Download proforma for registration of subjects for

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION.
1.
Name of the candidate and Address:
GIRISH.A.HAMPANNAVAR,
Dept. of Pharmaceutical Chemistry,
Govt. College of Pharmacy,
# 2, P.Kalinga Rao Road,
Subbaiah Circle,
Bangalore- 560 027.
2.
Name of the institution:
Government
College
of
Pharmacy,
Bangalore-560 027.
3.
Course of study and subject:
Master of Pharmacy in Pharmaceutical
Chemistry.
4.
Date of admission to the course:
5.
Title of the topic:
30th May 2007
“DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS
FOR QUANTITATIVE ESTIMATION OF ANTIRETROVIRAL DRUG IN
PHARMACEUTICAL DOSAGE FORM(S)”.
6.
Brief resume of the intended work:
6.1: Need for the study:
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 virus then replicates as the part of the cell’s DNA. This results
in Acquired immunodeficiency syndrome.
Antiretroviral drugs are medications for the treatment of infection by retroviruses,
primarily HIV. Different classes of antiretroviral drugs act at different stages of the HIV
life cycle. Combination of several (typically three or four) antiretroviral drugs is known
as Highly Active Anti-Retroviral Therapy (HAART). If an HIV infection becomes
resistant to standard HAART, there are limited options. One option is to take larger
combinations of antiretroviral drugs, an approach known as mega-HAART or salvage
therapy. 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 virus in the blood; this allows the immune system to recover.
Classification of antiretroviral agents:
i. Reverse transcriptase inhibitors (RTIs): target construction of viral DNA by
inhibiting activity of reverse transcriptase. There are three subtypes of RTIs with
different mechanisms of action: nucleoside analog reverse transcriptase inhibitors
(NRTI) are incorporated into the viral DNA leading to chain termination, the nonnucleoside reverse transcriptase inhibitor(NNRTI)distort the binding potential of
the reverse transcriptase enzyme and the nucleotide analog reverse transcriptase
inhibitors(NtRTI).E.g. Zidovudine, Stavudine, Didanosine, Zalcitabine
ii. Protease inhibitors (PIs): target viral assembly by inhibiting the activity of protease,
an enzyme used by HIV to cleave nascent proteins for final assembly of new
virons. E.g. Saquinavir, Indinavir, Retonavir, Nelfinavir etc.
iii. Fusion inhibitors: block HIV from fusing with a cell's membrane to enter and infect
it. There is currently only one FDA-approved drug in this class, enfuvirtide,
marketed as Fuzeon other examples are Acyclovir, Ganciclovir etc.
iv. Integrase inhibitors: inhibit the enzyme integrase, which is responsible for
integration of viral DNA into the DNA of the infected cell. There are several
integrase inhibitors currently under clinical trial, and raltegravir became the first to
receive FDA approval in October 2007.
v. Entry inhibitors: block HIV-1 from the host cell by binding CCR5, a molecule on
the host membrane termed a co-receptor that HIV-1 normally uses for entry into
the cell together with a primary receptor. Only one entry-inhibitor class drug is
available, maraviroc.
vi. Portmanteau inhibitors: A new way to combat HIV through the merging of two
antiviral agents into one drug, achieving the same effect as when two or more
drugs are taken separately.
The concept of analytical chemistry lies in the precise and accurate measurements.
This determination requires highly sophisticated instruments and methods like Mass
spectrophotometry, Gas chromatography, HPTLC, HPLC etc. HPLC method is
sensitive, accurate and precise and desirable for regular determination of drugs in
formulations, thereby is advantageous than volumetric methods. HPLC assay has been
developed and validated for the simultaneous quantitative determination of drugs.
On literature survey it was found that various above mentioned antiretroviral
drugs were estimated by several analytical methods either alone or in combination by
HPLC, HPLC coupled with electrospray mass spectrometry (HPLC-MS), first
derivative spectrophotometry and HPLC, HPLC with buffer, solid-phase extraction
coupled with HPLC, reversed phase HPLC, combined HPLC-Radioimmunoassay
procedure, etc. Since we are doing the project in collaboration with M/S. Strides
Arcolab Limited, Bangalore at Analytical service department, at this instance it is not
possible to disclose the name of the antiretroviral drug on which the analytical
methods were going to develop. In view of the need in the industry for routine analysis
of antiretroviral drugs in formulations attempts are being made to develop simple and
accurate instrumental method for estimation of anti retroviral drugs and extend it for
their determination in formulation.
6.2: Review of literature:

