**8.1. Preclinical development of anti-TB active principles**

Preclinical tests involve the use of animal models to prove the efficacy and safety of a given candidate before being tested in humans. Because of its management in terms of size, offer, maintenance, strength, and reproducibility, the mouse constitutes the preferred animal model for *in vivo* research of the TB infection [60]. Other possible animal models include rats, guinea pigs, and macaques. The amounts of viable mycobacteria and mortality and the possibility of organomegaly in the pulmonary tissue are evaluated during therapy, at the end of therapy and in the post-therapy period. Post antibiotic effect, relapses, and resistance development are examined. Antagonists, additives, or synergistic effects are also evaluated when the compound is administered in combination with other active principles, as well as its capability to sterilize lesions in experimentally infected animals. Finally, toxicological studies, which must be highly controlled and documented, are carried out forthe determination of the safety window in order to perform the subsequent clinical trials in humans [60].

**Survey of natural products:** Natural products represent an alternative for the search of new compounds. Different research institutescontinuously carry out screening of natural products (products from plants, fungi, and bacteria) with the hope of identifying compounds with anti-TB activity [5]. Some natural substances have shown significant anti-TB activity: saringosterol 24-epimers, esgosterol-5,8-endoperoxide, micromolide, ascididemin, the manzamines, and engelhardione, among others; however, there is lack of more research regarding selectivity

**Plants:** Drugs based on plants extracts have been used worldwide for the treatment of several diseases from ancient times.A great interest in phytomedicine and natural product structures are screened in order to measure their pharmacologic activity. In Colombia, there has been a

**Natural sea products:** oceans are outstanding sources of natural products, not only in inverte‐ brate species such as sponges, mollusks, bryozoos, but also in marine bacteria and marine sediments. The alkaloid (+)-8-hydroxymanzamine A was initially isolated from the *Pachypelli‐ na*spsponge[55].Inthesameway,irciniolAwasfoundinspongesfromtheIndianPacificproving to be a good candidate for further studies [56]. Aerothiononine isolated from the marine sponge *Aplysinagerardogreeni*marine sponge was active against clinical isolates of MDR-TB, despite of the resistance patterns, with MIC from 6.5 to 25 mg/L [57]. The alkaloid (+)-8-hydroxymanza‐

**Insects.** The immune system of invertebrates and vertebrates is made up by cytolitic peptides which act as antimicrobial agents during the invasion of eukaryotes and prokaryotes micro‐ organisms. Poison from arachnids (spiders and scorpions) contains toxic peptides of high molecular weight (2 – 12 kDa) with high specificity against prokaryote cells [59]. This type of

**Microorganisms.**Most of the major antibiotics drugs have been isolated frommicroorganisms. Streptomycin,the first effective anti-TB drug was identified in *Streptomyces griseus*. Besides streptomycin, aminogyicosides kanamycin, amikacin, and capreomycinhave been very important therapeutically as second-line agents [59]. Other important anti-TB drugs in TB treatment are the rifamycins, which constitute a group of semi-synthetic antibiotics isolated

**8. Preclinical and clinical development for new anti-tuberculosis drugs**

Preclinical tests involve the use of animal models to prove the efficacy and safety of a given candidate before being tested in humans. Because of its management in terms of size, offer, maintenance, strength, and reproducibility, the mouse constitutes the preferred animal model for *in vivo* research of the TB infection [60]. Other possible animal models include rats, guinea pigs, and macaques. The amounts of viable mycobacteria and mortality and the possibility of organomegaly in the pulmonary tissue are evaluated during therapy, at the end of therapy

mine A alkaloid showed potent inhibitory activity against *M. tuberculosis* H37Rv [58].

compounds may be very promising as a drug in the treatment of tuberculosis.

resurgent interest in the discovery of novel natural anti-TB drugs [50-54].

and toxicity [47-50].

342 Tuberculosis - Current Issues in Diagnosis and Management

from *Streptomyces mediterrani*[59].

**8.1. Preclinical development of anti-TB active principles**

The drugs regime must be administered for several months, using commonly between 100 – 150 mice per test, therefore requiring large amounts of space and resources for maintaining the animals. Model in mice is more effective regarding the cost-benefit relation, and most of the data obtained can be reproduced in clinical studies. The model of infection by TB in mice has served to predict the sterilizing potential of new compounds, the effectiveness of the combination of drugs, success in intermittent therapy and the duration of therapy necessary to avoid relapse. The effectiveness of the active principle is measured mainly the reduction of the colony forming units (CFU) in the lung and spleen. Several varieties of mice have been used in laboratories conducting this type of test and, to this date, no comparisons have been reported [61].

Genetically modified mice have been used in the in bioassay of compounds with antimycobacterial activity [62]. A mouse that does not express the interferon-γ gene (knock-out) is incapable of producing cytokine Th1 and therefore suffers a more acute infection. Bioassay with this mouse allows determining the initial efficacy of a chemical compound in six days. Because of their statistical power, substances with low antimycobacterial activity can be detected by a small decrease in the CFU count. The model has great usefulness in initial trials, when there is a limited amount of the chemical compound. Another model, still under development, has been proposed to study relapse. An animal that cannot produce the granulocyte-macrophage-colony stimulation factor (GM-CSF) is used.

Wayne's model, which indicates the effect of compounds against persistent bacilli, has also been used. Bacilli under anoxia conditions are used and they are directly inoculated in the mouse. The guinea pig model also allows observing the destruction of tissue by caseous necrosis where there is not oxygen contribution and bacteria go into a hypoxia state [61].

