Biomedical subjects

T K Venkatachalam

Publications and source records attributed to T K Venkatachalam.

At least 19 recordsLinked to original sources

PubMed2006

Effect of alkyl groups on the cellular hydrolysis of stavudine phosphoramidates.

We examined the effect of cellular metabolism of three alkyl-substituted amino acid ester phosphoramidate derivatives of stavudine in different cell lines. Marked cell-to-cell differences were found in both the rate of hydrolysis and chiral selectivity. This selectivity implies that different enzymes may be involved in the metabolism of these compounds depending on the cell type involved. Notably, both the methyl and ethyl substituted derivatives underwent hydrolysis in presence of various cell lines, whereas the tert-butyl substituted compound was resistant to hydrolysis implying that steric hindrance associated with this group along with electron density may play a key role in the hydrolysis profile of these compounds. Additionally we found this mimicked the hydrolysis profiles obtained for bacterial enzymes. Furthermore, our results suggest that the site of attack of the cellular enzymes is confined to the ester side chain of the molecule. This result is also consistent with our earlier observation using bacterial enzymes as well as using 'd' isomers.

Amides↗

PubMed2006

Synthesis and metabolism of naphthyl substituted phosphoramidate derivatives of stavudine.

The synthesis of naphthylphosphoramidate derivatives of stavudine was achieved using a four-step procedure. The derivatives were subjected to several different enzymes including lipase, esterase, Subtilisin Carlsberg, and Carica papaya, and their hydrolysis rates were determined. Based on the rates of hydrolysis, we were able to differentiate between the chiralities at the phosphorus center of the phosphoramidate compounds. In addition, lipase was found to distinguish between both alpha and beta forms of the compounds. The superior chiral selectivity shown by lipase toward the naphthyl substituted phosphoramidate derivatives is attributed to the restrictive binding pocket of the lipase.

Amides↗

PubMed2005

Effect of change in nucleoside structure on the activation and antiviral activity of phosphoramidate derivatives.

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.

Amides↗

PubMed2005

Zidampidine, an aryl phosphate derivative of AZT: in vivo pharmacokinetics, metabolism, toxicity, and anti-viral efficacy against hemorrhagic fever caused by Lassa virus.

The pharmacokinetics, metabolism, and toxicity of Zidampidine, an aryl phosphate derivative of AZT, 3'-azidothymidine-5'-[p-bromophenyl methoxyalaninyl phosphate] were investigated in CD-1 mice. Following iv injection, Zidampidine was rapidly converted to its metabolites Ala-AZT-MP and AZT. Zidampidine was not toxic to mice at doses up to 250mg/kg. We next examined the therapeutic effect of Zidampidine in CBA mice challenged with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or non-toxic doses of Zidampidine administered intraperitoneally 24h prior, 1h prior, and 24, 48, 72, and 96h after virus inoculation. The probability of survival following the Lassa challenge was significantly improved for Zidampidine-treated mice (Kaplan Meier, Log-Rank p value<0.0001). This pilot study provides the basis for future preclinical evaluation of Zidampidine and its potential as a new agent for the treatment of viral hemorrhagic fevers caused by Lassa virus.

Animals↗

PubMed2005

Enzymatic hydrolysis of stampidine and other stavudine phosphoramidates in the presence of mammalian proteases.

Mammalian proteases have not been implicated in the metabolism of any nucleoside phosphoramidate prodrug. The results presented herein provide unprecedented and conclusive experimental evidence that mammalian proteases are capable of hydrolyzing stavudine phosphoramidates. Specifically, cathepsin B and Proteinase K are able to metabolize stampidine and other phosphoramidate derivatives of stavudine. Additionally, cathepsin B exhibits chiral selectivity at the phosphorus center. The elucidation of the metabolic pathways leading to activation of stampidine may provide the basis for pharmacologic interventions aimed at modulating the metabolism and thereby improving the therapeutic window of stampidine as an anti-HIV agent.

Amides↗

PubMed2005

Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine.

Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.

Amides↗

PubMed2005

Stereochemical influence on lipase-mediated hydrolysis and biological activity of stampidine and other stavudine phosphoramidates.

Stampidine and other halogen substituted stavudine phosphoramidates can be activated by lipase-mediated hydrolysis. The target site for the lipase appears to be the methyl ester group of the L-alanine side chain. Accordingly, the D-amino acid substituted isomers {Rp or Sp}are resistant to lipase-mediated hydrolysis and exhibit substantially less anti-HIV activity. Molecular modeling results indicate that the L-amino acid configured isomers {Rp or Sp} are preferred in the lipase binding pocket.

