“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR - RILPIVIRINE BY HPLC AND OTHER INSTRUMENTAL METHODS”

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BENGALURU, KARNATAKA

BY

SUJITHA.T

I M. PHARM

DEPARTMENT OF PHARMACEUTICAL ANALYSIS

PESCOLLEGE OF PHARMACY

BENGALURU-560050

(2012-13)

UNDER THE GUIDENCE OF

VENKATESH PRASAD S.P.

ASST PROFESSOR

DEPARTMENT OF PHARMACEUTICAL ANALYSIS

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BENGALURU, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address: / SUJITHA.T
Department of Pharmaceutical Analysis,
PES College of Pharmacy,
50 Feet Road, Hanumanthanagar,
BSK I-Stage,
Bengaluru-560050.
Permanent Address
D/O T.V. Pratap Reddy,
Flat no.301, Kamaakshi residency,
Patwari enclave,Near Sandhya hospitals,
Opp I.D.P.L. Colony, Balanagar,
Hyderabad, Andhra Pradesh.
500037.
2. / Name of the institution: / PES College of Pharmacy
50 Feet Road, Hanumanthanagar,
BSK I- Stage,
Bengaluru -560050
3. / Course of study and subject: / MASTER OF PHARMACY IN
PHARMACEUTICAL ANALYSIS
4. / Date of the admission: / 12/07/2012
5. / Title of the topic:
“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR - RILPIVIRINEBY HPLC AND OTHER INSTRUMENTAL METHODS.”
6. BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
General Discussion :
Viruses have been producing an enormous health hazards continuously for the mankind since ages. These challenges were constantly met by the mankind by producing the effective drugs. There are number of new drug molecules that have been developed for the effective treatment of HIV infection or other viral infections. As recently the mankind has encountered some viral infections which are caused by newer viral strains like H1N1, Ebola virus, Hanta virus, Marburg virus etc which has caused havoc in both developed & developing countries posing newer threats & challenges to the mankind. These newer virus strains which have been recently identified are the main cause for the increased mortality rate in both humans and animals. All these infections require some new drugs so that these infections can be treated & prevented. So there is a need for development of newer antiretroviral drugs and formulations which in turn makes it obligatory for their quantifications either in APIs or in the formulations.
A retrovirus is any virus belonging to the viral family Retroviridae. They are enveloped viruses possessing a RNA genome, and replicate via a DNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA, which can then be integrated into the host's genome with an integrase enzyme. The virusthen replicates as part of the cells DNA. HIV or the human immunodeficiency virus causes HIV infection. Once a person becomes infected with this virus is infected for life resulting in AIDS (Acquired immunodeficiency syndrome).
Antiretroviral drugs are medications for the treatment of infection by retroviruses, primarily HIV.Antiretroviral treatment should be considered if an HIV infected adult has a CD4 count less than 200 cells, or has developed a serious (stage 4) illness. They lower the level of the virus in the blood. This allows the immune system to recover(1)
Rilpivirine is aNon-nucleoside reverse transcriptase inhibitors (NNRTI). NNRTI inhibit reverse transcriptase directly by binding to the enzyme and interfering with its function. Other drugs that comes under this category includesdelavirdine, efavirenz, etravirine, nevirapine(2)
Rilpivirine is an antiviral drug (NNRTI) of human immunodeficiency virus type-1 (HIV-) and inhibits HIV-1 replication by non-competitive inhibition of HIV-1 reverse transcriptase (RT)(3). Rilpivirine does not inhibit the human cellular DNA polymerases α, β and γ.
Drug profile:
Rilpivirine:Rilpivirine is the second generation of non-nucleoside reverse transcriptase inhibitors (NNRTIS) recently marketed for the treatment of HIV infection. It is superior to the first generation NNRTI(4-6) in that it is active against NNRTI resistant HIV-I.
. HCl
IUPAC NAME:4[(4[(4-(1E)-2 cyanoethenyl]-2,6- dimethyl phenyl] amino] 2 pyrimidimyl] amino benzonitrile mono hydrochloride
Empirical formula: C22H18N6.HCl
Molecular weight: 402.88
Nature: Rilpivarine hydrochloride is a white to almost white powder.
Solubility: Rilpivirine is practically insoluble in water over a wide pH range.
Availability: Rilpivirine is available as a white to off-white, film-coated, round, biconvex, 6.4 mm tablet for oral administration. Each tablet contains 27.5 mg of rilpivirine hydrochloride, which is equivalent to 25 mg of rilpivirine(7).
The concept of analytical chemistry lies in the precise and accurate measurements. This determination requires highly sophisticated instruments and methods like HPLC-dissolution studies(8),RP-HPLC(9), GC, HPTLC(10), UV – spectrophotometry(11) and other hyphenated techniques like GC-MS, LC-MS(12), HPLC-MS(13) etc..,.
Literature survey reveals that, Rilpivirine is not official in any of the pharmacopoeias like IP, BP, USP and European pharmacopoeia and it was found that above mentioned anti- retroviral drug was estimated by limited analytical methods like HPLC, UV- spectrophotometry, HPTLC,LC-MS methods were reported. In view of need for a suitable method for routine analysis in formulation, an attempt has been made to develop a simple, efficient and selective method for the determination of Rilpivirine as Rilpivirine hydrochloride in pharmaceutical dosage form. “We are carrying this Analytical Method Development work in collaboration with STRIDES ARCOLABS LIMITED, Advanced Research Centre, Bengaluru.”
Analytical validation is the corner stone of process validation. Without a proven measurement system it is impossible to confirm whether the manufacturing process has done what it purport to do. Hence, there is a need to validate the new methods developed.
6.2REVIEW OF THE LITERATURE:
  • Mohanreddy Chilukuri et al have developed Degradation pathway for Rilpivirine hydrochloride by validated stability indicating UP-LC method. Rilpivirine is subjected to stress conditions of acid, base, oxidation, thermal and photolysis and then six impurities are studied and major degradant is identified by LC-MS and spectral analysis. Chromatographic separation is achieved on a Shimpack XR-ODS-11 stationary phase with simple mobile phase combination and quantification is carried at 295nm with flow rate of 1.0ml/min. This developed LC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities and degradant determination.
  • D. Pranitha et al have published an research article on simultaneous estimation of emtricitabine, tenofovir, disoproxil fumerate and rilpivirine in bulk form by RP-HPLC method. The method employs Thermo Hypersil ODS C-18 column and flow rate of 1ml/min with load of 20μl. Acetonitrile and phosphate buffer (60:40) pH 3 was used as mobile phase. Detection carried out at 260nm and percentage recovery was found be 98-102%. This newly developed method was successfully utilized for the quantitative estimation of drugs in bulk form.
  • Konda Ravi Kumar et al have published a research article on a validated LC method for the estimation of rilpivirine in API and pharmaceutical dosage form. Chromatography was carried on symmetry Zodiac C18 at ambient temperature. Isocratic elution was carried with methanol:water:0.1% ortho phosphoric acid (80:10:10) as mobile phase with flow rate of 1.5ml/min and detection was carried out at 230nm. This method was successfully applied for routine analysis of Rilpivirine in tablet dosage form.
  • Ch Venkata Reddiah et al have developed effective estimation of Rilpivirine by HPLC method in tablet dosage forms and its invitro dissolution assessment(8).Solvents used was acetonitril : buffer (55 : 45) %v/v and absorption maxima of drug was found to be 280nm. Linear response was observed in range of 5.5 – 41.25μg/ml with R equal to 0.99. This method was successfully employed for quality control assay of compound simultaneously and dissolution data helpful in generating the further information regarding invivo absorption rate in tablet dosage form.
  • Christopher James et al have published a clinical review on Rilpivirine as a second generation non-nucleoside reverse transcriptase inhibitor.
  • T.Sudha et al have developed a reverse phase high performance and HPTLC methods for the determination of Rilpivirine bulk and in tablet dosage form. This method depends on RP-HPLC, the mobile phase used consists of mixed phosphate buffer : acetonitrile (60 : 40% v/v) with pH 6.8 and flow rate of 1.0ml/min in isocratic mode. Separation was carried out by UV – detector at 272nm. Percentage recovery was found as 100.53%. Second method depends on HPTLC and mobile phase used is ethyl acetate : methanol : chloroform (8:1:1% v/v/v). Densiometric analysis was carried out at 254nm and percentage recovery was found as 100.17%. This proposed method was validated statistically and recovery study for the determination of Rilpivirine in bulk and in tablet dosage form was performed.
  • Stephen H Hughes et al have proposed a research article on a comparison of the ability of Rilpivirine (TMC 278) and selected analogues to inhibit clinically relevant HIV-1 reverse transcriptase mutants.
  • Bunupuradha T et al have published a research article in European pubmed central about etravirine and rilpivinine resistance in HIV-1 subtype CRF01_AE-infected adults failing non-nucleoside reverse transcriptase inhibitor-based regimens.
  • Dr Calvin J Cohen et al have published the effect of rilpivirine versus efavirenz with two background nucleoside or nucleotide reverse transcriptase inhibitors in treatment-naive adults infected with HIV-1 (THRIVE): a phase 3, randomized, non-inferiority trial in the original The Lancet.
6.2 OBJECTIVES OF THE STUDY:
In the proposed work attempts shall be made to:
  • Develop new analytical method for the estimation of Rilpivirine drugby HPLC, other chromatographic and instrumental methods.