Pharmacokinetics and pharmacodynamics range from *in vitro* tests, *in vivo* tests in animal models, and finally clinical trials in humans [57]. The simplest pharmacodynamic measure is determining the MIC *in vitro,* used widely in the primary discovery of active principles. This measure can be roughly related to the maximum cut point of the active principle concentration in plasma (Cmax) and can aid in the prediction of *in vivo* pharmacodynamics among a series of structurally related agents. However, it does not represent the concentration at which the growth ceases, and, as we have already seen, does not allow distinguishing between bacteri‐ cide and bacteriostatic activity. Moreover, it does not allow obtaining information regarding the dynamic relationships *in vivo* either, since the growth conditions do not represent the ones of persistent organisms in the living tissue.

Animal models enable to evaluate the *in vivo* efficacy of novel active principles regimens. Protection experiments using monotherapyfor a certain amount of time and then performing lethal intravenous or aerosol inoculation can prove the efficacy and selection of a preliminary dose. Studies on the short term using colonies count in different homogenized organs allow estimating the bactericide capability of a medication or a combination of drugs, as well as the likely appearance of resistance [57]. However, in order to describe the sterilizing activity of a given compound, a larger study time is required as well as other techniques since negative cultures finalizing the therapy do not necessarily indicate that there was sterilization. Three months are required after the end of treatment to determine a durable cure and success of the sterilization.Cornell'smousemodelusesanintensivetherapyinordertoobtainnegativecultures and then evaluate the ability of individual active principles or their combinations to prevent relapse when the mouse is left untreated or when it is maintained immunocompromised[57].

sion of the disease for the 17 macaques studied were observed. Between 50 – 60% of infected primates developed active and chronic infection, characterized by clear signs of disease in thoracic x-rays and other tests. Approximately 40% of the initially infected macaques did not develop the disease in the 20 months of study. These primates showed clinical signs of latent TB. In summary, the study proves that it is possible to use the cynomolgus macaque in infection by *M. tuberculosis* because it presents the complete spectrum of infection in humans (rapid and lethal infection, chronic infection, and latent infection). This animal model is the only one that enables to study the latent forms of the infection. Its great advantage is the high pathologic similarity of the infection in macaques and humans, whereas the disadvantages are the cost and maintenance of the animals, particularly since they require facilities with Biosafety Level 3 [64]. This model has been proposed in final preclinical trials for the development of active principles for latent forms of TB [61].The following are examples of promising compounds in preclinical phase. Some of these substances are protected by patents and therefore access to

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The following are examples of promising compounds in preclinical phase. Some of these

**SQ609 dipiperidine:** this compound is a completely novel anti-TB active principle. It acts by interrupting the biosynthesis ofcell wall, but its specific mechanism is unknown [5]. It

**FAS20013 synthetase ATP inhibitor:** Inhibition of bacterial fatty acids synthesis (FASII) still represents a valid, target for the discovery of anti-TBdrugs. However, this novel compound was identified by Fasgen and it has as action target the inhibition of enzymes for biosynthesis

**Translocase inhibitors SQ641:** The pharmaindustryis developing a series of translocase inhibitors for the treatment of TB. The mycobacterial translocase I is an enzyme required for the biosynthesis of the cell wall, and the SQ641 compound has been reported as a selective

Identifying new anti-TB is a complex and highly regulated process carried out around a critical moment: when the new compound is tested in humans [5]. Currently, clinical images offer a support method for generation of drugs which enables to establish information about the biodistribution of the molecule, interaction of the target, and pharmacokinetics[68]. Clinical development of a promising substance is usually divided into four phases. The first phase is carried out in healthy human beings and it provides information regarding the chemical compound pharmacokinetic profile, and some preliminary information regarding safety [69]. Phase I trials are conducted in a small size, usually 15 to 30 subjects, and can be of single or multiple doses. Besides the phase I trials, researchers may consider incorporating the phar‐ macokinetics and safety studies to a wider population size (200 to 300 subjects). Phase II studies are conducted on patients diagnosed with active TB. The efficacy in monotherapy and

substances are protected by patents and therefore access to information is restricted.

of fatty acids in *Mycobacterium* [65]. It belongs to the β-sulfonylcarboxamides class.

demonstratedantymycobacterial activity in an*in vivo* mouse model.

information is restricted.

inhibitor of this enzyme [66,67].

**8.2. Clinical development of anti-TB active principles**

The following are the PK and PD parameters which are calculated in the trial with mice: the C/MIC quotient, defined as the ratio of the serum maximum concentration (Cmax) over the MIC; the AUC/MIC quotient, defined as the ratio of the area below the concentration-time curve (AUC) over the MIC in the serum during the total time of treatment(144 h) divided by 6 in order to obtain a daily value (AUC24/MIC); and the percentage of time above the MIC (T > MIC) estimated by the first order kinetic equation C = C0 e-kt, where C0 is the concentration to time 0, k is the constant and t the time, and it is defined as the percentage of the 144-hour time in which the medication concentration surpasses the MIC in the serum [63].

Recent studies of the PK and PD parameters for INH, RIF, and fluoroquinolones have improved the understanding of PK and PD properties of these drugs. Although the PK and PD parameters are characterized for antibacterial agents, a clear description of the efficacy is still lacking [63]. The parameter that best describes the bactericidal activity of anti-TB drugs in the mice model is AUC24 / MIC, with a correlation of 0.83. For INH when the value of AUC24 / MIC reaches 500, the maximum effect is observed with a decrease of 1.3 log CFU per mouse lung. In other words, the INH effect was the same when the total doses were admin‐ istered into 6, 12, or 18 doses divided equally during one week [63]. Mitchison observed that the administration of a single total dose of INH in infected guinea pigs had the same effect than if administered daily, every other day, or every four days during a six-week period. Therefore, the efficacy of INHwas dependent on the size of the doses but not on the regime [63]. Preclinical trials that establish pharmacodynamic and pharmacokinetic properties enable to obtain information regarding the optimal doses and regimens.