Amides↗

PubMed2005

In vitro anti-HIV potency of stampidine alone and in combination with standard anti-HIV drugs.

The purpose of the present study was compare the in vitro anti-HIV potency stampidine (CAS 217178-62-6), a novel aryl phosphate derivative of stavudine (CAS 3056-17-5), and drug combinations containing stampidine to the anti-HIV tency of the standard drugs zidovudine (CAS 30516-87-1), stavudine, lamivudine (CAS 134678-17-4), nelfinavir (CAS 159989-65-8), and nevirapine (CAS 129618-40-2) as well as their combinations. Stampidine inhibited the laboratory HIV-1 strain HTLV(IIIB) (B-envelope subtype) as well as the primary clinical HIV-1 isolates BR/92/025 (C-envelope subtype) and BR/93/20 (F-envelope sub-type) with subnanomolar IC50 values. Stampidine was as effective as zidovudine against HTLV(IIIB) and BR/92/025 and 3-logs more effective than zidovudine against BR/93/20. Stampidine was more effective than stavudine, lamivudine, nelfinavir, and nevirapine against all three HIV-1 isolates. The combination of stampidine with zidovudine + lamivudine was more effective than the combination of nelfinavir or nevirapine with zidovudine lamivudine against all three HIV-1 isolates. The combination of stampidine with nelfinavir was more effective than zidovudine + lamivudine as well as the combination of zidovudine + lamivudine with nelfinavir. The combination of stampidine with lamivudine + nelfinavir was more effective than the combination of zidovudine with lamivudine + nelfinavir. The combination of stampidine with lamivudine + nevirapine was more effective than the combination of stavudine with lamivudine + nevirapine. These findings demonstrate that (a) stampidine, as well as its combinations with the standard anti-HIV drugs zidovudine, lamivudine, nelfinavir or nevirapine, are potent inhibitors of HIV-1 replication in human peripheral blood mononuclear cells, and (b) replacement of either zidcovudine, zidovudine+lamivudine or stavudine in 3-drug cocktails with stampidine resulted in greater anti-HIV potency in vitro.

Anti-HIV Agents↗

PubMed2004

Effect of stereochemistry on the anti-HIV activity of chiral thiourea compounds.

Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside inhibitors of the reverse transcriptase (RT) enzyme (NNRTI) of the human immunodeficiency virus (HIV-1). Molecular modeling studies indicated that because of the asymmetric geometry of the NNRTI binding pocket, the R stereoisomers would fit the NNRTI binding pocket of the HIV-1 RT much better than the corresponding S stereoisomers, as reflected by their 10(4)-fold lower K1 values. The R stereoisomers of several PETT derivatives inhibited recombinant RT in vitro with lower IC(50) values than their enantiomers. The active compounds were further evaluated for their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells (PBMC). All the R isomers once again showed potent anti-HIV activity and inhibited the replication of the HIV-1 strain HTLVIIIB in peripheral blood mononuclear cells (PBMC) at nanomolar concentrations whereas their enantiomers were substantially less potent. The lead compounds in the respective groups were further tested against the NNRTI-resistant HIV strains, A17 (Y181C mutant), and A17Var (Y181C+K103N mutant) and RT MDR (V106N). The results showed that the lead compounds were several logs more potent than the standard NNRTI nevirapine. Structure-activity relationship studies also revealed a preference for the pyridyl unit with halo substitutions primarily at 5-position demonstrating the importance of regiochemistry. Our data provides experimental evidence that the stereochemistry as well as regiochemistry of NNRTI can profoundly affect their anti-HIV activity.

Anti-HIV Agents↗

PubMed2004

Lipase-mediated stereoselective hydrolysis of stampidine and other phosphoramidate derivatives of stavudine.

Enzymatic hydrolysis of stampidine and other aryl phosphate derivatives of stavudine were investigated using the Candida Antarctica Type B lipase. Modeling studies and comparison of the hydrolysis rate constants revealed a chiral preference of the lipase active site for the putative S-stereoisomer. The in vitro anti-HIV activity of these compounds correlated with their susceptibility to lipase- (but not esterase-) mediated hydrolysis. We propose that stampidine undergoes rapid enzymatic hydrolysis in the presence of lipase according to the following biochemical pathway: During the first step, hydrolysis of the ester group results in the formation of carboxylic acid. Subsequent step involves an intramolecular cyclization at the phosphorous center with simultaneous elimination of the phenoxy group to form a cyclic intermediate. In the presence of water, this intermediate is converted into the active metabolite Ala-d4T-MP. We postulate that the lipase hydrolyzes the methyl ester group of the l-alanine side chain to form the cyclic intermediate in a stereoselective fashion. This hypothesis was supported by experimental data showing that chloroethyl substituted derivatives of stampidine, which possess a chloroethyl linker unit instead of a methyl ester side chain, were resistant to lipase-mediated hydrolysis, which excludes the possibility of a direct hydrolysis of stampidine at the phosphorous center. Thus, our model implies that the lipase-mediated formation of the cyclic intermediate is a key step in metabolism of stampidine and relies on the initial configuration of the stereoisomers.

