P-bromophenyl Methoxyalaninyl Phosphate Research Articles

Stampidine as a Potent Epigenetic Silencer of Host HIV Dependency Factor Genes in HIV-Infected Cells

Here we report that our lead anti-retroviral (ARV) drug candidate Stampidine (2,'3'-didehydro-3'-deoxythymidine- 5'-(p-bromophenyl methoxy alaninyl phosphate)) results in methylation of a network of HIV-responsive regulatory genes in T-cells, including several genes for HIV-dependency factors (HDFs). Stampidine epigenetically modulates the host transcriptome in a unique manner, silences expression of a distinct set of genes encoding transcription factors and signal transduction molecules, and prevents HIV infection from distorting and disrupting key cellular transcriptional networks. At nanomolar concentrations that are 4-logs lower than those achieved at its non-toxic dose levels in mice, rats, cats, and dogs, Stampidine switched off genes for several HDFs that are required for HIV replication in T-cells. Notably, Stampidine reversed the effects of HIV exposure on the host transcriptome regardless of NRTI-sensitivity or RT mutations of the HIV isolate used and inhibited the replication of 17 NRTI-resistant HIV- 1 strains, including recombinant HIV clones containing common patterns of RT mutations responsible for NRTI resistance, in human peripheral blood mononuclear cells (PBMC) with subnanomolar-nanomolar IC 50 values (Mean ± SEM = 12.0 ± 3 .2 nM). Unlike available ARV agents that disrupt a specific step in the life-cycle of HIV, Stampidine has the potential to abrogate all steps in the life cycle of HIV.

Antileukemic Activity and Cellular Metabolism of the Aryl Phosphate Derivative of Bromo-Methoxy Zidovudine (Compound WHI-07)

The novel aryl phosphate derivative of bromo-methoxy zidovudine (ZDV/AZT) (compound WHI-07, CAS 213982-96-8) was found to be a potent antileukemic agent against human leukemia, lymphoma, and multiple myeloma cell lines in MTT and clonogenic assays with low micromolar IC50 values. In addition, WHI-07 was antimitotic, leading to cell fusion and developmental arrest in the Zebrafish model of rapid cell proliferation. WHI-07 was cytotoxic to drug-sensitive (NALM-6, MOLT-3, HL-60, P388) and multi-drug resistant (MDR) leukemia cell lines (HL-60/VCR, HL-60/ADR, P388/ ADR). Treatment of leukemia cells with WHI-07 showed rapid and dramatic depletion of all cellular nucleoside diphosphate and triphosphate (NDP/NTP) pools, which would contribute to the overall reduction of nucleic acid synthesis and cell death. WHI-07 was rapidly metabolized to alaninyl ZDV monophosphate (Ala-ZDV-MP), the levels of which inversely correlated with cytotoxic IC50 values of WHI-07. Glutathione was found to mediate the in vitro and in vivo detoxification pathway of WHI-07 to 3'-azidothymidine-5'-p-bromophenylmethoxyalaninyl phosphate and Ala-ZDV-MP, respectively. The proposed intracellular metabolic pathway for WHI-07 involves a thiol-mediated dehalogenation step followed by the paraoxon-sensitive carboxylesterase-mediated reaction leading to the formation of Ala-ZDV-MP as the major intracellular metabolite.

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.

