. The detector response was measured at 235 nm and the calibration plots(concentration versus peak area) were obtained using the linear regression method. The linearity data showed linearity over a concentration range of 0.03–31.25 μg/ml for amoxicillin and 0.24–15.6 μg/ml for clavulanic acid. Repeatable and intermediate precision of method was determined. During the same day with the same experimental condition repeatability of four determination (n = 4) at the same concentration was calculated. The results of assay were compared and evaluated on three different days by different analyst for intermediate precision. The precision values for amoxicillin and clavulanic acid were found to be 0.91 and 0.35% for intraday and 0.89 and 0.34% for inter-day respectively.

The recovery studies were carried out for the assurance of reliability and accuracy of the proposed method. A known quantity of the drug added with preanalyzed sample and then reanalyzed by the proposed method. The recovery studies for amoxicillin and clavulanic acid were performed at three different concentrations corresponded to 80, 100 and 120% of active ingredients. For each concentration mean %recovery were from 99.7 to 101.4 for amoxicillin and 100 to 101.4% for clavulanic acid.

Small variation in the method parameters was created to measure its reliability during routine usage and the robustness. Not any significant effect on the method performance was observed by changing the flow rate of mobile phase, column temperature and ratio of organic content in mobile phase indicated that the test method was robust for all variable conditions (**Table 1**).

#### **2.2. Pharmacokinetic evaluation of co-amoxiclav tablet**

In Pakistan Co-amoxiclav tablet of 375 mg marketed by a multinational company Code #1 Pakistan and a local company Code #2 (**Table 2**). Physico-chemical and potency determination


**Table 1.** Validation parameters of amoxicillin and clavulanic acid in mobile phase.


Note: This was a 2 year study and during this period we have taken products from different lots available in the market.

**Table 2.** Details of different amoxicillin/clavulanic acid products available in Pakistan.

from both sources of different batch were analyzed. This analysis was carried out just to compare the two different brands available in Pakistan as an added parameter. The main objective of study is to conduct pharmacokinetic study of Co-amoxiclav tablet from a multinational company in local population and to find out difference in the pharmacokinetic parameters with the previous reported data due to racial inconsistency. It was preferred to compare only multinational brand because this has been most commonly prescribed by the prescriber to treat infections and the local brand did not proved to be as efficacious as that of multinational brand.

Mobile Phase containing methanol (10 volume) and 0.02 M disodium hydrogen phosphate buffer (90 volume). The pH was adjusted to 3.0 by phosphoric acid. The mobile phase was filtered and degassed. An HPLC isocratic pump with UV–VIS detector was attached with RP 18e column

**Figure 2.** Linearity curve of clavulanic acid: conc vs. area showing linearity between standard solution and peak area.

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In a glass stoppered 15 ml centrifuge tube 0.75 ml of acetonitrile was added to 0.5 ml of plasma. After mixing(30s) the mixture centrifuged for 10 min at 5000 × g. Then 2.5 ml of dichloromethane was added to 300 μl of supernatant. After mixing (30 s) the mixture centrifuge for 10 min at 5000 × g. Then 20 μl of supernatant was injected into liquid chromatograph at 235 nm detection

There has been no study to compare the difference of blood concentration time curve of different formulations of co-amoxiclav in local population of Pakistan. Therefore author tried to focus on the Pharmacokinetic pattern especially of clavulanic acid. It is due to fact that absorption of clavulanic acid, after oral administration, is highly variable and may vary over a five-fold range between patients [9]. Based on the plasma amoxicillin and clavulanic acid concentrations of individual subjects, were calculated by applying both compartmental and noncompartmental method of analysis. As best fitted pharmacokinetic model one compartmental

**Figure 3.** Linearity curve of amoxicillin: conc vs. area showing linearity between standard solution and peak area.

**2.4. Pattern of variable absorption of clavulanic acid from different oral** 

**formulations of co-amoxiclav in healthy subjects**

(Hibar, 250 × 4.6 cm).

wave length.

The study was two treatments, two sequences, single dose and cross over design in 12 normal healthy volunteers. An equal number of volunteers were assigned to each sequence. The study covers to determine the pharmacokinetic parameters i.e. Cmax, Tmax, AUC, rate constant, Vd, total clearance and T½ of Co-amoxiclav in local population. The subjects engaged in the study were member of community at large and full-fill all of criteria to be included in the study. This criterion includes healthy males with normal vital signs, blood hematology and chemistry, non-smoker, able to consent and swallow. The study design was endorsed by the National Bioethics Committee, Ministry of Health, Government of Pakistan, Islamabad after critical ethical review and a written informed duly signed by volunteers has been taken. Four volunteers withdraw during study.

#### **2.3. Bioanalytical validation**

Plasma amoxicillin and clavulanic acid concentrations were determined using validated methods such as LC/MS/MS analysis (GTF) [8] (**Table 3**). The method was also validated according to International Council for Harmonization (ICH) guidelines (**Figures 2** and **3**).

The fundamental parameters of validation were Specificity, linearity, accuracy, precision, sensitivity, reproducibility, stability and robustness. All these parameters were determined and validated.


>> Amoxicillin and clavulanate potassium tablets contain equivalent of not less than 90.0% and not more than 120.0% of the labeled amount of amoxicillin and clavulanic acid (USP 28).

**Table 3.** HPLC assay of different brands of amoxicillin/clavulanic acid tablet (250/125 mg) available in Pakistan.

**Name of product Strength (mg) Date of mfg. Date of exp. Retail price (Rs) Industrial supplier**

Note: This was a 2 year study and during this period we have taken products from different lots available in the market.

from both sources of different batch were analyzed. This analysis was carried out just to compare the two different brands available in Pakistan as an added parameter. The main objective of study is to conduct pharmacokinetic study of Co-amoxiclav tablet from a multinational company in local population and to find out difference in the pharmacokinetic parameters with the previous reported data due to racial inconsistency. It was preferred to compare only multinational brand because this has been most commonly prescribed by the prescriber to treat infections and the local brand did not proved to be as efficacious as that of multinational brand. The study was two treatments, two sequences, single dose and cross over design in 12 normal healthy volunteers. An equal number of volunteers were assigned to each sequence. The study covers to determine the pharmacokinetic parameters i.e. Cmax, Tmax, AUC, rate constant, Vd, total clearance and T½ of Co-amoxiclav in local population. The subjects engaged in the study were member of community at large and full-fill all of criteria to be included in the study. This criterion includes healthy males with normal vital signs, blood hematology and chemistry, non-smoker, able to consent and swallow. The study design was endorsed by the National Bioethics Committee, Ministry of Health, Government of Pakistan, Islamabad after critical ethical review and a written informed duly signed by volunteers has been taken. Four

Plasma amoxicillin and clavulanic acid concentrations were determined using validated methods such as LC/MS/MS analysis (GTF) [8] (**Table 3**). The method was also validated according

The fundamental parameters of validation were Specificity, linearity, accuracy, precision, sensitivity, reproducibility, stability and robustness. All these parameters were determined and validated.

>> Amoxicillin and clavulanate potassium tablets contain equivalent of not less than 90.0% and not more than 120.0% of

**Table 3.** HPLC assay of different brands of amoxicillin/clavulanic acid tablet (250/125 mg) available in Pakistan.

to International Council for Harmonization (ICH) guidelines (**Figures 2** and **3**).

**Product name % labeled strength Product name % labeled strength**

AMCL1 106.1 CODE #1 113.38 AMCL2 119.8 CODE #2 109.9 Mean 112.9 111.64 S.D 9.6 2.46

the labeled amount of amoxicillin and clavulanic acid (USP 28).

(AMCL1) 250/125 02–12 08–13 75.00 Code#1 (AMCL2) 250/125 04–12 04–14 82.00 Code#2

**Table 2.** Details of different amoxicillin/clavulanic acid products available in Pakistan.

volunteers withdraw during study.

**2.3. Bioanalytical validation**

96 Medicinal Chemistry

**Figure 2.** Linearity curve of clavulanic acid: conc vs. area showing linearity between standard solution and peak area.

Mobile Phase containing methanol (10 volume) and 0.02 M disodium hydrogen phosphate buffer (90 volume). The pH was adjusted to 3.0 by phosphoric acid. The mobile phase was filtered and degassed. An HPLC isocratic pump with UV–VIS detector was attached with RP 18e column (Hibar, 250 × 4.6 cm).

In a glass stoppered 15 ml centrifuge tube 0.75 ml of acetonitrile was added to 0.5 ml of plasma. After mixing(30s) the mixture centrifuged for 10 min at 5000 × g. Then 2.5 ml of dichloromethane was added to 300 μl of supernatant. After mixing (30 s) the mixture centrifuge for 10 min at 5000 × g. Then 20 μl of supernatant was injected into liquid chromatograph at 235 nm detection wave length.

### **2.4. Pattern of variable absorption of clavulanic acid from different oral formulations of co-amoxiclav in healthy subjects**

There has been no study to compare the difference of blood concentration time curve of different formulations of co-amoxiclav in local population of Pakistan. Therefore author tried to focus on the Pharmacokinetic pattern especially of clavulanic acid. It is due to fact that absorption of clavulanic acid, after oral administration, is highly variable and may vary over a five-fold range between patients [9]. Based on the plasma amoxicillin and clavulanic acid concentrations of individual subjects, were calculated by applying both compartmental and noncompartmental method of analysis. As best fitted pharmacokinetic model one compartmental

model with lag time, first order absorption and first order elimination was selected for both amoxicillin and clavulanate potassium.

The maximum concentration Cmax of amoxicillin was achieved in 1.85 ± 0.01 h for amoxicillin in compartmental. Similarly Cmax for clavulanate potassium was achieved in 1.56 ± 0.01 h in

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The reported values of Vss after IV administration for amoxicillin is 0.28 ± 0.06 L/kg, and the Vss of clavulanic acid as 0.24 ± 0.06 L/kg, showing ratio for the volume of distribution between clavulanic acid and amoxicillin as 0.8571 [14] and therefore on the basis of this the ratio of amoxicillin to clavulanic acid AUCs should be 3.4. when co-amoxiclav is at dose of 250/125. The author observed in this study, the lowest AUCt amoxicillin/clavulanic acid ratio was 2.7 ± 0.50 at the lower doses used. This would assume equal absorption of both amoxicillin and clavulanic acid. But in the same dose amoxicillin/clavulanic acid AUCt ratios was higher that would suggest that with a similar amoxicillin absorption, clavulanic acid absorption must have been reduced. The reported absolute bioavailability of clavulanic acid, when co-administered with amoxicillin has been ranged from 31.4 to 98.8% [10]. Further it is reported that there is no major alteration in the mean AUCt of 125 mg clavulanate when it is administered along 500 mg of amoxicillin, but it creates marked impact on the coefficient of variation for the AUC which alter from 27.6% for clavulanic acid alone to 45.6% when given with amoxicillin [15]. Various other studies showed mean absorption up to 97% when clavulanic acid administered alone with minor inter-patient variability. It indicates interaction between absorption of amoxicillin and clavulanic acid [11, 12] . The author further found that there was no significant variation in the AUC observed for amoxicillin in this study either among subjects, on the basis of demographic data, or between formulations, once corrected for the dose. On the contrary, high variability was seen between subjects in the AUC of clavulanic acid (**Figure 5**). There were all healthy male subjects (with normal renal function), and it is difficult to explain the high variability seen in the clavulanic acid AUC on patient factors. However, it has been reported in other studies [13]. Also study observed broadened Tmax i.e. increase lag time indicating a rate limiting step in the absorption process. The authors being able to show that two different co-amoxiclav formulations each gave a variation in the absorption, or in the AUCt value, of clavulanic acid for the

In a study reported broadened Tmax with high dose of amoxicillin (875 mg) indicate a rate

In this study the authors tried to show variation in the absorption or in AUCt value of clavulanic acid at 125 mg dose with two formulations. Although we did not find any report of therapy failure among the patients due to this variation and its clinical efficacy has been maintained. It would suggest that it is more important to focus on the absolute or fixed amount of

**Formulation Dose (mg) T 1/2 (h) Tmax (h) Cmax (μg/L) AUC (μg.h/L)** AMCL1 125 1.20 ± 0.02 1.56 ± 0.01 2.60 ± 0.03 8.30 ± 0.06 AMCL2 125 1.21 ± 0.03 1.54 ± 0.02 1.98 ± 0.70 7.90 ± 0.13

limiting step in the absorption and support previous other studies [16].

clavulanic acid rather than on its plasma concentration.

**Table 4.** Pharmacokinetic parameters of clavulanic acid.

compartmental analysis (**Tables 4** and **5**).

same 125 mg dose.

The software Kinetica™ Ver 4.4.1. (Thermo Electron Corporation, USA) used to determine all parameters including both compartmental and non-compartmental analysis and interrelated for any variation in AUCt and demographic facts. The parameters determined were:

Cmax, Tmax (observed and calculated), Ka, Kel, T½ ka, T½α. AUC*0*−*t*, AUC*0*−*α,* ƛz, Vz, Vss, AUC last. AUC extrapolated, AUC total, %AUC extrapolated, AUMC and MRT.

where Cmax is maximum plasma concentration of drug(mg/L), Tmax is time required to achieve Cmax(h), Ka is absorption rate constant, AUC0−t, AUC0−α, AUC last with the help of linear trapezoidal method to find area under plasma-concentration time curve up to last measurable concentration (mg.h./L),Kel is elimination half-life (h), ƛz is terminal rate constant, Vz is apparent volume of distribution during terminal phase (L/kg), Vss is apparent volume of distribution at steady state (L/kg), AUMC is area under the first moment of concentrationtime curve from time zero to infinity (amount.(time)2 /volume) and MRT is mean residence time (h).

The mean ± plasma concentration time-curve of co-amoxiclav (250/125 mg) tablet of formulation 1 is shown in **Figure 4** in healthy volunteers (n = 8). The other formulation showed similar results. The half-life of all both formulation was 1.34 ± 0.06 h for amoxicillin and 1.20 ± 0.03 h for clavulanic acid.

The area under the concentration-time curve of clavulanic acid is the best measure of the absorption and beneficial effects in the recipient. Calculating the area under the curve using trough and peak blood levels versus using isolated readings for either of these levels alone is the most effective method of monitoring.

The mean AUC0−α values calculated through compartmental analysis were 26.81 ± 0.70 μg.h/ ml for amoxicillin while for clavulanate potassium 7.90 ± 0.13 μg.h/ml. The values of mean AUClast and AUCtot from non-compartmental analysis were 23.33 ± 0.70 and 27.96 ± 0.76 μg.h/ ml for amoxicillin. The clavulanate potassium showed the values of AUC last and AUC tot were 7.05 ± 0.11 and 7.70 ± 0.16 μg.h/ml.

**Figure 4.** Comparison of mean (±S.D) plasma-concentration time profile of amoxicillin and clavulanic acid after oral dose of 250/125 mg co-amoxiclav tablet (n-8: Formulation 1).

The maximum concentration Cmax of amoxicillin was achieved in 1.85 ± 0.01 h for amoxicillin in compartmental. Similarly Cmax for clavulanate potassium was achieved in 1.56 ± 0.01 h in compartmental analysis (**Tables 4** and **5**).

model with lag time, first order absorption and first order elimination was selected for both

The software Kinetica™ Ver 4.4.1. (Thermo Electron Corporation, USA) used to determine all parameters including both compartmental and non-compartmental analysis and interrelated

Cmax, Tmax (observed and calculated), Ka, Kel, T½ ka, T½α. AUC*0*−*t*, AUC*0*−*α,* ƛz, Vz, Vss,

where Cmax is maximum plasma concentration of drug(mg/L), Tmax is time required to achieve Cmax(h), Ka is absorption rate constant, AUC0−t, AUC0−α, AUC last with the help of linear trapezoidal method to find area under plasma-concentration time curve up to last measurable concentration (mg.h./L),Kel is elimination half-life (h), ƛz is terminal rate constant, Vz is apparent volume of distribution during terminal phase (L/kg), Vss is apparent volume of distribution at steady state (L/kg), AUMC is area under the first moment of concentration-

The mean ± plasma concentration time-curve of co-amoxiclav (250/125 mg) tablet of formulation 1 is shown in **Figure 4** in healthy volunteers (n = 8). The other formulation showed similar results. The half-life of all both formulation was 1.34 ± 0.06 h for amoxicillin and 1.20 ± 0.03 h

The area under the concentration-time curve of clavulanic acid is the best measure of the absorption and beneficial effects in the recipient. Calculating the area under the curve using trough and peak blood levels versus using isolated readings for either of these levels alone is

The mean AUC0−α values calculated through compartmental analysis were 26.81 ± 0.70 μg.h/ ml for amoxicillin while for clavulanate potassium 7.90 ± 0.13 μg.h/ml. The values of mean AUClast and AUCtot from non-compartmental analysis were 23.33 ± 0.70 and 27.96 ± 0.76 μg.h/ ml for amoxicillin. The clavulanate potassium showed the values of AUC last and AUC tot

**Figure 4.** Comparison of mean (±S.D) plasma-concentration time profile of amoxicillin and clavulanic acid after oral dose

/volume) and MRT is mean residence

for any variation in AUCt and demographic facts. The parameters determined were:

AUC last. AUC extrapolated, AUC total, %AUC extrapolated, AUMC and MRT.

amoxicillin and clavulanate potassium.

time curve from time zero to infinity (amount.(time)2

the most effective method of monitoring.

were 7.05 ± 0.11 and 7.70 ± 0.16 μg.h/ml.

of 250/125 mg co-amoxiclav tablet (n-8: Formulation 1).

time (h).

98 Medicinal Chemistry

for clavulanic acid.

The reported values of Vss after IV administration for amoxicillin is 0.28 ± 0.06 L/kg, and the Vss of clavulanic acid as 0.24 ± 0.06 L/kg, showing ratio for the volume of distribution between clavulanic acid and amoxicillin as 0.8571 [14] and therefore on the basis of this the ratio of amoxicillin to clavulanic acid AUCs should be 3.4. when co-amoxiclav is at dose of 250/125. The author observed in this study, the lowest AUCt amoxicillin/clavulanic acid ratio was 2.7 ± 0.50 at the lower doses used. This would assume equal absorption of both amoxicillin and clavulanic acid. But in the same dose amoxicillin/clavulanic acid AUCt ratios was higher that would suggest that with a similar amoxicillin absorption, clavulanic acid absorption must have been reduced. The reported absolute bioavailability of clavulanic acid, when co-administered with amoxicillin has been ranged from 31.4 to 98.8% [10]. Further it is reported that there is no major alteration in the mean AUCt of 125 mg clavulanate when it is administered along 500 mg of amoxicillin, but it creates marked impact on the coefficient of variation for the AUC which alter from 27.6% for clavulanic acid alone to 45.6% when given with amoxicillin [15]. Various other studies showed mean absorption up to 97% when clavulanic acid administered alone with minor inter-patient variability. It indicates interaction between absorption of amoxicillin and clavulanic acid [11, 12] . The author further found that there was no significant variation in the AUC observed for amoxicillin in this study either among subjects, on the basis of demographic data, or between formulations, once corrected for the dose. On the contrary, high variability was seen between subjects in the AUC of clavulanic acid (**Figure 5**). There were all healthy male subjects (with normal renal function), and it is difficult to explain the high variability seen in the clavulanic acid AUC on patient factors. However, it has been reported in other studies [13]. Also study observed broadened Tmax i.e. increase lag time indicating a rate limiting step in the absorption process. The authors being able to show that two different co-amoxiclav formulations each gave a variation in the absorption, or in the AUCt value, of clavulanic acid for the same 125 mg dose.

In a study reported broadened Tmax with high dose of amoxicillin (875 mg) indicate a rate limiting step in the absorption and support previous other studies [16].

In this study the authors tried to show variation in the absorption or in AUCt value of clavulanic acid at 125 mg dose with two formulations. Although we did not find any report of therapy failure among the patients due to this variation and its clinical efficacy has been maintained. It would suggest that it is more important to focus on the absolute or fixed amount of clavulanic acid rather than on its plasma concentration.


**Table 4.** Pharmacokinetic parameters of clavulanic acid.


**Author details**

Sheikh Abdul Khaliq3

2 Unilever Karachi, Pakistan

1982;**22**:353-357

Disease Journal. 1998;**17**:957-962

and Therapeutics. 1987;**10**:105-113

Biopharmaceutics. 2008;**70**(2):641-648

Analysis. 2007;**45**(3):531-534

\*, Mohammad Jiyad Shaikh<sup>2</sup>

\*Address all correspondence to: anabfatima@gmail.com

and Farah Khalid<sup>1</sup>

3 Faculty of Pharmacy, Hamdard University, Karachi, Pakistan

1 Faculty of Pharmacy, Dow University of Health Sciences, Karachi, Pakistan

, Hina Zahid<sup>1</sup>

Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-Lactamase Isolated from *Streptomyces clavuligerus*...

