Barbara Waurzyniak

Role of a JAK3-dependent Biochemical Signaling Pathway in Platelet Activation and Aggregation

Here we provide experimental evidence that identifies JAK3 as one of the regulators of platelet function. Treatment of platelets with thrombin induced tyrosine phosphorylation of the JAK3 target substrates STAT1 and STAT3. Platelets from JAK3-deficient mice displayed a decrease in tyrosine phosphorylation of STAT1 and STAT3. In accordance with these data, pretreatment of human platelets with the JAK3 inhibitor WHI-P131 markedly decreased the base-line enzymatic activity of constitutively active JAK3 and abolished the thrombin-induced tyrosine phosphorylation of STAT1 and STAT3. Following thrombin stimulation, WHI-P131-treated platelets did not undergo shape changes indicative of activation such as pseudopod formation. WHI-P131 inhibited thrombin-induced degranulation/serotonin release as well as platelet aggregation. Highly effective platelet inhibitory plasma concentrations of WHI-P131 were achieved in mice without toxicity. WHI-P131 prolonged the bleeding time of mice in a dose-dependent manner and improved event-free survival in a mouse model of thromboplastin-induced generalized and invariably fatal thromboembolism. To our knowledge, WHI-P131 is the first anti-thrombotic agent that prevents platelet aggregation by inhibiting JAK3.

In Vivo Antiretroviral Activity of Stampidine in Chronically Feline Immunodeficiency Virus-Infected Cats

ABSTRACT 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.

In Vivo Toxicity, Pharmacokinetics, and Anti-Human Immunodeficiency Virus Activity of Stavudine-5′-(p-Bromophenyl Methoxyalaninyl Phosphate) (Stampidine) in Mice

ABSTRACT 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.

Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats

ABSTRACT WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model. Genital transmission of FIV was monitored in recipient cats by the appearance of viral antibodies to FIV Gag proteins and by virus isolation of blood leukocytes as measured by FIV reverse transcriptase activity and FIV-specific PCR. Microbicidal activity was considered effective when the treated cats did not show evidence of FIV infection for up to 18 weeks postchallenge. An aggregate analysis of 46 specific-pathogen-free cats revealed that a single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina (100%) or rectum (66%). Pretreatment of the vagina or rectum with 2% WHI-07 alone or in combination with 0.25% VDDTC significantly (P = 0.004) protected cats from genital transmission by the highly infectious inoculum (7 million FIVBangston-infected feline T cells). Collectively, using the vaginal and rectal transmucosal model for FAIDS, our studies demonstrated that WHI-07 either alone or in combination with a vanadocene has clinical potential for the development of a dual-function anti-HIV microbicide for sexually active women.

Prognostic Significance of T-Lineage Leukemic Cell Growth in SCID Mice: A Children's Cancer Group Study

Contemporary intensive therapies are effective for the majority of pediatric T-lineage acute lymphoblastic leukemia (ALL) patients, thus current challenge is to identify patients who may benefit from alternative treatment modalities. Previously, we demonstrated that human leukemic cell growth in the severe combined immunodeficiency (SCID) mouse was a significant prognostic factor for very high risk B-lineage ALL patients. In the current report we show that primary leukemic cells from 24 of 88 (27%) T-lineage ALL patients (SCID+) caused histopathologically detectable leukemia in SCID mice. These SCID+ patients were similar to SCID- (n = 64) patients with respect to virtually all presenting features, including age, sex, race, and leukocyte count. Growth of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of T-lineage ALL patients. Two-year event-free survival (EFS) outcomes for SCID+ patient and SCID- patients were 76.2% (SD = 5.6%) and a 64.0% (SD = 10.4%; p = 0.20). Overall survival also was similar between the two groups (p = 0.36). Among the subset of patients with M1 or M2 marrow status by day 7 of induction chemotherapy (rapid early responders), those who were SCID+ had poorer outcomes than those who were SCID-, with a 2-year EFS of 68.4% (SD = 11.9%) vs. 85.7% (SD = 6.0%) and relative hazard rate of 3.06 (p = 0.06). These data suggest that leukemic cell growth in SCID mice may identify a subset of T-lineage ALL patients who are at higher risk for relapse despite achieving a rapid early response to induction chemotherapy.