Despite of the large use of the mouse model,this rodentdoes not develop the typical human lesions observed in pulmonary TB such as caseous necrosis or cavitations[57]. Also, one has to be very prudent conducting escalation in the doses of the agents between the mouse and the human due to the metabolic differences and possible pharmacokinetic interactions. The histological characteristics of guinea pigs in a TB infection are more similar to human pathol‐ ogy; but there is little experience in the chemotherapeutical use of this model. Preliminary studies suggest that the guinea pig model is capable of differentiating the sterilizing activities of INH and RIF [57].

A good model to study latent forms of TB is the cynomolgus macaque (*Macacafascicularis*) [61, 64]. All primates infected by bronchial instillation developed the infection, based on the tuberculin test and immune responses to *M. tuberculosis* antigens. Differences in the progres‐ sion of the disease for the 17 macaques studied were observed. Between 50 – 60% of infected primates developed active and chronic infection, characterized by clear signs of disease in thoracic x-rays and other tests. Approximately 40% of the initially infected macaques did not develop the disease in the 20 months of study. These primates showed clinical signs of latent TB. In summary, the study proves that it is possible to use the cynomolgus macaque in infection by *M. tuberculosis* because it presents the complete spectrum of infection in humans (rapid and lethal infection, chronic infection, and latent infection). This animal model is the only one that enables to study the latent forms of the infection. Its great advantage is the high pathologic similarity of the infection in macaques and humans, whereas the disadvantages are the cost and maintenance of the animals, particularly since they require facilities with Biosafety Level 3 [64]. This model has been proposed in final preclinical trials for the development of active principles for latent forms of TB [61].The following are examples of promising compounds in preclinical phase. Some of these substances are protected by patents and therefore access to information is restricted.

The following are examples of promising compounds in preclinical phase. Some of these substances are protected by patents and therefore access to information is restricted.

**SQ609 dipiperidine:** this compound is a completely novel anti-TB active principle. It acts by interrupting the biosynthesis ofcell wall, but its specific mechanism is unknown [5]. It demonstratedantymycobacterial activity in an*in vivo* mouse model.

**FAS20013 synthetase ATP inhibitor:** Inhibition of bacterial fatty acids synthesis (FASII) still represents a valid, target for the discovery of anti-TBdrugs. However, this novel compound was identified by Fasgen and it has as action target the inhibition of enzymes for biosynthesis of fatty acids in *Mycobacterium* [65]. It belongs to the β-sulfonylcarboxamides class.

**Translocase inhibitors SQ641:** The pharmaindustryis developing a series of translocase inhibitors for the treatment of TB. The mycobacterial translocase I is an enzyme required for the biosynthesis of the cell wall, and the SQ641 compound has been reported as a selective inhibitor of this enzyme [66,67].

#### **8.2. Clinical development of anti-TB active principles**

lethal intravenous or aerosol inoculation can prove the efficacy and selection of a preliminary dose. Studies on the short term using colonies count in different homogenized organs allow estimating the bactericide capability of a medication or a combination of drugs, as well as the likely appearance of resistance [57]. However, in order to describe the sterilizing activity of a given compound, a larger study time is required as well as other techniques since negative cultures finalizing the therapy do not necessarily indicate that there was sterilization. Three months are required after the end of treatment to determine a durable cure and success of the sterilization.Cornell'smousemodelusesanintensivetherapyinordertoobtainnegativecultures and then evaluate the ability of individual active principles or their combinations to prevent relapse when the mouse is left untreated or when it is maintained immunocompromised[57]. The following are the PK and PD parameters which are calculated in the trial with mice: the C/MIC quotient, defined as the ratio of the serum maximum concentration (Cmax) over the MIC; the AUC/MIC quotient, defined as the ratio of the area below the concentration-time curve (AUC) over the MIC in the serum during the total time of treatment(144 h) divided by 6 in order to obtain a daily value (AUC24/MIC); and the percentage of time above the MIC (T > MIC) estimated by the first order kinetic equation C = C0 e-kt, where C0 is the concentration to time 0, k is the constant and t the time, and it is defined as the percentage of the 144-hour time in

344 Tuberculosis - Current Issues in Diagnosis and Management

which the medication concentration surpasses the MIC in the serum [63].

to obtain information regarding the optimal doses and regimens.

of INH and RIF [57].

Recent studies of the PK and PD parameters for INH, RIF, and fluoroquinolones have improved the understanding of PK and PD properties of these drugs. Although the PK and PD parameters are characterized for antibacterial agents, a clear description of the efficacy is still lacking [63]. The parameter that best describes the bactericidal activity of anti-TB drugs in the mice model is AUC24 / MIC, with a correlation of 0.83. For INH when the value of AUC24 / MIC reaches 500, the maximum effect is observed with a decrease of 1.3 log CFU per mouse lung. In other words, the INH effect was the same when the total doses were admin‐ istered into 6, 12, or 18 doses divided equally during one week [63]. Mitchison observed that the administration of a single total dose of INH in infected guinea pigs had the same effect than if administered daily, every other day, or every four days during a six-week period. Therefore, the efficacy of INHwas dependent on the size of the doses but not on the regime [63]. Preclinical trials that establish pharmacodynamic and pharmacokinetic properties enable