[1] Cooper CE, Slocombe B, White AR. Effect of low concentrations of clavulanic acid on the invitro activity of amoxycillin against β-lactamase producing *Branhamella catharrhasis* and *Haemophilus influenzae*. The Journal of Antimicrobial Chemotherapy. 1990;**6**:371-380

[2] Adam D, de Visser I, Koeppe P. Pharmacokinetics of amoxicillin andclavulanic acid administered alone and in combination. Antimicrobial Agents and Chemotherapy.

[3] Reed MD. The clinical pharmacology of amoxicillin and clavulanic acid. Pediatric Infectious

[4] Soback S, Bor A, Kurtz B, Paz R, Ziv G. Clavulanate-potentiated amoxycillin: in vitro antibacterial activity and oral bioavailability in calves. Journal of Veterinary Pharmacology

[5] Todd PA, Benfield P. Amoxicillin/clavulanic acid: An update of its antibacterial activity,

[6] Vogelmann B, Gudmundsson S, Leggett J, Turnidge J, Ebert S, Craig WA. Correlation of antimicrobial pharmacokinetic parameters with therapeutically efficacy in an animal

[7] Weitschies W, Friedrich C, Wedemeyer RS, Schmidtmann M, et al. Bioavailability of amoxicillin and clavulanic acid from extended release tablets depends on intragastric tablet deposition and gastric emptying. European Journal of Pharmaceutics and

[8] Seyed Mohsen Foroutan, AfshinZarghi, Alireza Shafaati, Arash Khoddam et al.Simultaneous determination of amoxicillin and clavulanic acid in human plasma by isocratic reversed-phase HPLC using UV detection, Journal of Pharmaceutical and Biomedical

pharmacokinetic properties and therapeutic use. Drugs. 1990;**39**:264-307

model. The Journal of Infectious Diseases. 1998;**158**:831-847

, Ishart Younus<sup>3</sup>

,

http://dx.doi.org/10.5772/intechopen.79409

101

Anab Fatima<sup>1</sup>

**References**

**Table 5.** Pharmacokinetic parameters of amoxicillin.

**Figure 5.** Individual AUCts of amoxicillin plotted versus the AUCts of clavulanic acid. It can be seen that there is a variation in the AUCts of clavulanic acid, with little variation in those of amoxicillin (95% confidence interval).

## **3. Conclusions**

In conclusion, variable absorption nature of clavulanic acid has been highlighted with alteration in AUCt ratio of co-amoxiclav without any known cause. However, it is evident from clinical data that there is not any variability in the efficacy of co-amoxiclav and that the current dosage ratio of 4:1 holds a traditional value.

The study requires further evaluation to find out the reason for this variation.

## **Acknowledgements**

The authors are thankful to National Bioethics committee, Ministry of Health, Islamabad, Pakistan, for expert ethical review and guidance. Also humble co-operation from administrative and Paramedic staff of Imam Clinic Hospital Karachi and from all volunteers is highly acknowledged.

### **Conflict of interest**

There is no actual or potential conflict of interest between authors relative to this activity including financial relationship.

## **Author details**

Anab Fatima<sup>1</sup> \*, Mohammad Jiyad Shaikh<sup>2</sup> , Hina Zahid<sup>1</sup> , Ishart Younus<sup>3</sup> , Sheikh Abdul Khaliq3 and Farah Khalid<sup>1</sup>


## **References**

**3. Conclusions**

100 Medicinal Chemistry

**Acknowledgements**

**Conflict of interest**

including financial relationship.

acknowledged.

rent dosage ratio of 4:1 holds a traditional value.

**Table 5.** Pharmacokinetic parameters of amoxicillin.

In conclusion, variable absorption nature of clavulanic acid has been highlighted with alteration in AUCt ratio of co-amoxiclav without any known cause. However, it is evident from clinical data that there is not any variability in the efficacy of co-amoxiclav and that the cur-

**Figure 5.** Individual AUCts of amoxicillin plotted versus the AUCts of clavulanic acid. It can be seen that there is a variation in the AUCts of clavulanic acid, with little variation in those of amoxicillin (95% confidence interval).

**Formulation Dose (mg) T1/2 (h) Tmax (h) Cmax (μg/L) AUC (μg.h/L)** AMCL1 250 1.34 ± 0.06 1.85 ± 0.01 2.98 ± 0.27 26.81 ± 0.70 AMCL2 250 1.32 ± 0.05 1.83 ± 0.02 3.3 ± 1.12 26.98 ± 0.83

The authors are thankful to National Bioethics committee, Ministry of Health, Islamabad, Pakistan, for expert ethical review and guidance. Also humble co-operation from administrative and Paramedic staff of Imam Clinic Hospital Karachi and from all volunteers is highly

There is no actual or potential conflict of interest between authors relative to this activity

The study requires further evaluation to find out the reason for this variation.


[9] Vree TB, Dammers E, Exler PS. Identical pattern of highly variable absorption of clavulanic acid from four different oral formulations of co-amoxiclav in healthy subjects. The Journal of Antimicrobial Chemotherapy. 2003;**51**(2):373-378

**Chapter 7**

**Provisional chapter**

**New Antituberculosis Drug FS-1**

**New Antituberculosis Drug FS-1**

DOI: 10.5772/intechopen.80795

The new iodine complex (FS-1), including molecular iodine, which is coordinated by lithium, magnesium halides, and bioorganic ligands, possesses high bactericidal activity against various microorganisms, including *Mycobacterium* sp., *Staphylococcus aureus* MRSA and MSSA, *Escherichia coli*, *Pseudomonas aeruginosa*, etc. FS-1 has synergistic properties with a broad class of antibiotics. The experimental model of tuberculosis in guinea pigs caused by clinical multidrug-resistant strains of *Mycobacterium tuberculosis* shows antituberculosis, immunomodulatory, and anti-inflammatory activity. FS-1 is characterized by low acute toxicity and lack of genotoxicity and mutagenicity. FS-1 is well distributed to organs and tissues; its pharmacokinetics is linear. The maximum nontoxic dose is 100 mg/kg for rats after 28-day oral administration and 30 mg/kg for rabbits after 180-day oral administration.

**Keywords:** *Mycobacterium tuberculosis*, iodine, complex, antimicrobial activity, antimicrobial resistance, tuberculosis, antituberculosis drug, preclinical trials, toxicity

Antimicrobial resistance (AMR) has long been recognized as a global problem [1, 2]. At that, the solution to this problem is complicated by the fact that as soon as a new antibiotic appears, it is immediately reported about resistance to it. For example, some isolates of *Pseudomonas aeruginosa* and *Burkholderia cenocepacia* showed intermediate resistance to a new antibiotic eravacycline as early as at the stage of clinical trials, although the antibiotic effectiveness is very high against other bacteria, including the multiresistant ones [3]. Sometimes this resistance can be natural, for example, to pyrazinamide in *Mycobacterium bovis* [4, 5]. A widely practiced method of reducing AMR, the cyclic and mixed use of antibiotics that belong to

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Rinat Islamov, Bahkytzhan Kerimzhanova and

Rinat Islamov, Bahkytzhan Kerimzhanova

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

alternative classes, has proved to be ineffective [6].

http://dx.doi.org/10.5772/intechopen.80795

Alexander Ilin

and Alexander Ilin

**Abstract**

**1. Introduction**


**Chapter 7 Provisional chapter**

#### **New Antituberculosis Drug FS-1 New Antituberculosis Drug FS-1**

Rinat Islamov, Bahkytzhan Kerimzhanova and Alexander Ilin Rinat Islamov, Bahkytzhan Kerimzhanova and Alexander Ilin

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80795

#### **Abstract**

[9] Vree TB, Dammers E, Exler PS. Identical pattern of highly variable absorption of clavulanic acid from four different oral formulations of co-amoxiclav in healthy subjects. The

[10] Nilsson-Ehle I, Fellner H, Hedström SA, Nilsson-Ehle P, Sjövall J. Pharmacokinetics of clavulanic acid, given in combination with amoxicillin, in volunteers. Journal of Anti-

[11] Allen GD, Coates PE, Davies BE. On the absorption of clavulanic acid. Biopharmaceutics

[12] Bolton GC, Allen GD, Davies BE, Filer CW, Jeffery DJ. The disposition of clavulanic acid

[13] Elias Jones A, Barnes ND, Tasker TCG, Horton R. Pharmacokinetics of intravenous amo xycillin and potassium clavulanate in seriously ill children. Journal of Antimicrobial

[14] Horber FF, Frey FJ, Descoeurders C, Murray AT, Reubi FC. Differential effect of impaired renal function on the kinetics of clavulanic acid and amoxicillin. Antimicrobial Agents

[15] Witkowski G, Lode H, Höffken G, Koeppe P. Pharmacokinetic studies of amoxicillin, potassium clavulanate and their combination. European Journal of Clinical Microbiology.

[16] Chulavatnatol S, Charles BG. Determination of dose-dependent absorption of amoxycillin from urinary excretion data in healthy subjects. British Journal of Clinical Pharmacology.

Journal of Antimicrobial Chemotherapy. 2003;**51**(2):373-378

microbial Chemotherapy. 1985;**16**:491-496

and Drug Disposition. 1988;**9**:127-136

in man. Xenobiotica. 1986;**16**:853-863

Chemotherapy. 1990;**25**:269-274

1982;**1**:233-237

102 Medicinal Chemistry

1994;**38**:274-277

and Chemotherapy. 1986;**29**:614-619

The new iodine complex (FS-1), including molecular iodine, which is coordinated by lithium, magnesium halides, and bioorganic ligands, possesses high bactericidal activity against various microorganisms, including *Mycobacterium* sp., *Staphylococcus aureus* MRSA and MSSA, *Escherichia coli*, *Pseudomonas aeruginosa*, etc. FS-1 has synergistic properties with a broad class of antibiotics. The experimental model of tuberculosis in guinea pigs caused by clinical multidrug-resistant strains of *Mycobacterium tuberculosis* shows antituberculosis, immunomodulatory, and anti-inflammatory activity. FS-1 is characterized by low acute toxicity and lack of genotoxicity and mutagenicity. FS-1 is well distributed to organs and tissues; its pharmacokinetics is linear. The maximum nontoxic dose is 100 mg/kg for rats after 28-day oral administration and 30 mg/kg for rabbits after 180-day oral administration.

DOI: 10.5772/intechopen.80795

**Keywords:** *Mycobacterium tuberculosis*, iodine, complex, antimicrobial activity, antimicrobial resistance, tuberculosis, antituberculosis drug, preclinical trials, toxicity

#### **1. Introduction**

Antimicrobial resistance (AMR) has long been recognized as a global problem [1, 2]. At that, the solution to this problem is complicated by the fact that as soon as a new antibiotic appears, it is immediately reported about resistance to it. For example, some isolates of *Pseudomonas aeruginosa* and *Burkholderia cenocepacia* showed intermediate resistance to a new antibiotic eravacycline as early as at the stage of clinical trials, although the antibiotic effectiveness is very high against other bacteria, including the multiresistant ones [3]. Sometimes this resistance can be natural, for example, to pyrazinamide in *Mycobacterium bovis* [4, 5]. A widely practiced method of reducing AMR, the cyclic and mixed use of antibiotics that belong to alternative classes, has proved to be ineffective [6].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pulmonary tuberculosis poses a particular problem. The global WHO report announced the number of new tuberculosis cases detected in 2015—2110.4 million, of which 480,000 new cases of multidrug-resistant tuberculosis (MDR-TB) and 7579 cases of extensively drugresistant TB (XDR-TB). The greatest number of the disease cases is registered in six countries—India, Indonesia, China, Nigeria, Pakistan, and South Africa—60% of the worldwide incidence [7]. Despite the emergence of new antituberculosis drugs, the situation with high resistance of *M. tuberculosis* remains difficult [7, 8]. In this regard, the search and development of new anti-infectious drugs are extremely relevant.

Among the numerous substances with high antimicrobial activity, resistance to which was not detected or it remains at the minimum level, there are polymeric complexes of iodine [9, 10]. Iodine complexes have broad antimicrobial, anti-inflammatory, immunomodulatory, and antitumor activity [11–16]. Really interesting are nanocomposites with molecular iodine, which are superior in their antimicrobial activity to the widely known complex of polyvinylpyrrolidone and iodine (PVP-iodine) [17, 18]. The ability of molecular iodine to form complexes with a variety of properties and compositions with ligands of different nature makes it very promising to develop drugs based on iodine coordination compounds [19–24].

The new drug FS-1 relates to iodine coordination compounds with bioorganic ligands, magnesium, and lithium cations. The active center of FS-1 included α-dextrin helix with molecular iodine (I2 ) that is coordinated with lithium halogenides and amide groups of protein component. Such a structure protects I2 from interaction with bioorganic compounds after oral intake. Bioorganic compounds are only able to compete with I2 in complexing if donor activity is greater as against amide groups [24–26].

(isoniazid-resistant); and *M. bovis* 2, 3, and 5 showed the greatest susceptibility [36]. The MIC

FS-1 causes lysis of the bacterial cell, damaging the cell membrane [37]. A study on the membrane lytic activity of FS-1 on *M. smegmatis* by electron microscopy showed that the bacterial cell lysis occurs within 5–30 minutes at a concentration of FS-1 of 4 μg/mL. In addition, FS-1 inhibits DNA-dependent RNA polymerase (RNAP) of *M. tuberculosis* forming a complex

Most bacterial diseases including tuberculosis are treated with a combination of multiple drugs in a regimen. Synergistic effects with existing drugs are valuable characteristics of a new drug candidate. FS-1 was tested in combination with antibiotics and various first and second antituberculosis drugs (ATBD). Synergy between drugs and FS-1 was determined by chequerboard in vitro [27]. Testing showed that synergy between FS-1 and antituberculosis drugs (ATBD) was observed against both on susceptibility and on multidrug resistance MTB. Among the cephalosporins, only сefamandole showed synergy with FS-1 against clinical isolates *S. aureus*. The index of fractional inhibitory concentration was 0.62 [27]. Thus, according to the results of testing, in vitro the FS-1 is an effective compound of a class of

The most important stage of preclinical studies of new drugs is the evaluation of efficacy in animal models. There are various animal *M. tuberculosis* infection models. The most common are guinea pigs and mice. A classical guinea pig model of tuberculosis caused by highly

of FS-1 against *Y. pestis* and *B. anthracis* was 0.2 mg/mL [35].

*Staphylococcus aureus* ATCC 6538-Р В-RКМ 0039, *S. aureus* ATCC 29213 oxacillin-susceptible strain (MSSA), *S. aureus* ATCC 43300—methicillinand oxacillin-resistant (MRSA), *S. aureus* ATCC 29213, *S. aureus* ATCC 43300, *Escherichia coli* ATCC 25922, and ATCC BAA-196 and *Candida* 

\*\*\*At Kazakh Scientific Center for Quarantine and Zoonotic Diseases. \*\*\*\*At Institute of Experimental Pathology and Parasitology, Bulgaria.

\*\*At Kazakh Scientific Research Veterinary Institute.

*albicans* ATCC 18814

**Typified bacteria Clinical isolates**

\*Collections of microorganisms at the National Scientific Center for Pulmonology, Kazakhstan.

**Table 1.** List of bacteria and their characteristics used to examine antimicrobial activity of FS-1.

*M. tuberculosis* H37Rv\* *M. tuberculosis* MS-115 (MDR), SCAID

187.0 (MDR); 562, 892, 535, and 722\* (isoniazid-resistant); *M. bovis*\*\* 2, 3, and 5;

http://dx.doi.org/10.5772/intechopen.80795

New Antituberculosis Drug FS-1

105

*Yersinia pestis\*\*\*, Bacillus anthracis\*\*\*,* 

*S. aureus\*\*\*\**—114 MRSA and MSSA clinical isolates—*P. aeruginosa* № 4/32, *E. coli* O55

\*\*\*

№ 12, *C. albicans* 3/4

*M. avium* 780

*Brucella* sp*.*

between bacterial DNA and magnesium ion in RNAP [26].

iodine coordination compounds.

**3. In vivo antituberculosis activity**

The developed drug FS-1 possesses broad antimicrobial activity against antibiotic-resistant and antibiotic-susceptible Gram-positive and Gram-negative bacteria, mycobacteria, fungi, and viruses [27–33].

Both liquid and solid dosage forms of FS-1 were produced from the pharmaceutical development process [34]. Preclinical trials of the drug FS-1 were conducted according to the recommendations of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). This chapter will present the major results from preclinical trials of the new drug FS-1, conducted in 2004–2014.

## **2. In vitro antimicrobial activity**

The aim of these studies presented here was to assess the in vitro antimicrobial activity of FS-1. By using a serial dilution technique assay, the activities of FS-1 were tested against both on typified bacteria and on clinical isolates. **Table 1** shows a list of microorganisms that were used to examine the in vitro effectiveness of FS-1 [27–29, 33, 35, 36].

The minimum inhibitory concentration (MIC) was in the range of 0.02–0.3 mg/mL. At the same time, clinical isolates *M. tuberculosis* SCAID 187.0 (MDR); 562, 892, 535, and 722


**Table 1.** List of bacteria and their characteristics used to examine antimicrobial activity of FS-1.

(isoniazid-resistant); and *M. bovis* 2, 3, and 5 showed the greatest susceptibility [36]. The MIC of FS-1 against *Y. pestis* and *B. anthracis* was 0.2 mg/mL [35].

FS-1 causes lysis of the bacterial cell, damaging the cell membrane [37]. A study on the membrane lytic activity of FS-1 on *M. smegmatis* by electron microscopy showed that the bacterial cell lysis occurs within 5–30 minutes at a concentration of FS-1 of 4 μg/mL. In addition, FS-1 inhibits DNA-dependent RNA polymerase (RNAP) of *M. tuberculosis* forming a complex between bacterial DNA and magnesium ion in RNAP [26].

Most bacterial diseases including tuberculosis are treated with a combination of multiple drugs in a regimen. Synergistic effects with existing drugs are valuable characteristics of a new drug candidate. FS-1 was tested in combination with antibiotics and various first and second antituberculosis drugs (ATBD). Synergy between drugs and FS-1 was determined by chequerboard in vitro [27]. Testing showed that synergy between FS-1 and antituberculosis drugs (ATBD) was observed against both on susceptibility and on multidrug resistance MTB. Among the cephalosporins, only сefamandole showed synergy with FS-1 against clinical isolates *S. aureus*. The index of fractional inhibitory concentration was 0.62 [27]. Thus, according to the results of testing, in vitro the FS-1 is an effective compound of a class of iodine coordination compounds.

## **3. In vivo antituberculosis activity**

Pulmonary tuberculosis poses a particular problem. The global WHO report announced the number of new tuberculosis cases detected in 2015—2110.4 million, of which 480,000 new cases of multidrug-resistant tuberculosis (MDR-TB) and 7579 cases of extensively drugresistant TB (XDR-TB). The greatest number of the disease cases is registered in six countries—India, Indonesia, China, Nigeria, Pakistan, and South Africa—60% of the worldwide incidence [7]. Despite the emergence of new antituberculosis drugs, the situation with high resistance of *M. tuberculosis* remains difficult [7, 8]. In this regard, the search and development

Among the numerous substances with high antimicrobial activity, resistance to which was not detected or it remains at the minimum level, there are polymeric complexes of iodine [9, 10]. Iodine complexes have broad antimicrobial, anti-inflammatory, immunomodulatory, and antitumor activity [11–16]. Really interesting are nanocomposites with molecular iodine, which are superior in their antimicrobial activity to the widely known complex of polyvinylpyrrolidone and iodine (PVP-iodine) [17, 18]. The ability of molecular iodine to form complexes with a variety of properties and compositions with ligands of different nature makes it very promising to develop drugs based on iodine coordination com-

The new drug FS-1 relates to iodine coordination compounds with bioorganic ligands, magnesium, and lithium cations. The active center of FS-1 included α-dextrin helix with molecular

The developed drug FS-1 possesses broad antimicrobial activity against antibiotic-resistant and antibiotic-susceptible Gram-positive and Gram-negative bacteria, mycobacteria, fungi,

Both liquid and solid dosage forms of FS-1 were produced from the pharmaceutical development process [34]. Preclinical trials of the drug FS-1 were conducted according to the recommendations of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). This chapter will present the major

The aim of these studies presented here was to assess the in vitro antimicrobial activity of FS-1. By using a serial dilution technique assay, the activities of FS-1 were tested against both on typified bacteria and on clinical isolates. **Table 1** shows a list of microorganisms that were

The minimum inhibitory concentration (MIC) was in the range of 0.02–0.3 mg/mL. At the same time, clinical isolates *M. tuberculosis* SCAID 187.0 (MDR); 562, 892, 535, and 722

results from preclinical trials of the new drug FS-1, conducted in 2004–2014.

used to examine the in vitro effectiveness of FS-1 [27–29, 33, 35, 36].

