|Year : 2011 | Volume
| Issue : 4 | Page : 253-260
Antitubercular effect of 8-[(4-Chloro phenyl) sulfonyl]-7-Hydroxy-4-Methyl-2H-chromen-2-One in guinea pigs
Parvati B Patel1, Tejas K Patel1, Seema N Baxi2, Hemangini R Acharya1, Chandrabhanu Tripathi1
1 Department of Pharmacology, Government Medical College, Bhavnagar, Gujarat, India
2 Department of Pathology, Government Medical College, Bhavnagar, Gujarat, India
|Date of Web Publication||12-Oct-2011|
Department of Pharmacology, Government Medical College, Bhavnagar 364 001, Gujarat
| Abstract|| |
Objective: To evaluate the antitubercular efficacy and safety of New Chemical Entity (NCE): 8-[(4-Chloro phenyl) sulfonyl]-7-Hydroxy-4-Methyl-2H-chromen-2-One (CSHMC) in guinea pigs. Materials and Methods: This pilot study was carried out in guinea pigs. They were infected with M. tuberculosis H 37 Rv (1.5 × 10 4 cfu/guinea pig) via intramuscular route. After 30 days, infections were confirmed in 6 guinea pigs by histopathology of spleen, lung, and liver. After that CSHMC (5 and 20 mg/kg) was administered for 1 month and its effect was compared with vehicle, rifampicin (60 mg/kg) and isoniazid (30 mg/kg). Efficacy of CSHMC was evaluated on the basis of histopathologic scoring of lesion in lung, spleen, liver, and safety on the basis of measuring hemogram, liver and renal function parameters. Results: Isoniazid, rifampicin, and CSHMC (20 mg/kg) significantly reduce the median lesion score in lung, spleen, and liver as compared to disease control group. Reduction in median lesion score for lung and spleen were not statistically significant for CSHMC 5 mg/kg. CSHMC (20 and 5 mg/kg) did not produce any changes in hemogram, liver and renal function parameters with respect to normal values. Conclusions: CSHMC had shown significant antitubercular efficacy comparable to isoniazid and rifampicin and did not show hematological, hepato- and nephrotoxicity.
Keywords: Isoniazid, rifampicin, tuberculosis
|How to cite this article:|
Patel PB, Patel TK, Baxi SN, Acharya HR, Tripathi C. Antitubercular effect of 8-[(4-Chloro phenyl) sulfonyl]-7-Hydroxy-4-Methyl-2H-chromen-2-One in guinea pigs. J Pharmacol Pharmacother 2011;2:253-60
|How to cite this URL:|
Patel PB, Patel TK, Baxi SN, Acharya HR, Tripathi C. Antitubercular effect of 8-[(4-Chloro phenyl) sulfonyl]-7-Hydroxy-4-Methyl-2H-chromen-2-One in guinea pigs. J Pharmacol Pharmacother [serial online] 2011 [cited 2014 Oct 31];2:253-60. Available from: http://www.jpharmacol.com/text.asp?2011/2/4/253/85951
| Introduction|| |
Mycobacterium tuberculosis is the world's largest cause of death from a single microorganism after human immunodeficiency virus (HIV). Globally, there are almost nine million new cases of tuberculosis (TB) each year, two million of which results in death.  It is estimated that about 1/3 of the current global population is infected asymptomatically with M. tuberculosis of whom 5%-10% will develop clinical disease during their lifetime and approximately two million deaths are attributable annually. , TB continues to be a leading cause of mortality in spite of the availability of an effective chemotherapeutic regimen. Chemotherapy of TB consists of three or four drug regimen, administered for 6-9 months. The long duration of therapy results in poor compliance, produces unwanted side effects and therapeutic failure leading to emergence of multidrug resistant strains of M. tuberculosis. ,, Association with noncompliance, HIV, and increasing multidrug resistant tuberculosis (MDR-TB) appears to be a serious issue, especially for the developing nations.  Extensive drug resistant tuberculosis (XDR-TB) spreading all over the world is defined as a resistance to all first-line drugs and to fluoroquinolones and all of the injectables.  The XDR-TB strain is mixing with the acquired immuno deficiency syndrome (AIDS), causing nearly100% mortality. ,