Despite of the large use of the mouse model,this rodentdoes not develop the typical human lesions observed in pulmonary TB such as caseous necrosis or cavitations[57]. Also, one has to be very prudent conducting escalation in the doses of the agents between the mouse and the human due to the metabolic differences and possible pharmacokinetic interactions. The histological characteristics of guinea pigs in a TB infection are more similar to human pathol‐ ogy; but there is little experience in the chemotherapeutical use of this model. Preliminary studies suggest that the guinea pig model is capable of differentiating the sterilizing activities

A good model to study latent forms of TB is the cynomolgus macaque (*Macacafascicularis*) [61, 64]. All primates infected by bronchial instillation developed the infection, based on the tuberculin test and immune responses to *M. tuberculosis* antigens. Differences in the progres‐ Identifying new anti-TB is a complex and highly regulated process carried out around a critical moment: when the new compound is tested in humans [5]. Currently, clinical images offer a support method for generation of drugs which enables to establish information about the biodistribution of the molecule, interaction of the target, and pharmacokinetics[68]. Clinical development of a promising substance is usually divided into four phases. The first phase is carried out in healthy human beings and it provides information regarding the chemical compound pharmacokinetic profile, and some preliminary information regarding safety [69]. Phase I trials are conducted in a small size, usually 15 to 30 subjects, and can be of single or multiple doses. Besides the phase I trials, researchers may consider incorporating the phar‐ macokinetics and safety studies to a wider population size (200 to 300 subjects). Phase II studies are conducted on patients diagnosed with active TB. The efficacy in monotherapy and combination therapy is evaluated. One of the objectives of trials in phase I/II is to determine the optimal dose for the phase III studies.

Early Bactericidal Activity (EBA) is one of the fundamental parameters to determine the clinical efficacy of active principles [70]. It consists on a large trial conducted on patients recently diagnosed with pulmonary TB who are treated with active principles or combinations for a period of 2 to 14 days. Patients must not have used anti-TB drugs previously. During the treatment period, the amount of viable bacilli appearing in sputum samples is determined quantitatively. The traditional EBA unit is the logarithmic decrease of colony forming units (CFU/mL sputum/day during the first 48 hours). EBA studies have shown that there are differences between the fall of viable bacteria counts in the first two days of treatment in comparison with the following twelve [71]. Differences among several treatments were also more significant during the first two days. In the early therapy, the activity of INH was superior and dominant regarding the other active principles administered in combination. Any addition of INH to a regime leads to an increase of EBA but never higher than INH on its own. The addition of PZA to a regime of STR, INH, and RIF increased 0-2 days EBA from 0.415 to 0.472 [71]. The greatest disadvantage of determining the EBA is its inability to detect sterilizing activity. Some researchers have concluded that extended EBA trials (2 to 14 days) do not correlate to the sterilizing activity [72]. For example, the potent sterilizing activity of PZA was not detected in an extended EBA trial. STR showed potent activity in extended EBA, and it is known it has a very low sterilizing activity in randomized clinical trials. In extended EBA, EMB appears as antagonist; however, there is no clinical evidence that this drug interferes with the sterilizing effects of RIF and PZA [72].

In order to determine the sterilizing activity of the anti-TB active principles, and 8-week study has been proposed, and the ratio of patients whose sputum be negative is determined; this parameter correlates to the ratio of patients who suffer relapse after the treatment [73]. In these studies, frequent counts of the number of viable bacilli are carried out being known as "serial sputum CFU counts" or SSCC. This method enables to distinguish between differences in the organisms that divide rapidly from the persistent ones.These studies turn out to be appropriate to determine the possibility of a regime to decrease the time of treatment [73].

the trials, but there is also need to extend duplicates to local laboratories. Finally, validated relapse markers, which provide evidence of the sterilizing activity of an active principle or regime, are used. To this end, the most used method is determining the ratio of patients who have a negative sputum culture, 8 weeks after the beginning of the treatment compared to the standard treatment. Molecular methods using relapse markers require greater study and

**Figure 5. Pharmacological activity of RIF, INH and PZA targeting** *M. tuberculosis* **subpopulations.** Differential

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population of *M. tuberculosis* in the lesions, observed after 6 months of treatment.as log CFU in sputum

Phase IV studies include product development efforts such as patents, description of biologic activity, toxicity, safety profile in humans and demonstrated clinical efficacy. Best manufac‐ turing practices studies are conducted, as well as laboratory and clinical practice to ensure the marketing needs of the product. Post-marketing studies duringPhase IV are typically assess‐ ment of new regimens in comparison to thenormally used, and surveillance of likely adverse effects, including the development of resistance. The acceptance for use of the new active principle must be subscribed by the patient, and the economic benefits of the new drug must

**SQ109 diamine:** The lead was identified in a screening conducted in 2003 using a combinatory library based on EMB as pharmacophore. It shows an MIC value of 0.11 μg/ml. t remains equally active as EMB at 100 mg/kg when administered in the mouse model at a dose of 1 mg/ kg. However, SQ109 did not increase its effectiveness at higher doses (10 mg/kg, 25 mg/kg) and it was clearly less effective than INH[5]. Its effectiveness has been proved against MDR

validation in order to be employed successfully [60].

also be established [60].