) that is coordinated with lithium halogenides and amide groups of protein com-

from interaction with bioorganic compounds after oral

in complexing if donor activity

of new anti-infectious drugs are extremely relevant.

pounds [19–24].

104 Medicinal Chemistry

and viruses [27–33].

ponent. Such a structure protects I2

is greater as against amide groups [24–26].

**2. In vitro antimicrobial activity**

intake. Bioorganic compounds are only able to compete with I2

iodine (I2

The most important stage of preclinical studies of new drugs is the evaluation of efficacy in animal models. There are various animal *M. tuberculosis* infection models. The most common are guinea pigs and mice. A classical guinea pig model of tuberculosis caused by highly virulent clinical strains of *M. tuberculosis* MS-115 and SCAID 187.0 isolated from patients with MDR-TB was used in the study on the therapeutic effectiveness of FS-1 [8, 29, 38].

provoked minimal damage to the gastric mucosa. Histological examination of thyroid gland

New Antituberculosis Drug FS-1

107

http://dx.doi.org/10.5772/intechopen.80795

Toxicokinetics (TK) of FS-1 liquid form was examined in male and female rats after single oral administration at doses of 233, 116, and 30 mg/kg [48]. The content of FS-1 was measured in the blood, heart, lungs, liver, kidneys, and spleen. The primary TK parameters were calculated by non-compartmental method. The maximum concentration of FS-1 in the blood (Cmax) and the time it was reached (tmax) were found visually on the graph. The TK parameters for

FS-1 is absorbed quite rapidly from the gastrointestinal tract; the maximum values of its concentrations in the blood were reached within 1–1.5 hours after its administration. During the first 10–30 minutes, the concentration of FS-1 in the blood increased and reached a maximum after 2 hours. The level of the drug was further reduced, and at the end of 96 hours, the detection limit was reached. High values of the volume of distribution, according to the generally accepted point of view, can be interpreted as a sign of a wide distribution of the drug in the body. At the same time, the rate of distribution of FS-1 in the organs was lower than in the blood, therefore, accumulation does not take place. The nature of the relationship between the dose of FS-1 and the area under the pharmacokinetic curve (AUC) in the blood, as well as the constancy of the invariant TK parameters, indicate that the dynamics of drug absorption, distribution, and elimination obey the basic principles of linear kinetics. The

primary metabolites of FS-1 include iodides, which are excreted in the urine [48].

AUC(mg·h/L) 138.2 ± 19.5 382.4 ± 49.6 669.5 ± 103.1 Cl (L·h/kg) 0.22 ± 0.03 0.30 ± 0.04 0.35 ± 0.05 MRT (h) 24.6 ± 3.5 20.2 ± 2.7 23.1 ± 3.6

) 0.026 ± 0.003 0.025 ± 0.004 0.026 ± 0.004

 (L/kg) 8.2 ± 1.2 12.0 ± 1.6 13.5 ± 2.1 Cmax (mg/L) 9.0 ± 0.2 34.1 ± 5.0 45.1 ± 9.6

(h) 26.6 ± 3.7 26.6 ± 3.6 26.6 ± 3.7

**Table 2.** Toxicokinetic parameters of FS-1 after single oral administration of three doses to rats by non-compartmental

max (h) 2 1 1

In addition, chronic toxicity was examined during 180-day oral administration in rabbits. The characteristic symptoms of iodine toxicity in rabbits were not detected at a dose of 30 mg/kg

Despite the fact that iodine does not have mutagenic properties, there is evidence of the effect of high iodide doses on the development of mice. Although the mechanisms of toxic effect of

**30 116 233**

did not reveal the pathological changes [47].

oral doses of FS-1 are given in **Table 2**.

**TK parameter Dose, mg/kg**

[47–51].

β (h−<sup>1</sup>

Vβ

t

t 1/2(β)

method, n = 6.

The efficacy result of the tuberculosis model showed that the FS-1 combination regimen reduced the bacterial load in comparison with standard therapy. A primary characteristic of the therapeutic activity of FS-1 was an increase in the effectiveness when combined with ATBD including isoniazid, rifampicin, pyrazinamide, cycloserine, protionamide, capreomycin, and amikacin [29, 30]. It was noted that in the treatment of tuberculosis in guinea pigs with FS-1 *M. tuberculosis* acquired susceptibility to first-line ATBD. Apparently this is due to the effect of FS-1 on the *M. tuberculosis* genome [30]. In the studies on other bacteria, *Streptococcus mutans* and *S. aureus* and *S. epidermidis*, iodine complexes have been shown to influence the transcription activity in microbial genome [39–41].

The therapeutic effect on the guinea pig body has also been noted. In particular, the exposure to FS-1 inhibits the development of inflammation in tuberculosis and increases the airiness of the lung parenchyma and permeability of capillaries [29]. This is due to the ability of iodine complexes to inhibit the production of NO and TNF-alpha and increase mucociliary clearance in the respiratory tract [15, 42]. In addition, combination therapy with FS-1 and ATBD reduces the incidence of adverse reactions and toxic effects of the drugs in animals [30].

In the studies presented here, the combination of FS-1 in MDR-TB animal models is significantly more effective than therapy only with ATBD.

## **4. Toxicity studies**

The toxicity of FS-1 was examined in both liquid form and tablets [34, 43].

The median cytotoxic concentration (CC50) of FS-1 on the MDCK cells exceeds 5 mg/mL [44]. In vitro and in vivo genotoxicity and mutagenic potential of FS-1 were examined in mice with Ames test, micronucleus, and comet assays. According to the results from the studies, FS-1 does not induce gene mutations or chromosomal abnormalities [45, 46].

Acute toxicity study of FS-1 liquid dosage form was carried out after intravenous administration to rodents, mice (CD-1), and rats (Wistar) [47]. The median lethal dose (LD50) was 65 mg/kg in mice and 100 mg/kg in rats. After the administration of FS-1, the following signs were observed in animals: exophthalmos, piloerection, impaired motor coordination, paroxysmal convulsions of lower limbs, bradycardia, and tachypnea. Death occurred within 24 hours after the administration of FS-1. Necropsy revealed a disturbance of blood circulation in the heart, liver, and kidneys and pulmonary edema in dead animals. Microscopic examination showed plethora and inflammatory infiltration in the lungs, liver, and kidneys and diapedesis hemorrhages in the myocardium. At the same time, there were no changes in the thyroid gland [47].

After oral administration of FS-1 liquid form to rats (Wistar), LD50 was not achieved. The maximum administered dose of FS-1 was 466 mg/kg. The minimal dose of FS-1 did not cause damage to the mucous membrane of the gastrointestinal tract (GIT). Medium and high doses provoked minimal damage to the gastric mucosa. Histological examination of thyroid gland did not reveal the pathological changes [47].

virulent clinical strains of *M. tuberculosis* MS-115 and SCAID 187.0 isolated from patients with

The efficacy result of the tuberculosis model showed that the FS-1 combination regimen reduced the bacterial load in comparison with standard therapy. A primary characteristic of the therapeutic activity of FS-1 was an increase in the effectiveness when combined with ATBD including isoniazid, rifampicin, pyrazinamide, cycloserine, protionamide, capreomycin, and amikacin [29, 30]. It was noted that in the treatment of tuberculosis in guinea pigs with FS-1 *M. tuberculosis* acquired susceptibility to first-line ATBD. Apparently this is due to the effect of FS-1 on the *M. tuberculosis* genome [30]. In the studies on other bacteria, *Streptococcus mutans* and *S. aureus* and *S. epidermidis*, iodine complexes have been shown to influence the

The therapeutic effect on the guinea pig body has also been noted. In particular, the exposure to FS-1 inhibits the development of inflammation in tuberculosis and increases the airiness of the lung parenchyma and permeability of capillaries [29]. This is due to the ability of iodine complexes to inhibit the production of NO and TNF-alpha and increase mucociliary clearance in the respiratory tract [15, 42]. In addition, combination therapy with FS-1 and ATBD reduces

In the studies presented here, the combination of FS-1 in MDR-TB animal models is signifi-

The median cytotoxic concentration (CC50) of FS-1 on the MDCK cells exceeds 5 mg/mL [44]. In vitro and in vivo genotoxicity and mutagenic potential of FS-1 were examined in mice with Ames test, micronucleus, and comet assays. According to the results from the studies, FS-1

Acute toxicity study of FS-1 liquid dosage form was carried out after intravenous administration to rodents, mice (CD-1), and rats (Wistar) [47]. The median lethal dose (LD50) was 65 mg/kg in mice and 100 mg/kg in rats. After the administration of FS-1, the following signs were observed in animals: exophthalmos, piloerection, impaired motor coordination, paroxysmal convulsions of lower limbs, bradycardia, and tachypnea. Death occurred within 24 hours after the administration of FS-1. Necropsy revealed a disturbance of blood circulation in the heart, liver, and kidneys and pulmonary edema in dead animals. Microscopic examination showed plethora and inflammatory infiltration in the lungs, liver, and kidneys and diapedesis hemorrhages in the myocardium. At the same time, there were no changes in the thyroid gland [47].

After oral administration of FS-1 liquid form to rats (Wistar), LD50 was not achieved. The maximum administered dose of FS-1 was 466 mg/kg. The minimal dose of FS-1 did not cause damage to the mucous membrane of the gastrointestinal tract (GIT). Medium and high doses

the incidence of adverse reactions and toxic effects of the drugs in animals [30].

The toxicity of FS-1 was examined in both liquid form and tablets [34, 43].

does not induce gene mutations or chromosomal abnormalities [45, 46].

MDR-TB was used in the study on the therapeutic effectiveness of FS-1 [8, 29, 38].

transcription activity in microbial genome [39–41].

cantly more effective than therapy only with ATBD.

**4. Toxicity studies**

106 Medicinal Chemistry

Toxicokinetics (TK) of FS-1 liquid form was examined in male and female rats after single oral administration at doses of 233, 116, and 30 mg/kg [48]. The content of FS-1 was measured in the blood, heart, lungs, liver, kidneys, and spleen. The primary TK parameters were calculated by non-compartmental method. The maximum concentration of FS-1 in the blood (Cmax) and the time it was reached (tmax) were found visually on the graph. The TK parameters for oral doses of FS-1 are given in **Table 2**.

FS-1 is absorbed quite rapidly from the gastrointestinal tract; the maximum values of its concentrations in the blood were reached within 1–1.5 hours after its administration. During the first 10–30 minutes, the concentration of FS-1 in the blood increased and reached a maximum after 2 hours. The level of the drug was further reduced, and at the end of 96 hours, the detection limit was reached. High values of the volume of distribution, according to the generally accepted point of view, can be interpreted as a sign of a wide distribution of the drug in the body. At the same time, the rate of distribution of FS-1 in the organs was lower than in the blood, therefore, accumulation does not take place. The nature of the relationship between the dose of FS-1 and the area under the pharmacokinetic curve (AUC) in the blood, as well as the constancy of the invariant TK parameters, indicate that the dynamics of drug absorption, distribution, and elimination obey the basic principles of linear kinetics. The primary metabolites of FS-1 include iodides, which are excreted in the urine [48].

In addition, chronic toxicity was examined during 180-day oral administration in rabbits. The characteristic symptoms of iodine toxicity in rabbits were not detected at a dose of 30 mg/kg [47–51].


Despite the fact that iodine does not have mutagenic properties, there is evidence of the effect of high iodide doses on the development of mice. Although the mechanisms of toxic effect of

**Table 2.** Toxicokinetic parameters of FS-1 after single oral administration of three doses to rats by non-compartmental method, n = 6.

high iodide doses on pregnant females and embryos have not been revealed, it is assumed that this is due to impaired thyroid function in pregnant females. Thyroid hormones in turn affect sex glands of animals [52]. An evaluation of the embryotoxic properties of FS-1 was performed on a bird model using *Gallus gallus* chicken embryos at different developmental stages [53]. Embryotoxicity was assessed by the effect of FS-1 on survival and developmental pathology. At a concentration of 3 mg/mL, the mortality rate of 10-day embryos was 100%, whereas for 12- and 18-day-old embryos, it was 80 and 50%, respectively. At the same time, developmental abnormalities were not detected. The toxicity of FS-1 toward chicken embryos depends on the concentration and developmental stage [54]. The Spearman's correlation coefficient was greater than 0.976 at p < 0.001, which indicates a high dependence. It is known that iodides can exhibit toxicity to the reproductive function in two ways: through oxidative stress in the testis of rats and thyroid dysfunction in pregnant females [52, 55].

Toxicology of iodides and iodates was well studied in numerous animal species, as well as in humans, whereas the data on iodine complexes including Lugol's solution and PVP-iodine

New Antituberculosis Drug FS-1

109

http://dx.doi.org/10.5772/intechopen.80795

The toxicity of FS-1 tablets associated with repeated administration was examined in rats (Wistar) after 28-day oral administration. It was found that the organs for the damaging action of FS-1 included the thyroid gland, liver, and kidneys. There were changes in the blood parameters: the levels of leukocytes and lymphocytes and alanine aminotransferase and aspartate aminotransferase increased. But these changes were reversible, and after 28 days of

It is known that prolonged exposure to or intake of high doses of the iodine-containing drugs is accompanied by thyrotoxic reactions in the form of iodine-induced hypothyroidism [64]. Due to excessive intake of iodine, the Wolff-Chaikoff effect occurs, which develops within a few days [65]. This is accompanied by a temporary reduction of the thyroid hormone level due to a decrease in the Na+/I-symporter activity (NIS) [66]. The Wolff-Chaikoff effect is transient, and the level of hormones with the withdrawal of the iodine-containing drugs is restored in a few days. It was noted that the chronic effect of iodine doses (in Japan, for example, up to several milligrams per day are consumed with food, exceeding the WHO recommended standards (90–200 μg)) frequently does not cause any hypothyroid or thyrotoxic conditions [67]. Therefore, rats were also analyzed for the level of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3). The findings of the studies have shown that the levels of TSH, T3, and T4 in the blood serum of rats did not change. It has been established that the T3/T4 ratio in all studied groups was in the range of 0.20–0.50. The absence of significant changes in the T3/T4 ratio in animals of all groups indicates a normal rate of deiodization. As a result, the highest nontoxic dose (NOAEL) of FS-1 was established, which was of 100 mg/kg [43]. Summarizing the results obtained, it can be noted that high doses of FS-1 lead to the development of thyrotoxicosis in rats and not hypothyroidism, as with excessive intake of

FS-1 is highly effective against various Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. MIC varied over a wide range from 0.02 to 0.3 mg/mL. At that, FS-1 has a synergistic effect with some antimicrobial agents. The main mechanism of action of FS-1 consists in membrane lytic activity. In addition, the investigational drug affects the gene expression in bacteria. The effectiveness of FS-1 in combination with ATBD of the first- and second-line was examined in the guinea pig models of tuberculosis. FS-1 has pronounced anti-inflammatory and antitoxic effects. The CC50 value in MDCK cells is more than 5 mg/mL. This is 16 times higher than the MIC. FS-1 does not have mutagenic and genotoxic properties. The acute oral toxicity of FS-1 in rats is LD50 of more than 2000 mg/kg. The extent of FS-1 distribution in the organs is not greater than in the blood. Its kinetics is linear. The primary metabolites include iodides. At the same time, FS-1 possesses embryonic toxicity in

are very scarce [51, 63].

recovery period, all parameters were normalized [43].

iodides into the animal organism [51].

**5. Conclusion**

One of the problems encountered in the clinical trials of new drugs is that there could be various unforeseen immunotoxic reactions [56, 57]. As already noted, iodine complexes have immunotropic properties [15, 58]. Therefore, the immunotoxicity and allergenicity of FS-1 were studied in various tests:


The study found that FS-1 does not possess anaphylactogenicity, does not cause type I allergic reaction and does not influence the formation of a delayed-type hypersensitivity reaction, does not have immunopathological and immunotoxic effects, and does not cause disorders and/or dysfunctions in the processes involved in normal maintenance of immune status in the tested doses, even against the background of antigenic stimulus [59, 60].

The preparation of a tablet dosage form of FS-1 is the most important stage in the pharmaceutical development of novel drug [34, 61, 62].

After the oral administration of FS-1 tablets to rats (Wistar), LD50 was found to exceed 2000 mg/kg [43]. The observed clinical and pathomorphological signs of damage to the body of animals with FS-1 are similar to the symptoms of poisoning with iodine solutions [50]. Toxicology of iodides and iodates was well studied in numerous animal species, as well as in humans, whereas the data on iodine complexes including Lugol's solution and PVP-iodine are very scarce [51, 63].

The toxicity of FS-1 tablets associated with repeated administration was examined in rats (Wistar) after 28-day oral administration. It was found that the organs for the damaging action of FS-1 included the thyroid gland, liver, and kidneys. There were changes in the blood parameters: the levels of leukocytes and lymphocytes and alanine aminotransferase and aspartate aminotransferase increased. But these changes were reversible, and after 28 days of recovery period, all parameters were normalized [43].

It is known that prolonged exposure to or intake of high doses of the iodine-containing drugs is accompanied by thyrotoxic reactions in the form of iodine-induced hypothyroidism [64]. Due to excessive intake of iodine, the Wolff-Chaikoff effect occurs, which develops within a few days [65]. This is accompanied by a temporary reduction of the thyroid hormone level due to a decrease in the Na+/I-symporter activity (NIS) [66]. The Wolff-Chaikoff effect is transient, and the level of hormones with the withdrawal of the iodine-containing drugs is restored in a few days. It was noted that the chronic effect of iodine doses (in Japan, for example, up to several milligrams per day are consumed with food, exceeding the WHO recommended standards (90–200 μg)) frequently does not cause any hypothyroid or thyrotoxic conditions [67]. Therefore, rats were also analyzed for the level of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3). The findings of the studies have shown that the levels of TSH, T3, and T4 in the blood serum of rats did not change. It has been established that the T3/T4 ratio in all studied groups was in the range of 0.20–0.50. The absence of significant changes in the T3/T4 ratio in animals of all groups indicates a normal rate of deiodization. As a result, the highest nontoxic dose (NOAEL) of FS-1 was established, which was of 100 mg/kg [43]. Summarizing the results obtained, it can be noted that high doses of FS-1 lead to the development of thyrotoxicosis in rats and not hypothyroidism, as with excessive intake of iodides into the animal organism [51].

## **5. Conclusion**

high iodide doses on pregnant females and embryos have not been revealed, it is assumed that this is due to impaired thyroid function in pregnant females. Thyroid hormones in turn affect sex glands of animals [52]. An evaluation of the embryotoxic properties of FS-1 was performed on a bird model using *Gallus gallus* chicken embryos at different developmental stages [53]. Embryotoxicity was assessed by the effect of FS-1 on survival and developmental pathology. At a concentration of 3 mg/mL, the mortality rate of 10-day embryos was 100%, whereas for 12- and 18-day-old embryos, it was 80 and 50%, respectively. At the same time, developmental abnormalities were not detected. The toxicity of FS-1 toward chicken embryos depends on the concentration and developmental stage [54]. The Spearman's correlation coefficient was greater than 0.976 at p < 0.001, which indicates a high dependence. It is known that iodides can exhibit toxicity to the reproductive function in two ways: through oxidative stress

One of the problems encountered in the clinical trials of new drugs is that there could be various unforeseen immunotoxic reactions [56, 57]. As already noted, iodine complexes have immunotropic properties [15, 58]. Therefore, the immunotoxicity and allergenicity of FS-1

in the testis of rats and thyroid dysfunction in pregnant females [52, 55].

**f.** Analysis of changes in mass and cellularity of the popliteal lymph node

**h.** Analysis of the relative count of basophils and eosinophils in human peripheral blood

**i.** Determination of mass and cellularity of immune organs and antibody response in guinea

The study found that FS-1 does not possess anaphylactogenicity, does not cause type I allergic reaction and does not influence the formation of a delayed-type hypersensitivity reaction, does not have immunopathological and immunotoxic effects, and does not cause disorders and/or dysfunctions in the processes involved in normal maintenance of immune status in the

The preparation of a tablet dosage form of FS-1 is the most important stage in the pharmaceu-

After the oral administration of FS-1 tablets to rats (Wistar), LD50 was found to exceed 2000 mg/kg [43]. The observed clinical and pathomorphological signs of damage to the body of animals with FS-1 are similar to the symptoms of poisoning with iodine solutions [50].

