A 13-week subchronic intravaginal toxicity study of the novel broad-spectrum anti-HIV and spermicidal agent, N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (PHI-346) in mice.

The nonnucleoside inhibitor (NNI) of HIV-1 reverse transcriptase, PHI-346 (N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea), is a dual-function spermicidal agent with potent anti-HIV activity against drug-sensitive as well as drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. PHI-346 was formulated via a lipophilic gel-microemulsion for intravaginal use as a potential dual-function microbicide. To evaluate the toxicity potential of short-term intravaginal exposure to PHI-346, groups of 15 female B6C3F1 and CD-1 mice were exposed intravaginally to a gel-microemulsion containing 0, 0.

Pre-clinical safety evaluation of novel nucleoside analogue-based dual-function microbicides (WHI-05 and WHI-07).

Compounds WHI-05 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl)-methoxyalaninyl phosphate] and WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate] are aryl phosphate derivatives of zidovudine (ZDV) with anti-HIV and contraceptive activity. WHI-05 and WHI-07 differ fundamentally from currently used surfactant microbicides that are cytotoxic to genital tract epithelial cells at spermicidal concentrations. These drugs were rationally designed to bypass the thymidine kinase dependency of ZDV activation in genital tract secretions, as well as to achieve spermicidal activity.

Metvan: a novel oxovanadium(IV) complex with broad spectrum anticancer activity.

Among the 25 bis(cyclopentadienyl)vanadium(IV) and 14 oxovanadium(IV) compounds synthesised and evaluated for anticancer activity, bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (metvan) was identified as the most promising multitargeted anticancer vanadium complex with apoptosis-inducing activity. At nanomolar and low micromolar concentrations, metvan induces apoptosis in human leukaemia cells, multiple myeloma cells and solid tumour cells derived from breast cancer, glioblastoma, ovarian, prostate and testicular cancer patients. It is highly effective against cisplatin-resistant ovarian cancer and testicular cancer cell lines.

Effects of aryl substituents on the anti-HIV activity of the arylphosphoramidate derivatives of stavudine.

We compared the anti-HIV activity of 13 phenyl phosphate derivatives of stavudine (2',3'-didehydro-2',3'-dideoxythymidine/d4T) by examining their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells. Our results show that the introduction of electron-withdrawing substituents enhances the activity of these phosphoramidate derivatives. The rate of chemical hydrolysis under alkaline conditions (but not the lipophilicity) predicted the potency of the compounds.

Structural requirements for potent anti-human immunodeficiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase.

The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs.

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.

Genomic studies of the spleen protein tyrosine kinase locus reveal a complex promoter structure and several genetic variants.

Here we show that the gene of the cytoplasmic tyrosine kinase SYK spans a region of 90kb with 13 coding exons, an alternative exon 14 and at least two 5' untranslated regions exons 1a and 1b. 5' RACE (Rapid amplification of cDNA ends) of human Syk cDNAs demonstrated a complex promoter usage and splicing pattern. We identified three common single nucleotide polymorphisms in the exon la promoter region of the Syk gene as well as a variant Syk cDNA haplotype.

Role of the leukemia-associated transcription factor STAT3 in platelet physiology.

Actinomycin D, a transcriptional inhibitor, was found to inhibit platelet potentiation by thrombopoietin (TPO), suggesting that TPO stimulation of platelets involves mitochondrial transcription. We sought to determine a possible role for leukemia-associated signal transducers and activators of transcription (STAT) proteins as mitochondrial transcription factors, focusing specifically on STAT3 in human platelets. We found TPO stimulation of platelets activated STAT3 in vitro, that STAT3 was present in platelet mitochondrial-rich fractions as determined by Western Blot analysis and was capable of binding to the regulatory D-loop region of human mitochondrial DNA upon activation.

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.

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.

Two-year toxicity and carcinogenicity studies in B(6)C(3)F(1) mice with 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), a novel anti-HIV and contraceptive agent.