Phase III clinical trials are carried out at large scale; they are randomized and they are conducted to demonstrate the improved or equivalent efficacy of a new treatment against standard treatments [60]. Around 1000 patients are enrolled per study for TB and the cure on treated patients is bacteriologically observed during certain amount of time as well as the ratio of relapse. The accepted end point to demonstrate efficacy is 2 years. The experimental design of Phase III trials must be designed cautiously clearly definingcritical primary and secondary end points, the size of the sample, the intervalsof confidence, and the statistical methods that will be used to obtain the data [60]. It is fundamental that microbiologic assessments are being conducted during an appropriate time, with the aim of determining the real activity of the researched agent. To ensure a sufficient population in Phase III studies, trials may be conducted in countries with high incidence rate of TB. Countries possessing a robust and expansive TB control program that provides essential information such as annual incidence (location of the disease, comorbility, resistance) are preferred. A reference laboratory is required for most of

combination therapy is evaluated. One of the objectives of trials in phase I/II is to determine

Early Bactericidal Activity (EBA) is one of the fundamental parameters to determine the clinical efficacy of active principles [70]. It consists on a large trial conducted on patients recently diagnosed with pulmonary TB who are treated with active principles or combinations for a period of 2 to 14 days. Patients must not have used anti-TB drugs previously. During the treatment period, the amount of viable bacilli appearing in sputum samples is determined quantitatively. The traditional EBA unit is the logarithmic decrease of colony forming units (CFU/mL sputum/day during the first 48 hours). EBA studies have shown that there are differences between the fall of viable bacteria counts in the first two days of treatment in comparison with the following twelve [71]. Differences among several treatments were also more significant during the first two days. In the early therapy, the activity of INH was superior and dominant regarding the other active principles administered in combination. Any addition of INH to a regime leads to an increase of EBA but never higher than INH on its own. The addition of PZA to a regime of STR, INH, and RIF increased 0-2 days EBA from 0.415 to 0.472 [71]. The greatest disadvantage of determining the EBA is its inability to detect sterilizing activity. Some researchers have concluded that extended EBA trials (2 to 14 days) do not correlate to the sterilizing activity [72]. For example, the potent sterilizing activity of PZA was not detected in an extended EBA trial. STR showed potent activity in extended EBA, and it is known it has a very low sterilizing activity in randomized clinical trials. In extended EBA, EMB appears as antagonist; however, there is no clinical evidence that this drug interferes with

In order to determine the sterilizing activity of the anti-TB active principles, and 8-week study has been proposed, and the ratio of patients whose sputum be negative is determined; this parameter correlates to the ratio of patients who suffer relapse after the treatment [73]. In these studies, frequent counts of the number of viable bacilli are carried out being known as "serial sputum CFU counts" or SSCC. This method enables to distinguish between differences in the organisms that divide rapidly from the persistent ones.These studies turn out to be appropriate

Phase III clinical trials are carried out at large scale; they are randomized and they are conducted to demonstrate the improved or equivalent efficacy of a new treatment against standard treatments [60]. Around 1000 patients are enrolled per study for TB and the cure on treated patients is bacteriologically observed during certain amount of time as well as the ratio of relapse. The accepted end point to demonstrate efficacy is 2 years. The experimental design of Phase III trials must be designed cautiously clearly definingcritical primary and secondary end points, the size of the sample, the intervalsof confidence, and the statistical methods that will be used to obtain the data [60]. It is fundamental that microbiologic assessments are being conducted during an appropriate time, with the aim of determining the real activity of the researched agent. To ensure a sufficient population in Phase III studies, trials may be conducted in countries with high incidence rate of TB. Countries possessing a robust and expansive TB control program that provides essential information such as annual incidence (location of the disease, comorbility, resistance) are preferred. A reference laboratory is required for most of

to determine the possibility of a regime to decrease the time of treatment [73].

the optimal dose for the phase III studies.

346 Tuberculosis - Current Issues in Diagnosis and Management

the sterilizing effects of RIF and PZA [72].

**Figure 5. Pharmacological activity of RIF, INH and PZA targeting** *M. tuberculosis* **subpopulations.** Differential population of *M. tuberculosis* in the lesions, observed after 6 months of treatment.as log CFU in sputum

the trials, but there is also need to extend duplicates to local laboratories. Finally, validated relapse markers, which provide evidence of the sterilizing activity of an active principle or regime, are used. To this end, the most used method is determining the ratio of patients who have a negative sputum culture, 8 weeks after the beginning of the treatment compared to the standard treatment. Molecular methods using relapse markers require greater study and validation in order to be employed successfully [60].

Phase IV studies include product development efforts such as patents, description of biologic activity, toxicity, safety profile in humans and demonstrated clinical efficacy. Best manufac‐ turing practices studies are conducted, as well as laboratory and clinical practice to ensure the marketing needs of the product. Post-marketing studies duringPhase IV are typically assess‐ ment of new regimens in comparison to thenormally used, and surveillance of likely adverse effects, including the development of resistance. The acceptance for use of the new active principle must be subscribed by the patient, and the economic benefits of the new drug must also be established [60].

**SQ109 diamine:** The lead was identified in a screening conducted in 2003 using a combinatory library based on EMB as pharmacophore. It shows an MIC value of 0.11 μg/ml. t remains equally active as EMB at 100 mg/kg when administered in the mouse model at a dose of 1 mg/ kg. However, SQ109 did not increase its effectiveness at higher doses (10 mg/kg, 25 mg/kg) and it was clearly less effective than INH[5]. Its effectiveness has been proved against MDR strains. Preclinical toxicology studies have been completed and further phase II clinical studies are underway [67].

did not achieve total eradication in any of the mice treated. The 6-month regime of PA-824 in combination with RIF, INH, and PZA in mice proved to be superior to the standard regime regarding quickness of eradication and lower relapse rate. This compound has been widely evaluated in animals and humans; currently it is under phase II clinical trials as part of an

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**Nitroimidazole-oxazol OPC67683:**There is very little public information regarding this compound. It belongs to a subclass of mycolic acids synthesis inhibitors. It has shown *in vitro* activity against standard and resistant strains, showing a MICof 12 ng/ml [80]. It has not shown cross resistance with any other medication. The compound shown activity against bacilli residing within human macrophages and type II pneumocytes. The chronic TB trial in mice demonstrated an activity 6 – 7 times more effective than the one observed for first line INH and RIFdrugs. Favorable oral absorption and distribution have been reported. Currently