**g.** Assessment of specific lysis of human peripheral blood leukocytes

tested doses, even against the background of antigenic stimulus [59, 60].

were studied in various tests:

108 Medicinal Chemistry

**c.** mast cell degranulation

**d.** Anaphylactogenicity

pigs

**a.** Intradermal and conjunctival test

**b.** Active and passive (ovary) cutaneous anaphylaxis

**e.** Induction of delayed-type hypersensitivity reaction

tical development of novel drug [34, 61, 62].

FS-1 is highly effective against various Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. MIC varied over a wide range from 0.02 to 0.3 mg/mL. At that, FS-1 has a synergistic effect with some antimicrobial agents. The main mechanism of action of FS-1 consists in membrane lytic activity. In addition, the investigational drug affects the gene expression in bacteria. The effectiveness of FS-1 in combination with ATBD of the first- and second-line was examined in the guinea pig models of tuberculosis. FS-1 has pronounced anti-inflammatory and antitoxic effects. The CC50 value in MDCK cells is more than 5 mg/mL. This is 16 times higher than the MIC. FS-1 does not have mutagenic and genotoxic properties. The acute oral toxicity of FS-1 in rats is LD50 of more than 2000 mg/kg. The extent of FS-1 distribution in the organs is not greater than in the blood. Its kinetics is linear. The primary metabolites include iodides. At the same time, FS-1 possesses embryonic toxicity in chicken embryos but does not lead to developmental abnormalities. FS-1 does not cause allergic reactions and does not possess immunotoxic properties. The liver, kidney, and thyroid gland are the target organs for toxic injuries induced by repeated administration of FS-1. At the same time, thyrotoxicosis develops but not hypothyroidism. NOAEL value is 100 mg/kg of rats after 28-day oral administration and 30 mg/kg in rabbits after 180-day administration.

[7] Global Tuberculosis Report. 2016. Available from: http://apps.who.int/medicinedocs/

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## **Conflict of interest**

Authors report grants from Ministry of Investments and Development of the Republic of Kazakhstan, during the conduct of the studies.

## **Author details**

Rinat Islamov\*, Bahkytzhan Kerimzhanova and Alexander Ilin

\*Address all correspondence to: renatislamov@gmail.com

Scientific Center for Anti-Infectious Drugs, Almaty, Kazakhstan

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Authors report grants from Ministry of Investments and Development of the Republic of

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resistant *Mycobacterium tuberculosis* induced by a nanomolecular iodine-containing complex FS-1. Frontiers in Cellular and Infection Microbiology. 2017;**7**:151. DOI: 10.3389/ fcimb.2017.00151

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[21] Mahmoud KR, Refat MS, Sharshar T, Adam MA, Manaaa E-SA. Synthesis of amino acid iodine charge transfer complexes in situ methanolic medium: Chemical and physical investing. Journal of Molecular Liquids. **222**:1061-1067. DOI: 10.1016/j.molliq.2016.07.138

[22] Berdibay SB, Paretskaya NA, Sabitov AN, Islamov RA, Tamazyan RA, Tokmoldin SZ, et al. Phenylalanine – Iodine complex and its structure. News of the National Academy of Sciences of the Republic of Kazakhstan Physico-Mathematical Series. 2017;**2**:5-9 [23] Barinov D, Bekesheva K, Ustenova G, Kurmanalieva AR, Kalykova AS, Ilin AI. IR spectroscopic study of substances containing iodine adduct. Research Journal of

[24] Yuldasheva GA, Zhidomirov GM, Ilin AI. Effect of α-dextrin nanoparticles on the structure of iodine complexes with polypeptides and alkali metal halogenides, and on the mechanisms of their anti-human immunodeficiency virus and anticancer activity. In: Grumezescu A, editor. Design and Development of New Nanocarriers. Elsevier Inc;

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[65] Wolff J, Chaikoff IL. The inhibitory action of excessive iodide upon the synthesis of diiodotyrosine and of thyroxine in the thyroid gland of the normal rat. Endocrinology. 1948;**43**:174-179. DOI: 10.1210/endo-43-3-174

**Chapter 8**

**Provisional chapter**

**Clinical Relevance of Medicinal Plants and Foods of**

**Clinical Relevance of Medicinal Plants and Foods of** 

DOI: 10.5772/intechopen.79971

**Vegetal Origin on the Activity of Cytochrome P450**

Drug metabolism is a pharmacokinetic process whose main objective is to modify the chemical structure of drugs to easily excretable compounds. This process is carried out through phase I and phase II reactions. The enzymes of cytochrome P450 (CYP450) participate in phase I reactions, and their activity can be inhibited or induced by xenobiotics. The aim of this chapter is to study the clinical relevance of the induction and inhibition of CYP450, by describing the effect that some bioactive compounds present in medicinal plants or foods can modify, either increasing or decreasing the activity of CYP450 enzymes and with it modify the bioavailability and depuration of drugs. Examples will be described on the interaction of medicinal plants and foods of vegetal origin that when combined with some drugs can generate toxicity or therapeutic failure; this will allow gathering relevant information on the adequate pharmacological management in differ-

**Keywords:** cytochrome P450, drug metabolism, medicinal plants, foods of vegetal

When a patient is in pharmacological treatment, and at some point a pharmacological response different from the expected one is observed, it is possible to think that a pharmacological interaction occurred. This occurs when a drug is administered or consumed in combination

**Vegetal Origin on the Activity of Cytochrome P450**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Sandra N. Jiménez-García, Vicente Beltrán Campos,

Sandra N. Jiménez-García, Vicente Beltrán Campos,

Gabriel Herrera Pérez and Rafael Vargas-Bernal

Gabriel Herrera Pérez and Rafael Vargas-Bernal

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Xóchitl S. Ramírez-Gómez,

Xóchitl S. Ramírez-Gómez,

Esmeralda Rodríguez Miranda,

Esmeralda Rodríguez Miranda,

http://dx.doi.org/10.5772/intechopen.79971

**Abstract**

ent clinical situations.

**1. Introduction**

origin toxicity, therapeutic failure


#### **Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450 Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450**

DOI: 10.5772/intechopen.79971

Xóchitl S. Ramírez-Gómez, Sandra N. Jiménez-García, Vicente Beltrán Campos, Esmeralda Rodríguez Miranda, Gabriel Herrera Pérez and Rafael Vargas-Bernal Xóchitl S. Ramírez-Gómez, Sandra N. Jiménez-García, Vicente Beltrán Campos, Esmeralda Rodríguez Miranda, Gabriel Herrera Pérez and Rafael Vargas-Bernal

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79971

#### **Abstract**

[65] Wolff J, Chaikoff IL. The inhibitory action of excessive iodide upon the synthesis of diiodotyrosine and of thyroxine in the thyroid gland of the normal rat. Endocrinology.

[66] Eng PH, Cardona GR, Fang SL, Previti M, Alex S, Carrasco N, et al. Escape from the acute Wolff-Chaikoff effect is associated with a decrease in thyroid sodium/iodide symporter messenger ribonucleic acid and protein. Endocrinology. 1999;**140**:3404-3410. DOI:

[67] Larsen PR, Davies TF, Schlumberger MJ, Hay ID. Thyroid physiology and diagnostic evaluation of patients with thyroid disorders. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, editors. Williams Textbook of Endocrinology. 10th ed. Philadelphia: WB

1948;**43**:174-179. DOI: 10.1210/endo-43-3-174

10.1210/endo.140.8.6893

116 Medicinal Chemistry

Saunders Company. pp. 331-373

Drug metabolism is a pharmacokinetic process whose main objective is to modify the chemical structure of drugs to easily excretable compounds. This process is carried out through phase I and phase II reactions. The enzymes of cytochrome P450 (CYP450) participate in phase I reactions, and their activity can be inhibited or induced by xenobiotics. The aim of this chapter is to study the clinical relevance of the induction and inhibition of CYP450, by describing the effect that some bioactive compounds present in medicinal plants or foods can modify, either increasing or decreasing the activity of CYP450 enzymes and with it modify the bioavailability and depuration of drugs. Examples will be described on the interaction of medicinal plants and foods of vegetal origin that when combined with some drugs can generate toxicity or therapeutic failure; this will allow gathering relevant information on the adequate pharmacological management in different clinical situations.

**Keywords:** cytochrome P450, drug metabolism, medicinal plants, foods of vegetal origin toxicity, therapeutic failure

#### **1. Introduction**

When a patient is in pharmacological treatment, and at some point a pharmacological response different from the expected one is observed, it is possible to think that a pharmacological interaction occurred. This occurs when a drug is administered or consumed in combination

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

with other drugs, foods, or medicinal plants. In this context, changes in responses to drugs can be positive or negative for the patient. However, it is of particular interest to study the negative changes in pharmacological responses such as intoxication or therapeutic failure.

In this chapter, we focus on describing the effect of the interaction between drugs, medicinal plants, and foods of vegetable origin on the activity of cytochrome P450. Due to the natural products may modify the plasmatic concentrations of the drugs, either by inhibition or induction enzymatic, respectively.

In clinical practice, it is very important to know this topic to identify which medicinal plants and foods of vegetable origin should not be consumed when the patient is in pharmacological treatment and to avoid suffering a change in the response to medications that they consume by prescription and that could put their lives at risk.

## **2. General aspect of pharmacokinetics**

To understand the effect of the chemical compounds, present in some medicinal plants and foods of vegetable origin on the activity of cytochrome P450 (CYP450), we will start with a brief description of the pharmacokinetics because the CYP450 participates in the phase I reactions of drug metabolism.

Pharmacokinetics is the branch of pharmacology that is responsible for studying and explaining the processes by which drugs are absorbed, distributed, metabolized, and eliminated from organism [1, 2]. It is important to know these pharmacokinetics processes and how they influence the bioavailability of drugs [2].

Bioavailability refers to the amount of drug found in the bloodstream and is available to exert its pharmacological effect [3]. However, if the plasma quantity of a drug is modified, the pharmacological response will be modified [1–3]. The four pharmacokinetic processes influence the bioavailability of the drugs. In the process of metabolism, the plasma concentrations of the drugs can be modified, either by inhibition or by induction of different CYP isoenzymes, as shown in **Figure 1**.

time (*tmax*). These parameters are specific for each drug [6]. The *Cmax* of a drug is within the

Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450

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119

On the other hand, after several administrations of a drug, the final concentration begins to increase due to the remainder of the previous dose, until reaching a constant concentration called the equilibrium state [1]. Steady state is usually achieved after four to five half-lives [1, 2]. The half-

1/2) is defined as the time required by a drug to decrease its initial concentration by half [1]. In the equilibrium state, the drug plasmatic concentrations are within the range of therapeutic effect. If the patient suspends the administration of the drug, the plasma levels fall to concentrations below the therapeutic level causing therapeutic failure. Generally, the elimination of

When the patient does not remember if took the dose of the drug and decides to take the dose thinks was needed, the concentration of that drug will accumulate, then the plasma concentration reaches levels above the therapeutic concentration, and additionally, some signs of toxicity begin

In this chapter, we will focus on describing the effect that some compounds present in medicinal plants and some foods of vegetable origin can have on the activity of cytochrome P450 enzymes. The World Health Organization (WHO) estimates that more than 80% of

therapeutic range [4].

a drug is carried out after four to five half-lives [1–3].

**Figure 1.** Effect of drug metabolism on the pharmacological response.

life (*t*

to appear [5].

The following example makes it easier to understand the importance of adherence to treatment to avoid fluctuations in plasmatic concentration. When patients are in pharmacological treatment, it is important that dosage regimen be complied. For example, if the prescription is 500 mg of acetaminophen every 8 h, this patient should be taken exactly three tablets of 500 mg of acetaminophen per day.

In order for patient has an adequate pharmacological response to acetaminophen, and a lower probability of presenting adverse effects or therapeutic failure, the amount of drug and the time of administration indicated in each shot must be respected. If, patient modifies any of these two variables, the plasma concentration of the drug changes and with it its response also changes [4].

When a single dose of drug is administered orally, after a certain time, the plasma concentrations of the drug are enlarged until reaching a maximum level. This maximum point is known as maximum plasma concentration (*Cmax*), and it is reached in a determined maximum Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450 http://dx.doi.org/10.5772/intechopen.79971 119

**Figure 1.** Effect of drug metabolism on the pharmacological response.

with other drugs, foods, or medicinal plants. In this context, changes in responses to drugs can be positive or negative for the patient. However, it is of particular interest to study the negative changes in pharmacological responses such as intoxication or therapeutic failure.

In this chapter, we focus on describing the effect of the interaction between drugs, medicinal plants, and foods of vegetable origin on the activity of cytochrome P450. Due to the natural products may modify the plasmatic concentrations of the drugs, either by inhibition or induc-

In clinical practice, it is very important to know this topic to identify which medicinal plants and foods of vegetable origin should not be consumed when the patient is in pharmacological treatment and to avoid suffering a change in the response to medications that they consume

To understand the effect of the chemical compounds, present in some medicinal plants and foods of vegetable origin on the activity of cytochrome P450 (CYP450), we will start with a brief description of the pharmacokinetics because the CYP450 participates in the phase I reac-

Pharmacokinetics is the branch of pharmacology that is responsible for studying and explaining the processes by which drugs are absorbed, distributed, metabolized, and eliminated from organism [1, 2]. It is important to know these pharmacokinetics processes and how they

Bioavailability refers to the amount of drug found in the bloodstream and is available to exert its pharmacological effect [3]. However, if the plasma quantity of a drug is modified, the pharmacological response will be modified [1–3]. The four pharmacokinetic processes influence the bioavailability of the drugs. In the process of metabolism, the plasma concentrations of the drugs can be modified, either by inhibition or by induction of different CYP isoenzymes,

The following example makes it easier to understand the importance of adherence to treatment to avoid fluctuations in plasmatic concentration. When patients are in pharmacological treatment, it is important that dosage regimen be complied. For example, if the prescription is 500 mg of acetaminophen every 8 h, this patient should be taken exactly three tablets of

In order for patient has an adequate pharmacological response to acetaminophen, and a lower probability of presenting adverse effects or therapeutic failure, the amount of drug and the time of administration indicated in each shot must be respected. If, patient modifies any of these two variables, the plasma concentration of the drug changes and with it its response

When a single dose of drug is administered orally, after a certain time, the plasma concentrations of the drug are enlarged until reaching a maximum level. This maximum point is known as maximum plasma concentration (*Cmax*), and it is reached in a determined maximum

tion enzymatic, respectively.

118 Medicinal Chemistry

tions of drug metabolism.

as shown in **Figure 1**.

also changes [4].

500 mg of acetaminophen per day.

by prescription and that could put their lives at risk.

**2. General aspect of pharmacokinetics**

influence the bioavailability of drugs [2].

time (*tmax*). These parameters are specific for each drug [6]. The *Cmax* of a drug is within the therapeutic range [4].

On the other hand, after several administrations of a drug, the final concentration begins to increase due to the remainder of the previous dose, until reaching a constant concentration called the equilibrium state [1]. Steady state is usually achieved after four to five half-lives [1, 2]. The halflife (*t* 1/2) is defined as the time required by a drug to decrease its initial concentration by half [1].

In the equilibrium state, the drug plasmatic concentrations are within the range of therapeutic effect. If the patient suspends the administration of the drug, the plasma levels fall to concentrations below the therapeutic level causing therapeutic failure. Generally, the elimination of a drug is carried out after four to five half-lives [1–3].

When the patient does not remember if took the dose of the drug and decides to take the dose thinks was needed, the concentration of that drug will accumulate, then the plasma concentration reaches levels above the therapeutic concentration, and additionally, some signs of toxicity begin to appear [5].

In this chapter, we will focus on describing the effect that some compounds present in medicinal plants and some foods of vegetable origin can have on the activity of cytochrome P450 enzymes. The World Health Organization (WHO) estimates that more than 80% of the population of developed and underdeveloped countries use medicinal plants as a first resource for their health care and, on the other hand, there is a context cultural acceptance of the traditional practice with herbs, which makes its use popular and that in many cases patients combine their pharmacological treatment with herbal treatment [6–10].

the amount of drug that was not biotransformed enters the liver through the portal circulation, and there in the hepatocytes the metabolism process *per se* is carried out [24]. The amount of drug remaining after liver extraction is bioavailable to give an adequate pharmacological response. It is important not to modify this bioavailability because the effective doses of the drugs used in the clinical ready are considered as the effect of metabolism of the first step. Above all, caution should be exercised in drugs with a narrow safety margin, such as barbi-

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When drugs are biotransformed by phase I reactions, and the molecule obtained is not polar enough to be eliminated, their biotransformation continues through phase II reactions. In this phase, the metabolites are generally conjugated with glucuronide acid, giving a polar molecule with a higher molecular weight [1, 2]. These conjugates are secreted from the hepatocyte into the bile and stored there in the form of a drug-glucuronide complex; when the bile is secreted in the intestine by some stimulus, the drug-bile complex is eliminated through the feces [25]. However, intestinal microorganisms produce various enzymes, such as betaglucuronidases, which break the bond between the drug and glucuronide acid, leaving the drug free again, where it can be reabsorbed through the small intestine and enter the general

In children and older adults, the metabolic rate is decreased compared to the metabolic rate of a young adult [1]. In the child, the microsomal enzymes are not yet fully induced [26]. In elderly people, the number of hepatocytes and blood flow that reaches this organ is reduced [27]. So, there are fewer cytochrome P450 enzymes available to metabolize drugs. In pregnancy, there is greater hepatic flow and greater activity of cytochrome P450, which increases

The number of hepatocytes decreases, and the metabolic rate also decreases. In addition, there is an increase in the plasmatic concentrations and half-life of drugs. Therefore, it is necessary

Some drugs and phytochemical compounds present in medicinal plants and foods of plant

Biotransformation reactions of drugs are divided into reactions of phase I or functionalization

circulation. In this case, the half-life of the drugs is increased [25].

origin can induce or inhibit the activity of cytochrome P450 [29].

*2.1.3. Factors that affect the metabolism of drugs*

*2.1.3.1. Physiological factors: age and pregnancy*

turates [1, 2].

*2.1.2. Enterohepatic circuit*

the metabolic rate [28].

*2.1.3.2. Pathological factors: liver disease*

to adjust the dose, to prevent toxicity [2].

*2.1.3.3. Drugs, medicinal plants, and foods*

and reactions of phase II or conjugation [2].

**2.2. Phases of drug metabolism**

On the other hand, the consumption of certain foods of vegetable origin with nutraceutical properties has increased considerably in recent years, especially to treat and prevent conditions such as cancer, diabetes, hypertension, hypercholesterolemia, obesity, among others. Therefore, by combining these foods with the pharmacological treatment indicated in the abovementioned conditions, they can significantly modify the plasma levels of some drugs and put the patient's life at risk, either due to therapeutic failure (decrease in plasma concentration) or toxicity (increased plasma concentration) [11–15].

#### **2.1. Pharmacokinetic process of drug metabolism**

The drugs are defined chemically as acids or weak bases, and during the absorption process, the nonionized fraction of a drug is the one that crosses the biological membranes, due to its lipid solubility. Until the condition of lipid solubility is not lost, the drug will continue remaining in the body, by means of processes of reabsorption at the renal level or the enterohepatic circuit and redistribution from drug deposits in adipose tissue [16–19].

If this lipid solubility condition is not lost, the drug will not be able to be eliminated [2, 18]. Fortunately, the pharmacokinetic process of the metabolism helps to modify the chemical structure of drugs into structures more polar, so that these can be more easily excreted [2, 18, 19].

The main organ that participates in the metabolism of drugs and other xenobiotics is the liver. However, other tissues also have metabolic capacity such as the gastrointestinal tract, lungs, skin, kidneys, and brain [20–23].

The functional unit of this organ is the hepatocyte, and it contains different enzymes that are in the mitochondria, smooth and rough reticulum membrane, cytosol, etc. [23].

During this process of drug metabolism, the following may occur:


It is important to mention that there are drugs that do not transform. Their chemical structure is not modified, and they are eliminated unaltered [2].

#### *2.1.1. Effect of metabolism of the first step*

When drugs are administered orally, they suffer a phenomenon of elimination prior to the process *per se* of the metabolism. This effect is known as first-pass metabolism and occurs in the epithelial cells of the gastrointestinal tract mainly in the small intestine [24]. Subsequently, the amount of drug that was not biotransformed enters the liver through the portal circulation, and there in the hepatocytes the metabolism process *per se* is carried out [24]. The amount of drug remaining after liver extraction is bioavailable to give an adequate pharmacological response. It is important not to modify this bioavailability because the effective doses of the drugs used in the clinical ready are considered as the effect of metabolism of the first step. Above all, caution should be exercised in drugs with a narrow safety margin, such as barbiturates [1, 2].

