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Middle and low income countries are more prone to HIV, where there are more than 4 million people infected and receiving ART (anti retroviral therapy). As per the current report as on 30th september 2009, WHO director stated that still there are 5 million people who are exposed to the life prolonging treatment and care. So there are various studies being carried out to get the better treatment available(1).
In vitro techniques are the methods used for testing cell lines or cultures outside the organ of interest by keeping under controlled temperature and pressure conditions. These techniques being devoid of use of animal models play an important role in medical research as they tend to reduce chances of ethical complications by keeping animals away from test environments. The added advantage of using in vitro methods to test recent developments in field of research is that they provide homogeneity in results with increased evidence of therapeutic efficiency.
ARV therapy means treating viral infections like HIV with drugs. The drugs do not kill the viruses. However they slow down the growth of viruses. When the virus is slowed down, so is HIV decreased. Antiretroviral drugs are referred to as ARV. ARV therapy is referred to as ART(2). As such there is no cure for AIDS. However these drugs can slow down the progress of HIV and thus slow down the damage to your immune system. The goal of ARV treatment are
To ensure maximum and lasting control of the amount HIV in the body
To restore and protect the immune functioning of the body by allowing the CD4 cells to replenish their number
To reduce HIV related illness and deaths
In the long run to improve the quality of life for people leaving with AIDS(3)
Anti retroviral therapy being too complex therapy, and the single dose not being so effective there are various other combinations tried with the same as well as with different anti viral classes of drugs. Despite the increasing number of anti retroviral compounds, the number of useful drugs is limited owing to high frequency of cross resistance.
The search for anti retroviral drug has been started since years back, in 1992 already new anti HIV drugs were in developmental phase and variety of drugs were still undergoing clinical testing. Simultaneously there were various different combinations tried, which therapies enhances the anti retroviral activity and reduce the adverse effect of each drug(4). In the addition to the development of new drug resistance, different HIV variants with the help of different combination therapies may also be delayed(4). In the study that was conducted here were, 10 C2 symmetry based or pseudo base HIV protease inhibitors, which were tested against Human Immunodeficiency Virus Type - 1 for its anti retroviral activity, by using cytopathic effect inhibition assay with the help of ATH8 cell line(4). The various combinations which were tried as explained below, amongst those all showed the positive effect against HIV-1. The same protease inhibitors showed synergistic activity in combination with analogs AZT and ddI(4).
Later on with the progression of the time treatment of HIV was modified, in which it was proved with the help of in vitro techniques that combination therapies are more useful as compared to that of single dose therapy and helps in decreasing the viral load(5). And there were evidences which also stated that any two combinations can also be used and can give good results in decreasing the viral load like nucleoside analogues, protease inhibitors or combination of reverse transcriptase inhibitors and protease inhibitors(5). This was the study which compared two different analysis methods and also different combinations of RT and protease inhibitors were tried in CEM-SS cells and PBMC's. In this study there was no antagonism seen in one cell type and synergism in another(5). Good synergism was observed when higher protease inhibitors to nucleoside analogues were tried. Also in combination with protease inhibitors with saquinavir and indinavir with both the cell types it showed lover effective level and antagonism at 50% inhibition of virus replication respectively(5).
Further studies were conducted on various other combinations like trying more then two classes or more than two drugs, nelfinavir mesylate (formerly AG1343) was the main drug with which the other class or other two classes of drug were combined and tried. Combination of three also gave minimal cellular cytotoxicity and also combination of two showed a good synergistic interaction(6). Simultaneously also different range of dose responses were also tried out using nelfinavir, here for nelfinavir five successive two fold dilutions were used with the similar dilution of the other drug(6). Types and intensity for significant quantitative measure was done for various combinations of the drug, as per Greco et al value of α is positive or negative or equal to zero it indicates synergy, antagonism and additive, respectively(6). In this particular study it was clearly indicated that combination of drug with nelfinavir with reverse transcriptase inhibitor eg. ZVD, 3TC, etc. added to additive synergistic effect and similar results were obtained in the triple combination. In case of triple combination it was proved to be more potent and very effective as anti retroviral agent and at lower concentrations(6). A combination-therapy strategy may also lead to the suppression of phenotypic resistance conferred by the selection of an additional mutation following the treatment with a second regimen, as has been described for ZDV and 3TC combination protocols(6). Preliminary results which describe the sensitivity of nelfinavir-resistant HIV isolates to other protease inhibitors suggest that nelfinavir may be a potential candidate for inclusion in combination or sequential therapeutic regimens involving other protease inhibitors(6).