**Pyrrole LL-3858:** Some pyrroles derivatives have been found to have *in vitro* activity against *M. tuberculosis.* The MIC of pyrrole LL-3858 is between 0.025 and 0.12 μg/ml. The LL-3858 derivate identified by Lupin Limited showed greater bactericidal activity in the lungs of mice infected in monotherapy than INH. The trial of LL-3858 in combination with INH and RIF, or with INH, RIF, and PZA sterilized the totality of mice in 3 months [5]. Currently, the compound

In the dawn of the 21st century, pathogenesis of the infectious disease appears as a competition between the host and the pathogen involving short term adaptations and co evolution of the genomes [81]. The pathogen and the host constantly exercise selective pressure over each other,

In latent tuberculous infection (LTBI), most of bacilli are not replicating, whereas in a phase of active disease most of the population is on active growth. Chemotherapy must take this metabolic adversity to favor the host.A durable cure must eliminate both the replicative and the persistent bacilli.Eradication of the persistent bacilli onchemotherapylasting between 6 – 24 months has been proposed in order to avoid relapse. However, such a long treatment is difficult to sustain and there is always resistance-associated risks in interrupted regimens [81].A philosophy of mycobacterial infection states that the essential genes for infection in mice

The proteasome of *M. tuberculosis* is a set of proteins that provide a quick adaptation to changing conditions [82]. Two genes, *mpa (Mycobacterium* proteosomal ATPase) and *pafA (*proteasome accessory factor) were identified as important in the survival of *M. tuberculosis*to exposure against reactive nitrogen species (RNS) *in vitro* and required for active disease *in vivo* [82]. These genes codify for proteins involved in the bacteria proteasomal function. Proteasomes are barrel-shaped proteases consisting of 14 α units and 14 β units [82]. Mpa is similar to ATPases found in the proteosome of eukaryotes cells, and chemical inhibition of the protease activity of the *M. tuberculosis* proteosome causes sensitization of the wild strain to

making the environment in test tube completely different from that within the host.

**8.3. New approaches for the development of anti-TB active principles**

initial scheme (PA-824, moxifloxacin, and PZA) containing new anti-TB drugs[79].

it is under phase II clinical trials.

is under phase II clinical trials [67].

include genes that are not essential *in vitro.*

**TMC-207 diarylquinoline:** This agent is a promising agent as a new kind of antimycobacterial agent. Twenty diarylquinolines have been reported on the series with MIC lower than 0.5 mg/L against H37Rv. Antimycobacterial activity was confirmed *in vivo* for three compounds of this class. The most effective agent was TMC-207, which had a MIC of 0.06 mg/L against H37Rv and its spectrum was unique in specificity against mycobacteria [74]. TMC-207 inhibits the ATP synthetase leading to a decrease of ATP and a pH imbalance. This compound has a potent EBA in the murine infection model, superior or similar to INH. The combinations TMC-207, INH, and PZA cleansed the bacilli present in the lungs of all mice after two months. Trials have also been conducted in mice with combinations of second line agents. Preliminary studies have proved*invivo*sterilizingactivityinthemicemodel,anddecreaseinthetreatmenttime.Currently it is in phase IIa. Therefore, it is the most promising drug candidate in the last 30 years.

**Gatifloxacin:** It has been reported bactericidal activity *in vitro* and *in vivo* against *M. tubercu‐ losis* to this compound. Its MIC has been reported between 0.25 mg/L and 0.03 mg/L against H37Rv [76]. In an *in vitro* study on bacilli in stationary phase, gatifloxacin showed the greatest bactericidal activity in the first two days, but none afterwards. In mice studies, the combination of gatifloxacin with EMB and PZA cleansed the lungs of infected animals after two months of treatment. Currently, gatifloxacin is under phase III to prove the efficacy and safety of a fourmonth regime for the treatment of pulmonary TB supervised by the European Commission Oflotub Consortium, WHO-TDR, Tuberculosis Research Unit (TBRU), National Institute of Allergy and Infectious Diseases (NIAID), Tuberculosis Research Centre [5,67].

**Moxifloxacin:** Moxifloxacin is the most promising fluorquinolone against *M. tuberculosis.* Its activity *in vitro* seems to affect bacilli unaffected by RIF. Its MIC reported *in vitro* is 0.5 mg/L [77]. It seems that moxifloxacin interferes with the protein synthesis on bacteria with low metabolic activity by a mechanism different to the one of RIF. However, the specific action mechanism is unknown. In the mouse model, the effectiveness of moxifloxacin is comparable to INH. When administered in combination with PZA, moxifloxacin has a greater bactericidal activity tan the INH + RIF + PZA regime. In fact, the combination RIF + moxifloxacin + PZA decreases completely bacilli count within four months, whereas the combination RIF + INH + PZA requires 6 months. It is likely that there is synergism among the three drugs, and the alternative regime replacing INH by moxifloxacin has been proposed. Moxifloxacin is under phase III [67]. Clinical studies have not proved a greater sterilizing activity of a regime containing moxifloxacin in comparison with the standard regime; however, it has increased activity in early points [5,75].