#### *2.1.2. Enterohepatic circuit*

the population of developed and underdeveloped countries use medicinal plants as a first resource for their health care and, on the other hand, there is a context cultural acceptance of the traditional practice with herbs, which makes its use popular and that in many cases

On the other hand, the consumption of certain foods of vegetable origin with nutraceutical properties has increased considerably in recent years, especially to treat and prevent conditions such as cancer, diabetes, hypertension, hypercholesterolemia, obesity, among others. Therefore, by combining these foods with the pharmacological treatment indicated in the abovementioned conditions, they can significantly modify the plasma levels of some drugs and put the patient's life at risk, either due to therapeutic failure (decrease in plasma concen-

The drugs are defined chemically as acids or weak bases, and during the absorption process, the nonionized fraction of a drug is the one that crosses the biological membranes, due to its lipid solubility. Until the condition of lipid solubility is not lost, the drug will continue remaining in the body, by means of processes of reabsorption at the renal level or the entero-

If this lipid solubility condition is not lost, the drug will not be able to be eliminated [2, 18]. Fortunately, the pharmacokinetic process of the metabolism helps to modify the chemical structure of drugs into structures more polar, so that these can be more easily excreted [2, 18, 19].

The main organ that participates in the metabolism of drugs and other xenobiotics is the liver. However, other tissues also have metabolic capacity such as the gastrointestinal tract, lungs,

The functional unit of this organ is the hepatocyte, and it contains different enzymes that are

It is important to mention that there are drugs that do not transform. Their chemical structure

When drugs are administered orally, they suffer a phenomenon of elimination prior to the process *per se* of the metabolism. This effect is known as first-pass metabolism and occurs in the epithelial cells of the gastrointestinal tract mainly in the small intestine [24]. Subsequently,

hepatic circuit and redistribution from drug deposits in adipose tissue [16–19].

in the mitochondria, smooth and rough reticulum membrane, cytosol, etc. [23].

During this process of drug metabolism, the following may occur:

patients combine their pharmacological treatment with herbal treatment [6–10].

tration) or toxicity (increased plasma concentration) [11–15].

**2.1. Pharmacokinetic process of drug metabolism**

skin, kidneys, and brain [20–23].

120 Medicinal Chemistry

**1.** Transform to a more active molecule [1–3].

**3.** Transform to an inactive molecule [1–3].

**4.** Transform to a toxic molecule [1–3].

*2.1.1. Effect of metabolism of the first step*

**2.** Transform to give biological activity (prodrug) [1–3].

is not modified, and they are eliminated unaltered [2].

When drugs are biotransformed by phase I reactions, and the molecule obtained is not polar enough to be eliminated, their biotransformation continues through phase II reactions. In this phase, the metabolites are generally conjugated with glucuronide acid, giving a polar molecule with a higher molecular weight [1, 2]. These conjugates are secreted from the hepatocyte into the bile and stored there in the form of a drug-glucuronide complex; when the bile is secreted in the intestine by some stimulus, the drug-bile complex is eliminated through the feces [25]. However, intestinal microorganisms produce various enzymes, such as betaglucuronidases, which break the bond between the drug and glucuronide acid, leaving the drug free again, where it can be reabsorbed through the small intestine and enter the general circulation. In this case, the half-life of the drugs is increased [25].

#### *2.1.3. Factors that affect the metabolism of drugs*

#### *2.1.3.1. Physiological factors: age and pregnancy*

In children and older adults, the metabolic rate is decreased compared to the metabolic rate of a young adult [1]. In the child, the microsomal enzymes are not yet fully induced [26]. In elderly people, the number of hepatocytes and blood flow that reaches this organ is reduced [27]. So, there are fewer cytochrome P450 enzymes available to metabolize drugs. In pregnancy, there is greater hepatic flow and greater activity of cytochrome P450, which increases the metabolic rate [28].

#### *2.1.3.2. Pathological factors: liver disease*

The number of hepatocytes decreases, and the metabolic rate also decreases. In addition, there is an increase in the plasmatic concentrations and half-life of drugs. Therefore, it is necessary to adjust the dose, to prevent toxicity [2].

#### *2.1.3.3. Drugs, medicinal plants, and foods*

Some drugs and phytochemical compounds present in medicinal plants and foods of plant origin can induce or inhibit the activity of cytochrome P450 [29].

### **2.2. Phases of drug metabolism**

Biotransformation reactions of drugs are divided into reactions of phase I or functionalization and reactions of phase II or conjugation [2].

The chemical reactions of phase I allow the introduction of functional groups such as –OH, –COOH, –SH, –O–, or –NH2 . Phase I reactions are very simple chemical reactions such as oxidation, reduction, hydrolysis, alkylation, and dealkylation [2]. Of these chemical reactions, the most important in the metabolism of drugs and that occur more frequently are the oxidation reactions performed by the cytochrome P450 enzymes (CYP450). These enzymes are located mainly in the smooth endoplasmic reticulum [1, 2].

CYP2D6, CYP2E1, CYP3A4,5,7 [1–3]. CYP3A4,5,7 is the most abundant and participates in the

Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450

http://dx.doi.org/10.5772/intechopen.79971

123

Many substances such as drugs, environmental toxins, and phytochemicals present in medicinal plants and some foods of plant origin contain substances that act as inhibitors or inducers of cytochrome P450 enzymes; this induction and inhibition can be strong or weak so it can sometimes have relevant clinical implications such as producing toxicity or therapeutic failure [36].

Enzymatic induction refers to the increase of enzymes and/or their activity. Additionally, it increases the metabolic rate of CYP450, and therefore, the concentrations of the drug in blood will decrease, which can cause a decrease in pharmacological effects and with it a therapeutic

In enzymatic inhibition, the number of enzymes and/or their activity decreases. There are fewer enzymes available to biotransform the drugs and increase their plasma levels with each

It is important not to induce or inhibit the activity of CYP450; they directly influence the bioavailability of the drugs. On the other hand, the genetic polymorphism of CYP450 is also responsible for the variability in the response to drugs between each individual [34, 35]. Genetic variability, especially of CYP2C9, CYP2C19, CYP2D6, and CYP3A5, is known to have an important clinical impact on drugs that are metabolized by these enzymes [38–40].

**2.5. Effect of bioactive compounds of medicinal plants and foods of vegetable origin** 

In the literature, there is a lot of information about the effect of drugs to inhibit or induce certain CYP450 isoenzymes. Recently the study of the effect of some phytochemical components that are present in medicinal plants and foods of vegetable origin on the activity of CYP450 has been increasing, because the population makes use of herbal medicine in its traditional practice and, on the other hand, it consumes foods with nutraceutical properties, either to

administration of the drug will produce toxicity (**Figure 2**) [33].

**Figure 2.** Effect of medicinal plants and foods of vegetable origin on CYP450 activity.

metabolism of more than 50% of the drugs currently used in the clinic [1–3, 35].

**2.4. Induction and Enzymatic inhibition**

failure (**Figure 2**) [37].

**on the activity of CYP450**

prevent or to control any disease.

When the addition of the functional groups (–OH, –COOH, –SH, –O–, –NH2 ) to the drug molecule is not enough to transform it to a more polar molecule, the molecule continues its modification through reactions of phase II. Phase II reactions are called also conjugation reactions. In these reactions, the molecule of the drug or metabolite previously formed in the reactions of phase I is conjugated with a large molecule of polar nature (hydrophilic) as the acid glucuronide, or acetyl Co-A, glycine, glutathione, phosphoadenosyl phosphosulfate, and S-adenosylmethionine [2]. These reactions are carried out by means of specific enzymes called transferases that are generally located in the microsomes and in the cytosol [1–3].

#### **2.3. Role of cytochrome P450 (CYP450) in drug metabolism**

Cytochrome P450 (CYP450) is a superfamily of enzymes that contain a heme group, so they are hemoproteins. The iron in the heme group is reduced and forms complexes with the carbon monoxide that absorbs light at a wavelength of 450 nm [30]. They have identified more than 8700 genes that code for their proteins and are found in eukaryotic and prokaryotic cells [31]. They are responsible for metabolizing or biotransforming endogenous substances in the body such as hormones, and different xenobiotics such as drugs. These enzymes perform oxidation reactions and participate in the phase I reactions of drug metabolism [1–3]. They are also known as mixed function oxidases or monooxygenases; they require a reducing agent such as NADPH and molecular oxygen [32].

They have different patterns of specificity for the substrate; for example, acetaminophen is a substrate of both CYP1A2 and CYP2E1, while halogenated anesthetics are substrate only of CYP2E1 [2, 34–36]. This enzyme system is found in different tissues such as kidney, lung, skin, brain, adrenal cortex, placenta, testicles, and other tissues, but the liver and small intestine are the organs that have more CYP450 [33, 34].

#### *2.3.1. Nomenclature of CYP450*

The CYP450 is grouped into families and subfamilies depending on the analogy in their amino acid sequences, such that CYPs that present 40% homology in their amino acids belong to a family, and when the analogy is greater than 55%, they form a subfamily, are named with the prefix CYP, and followed by the family number, a capital letter indicating the subfamily, and a number that marks the individual form: for example, CYP1A1, in this way, represents the individual form 1 of subfamily A of family 1 [35]. Eighteen families, 42 subfamilies, and more than 50 individual genes of human origin have been described. However, the most important in the metabolism of drugs are CYP1A1/2, CYP1B1, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, CYP3A4,5,7 [1–3]. CYP3A4,5,7 is the most abundant and participates in the metabolism of more than 50% of the drugs currently used in the clinic [1–3, 35].

#### **2.4. Induction and Enzymatic inhibition**

The chemical reactions of phase I allow the introduction of functional groups such as –OH,

oxidation, reduction, hydrolysis, alkylation, and dealkylation [2]. Of these chemical reactions, the most important in the metabolism of drugs and that occur more frequently are the oxidation reactions performed by the cytochrome P450 enzymes (CYP450). These enzymes are

molecule is not enough to transform it to a more polar molecule, the molecule continues its modification through reactions of phase II. Phase II reactions are called also conjugation reactions. In these reactions, the molecule of the drug or metabolite previously formed in the reactions of phase I is conjugated with a large molecule of polar nature (hydrophilic) as the acid glucuronide, or acetyl Co-A, glycine, glutathione, phosphoadenosyl phosphosulfate, and S-adenosylmethionine [2]. These reactions are carried out by means of specific enzymes called transferases that are generally located in the microsomes

Cytochrome P450 (CYP450) is a superfamily of enzymes that contain a heme group, so they are hemoproteins. The iron in the heme group is reduced and forms complexes with the carbon monoxide that absorbs light at a wavelength of 450 nm [30]. They have identified more than 8700 genes that code for their proteins and are found in eukaryotic and prokaryotic cells [31]. They are responsible for metabolizing or biotransforming endogenous substances in the body such as hormones, and different xenobiotics such as drugs. These enzymes perform oxidation reactions and participate in the phase I reactions of drug metabolism [1–3]. They are also known as mixed function oxidases or monooxygenases; they require a reducing agent

They have different patterns of specificity for the substrate; for example, acetaminophen is a substrate of both CYP1A2 and CYP2E1, while halogenated anesthetics are substrate only of CYP2E1 [2, 34–36]. This enzyme system is found in different tissues such as kidney, lung, skin, brain, adrenal cortex, placenta, testicles, and other tissues, but the liver and small intes-

The CYP450 is grouped into families and subfamilies depending on the analogy in their amino acid sequences, such that CYPs that present 40% homology in their amino acids belong to a family, and when the analogy is greater than 55%, they form a subfamily, are named with the prefix CYP, and followed by the family number, a capital letter indicating the subfamily, and a number that marks the individual form: for example, CYP1A1, in this way, represents the individual form 1 of subfamily A of family 1 [35]. Eighteen families, 42 subfamilies, and more than 50 individual genes of human origin have been described. However, the most important in the metabolism of drugs are CYP1A1/2, CYP1B1, CYP2A6, CYP2B6, CYP2C9,

When the addition of the functional groups (–OH, –COOH, –SH, –O–, –NH2

. Phase I reactions are very simple chemical reactions such as

) to the drug

–COOH, –SH, –O–, or –NH2

122 Medicinal Chemistry

and in the cytosol [1–3].

located mainly in the smooth endoplasmic reticulum [1, 2].

**2.3. Role of cytochrome P450 (CYP450) in drug metabolism**

such as NADPH and molecular oxygen [32].

tine are the organs that have more CYP450 [33, 34].

*2.3.1. Nomenclature of CYP450*

Many substances such as drugs, environmental toxins, and phytochemicals present in medicinal plants and some foods of plant origin contain substances that act as inhibitors or inducers of cytochrome P450 enzymes; this induction and inhibition can be strong or weak so it can sometimes have relevant clinical implications such as producing toxicity or therapeutic failure [36].

Enzymatic induction refers to the increase of enzymes and/or their activity. Additionally, it increases the metabolic rate of CYP450, and therefore, the concentrations of the drug in blood will decrease, which can cause a decrease in pharmacological effects and with it a therapeutic failure (**Figure 2**) [37].

In enzymatic inhibition, the number of enzymes and/or their activity decreases. There are fewer enzymes available to biotransform the drugs and increase their plasma levels with each administration of the drug will produce toxicity (**Figure 2**) [33].

It is important not to induce or inhibit the activity of CYP450; they directly influence the bioavailability of the drugs. On the other hand, the genetic polymorphism of CYP450 is also responsible for the variability in the response to drugs between each individual [34, 35]. Genetic variability, especially of CYP2C9, CYP2C19, CYP2D6, and CYP3A5, is known to have an important clinical impact on drugs that are metabolized by these enzymes [38–40].

#### **2.5. Effect of bioactive compounds of medicinal plants and foods of vegetable origin on the activity of CYP450**

In the literature, there is a lot of information about the effect of drugs to inhibit or induce certain CYP450 isoenzymes. Recently the study of the effect of some phytochemical components that are present in medicinal plants and foods of vegetable origin on the activity of CYP450 has been increasing, because the population makes use of herbal medicine in its traditional practice and, on the other hand, it consumes foods with nutraceutical properties, either to prevent or to control any disease.

**Figure 2.** Effect of medicinal plants and foods of vegetable origin on CYP450 activity.


**Medicinal plant**

*Camellia sinensis*

*Piper methysticum*

*Hypericum perforatum*

**VI:** Nicotine.

*Panax ginseng* Is believed to

enhance cognitive ability and to lower blood sugar levels Ginsenosides and gintonin

Ginsenoside F2 and protopanaxadiol

It is consumed to treat cancer, cardiovascular disease, dyslipidemia, inflammation, and weight loss

Is used to treat anxiety and depression

ondansetron, haloperidol, naproxen, propanolol.

tolbutamide, glipizide, glibenclamide, fluoxetine, tamoxifen.

**Table 1.** Effect of medicinal plants on CYP450 activity.

**VII:** Bupropion, cyclophosphamide, efavirenz, ifosfamide, methadone. **VIII**: Paclitaxel, torsemide, amodiaquine, cerivastatin, repaglinide.

**Tradicional uses Phytochemistry** 

**compounds**

Catechin

gallate

Anxiolytic effect Flavokawain A ↑CYP2C9

(-)-Epigallocatechin -3-

Ginsenosides ↓CYP2C9

**Activity on CYP450**

Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450

↓CYP3A4

↓CYP1A2 ↓CYP2B6 ↓CYP2C8 ↓CYP2C9 ↓CYP2D6 ↓CYP3A4

↓CYP1A2 ↓CYP3A4

↑CYP3A4

Hyperforin ↑CYP2C9

**I:** Omeprazole, pantoprazole, diazepam, S-mephenytoin, amitriptyline, carisoprodol, citalopram, chloramphenicol,

**II:** Acetaminophen, amitriptyline, phenacetin, tacrine, theophylline, tamoxifen, (R)warfarin, caffeine, verapamil,

**III**: Propoxyphene, codeine, oxycodone, dextromethorphan, clozapine, timolol, tamoxifen, tramadol, seleglinide,

**IV**: Amitriptyline, celcoxib, ibuprofen, diclofenac, meloxicam, hexobarbital, losartan, S-warfarin, fluvastation, phenytoin,

**V**: Acetaminophen, amiodarone, cisapride, astemizole, cocaine, cyclosporine, dapsone, diazepam, dihydroergotamine, diltiacem, felodipine, nifedipine, erythromycin, indinavir, lidocaine, methadone, miconazole, quinidine, paclitaxel,

clomipramine, cyclophosphamide, indomethacin, moclobemide, nelfinavir, propranolol, progesterone.

mifepristone, spironolactone, verapamil, trazolam, desametaxone, ritonavir, lovastatin, hydrocortisone.

fluoxetine, phenformin, paroxetine, risperidone, metoprolol, tricyclic antidepressants.

**Clinical effect on substrates of CYP450**

http://dx.doi.org/10.5772/intechopen.79971

↑Plasmatic concentration of IV ↑Plasmatic concentration of V

↑Plasmatic concentration of II ↑Plasmatic concentration of VII ↑Plasmatic concentration of VIII ↑Plasmatic concentration of IV ↑Plasmatic concentration of III ↑Plasmatic concentration of V

↓Plasmatic concentration of IV ↑Plasmatic concentration of II ↑Plasmatic concentration of V

↓Plasmatic concentration of IV ↓Plasmatic concentration of V **References**

125

[3, 57]

[3, 58–60]

[3, 61–64]

[3, 65–67]


**I:** Omeprazole, pantoprazole, diazepam, S-mephenytoin, amitriptyline, carisoprodol, citalopram, chloramphenicol, clomipramine, cyclophosphamide, indomethacin, moclobemide, nelfinavir, propranolol, progesterone.

**II:** Acetaminophen, amitriptyline, phenacetin, tacrine, theophylline, tamoxifen, (R)warfarin, caffeine, verapamil, ondansetron, haloperidol, naproxen, propanolol.

**III**: Propoxyphene, codeine, oxycodone, dextromethorphan, clozapine, timolol, tamoxifen, tramadol, seleglinide, fluoxetine, phenformin, paroxetine, risperidone, metoprolol, tricyclic antidepressants.

**IV**: Amitriptyline, celcoxib, ibuprofen, diclofenac, meloxicam, hexobarbital, losartan, S-warfarin, fluvastation, phenytoin, tolbutamide, glipizide, glibenclamide, fluoxetine, tamoxifen.

**V**: Acetaminophen, amiodarone, cisapride, astemizole, cocaine, cyclosporine, dapsone, diazepam, dihydroergotamine, diltiacem, felodipine, nifedipine, erythromycin, indinavir, lidocaine, methadone, miconazole, quinidine, paclitaxel, mifepristone, spironolactone, verapamil, trazolam, desametaxone, ritonavir, lovastatin, hydrocortisone.

**VI:** Nicotine.

**Medicinal plant**

124 Medicinal Chemistry

*Artemisia annua L.*

*Cimicifuga racemosa*

*Echinacea purpurea* (L.)

*Garcinia cambogia*

*Gardenia jasminoides* Ellis.

**Tradicional uses Phytochemistry** 

Are used as a hormone replacement and antiinflammatory

healing and maintaining normal blood pressure.

anticancer and antiarthritic effect.