Amides↗

PubMed2004

Phenyl phosphoramidate derivatives of stavudine as anti-HIV agents with potent and selective in-vitro antiviral activity against adenovirus.

Adenoviruses are responsible for a broad range of clinical diseases that may be associated with high mortality, including pneumonia, hepatitis, encephalitis, hemorrhagic cystitis, nephritis, and gastroenteritis in immunocompromised patients, including HIV-infected individuals. Here we report the identification of halo-substituted stavudine phenyl phosphoramidate derivatives as a new class of dual-function anti-HIV agents with potent and selective anti-adenovirus (ADV) activity. We examined the investigational stavudine phenyl phosphoramidate derivative stampidine and 12 structurally similar stavudine derivatives for anti-ADV activity. All 13 derivatives of stavudine, including stampidine, were substantially more potent than stavudine and inhibited ADV-induced plaque formation at nanomolar IC(50) values. Compounds with halo substitutions in the phenyl ring as well as the unsubstituted compound 607 were more potent than compounds with methoxy, methyl, or cyano substitutions. Compound 113 (stampidine) with a 4-Br substitution and compound 609 with a 4-Cl substitution were identified as the most potent lead anti-ADV agents. Compound 113/Stampidine inhibited ADV-induced plaque formation in skin fibroblasts in a concentration-dependent fashion with a mean (+/-S.E.M.) IC(50) value of 17 +/- 2 nM without any evidence of cytotoxicity even at 100 microM. Similarly, compound 609 inhibited ADV-induced plaque formation with an IC(50) value of 27 +/- 3 nM. We next sought to determine if the lead compounds 113 and 609 can also inhibit other viruses. Both compounds exhibited potent anti-HIV activity at nanomolar concentrations. However, neither compound exhibited any antiviral activity against non-HIV viruses, including Cytomegalovirus (CMV), Type I or Type II herpes simplex viruses (HSV-1, HSV-2), enterovirus ECHO 30, or respiratory syncytial virus (RSV) (IC(50) > 100 microM). The remarkable anti-ADV potency of the lead compounds stampidine and compound 609 warrants the further development of these promising new antiviral agents for possible clinical use in ADV infected patients.

Adenoviridae↗

PubMed2004

A comparative study of the hydrolysis pathways of substituted aryl phosphoramidate versus aryl thiophosphoramidate derivatives of stavudine.

A comparative study of aryl phosphoramidate and aryl thiophosphoramidate derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) was performed. The study focused on the nature of the substituents and the influence of a thiophosphoramidate in the structure of these derivatives. The rate of alkaline hydrolysis of these two types of d4T derivatives indicated that replacement of oxygen with sulfur decreases the rate of hydrolysis by twofold. Additionally, the activation energy (E(a)) for the sulfur analogs is comparatively higher than that of the oxygen analogs. Notably, an intermediate was formed in the hydrolysis reaction of the sulfur analogs of d4T that was absent in the case of the oxygen analog, and the tentative structure of the intermediate was proposed based on LC/mass spectroscopy data. Using both HPLC and (31)P-NMR techniques, we identified the hydrolysis product of the phosphoramidate derivatives and were able to show in in vitro studies that porcine liver esterase can hydrolyze the methyl ester portion of the phosphoramidate derivatives. Aryl phosphoramidate derivatives of d4T were 1000-fold more active than the corresponding aryl thiophosphoramidate derivatives, indicating that the energy of activation of hydrolysis of these phosphoramidate derivatives plays a significant role in their biological potency.

Amides↗

PubMed2004

Rational drug design of multifunctional phosphoramidate substituted nucleoside analogs.

