Saturday, August 17, 2019
HIV (Human Immunodeficiency Virus) Essay
Ã Ã Ã Ã Ã Ã HIV (Human Immunodeficiency Virus) causes AIDS (Acquired Immune Deficiency Syndrome) that disables the immune system.Ã It was discovered in 1983 .HIV enters the body through the bloodstream and duplicates itself rapidly.Ã The victim is susceptible to infectious diseases that eventually are fatal (1). Statistically, HIV/AIDS is the number one killer of African-American women ages 25 to 34, according to the Centers for Disease Control and Prevention.Ã Between 2000 and 2003, they were nineteen times more likely than White females and five times more likely than Hispanic females to contract the disease.Ã African-American men were seven times more likely than White men and three times the rate of Hispanic men to contract HIV/AIDS (2). In 2008, there is still not a cure for AIDS.Ã Instead, scientists have discovered drugs that can slow down the progression of the disease. Protease inhibitors (PIs) are antiviral drugs that slow down the spread of HIV (1). The virus produces a protein called protease so that it can replicate itself.Ã Ã¢â¬Å"Protease cuts long chains of proteins and enzymes into shorter chains, the first step in the process by which HIV infects a cellÃ¢â¬ (1).Ã If this doesnÃ¢â¬â¢t happen, replication does not continue. In 1987, AZT was the first anti-HIV medication that was created.Ã It showed hope and kept people healthier longer.Ã However, the side effects were very high for men.Ã Since that time, more HIV medications have evolved. Ã Antiretroviral therapy usually consists of combinations of nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, or fusion inhibitors, which are prescribed in the later stages of HIV (19). Dual protease inhibitor therapy is also being used clinically (3).Ã They are also associated with improving morbidity and mortality of HIV-positive persons (5).Ã Although these drugs are expensive, they have proven to be the most successful therapy in managing HIV.Ã However, some patients donÃ¢â¬â¢t comply 100% with their treatments.Ã This is largely due to the side effects. Sometimes they make a patient feel worse than the actual disease. The most commonly reported ones are abdominal pain, abnormal bowel movements, diarrhea, fatigue, headache, and nausea.Ã Children usually develop a skin rash. The more serious side effects are liver problems and pancreatitis.Ã Some patients have also seen large increase in triglyceride ad cholesterol levels.Ã Diabetics saw an increase in their blood sugar levels.Ã Other patients developed diabetes while taking protease inhibitors (16). Presently, nine PIs have been approved for use in the United States and Europe:Ã amprenavir, atazanavir, fosamprenavir, lopinavir, indinavir, ritonavir, saquinavir, tipranavir, and nelfinavir. Three non-nucleoside reverse transcriptase inhibitors (NNRTIs) are used for treatment of HIV:Ã nevirapine, delavirdine, and efavirenz (7). Drug treatment selection depends on factors such as drug resistance, tolerability, drug interactions, and effectiveness. Therapeutic drug monitoring (TDM) Ã¢â¬Å"has been proposed so that practitioners may better maintain appropriate plasma concentrations of drugs in their patients by identifying interactions with other medications and assessing medication adherenceÃ (7). Figure 1 shows the structures of these compounds. Fig. 1. Chemical structures of protease inhibitors and non-nucleoside reverse transcriptase inhibitors. About 50% of Ã¢â¬Å"treatment-naÃ ¯ve patientsÃ¢â¬ donÃ¢â¬â¢t have continued antiviral response after one year of therapy (18). In some cases, there is a development of drug resistance and metabolic complications.Ã Also, Ã¢â¬Å"there is increasing evidence that virological treatment failure is correlated with variations in the pharmacokinetic parameters of drugs (20).Ã This can be due to drug interactions, low bioavailability, and variations in metabolic enzyme activity.Ã Atazanavir (ATV) has good oral bioavailability and a favorable pharmacokinetics profile (18).Ã With this in mind, patients can mostly take a once-daily dose. A separate analytical method has been recently published for quantifying ATV in human plasma using solid phase extraction and HPLC with PDA (photodiode array) detection at 201 nm (18).Ã This method provides excellent separation of ATV from its internal standard, clozapine (CLZ) and the other PIs, thus, obtaining an accurate measurement of the drug (see figure 2). CLZ elutes at 8.9 minutes, and ATV elutes a 24.4 minutes. A 40-ml injection resulted in a recovery yield of 100%. Fig. 2.