It is used to treat colds, upper respiratory infections, and dermatologic issues

Is used as an antioxidant, hypoglycemic , antithrombotic, antiinflammatory, antidepression effect, and improved sleeping quality

hypertensive as well as to treat macular degeneration and tinnitus. Are effective in treating cerebral infarction

*Gingko biloba* It is used as an anti-

*Centella asiatica* Used for wound

*Curcuma longa* Antiinflammatory,

**compounds**

Triterpene glycosides Fukinolic

Flavonoids: Quercetin Kaempferol

Curcuminoids: Curcumin Methoxycurcumin, Bisdemethoxycurcumin

Cichoric acid Caftaric acid Echinacoside Alkylamides

Geniposide Genipin

Ginkgolide A Ginkgolide B Bilobalide Quercetin kaempferol

Obesity treatment Extract crude ↓CYP2B6 ↑Plasmatic

Cimicifugic acid A Cimicifugic acid B

acid

Antimalaria effect Artemisinin ↑CYP2C19 ↓Plasmatic

**Activity on CYP450**

↓CYP1A2 ↓CYP2D6 ↓CYP2C9 ↓CYP3A4

↓CYP2D6 ↓CYP2C9 ↓CYP3A4

↓CYP1A2 ↑CYP2A6 ↓CYP2C9 ↓CYP3A4

↑CYP1A2 ↓CYP3A4

↑CYP2D6 ↓CYP2C19 ↓CYP3A4

↓CYP2B6 ↑CYP1A2 ↑CYP3A4 **Clinical effect on substrates of CYP450**

concentration of I

↑Plasmatic concentration of II ↑Plasmatic concentration of III ↑Plasmatic concentration of IV ↑Plasmatic concentration of V

↑Plasmatic concentration of III ↑Plasmatic concentration of IV ↑Plasmatic concentration of V

↑Plasmatic concentration of II ↓Plasmatic concentration of VI ↑Plasmatic concentration of IV ↑Plasmatic concentration of V

↓Plasmatic concentration of II ↑Plasmatic concentration of V

concentration of VII

↓Plasmatic concentration of III ↑Plasmatic concentration of I ↑Plasmatic concentration of V

↑Plasmatic concentration of VII ↓Plasmatic concentration of II ↓Plasmatic concentration of V **References**

[3, 41, 42]

[3, 44, 45]

[3, 46–48]

[3, 49, 50]

[3, 51]

[3, 52, 53]

[3, 54–56]

[3, 43]

**VII:** Bupropion, cyclophosphamide, efavirenz, ifosfamide, methadone.

**VIII**: Paclitaxel, torsemide, amodiaquine, cerivastatin, repaglinide.

**Table 1.** Effect of medicinal plants on CYP450 activity.


**Author details**

Xóchitl S. Ramírez-Gómez1

Esmeralda Rodríguez Miranda3

of Guanajuato, Celaya, Guanajuato, Mexico

Guanajuato, León, Guanajuato, Mexico

McGraw-Hill; 2013. pp. 37-68

McGraw-Hill; 2014. pp. 46-105

10.3390/ijms18112353

Irapuato, Guanajuato, Mexico

**References**

\*, Sandra N. Jiménez-García2

\*Address all correspondence to: xosofira2002@yahoo.com.mx

University of Guanajuato, Celaya, Guanajuato, Mexico

, Gabriel Herrera Pérez4

2 Department of Nursing and Obstetrics, Division of Health Sciences and Engineering,

3 Department of Medicine and Nutrition, Division of Health Sciences, University of

4 Department of Materials Engineering, Instituto Tecnológico Superior de Irapuato,

of Therapeutics. 12th ed. United States: McGraw-Hill; 2012. pp. 123-143

Impotence Research. 2007;**19**:253-264. DOI: 10.1038/sj.ijir.3901522

Journal of the Association of Physicians of India. 2016;**64**:68-72

[1] Brunton LL, Chabner BA, Knollman B. Goodman & Gilman's The Pharmacological Basis

[2] Katzung BG, Masters SB, Trevor AJ. Basic & Clinical Pharmacology. 12th ed. United States:

[3] Flórez J, Armijo JA, Mediavilla A. Human Pharmacology. 6th ed. Barcelona, Spain:

[4] Mehrotra N, Gupta M, Kovar A, Meibohm B. The role of pharmacokinetics and pharmacodynamics in phosphodiesterase-5 inhibitor therapy. International Journal of

[5] Chawla PK, Udwadia ZF, Soman R, Mahashur AA, Amale RA, Dherai AJ, Lokhande RV, Naik PR, Ashavaid TF. Importance of therapeutic drug monitoring of rifampicin. The

[6] World Health Organization. Traditional Medicine Strategy 2014-2023 [Internet]. Available from: http://apps.who.int/medicinedocs/documents/s21201en/s21201en.pdf [Accessed: April 01, 2018]

[7] Brewer CT, Chen T. Hepatotoxicity of herbal supplements mediated by modulation of cytochrome P450. International Journal of Molecular Sciences. 2017;**18**:E2353. DOI:

[8] Hua S, Zhang Y, Liu J, Dong L, Huang J, Lin D, Fu X. Ethnomedicine, phytochemistry and pharmacology of *Smilax glabra*: An important traditional Chinese medicine. The American

Journal of Chinese Medicine. 2018;**46**:261-297. DOI: 10.1142/S0192415X18500143

1 Department of Clinical Nursing, Division of Health Sciences and Engineering, University

Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450

, Vicente Beltrán Campos1

http://dx.doi.org/10.5772/intechopen.79971

and Rafael Vargas-Bernal4

,

127

**II:** Acetaminophen, amitriptyline, phenacetin, tacrine, theophylline, tamoxifen, (R)warfarin, caffeine, verapamil, ondansetron, haloperidol, naproxen, propanolol.

**III:** Propoxyphene, codeine, oxycodone, dextromethorphan, clozapine, timolol, tamoxifen, tramadol, seleglinide, fluoxetine, phenformin, paroxetine, risperidone, metoprolol, tricyclic antidepressants.

**IV:** Amitriptyline, celcoxib, ibuprofen, diclofenac, meloxicam, hexobarbital, losartan, S-warfarin, fluvastation, phenytoin, tolbutamide, glipizide, glibenclamide, fluoxetine, tamoxifen.

**V:** Acetaminophen, amiodarone, cisapride, astemizole, cocaine, cyclosporine, dapsone, diazepam, dihydroergotamine, diltiacem, felodipine, nifedipine, erythromycin, indinavir, lidocaine, methadone, miconazole, quinidine, paclitaxel, mifepristone, spironolactone, verapamil, trazolam, desametaxone, ritonavir, lovastatin, hydrocortisone.

**Table 2.** Effect of fruits or vegetables on CYP450 activity.

**Tables 1** and **2** show the effect of the phytochemical compounds present in medicinal plants and foods of vegetable origin. We mentioned principally those natural products that have an important effect on the induction and inhibition of different CYP450 isoenzymes and that have clinical relevance to produce toxicity or therapeutic failure.

## **3. Conclusions**

The induction and inhibition of CYP450, by some bioactive compounds present in medicinal plants or foods, can modify the bioavailability of drugs. The changes in the bioavailability are important in the efficacy and safety of pharmacological management. It is important to consider that when a patient will be in a pharmacologic treatment, the patient should not use any medicinal plants or foods of vegetable origin that can induce or inhibit any CYP450 isoenzymes.

Especially, they should not use the St. John's wort and grapefruit, as their phytochemical compounds have a potent effect to induce or inhibit, respectively, the activity of CYP3A4 with important clinical relevance.

## **Author details**

Xóchitl S. Ramírez-Gómez1 \*, Sandra N. Jiménez-García2 , Vicente Beltrán Campos1 , Esmeralda Rodríguez Miranda3 , Gabriel Herrera Pérez4 and Rafael Vargas-Bernal4

\*Address all correspondence to: xosofira2002@yahoo.com.mx

1 Department of Clinical Nursing, Division of Health Sciences and Engineering, University of Guanajuato, Celaya, Guanajuato, Mexico

2 Department of Nursing and Obstetrics, Division of Health Sciences and Engineering, University of Guanajuato, Celaya, Guanajuato, Mexico

3 Department of Medicine and Nutrition, Division of Health Sciences, University of Guanajuato, León, Guanajuato, Mexico

4 Department of Materials Engineering, Instituto Tecnológico Superior de Irapuato, Irapuato, Guanajuato, Mexico

## **References**

**Tables 1** and **2** show the effect of the phytochemical compounds present in medicinal plants and foods of vegetable origin. We mentioned principally those natural products that have an important effect on the induction and inhibition of different CYP450 isoenzymes and that

**Activity on CYP450**

↓CYP2D6

↓CYP2C9 ↓CYP3A4

Sevillian orange Furanocoumarin ↓CYP3A4 ↑Plasmatic concentration of V [3, 73]

Star fruit Catechin Epicatechin ↓CYP3A4 ↑Plasmatic concentration of V [3, 74–76]

**II:** Acetaminophen, amitriptyline, phenacetin, tacrine, theophylline, tamoxifen, (R)warfarin, caffeine, verapamil,

**III:** Propoxyphene, codeine, oxycodone, dextromethorphan, clozapine, timolol, tamoxifen, tramadol, seleglinide,

**IV:** Amitriptyline, celcoxib, ibuprofen, diclofenac, meloxicam, hexobarbital, losartan, S-warfarin, fluvastation, phenytoin,

**V:** Acetaminophen, amiodarone, cisapride, astemizole, cocaine, cyclosporine, dapsone, diazepam, dihydroergotamine, diltiacem, felodipine, nifedipine, erythromycin, indinavir, lidocaine, methadone, miconazole, quinidine, paclitaxel,

mifepristone, spironolactone, verapamil, trazolam, desametaxone, ritonavir, lovastatin, hydrocortisone.

Grapefruit Furanocoumarin ↓CYP3A4 ↑Plasmatic concentration of V [3, 65, 70, 71]

**Clinical effect on substrates** 

↓Plasmatic concentration of II ↑Plasmatic concentration of III

↑Plasmatic concentration of IV ↑Plasmatic concentration of V

**References**

[3, 68, 69]

[3, 72]

**of CYP450**

The induction and inhibition of CYP450, by some bioactive compounds present in medicinal plants or foods, can modify the bioavailability of drugs. The changes in the bioavailability are important in the efficacy and safety of pharmacological management. It is important to consider that when a patient will be in a pharmacologic treatment, the patient should not use any medicinal plants or foods of vegetable origin that can induce or inhibit any CYP450

Especially, they should not use the St. John's wort and grapefruit, as their phytochemical compounds have a potent effect to induce or inhibit, respectively, the activity of CYP3A4 with

have clinical relevance to produce toxicity or therapeutic failure.

fluoxetine, phenformin, paroxetine, risperidone, metoprolol, tricyclic antidepressants.

**3. Conclusions**

**Fruit or vegetable Phytochemistry** 

126 Medicinal Chemistry

Pomegranate Flavonoids

**compound**

Broccoli Sulforaphane ↑CYP1A2

Tannins Phenolic acids

ondansetron, haloperidol, naproxen, propanolol.

tolbutamide, glipizide, glibenclamide, fluoxetine, tamoxifen.

**Table 2.** Effect of fruits or vegetables on CYP450 activity.

isoenzymes.

important clinical relevance.


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**Section 4**

**Effects on Biological Systems: In Vivo Testing**

**Effects on Biological Systems: In Vivo Testing**

**Chapter 9**

Provisional chapter

**The Pragmatic Strategy to Detect Endocrine-Disrupting**

DOI: 10.5772/intechopen.81030

Endocrine-disrupting activity induced by xenobiotics might pose a possible health threat. Facing so many chemicals, there is an issue on how we detect them precisely and effectively. The whole embryo culture (WEC) test, an ex vivo exposure lasting 48 hours with rat embryos of 10.5 days old, is used to detect prenatal developmental toxicity. We extended the WEC function to detect the endocrine-disrupting activity induced by environmental chemicals. Results showed that in the development of rat embryo, basically 17ß-estradiol, triiodothyronine, triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crown-rump length, head length, and developmental score. In the immunohistochemistry, the positive control of 17ß-estradiol showed positive effect for its receptor expressions. These three triazoles induced expressions of ERα and ERß in WEC. This result basically meets the mode of action that triazoles were designed to disrupt the synthesis of steroid hormone. Here we gave a strategy to detect possible endocrine-disrupting activity induced by xenobiotics in food. This strategy is quick to initiate the whole rat embryo culture with 10.5 days to detect the hormone receptors such as androgen, estrogen, thyroid, aromatase activity and its related receptors.

Keywords: whole embryo culture, xenobiotic, receptors, ex vivo, in vivo,

As we know, there are many pesticides identified as endocrine disruptors, but the degree of endocrine-disrupting activity (EDA) is different [1–5]. The different disrupting activities are

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

The Pragmatic Strategy to Detect Endocrine-Disrupting

**Activity of Xenobiotics in Food**

Activity of Xenobiotics in Food

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81030

endocrine-disrupting activity

1. Introduction

Chen-Yi Weng

Chen-Yi Weng

Abstract

#### **The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food** The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

DOI: 10.5772/intechopen.81030

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and Chen-Yi Weng Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and Chen-Yi Weng

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81030

#### Abstract

Endocrine-disrupting activity induced by xenobiotics might pose a possible health threat. Facing so many chemicals, there is an issue on how we detect them precisely and effectively. The whole embryo culture (WEC) test, an ex vivo exposure lasting 48 hours with rat embryos of 10.5 days old, is used to detect prenatal developmental toxicity. We extended the WEC function to detect the endocrine-disrupting activity induced by environmental chemicals. Results showed that in the development of rat embryo, basically 17ß-estradiol, triiodothyronine, triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crown-rump length, head length, and developmental score. In the immunohistochemistry, the positive control of 17ß-estradiol showed positive effect for its receptor expressions. These three triazoles induced expressions of ERα and ERß in WEC. This result basically meets the mode of action that triazoles were designed to disrupt the synthesis of steroid hormone. Here we gave a strategy to detect possible endocrine-disrupting activity induced by xenobiotics in food. This strategy is quick to initiate the whole rat embryo culture with 10.5 days to detect the hormone receptors such as androgen, estrogen, thyroid, aromatase activity and its related receptors.

Keywords: whole embryo culture, xenobiotic, receptors, ex vivo, in vivo, endocrine-disrupting activity

#### 1. Introduction

As we know, there are many pesticides identified as endocrine disruptors, but the degree of endocrine-disrupting activity (EDA) is different [1–5]. The different disrupting activities are

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

involved in pesticide management. Because the potential endocrine-disrupting pesticides should be prohibited, low EDA will be accepted under the control of below maximum residue level (MRL). The development of new pesticide is based on its chemical functional groups for pests including fungicides, insecticides, herbicides, and others. Due to the objective of pest control of diseases, insects, and weeds, the side effect of pesticides will be appropriately managed in order not to pose risk to the human and environment. It is reported that 105 pesticides could be listed in the endocrine-disrupting chemical (EDC) group (Table 1) [6–54]. Among these 105 pesticides, 31% are fungicides, 21% herbicides, and 46% insecticides; some of these were withdrawn from use several years ago; even a little still can be detected in the environment such as dichloro-diphenyl-trichloroethane (DDT) and atrazine in some countries.

Pesticides EDC related Pesticides EDC related 2,4-D (H) AR [6] Heptachlor (I) ER, AR [25, 46]

Aldrin (I) AR [13] Malathion (I) TR [10, 48]

Bendiocarb (I) Estrogen effect [10] Methomyl (I) Aromatase activity,

Bupirimate (F) Pregnane X cellular receptor [5] Mirex (I) Estrogen effect [10] Captan (F) Estrogen action [21] Molinate (H) Reduction of fertility [10] Carbaryl (I) Estrogen effect [10] Myclobutanil (F) Estrogen, androgen, ER, AR,

Carbendazim (F) Estrogen and aromatase activity [18] Nitrofen (H) Estrogen, androgen [21]

Chlorothalonil (F) Androgen-sensitive [23] Parathion (I) Melatonin, gonadotrophic

Chlordane (I) ER [10], AR [13] Penconazole (F) Estrogenic effect, aromatase

Chlorfenvinphos (I) Estrogen effect [26] Permethrin (I) Estrogen-sensitive [19, 29]

Cypermethrin (I) Estrogenic effect [28, 29] Prochloraz (F) Pregnane X cellular receptor, AR,

Chlorpyrifos methyl (I) AR [27] Phenylphenol (F) Estrogen [50]

Deltamethrin (I) Estrogenic activity [2] Propanil (H) Estrogen [52]

(H, F, I)

Procymidone (F) AR [25]

Chlordecone (I) AE, ER [21, 24, 25] Pentachlorophenol

AR, androgen-sensitive, ER, PR [13, 23, 24, 30]

Benomyl (F) Estrogen, aromatase activity [18] Methoxychlor (I) Estrogenic effect, AR, pregnane X

Bioallethrin (I) Estrogen-sensitive [19] Metolachlor Pregnane X cellular receptor [5]

Aldicarb (I) 17 Beta-estradiol,

Atrazine (H) Androgen, aromatase activity,

Bitertanol (F) Aromatase activity, estrogens, androgen [20]

Carbofuran (I) Progesterone, cortisol, estradiol, testosterone [22]

Cyproconazole (F) Aromatase activity, estrogens, androgens [20]

DDT and metabolites

(I)

progesterone [10, 12]

estrogen, luteinizing hormone, prolactin [10, 14–17]

Acephate (I) Hypothalamus [7] Hexaconazole (F) Aromatase activity, estrogens,

Acetochlor (H) ER, TR [8, 9] Isoproturon (H) Pregnane X cellular receptor [5] Alachlor (H) ER, PR [10, 11] Iprodione (F) Aromatase activity, estrogen [2]

androgens [20]

http://dx.doi.org/10.5772/intechopen.81030

139

estrogen [2, 10]

somatotropin [49]

aromatase [20, 21, 35]

hormone [10]

activity, estrogens, androgens [20, 35]

ER, AhR, aromatase activity [2, 5, 36, 51]

Propamocarb (F) Aromatase activity, estrogen [2]

Estrogenic, androgenic affect [10]

cellular receptor [10, 11, 13]

Linuron (H) AR, TR [25, 47]

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

Methiocarb (H) Androgen, estrogen [2]

Metribuzin (H) Hyperthyroidism,

Oxamyl (I) Estrogen effect [10]

EDCs focused on interfering with endogenous hormones possible by binding to and activating various hormone receptors including estrogen, androgen, thyroid receptors, and aromatase enzymes and mimic the hormone or enzyme activities including agonistic and antagonistic actions. Basically, EDA is mainly related to the reproductive and developmental toxicity. Also the major endocrine pathways would be hypothalamus-pituitary-gonadal and hypothalamuspituitary-thyroid, and the involving hormones are estrogen, androgen, and thyroid. The Organization for Co-operation and Development (OECD) test guidelines for reproductive and developmental toxicity and EDA are listed in Table 2 [55, 56]. United States Environmental Protection Agency (US EPA) test guidelines for reproductive and developmental toxicity and EDA are as follows. Guidelines are 870.3550 reproduction/development toxicity screening test, 870.3650 combined repeated dose toxicity with the reproduction/development toxicity screening test, 870.3700 prenatal developmental toxicity study, 870.3800 reproduction and fertility effects, and 870.6300 developmental neurotoxicity study. USEPA Series 890 endocrine disruptor screening program test guidelines are isolated from OPPTS 870 Series. The final endocrine disruptor screening program test guidelines are generally intended to meet testing requirements under Toxic Substances Control Act (TSCA); Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); and Federal Food, Drug, and Cosmetic Act (FFDCA) to determine if a chemical substance may pose a risk to human health or the environment due to the disruption of the endocrine system. Group A —EDSP Tier 1 and Group B—EDSP Tier 2 test guidelines are listed in Table 3.

The main shortcomings of above guidelines are that they are expensive and time-consuming and the need of a lot of number of laboratory animals. It is reported that cost and the minimum number of laboratory animals are requested for applying OECD test guidelines to test toxicity to reproductive and developmental toxicity. Table 2 shows the cost and minimum number of laboratory animals [55, 56]. Besides, the associated bioethical and social concerns are becoming a challenge. Nowadays, the common knowledge of using laboratory animals is reduce, refine, and replace (3Rs). Facing these situations, we should take cheap and reliable alternatives to screen the reproductive and developmental toxicity and EDA and decide the next steps for necessities of toxicity tests.

It is reported that a widely used technique for screening prenatal developmental toxicity is by monitoring organogenesis during gestational days (GD) 10–12 [57]. In support to whole rat embryo culture (rat WEC), a variety of morphological endpoints is integrated in the total morphological score (TMS) [58]. When applying the TMS in rat WEC, effects of pesticides on


involved in pesticide management. Because the potential endocrine-disrupting pesticides should be prohibited, low EDA will be accepted under the control of below maximum residue level (MRL). The development of new pesticide is based on its chemical functional groups for pests including fungicides, insecticides, herbicides, and others. Due to the objective of pest control of diseases, insects, and weeds, the side effect of pesticides will be appropriately managed in order not to pose risk to the human and environment. It is reported that 105 pesticides could be listed in the endocrine-disrupting chemical (EDC) group (Table 1) [6–54]. Among these 105 pesticides, 31% are fungicides, 21% herbicides, and 46% insecticides; some of these were withdrawn from use several years ago; even a little still can be detected in the environment such as dichloro-diphenyl-trichloroethane (DDT) and atrazine in some countries. EDCs focused on interfering with endogenous hormones possible by binding to and activating various hormone receptors including estrogen, androgen, thyroid receptors, and aromatase enzymes and mimic the hormone or enzyme activities including agonistic and antagonistic actions. Basically, EDA is mainly related to the reproductive and developmental toxicity. Also the major endocrine pathways would be hypothalamus-pituitary-gonadal and hypothalamuspituitary-thyroid, and the involving hormones are estrogen, androgen, and thyroid. The Organization for Co-operation and Development (OECD) test guidelines for reproductive and developmental toxicity and EDA are listed in Table 2 [55, 56]. United States Environmental Protection Agency (US EPA) test guidelines for reproductive and developmental toxicity and EDA are as follows. Guidelines are 870.3550 reproduction/development toxicity screening test, 870.3650 combined repeated dose toxicity with the reproduction/development toxicity screening test, 870.3700 prenatal developmental toxicity study, 870.3800 reproduction and fertility effects, and 870.6300 developmental neurotoxicity study. USEPA Series 890 endocrine disruptor screening program test guidelines are isolated from OPPTS 870 Series. The final endocrine disruptor screening program test guidelines are generally intended to meet testing requirements under Toxic Substances Control Act (TSCA); Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); and Federal Food, Drug, and Cosmetic Act (FFDCA) to determine if a chemical substance may pose a risk to human health or the environment due to the disruption of the endocrine system. Group A

—EDSP Tier 1 and Group B—EDSP Tier 2 test guidelines are listed in Table 3.

necessities of toxicity tests.