Later on coming to 21st century there were other several studies carried out in testing the efficacy of the combination therapies with second generation of PI's but were not found to be more effective(7). In this study in vitro studies were conducted using second generation PI's to test for anti retroviral activity and also for cytotoxic effect, combinations of the first generation PI's were taken as a standards, the results were no cellular toxicity was observed throughout the process(7). Previous studies which were conducted on the same sowed additive effect using the similar various combinations of second generation drugs. Simultaneously it had also stated that in case of low concentration it showed some additive and synergistic property(7).
The later on studies basically focused on the concentrations of the dose and its effectiveness, here in this study the drug used was TMC 114, it is considered to be potent anti retroviral drug wich showed its activity with 50% effective concentration of 1 - 5nM and 90% effective concentration of 2.7 - 13nM(8). This was the drug which did not show any cytotoxicity at concentration upto 100µM and also no evidence of antagonism between TMC 114 and other available PI or RT inhibitors, similarly combination with other anti retroviral drugs like amprenavir, ritonavir, nefinavir, etc showed evidence of synergism(8). It also had a very good binding capacity to wild type HIV-1 protease as compared to the previous drugs. The potential activity of TMC114 against a very large range of PI-resistant viruses is assured(8). It showed good activity against a selected 19 recombinant clinical isolates carrying multiple protease mutations and demonstrating resistance to an average of five other PIs. HIV was slower as compared to other second generation drugs in presence of TMC 114 and also it showed its effect on virus replication(8). Combination therapy is the current standard of care for antiretroviral therapy. Since combinations of antiretroviral agents may be synergistic, additive, or antagonistic, it is important to test developmental compounds in combination with all the currently prescribed antiretrovirals(8).
HIV protease inhibitors are being one of the most potent and effective antiretroviral as they are considered to be essential components of successful combination therapy or Highly active antiretroviral therapy (HAART) to treat HIV(9). Although the treatment options include protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non nucleoside reverse transcriptase inhibitors (NNRTIs), emergence of viral resistance to protease inhibitors and cross resistance between the members of the protease inhibitors class of drugs are found to be the major factors influencing the failure of HIV clinical management(9). This has thus led to need for development of novel inhibitors targeting critical sites on specifically protease enzymes which have not yet been targeted by the already present protease inhibitors class of drugs(9).
For the very same cause, a pharmacological comparative study was conducted by Hazen et al on the various protease inhibitors available in the market with the low nanomolecular activity providing PI resistant antiretroviral drug Brecanavir(9). The main goal behind conducting this study was to identify the broad spectrum, mutant HIV 1 strain inhibitors which would be compatible with the already present protease inhibitor class of drugs and also with the existing anti retroviral treatment options(9).
This study clearly identifies the following purposes:
Detecting and identifying a variety of clinically relevant mutant HIV 1 strains
Understanding the compatibility of the new PI inhibitor with other anti HIV agents
Highlighting the unique resistant profiles of each anti retroviral in comparison with the other on various HIV 1 strains available for testing(9).
The study makes use of a multitude of protease inhibitors in comparison to NNRTIs and NRTIs, showing equal or almost similar effects when tested on MT-4 cells that are human T-cell leukemia virus type 1 transformed T cell lines. Each of the strains shortlisted for this study was separately assayed and anti-HIV tests were conducted for each combination of drugs with the drug under inspection, Brecanavir(9). The various drug combinations were tested by extracting their EC50 concentrations which would provide exact estimates of the possible strength of each drug and its interaction. Inhibition of each drug was also tested using the same EC50 concentrations(9).
Brecanavir proved to be more potent than most of the protease inhibitors due to production of equivalent EC50. Inhibition of HIV-1 strain IIIB and strain Ba-L in subnano molar concentrations was seen with brecanavir as compared to the other antiretrovirals, in case of the antiviral activity in peripheral blood mononuclear cells BCV stood out as the most potent of all. A seven fold increase in potency was observed when BCV was compared to DRV antiviral potency at exactly equimolar concentrations. However the BCV to the TPV potency increased upto 58 fold with the addition of human serum proteins(9).