Ã Chromatogram of ATV with PIs/NNRTIs (8000 ng/ml) spiked with CLZ Nelfinavir mesylate has been shown in phase III controlled clinical trials to significantly reduce viral load and increase CD4+ cell counts when used with reverse transcriptase inhibitors. ItÃ¢â¬â¢s prescribed as part of triple drug combination therapy (9). CD4+ are helper T cells.Ã They are important for immune reconstitution in patients that are receiving antiretroviral therapy (10).Ã After a large number of these cells are destroyed, AIDS develops (7). Six clinical trials was conducted in the past ten years using 2, 148 HIV-infected children enrolled in the Pediatric AIDS Clinical Trials Group treatment trials (10).Ã Patient ages varied from 2 to 7 years of age.Ã The focus was to observe short-term variability of CD4 percentages.Ã The study found that 49% of patients had CD4 percentages above 25%.Ã 32% of patients had CD4 counts between 15% and 24%; 19% were less than 15%.Ã Finally, 5.4% had a CD4% of less than 5% (10). In June 2006, Darunavir (DRV) was licensed in the United States.Ã It is a promising PI that is active against HIV strains that are resistant to the other PIs that currently on the market (17).Ã It is prescribed in 600-mg doses that are taken twice daily with 100 mg of ritonavir that acts as a booster. Protease inhibitors are associated with unfavorable pharmacokinetics and many side effects such as gastrointestinal disturbances and lipid abnormalities (5).Ã Four of the most common PIs used are indinavir, ritonavir, saquinavir, and nelfinavir (3).Ã Ã¢â¬Å"Monitoring blood concentration of PIs, which can indicate both therapeutic and toxic levels of the drugs as well as patient noncompliance with the medication, may improve the care of both HIV-infected adults and childrenÃ¢â¬ (3). When ritonavir was first introduced, it was given in doses of 600 mg every 12 hours. Ã¢â¬Å"However, patient intolerance of full doses led to its primary use as a pharmacologic enhancer to increase the concentrations in plasma of a second protease inhibitor to improve the convenience of antiretroviral regimens by extending the dosing interval, reducing pill burden, and /or eliminating food-induced reductions in pharmacokinetic exposureÃ¢â¬ (8). Ã Ã Ã Ã Ã Ã Researchers used High Performance Liquid Chromatography (HPLC) to determine concentrations of PIs in blood.Ã Various methods have been used to study the pharmacokinetics and drug interactions. Interpreting plasma levels can be used to Ã¢â¬Å"individualize drug dosage of antiretrovirals (4). Quality control (QC) procedures must be done to ensure that these methods are accurate and precise.Ã Ã¢â¬Å"Such procedures usually include intralaboratory (internal) method validation, intralaboratory QC procedures, and participation in an interlaboratory QC program for antiretroviral drugsÃ¢â¬ (4). Since the latter hadnÃ¢â¬â¢t been done before, it was established so that laboratories can obtain better measurement results of antiretroviral drugs. Nine laboratories participated in the first part of the program.Ã The first part of the experiment involved the measurement of the protease inhibitors:Ã indinavir, nelfinavir, ritonavir, and saquinavir.Ã All had a specified purity of 99% or higher.Ã QC samples were prepared by spiking blank plasma from HIV-negative volunteers with PI standard. The low concentrated standards ranged from 0.087 to 0.15 mg/L while the intermediate concentrated standards contained ranged from 2 to 3 mg/L of all four PIs.Ã Finally, the high-concentrated standards contained approximately 5 to 11 mg/L of drug.Ã All drugs were dissolved in methanol following accurate weighings and diluted with blank plasma (4). High Performance Liquid Chromatography (HPLC) was used to analyze twelve samples.Ã All laboratories measuring more than one protease inhibitor used as assay for Ã¢â¬Å"simultaneous determinationÃ¢â¬ (4).Ã Six laboratories used HPLC/UV and three labs used HPLC-MS/MS. Mass spectrometry detection is often recommended for measurement of low concentration levels.Ã Also, this type of analysis is usually faster and Ã¢â¬Å"does not require complete resolution of drugs for detection and quantificationÃ¢â¬ (5).Ã Only five laboratories were able to measure all four PIs.Ã Three laboratories were not able to determine nelfinavir.Ã One laboratory only measured indinavir (4). Acceptable accuracy results are between 80% and 120%.Ã Only indinavir resulted in an acceptable accuracy of 80%.Ã The remaining PIs had between 36% and 74% accuracy.