138 Medicinal Chemistry

The main shortcomings of above guidelines are that they are expensive and time-consuming and the need of a lot of number of laboratory animals. It is reported that cost and the minimum number of laboratory animals are requested for applying OECD test guidelines to test toxicity to reproductive and developmental toxicity. Table 2 shows the cost and minimum number of laboratory animals [55, 56]. Besides, the associated bioethical and social concerns are becoming a challenge. Nowadays, the common knowledge of using laboratory animals is reduce, refine, and replace (3Rs). Facing these situations, we should take cheap and reliable alternatives to screen the reproductive and developmental toxicity and EDA and decide the next steps for

It is reported that a widely used technique for screening prenatal developmental toxicity is by monitoring organogenesis during gestational days (GD) 10–12 [57]. In support to whole rat embryo culture (rat WEC), a variety of morphological endpoints is integrated in the total morphological score (TMS) [58]. When applying the TMS in rat WEC, effects of pesticides on


I, insecticides; F, fungicides; H, herbicides

Table 1. The summary of reported endocrine disruptor pesticides and their related EDC activity.

the embryonic toxicity could be investigated with qualitative and quantitative endpoints. As we know, azoles are antifungal agents for clinical and agricultural use. Penconazole, propiconazole, and triadimefon were most common triazole pesticides in Taiwan. A report showed that triazole chemicals antagonized the aromatase, which transfer testosterone into 17ß-estradiol in mammals. Triazole chemicals were designed to disrupt the Cyp51 enzyme, which catalyzes the conversion of lanosterol to ergosterol on the fungal cell membrane, and led to cell death when attacked [59]. Though in the respect of mammalian systems Cyp51 is less

Topic Animals Estimated

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

414 Prenatal development toxicity 784 63,100 (rats)

416 Reproductive toxicity in two generations 3200<sup>a</sup> 328,00 421 Screening test for reproductive and developmental toxicity 560 54,600

426 Neurodevelopmental toxicity study 1400 1100

Table 2. Economical cost and number of animals needed to apply the OECD guidelines for testing reproductive

890.1300 Estrogen receptor transcriptional activation (human cell line HeLa-9903)

422 Combined repeated dose toxicity study with the reproduction/developmental

890.1100 Amphibian metamorphosis (frog)

890.1200 Aromatase (human recombinant) 890.1250 Estrogen receptor binding

890.1350 Fish short-term reproduction

890.1400 Hershberger (rat) 890.1450 Female pubertal (rat) 890.1500 Male pubertal (rat)

890.1600 Uterotrophic (rat)

Table 3. USEPA Tier 1 and Tier 2 test guidelines.

890.1150 Androgen receptor binding (rat prostate)

890.1550 Steroidogenesis (human cell line—H295R)

890.2100 Avian two-generation toxicity test in the Japanese quail 890.2200 Medaka-extended one-generation reproduction test

890.2300 Larval amphibian growth and development assay (LAGDA)

toxicity screening test

All the animals including discarded pups.

Data came from Rovida and Hartung [55]; Sogorb et al. [56].

OPPTS 890 series Topic

cost (€)

141

92,500 (rabbits)

412 92,000

http://dx.doi.org/10.5772/intechopen.81030

OECD guideline

a

toxicology.

Group A—EDSP Tier 1

Group B—EDSP Tier 2


a All the animals including discarded pups.

Table 2. Economical cost and number of animals needed to apply the OECD guidelines for testing reproductive toxicology.


Table 3. USEPA Tier 1 and Tier 2 test guidelines.

the embryonic toxicity could be investigated with qualitative and quantitative endpoints. As we know, azoles are antifungal agents for clinical and agricultural use. Penconazole, propiconazole, and triadimefon were most common triazole pesticides in Taiwan. A report

HCB (F) Thyroid hormone, androgen [43, 44] Vinclozolin (F) AR, pregnane X cellular receptor,

Pesticides EDC related Pesticides EDC related

Dieldrin (I) AR, estrogenic effect, ER [2, 13, 24, 32] Prothiophos (I) Estrogenic effect [31]

Diuron (H) Androgen action [17] Pyriproxyfen (I) Estrogenic effect [31]

Endrin (I) AR [13] Simazine (H) Aromatase activity, estrogen [15]

Diflubenzuron (I) Pregnane X cellular receptor [5] Pyridate (H) ER, AR [21]

Dicofol (I) Androgen synthesis, estrogens

140 Medicinal Chemistry

Dimethoate (I) Thyroid hormones, insulin,

Epoxiconazole (F) Aromatase activity, estrogen,

Fenarimol (F) Androgenic action, aromatase,

Fenbuconazole (F) Thyroid hormones, pregnane X

Fenvalerate (I) Estrogen-sensitive,

Fluvalinate (I) Human sex hormone,

Flusilazole (F) Aromatase activity, estrogens, androgens [20]

HCH (lindane) (I) Estrous cycles, luteal progesterone,

PR [33, 45]

I, insecticides; F, fungicides; H, herbicides

insulin, estradiol, thyroxine, AR, ER,

Table 1. The summary of reported endocrine disruptor pesticides and their related EDC activity.

Endosulfan (I) AR, estrogenic effect, ER, aromatase activity [2, 13, 30, 32]

androgens [20, 35]

cellular receptor [5, 10]

progesterone [18, 39]

progesterone [40, 41]

pregnane X cellular receptor [2, 5, 36]

Fenitrothion (I) AR, estrogens [21, 37] Tolclofos-methyl (I) ER [36]

Fenoxycarb (I) Testosterone [38] Toxaphene (I) Estrogen-sensitive,

Flutriafol (F) Estrogen [35] Trichlorfon (I) Thyroid function [54] Glyphosate (H) Aromatase activity, estrogens [42] Trifluralin (H) Pregnane X cellular receptor,

synthesis, ER [17, 21]

luteinizing hormone [33, 34]

Diazinon (I) Estrogenic effect [31] Propazine (H) Aromatase activity, estrogen [15] Dichlorvos (I) AR [2] Propiconazole (F) Estrogen, aromatase activity,

androgens [20, 35]

progesterone [19, 39]

corticosterone [10, 32]

activity, androgens [21]

Estrogenic effect [2]

steroid hormone [11]

steroid hormone [2, 11, 25]

activity, androgens [20, 21]

Tebuconazole (F) Aromatase activity, estrogens, androgens [20]

Triadimefon (F) Estrogenic effect, aromatase

Triadimenol (F) Estrogenic effect, aromatase

Propoxur (I) Estrogenic effect [10]

Pyrifenox (F) Estrogen [35]

Resmethrin (I) Sex hormone [40]

Sumithrin (I) Estrogen-sensitive,

Tetramethrin (I) Estrogen [53]

Tribenuron-methyl

(H)

showed that triazole chemicals antagonized the aromatase, which transfer testosterone into 17ß-estradiol in mammals. Triazole chemicals were designed to disrupt the Cyp51 enzyme, which catalyzes the conversion of lanosterol to ergosterol on the fungal cell membrane, and led to cell death when attacked [59]. Though in the respect of mammalian systems Cyp51 is less sensitive to azoles, it was still critical for the sterol biosynthesis pathway and might be related to the thyroid function. In this study, we will take triazoles penconazole, propiconazole, and triadimefon as an example for the alternative of endocrine-disruptor screening.

stages, which were considered death if the embryonic yolk sac circulation system or the heart stopped beating. Finally, the carcass head-tail length, developmental grade, head length, number of body segments, and yolk sac diameter were analyzed by t-test and related measurements according to statistical methods; death and abnormal embryos were determined by chi-square. Half of the evaluated embryos were preserved in neutral formalin solution for immunostaining, and the other half were stored in PBS for WB analysis to detect antibody responses related to hormone receptor or enzyme antibodies including AR, ERα, ERß, TRα,

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

http://dx.doi.org/10.5772/intechopen.81030

143

This study aimed to investigate the effect of these three pesticides on estrogen receptor (ERα and ERß), thyroid receptor (TRα and TRß), and aromatase activities in whole rat embryo culture (rat WEC) on gestation day (GD) 10.5. The concentrations of WEC were 3.1E-5, 6.2E-5, and 1.2E-4 M of penconazole, propiconazole, and triadimefon. The culture period was 48 hours. After culture the embryo morphology was assessed according to the TMS system [62], we graded the endpoint as no effect (), little effect (), effect (+), and potential effect (++). After evaluation of embryo development, it was fixed in formalin or kept in HBSS for

The embryos were treated by penconazole, propiconazole, and triadimefon with concentrations of 3.1E-5, 6.2E-5, and 1.2E-4 M. Embryos from control and pesticide treatments were fixed in 10% neutral buffered formalin for 1 week. The embryos were then dehydrated with increasing concentrations of ethanol, cleared in toluene, and embedded in paraffin. All the sections were cut into 5 mm slices and deparaffinized, hydrated, and treated with 0.3% H2O2 in PBS (pH 7.6) for 30 minutes to block endogenous peroxidase activity and finally treated with a protein-blocking solution (5% goat serum diluted in phosphate-buffered saline). All these steps were followed by heating the sections in a microwave oven for antigen retrieval using a 0.01 M citrate buffer solution (pH 5.5). Tissue sections were immunostained with rabbit anti-AR(N-20), anti-ER (MC) antibody (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P)(Acris Antibodies, Inc., San Diego, CA), which was diluted 1:250 in phosphate-buffered saline and 0.25% bovine serum albumin and maintained at room temperature overnight. The tissue sections were then developed with a streptavidin-HRP kit (Chemicon IHC Select® CA, USA), using diaminobenzidine as the chromogen, and were counterstained with hematoxylin. All images were optimized by using an inverted microscope (Leica, Wetzlar GmbH, Germany). To quantify the relative amount of activity of ER, TR, and aromatase in the IHC, 200 nuclei stained per field in a slide, 5 fields per slide, and 5 slides per dose were counted. The intensity of AR, ER, TR, and aromatase proteins stained in nucleus was graded as (0, negative), + (1, mild), ++ (2, moderate), +++ (3, intense), ++++ (4, more intense), or +++++ (5, very intense). The measurements were

2.4. Pesticide treatment and evaluation of embryo morphology

immunohistochemistry (IHC) and western blot (WB), respectively.

control group adjusted, and the values were statistically analyzed.

2.5. Immunohistochemical (IHC) evaluation

TRß, and aromatase.

## 2. Materials and methods

#### 2.1. Animals

The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Taiwan Agricultural Chemicals and Toxic Substances Research Institute. Five-week-old male and female Wistar rats were purchased from BioLASCO (Taipei, Taiwan, ROC). The rats were acclimated to the laboratory environment and reared under a controlled temperature (21 2C), humidity (40–70%), frequency of ventilation (at least 10/h), and alternating 12 hour cycles of light and darkness. The rats were administered a pellet rodent diet and water ad libitum until they were sacrificed. At 12 weeks of age, the 4 male and 20 female rats were allowed to mate with 2 males to 2 females per day. Gestation day (GD) 0 was defined as the day that sperm was observed in the vagina of the female following mating.

#### 2.2. Chemicals

Materials were obtained from the following manufacturers: DMSO (dimethyl sulfoxide), T3 (triiodothyroxine), Tria (triadimefon), Penc (penconazole), and Prop (propiconazole). All these chemicals with 97% pure at least were purchased from Sigma Chemical Co. (St. Louis, MO).

#### 2.3. Rat whole embryo culture

Five-week-old female and male rats were purchased and reared in the first animal house breeding room until 11–12 weeks of age. Two males and two females were bred in the same cage. The female rats were examined for vaginal plugs on the next day. The occurrence was considered as successful breeding. From the date of pregnancy to the 10.5th day, the embryos were dissected. Reichert's membrane was removed according to the method described by Andrews et al. [60] and Dimopoulou et al. [61], and the embryos containing the intact yolk sac placenta and the urinary membrane were removed and randomly placed in a 4 mL culture medium HBSS solution containing 50 IU of penicillin G/mL and 50 μg streptomycin/mL. The sample was added to a 25 T culture flask containing filter-sterilized rat serum and subjected to complement deactivation and cultured in a constant temperature incubator at 37C for 48 hours. The culture solution was initially inflated with a mixed gas of 5% O2, 5% CO2, and 90% N2 for 1 minute, and after about 16 hours of culture, 10% O2, 5% CO2, and 85% N2, inflated for 1 minute, and were cultured until the 24th hour. Inflate for 1 minute with 20% O2, 5% CO2, and 75% N2. Each treatment dose was inflated for 1 minute at 40% O2, 5% CO2, and 55% N2 at 40 hours, and the embryos were measured for growth, development, and morphology at the end of 48 hours of culture. Embryonic development was modified according to Brown and Fabro [62], and the evaluation included embryo growth traits and developmental stages, which were considered death if the embryonic yolk sac circulation system or the heart stopped beating. Finally, the carcass head-tail length, developmental grade, head length, number of body segments, and yolk sac diameter were analyzed by t-test and related measurements according to statistical methods; death and abnormal embryos were determined by chi-square. Half of the evaluated embryos were preserved in neutral formalin solution for immunostaining, and the other half were stored in PBS for WB analysis to detect antibody responses related to hormone receptor or enzyme antibodies including AR, ERα, ERß, TRα, TRß, and aromatase.

#### 2.4. Pesticide treatment and evaluation of embryo morphology

This study aimed to investigate the effect of these three pesticides on estrogen receptor (ERα and ERß), thyroid receptor (TRα and TRß), and aromatase activities in whole rat embryo culture (rat WEC) on gestation day (GD) 10.5. The concentrations of WEC were 3.1E-5, 6.2E-5, and 1.2E-4 M of penconazole, propiconazole, and triadimefon. The culture period was 48 hours. After culture the embryo morphology was assessed according to the TMS system [62], we graded the endpoint as no effect (), little effect (), effect (+), and potential effect (++). After evaluation of embryo development, it was fixed in formalin or kept in HBSS for immunohistochemistry (IHC) and western blot (WB), respectively.

#### 2.5. Immunohistochemical (IHC) evaluation

sensitive to azoles, it was still critical for the sterol biosynthesis pathway and might be related to the thyroid function. In this study, we will take triazoles penconazole, propiconazole, and

The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Taiwan Agricultural Chemicals and Toxic Substances Research Institute. Five-week-old male and female Wistar rats were purchased from BioLASCO (Taipei, Taiwan, ROC). The rats were acclimated to the laboratory environment and reared under a controlled temperature (21 2C), humidity (40–70%), frequency of ventilation (at least 10/h), and alternating 12 hour cycles of light and darkness. The rats were administered a pellet rodent diet and water ad libitum until they were sacrificed. At 12 weeks of age, the 4 male and 20 female rats were allowed to mate with 2 males to 2 females per day. Gestation day (GD) 0 was defined as the day that sperm was observed in the vagina of the female following mating.

Materials were obtained from the following manufacturers: DMSO (dimethyl sulfoxide), T3 (triiodothyroxine), Tria (triadimefon), Penc (penconazole), and Prop (propiconazole). All these chemicals with 97% pure at least were purchased from Sigma Chemical Co. (St. Louis, MO).

Five-week-old female and male rats were purchased and reared in the first animal house breeding room until 11–12 weeks of age. Two males and two females were bred in the same cage. The female rats were examined for vaginal plugs on the next day. The occurrence was considered as successful breeding. From the date of pregnancy to the 10.5th day, the embryos were dissected. Reichert's membrane was removed according to the method described by Andrews et al. [60] and Dimopoulou et al. [61], and the embryos containing the intact yolk sac placenta and the urinary membrane were removed and randomly placed in a 4 mL culture medium HBSS solution containing 50 IU of penicillin G/mL and 50 μg streptomycin/mL. The sample was added to a 25 T culture flask containing filter-sterilized rat serum and subjected to complement deactivation and cultured in a constant temperature incubator at 37C for 48 hours. The culture solution was initially inflated with a mixed gas of 5% O2, 5% CO2, and 90% N2 for 1 minute, and after about 16 hours of culture, 10% O2, 5% CO2, and 85% N2, inflated for 1 minute, and were cultured until the 24th hour. Inflate for 1 minute with 20% O2, 5% CO2, and 75% N2. Each treatment dose was inflated for 1 minute at 40% O2, 5% CO2, and 55% N2 at 40 hours, and the embryos were measured for growth, development, and morphology at the end of 48 hours of culture. Embryonic development was modified according to Brown and Fabro [62], and the evaluation included embryo growth traits and developmental

triadimefon as an example for the alternative of endocrine-disruptor screening.

2. Materials and methods

2.1. Animals

142 Medicinal Chemistry

2.2. Chemicals

2.3. Rat whole embryo culture

The embryos were treated by penconazole, propiconazole, and triadimefon with concentrations of 3.1E-5, 6.2E-5, and 1.2E-4 M. Embryos from control and pesticide treatments were fixed in 10% neutral buffered formalin for 1 week. The embryos were then dehydrated with increasing concentrations of ethanol, cleared in toluene, and embedded in paraffin. All the sections were cut into 5 mm slices and deparaffinized, hydrated, and treated with 0.3% H2O2 in PBS (pH 7.6) for 30 minutes to block endogenous peroxidase activity and finally treated with a protein-blocking solution (5% goat serum diluted in phosphate-buffered saline). All these steps were followed by heating the sections in a microwave oven for antigen retrieval using a 0.01 M citrate buffer solution (pH 5.5). Tissue sections were immunostained with rabbit anti-AR(N-20), anti-ER (MC) antibody (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P)(Acris Antibodies, Inc., San Diego, CA), which was diluted 1:250 in phosphate-buffered saline and 0.25% bovine serum albumin and maintained at room temperature overnight. The tissue sections were then developed with a streptavidin-HRP kit (Chemicon IHC Select® CA, USA), using diaminobenzidine as the chromogen, and were counterstained with hematoxylin. All images were optimized by using an inverted microscope (Leica, Wetzlar GmbH, Germany). To quantify the relative amount of activity of ER, TR, and aromatase in the IHC, 200 nuclei stained per field in a slide, 5 fields per slide, and 5 slides per dose were counted. The intensity of AR, ER, TR, and aromatase proteins stained in nucleus was graded as (0, negative), + (1, mild), ++ (2, moderate), +++ (3, intense), ++++ (4, more intense), or +++++ (5, very intense). The measurements were control group adjusted, and the values were statistically analyzed.

#### 2.6. Western blot

The embryo homogenates were then centrifuged at 3000 g for 30 minutes at 4C. The supernatants were aliquoted and stored at 86C before use. Before western blotting, protein contents were measured by BCA protein assay (Cat. No. 23225, Pierce). Equal amounts of protein were loaded onto each polyacrylamide gel. The antibody dilutions were 1:200 for the anti-AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P) (Acris Antibodies, Inc., San Diego, CA) and 1:5000 for the horseradish peroxidase-conjugated goat anti-rabbit IgG (AP132P, Chemicon International). For each treatment group, five samples were analyzed in two separate blots. Total protein extracts from the embryo homogenates were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes. The membranes were then blocked for non-specific binding and incubated with polyclonal primary antibodies for AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), aromatase (SM2222P) (Acris Antibodies, Inc., San Diego, CA), and ß-actin (AP132P, Chemicon International). After incubation with primary antibody, the membranes were incubated with horseradish peroxidase-linked antigoat IgG secondary antibody and visualized on film exposed to enhanced chemiluminescence (VisualizerTM Western Blot Detection Kit, Millipore, MA, USA). The relative amount of protein in the resulting immunoblot bands was estimated by measuring the optical densities of the bands on exposed films using a FOTO/Analyst® Investigator System (Fotodyne Incorporated, WI, USA). The measurements were background adjusted, and the values were statistically analyzed. Protein for ß-actin served as an internal standard.

#### 2.7. Statistical analysis

The values of ER, TR, and aromatase in western blot were normalized against ß-actin. All results were statistically analyzed with the t-test, and p < 0.05 was considered statistically significant. The other data were expressed as mean SE. Data were subjected to ANOVA followed by t-test. The level of significance was set at p < 0.05.