While comparing with the NRTIs and combining BCV, the activity of BCV proved to be additive rather than antagonistic, while that when combines with NNRTIs proved to be synergistic(9). Thus the following conclusions have been deduced from the study:
BCV has proven to show subnanomolar in vitro antiretroviral activity against most of the HIV1 infectious strains shortlisted for the study.
The potency of BCV however has proved to be much more when compared to the other protease inhibitors when tested against certain HIV receptors like CCR5.
Becanavir has shown to produce significant potency over other protease inhibitors even in presence of human serum proteins.
In combination with the other antiretroviral drugs, additive and synergistic interactions were observed with NRTIs and NNRTIs respectively.
In case of viruses extracted from protease inhibitor treatment experienced patients, a subnanomolar potency was observed with becanavir(9).
Thus, becanavir has proved to be the most potent protease inhibitor and broadly active against most of the HIV1 strains when tested in vitro(9).
The studied case was fully based on the newly searched anti retroviral drug Brecanavir which is a protease inhibitor and said to be very potent, the compound potently inhibited HIV-1 in cell culture assay with 50% effective concentration of 0.2 - 0.53nM, while that in the previous studies with TMC 114 its was 1 - 5 nM. BCV has a high potency against the wild type protease enzyme, in competitive binding assay it was found to be 2000 fold more potent than APV where as TMC 114 was just 100 folds more potent than indinavir. When there was a comparison with other PI's against a particular stain in PBMC's, it was found that BCV is more potent than any other strain. In the previous studies it was found that the particular drug gave additive effect or synergistic effect with a particular class of drug but in case of BCV it is different, in vitro results with BCV gave un-expected results like in combination with one drug of one class it gave additive effect and with the different drug of the same class it gave synergistic effect. Example BCV in combination with EFV was additive at the same time with NVP was synergistic. BCV is a drug with strong activity and especially in case of HAART high pill burden is a big problem to be faced and no proper adherence which leads to the progression of the disease, in the previous studies it was suggested that single dose therapy was not effective as compared to that of combination dose therapy and hence this is a better option to avoid the combination as the single dose might prove to be more effective and in combination with other doses it may give the best possible result. Previous studies were dealing with the combination therapy amongst the same class of the drug, but in case of BCV as seen it was managed with the different drugs of the same class and the observed results were un-expected but might be helpful (not concluded in the study). There were various combinations tried out with major anti viral drug classes, when tested in combination with NRTI's (nucleotide reverse transcriptase inhibitors) it showed synergistic effect and additive effect with stavudine and zidovidune respectively, in case of NNRTI's (non-nucleoside reverse transcriptase inhibitors) it gave additive effect with nevirapine and delavirdine and in case of other combinations with PI's it had additive effect to the activities of the drugs.
From the studied case and the reviewed articles based on the in vitro studies carried out since the start of the invention of the drugs for Human Immunodeficiency Virus Type - 1 there are certain conclusions that can be pointed out, and can be divided under three main headings viz.
Conclusions from the reviewed articles
Single dose id less preferable as combination therapy is more effective.
All the combinations which were tried out gave additive or synergistic effect as per the combinations.
Antiviral effect of drug added in combination had resulted in inhibitory effects than expected on the basis of the effect observed for each drug and also in case of different concentrations.
TMC 114 was the drug which was found to be highly potent to HIV-1, it had high affinity to bind to HIV-1 protease
All the studies concluded that these data can be of a choice for the further clinical evaluation
Conclusions from the case study
More potent than the above mentioned drug, based on the various in Vito anti retroviral activities, with different HIV strains
More potent than other PI's available
Increased effect in the potency when added with human serum or serum protein
It is additive as well as synergistic as per the combination varies in different combinations
To the end it can be concluded, as the time passed on there were different drugs being developed and at present BCV is the drug of choice as it had to good action against the anti retroviral activity, and is a most potent drug (according to the in vitro studies). It combination therapies are more preferred and even in case of BCV combination therapy has showed more potency and both the possible effect additive as well as synergistic.