**Nitroimidazole PA-824:** This bicyclical nitroimidazole is under development by the TB Alliance, which has the proprietary rights. The *in vitro* MIC of the PA-824 compound is between 0.15 and 0.3 μg/ml [78].After an activation by the F420 factor of *M. tuberculosis,* the PA-824 compound inhibits biosynthesis of the cell wall components by means of mechanisms still to be established. It has proved bactericidal activity against replicative and static bacilli *in vitro.* Although PA-824 was more efficient than INH or moxifloxacin, during the continuation phase it was not better than the RIF + INHH combination. On the long term, the 12-month treatment did not achieve total eradication in any of the mice treated. The 6-month regime of PA-824 in combination with RIF, INH, and PZA in mice proved to be superior to the standard regime regarding quickness of eradication and lower relapse rate. This compound has been widely evaluated in animals and humans; currently it is under phase II clinical trials as part of an initial scheme (PA-824, moxifloxacin, and PZA) containing new anti-TB drugs[79].

strains. Preclinical toxicology studies have been completed and further phase II clinical studies

**TMC-207 diarylquinoline:** This agent is a promising agent as a new kind of antimycobacterial agent. Twenty diarylquinolines have been reported on the series with MIC lower than 0.5 mg/L against H37Rv. Antimycobacterial activity was confirmed *in vivo* for three compounds of this class. The most effective agent was TMC-207, which had a MIC of 0.06 mg/L against H37Rv and its spectrum was unique in specificity against mycobacteria [74]. TMC-207 inhibits the ATP synthetase leading to a decrease of ATP and a pH imbalance. This compound has a potent EBA in the murine infection model, superior or similar to INH. The combinations TMC-207, INH, and PZA cleansed the bacilli present in the lungs of all mice after two months. Trials have also been conducted in mice with combinations of second line agents. Preliminary studies have proved*invivo*sterilizingactivityinthemicemodel,anddecreaseinthetreatmenttime.Currently

it is in phase IIa. Therefore, it is the most promising drug candidate in the last 30 years.

Allergy and Infectious Diseases (NIAID), Tuberculosis Research Centre [5,67].

**Gatifloxacin:** It has been reported bactericidal activity *in vitro* and *in vivo* against *M. tubercu‐ losis* to this compound. Its MIC has been reported between 0.25 mg/L and 0.03 mg/L against H37Rv [76]. In an *in vitro* study on bacilli in stationary phase, gatifloxacin showed the greatest bactericidal activity in the first two days, but none afterwards. In mice studies, the combination of gatifloxacin with EMB and PZA cleansed the lungs of infected animals after two months of treatment. Currently, gatifloxacin is under phase III to prove the efficacy and safety of a fourmonth regime for the treatment of pulmonary TB supervised by the European Commission Oflotub Consortium, WHO-TDR, Tuberculosis Research Unit (TBRU), National Institute of

**Moxifloxacin:** Moxifloxacin is the most promising fluorquinolone against *M. tuberculosis.* Its activity *in vitro* seems to affect bacilli unaffected by RIF. Its MIC reported *in vitro* is 0.5 mg/L [77]. It seems that moxifloxacin interferes with the protein synthesis on bacteria with low metabolic activity by a mechanism different to the one of RIF. However, the specific action mechanism is unknown. In the mouse model, the effectiveness of moxifloxacin is comparable to INH. When administered in combination with PZA, moxifloxacin has a greater bactericidal activity tan the INH + RIF + PZA regime. In fact, the combination RIF + moxifloxacin + PZA decreases completely bacilli count within four months, whereas the combination RIF + INH + PZA requires 6 months. It is likely that there is synergism among the three drugs, and the alternative regime replacing INH by moxifloxacin has been proposed. Moxifloxacin is under phase III [67]. Clinical studies have not proved a greater sterilizing activity of a regime containing moxifloxacin in comparison with the standard regime; however, it has increased

**Nitroimidazole PA-824:** This bicyclical nitroimidazole is under development by the TB Alliance, which has the proprietary rights. The *in vitro* MIC of the PA-824 compound is between 0.15 and 0.3 μg/ml [78].After an activation by the F420 factor of *M. tuberculosis,* the PA-824 compound inhibits biosynthesis of the cell wall components by means of mechanisms still to be established. It has proved bactericidal activity against replicative and static bacilli *in vitro.* Although PA-824 was more efficient than INH or moxifloxacin, during the continuation phase it was not better than the RIF + INHH combination. On the long term, the 12-month treatment

are underway [67].

348 Tuberculosis - Current Issues in Diagnosis and Management

activity in early points [5,75].

**Nitroimidazole-oxazol OPC67683:**There is very little public information regarding this compound. It belongs to a subclass of mycolic acids synthesis inhibitors. It has shown *in vitro* activity against standard and resistant strains, showing a MICof 12 ng/ml [80]. It has not shown cross resistance with any other medication. The compound shown activity against bacilli residing within human macrophages and type II pneumocytes. The chronic TB trial in mice demonstrated an activity 6 – 7 times more effective than the one observed for first line INH and RIFdrugs. Favorable oral absorption and distribution have been reported. Currently it is under phase II clinical trials.

**Pyrrole LL-3858:** Some pyrroles derivatives have been found to have *in vitro* activity against *M. tuberculosis.* The MIC of pyrrole LL-3858 is between 0.025 and 0.12 μg/ml. The LL-3858 derivate identified by Lupin Limited showed greater bactericidal activity in the lungs of mice infected in monotherapy than INH. The trial of LL-3858 in combination with INH and RIF, or with INH, RIF, and PZA sterilized the totality of mice in 3 months [5]. Currently, the compound is under phase II clinical trials [67].

#### **8.3. New approaches for the development of anti-TB active principles**

In the dawn of the 21st century, pathogenesis of the infectious disease appears as a competition between the host and the pathogen involving short term adaptations and co evolution of the genomes [81]. The pathogen and the host constantly exercise selective pressure over each other, making the environment in test tube completely different from that within the host.