Ã These results should encourage laboratories to improve their analytical methods and QC procedures.Ã Other PIs, such as amprenavir and lopinavir, can be analyzed as well (4). HIV-positive plasma samples are heat inactivated before analysis, approximately 58Ã °C for 40 minutes, to decrease the risk of infection to the operator.Ã They may also go through a freeze/thaw cycle. As with the QC study, blank plasma was spiked with seven PIs (indinavir, amprenavir, atazanavir, ritonavir, saquinavir, lopinavir, and nelfnavir) at low, intermediate, and high concentrations measured in ng/ml.Ã Certain assays only require 100 Ã µl of plasma for analysis.Ã Ã¢â¬Å"This is advantageous when measuring PI concentrations as part of clinical studies as they often necessitate hourly sampling to generate complete PK profiles; therefore less blood can be drawn from the patientÃ¢â¬ (5).Ã In addition, seven PIs can be quantified in one assay, but impossible to assay all seven in a single preparation. Ã¢â¬Å"The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) has emerged as the developmental method of choice supporting clinical and pre-clinical pharmacokinetic studiesÃ¢â¬ (13). Recovery for the HPLC-MS/MS methods was above 87% for all seven drugs at all three concentration levels (5).Ã It was successful in quantifying seven PI concentrations in plasma of HIV positive persons that participated in a run time of nine minutes.Ã Therefore, the assay may be used for determining PI concentrations in semen, lymphocytes, and cerebrospinal fluid (5). A fast and highly-sensitive LC-MS-MS method was developed that could analyze five protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) in one run using an internal standard.Ã Sample sizes were small (ng/ml) and run times were approximately 5 minutes.Ã Recoveries for all five PIs were between 87% and 92%(11). Tipranavir is part of a class of non-peptidic PIs that works against both Ã¢â¬Å"wild-type virus and variants resistant to current PIsÃ¢â¬ (6).Ã It also has a high genetic barrier.Ã Tipranavir is prescribed in a 500-mg dose taken in combination with 200 mg of ritonavir twice daily as part of antiretroviral therapy for patients with HIV-1 strains that are resistant to multiple PIs (6). Fig. 3. Tipranavir chemical structure Ã Ã Ã Ã Ã Ã An HPLC-UV method has been validated and is currently applied when monitoring tipranavir (TPV) plasma levels in HIV patients.Ã Samples were prepared for solid phase extraction (SPE) by conditioning the cartridges with 0.1% phosphoric acid, pH 7.Ã TPV stock solutions with a concentration of 5 mg/ml were diluted from with 50% methanol. Samples were spiked with TPV at 1.875, 7.5, 18.75, 37.5, 60, and 75 Ã µg/ml in triplicate. QC samples were diluted with blank plasma and phosphate buffer to 5.625, 22.5, and 67.5 Ã µg/ml.Ã Clozapine was used as the internal standard (6).Ã 5-ml aliquots of blood samples were obtained from HIV infected patients.Ã The plasma obtained from centrifugation was heated at 60Ã ° C for one hour in a water bath. TPV in plasma was measured at a UV absorbance of 201 nm with a retention time of 32.2 minutes. Its internal standard, clozapine (CLZ) has a retention time of 8.3 minutes (6). Figure 4 below shows the chromatogram of TPV, its internal standard, and all other PIs and NNRTIsÃ present.Ã Figure 5 shows TPV and its internal standard CLZ only. Fig. 4. Chromatogram of plasma control sample of TVP (22.5 mg/ml) spiked with internal standard and all PIs and NNRTIs. Fig. 5. Chromatogram of calibration sample of TVP (37.5 mg/ml) spiked with internal standard CLZ. Isocratic HPLC methods combined with the use of UV and fluorescence detection produces more sensitivity. Amprenavir is a fluorescent compound.Ã Its internal standard PR25 can also be seen under fluorescence (see figure 6). Fig. 6.Ã Blank plasma with 100 ng/ml amprenavir and 1000 ng/ml of PR25 seen under fluorescence. In conclusion, much progress has been made in the development of protease inhibitors and other antiretroviral therapy. HPLC with UV detection has been the most commonly used method of analysis.Ã It is rapid, simple, and highly sensitive.Ã LC-MS-MS has been noted at the developmental method of choice for clinical and pre-clinical pharmacokinetic studies (13).Ã More compounds can be analyzed in less time.Ã Also, they can be used for methods using other human biological matrices. For the majority of protease inhibitors, all side effects are not known. 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