Slusher JT, et al1., developed a specific and sensitive combined high-pressure
liquid chromatography (HPLC)-radioimmunoassay (RIA) procedure for the
determination of Zidovudine (ZDV) and its phosphates in peripheral blood mono
nuclear cells of HIV infected patients. ZDV and its anabolites were separated by
HPLC and measured by using RIA protocol. This method provides a useful tool
for evaluating in vivo pharmacokinetics of ZDV anabolites.

Moore JD, et al2., A high-performance liquid chromatography (HPLC) method
utilizing triple quadrupole mass spectrometry (MS) detection was developed and
validated for the simultaneous measurement of the intracellular nucleoside 59triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP), and
stavudine (d4T-TP). This method can be utilized to measure the intracellular 59triphosphate levels in HIV infected patients receiving antiretroviral therapy
containing the nucleoside reverse transcriptase inhibitors 3TC, d4T, or ZDV.

Gunawan S*, Griswold MP, Kahn DG. 3.,developed a method for quantitation of
amprenavir (agenerase) in human immunodeficiency virus type-1 infected patient
serum or plasma using liquid chromatography–tandem mass spectrometry (LC–
MS–MS). Amprenavir and an internal standard (reserpine) are extracted by
liquid–liquid extraction and chromatographically separated by a reversed-phase
C18 -analytical column. The triple quadrupole LC–MS–MS system is operated in
the positive-ion mode and 18 multiple reaction monitoring is used for drug
quantitation.

Rentsch KM *4., developed a method for quantifying the different proteinase
inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir) and
non-nucleoside reverse transcriptase inhibitors (efavirenz, nelfinavir). The
antiretroviral agents were separated and detected using LC–MS and atmospheric
pressure chemical ionization. After solid-phase extraction, the antiretrovirals were
separated within 21 min using gradient elution.

Rouzes Aa, et al5., developed an method for selective and accurate assay for the
simultaneous quantitation of four protease inhibitors and a non-nucleoside reverse
transcriptase inhibitor in human peripheral blood mononuclear cells using highperformance liquid chromatography–mass chromatography (LC/MS) has been
developed and validated however this is applied for therapeutic drug monitoring
and pharmacokinetic studies.

Dailly Ea, Raffi Fb, Jolliet Pa 6., developed an method for therapeutic drug
monitoring of atazanavir and non nucleoside reverse transcriptase inhibitors, all
drugs are extracted after a liquid–liquid extraction and separated on a C18 column
with a binary gradient elution except lopinavir which is separated without this
gradient.

Rezk NL, Crutchley RD, Angela DM, Kashuba7., developed an method for
sensitive and simple reverse-phase (RP) high-performance liquid chromatography
(HPLC) assay for the simultaneous quantitative determination of emtricitabine
and tenofovir in human blood plasma.

Bezy Va, et al8., developed a method for quantitative recovery of drugs from rat
plasma by solid-phase extraction followed by optimised HPLC separation on dC18
column with acetic acid–hydroxylamine buffer. Hence a new buffer, obtained
with acetic acid and hydroxylamine, has been tested in HPLC/ESIMS/MS and
appears to be an efficient volatile buffer in the medium 5–7 pH range.

Wang PG, et al9., developed a method for simultaneous quantification of lopinavir
and ritonavir in human plasma using semi-automated 96-well liquid–liquid
extraction.