Stampidine is a potential nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Objective Stampidine (2,′3′-didehydro-3′-deoxythymidine-5′-( p-bromophenyl methoxy alaninyl phosphate) is a novel aryl phosphate derivative of stavudine/d4T with broad-spectrum anti-HIV activity in vitro and in vivo. This study investigated the potential utility of stampidine as a nonspermicidal microbicide. Design Prospective, controlled study. Setting Center for Advanced Preclinical Sciences and Reproductive Biology Department. Patient(s) Seven sperm donors. Animal(s) Fifty-two sexually mature, female and twenty-four male New Zealand white rabbits. Intervention(s) Human semen and genital tract epithelial cells were exposed to stampidine (up to 1 mM). Ovulated does in subgroups of 12 were artificially inseminated with rabbit semen pretreated with stampidine (1 mM) or vehicle. Does in subgroups of four and three, respectively, were exposed intravaginally to a gel or a thermoreversible ovule formulation with and without 0.5%, 1.0%, or 2.0% stampidine (9 to 36 mM) for 14 days. Main outcome measure(s) Effect of stampidine on human sperm motility, kinematics, penetration through cervical mucus, and epithelial cell viability. Reproductive parameters on gestation day 8. Vaginal tissues were histologically scored 24 hours after dosing. Result(s) Exposure of human sperm to stampidine even at a concentration 10 6-times higher than its in vitro anti-HIV-1 activity (50% inhibitory concentration = 1 nM) had no adverse effect on sperm motility, kinematics, cervical mucus penetrability, or the viability of vaginal and endocervical epithelial cells. Reproductive indices of pregnancy rate, embryo implantation, and preimplantation losses were not affected by pretreatment of rabbit semen with stampidine. Gel formulations of 0.5% to 2.0% stampidine (9 to 36 mM) lacked mucosal toxicity. Conclusion(s) The broad-spectrum anti-HIV agent stampidine had no adverse effect on sperm functions, was not cytotoxic, and did not induce mucosal toxicity. Stampidine has clinical potential as a prophylactic microbicide without contraceptive activity.

Stampidine: A potential nonspermicidal broad-spectrum anti-HIV microbicide

Objective: Stampidine (2,′3′-didehydro-3′-deoxythymidine-5′-(p-bromophenyl methoxy alaninyl phosphate) is a novel aryl phosphate derivative of stavudine/d4T with broad-spectrum anti-HIV activity in vitro and in vivo. Stampidine is 10- to 100-times more active than d4T against B envelope type as well as non-B envelope HIV-1 subtypes (A, C, F, and G). Further, stampidine is effective against genotypically and phenotypically non-nucleoside and nucleoside analogue reverse transcriptase inhibitor-resistant HIV-1 isolates at nanomolar concentrations. Because of its potent and broad-spectrum anti-HIV activity, we investigated the potential utility of stampidine as a noncontraceptive microbicide for prevention of sexual transmission of HIV via semen.

Development and evaluation of a thermoreversible ovule formulation of stampidine, a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide.

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5-2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.

In Vivo Evaluation of a Gel Formulation of Stampidine, a Novel Nonspermicidal Broad-Spectrum Anti-HIV Microbicide

IntroductionStampidine (2′,3′-didehydro-2′,3′-dideoxythymidine 5′-[p-bromophenyl methoxyalaninyl phosphate]), a novel aryl phosphate derivative of stavudine (STV/d4T), is a potent, broad-spectrum anti-HIV agent with potential as a new class of nonspermicidal microbicide. As part of an effort to develop an acceptable formulation for the clinical use of stampidine as a microbicide, we developed a vaginal gel formulation of stampidine and evaluated its potential to cause mucosal toxicity in the New Zealand white rabbit vaginal irritation model.MethodsStampidine was solubilized in a gel formulation composed of microcrystalline cellulose, xanthan gum, macrogol (polyethylene glycol [PEG]) 400, sorbitol, and polysorbate 80. Rabbits in groups of four were exposed intravaginally to a gel with 0.5, 1, or 2% stampidine or no active drug for 14 consecutive days. The rabbits were euthanized on day 15 and their vaginal tissues were evaluated for histologic evidence of mucosal toxicity and immunohistochemical evidence of cellular inflammation or hyperplasia. Additionally, rabbits in groups of three were exposed intravaginally to a 2% stampidine gel, and plasma samples collected at various timepoints were assayed for stampidine and its major metabolites, alaninyl-stavudine-monophosphate and stavudine by analytical high performance liquid chromatography (HPLC).ResultsWith application of 0.5–2.0% stampidine, only minimal-to-mild vaginal irritation was observed, which is within the acceptable range for clinical trials (total score <4 out of a possible 16). Repeated intravaginal treatment with stampidine gel caused no inflammation or hyperplasia in the vaginal epithelium and submucosa. Stampidine and its major metabolites were not detectable in the plasma of rabbits given 2% stampidine-containing gel intravaginally.ConclusionsThe favorable toxicity profile of intravaginally administered stampidine-containing gel may provide the foundation for its clinical development as a safe and effective broad-spectrum anti-HIV microbicide without contraceptive properties.