This review focuses on our approach to the study of the effect of a series of phosphoramidate substituted nucleoside analogs on model systems for cancer, HIV and fertility. This approach allowed the development of compound WHI-07, an arylphosphoramidate derivative of zidavudine. This compound is a multifunctional agent showing potent activity in the above mentioned model systems. Our rational drug design provided such a powerful derivative with all the necessary characteristic of a drug candidate. Importantly, we have experimental evidence that each of the groups associated with the molecular frame of WHI-07 imparts the multifunctional ability for this agent. In addition, we have also suggested a possible biological pathway for WHI-07 including various products with their therapeutic targets that are formed during the course of its metabolism inside the cell. We also propose which individual moieties in the structure of WHI-07 are responsible for the biological activity from the formation of these metabolites. A detailed structure-activity relationship is presented in the review in connection with various structural modifications of the agent. Application of this active agent in animal models shows the potential usefulness of this agent as a drug candidate. We further plan to utilize gene-chip technology to identify new targets and modes of action using microarrays to measure expression changes in thousands of gene products. In conclusion, we have demonstrated the power of multifunctional drug design to discover drugs to combat various diseases. We believe this is the future direction of the drug discovery process.

Amides↗

PubMed2003

Substituted heterocyclic thiourea compounds as a new class of anti-allergic agents inhibiting IgE/Fc epsilon RI receptor mediated mast cell leukotriene release.

Mast cell derived leukotrienes (LT's) play a vital role in pathophysiology of allergy and asthma. We synthesized various analogues of indolyl, naphthyl and phenylethyl substituted halopyridyl, thiazolyl and benzothiazolyl thioureas and examined their in vitro effects on the high affinity IgE receptor/Fc epsilon RI-mediated mast cell leukotriene release. Of the 22 naphthylethyl thiourea compounds tested, there were 7 active compounds and N-[1-(1-naphthyl)ethyl]-N'-[2-(ethyl-4-acetylthiazolyl)]thiourea (17 and 16) (IC(50)=0.002 microM) and N-[1-(1R)-naphthylethyl]-N'-[2-(5-methylpyridyl)]thiourea (compound 5) (IC(50)=0.005 microM) were identified as the lead compounds. Among the 11 indolylethyl thiourea compounds tested, there were seven active compounds and the halopyridyl compounds N-[2-(3-indolylethyl)]-N'-[2-(5-chloropyridyl)]thiourea (24) and N-[2-(3-indolylethyl)]-N'-[2-(5-bromopyridyl)]thiourea (25) were the most active agents and inhibited the LTC(4) release with low micromolar IC(50) values of 4.9 and 6.1 microM, respectively. The hydroxylphenyl substituted compounds N-[2-(4-hydroxyphenyl)ethyl]-N'-[2-(5-chloropyridyl)]thiourea (37; IC(50)=12.6 microM), N-[2-(4-hydroxyphenyl)ethyl]-N'-[2-(5-bromopyridyl)]thiourea (50; IC(50)=16.8 microM) and N-[2-(4-hydroxyphenyl)ethyl]-N'-[2-(pyridyl)]thiourea (35; IC(50)=8.5 microM) were the most active pyridyl thiourea agents. Notably, the introduction of electron withdrawing or donating groups had a marked impact on the biological activity of these thiourea derivatives and the Hammett sigma values of their substituents were identified as predictors of their potency. In contrast, experimentally determined partition coefficient values did not correlate with the biological activity of the thiourea compounds which demonstrates that their liphophilicity is not an important factor controlling their mast cell inhibitory effects. These results establish the substituted halopyridyl, indolyl and naphthyl thiourea compounds as a new chemical class of anti-allergic agents inhibiting IgE receptor/Fc epsilon RI-mediated mast cell LTC(4) release. Further lead optimization efforts may provide the basis for new and effective treatment as well as prevention programs for allergic asthma in clinical settings.

Animals↗

PubMed2003

Evaluating dissolution profiles of an anti-HIV agent using ANOVA and non-linear regression models in JMP software.

A powerful statistical method was designed using JMP software to detect factors contributing to differences in the dissolution process of an antiviral drug delivered in an oral dosage form. Due to the large number of dissolution media available for solid dosage forms, a statistical method to choose the appropriate medium is critical for testing solid dosage forms. We have developed an analysis of variance model to analyze the overall dissolution profile obtained from the various media. In vitro tests were performed using a standard USP basket apparatus (Vankel Inc., Cary, NC), and the analysis used the restricted/residual maximum likelihood method (JMP software) to partition the variance due to media (pH 1.2 and 6.8, +SDS, water alone and at pH 1.2 with pepsin), time (repeated measure) and capsule (random effect). This allowed correct standard error estimates to be used to compare dissolution in different media using planned linear contrasts. The model provided us with statistically powerful criteria to identify significant differences in capsule dissolution across time and to quantify capsule-to-capsule population variance estimate. The time specific linear contrasts showed the largest sum of square values (SS) occurred at 180 min (SS=0.268) for the simulated SIF (pH 6.8) versus SGF (pH 1.2) comparison (DF=166, MSE=3.92 x 10(-3)). The dissolution processes were further characterized using a non-linear regression fit of a power law function to the data for each capsule. This resulted in a method to statistically differentiate between the dissolution processes of the capsules in different media.