In latent tuberculous infection (LTBI), most of bacilli are not replicating, whereas in a phase of active disease most of the population is on active growth. Chemotherapy must take this metabolic adversity to favor the host.A durable cure must eliminate both the replicative and the persistent bacilli.Eradication of the persistent bacilli onchemotherapylasting between 6 – 24 months has been proposed in order to avoid relapse. However, such a long treatment is difficult to sustain and there is always resistance-associated risks in interrupted regimens [81].A philosophy of mycobacterial infection states that the essential genes for infection in mice include genes that are not essential *in vitro.*

The proteasome of *M. tuberculosis* is a set of proteins that provide a quick adaptation to changing conditions [82]. Two genes, *mpa (Mycobacterium* proteosomal ATPase) and *pafA (*proteasome accessory factor) were identified as important in the survival of *M. tuberculosis*to exposure against reactive nitrogen species (RNS) *in vitro* and required for active disease *in vivo* [82]. These genes codify for proteins involved in the bacteria proteasomal function. Proteasomes are barrel-shaped proteases consisting of 14 α units and 14 β units [82]. Mpa is similar to ATPases found in the proteosome of eukaryotes cells, and chemical inhibition of the protease activity of the *M. tuberculosis* proteosome causes sensitization of the wild strain to reactive nitrogen species (RNS). The PafA protein does not share homology with any protein of known function [82]. Two specific proteasomeinhibitors, epoxomicin and a peptidicboronate prevented the growth of *M. tuberculosis* and turned out to be bactericidal during the recovery of the mycobacterium against exposure to RNS [81]. The operon that codifies for the proteasome was knocked out by using conditional gene silencing and it was proved that bacteria require it to survive during the chronic infection in mice and its silencing allowed the mouse to be free of the persistent infection [83]. Whereas the proteasome of the mycobacterium is essential for the infection of a host, it is not required to grow in a rich and aerated medium such as Middlebrook 7H9 broth [81].

pandemics through simplification and shortening in the treatment time of the disease world‐ wide. The combination is currently in phase II of clinical trials and contains PA-824 and moxifloxacin together with PZA. Researchers have reported that preclinical data reveal a decrease in the treatment time both for DS-TB and MDR-TB patients, and possibly XDR-TB

Research and Development of New Drugs Against Tuberculosis

http://dx.doi.org/10.5772/54278

351

Nanoparticles can create new directions in the diagnosis, treatment, and prevention of TB. A significant application in the progress of this technology is using drug carriers.This system has been found to be advantageous, as it gives high stability of the drug, high load capacity (many molecules of the medication can be incorporated in the matrix of the particle), easiness to incorporate hydrophilic and hydrophobic substances, possibility of being administered orally or via inhalation. Perhaps more importantly, the anti-TB drug release in a controlled manner from the matrix enables to improve the bioavailability and reduction of the doses frequency. Load or delivery systems such as liposome or microspheres have been developed for the sustained release of anti-TB drugs, and better chemotherapeutical efficacy has been found when the system is researched in animal models (e.g. mice) [85,86].In 2005, the efficacy of nanoparticles was assessed in the distribution of anti-TBdrugs administered every 10 days versus the non capsulated form of aerosol administration of drugs against *M. tuberculosis* in guinea pigs; in both cases the treatments reduced the bacteria count. These findings suggest that the distribution of drugs by nanoparticles has a great potential in the treatment of TB [86].

Currently, devastating diseases in the world such as tuberculosis get the attention of author‐ ities with the aim of supporting breakthroughs which provide alternatives for their control. The development of active principles against *M. tuberculosis* is nowadays a worldwide priority due to the appearance of strains resistant to medications used in current therapeutic schemes, thus existing the need to articulate in an expedite manner the basic research looking for new therapeutic choices, along with clinical research and its articulation with the industry in order to guarantee a quick production of novel alternatives which overcome the limitations of

The concern in many sectors devoted to tuberculosis control is that there are not sufficient alternatives that can join rapidly the treatment against tuberculosis, and they convey discour‐ aging estimations regarding the degree of resistance that each one of these molecules will have at the moment of entering the therapeutic scheme deduced from natural resistant bacilli. These justifications have promoted research around the world towards finding new molecules, based on investigations of natural sources such as plants, insects, marine microorganisms, synthetic molecules deduced from the modification or substitutions made on the structure of already

ones with a simple three-drug treatment scheme [67].

**11. Conclusions**

current treatment schemes.

**10. Nano-particles: A projection towards the future**

Unlike other bacteria, *M. tuberculosis* possesses a unique defense system that relates the antioxidant and metabolic activities [81]. The system includes a peroxyredoxin, the C subunit of an alkylhydroperoxy-reductase (AhpC), a thioredoxin type protein (AhpD), dihydrolipoa‐ mideacyltransferase (DlaT), and lipoamide dehydrogenase (Lpd), and the four enzymes together work as peroxydases and peroxynitroreductases and peroxynitroreductasesdepend‐ ant of NADH [81]. The dual functionality of these enzymes is interesting as potential targets for the development of anti-TB active principles.

Moreover,theDosRsystem,discovered15yearsago,regulates thedevelopmentofaformofnoneplicative survival without morphologic differentiation in *M. tuberculosis* (known as latency state).Thisstateofphysiologicquiescencemaintainedviablethemicroorganismforlongperiods of time, contributing with two key characteristicsof TB: the symptom-free latent infection state and the persistence of the active disease of the tubercular infection in spite of the prolonged therapy time. Due to the importance of the bacilli latency state in the pathophysiology and chemotherapy of the disease, researchers have set their interest in the DosR system. Drugs that attack the latent state of the bacterium not only would be the key for eradication of the latent infection, but also shortening the time of treatment of active infection [84].