In the immunohistochemistry (IHC), the 17ß-estradiol (ERα and ERß) positive control showed the respective results of receptor expressions. Our results showed that penconazole, propiconazole, and triadimefon induced expressions of ERα (Figure 2) and ERß (Figure 3) in WEC. This result basically meets the mechanisms of triazoles designed to disrupt the synthesis of steroid hormone. Also, results showed that penconazole, propiconazole, and triadimefon induced expressions of TRß (data not shown), but not in TRα (data not shown) with WEC. The relationship among TRß and AR and ER still needs to be investigated. Also, we need to study the antagonistic effects by adding the antagonists for the receptor expression. These three pesticides did not affect significantly AR (data not shown) and aromatase activity (data not shown). In the western blot (WB) data, these three pesticides did not affect significantly AR, ERα, ERß, TRα, TRß, and aromatase expressions in WEC (data not shown). The difference between IHC and WB induced by these three pesticides might be the sensitivity of detecting method. WB needs some embryos for the protein quantitative, while IHC can detect activity in

Table 4. Effect of treatment with triazole pesticides on some developmental scores of rat embryo culture of day 10.5 for

Allantois Flexion Heart Caudal neural

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

DMSO 3.1 0.3 4.0 0.0 2.0 0.8 2.6 0.7 3.7 1.3 2.8 0.8 2.8 0.8 E2 3.0 0.8 4.0 0.0 3.0 1.8 2.8 0.5 4.3 1.0 2.0 1.2 2.5 1.0 T3 3.0 0.0 4.0 0.0 2.5 2.1 2.0 1.4 3.0 0.0 3.0 0.0 3.0 0.0

L 2.5 0.6 4.0 0.0 3.0 0.8 3.0 0.0 4.0 0.0 1.0 0.0 3.0 0.0 M 2.8 0.5 4.0 0.0 2.5 0.6 3.0 0.0 4.3 0.5 2.5 1.0 2.5 1.0 H 3.0 0.0 4.0 0.0 1.8 1.0 2.8 0.4 4.0 0.9 2.7 0.8 2.7 1.0

<sup>L</sup> 3.7 0.6\* 4.0 0.0 3.7 1.2\* 3.0 0.0 4.3 1.2 3.0 0.0 3.0 0.0 <sup>M</sup> 3.6 0.6\* 4.0 0.0 2.7 0.6 3.0 0.0 4.0 0.0 3.0 0.0 3.0 0.0 H 3.4 0.5 4.0 0.0 3.0 1.9 2.4 0.9 3.8 0.8 2.2 1.1 2.4 0.9

L 3.0 0.0 4.0 0.0 3.0 2.0 3.0 0.0 3.7 0.6 2.3 1.2 2.3 1.2 M 2.8 0.4 3.8 0.4 2.4 0.9 3.0 0.0 4.0 0.7 2.8 0.4 2.8 0.5 H 3.0 0.8 4.0 0.0 2.5 1.7 3.0 0.0 3.8 1.0 3.0 0.0 3.0 0.0 All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations, 1.2E-

tube

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Hindbrain Midbrain

145

an embryo.

Treatment Yolk sac circulatory

Triadimefon

Penconazole

Propiconazole

4 M. \* P < 0.05.

48 hours.

system

#### 3. Results

In the development of rat embryo, 17ß-estradiol (E2), triiodothyronine (T3), triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crownrump length, head length, and developmental score (Tables 4–6; Figure 1).


2.6. Western blot

144 Medicinal Chemistry

2.7. Statistical analysis

3. Results

The embryo homogenates were then centrifuged at 3000 g for 30 minutes at 4C. The supernatants were aliquoted and stored at 86C before use. Before western blotting, protein contents were measured by BCA protein assay (Cat. No. 23225, Pierce). Equal amounts of protein were loaded onto each polyacrylamide gel. The antibody dilutions were 1:200 for the anti-AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), and aromatase (SM2222P) (Acris Antibodies, Inc., San Diego, CA) and 1:5000 for the horseradish peroxidase-conjugated goat anti-rabbit IgG (AP132P, Chemicon International). For each treatment group, five samples were analyzed in two separate blots. Total protein extracts from the embryo homogenates were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes. The membranes were then blocked for non-specific binding and incubated with polyclonal primary antibodies for AR (N-20), ERα (MC-20), ERß (H-150) (Santa Cruz Co., CA), TRα (C0345), TRß (C0346) (Assay Biotechnology Co. Sunnyvale, CA), aromatase (SM2222P) (Acris Antibodies, Inc., San Diego, CA), and ß-actin (AP132P, Chemicon International). After incubation with primary antibody, the membranes were incubated with horseradish peroxidase-linked antigoat IgG secondary antibody and visualized on film exposed to enhanced chemiluminescence (VisualizerTM Western Blot Detection Kit, Millipore, MA, USA). The relative amount of protein in the resulting immunoblot bands was estimated by measuring the optical densities of the bands on exposed films using a FOTO/Analyst® Investigator System (Fotodyne Incorporated, WI, USA). The measurements were background adjusted, and the values were statistically

The values of ER, TR, and aromatase in western blot were normalized against ß-actin. All results were statistically analyzed with the t-test, and p < 0.05 was considered statistically significant. The other data were expressed as mean SE. Data were subjected to ANOVA

In the development of rat embryo, 17ß-estradiol (E2), triiodothyronine (T3), triadimefon, penconazole, and propiconazole exhibited no significant effect on yolk sac circulatory system, allantois, flexion, heart caudal neural tube, hindbrain, midbrain, forebrain, otic system, optic system, olfactory system, maxillary process, forelimb, hind limb, yolk sac diameter, crown-

rump length, head length, and developmental score (Tables 4–6; Figure 1).

analyzed. Protein for ß-actin served as an internal standard.

followed by t-test. The level of significance was set at p < 0.05.

All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations, 1.2E-4 M. \* P < 0.05.

Table 4. Effect of treatment with triazole pesticides on some developmental scores of rat embryo culture of day 10.5 for 48 hours.

In the immunohistochemistry (IHC), the 17ß-estradiol (ERα and ERß) positive control showed the respective results of receptor expressions. Our results showed that penconazole, propiconazole, and triadimefon induced expressions of ERα (Figure 2) and ERß (Figure 3) in WEC. This result basically meets the mechanisms of triazoles designed to disrupt the synthesis of steroid hormone. Also, results showed that penconazole, propiconazole, and triadimefon induced expressions of TRß (data not shown), but not in TRα (data not shown) with WEC. The relationship among TRß and AR and ER still needs to be investigated. Also, we need to study the antagonistic effects by adding the antagonists for the receptor expression. These three pesticides did not affect significantly AR (data not shown) and aromatase activity (data not shown). In the western blot (WB) data, these three pesticides did not affect significantly AR, ERα, ERß, TRα, TRß, and aromatase expressions in WEC (data not shown). The difference between IHC and WB induced by these three pesticides might be the sensitivity of detecting method. WB needs some embryos for the protein quantitative, while IHC can detect activity in an embryo.


All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-4 M.

4. Discussion

day 10.5 for 48 hours.

Treatment Forelimb Hind

Propiconazole

4 M. \* P < 0.05. limb

Yolk sac diameter (A) (mm)

detect EDCs in food.

5. Future work and recommendations

WEC was used to study the prenatal developmental toxicity induced by environmental chemicals including phthalate and methoxyacetic acid [63, 64], aliphatic amides [65], and triazole pesticides [66, 67]. In respect of the 3Rs principle of animal study, WEC is an alternative to screen the potential of prenatal developmental toxicity of environmental compounds. Although ex vivo exposure of WEC was used limitedly without metabolisms of chemicals, most chemicals exhibited their action by parent compound. In this study, we found that in combination with IHC and WB, WEC will be a robust way to detect the endocrine-disrupting activity induced by environmental chemicals. In this study, we used WEC to detect the important receptors including AR, ERα, ERß, TRα, and TRß and enzyme aromatase activity potential induced by triadimefon, penconazole, and propiconazole. There is one shortcoming of WEC to be addressed. Due to the small amount of embryo, WB is hard to quantify the proteins of hormone receptors. The solution to the problem is to pool the embryo treated by one dose and analyze it. Also, we knew that fortunately nowadays IHC quantification is available. Finally, we concluded that in combination with IHC and WB, WEC will be a robust way to

Table 6. Effect of co-treatment with triazole pesticides on developmental parameters and scores of rat embryo culture of

Yolk sac diameter (B) (mm)

L 0.7 0.6 0.7 0.6 6.0 1.4 5.4 0.8 5.0 0.4 2.1 0.7 38 9 M 0.6 0.5 0.6 0.5 4.2 0.8\* 4.5 1.0\* 4.5 1.1 1.8 0.4 40 <sup>5</sup> H 0.7 0.6 0.8 0.5 5.5 2.2 4.8 0.5 4.2 1.4 1.9 0.8 38 6 All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-

Crown-rump length (mm)

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Head length (mm)

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Developmental score

147

In order to meet the 3Rs including reduction, refine, and replace and precise risk assessment, adverse outcome pathway (AOP) is extensively developed by OECD. By tier screening for EDCs, the molecular initiating event (MIE), key event (KE), key event relationship (KER), and adverse outcome (AO) will be studied. As the guideline stated, the AOP framework made clear the mechanisms from MIE, KE, and KER to AO will meet the criteria of 3Rs of the animal study and provide a quick and precise way to regulatory protection goals and decision-making.

Table 5. Effect of treatment with triazole pesticides on some other developmental scores of rat embryo culture of day 10.5 for 48 hours.



All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-4 M. \* P < 0.05.

Table 6. Effect of co-treatment with triazole pesticides on developmental parameters and scores of rat embryo culture of day 10.5 for 48 hours.

## 4. Discussion

Treatment Forebrain Otic

Triadimefon

146 Medicinal Chemistry

Penconazole

Propiconazole

4 M.

for 48 hours.

Triadimefon

Penconazole

Treatment Forelimb Hind

limb

Yolk sac diameter (A) (mm)

system

Optic system Olfactory system

DMSO 2.7 0.7 1.8 0.4 2.8 1.3 1.5 0.7 1.4 0.5 0.9 0.3 2.0 0.0 E2 2.8 1.3 2.0 0.8 3.0 1.4 1.0 0.0 1.0 0.0 1.0 0.0 2.0 0.0 T3 3.0 0.0 1.5 0.7 2.5 2.1 1.5 0.7 1.5 0.7 1.0 0.0 2.0 0.0

L 2.8 0.6 1.5 0.6 3.5 1.0 1.8 0.5 1.3 0.5 1.0 0.0 2.0 0.0 M 2.3 1.0 1.5 0.6 3.3 1.0 1.0 0.0 1.5 0.6 1.0 0.0 2.0 0.0 H 2.7 1.0 1.7 0.5 3.3 0.8 1.5 0.5 1.2 0.4 1.0 0.0 2.0 0.0

L 3.3 0.6 1.7 0.6 3.3 1.2 1.7 0.6 1.3 0.6 1.0 0.0 2.0 0.0 M 3.7 0.6 1.7 0.6 4.0 0.0 1.7 0.6 1.7 0.6 1.0 0.0 2.0 0.0 H 2.6 1.5 2.0 1.0 3.2 1.8 1.2 0.8 1.2 0.4 1.0 0.0 2.0 0.0

L 2.7 1.5 1.7 1.2 3.3 2.1 1.3 0.6 1.3 0.6 1.0 0.0 2.0 0.0 M 2.8 0.4 1.4 0.5 2.6 1.1 1.2 0.4 1.2 0.4 1.0 0.0 2.0 0.0 H 3.3 0.5 1.5 0.6 2.5 1.7 1.8 0.5 1.0 0.0 1.0 0.0 2.0 0.0 All pesticide concentrate are 3.1E-5 M (low concentration, L), 6.2E-5 M (middle concentration, M), and 1.2E-4 M (high concentration, H). Dimethyl sulfoxide, DMSO; 17ß-estradiol, E2; and triiodothyronine, T3. E2 and T3 concentrations: 1.2E-

Table 5. Effect of treatment with triazole pesticides on some other developmental scores of rat embryo culture of day 10.5

Yolk sac diameter (B) (mm)

DMSO 0.7 0.5 0.7 0.5 6.4 1.2 5.7 1.0 5.2 1.1 1.9 0.6 38 7 E2 0.8 0.5 0.8 0.5 6.6 1.4 5.2 1.7 4.4 1.4 2.2 0.7 38 8 T3 1.0 0.0 1.0 0.0 5.8 0.1 4.9 1.6 4.0 1.8 1.7 0.8 38 6

L 0.5 0.6 0.8 0.5 4.8 1.0 4.8 0.6 4.9 0.4 1.7 0.3 40 3 M 0.5 0.6 1.0 0.0 5.0 0.7 5.0 0.7 5.4 1.0 1.9 0.3 38 2 H 0.7 0.5 0.8 0.4 4.7 0.9\* 5.3 1.2 4.9 0.9 1.8 0.5 39 <sup>4</sup>

L 0.7 0.6 1.3 0.6 7.1 1.7 6.4 1.6 6.0 1.0 2.5 0.6 43 4 M 1.0 0.0 0.7 0.6 6.9 0.5 5.7 1.1 5.8 0.7 3.0 0.5 43 3 H 0.6 0.5 1.0 0.7 6.3 1.2 6.2 1.2 4.6 1.7 2.1 1.1 39 9

Crown-rump length (mm)

Head length (mm)

Developmental score

Branchial bars

Maxillary process

Mandibular process

> WEC was used to study the prenatal developmental toxicity induced by environmental chemicals including phthalate and methoxyacetic acid [63, 64], aliphatic amides [65], and triazole pesticides [66, 67]. In respect of the 3Rs principle of animal study, WEC is an alternative to screen the potential of prenatal developmental toxicity of environmental compounds. Although ex vivo exposure of WEC was used limitedly without metabolisms of chemicals, most chemicals exhibited their action by parent compound. In this study, we found that in combination with IHC and WB, WEC will be a robust way to detect the endocrine-disrupting activity induced by environmental chemicals. In this study, we used WEC to detect the important receptors including AR, ERα, ERß, TRα, and TRß and enzyme aromatase activity potential induced by triadimefon, penconazole, and propiconazole. There is one shortcoming of WEC to be addressed. Due to the small amount of embryo, WB is hard to quantify the proteins of hormone receptors. The solution to the problem is to pool the embryo treated by one dose and analyze it. Also, we knew that fortunately nowadays IHC quantification is available. Finally, we concluded that in combination with IHC and WB, WEC will be a robust way to detect EDCs in food.

### 5. Future work and recommendations

In order to meet the 3Rs including reduction, refine, and replace and precise risk assessment, adverse outcome pathway (AOP) is extensively developed by OECD. By tier screening for EDCs, the molecular initiating event (MIE), key event (KE), key event relationship (KER), and adverse outcome (AO) will be studied. As the guideline stated, the AOP framework made clear the mechanisms from MIE, KE, and KER to AO will meet the criteria of 3Rs of the animal study and provide a quick and precise way to regulatory protection goals and decision-making.

Figure 2. Effect of penconazole, propiconazole, and triadimefon on ERalpha activity in WEC.

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

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149

Figure 1. The rat whole embryo culture.

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food http://dx.doi.org/10.5772/intechopen.81030 149

Figure 1. The rat whole embryo culture.

148 Medicinal Chemistry

Figure 2. Effect of penconazole, propiconazole, and triadimefon on ERalpha activity in WEC.

Figure 4. Suggestion of flow chart for assessment of endocrine disrupters.

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

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151

Figure 4. Suggestion of flow chart for assessment of endocrine disrupters.

Figure 3. Effect of penconazole, propiconazole, and triadimefon on ERbeta activity in WEC.

150 Medicinal Chemistry

The overall weight of evidence (WoE) and level of certainty underlying the inference and extrapolation will in turn dictate the most suitable application of the AOP.

AR androgen receptor

WB western blot

Author details

Shui-Yuan Lu1

R.O.C.

References

1160-1169

ERα estrogen receptor alpha ERß estrogen receptor beta TRα thyroid receptor alpha TRß thyroid receptor beta IHC immunohistochemistry

\*, Pinpin Lin<sup>2</sup>

(NHRI), Miaoli County, Taiwan, R.O.C.

Pharmacology. 2002;179:1-12

ical Sciences. 2006;91:501-509

\*Address all correspondence to: lusueyen@tactri.gov.tw

, Wei-Ren Tsai1 and Chen-Yi Weng<sup>2</sup>

The Pragmatic Strategy to Detect Endocrine-Disrupting Activity of Xenobiotics in Food

http://dx.doi.org/10.5772/intechopen.81030

153

2 National Institute of Environmental Health Sciences, National Health Research Institutes

[1] Vinggaard AM, Hnida C, Breinholt V, Larsen JC. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicology In Vitro. 2000;14:227-234

[2] Andersen HR, Cook SJ, Waldbillig D. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied

[3] Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environmental Health Perspectives. 2004;112:524-531

[4] Lemaire G, Mnif W, Mauvais P, Balaguer P, Rahmani R. Activation of alpha- and betaestrogen receptors by persistent pesticides in reporter cell lines. Life Sciences. 2006;79:

[5] Lemaire G, Mnif W, Pascussi JM, Pillon A, Rabenoelina F, Fenet H, et al. Identification of new human PXR ligands among pesticides using a stable reporter cell system. Toxicolog-

1 Applied Toxicology Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI), Council of Agriculture, Executive Yuan, Taichung, Taiwan,

## 6. Diagram/schematic figure

The pragmatic strategy to detect EDA of xenobiotics in food is to take a tier screening. Figure 4 showed the suggestion of flow chart for assessment of endocrine disruptors. Basically rat embryo culture could be the first screening method except for chemical structure-activity relationship.

## 7. Conclusions

Penconazole, propiconazole, and triadimefon significantly induced the estrogen receptor expressions. It seems that WEC can be used as a robust method of endocrine-disrupting screening for estrogen receptors.

## Acknowledgements

The study was supported partly by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, ROC (105AS-10.7.1-PI-P2), and partly by National Health Research Institutes, Zhunan, Miaoli County 35053, ROC (NHRI-106A1- PDCO-3416181).

## Conflicts of interest

The authors declare no conflicts of interest.

## Abbreviations



## Author details

The overall weight of evidence (WoE) and level of certainty underlying the inference and

The pragmatic strategy to detect EDA of xenobiotics in food is to take a tier screening. Figure 4 showed the suggestion of flow chart for assessment of endocrine disruptors. Basically rat embryo culture could be the first screening method except for chemical structure-activity

Penconazole, propiconazole, and triadimefon significantly induced the estrogen receptor expressions. It seems that WEC can be used as a robust method of endocrine-disrupting

The study was supported partly by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, ROC (105AS-10.7.1-PI-P2), and partly by National Health Research Institutes, Zhunan, Miaoli County 35053, ROC (NHRI-106A1-

extrapolation will in turn dictate the most suitable application of the AOP.

6. Diagram/schematic figure

screening for estrogen receptors.

Acknowledgements

Conflicts of interest

The authors declare no conflicts of interest.

MRL maximum residue level

rat WEC whole rat embryo culture

EDCs endocrine-disrupting chemicals

OECD Organization for Economic Co-operation and Development OPPTS The Office of Prevention, Pesticides, and Toxic Substances

PDCO-3416181).

Abbreviations

relationship.

152 Medicinal Chemistry

7. Conclusions

Shui-Yuan Lu1 \*, Pinpin Lin<sup>2</sup> , Wei-Ren Tsai1 and Chen-Yi Weng<sup>2</sup>

\*Address all correspondence to: lusueyen@tactri.gov.tw

1 Applied Toxicology Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI), Council of Agriculture, Executive Yuan, Taichung, Taiwan, R.O.C.

2 National Institute of Environmental Health Sciences, National Health Research Institutes (NHRI), Miaoli County, Taiwan, R.O.C.

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## *Edited by Janka Vašková and Ladislav Vaško*

The area covered by this book undoubtedly includes a multidisciplinary approach. It combines and uses the wide range of methods and knowledge from a variety of disciplines in chemistry, pharmacology, and biology to synthesize new or extracted natural substances and their characterization, in terms of bioefficiency in different systems, pharmacokinetics, and pharmacodynamics. Importance is placed on revealing the interactions and effects on organisms. The process is long term, ranging from synthesis to potential testing of substances in animal studies, followed by monitoring effects on patients. The purpose is to define molecular targets of the highest efficacy of the prepared drugs, minimizing the undesirable effects. The content of this book is conceived with these intentions.

Published in London, UK © 2019 IntechOpen © afreydin / iStock

Medicinal Chemistry

Medicinal Chemistry

*Edited by Janka Vašková and Ladislav Vaško*