Chemotherapy is being the mainstay of TB control. Development and evaluation of new chemical entity (NCE) against TB is a challenging task. 8-[(4-Chloro phenyl) sulfonyl]-7-Hydroxy-4-Methyl-2H-chromen-2-One (CSHMC) used in the present study is derivative of coumarin [Figure 1]a and heterocyclic analogue of dapsone (diaminodiphenyl sulfone [DADS]). Coumarin has a wide range of pharmacological properties, such as anticoagulant, antibacterial, diuretic, vasodilator and antiallergic.  Several coumarin substitutions have shown antitubercular activity. ,, Dapsone [Figure 1]b was found to be effective in suppressing experimental infections with tubercle bacilli. ,,, Minimum inhibitory concentration (MIC) of dapsone against M. tuberculosis, M. avium complex (MAC), and M. leprae are 50-250 mg/L, 2-100 mg/L, and 1.5-4 mg/L, respectively. ,, So, a higher dose of dapsone is required in the treatment of tuberculosis. Metabolism of dapsone by hepatic oxidation to hydroxylamine and its subsequent reduction to amine within red cells leads to persistence of drugs in red cells and which is suspected for its hematological toxicity.  An attempt was made to reduce its toxicity by replacing free amino (NH2) group with chloro group. Due to antibacterial activity against a variety of microorganisms by sulfones, 7-hydroxy-4-methyl coumarin was selected for the synthesis of CSHMC. It has been shown to have an antitubercular activity in vitro on culture of sensitive strain of M. tuberculosis in Lowenstein-Jensen Media. The MIC was found to be 0.99 mg/L.  The present study was planned to evaluate antitubercular efficacy and safety of CSHMC in isoniazid and rifampicin-sensitive strain in guinea pigs. If infected with M. tuberculosis, they show striking similarities to natural infections in humans, thus making it a good model for testing the effects of drug therapy. ,,
|Figure 1: Chemical structure of (a) coumarin; (b) dapsone (diaminodiphenyl sulfone [DADS]); (c) 8-[(4-Chloro phenyl) sulfonyl]-7-hydroxy-4-methyl-2H-chromen-2-One|
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| Materials and Methods|| |
The study (experiment protocol no. 05/2007) was approved by the Institutional Animal Ethics Committee, Government Medical College, Bhavnagar, Gujarat, India.
Drugs and chemicals
Isoniazid (Alfa Aesar, A Johnson Mathey Company, Lancaster), rifampicin (Tokyo Chemical Industry Co. Ltd., Japan), purified protein derivative (PPD RT 23 Tween 80; Radiant Parenterals Ltd., Vaghodia, India) were purchased and used in the study. CSHMC was a kind free gift sample from the Department of Chemistry, Bhavnagar University, Bhavnagar, India. Its chemical structure is shown in [Figure 1]c. Chemical formula and molecular weight of CSHMC are C 16 H 11 O 5 SCL and 350.5, respectively. Spectral data (IR) confirmed the structure of the synthesized compound.
Bacterial strain and culture media
M . tuberculosis H 37 Rv in Middle Brook 7H9 broth media was obtained from State Tuberculosis Demonstration Centre (STDC), Ahmedabad, Gujarat, India, as a free gift sample.
Dunkin-Hartley guinea pigs (250-300 g) and Swiss albino mice (20-30 g) of both genders were obtained from central animal house of the institute. Male and female guinea pigs were housed in separate stainless steel cages and mice in polypropylene cages under standard condition; temperature-controlled room (24°C ± 2°C) with 12 h light:dark cycles and were given standard laboratory diet and water ad libitum.