The zidovudine derivative, 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxy alaninyl phosphate (WHI-07), is a dual-function spermicidal and anti-HIV agent with contraceptive and microbicidal activity. In previous two subchronic toxicity studies, intravaginal application of 0.5-2.

Structure-based design of novel anticancer agents.

Recently identified agents that interact with cytoskeletal elements such as tubulin include synthetic spiroketal pyrans (SPIKET) and monotetrahydrofuran compounds (COBRA compounds). SPIKET compounds target the spongistatin binding site of beta-tubulin and COBRA compounds target a unique binding cavity on alpha-tubulin. At nanomolar concentrations, the SPIKET compound SPIKET-P causes tubulin depolymerization and exhibits potent cytotoxic activity against cancer cells.

Expression of a unique helios isoform in human leukemia cells.

The purpose of the present study was to characterize the human Helios gene products expressed in leukemia cells. A 3.5 kb human Helios cDNA clone was isolated from a human T-cell cDNA library derived from the human T-acute lymphoblastic leukemia (ALL) cell line JURKAT.

A dual function anti-leukemic agent with anti-thrombotic activity.

Here we report that treatment with the anti-leukemic compound, LFM-A13 resulted in SYK kinase activation and caused distinct shape changes in platelets. Also provided is electron microscopic evidence that similar shape changes are observed in platelets from XID mice. We propose that LFM-A13 induces a conformational change in the PH domain of BTK and causes BTK to associate with PIP2 which effects actin bundling and shape change.

Janus kinase 3 inhibitor WHI-P131/JANEX-1 prevents graft-versus-host disease but spares the graft-versus-leukemia function of the bone marrow allografts in a murine bone marrow transplantation model.

The purpose of the present study was to evaluate the effects of graft-versus-host disease (GVHD) prophylaxis with the Janus kinase 3 (JAK3) inhibitor WHI-P131/JANEX-1 on the graft-versus-leukemic (GVL) function of marrow allografts in mice undergoing bone marrow transplantation (BMT) after being challenged with an otherwise invariably fatal dose of BCL-1 leukemia cells. GVHD prophylaxis using WHI-P131 markedly improved the survival outcome after BMT. The probability of survival at 30 days after BMT was 11% +/- 6% for vehicle-treated recipients (median survival time, 25 days) versus 63% +/- 12% for recipients treated with WHI-P131 (median survival time, 36 days; P <.

In vivo pharmacokinetic features, toxicity profile, and chemosensitizing activity of alpha-cyano-beta-hydroxy-beta- methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase.

The purpose of the present study was to examine the in vivo pharmacokinetics and activity of alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase (BTK). We have applied an analytical high-performance liquid chromatography method for the quantitative detection of LFM-A13 in plasma samples. Our findings indicate that LFM-A13 is quickly absorbed, with the time required to reach the maximum plasma drug concentration (t(max)) being 10-18 min after i.

Regulation of mast cell-mediated innate immunity during early response to bacterial infection.

Although the area of research on the role of MCs in innate immunity is relatively new, a number of studies that are reviewed here provide substantial evidence that MCs play a critical role in host immune defense against gram-negative bacteria. The studies show that mast cells have the ability to recognize and engulf bacteria and they release a number of inflammatory mediators including interleukin (IL)-4, IL-6, IL-10, TNF alpha, and leukotrienes in response to bacterial challenge. MC-derived TNF alpha and leukotrienes are shown to be important for bacterial clearance and early recruitment of phagocytic help at the site of infection.

Human sperm immobilizing activity of aminophenyl arsenic acid and its N-substituted quinazoline, pyrimidine, and purine derivatives: protective effect of glutathione.