Sarkar M, Khandavilli S, Panchagnula R10.,developed a method for the
quantitative determination of three antiretroviral drugs by isocratic technique on a
reversed-phase C-18 SYMMETRY column with mobile phase based and
optimized depending on the polarity of the molecules. The proposed methods
were successfully applied for the reliable quantification of API content in the
commercial formulations of Lamivudine, Stavudine and Nevirapine.

Kapoor N, Khandavilli S, Panchagnula R11.,developed a method for Simultaneous
determination of lamivudine and stavudine in antiretroviral fixed dose
combinations by first derivative spectrophotometry and high performance liquid
chromatography.

Antonio DA, et al12., developed a method for the simultaneous quantification of
the new HIV protease inhibitor darunavir, and 11 other antiretroviral agents in
plasma of HIV-infected patients using HPLC–MS method.

I.Lefebvrea, J.-Y.Puya, C.Perrinb, C.Perigauda
13
.,developed an method for
quantification of Zidovudine and its monophosphate in cell extracts by on-line
solid-phase extraction coupled to liquid chromatography. This method was used
to study the decomposition pathway of a model pronucleotide in an in vitro
approach.

Nandini P and Desai AD14., developed an RP-HPLC method for simultaneous
estimation of lamivudine, Zidovudine and nevirapine from tablets by external
standard method.
6.3: Main objectives of the study:
In the proposed work attempts shall be made to:

To develop new analytical methods for the estimation of antiretroviral drugs
preferably by spectrophotometric and chromatographic methods.

Validate the proposed method in accordance with BP, USP and ICH guidelines
for the intended analytical application.

Apply the proposed methods for analysis of these drugs in their dosage form and
in raw materials.
7
Materials and methods:
7.1: Source of the data:
The preliminary data required for the experimental study was obtained from
1. Library of Govt.College of Pharmacy, Bangalore.
2. JRD TATA Memorial Library IISc, Bangalore.
3. Library and Information Centre, Rajiv Gandhi University of Health Sciences,
Bangalore.
4. Internet sources.
5. http://www.rguhs.ac.in/HelinetHome/
7.2 Methods of collection of the data (including sampling procedure if any)