Acute oral toxicity determination
The nonpregnant, nulliparous female albino mice of 8-12 weeks old and 20-25 g body weight were selected to find out the acute oral toxicity of the CSHMC. The mice were fasted for 3 h prior to the experiment and were administered with CSHMC in the range of doses 300, 1000, and 2000 mg/kg and observed for mortality up to 14 days according to OECD guidelines.  The LD 50 of CSHMC was found to be 300 mg/kg. Equivalent dose for guinea pig (200 mg/kg) was calculated as per Ghosh.  1/10 th (20 mg/kg) and 1/40 th (5 mg/kg) dose of it were selected for further study.
All the guinea pigs were tested with Mantoux test using 0.1 mL of purified protein derivatives (PPD RT 23 Tween 80) injected intradermally into the lower left side of abdomen. All the guinea pigs examined (up to 72 h) were Mantoux negative and hence used for experiment. 
Antitubercular study was carried out on guinea pigs and they were divided into 6 groups of either gender with 6 in each group.
Cultures of M. tuberculosis H 37 Rv were harvested from a 2-week culture in Middle Brooke 7H9 broth media. All the guinea pigs were infected with 0.1 mL (1.5 × 10 4 cfu/guinea pig) suspension via intramuscular route in the left thigh muscle. Qurrat-ul-Ain et al. and Challu et al. have produced a tuberculosis infection in guinea pigs via intramuscular route. ,, This suspension was matched with McFarland standard. After 30 days, infections were confirmed in 6 guinea pigs by histopathology of spleen, lung, and liver. Other 30 guinea pigs were randomly divided into 5 groups (Groups II-VI) by random allocation software version 1.0, May 2004, by simple randomization. Drugs and CSHMC were administered by mouth once a day for 30 days in Groups III-VI. Human equivalent dose of isoniazid (30 mg/kg) and rifampicin (60 mg/kg) was calculated by extrapolation as mentioned by Ghosh.  Shang et al. took a human-equipotent dose of isoniazid and rifampicin in guinea pigs as 30 and 50 mg/kg, respectively.  Group II received vehicle (10% ethanol) by mouth once a day for 30 days. Food was withdrawn 12 h before the experiments.
Group I: Normal control group: Did not receive the injection of M. tuberculosis H 37 Rv and received vehicle (10% ethanol) by mouth once a day for 30 days.
Group II: Disease control group: Received vehicle by mouth once a day for 30 days.
Group III: Standard control group: Received isoniazid (30 mg/kg) by mouth once a day for 30 days.
Group IV: Standard control group: Received rifampicin (60 mg/kg) by mouth once a day for 30 days.
Group V: Test compound group: Received CSHMC (20 mg/kg) by mouth once a day for 30 days.
Group VI: Test compound group: Received CSHMC (5 mg/kg) by mouth once a day 30 for days.
Blood samples were collected in ethylene diamino tetra acetic acid (EDTA) and plain bulb from the carotid artery under pentobarbitone anesthesia intraperitoneally (30 mg/kg) 24 h after the last dose of drug. EDTA sample was used for estimation of hemoglobin (Hb), total white blood cell (WBC) count, total red blood cell (RBC) count, total platelet count, differential count (polymorphs, lymphocytes, eosinophils, monocytes), blood indices (packed cell volume, mean corpuscle volume, mean corpuscle hemoglobin, mean corpuscle hemoglobin concentration, red cell distribution width, and erythrocyte sedimentation rate). Complete blood count was analyzed by using celltac alpha cell counter (Nihontohdem, Japan). Serum was separated for estimation of biochemical parameters: Serum glutamic oxaloacetic transaminase (SGOT),  serum glutamic pyruvic transaminase (SGPT),  alkaline phosphatase (ALP)  by enzyme kinetic method, bilirubin  by Jendrassik-Grof method (total, direct, and indirect) and serum creatinine  by Jaffé creatinine method in a fully automated analyzer.