We examined the potential toxicity of pentavalent organic arsenicals for human sperm. We used computer-assisted sperm analysis to examine the effects of three aminophenyl arsenicals and their nine N-substituted quinazoline, pyrimidine, and purine derivatives on human sperm motility and kinematics in human semen and medium. Among the arsenicals examined, (aminophenylazo)-phenyl arsonic acid and its N-substituted pyrimidine derivative PHI-370 (2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine) exhibited rapid sperm immobilizing activity in medium with EC(50) values of 77 and 82 microM, respectively, and t(1/2) of < 3 min.

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors.

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid-liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity.

Stereochemistry as a major determinant of the anti-HIV activity of chiral naphthyl thiourea compounds.

Eleven chiral naphthyl thiourea (CNT) compounds were synthesized as non-nucleoside inhibitors (NNI) of the reverse transcriptase (RT) enzyme of HIV-1. Molecular modelling studies indicated that, because of the asymmetric geometry of the NNI binding pocket, the 'R' stereoisomers would fit the NNI binding pocket of the HIV-1 RT much better than the corresponding 'S' stereoisomers, as reflected by their 10(4)-fold lower Ki values. The 'R' stereoisomers of all 11 compounds inhibited the recombinant RT in vitro with lower IC50 values than their enantiomers.

Role of STAT6 in IgE receptor/FcepsilonRI-mediated late phase allergic responses of mast cells.

In this study we show that IgE receptor engagement triggers activation of STAT6 in mast cells. We sought to determine the role of STAT6 activation in IgE receptor-mediated mast cell responses using STAT6 knockout mice. After IgE receptor engagement, bone marrow mast cells from STAT6(-/-) mice exhibited normal histamine and leukotriene C(4) release, but their cytokine release was markedly reduced.

Subchronic (13-week) toxicity studies of intravaginal administration of spermicidal vanadocene acetylacetonato monotriflate in mice.

Bis-cyclopentadienyl complexes of vanadium(IV) or vanadocenes are rapid and potent inhibitors of human sperm motility with potential as a new class of contraceptive agents. In this study, groups of 10 B(6)C(3)F(1) and 20 CD-1 female mice were exposed intravaginally to a gel-microemulsion containing 0, 0.06, 0.

2,4,6-Trihydroxy-alpha-p-methoxyphenylacetophenone (Compound D-58) is a potent inhibitor of allergic reactions.

The authors investigated the effects of 2,4,6-trihydroxy-alpha-p-methoxyphenylacetophenone (compound D-58), a potent inhibitor of protein tyrosine kinases SYK and Bruton's tyrosine kinase (BTK), on IgE receptor/FcepsilonRI-triggered mast cell-mediated acute allergic responses in vitro and in vivo. Compound D-58 abrogated IgE receptor/FcepsilonRI-mediated SYK and BTK activation as well as calcium mobilization in mast cells. Mast-cell degranulation and leukotriene (LT) C(4) release was inhibited by compound D-58 in a concentration-dependent fashion.

Subchronic (13-week) toxicity studies of intravaginal administration of spermicidal vanadocene dithiocarbamate in mice.

Spermicidal organometallic complexes of vanadium(IV) with bis(cyclopentadienyl) rings or vanadocenes are a new class of experimental contraceptive agents. In a systematic search for vanadocenes with selective spermicidal activity, we identified vanadocene dithiocarbamate (VDDTC) as the most potent and stable spermicidal compound. In this study, groups of 10 B(6)C(3)F(1) and 20 female CD-1 mice were exposed intravaginally to a gel-microemulsion containing 0, 0.

Gel-microemulsions as vaginal spermicides and intravaginal drug delivery vehicles.

There is a need for novel formulations to improve the bioavailability through the vaginal/rectal mucosa of microbicidal drug substances against sexually transmitted diseases. In addition, there is a need for more effective and less toxic vaginal spermicides. Here we review our recent discovery of novel gel-microemulsions (GM) as nontoxic, dual-function intravaginal spermicides, which can be used as delivery vehicles for lipophilic drug substances targeting sexually transmitted pathogens.