Analysis of Variance↗

PubMed2003

In vivo antiretroviral activity of stampidine in chronically feline immunodeficiency virus-infected cats.

Here we report the antiretroviral activity of the experimental nucleoside reverse transcriptase inhibitor (NRTI) compound stampidine in cats chronically infected with feline immunodeficiency virus (FIV). Notably, a single oral bolus dose of 50 or 100 mg of stampidine per kg resulted in a transient >/=1-log decrease in the FIV load of circulating peripheral blood mononuclear cells in five of six FIV-infected cats and no side effects. A 4-week stampidine treatment course with twice-daily administration of hard gelatin capsules containing 25 to 100 mg of stampidine per kg was also very well tolerated by cats at cumulative dose levels as high as 8.4 g/kg and exhibited a dose-dependent antiretroviral effect. One of three cats treated at the 25-mg/kg dose level, three of three cats treated at the 50-mg/kg dose level, and three of three cats treated at the 100-mg/kg dose level (but none of three control cats treated with placebo pills) showed a therapeutic response, as evidenced by a >/=1-log reduction in the FIV load in peripheral blood mononuclear cells within 2 weeks. The previously documented in vitro and in vivo antiretroviral activity of stampidine against primary clinical human immunodeficiency virus type 1 isolates with genotypic and/or phenotypic NRTI resistance, together with its favorable animal toxicity profile, pharmacokinetics, and in vivo antiretroviral activity in FIV-infected cats, warrants further development of this promising new NRTI compound.

Animals↗

PubMed2002

Metabolism of stavudine-5'-[p-bromophenyl methoxyalaninyl phosphate], stampidine, in mice, dogs, and cats.

We examined the pharmacokinetics and metabolism of the experimental nucleoside reverse transcriptase inhibitor compound stampidine in mice, dogs, and cats. Also reported is the identification of p-bromophenyl sulfate (p-Br-Ph-S) as a major in vivo phase II metabolite of stampidine. Liver cytosol was shown to take part in the hydrolysis of stampidine to form alaninyl-STV-monophosphate (Ala-STV-MP), 2',3'-didehydro-3'-deoxythymidine (STV), and p-bromophenol; p-bromophenol was further sulfonated by sulfotransferase to form p-Br-Ph-S. Notably, plasma concentrations of stampidine >4 logs higher than its IC(50) value can be achieved in both dogs and cats after its p.o administration at a 100-mg/kg dose level. In dogs as well as cats, stampidine was metabolized to yield micromolar concentrations of the active metabolites ala-STV-MP and STV, which is similar to the metabolism of stampidine in mice. These findings encourage the further development of this new antiviral agent for possible clinical use in human immunodeficiency virus-infected patients.

Animals↗

PubMed2002

In vivo toxicity, pharmacokinetics, and anti-human immunodeficiency virus activity of stavudine-5'-(p-bromophenyl methoxyalaninyl phosphate) (stampidine) in mice.

We have evaluated the clinical potential of stavudine-5'-(p-bromophenyl methoxyalaninyl phosphate(stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, as a new anti-human immunodeficiency virus (anti-HIV) agent, by examining its acute, subacute, and chronic toxicity profile in mice as well as by testing its antiviral activity in a surrogate human peripheral blood lymphocyte (Hu-PBL)-SCID mouse model of human AIDS. STAMP was very well tolerated in BALB/c and CD-1 mice, without any detectable acute or subacute toxicity at single intraperitoneal or oral bolus doses as high as 500 mg/kg of body weight. Notably, daily administration of STAMP intraperitoneally or orally for up to 8 consecutive weeks was not associated with any detectable toxicity at cumulative dose levels as high as 6.4 g/kg. Micromolar concentrations of the active STAMP metabolite in plasma were rapidly achieved and maintained for more than 4 h after parenteral as well as oral administration of a nontoxic 100-mg/kg bolus dose of STAMP. In accordance with its favorable pharmacokinetic profile and in vitro potency, STAMP exhibited dose-dependent and potent in vivo anti-HIV activity in Hu-PBL-SCID mice against a genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor (NRTI)-resistant clinical HIV type 1 (HIV-1) isolate (BR/92/019; D67N, L214F, T215D, K219Q) at nontoxic dose levels. The remarkable in vivo safety and potency of STAMP warrants the further development of this promising new antiretroviral agent for possible clinical use in patients harboring NRTI-resistant HIV-1.

Administration, Oral↗