Histopathologic lesion analysis
The animals were sacrificed soon after blood collection. Thorax and abdomen were cut open to remove lung, spleen, and liver. Their gross appearance was noted and they were placed in 10% formalin for 24 h. Then 5 mm thick pieces of the lung, spleen, and liver were embedded in paraffin, cut into 5 mm thick sections, stained using hematoxylin-eosin dye (H and E), and finally mounted in dibutyl diesterate parathylate xylene.  Each slide was coded and observer masked to evaluate histopathological changes in the lung, spleen, and liver. The scoring was done based on a previously described method and photomicrographs were taken.  The guinea pig lungs were scored based on the following three criteria: (1) Primary lesion (epithelioid cells and other inflammatory cells): 0, no primary lesions present; 1, single primary lesion; 2, two or more primary lesions; multifocal; 3, two or more primary lesions; multifocal to coalescing; 4, multiple primary lesions, coalescing, and extensive. (2) Secondary lesion (granuloma formation, giant cells, and caseous necrosis): 0, no secondary lesion present; 1, up to 25% of lung involved; 2, up to 50% of lung involved; 3, up to 75% of lung involved; 4, above 75% of lung involved. (3) Pneumonitis (alveolar wall thickness, interstitial, and peribronchial inflammation): 0, alterations in less than 10% of the fields; 1, alterations in 10%-30% of the fields; 2, alterations in 30%-50% of the fields; 3, alterations in 50%-70% of the fields; 4, alterations in more than 70% of the fields are scored based on severity as follows: 0, none; 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. All individual scores were added for the final total score for each organ.
The spleen was scored based on two criteria (same scales as lung); (1) Primary lesion and (2) Secondary lesion. The liver was scored based on one criterion; ballooning degeneration: 0, no ballooning degeneration; 1, minimal enlargement in few hepatocytes; 2, mild enlargement in many hepatocytes; 3, moderate enlargement in most hepatocytes; 4, severe enlargement in most hepatocytes.
The histopathologic score of organs are presented as Median (Interquartertile Range). The nonparametric Kruskal-Wallis test followed by Dunnett's test was used to assess statistical significance between total lesion score of lung, spleen, and liver. The hematological, liver function, and renal function parameters are expressed as mean ± standard error of mean (SEM). Statistical differences between means were determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests using GraphPad Prism software demo version. P<0.05 was considered as significant.
| Results|| |
In normal control group (Group I); gross examination and histology of lung [Figure 2]a, spleen [Figure 3]a, and liver [Figure 4]a of guinea pig showed the normal morphology.
|Figure 2: Histopathology of lung (H and E stain, ×40). (a) Normal control group— normal alveolar morphology; (b) disease control group— severe (score 6) epithelioid cells (E), lymphocyte (L), alveolar wall infi ltration (A), caseation necrosis (CN), and giant cells (G); (c– e) isoniazid, rifampicin, and CSHMC (20 mg/kg) treated group— minimal (score 0.5– 1) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell; (f) CSHMC (5 mg/kg) treated group— mild (score 1.5) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell|
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|Figure 3: Histopathology of spleen (H and E stain, ×40). (a) Normal control group— normal morphology; (b) disease control group— severe (score 6) epithelioid cells (E), lymphocyte (L) infi ltrate with caseation necrosis (CN), and giant cells (G); (c– e) isoniazid, rifampicin, and CSHMC (20 mg/kg) treated group— minimal to mild (score 1– 1.5) epithelioid cells infi ltration with no caseation necrosis and giant cell; (f) CSHMC (5 mg/kg) treated group— mild to moderate (score 2.5) epithelioid cells infi ltration with no caseation necrosis and giant cell|
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|Figure 4: Histopathology of liver (H and E stain, ×40). (a) Normal control group— liver shows normal morphology; (b) disease control group— a mild (score 2) ballooning degeneration (BD); (c– f) isoniazid, rifampicin, and CSHMC (20 and 5 mg/kg) treated group— a minimal or no (score 0.5)|
ballooning degeneration (BD)
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Isoniazid, rifampicin, and CSHMC treated groups showed less white caseous granulomatous foci as compared with disease control group in lung and spleen by gross examination. Liver did not show any gross changes in Groups II-VI.