Evaluation of subchronic (13-week) and reproductive toxicity potential of intravaginal gel–microemulsion formulation of a dual-function phenyl phosphate derivative of bromo-methoxy zidovudine (compound whi-07) in B6C3F1 mice

Heterosexual transmission of human immunodeficiency virus (HIV) accounts for 90% of all new infections worldwide and significantly contributes to new acquired immunodeficiency syndrome (AIDS) cases in the USA. In a systematic effort to develop a microbicidal contraceptive capable of preventing HIV transmission as well as providing fertility control, we previously identified novel phenyl phosphate derivatives of 3′‐azido‐3′‐deoxythymidine (zidovudine) that exhibit potent anti‐HIV and spermicidal activities. This study reports the preclinical studies of our lead compound WHI‐07, 5‐bromo‐6‐methoxy‐5,6‐dihydro‐3′‐azidothymidine‐5′‐(p‐bromophenyl) methoxyalaninyl phosphate, for use as a dual‐function topical microbicide. In vivo toxicity studies in non‐human primates and rodents given WHI‐07 (20 mg kg−1) intravenously and intraperitonealy, respectively, had no detectable adverse effects on hematological and clinical chemistry profiles. The 13‐week subchronic and reproductive toxicity potential of an intravaginal gel–microemulsion formulation of WHI‐07 was studied in mice to support its further development as a dual‐function microbicide. Groups of ten female B6C3F1 mice were exposed intravaginally to a gel–microemulsion formulation containing 0, 0.5, 1.0 or 2.0% WHI‐07, 5 days a week, for 13 consecutive weeks. On a molar basis, these concentrations represent 1400–5700 times their in vitro spermicidal potency EC50) and 1.4 × 106–5.7 × 106 times their in vitro anti‐HIV activity50). After 13 weeks of intravaginal treatment, half of the treated mice were evaluated for toxicity and the other half were mated with untreated males to evaluate potential reproductive and developmental effects. The endpoints that were evaluated included survival, body weight gain, hematological and clinical chemistries, absolute and relative organ weights and histopathology. The WHI‐07 applications did not cause weight loss, morbidity, mortality or specific tissue lesions detectable by histopathology. Repeated intravaginal exposure of mice to WHI‐05 for 13 weeks had no adverse effects on subsequent reproductive performance (100% fertile), neonatal survival (>95%) or pup development. These findings collectively show that the experimental dual‐function anti‐HIV and contraceptive agent WHI‐07 did not cause significant acute or subchronic toxicity. Copyright

Contraceptive efficacy and safety studies of a novel microemulsion-based lipophilic vaginal spermicide