  • Validate the proposed method in accordance with ICH and Pharmacopoeial guidelines for the intended analytical application.
  • Apply the proposed methods for analysis of Rilpivirine as API and in dosage form.
7. MATERIALS AND METHODS:
7.1 Source of data:
  • Literature survey was done at P.E.S. College of Pharmacy using Internet facilities
(RGUHS HELINET) and at Library.
  • Reference from library at RGUHS, Bangalore.
  • Indian Institute of Sciences, Bangalore.
  • Department of Drug testing Laboratories, Bangalore.
7.2Method of collection of data:
Analytical method development on Rilpivirine drug shall be carried out in collaboration with M/s STRIDES ARCOLAB LIMITED. The analytical methods will be developed by using instruments like UV visible spectrometer, HPLC, and HPTLC. The data so obtained is treated statistically to determine the compliance of the experimental result as per ICH and Pharmacopoeial guidelines and for the routine use of the developed analytical methods in industry.
JOURNALS:
  1. Indian Journal of Pharmaceutical Sciences.
  2. Journal of Chromatography A
  3. Journal of Chromatography B.
  4. Asian Journal of Pharmaceutical and Clinical Research.
  5. Eurasian Journal of Pharma Chemistry.
  6. International Journal of Pharma Research and Development.
  7. Journal of Medical Microbiology.
  8. Virology Journal.
  9. Journal of Young Pharmacist.
  10. World Journal of Pharmaceutical research.
  11. International Journal of Pharmacy and Pharmaceutical sciences.
  12. International Journal of Clinical Pharmacology and Toxicology.
7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
NOT APPLICABLE
7.4. HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
NOT APPLICABLE.
8. REFERENCES:
1.Laurence L, John S, Keith L. The Pharmacological Basis of Therapeutics. 2006.
2.Scientific Discussion.
3.Barry CJ, Gary TP, Ganesha R, Disha P, Joseph DB, Heather LB, et al. A comparison of the ability of rilpivirine (TMC278) and selected analogues to inhibit clinically relevant HIV-1 reverse transcriptase mutants. BioMed Central. [Research]. 2012(10.1186/1742-4690-9-99).
4.Calvin JC, Jaime A-V, Bonaventura C, Jan F, Margaret AJ, Kiat R, et al. Rilpivirine versus efavirenz with two background nucleoside or nucleotide reverse transcriptase inhibitors in treatment-naive adults infected with HIV-1 (THRIVE): a phase 3, randomised, non-inferiority trial. 2011 16;378(9787):229 - 37.
5.Cohen C, Molina J, Cahn P, Clotet B, Fourie J, Grinsztejn B, et al. Efficacy and safety of rilpivirine (TMC278) versus efavirenz at 48 weeks in treatment-naive HIV-1-infected patients: pooled results from the phase 3 double-blind randomized ECHO and THRIVE Trials. Journal Acquir Immune Deficiency Syndrome. 2012;1;60(1):33-42.
6.Bunupuradah T, Ananworanich J, Chetchotisakd P, Kantipong P, Jirajariyavej S, Sirivichayakul S, et al. Etravirine and rilpivirine resistance in HIV-1 subtype CRF01_AE-infected adults failing non-nucleoside reverse transcriptase inhibitor-based regimens. European pubmed central. [Journal]. 2011;16(7):1113-21.
7.Therapeutics. T. Edurant (rilpivirine) tablets prescribing information. NJ. 2011.
8.Venkata Reddiah C, Rama Devi P, Mukkanti K. Effective estimation of Rilpivirine by HPLC method in tablet dosage forms and its invitro dissolution assessment. International Journal of Pharmacy and Pharmaceutical Sciences. [Research]. 2012 27;4(3).
9.Pranitha D, Vanitha C, Prince F, Alagar Raja M, Vishnu vardan P, Surendar M, et al. Simultaneous estimation of emtricitabine, tenofovir disoproxil fumerate, and rilpivirine in bulk form by RP-HPLC method. Journal of Pharmacy Research. [Research]. 2012 29;5(8):4600-2.
10.Sudha T, Shanmugasundram P. Reverse phase High performance and HPTLC methods for the determination of Rilpivirine bulk and in tablet dosage form. World Journal of Pharmaceutical research. [Research]. 2012 27;1 (4):1183-96.
11.Committee for Medicinal Products for Human Use. European medicines agencies. 2011 22.
12.Konda RK, Suneetha A, Srilakshmi N. A validated LC Method for the Estimation of Rilpivirine in API and Pharmaceutical Dosage Form. Journal of Pharmacy Research. [Research]. 2012 28;5(8):4434-6.
13.Mohanareddy C, K. HRP, Narayanareddy P, Venkataramana M. Degradation pathway for Rilpivirine hydrochloride by validated stability indicating UP-LC method. International Journal of Clinical Pharmacology & Toxicology. 2012 16.
14.Websites:







9. / Signature of the candidate / (Sujitha.T)
10. / Remarks of the guide
11. / Name and Designation of
11.1 Guide / Venkatesh Prasad S.P.
Asst Professor,
Department of Pharmaceutical Analysis,
PES College of Pharmacy,
Hanumanthanagar,
Bangalore-560050
11.2 Signature
11.3 Head of the department / Dr. Nagaraj
Professor & Head
Department of Pharmaceutical Analysis,
PES College of Pharmacy, Hanumanthanagar,
Bangalore-560050
11.4 Signature
12. / Remarks of the Chairman and Principal / Prof. Dr. S. Mohan
Principal & Director,
PES College of Pharmacy, Hanumanthanagar,
Bangalore-560050
12.1 Signature

1