As shown in [Figure 2]b, lesion of lung consisted of epithelioid cells, lymphocytic infiltration, multinucleated giant cells, caseation necrosis, granuloma, and pneumonitis. Isoniazid [Figure 2]c, rifampicin [Figure 2]d, and CSHMC (20 mg/kg) [Figure 2]e treated group had shown a minimal epithelioid cells and alveolar wall infiltration with no caseation necrosis and giant cell. The lesion of spleen consisted of epithelioid cells, lymphocytic infiltration, multinucleated giant cells, caseation necrosis, and granuloma [Figure 3]b. Isoniazid [Figure 3]c, rifampicin [Figure 3]d, and CSHMC (20 mg/kg) [Figure 3]e treated group had minimal to mild epithelioid cells infiltration with no caseation necrosis and giant cell. As shown in [Table 1], isoniazid, rifampicin, and CSHMC (20 mg/kg) significantly reduces the median lesion score in lung and spleen. Reduction in median lesion score of lung and spleen were not statistically significant for CSHMC (5 mg/kg) [Figure 2]f and [Figure 3]f. The lesion of liver consisted of ballooning degeneratin [Figure 4]. CSHMC (20 and 5 mg/kg) treated group had shown a minimal or no ballooning degeneration. Isoniazid [Figure 4]c, rifampicin [Figure 4]d, and CSHMC [Figure 4]e and f significantly reduce the median lesion scores of liver [Table 1].
|Table 1: The lesion scores of lungs, spleen, and liver were calculated in all the 5 groups after 30 days of the treatment|
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The hemogram [Table 2], liver [Table 3], and renal [Table 3] function parameters were monitored after 30 days of isoniazid, rifampicin, and CSHMC treatment. The result indicates that CSHMC did not produce any changes in hemogram, liver functions, and renal function parameters with respect to normal values.
|Table 2: Effect of isoniazid, rifampicin, and CSHMC on hemogram parameters in blood of guinea pigs after 30 days of treatment|
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|Table 3: Effect of isoniazid, rifampicin, and CSHMC on liver and renal function parameters in blood of guinea pigs after 30 days of treatment|
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| Discussion|| |
The main drawback of antitubercular chemotherapy is the lack of patient compliance, therapeutic failure, long duration of therapy, emergence of multidrug and extensive drug resistance tuberculosis, increasing incidence of HIV and toxic side effects, such as hepatotoxicity.  The XDR-TB is virtually untreatable with high mortality and now treatment of it will depend on sensitivity of drugs. The second-line drugs are less efficacious, less convenient, more toxic, and more expensive. These drugs have to be given for 18-24 months.  In view of this, we investigated the antitubercular efficacy and safety of coumarin derivatives: CSHMC in guinea pig model. The antitubercular efficacy was evaluated in terms of histopathological score of the organs of infected guinea pigs after daily administration of CSHMC for 30 days. Higher reduction of the histopathological score of lesion from the lung, spleen, and liver with CSHMC in the dose of 20 mg/kg than with 5 mg/kg suggests clearance of tubercle bacilli from the lesion in a dose-dependent manner. CSHMC-20 mg/kg had shown similar reduction in median score lesion of lung, spleen, and liver as compared to isoniazid and rifampicin. Antitubercular efficacy can be better measured by colony counting in the culture of the organs (lung, spleen, and liver) of the guinea pigs infected with M. tuberculosis. This is the limitation of our study.
