Abstract

EVALUATION OF POTENTIALITY OF ANTIMICROBIAL ACTIVITY OF TRIAZAPENTACYCLO PIPERIDINE CONTAINING BROAD SPECTRUM ANTIBIOTIC RIFABUTIN BY MINIMAL INHIBITORY CONCENTRATION TO TREAT MYCOBACTERIUM AVIUM COMPLEX INFECTION IN HIV PATIENT AND QUANTIFICATION OF PHAR

Pallab Mandal, Soumya Chakraborty, Chiranjit Saha, Nilendra Chakraborty, Sanmoy Karmakar and Tapan Kumar Pal*

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Abstract

Mycobacterium has caused epic diseases: tuberculosis is still the more important infectious killer of humans. Mycobacterium aviumintracellulare (or Mycobacterium avium complex; MAC) continues to be difficult to treat. Rifabutin is an antibiotic used as first line treatment to treat tuberculosis and prevent and treat Mycobacterium avium complex. This derivative of macrocyclic antibiotic rifampin is typically only used in those who cannot tolerate rifampin such as people with HIV/AIDS on antiretroviral and for active tuberculosis it is used with other antimycobacterial medications also used for latent tuberculosis when exposed to drug resistant TB. Rifabutin inhibits the growth of most MAC isolates at concentration ranging from 0.25- 1.0mg/L and also inhibit the growth of many strains of M. tuberculosis at concentration of 0.015to 0.125μg/Ml and it had been reported from Pfizer Mycobutin hard gelatin capsule 150mg that rifabutin in vitro MIC99 values of ≤0.5μg/ml, determined by agar dilution method for Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium marinum that in case of in invitro has better MICs than rifampin. Rifabutin (CAS NO.-72559-06-9), is similar in structure and activity of rifampin, which is semi synthetic rifamycin. The chemical name of rifabutin is 1, 4-didehydro-1-deoxy-1-4-dihydro-5’-(2-methyl propyl)-1-oxorifamycin XIV. Molecular formula of rifabutin is C46H62N4O11, belonging to the class of ansamycins. It is found that rifabutin inhibits DNA –dependent RNA polymerase, which interact with and to penetrate the outer layers of Escherichia coli (E. coli) and Bacillus subtilis. But mammalian cells are not affected. This enzyme did not inhibited by resistant strains of E. coli, rifabutin, like rifampin. Advanced HIV patients are administered rifabutin for prevention of disseminated Mycobacterium avium complex. It is not known how rifabutin inhibits this reaction. Rifabutin also inhibited the DNA synthesis of rifampin resistant Mycobacterium tuberculosis by inhibiting thymidine incorporation into DNA. In the present study efforts were given to develop and validate a bioanalytical method for estimation of rifabutin in human plasma by LC-MS/MS (API-4000). The calibration concentrations of rifabutin were 62.5-4000 ng/ml which accuracy was 101.36-106.54%, 100.56-105.79%, 97.73-107.59%, 100.55-108.15% and 97.13-103.42% for freeze thaw, short term, bench top, auto sampler stability and long term stability respectively and recovery was 98.53-99.56%, matrix factor was 0.94-0.96.The developed method used for determination and quantification of pharmacokinetic parameter of rifabutin in human plasma was also validated as per the US-FDA guidelines. The validation parameters found within the specified regulatory limit, hence acceptable. The present method also has a short run time (3.50 min) and easy plasma extraction process by protein precipitation technique. This method was simple, specific, highly selective, sensitive and reproducible. The developed and validated LC-ESI-MS/MS assay method was applied to compare the oral bioavailability of two formulations (test and reference) by conducting the single oral dose, open label, randomized, two period, two sequence, crossover study of 24 healthy Indian volunteers (male) with an average age of 28.08±4.92years and average BMI of 21.88±1.54Kg/m2 under fasting condition. The pharmacokinetic parameters like Cmax, Tmax, AUC0-t, AUC0-∞, Kel, T1/2 are determined for rifabutin to calculate the relative bioavailability of test preparation rifabutin 150mg tablet over the reference preparation of same dosage after oral administration to healthy human volunteers. In vitro dissolution of reference and test preparation showed that more than 90% drug was released for reference preparation and test preparation after 45 minute. In vitro dissolution test of rifabutin showed that after 45 minutes 93.51 (for reference), and 96.47 (for test) drug released in appropriate test condition. From the study of pharmacokinetic application of rifabutin it was observed that after oral administration of 150mg rifabutin capsule dissolution occurs in 45min. and absorb into the systemic circulation and peak plasma levels were achieved between 2.0-4.0hrs for reference preparation and 1.0-3.0 hrs for test preparation. The mean peak plasma levels of rifabutin with reference preparation, on the study day ranged between 233.940 – 385.640 ng/ml. While the rifabutin test preparation of ranged between 219.570 – 394.790 ng/ml. The value of pharmacokinetic parameters of rifabutin are Cmax 302.464±41.612 for reference drug and 298.270±56.790 for test drug; Tmax 2.813±0.845 for reference drug and 1.979±0.667 for test drug; AUC0-∞ 1937.949±521.650 for reference drug and 1960.553±445.403 for test drug; Kel 0.0205±0.075 for reference drug and 0.173±0.041 for test drug; T1/2 3.815±1.318 for reference drug and 4.272 ±1.171 for test drug. On the basis of comparison of the AUC0-t for rifabutin 150 mg capsule after single dose administration, the relative bioavailability of the test preparation of rifabutin 150mg was 100.22% of that of the reference preparation. According to WHO report the critical concentration (MIC) of rifabutin 500ng/ml and below this concentration rifabutin is highly sensitive. According to WHO report 13% MAC isolates from MAC infected HIV patient after treating by 500ng/mL concentrated rifabutin , so after treating test preparation rifabutin capsule approximately less than 10% MAC will be isolated from MAC infected HIV patient within 1.0-3.0 hrs. It was concluded that rifabutin 150mg test preparation highly sensitive than reference preparation in case of MAC infected HIV patient.

Keywords: Rifabutin, Antimicrobial activity of Rifabutin, LCMS/MS, Pharmacokinetic application of Rifabutin.