Data pertaining to the present study will be obtained from the experiments
performed
in
STRIDES
ARCOLAB
LIMITED
(Analytical
service
department). They are adequately equipped with necessary analytical
instrumental setup to carry out the desired work.
7.3:
Does the study require any investigations or intervention to be conducted on
Patients other human or animals? If so, please describe briefly:
-NO-
7.4: Has ethical clearance been obtained from your institute in case of 7.3
-NOT APPLICABLE-
8
List of references:
1) Slusher JT1,2 , Kuwahara SK1,2* , Hamzeh FM,1, Lewis LD,1,2, Kornhauser
DM1,2,3,AND Lietman PS1,2,3., Intracellular Zidovudine(ZDV) and ZDV
Phosphates as Measured by a validated combined High-pressure Liquid
chromatography-radio immunoassay procedure. Antimicrobial Agents And
Chemotherapy Nov.1992; 36 (11): 2473-77.
2) Moore JD, Valette G, Darque A, Zhou XJ, Sommadossi JP., Simultaneous
Quantitation of the 5’-Triphosphate Metabolites of Zidovudine, Lamivudine, and
Stavudine in Peripheral Mononuclear Blood Cells of HIV Infected Patients by
High-Performance
Liquid
Chromatography
Tandem
Mass
Spectrometry.
American Society for Mass Spectrometry 2000; 11: 1134–43.
3) Gunawan S*, Griswold MP, Kahn DG., Liquid chromatographic–tandem mass
spectrometric determination of amprenavir (agenerase) in serum/plasma of human
immunodeficiency virus type-1 infected patients receiving
combination
antiretroviral therapy. Journal of Chromatography A, 2001; 914: 1–4.
4) Rentsch KM., Sensitive and specific determination of eight antiretroviral agents in
plasma by high-performance liquid chromatography–mass Spectrometry. Journal
of Chromatography B, 2003; 788: 339–50.
5) Rouzes Aa
et al., Simultaneous determination of the antiretroviral agents:
amprenavir, lopinavir, ritonavir, saquinavir and efavirenz in human peripheral
blood mononuclear cells by high-performance liquid chromatography–mass
spectrometry. Journal of Chromatography B, 2004; 813: 209–16
6) Dailly Ea, Raffi Fb, Jolliet Pa., Determination of atazanavir and other antiretroviral
drugs (indinavir, amprenavir, nelfinavir and its active metabolite M8, saquinavir,
ritonavir, lopinavir, nevirapine and efavirenz) plasma levels by high performance
liquid chromatography with UV detection. Journal of Chromatography B, 2004;
813: 353–58.
7) Rezk NL, Crutchley RD, Angela DM, Kashuba7., Simultaneous quantification of
emtricitabine and tenofovir in human plasma using high-performance liquid
chromatography after solid phase extraction. Journal of Chromatography B, 2005;
822: 201–8.
8) Bezy Va, Morin Pa,, Couerbe Pb, Leleu Gc, Agrofoglio La., Simultaneous analysis
of several antiretroviral nucleosides in rat-plasma by high-performance liquid
chromatography with UV using acetic acid/hydroxylamine buffer Test of this new
volatile medium-pH for HPLC–ESI-MS/MS. Journal of Chromatography B, 2005;
821: 132–43.
9) Wang PG*, Wei JS, Kim G, Chang M, El-Shourbagy T., Validation and
application of a high-performance liquid chromatography–tandem mass
spectrometric method for simultaneous quantification of lopinavir and ritonavir in
human plasma using semi-automated 96-well liquid–liquid extraction. Journal of
Chromatography A, 2006; 1130: 302–7.
10) Sarkar M, Khandavilli S, Panchagnula R*. Development and validation of RPHPLC and ultraviolet spectrophotometric methods of analysis for the quantitative
estimation of antiretroviral drugs in pharmaceutical dosage forms. Journal of
Chromatography B, 2006; 830: 349–54.
11) Kapoor N, Khandavilli S, Panchagnula R11*. Simultaneous determination of
lamivudine and stavudine in antiretroviral fixed dose combinations by first
derivative spectrophotometry and high performance liquid chromatography.
Journal of Pharmaceutical and Biomedical Analysis, 2006; 41: 761–65.
12) Antonio DA* et al., HPLC–MS method for the simultaneous quantification of the
new HIV protease inhibitor darunavir, and 11 other antiretroviral agents in plasma
of HIV-infected patients. Journal of Chromatography B, 2007; 859: 234-40.
13) I.Lefebvrea, J.-Y.Puya, C.Perrinb, C.Perigauda., Quantification of Zidovudine and
its monophosphate in cell extracts by on-line solid-phase extraction coupled to
liquid chromatography. Journal of Chromatography B, 2007; 858: 2-7.
14) Nandini P, Desai AD., Simultaneous Reverse Phase HPLC Estimation of Some
Antiretroviral Drugs from Tablets. Indian Journal of Pharmaceutical Sciences,
2007; 69(1):118-120.
9
Signature of the candidate:
(GIRISH.A.HAMPANNAVAR )
1O
Remarks of the guide:
11.
Name and designation of
11.1 Guide:
CHANDRASHEKAR JAVALI,
Asst. Professor,
Dept. of Pharmaceutical Chemistry,
Govt. College of Pharmacy,
Subbaiah circle,
Bangalore- 560 027.
11.2 Signature
11.3 Co-guide
NOT APPLICABLE
11.4 Signature
NOT APPLICABLE
11.5 Head of the Department
M S NIRANJAN,
Professor,
Dept. of Pharmaceutical Chemistry,
Govt. College of Pharmacy,
Subbaiah circle,
Bangalore- 560 027.
11.6 Signature
12.
12.1 Remarks of the Chairman and
Principal
12.2 Signature
Dr. S SHASHIDHARA
PRINCIPAL
GOVT COLLEGE OF PHARMACY,
BANGALORE- 560 027