The safety of CSHMC was evaluated in terms of alteration on hematological, liver and renal function parameters of the infected guinea pigs. Antitubercular drugs, such as isoniazid, rifampicin, and ethambutol have been shown to induce hematological toxicity.  In the present study, no alteration in hematological parameters with NCE in the dose of 20 and 5 mg/kg compared to normal control group. Most of the antitubercular drugs, such as isoniazid, rifampicin, and pyrizinamide have been shown to induce hepatotoxicity particularly when used in combinations.  Levels of SGPT, SGOT, ALP, and total bilirubin were monitored as an index of hepatotoxicity. In the present study, no alteration in the levels of liver function parameters with CSHMC in the dose of 20 and 5 mg/kg were observed compared to normal control group. Antitubercular drugs, such as streptomycin and ethambutol cause nephrotoxicity. , In this study, serum creatinine was used to detect nephrotoxic potential of CSHMC. No alteration in the level of renal function parameters with CSHMC in the dose of 20 and 5 mg/kg were observed compared to normal control group. This study indicates the CSHMC in the dose of 20 and 5 mg/kg did not show the hematological toxicity, hepatotoxicity, and nephrotoxicity.
NCE is a coumarin derivative and heterocyclic analogue of dapsone (DADS) was attached to it. In DADS free amino group was replaced by chloro group, which may reduce the toxicity. Antimycobacterial effect of this compound may be due to its 8-[(4-Chloro phenyl) sulfonyl] group and/or due to coumarin ring. This study supports the role of chlorophenyl moiety at C4 for the antimycobacterial activity.  Quantitative structure-activity relationship (QSAR) study remains open for the further studies.
| Conclusions|| |
CSHMC has shown an antitubercular effect. CSHMC in the dose of 20 mg/kg had shown more efficacies against the M. tuberculosis infection in guinea pigs compared with a dose of 5 mg/kg, which is comparable to isoniazid and rifampicin by histopathological data. In both doses, it did not produce hematological, hepatic and renal function toxicity.
| Acknowledgment|| |
Authors are sincerely thankful to Dr. K. R. Joshi, Senior officer, Research and Development Department, Excel Crop Care Limited, Bhavnagar, and Dr. N. K. Undavia, Ex-Professor and Head, Department of Chemistry, Bhavnagar University, Bhavnagar for providing NCE for research work; Dr. Nabendu Joshi, M. D., State Tuberculosis Demonstration Centre, Ahmedabad, for providing M. tubercle bacilli H 37 Rv strain.
| References|| |
|1.||Vasantha M, Gopi PG, Subramani R. Survival of tuberculosis patients treated under dots in a rural Tuberculosis unit (tu), south India. Indian J Tuberc 2008;55:64-9. |
|2.||Tomioka H, Namba K. Development of antituberculous drugs: Current status and future prospects. Kekkaku 2006;81:753-74. |
|3.||Dutt M, Khuller GK. Chemotherapy of Mycobacterium tuberculosis infections in mice with a combination of Isoniazide and Rifampicin entrapped in Poly (Dl-Lactide-Co-glycolide) microparticles. J Antimicrob Chemother 2001;47:829-35. |
|4.||Caminero JA. Management of multidrug-resistant tuberculosis and patients in retreatment. Eur Respir J 2005;25:928-36. |
|5.||Furin JJ, Johnson JL. Recent advances in the diagnosis and management of tuberculosis. Curr Opin Pulm Med 2005;11:189-94. |
|6.||Chakraborty AK. Epidemiology of tuberculosis: Current status in India. Indian J Med Res 2004;120:248-76. |
|7.||Singhal KC, Grover JK, Moideen R, Kamini V. Incidence of adverse reactions to antitubercular drugs. Ind J Pharmacol 1990;5:1413-5. |
|8.||Caminero JA. Extensively drug-resistant tuberculosis: Is its definition correct? Eur Respir J 2008;32:928-36. |
|9.||Singh MM. XDR-TB- Danger Ahead. Indian J Tuberc 2007;54:1-2. |
|10.||Chauhan LS. Drug resistant TB- RNTCP response. Indian J Tuberc 2008;55:5-8. |
|11.||Wu L, Wang X, Xu W, Farzaneh F, Xu R. The structure and pharmacological functions of coumarins and their derivatives. Curr Med Chem 2009;16:4236-60. |
|12.||Manvar A, Malde A, Verma J, Virsodia V, Mishra A, Upadhyay K, et al. Synthesis, anti-tubercular activity and 3D-QSAR study of coumarin-4-acetic acid benzylidene hydrazides. Eur J Med Chem 2008;43:2395-403. |
|13.||Siddiqui AA, Rajesh R, Mojahid-Ul-Islam, Alagarsamy V, Meyyanathan SN, Kumar BP, et al. Synthesis, antiviral, antituberculostic and antibacterial activities of some novel, 4-(4'- substituted phenyl)-6-(4"- hydroxyphenyl)-2-(substituted imino) pyrimidines. Acta Pol Pharm 2007;64:17-26. |
|14.||Schinkovitz A, Gibbons S, Stavri M, Cocksedge MJ, Bucar F. Ostruthin: An antimycobacterial coumarin from the roots of Peucedanum ostruthium. Planta Med 2003;69:369-71. |
|15.||Meindl WR, Von Angerer E, Schönenberger H, Ruckdeschel G. Benzylamines: Synthesis and evaluation of antimycobacterial properties. J Med Chem 1984;27:1111-8. |
|16.||Wolf R, Matz H, Orion E, Tuzun B, Tuzun Y. Dapsone. Dermatol Online J 2002;8:37-53. |
|17.||Francis J. The Treatment of Tuberculosis in the Guinea pig with Streptomycin alone or in Combination with -Dapsone, Hiosemicarbazones, Pas, or Sodium Iodide. Brit J Pharmacol 1953;8:259-62. |
|18.||Shah LM, Destefano MS, Cynamon MH. Enhanced In Vitro Activity of WR99210 in Combination with Dapsone against Mycobacterium avium Complex. Antimicrob Agents Chemother 1996;40:2644-5. |
|19.||Rastogi N, Goh KS, Labrousse V. Activity of subinhibitory concentrations of dapsone alone and in combination with cell-wall inhibitors against Mycobacterium avium complex organisms. Eur J Clin Microbiol Infect Dis 1993;12:954-8. |
|20.||Peters JH, Gordon GR, Murray JF Jr, Fieldsteel AH, Levy L. Minimal inhibitory concentration of dapsone for Mycobacterium leprae in rats. Antimicrob Agents Chemother 1975;8:551-7. |
|21.||George J, Balakrishnan S. Blood dapsone levels in leprosy patients treated with acedapsone. Indian J Lepr 1986;58:401-6. |
|22.||Joshi KR. A Study of New Sulfur Dyes. PhD Thesis. Bhavnagar, Gujarat, India: Department of Chemistry, Bhavnagar University; 2007. |
|23.||Ordway DJ, Shanley CA, Caraway ML, Orme EA, Bucy DS, Hascall-Dove L, et al. Orme. Evaluation of Standard Chemotherapy in the Guinea Pig Model of Tuberculosis. Antimicrob Agents Chemother 2010;54:1820-33. |
|24.||Ahmad Z, Nuermberger EL, Tasneen R, Pinn ML, Williams KN, Peloquin CA, et al. Comparison of the 'Denver regimen' against acute tuberculosis in the mouse and guinea pig. J Antimicrob Chemother 2010;65:729-734. |
|25.||Ahmad Z, Fraig MM, Bisson GP, Nuermberger EL, Grosset JH, Karakousis PC. Dose-Dependent Activity of Pyrazinamide in Animal Models of Intracellular and Extracellular Tuberculosis Infections. Antimicrob Agents Chemother 2011;55:1527-32. |
|26.||OECD Guideline for Testing of Chemical. Acute Oral Toxicity- Fixed dose procedure. adopted: 17 th December 2001. Available from: http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD _GL420.pdf. [Last updated on 2010 May 11; last cited on 2010 Aug 10]. |
|27.||Ghosh MN. Toxicity Studies. Fundamentals of Experimental Pharmacology.2nd ed. Kolkata: Hilton and Company; 2008. p. 184-9. |
|28.||Ganguly NK, Chitkara NL. Comparative Studies of Cultural and Histopathological Aspects of Experimental H37rv Infection in Guineapigs. Indian J Tuberc 1975;22:76-80. |
|29.||Qurrat-ul-Ain, Sharma S, Khuller GK, Garg SK. Alginate-based oral drug delivery system for tuberculosis: Pharmacokinetics and therapeutic effects. J Antimicrob Chemother 2003;51:931-8. |
|30.||Challu VK, Chandrashekaran S, Chauhan MM. Haematogenous Dissemination Of Pulmonary Isolates of M. Tuberculosis in Animal Model-A Quantitative Measurement. Indian J Tub 1998;45:23-7. |
|31.||Challu VK, Chandrasekaran S, Mahadev B, Jones B, Rajalakshmi R. Behaviour of a south Indian variant of M. tuberculosis during eight years of animal passage. Indian J Tuberc 1993;40:191. |
|32.||Shang S, Shanley CA, Caraway ML, Orme EA, Henao-Tamayo M, Hascall-Dove L, et al. Activities of TMC 207, rifampin, and pyrazinamide against Mycobacterium tuberculosis infection in guinea pigs. Antimicrob Agents Chemother 2011;55:124-31. |
|33.||Bergmeyer HU, Horder M, Rej R. International Federation of Clinical Chemistry (IFCC) Scientific Committee, Analytical Section: Approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase, EC 220.127.116.11). J Clin Chem Clin Biochem 1986;24:481-95. |
|34.||Teitz NW, Rinker AD, Shaw LM. IFCC methods for the measurement of catalytic concentration of enzymes. Part 5. IFCC method for alkaline phosphatise. J Clin Chem Clin Biochem 1983;21:731-48. |
|35.||Garber CC. Jendrassik-Grof analysis for total and direct bilirubin in serum with centrifugal analysis. Clin Chem 1981;27:1410-6. |
|36.||Lo SC, Tsai KS. Glucose interference in Jaffé creatinine method: Effect of calcium from peritoneal dialysate. Clin Chem 1994;40:2326-7. |
|37.||Galigher AE, Kozloff EN. Essentials of practicle microtechnique. Philadelphia: Lea and Febriger; 1971. p. 77. |
|38.||Palanisamy GS, Smith EE, Shanley CA, Ordway DJ, Orme IM, Basaraba RJ. Disseminated disease severity as a measure of virulence of Mycobacterium tuberculosis in the guinea pig model. Tuberculosis 2008;88:295-306. |
|39.||Tomioka H. Current status of some antituberculosis drugs and the development of new antituberculous agents with special reference to their in vitro and in vivo antimicrobial activities. Curr Pharm Des 2006;12:4047-70. |
|40.||Alcaide F, Santin M. Multidrug-resistant tuberculosis. Enferm Infecc Microbiol Clin 2008;13:54-60. |
|41.||Nagayama N, Shishido Y, Masuda K, Baba M, Tamura A, Nagai H, et al. Leukopenia due to anti-tuberculous chemotherapy including rifampicin and isoniazid. Kekkaku 2004;79:341-8. |
|42.||Tostmann A, Boeree MJ, Aarnoutse RE, de Lange WC, van der Ven AJ, Dekhuijzen R. Antituberculosis drug-induced hepatotoxicity: Concise up-to-date review. J Gastroenterol Hepatol 2008;23:192-202. |
|43.||De Jager P, van Altena R. Hearing loss and nephrotoxicity in long-term aminoglycoside treatment in patients with tuberculosis. Int J Tuberc Lung Dis 2002;6:622-7. |
|44.||Collier J, Joekes AM, Philalithis PE, Thompson FD. Two cases of ethambutol nephrotoxicity. Br Med J 1976;2:1105-6. |
|45.||Castagnolo D, Manetti F, Radi M, Bechi B, Pagano M, De Logu A, et al. Synthesis, biological evaluation, and SAR study of novel pyrazole analogues as inhibitors of Mycobacterium tuberculosis: Part 2. Synthesis of rigid pyrazolones. Bioorg Med Chem 2009;17:5716-21. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]