**5. The concept of the drug resistance reversion and implementation thereof**

The concept of drug resistance reversion was applied in recent studies [7, 41]. Drug resistance mutations are often incompatible with one another, as shown by negative linkage disequilibrium values. This suggests that the cumulative fitness cost of mutations is often too high for the resulting strain to be viable. FS-1 is a new drug which seems to exploit this tendency. Active units of FS-1 are aggregated micelles containing complexes of tri-iodide molecules coordinated by metal ions and integrated into a dextrin-polypeptide moiety. The basic formula of the micelle is:

$$\left[ \left\{ \left( \text{L}\_{\text{n}} \left( \text{MeJ}\_{\text{s}} \right)^{\*} \right)\_{\text{y}} \left[ \text{Me(L}\_{\text{m}} \text{J)} \right]^{+} \_{\text{x}} \right\} \left( \text{Cl}^{-} \right)\_{\text{y} \star \text{x} \star \text{k}} \right] .$$

where *L*—dextrin-polypeptide ligand; *Me*—Li/Mg ions; *n*, *m*, *x*, *y* and *k*—variable integers ≥1; molecular mass of the micelles is in the range of 30–300 kD. This molecular complex was designed to prolong the residence time of moderately oxidative iodine molecules in an organism and facilitate their transportation to inner tissues.

Studies of XDR-TB infection in animal models showed the reversion of Mtb pathogens to a more drug sensitive phenotype after treatment with FS-1 despite the remaining DR related mutations in their genomes [7]. Drug resistance reversion was also confirmed on an *in vitro* model with a XDR-TB clinical isolate SCAID 187.0 when cultivated for 60 days in six passages on a medium with a sub-lethal dose of ¼ MIC of FS-1. Reduction of the antibiotic resistance of XDR-TB isolates obtained during the clinical trial of FS-1 was consistent with the results of the above-mentioned laboratory experiments. It was concluded that the DR phenotype requires multiple genes to be in specific activity states controlled either by transcription regulation or resulting from specific mutations. A combination of genetic variants creates a genomic context of drug resistance.

dependence is highly symmetric: in more than 90% of cases both mutations co-occur in the same genomes. It may indicate a genetic drift event when the DR phenotype is characteristic for a sub-lineage of isolates sharing common ancestry and the neutral mutation in the hyper-

**Table 2.** Attributable risk of acquisition of DR mutations in sub-populations of Mtb with secondary mutations.

Polymorphic sites are denoted by names of genes and pairs of amino acid substitutions from the most common allele to

Deletions are marked by asterisks (\*).Values *Xmin to Xmax* in cells represent confidence ranges estimated for *p*-value ≤0.05

**Drug resistance mutations Annotation**

**EmbC V981 L (EMB)**

**RpsL K43R (SM)**

72.9 to 95.0

73.8 to 95.2

68.5 to 90.8

76.2 to 94.2

83.3 to 98.1

83.1 to 98.9

82.9 to 98.8

85.3 to 99.7

82.6 to 98.8

79.5 to 96.9

**GidB E92D (SM)**

74.8 to 96.2

75.6 to 96.4

78.8 to 99.5

79.5 to 96.9

**GidB L16R (SM)**

66.8 to 85.9

70.6 to 88.3

82.3 to 96.8

80.6 to 95.9

83.4 to 95.3

84.9 to 95.1

82.5 to 94.0

88.2 to 97.8

89.4 to 98.4

89.7 to 98.5

89.2 to 98.4

83.4 to 94.9

Type VII secretion

Phosphate-transport integral membrane ABC transporter

protein

Hypothetical protein

23S rRNA methyl-transferase

Phenylalaninerepressible DAHP synthetase

Osmoprotectant

Drug efflux protein

Isocitratelyase

Galactose-1 phosphate uridylyl-transferase

Transcriptional regulator

ESX-1 secretion-Associated protein

Sugar ABC transporter

**ThyA T202A (PAS)**

70.2 to 93.6

71.1 to 93.8

79.8 to 99.5

80.1 to 99.5

73.3 to 93.5

For further analysis, only those secondary polymorphisms which influenced the DR mutations significantly, but were independent, were selected; i.e. cases were selected when confidence

mutable PPE35 protein is a genetic marker of the sublineage.

**Secondary mutations**

EccC3 P214R

Rv2542 T211A

TsnR L232P

AroG D265E

*UspA* V127 L

AceAa G179D

GalTb T174A

Rv0324 T168A

EspK C729S

(Eq. (1)).

PstA1 M5T 87.2 to 95.3

**GyrA S95 T (FLQ)**

77.6 to 87.4

71.8 to 82.4

88.6 to 96.2

ProX L85P 61.4 to

Stp D69Y 53.8 to

**KatG S315 T,N (INH)**

90 Basic Biology and Applications of Actinobacteria

79.7 to 92.5

81.3 to 93.4

74.6 to 90.6

72.4 to 89.0

81.7 to 92.8

75.4

71.1

53.2 to 70.8

54.0 to 71.1

54.8 to 72.3

54.0 to 70.5

one or several alternative allelic states.

59.2 to 74.3

**KatG R463L (INH)**

71.2 to 87.8

66.4 to 84.1

82.9 to 96.4

79.9 to 94.7

81.5 to 93.4

81.2 to 92.1

82.1 to 93.2

90.9 to 98.6

90.8 to 98.6

84.6 to 95.3

87.2 to 96.9

83.6 to 94.4

**AccD6 D229G (INH)**

65.1 to 84.8

64.9 to 84.4

84.1 to 97.6

76.6 to 93.8

77.3 to 91.6

80.0 to 92.1

78.2 to 91.3

90.7 to 99.0

87.9 to 97.8

87.1 to 97.3

90.5 to 98.9

85.5 to 96.2

Clinical trials of FS-1 has been undertaken in Kazakhstan and registered in the Clinical Trial database (www.ClinicalTrials.gov) under an accession number NCT02607449. It was found that FS-1 had a high absorption rate after per-oral administration, which was not affected by food intake. Peak plasma concentration of FS-1 was observed within 1–2 h after administration. Gastric juice activated the infusion of FS-1 in stomach. Pharmacokinetic study of FS-1 showed a long residence time of the drug in the blood stream and an elevated accumulation in the liver. The drug was excreted from the test organism mainly by the kidneys.

creatinine in blood; dermatomycosis; mental disorders; hypothyroidism; any allergies, especially an allergy to iodine-containing preparations; and any other cardiovascular, kidney or

Clade-Specific Distribution of Antibiotic Resistance Mutations in the Population…

http://dx.doi.org/10.5772/intechopen.75181

93

The therapeutic efficacy of the drug was evaluated by bacteriological examination of sputum samples of patients on Lowenstein-Jensen medium for the presence of Mtb isolates. Other tests performed during the trial were: microscopic examination of sputum smears; controlling the positive dynamics of recovery by regular X-ray examinations and by general clinical tests; positive body weight dynamics; and the efficacy of prevention of disease relapses. The efficacy and safety of the regimen of per-oral administration of FS-1 in concentrations of 2.0–5.0 mg/kg during the 6 months in combination with commonly prescribed antibiotics against XDR-TB were confirmed in the second phase of the trial. No serious side effects of the treatment were recorded. In particular, thyroid gland function was monitored for adverse effects. No statistically reliable alterations in the concentration of thyroid hormones in blood were observed, which indicated no deleterious effect of this iodine-containing drug on thyroid gland functions. The time of complete recovery from XDR-TB was reduced, with no disease relapses during the 12 months surveillance, resulting in a significant reduction of the

Mtb isolates were collected on a regular basis during the second phase of the FS-1 clinical trials. It was found that the percentage of drug resistant isolates decreased continuously during the treatment course with FS-1 despite the administration of the antibiotics. It was hypothesized that the therapeutic activity of FS-1 may be associated with the reversion of antibiotic resistance [37]. This hypothesis was then confirmed in an *in vivo* experiment on guinea pigs,

The third phase of clinical trials began in 2014 and is still in progress. The drug FS-1 has been approved as an antibacterial medicine for per-oral administration in a complex of commonly prescribed anti-tuberculosis drugs for the treatment of XDR-TB in Kazakhstan (approval cer-

**XDR TB treatment expenses Conventional antibiotic therapy Combined therapy by** 

TB relapses

**Table 3.** Summarized efficacy of application of FS-1 in the second phase of the clinical trial in terms of reduction of the

Daily therapy cost in clinics of Kazakhstan \$ 11.7 \$ 12.5

12–24 months 3–6 months

46.1% Not observed

\$ 4274 or up to \$ 8548 in the case of

**antibiotics with FS-1**

\$ 2256 (no TB relapses were

recorded)

liver decompensated concomitant diseases.

average cost of XDR-TB treatment (**Table 3**).

which has been recently published [7].

tificate РК-ЛС-5№021305 from 08-04-2015).

Time of 100% sanation from *M. tuberculosis*

Percentage of relapses in 12 month surveillance

Full cost of the treatment course including the

isolation from sputum

treatment of disease relapses

treatment course duration and cost.

period

The preclinical trial of FS-1 included pharmacological studies (primary and secondary pharmacokinetics); general toxicity determination; tests for mutagenesis, inhibition of reproductive performance, immune toxicity, mucous membrane irritation and several other general physicochemical studies of the compound. FS-1 caused no irritation of the stomach mucosa when applied in concentrations of up to 5.0 mg/kg. No ulcerogenic, allergenic, immune toxic, mutagenic or carcinogenic side effects were observed after repeated administration of FS-1. Also, no cytotoxicity or embryonic toxicity was observed. Toxicological studies attributed FS-1 to low toxicity compounds with a reduced accumulation in an organism (drug accumulation coefficient was 1.85). The maximum endurable dose of FS-1 identified in rats was 496 mg/kg, and in mice, 993 mg/kg. The average lethal dose (LD50) in rats was found to be 992 mg/kg for both male and female individuals. Therapeutic doses of FS-1 in clinical trials on humans for the treatment of patients with lung XDR-TB infection ranged from 1.0 to 5.0 mg/kg. During the clinical trials, FS-1 was administrated for up to 6 months in combination with the antibiotics commonly prescribed for XDR-TB treatment. Currently, in the third stage of the clinical trials, FS-1 is administrated at a concentration of 2.5 mg/ml for 6 months. Clinical studies complied with the regulations and recommendations of the Ministry of Health of Kazakhstan and were approved by the respective committees of the Ministry.

The first phase of clinical trials was undertaken in 2009–2010 at the Central Clinical Hospital of the Executive Officer of the President of Kazakhstan. During this phase, the drug tolerance and safety of a unitary and repeated per-oral intake of the drug by healthy volunteers were determined. Hematological parameters, including measuring the concentrations of important microelements, i.e., potassium, sodium, magnesium and calcium; functions of liver and kidney, electro-physiological parameters of myocardium, metabolism of proteins, hydrocarbons and lipids, were monitored. Biochemical parameters of the blood plasma of volunteers remained normal during the study. It was found that the administration of FS-1 activated cellular immunity and synthesis of γ-interferon.

The second phase of clinical trials was conducted in 2010–2012 at the Municipal anti-tuberculosis clinic in Almaty, at the National Centre of Tuberculosis in Almaty and at the Regional anti-tuberculosis clinic of the Karaganda region in Kazakhstan. In total, 220 volunteer patients with active XDR-TB lung tuberculosis were involved in this phase of trials. The volunteers ranged from 18 to 65 years old, with a body mass within 10% of the average body weight of male and female adults, with no contraindications to the common MDR-TB antibiotic therapy. Informed consent principles, which imply voluntariness of participation and understanding of the matter of the trial, were complied with. Contraindications to participation in the trial were: pregnancy; oncological diseases; HIV; 3-fold higher than normal ALT/AST or increased creatinine in blood; dermatomycosis; mental disorders; hypothyroidism; any allergies, especially an allergy to iodine-containing preparations; and any other cardiovascular, kidney or liver decompensated concomitant diseases.

Clinical trials of FS-1 has been undertaken in Kazakhstan and registered in the Clinical Trial database (www.ClinicalTrials.gov) under an accession number NCT02607449. It was found that FS-1 had a high absorption rate after per-oral administration, which was not affected by food intake. Peak plasma concentration of FS-1 was observed within 1–2 h after administration. Gastric juice activated the infusion of FS-1 in stomach. Pharmacokinetic study of FS-1 showed a long residence time of the drug in the blood stream and an elevated accumulation

The preclinical trial of FS-1 included pharmacological studies (primary and secondary pharmacokinetics); general toxicity determination; tests for mutagenesis, inhibition of reproductive performance, immune toxicity, mucous membrane irritation and several other general physicochemical studies of the compound. FS-1 caused no irritation of the stomach mucosa when applied in concentrations of up to 5.0 mg/kg. No ulcerogenic, allergenic, immune toxic, mutagenic or carcinogenic side effects were observed after repeated administration of FS-1. Also, no cytotoxicity or embryonic toxicity was observed. Toxicological studies attributed FS-1 to low toxicity compounds with a reduced accumulation in an organism (drug accumulation coefficient was 1.85). The maximum endurable dose of FS-1 identified in rats was 496 mg/kg, and in mice, 993 mg/kg. The average lethal dose (LD50) in rats was found to be 992 mg/kg for both male and female individuals. Therapeutic doses of FS-1 in clinical trials on humans for the treatment of patients with lung XDR-TB infection ranged from 1.0 to 5.0 mg/kg. During the clinical trials, FS-1 was administrated for up to 6 months in combination with the antibiotics commonly prescribed for XDR-TB treatment. Currently, in the third stage of the clinical trials, FS-1 is administrated at a concentration of 2.5 mg/ml for 6 months. Clinical studies complied with the regulations and recommendations of the Ministry of Health of Kazakhstan and were approved by the respective committees of the Ministry.

The first phase of clinical trials was undertaken in 2009–2010 at the Central Clinical Hospital of the Executive Officer of the President of Kazakhstan. During this phase, the drug tolerance and safety of a unitary and repeated per-oral intake of the drug by healthy volunteers were determined. Hematological parameters, including measuring the concentrations of important microelements, i.e., potassium, sodium, magnesium and calcium; functions of liver and kidney, electro-physiological parameters of myocardium, metabolism of proteins, hydrocarbons and lipids, were monitored. Biochemical parameters of the blood plasma of volunteers remained normal during the study. It was found that the administration of FS-1 activated

The second phase of clinical trials was conducted in 2010–2012 at the Municipal anti-tuberculosis clinic in Almaty, at the National Centre of Tuberculosis in Almaty and at the Regional anti-tuberculosis clinic of the Karaganda region in Kazakhstan. In total, 220 volunteer patients with active XDR-TB lung tuberculosis were involved in this phase of trials. The volunteers ranged from 18 to 65 years old, with a body mass within 10% of the average body weight of male and female adults, with no contraindications to the common MDR-TB antibiotic therapy. Informed consent principles, which imply voluntariness of participation and understanding of the matter of the trial, were complied with. Contraindications to participation in the trial were: pregnancy; oncological diseases; HIV; 3-fold higher than normal ALT/AST or increased

cellular immunity and synthesis of γ-interferon.

in the liver. The drug was excreted from the test organism mainly by the kidneys.

92 Basic Biology and Applications of Actinobacteria

The therapeutic efficacy of the drug was evaluated by bacteriological examination of sputum samples of patients on Lowenstein-Jensen medium for the presence of Mtb isolates. Other tests performed during the trial were: microscopic examination of sputum smears; controlling the positive dynamics of recovery by regular X-ray examinations and by general clinical tests; positive body weight dynamics; and the efficacy of prevention of disease relapses. The efficacy and safety of the regimen of per-oral administration of FS-1 in concentrations of 2.0–5.0 mg/kg during the 6 months in combination with commonly prescribed antibiotics against XDR-TB were confirmed in the second phase of the trial. No serious side effects of the treatment were recorded. In particular, thyroid gland function was monitored for adverse effects. No statistically reliable alterations in the concentration of thyroid hormones in blood were observed, which indicated no deleterious effect of this iodine-containing drug on thyroid gland functions. The time of complete recovery from XDR-TB was reduced, with no disease relapses during the 12 months surveillance, resulting in a significant reduction of the average cost of XDR-TB treatment (**Table 3**).

Mtb isolates were collected on a regular basis during the second phase of the FS-1 clinical trials. It was found that the percentage of drug resistant isolates decreased continuously during the treatment course with FS-1 despite the administration of the antibiotics. It was hypothesized that the therapeutic activity of FS-1 may be associated with the reversion of antibiotic resistance [37]. This hypothesis was then confirmed in an *in vivo* experiment on guinea pigs, which has been recently published [7].

The third phase of clinical trials began in 2014 and is still in progress. The drug FS-1 has been approved as an antibacterial medicine for per-oral administration in a complex of commonly prescribed anti-tuberculosis drugs for the treatment of XDR-TB in Kazakhstan (approval certificate РК-ЛС-5№021305 from 08-04-2015).


**Table 3.** Summarized efficacy of application of FS-1 in the second phase of the clinical trial in terms of reduction of the treatment course duration and cost.