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.

Stampidine is a potent inhibitor of Zidovudine- and nucleoside analog reverse transcriptase inhibitor-resistant primary clinical human immunodeficiency virus type 1 isolates with thymidine analog mutations.

We report the antiretroviral activity of stavudine-5'-(p-bromophenyl methoxyalaninyl phosphate) (stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, against primary clinical human immunodeficiency virus type 1 (HIV-1) isolates. STAMP inhibited each one of nine clinical HIV-1 isolates of non-B envelope subtype and 20 genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor-resistant HIV-1 isolates at subnanomolar to low-nanomolar concentrations.

AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) as a potent and non-toxic anti-human immunodeficiency virus agent.

The potency and selectivity index of the AZT-phenyl phosphate derivatives in thymidine kinase (TK)-deficient T cells were substantially enhanced by introducing a single para-bromo substitutent in the phenyl moiety. AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) was 43-fold more potent than AZT-5'-(phenyl methoxyalaninyl phosphate) and was fivefold more potent than AZT in inhibiting human immunodeficiency virus (HIV) replication in TK-deficient CEM cells.

Synthesis of dual function (5R,6R)- and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-5'-(para-bromophenyl methoxyalaninyl phosphate) as novel spermicidal and anti-HIV agents.

We synthesized a novel compound, 5-bromo-6-methoxy-5,6-dihydro-AZT-5'- (p-bromophenyl methoxyalaninyl phosphate), which had an EC50 value of 5 microM in sperm motility assays. This is > 1 log10 better than that of the detergent spermicide nonoxynol-9 (EC50 81 microM). The compound also displayed a potent anti-human immunodeficiency virus (HIV) activity with an IC50 value of 0.005 microM in HIV replication assays, which was virtually identical to that of AZT (IC50 0.006 microM) and > 2 log10 more potent than that of nonoxynol-9 (IC50 2.2 microM). The promising results reported herein recommend the further development of the dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk of acquiring HIV by heterosexual transmission. These dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.

Enhancing effects of a mono-bromo substitution at the para position of the phenyl moiety on the metabolism and anti-HIV activity of D4T-phenyl methoxyalaninyl phosphate derivatives

d4T-5′-[ p-Bromophenyl methoxyalaninyl phosphate] (d4T-pBPMAP), a novel phenyl phosphate derivative of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) that has an enhanced ability to undergo hydrolysis due to the electron withdrawing properties of its single bromo substituent at the para-position of the phenyl moiety, was found to yield substantially more of the key metabolite alaninyl d4T monophosphate (A-d4T-MP) than the unsubstituted d4T-5′-phenyl methoxyalaninyl phosphate or para-methoxy substituted d4T-5′-phenyl methoxyalaninyl phosphate. d4T-pBPMAP was tested for its anti-HIV-1 activity in peripheral blood mononuclear cells (PBMNC) and thymidine kinase (TK)-deficient CEM T-cells. d4T-pBPMAP was 12.6-fold more potent than the parent compound d4T in inhibiting p24 production (IC 50 values: 44 nM vs 556 nM) and 41.3-fold more potent than d4T in inhibiting the reverse transcriptase (RT) activity (IC 50 values: 57 nM vs 2355 nM) in HIV-1-infected TK-deficient CEM cells. Similarly, d4T-pBPMAP was more potent than the unsubstituted or para-methoxy substituted phenyl methoxyalaninyl phosphate derivatives of d4T. d4T-pBPMAP did not exhibit any detectable cytotoxicity to PBMNC or CEM cells at concentrations as high as 10,000 nM. Notably, d4T-pBPMAP was capable of inhibiting the replication of a zidovudine (ZDV/AZT)-resistant HIV-1 strain as well as HIV-2 in PBMNC at nanomolar concentrations. To our knowledge, this is the first demonstration that the potency of the d4T-aryl-phosphate derivatives can be substantially enhanced by introducing a single para-bromo substituent in the aryl moiety.