OBJECTIVE To investigate the in vivo contraceptive potency and safety of a novel microemulsion-based lipophilic vaginal spermicide. DESIGN In vitro and in vivo spermicidal activity and safety of a submicron-particle-size, lipophilic gel-microemulsion (GM-4). SETTING Center for Advanced Preclinical Sciences at the Parker Hughes Institute. PATIENT(S) Nine male volunteer sperm donors. INTERVENTION(S) Motile human sperm in semen and medium were exposed to eight GM-4 components or GM-4 formulation. Forty-eight ovulated NZW rabbits in subgroups of 16 with or without intravaginal administration of GM-4 or nonoxynol-9 gel (N-9; Gynol II) were artificially inseminated and allowed to complete pregnancy. Eleven rabbits were exposed to daily intravaginal application of GM-4 with and without N-9 for 10 consecutive days. Ten of 20 B(6)C(3)F(1) mice were given repetitive intravaginal application of GM-4 for 5 days/week over 13 consecutive weeks. MAIN OUTCOME MEASURE(S) The motility of human sperm treated with GM-4 components and GM-4. Term pregnancy in rabbits and histopathological grading of rabbit vaginal tissue for irritation. Evaluation of mice for survival, growth, hematologic parameters, blood-chemistry profiles, absolute and relative organ weights, and histopathology. RESULT(S) The individual components of GM-4 lacked spermicidal activity in human semen, whereas the GM-4 formulation containing all the eight pharmacological excipients exhibited potent spermicidal activity with rapid kinetics. GM-4 showed remarkable contraceptive activity in the rigorous rabbit model. None of the 16 (0%) rabbits given GM-4 intravaginally before artificial insemination became pregnant. By contrast, 15 of 16 (93.7%) control rabbits and 5 of 16 (31.2%) Gynol II-treated rabbits became pregnant and delivered newborns. Thus, GM-4 was a significantly more effective contraceptive than a commercially available N-9 gel [100% vs. 68.7% protection; P< 0.05, Fishers exact test]. Unlike the rabbits treated with N-9, none of the rabbits that were given GM-4 intravaginally for 10 consecutive days developed epithelial ulceration, edema, leukocyte influx, or vascular congestion characteristic of inflammation. Furthermore, repeated intravaginal application of GM-4 for up to 13 weeks in mice had no adverse effects on survival, growth, metabolism, or organ function. CONCLUSION We conclude that the novel spermicidal GM-4 formulation is safe and significantly more effective than N-9 in preventing conception.

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, 0.06, 0.12, and 0.25% VDDTC 5 days per week for 13 consecutive weeks. The doses of VDDTC used were nearly 1250- to 5000-fold higher than its in vitro spermicidal EC(50) value. After 13 weeks of intravaginal treatment, B(6)C(3)F(1) mice were evaluated for survival, body weight gain, absolute and relative organ weights, and systemic toxicity. Blood was analyzed for hematologic and clinical chemistry parameters. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Vanadium content in tissues was determined by atomic absorption spectroscopy. Placebo control and VDDTC-dosed female CD-1 mice were mated with untreated males to evaluate whether VDDTC has any deleterious effects on the reproductive performance. There were no treatment-related effects on survival and mean body weight and mean body weight gain during the dosing period. The blood chemistry or hemogram profiles did not reveal any toxicologically significant changes that could be attributed to VDDTC treatment. No clinically significant changes in absolute and relative organ weights were noted in VDDTC dose groups. Extensive histopathological examination of tissues revealed no treatment-related abnormalities in any of the three VDDTC dose groups. The vanadium content of all mouse tissue analyzed was <1 microg/g. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of VDDTC for 13 weeks had no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%), or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of VDDTC to yield effective spermicidal concentrations (<0.1%) in the vagina was not associated with systemic toxicity and did not adversely affect the reproductive performance in mice. VDDTC may have clinical utility as an active ingredient of non-detergent type, safe, vaginal spermicidal contraceptives.

In Vivo., Toxicity and Pharmacokinetic Features of B43(Anti-CD19)-Genistein Immunoconjugate

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase inhibitory isoflavone to the membrane-associated anti-apoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-Genistein as well as unconjugated genistein were evaluated in mice. B43-Genistein and genistein were administered either as single bolus injections or daily injections for 10 consecutive days via the intraperitoneal route to mice. Genistein was not toxic to mice at the highest dose of 40 mg/kg and no test article-related histopathological lesions were found in any of the 64 genistein-treated mice. B43-Genistein had a significantly longer elimination half-life and slower plasma and tissue clearance than unconjugated genistein. B43-Genistein was not toxic to mice at the highest single dose of 40 mg/kg or highest cumulative dose of 100 mg/kg and no test article-related histopathological lesions were found in any of the 108 mice treated with B43-genistein. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate.

Structure-based design and engineering of a nontoxic recombinant pokeweed antiviral protein with potent anti-human immunodeficiency virus activity.

ABSTRACT A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102(151AA152) and FLP-105(191AA192) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents.