Biomedical subjects

F M Uckun

Publications and source records attributed to F M Uckun.

At least 127 records · Page 7Linked to original sources

PubMed1999

Pokeweed antiviral protein isoforms PAP-I, PAP-II, and PAP-III depurinate RNA of human immunodeficiency virus (HIV)-1.

Pokeweed antiviral protein (PAP) is a naturally occurring broad-spectrum antiviral agent with potent anti-human immunodeficiency virus (HIV)-1 activity by an as yet undeciphered molecular mechanism. In the present study, we sought to determine if PAP is capable of recognizing and depurinating viral RNA. Depurination of viral RNA was monitored by directly measuring the amount of the adenine base released from the viral RNA species using quantitative high-performance liquid chromatography. Our findings presented herein provide direct evidence that three different PAP isoforms from Phytolacca americana (PAP-I from spring leaves, PAP-II from early summer leaves, and PAP-III from late summer leaves) cause concentration-dependent depurination of genomic RNA (63 to 400 pmols of adenine released per micrograms of RNA) purified from human immunodeficiency virus type-I (HIV-I), plant virus (tobacco mosaic virus (TMV), and bacteriophage (MS 2). In contrast to the three PAP isoforms, ricin A chain (RTA) failed to cause detectable depurination of viral RNA even at 5 microM, although it was as effective as PAP in inhibiting protein synthesis in cell-free translation assays. PAP-I, PAP-II, and PAP-III (but not RTA) inhibited the replication of HIV-1 in human peripheral blood mononuclear cells with IC(50) values of 17 nM, 25 nM, and 16 nM, respectively. These findings indicate that the highly conserved active site residues responsible for the depurination of rRNA by PAP or RTA are not sufficient for the recognition and depurination of viral RNA. Our study prompts the hypothesis that the potent antiviral activity of PAP may in part be due to its unique ability to extensively depurinate viral RNA, including HIV-1 RNA.

Anti-HIV Agents↗

PubMed1999

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.

Adenosine Triphosphate↗

PubMed1999

Structural and functional characterization of seven spermicidal vanadium(IV) complexes: potentiation of activity by methyl substitution on the cyclopentadienyl rings.

In a systematic effort to identify and develop effective vanadocene(IV) complexes as a new class of contraceptive agents, the effect of methyl substitution in the cyclopentadienyl rings of Cpx2VCl2 on their spermicidal activity has been examined. The spermicidal activities of compounds Cpx2VCl2 [Cpx = Me5Cp (Cp*) (1), Cp (3), MeCp (Cp') (5)], as well as two of their corresponding vanadium(V) oxidation products Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), were examined by computer-assisted sperm analysis (CASA). These analyses have established that penta-substitution of the Cp ring by electron-donating methyl groups augments the spermicidal activity 10-fold. The corresponding V(V) oxo compounds, Cp*V(O)Cl2 (2) and CpV(O)Cl2 (4), tested under identical conditions did not show as effective spermicidal activity even though these complexes have a pseudo-tetrahedral geometry similar to the active vanadocene(IV) dichlorides. Two pseudo-octahedral V(IV) complexes with tris-pyrazolyl borate as ligand, (HBpz3)V(O)Cl.DMF (6) and (HB(3,5-Me2pz)3)V(O)Cl.DMF (7), were also found to exhibit potent spermicidal activity. Although some vanadium(IV) complexes may immobilize sperm due to the generation of .OH radicals, the lack of spermicidal activity of VOSO4 which generates .OH radicals, and the potent spermicidal activity of [Cp2V(acac)][O3SCF3] (8), and [Cp2V(DeDtc)][O3SCF3] (9) which do not generate .OH radicals, indicate that .OH radical mediated reactions may not be essential for the spermicidal activity of vanadium(IV) complexes.

Electron Spin Resonance Spectroscopy↗

PubMed1999

Rational design of N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-236) as a potent non-nucleoside inhibitor of drug-resistant human immunodeficiency virus.

The novel thiourea compound N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thi ourea (HI-236) targeting the non-nucleoside inhibitor (NNI) binding pocket of HIV-1 reverse transcriptase (RT) was rationally designed using a computer model of the NNI binding pocket. The NNI binding pocket model takes into consideration changes in binding pocket size, shape, and changes in residue character that result from clinically-observed NNI resistance-associated mutations of HIV RT. RT assays revealed that HI-236 was not only more potent than trovirdine, MKC-442, and AZT against the drug-sensitive HIV-1 strain HTLV(IIIB), it was also 50-100 times more effective than delavirdine or nevirapine and twice as effective as our recently reported lead compound N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240) against the NNI-resistant Y181C mutant HIV-1 strain A17. Most importantly, HI-236 was highly effective against the multidrug-resistant HIV-1 strain RT-MDR with multiple mutations involving the RT residues 74V, 41L, 106A, and 215Y. The activity of HI-236 against RT-MDR was superior to that of other anti-HIV agents tested, which are listed in the following order: HI-236 (IC50: 5 nM) > HI-240 (IC50: 6 nM) > trovirdine (IC50: 20 nM) > AZT (IC50: 150 nM) > MKC-442 (IC50: 300 nM) > delavirdine (IC50: 400 nM) > nevirapine (IC50: 5 microM).

Anti-HIV Agents↗

PubMed1999

Genistein is neuroprotective in murine models of familial amyotrophic lateral sclerosis and stroke.

Amyotrophic lateral sclerosis (ALS), whether sporadic or familial (FALS), is a progressive, fatal neurodegenerative disorder involving the motor neurons of the cortex, brain stem, and spinal cord. In some studies, the male/female ratio of ALS patients was as high as 2 to 1. In FALS mice, disease onset and mortality were earlier among males than among females. This sexual dimorphism was due to estrogen, as treatment with genistein, a phytoestrogen, eliminated the observed sexual dimorphism in FALS mice. Genistein treatment also protected against oxygen singlet-induced cerebral damage in vivo. However, sexual dimorphism was not observed in this model of stroke; and genistein was equally effective in males and females. These data suggest that genistein has both estrogen-dependent and estrogen-independent neuroprotective activities and it should be investigated as a prophylactic agent against pathologic conditions such as ALS and stroke.

Amyotrophic Lateral Sclerosis↗

PubMed1999

Quantitative high-performance liquid chromatographic method for pharmacokinetic studies of the potent mast cell inhibitor 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131).

The novel quinazoline derivative 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131) has recently been identified as a potent mast cell inhibitor capable of preventing IgE/antigen induced cutaneous as well as systemic fatal anaphylaxis in mice. Here we describe a sensitive high-performance liquid chromatography (HPLC)-based quantitative detection method for measurement of WHI-P131 levels in plasma as well as in target mast cells. The average extraction recovery for WHI-P131 was 88.4% for plasma and 75.7% for RBL-2H3 mast cell lysates. Good linearity (r>0.999) was observed throughout the concentration range of 0.1-20 microM in plasma and 0.01-5 nmol in 5 x 10(6) cells (0.5-238 microM per cell) for WHI-P131. Intra- and inter-assay variabilities were <7% and the lowest detection limit of WHI-P131 was 0.05 microM in plasma and 0.005 nmol in 5 million cells, respectively, at a signal-to-noise ratio of approximately 2. The practical utility of this new HPLC method was confirmed in a pilot pharmacokinetic study in BALB/c mice as well as in a cellular drug uptake and disposition study in RBL-2H3 mast cells. After intraperitoneal administration of a non-toxic 40 mg/kg bolus dose of WHI-P131, the estimated maximum plasma concentration was 92.7 microM, which is approximately 1-log higher than the effective in vitro mast cell inhibitory concentrations of WHI-P131. The drug absorption was rapid with an absorption half-life of only 2.9 min and the estimated time to reach the maximum plasma concentration was 8.3 min. WHI-P131 was cleared with an apparent systemic clearance rate of 2586 ml/h/kg and an elimination half-life of 1.8 h. An intracellular exposure level (AUC) of 55 microM x h was obtained after in vitro treatment of RBL-2H3 mast cells with WHI-P131 at a 33.6 microM final concentration in culture medium. The availability of the described quantitative HPLC detection method for WHI-P131 provides the basis for further development of WHI-P131 as an anti-allergic drug through detailed pharmacodynamic studies in preclinical animal models.

Animals↗

PubMed1999

Genetic and biochemical evidence for a critical role of Janus kinase (JAK)-3 in mast cell-mediated type I hypersensitivity reactions.

We investigated the role of JAK3 in IgE receptor/FcepsilonRI-mediated mast cell responses. IgE/antigen induced degranulation and mediator release were substantially reduced with Jak3-/- mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells. Further, treatment of mast cells with 3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154), a potent inhibitor of JAK3, inhibited degranulation and proinflammatory mediator release after IgE receptor/ FcepsilonRI crosslinking. Thus, JAK3 plays a pivotal role in IgE receptor/ FcepsilonRI-mediated mast cell responses and targeting JAK3 may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

Animals↗

PubMed1999

Apolipoprotein(a), a link between atherosclerosis and tumor angiogenesis.

Lipoprotein (a) [Lp(a)] is a LDL-like particle with one apolipoprotein(a) [apo(a)] covalently bound to apolipoprotein B, the structural protein of Low Density Lipoprotein (LDL). Lewis Lung Carcinoma (LL/2) cells exhibited delayed growth and reduced angiogenesis in apo(a) transgenic mice, expressing a recombinant apo(a) [r-apo(a)] with 18 kringle 4 repeats. The mean microvessel density of subcutaneous LL/2 tumors from apo(a) transgenic mice was significantly lower than that of tumors from control wild type mice. CHO cells secreting a truncated apo(a) protein with only six kringle 4 repeats did not exhibit delayed tumor growth nor did it impair angiogenesis. These data point to an unappreciated role of human apo(a) in angiogenesis and cancer biology. As angiogenesis is necessary for reendothelialization following vascular injury, suppression of angiogenesis by apo(a) may also contribute to the atherogenicity of apo(a). The differences between the truncated apo(a) and r-apo(a) are consistent with the higher atherogenicity of higher molecular weight isoforms.

Animals↗

PubMed1999

Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide].

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1999

Quantitative high-performance liquid chromatography-based detection method for calphostin C, a naturally occurring perylenequinone with potent antileukemic activity.

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca2+-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250x4 mm LiChrospher 100, RP-18 (5 microm) in conjunction with a 4x4 mm LiChrospher 100, RP-18 guard column (5 microm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile-water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05-40 microM for calphostin C in 50 microl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 microl plasma was 0.02 microM at a signal-to-noise ratio of approximately 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.

Animals↗

PubMed1999

Low density lipoprotein (LDL)-mediated suppression of Lewis lung carcinoma in hypercholesterolemic LDL receptor-deficient mice.

An inverse relationship has been reported between cancer risk and cholesterol level, prompting the hypothesis that hypercholesterolemia may be protective against cancer. We tested this hypothesis by evaluating the growth of Lewis lung carcinoma in three different murine models of hypercholesterolemia: Pluronic treated mice, apolipoprotein E (ApoE) deficient mice, and low density lipoprotein receptor (LDL-R) deficient mice. Only the accumulation of LDL-cholesterol in LDL-R deficient mice suppressed tumor growth. Accumulation of chylomicrons, very low density lipoproteins (VLDL), and cholesterol-enriched remnants in the Pluronic treated mice and ApoE deficient mice did not inhibit tumor growth, even though mice in all three models were equally hypercholesterolemic. Taken together, the experimental evidence from our studies indicate that high plasma cholesterol in the form of LDL-cholesterol could have a beneficial effect against cancer in vivo.

Animals↗

PubMed1999

Molecular genotoxicity profiles of apoptosis-inducing vanadocene complexes.

Metallocene complexes containing vanadium induce apoptosis in human cancer cells by an as yet unknown mechanism and may therefore be useful as a new class of cytotoxic anticancer drugs. Ultrastructural studies showing the formation of metallocene-DNA complexes prompted the hypothesis that their mechanism of action may resemble the DNA damage induced by cisplatin. Molecular genotoxicity testing provides insights into the mechanisms of action of new chemotherapeutic agents. Therefore, we determined the effects of three cytotoxic vanadocene complexes, vanadocene dichloride, vanadocene dithiocyanate, and vanadocene dioxycyanate, on genomic stability using the yeast DEL recombination assay and transcriptional activation of genotoxic stress-specific promoters in human HepG2 cells using the CAT-Tox(L) assay. Cisplatin caused an 11-fold increase of recombination frequency in yeast and induced transcriptional activation of the DNA damage-associated promoters such as the minimum promoter containing p53 response elements and the GADD45 promoter in addition to activating the promoters for c-fos, heat shock protein 70, metallothionine IIa, and the minimum promoter containing nuclear factor kappa(kappa)B response elements. In contrast to cisplatin, vanadocene complexes did not increase the DEL recombination frequency in yeast nor did they activate any of the DNA damage-associated promoters in HepG2 cells. Vanadocene complexes triggered activation of the c-fos promoter without affecting the minimum promoter containing p53 response elements or the GADD45 promoter. These results indicate that the apoptotic signal of vanadocene complexes is not triggered by primary DNA damage and it does not require p53 induction, thereby disproving the hypothesis that it mechanistically resembles the cytotoxic action of cisplatin.

Antineoplastic Agents↗

PubMed1999

Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia.

Ikaros, a zinc finger-containing DNA-binding protein, is required for normal lymphocyte development, and germline mutant mice that express only non-DNA binding dominant-negative "leukemogenic" Ikaros isoforms lacking critical N-terminal zinc fingers develop an aggressive form of lymphoblastic leukemia 3-6 months after birth. Therefore, we sought to determine whether molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in infants. Primary leukemic cells were freshly obtained from 12 infants (<1 year of age) with newly diagnosed ALL. In leukemic cells from each of the 12 infants with ALL, we found high level expression of dominant-negative isoforms of Ikaros with abnormal subcellular compartmentalization patterns. PCR cloning and nucleotide sequencing were used to identify the specific Ikaros isoforms and detect Ikaros gene mutations in these cells. Leukemic cells from seven of seven infants with ALL, including five of five MLL-AF4(+) infants, expressed dominant-negative Ikaros isoforms Ik-4, Ik-7, and Ik-8 that lack critical N-terminal zinc fingers. In six of seven patients, we detected a specific mutation leading to an in-frame deletion of 10 amino acids (Delta KSSMPQKFLG) upstream of the transcription activation domain adjacent to the C-terminal zinc fingers of Ik-2, Ik-4, Ik-7, and Ik-8. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal nuclear localization were found in normal infant bone marrow cells and infant thymocytes. These results implicate the expression of dominant-negative Ikaros isoforms and the disruption of normal Ikaros function in the leukemogenesis of ALL in infants.

Amino Acid Sequence↗

PubMed1999

Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex.

Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases. Mutations in the btk gene have been linked to severe developmental blocks in human B-cell ontogeny leading to X-linked agammaglobulinemia. Here, we provide unique biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), and kinase domains of BTK are all individually important and apparently indispensable, but not sufficient, for its function as a negative regulator of Fas-mediated apoptosis. BTK associates with Fas via its kinase and PH domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout underwent apoptosis after Fas ligation, but wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with wild-type human btk gene did not. Introduction of an Src homology 2 domain, a PH domain, or a kinase domain mutant human btk gene into BTK-deficient cells did not restore the resistance to Fas-mediated apoptosis. Introduction of wild-type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation. BTK-deficient RAMOS-1 human Burkitt's leukemia cells underwent apoptosis after Fas ligation, whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells expressing similar levels of Fas did not. Treatment of the anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite analog alpha-cyano-beta-methyl-beta-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-mediated apoptosis. The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal B-cell ontogeny may be reciprocally regulated by Fas and BTK.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1999

Innovative treatment programs against cancer: II. Nuclear factor-kappaB (NF-kappaB) as a molecular target.

Nuclear factor-kappaB (NF-kappaB) activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as to ionizing radiation. Preventing the protective function of NF-kappaB may result in chemo- and radio-sensitization of cancer cells. Therefore, NF-kappaB has emerged as one of the most promising molecular targets in rational drug design efforts of translational cancer research programs.

Animals↗

PubMed1999

High-level expression and purification of biologically active recombinant pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes. The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source. Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system. Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E. coli. Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E. coli.

Base Sequence↗

PubMed1999

WHI-05, a novel bromo-methoxy substituted phenyl phosphate derivative of zidovudine, is a dual-action spermicide with potent anti-HIV activity.

Heterosexual transmission of HIV to women is the fastest-growing mode of transmission. In a systematic effort to develop a microbicide capable of preventing HIV transmission as well as providing fertility control, novel phenyl phosphate derivatives of 3'-azido-3'-deoxythymidine (zidovudine, ZDV) have been identified that exhibit potent anti-HIV and spermicidal activities. This study reports the synthesis, characterization, and preclinical formulation of compound WHI-05, 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl) methoxyalaninyl phosphate. The anti-HIV activities of WHI-05 and ZDV were compared by measuring p24 antigen production and reverse transcriptase activity as markers of viral replication using human peripheral blood mononuclear cells (PBMC) infected with both ZDV-sensitive and ZDV-resistant strains of HIV. The sperm immobilizing activity (SIA) of WHI-05 was compared with that of ZDV and nonoxynol-9 (N-9) by computer-assisted sperm analysis (CASA). The effect of WHI-05 on sperm membrane integrity was examined by high resolution, low voltage scanning electron microscopy (HR-LVSEM). The in vitro cytotoxicity profile of WHI-05 versus N-9 were compared using normal human vaginal, ectocervical, and endocervical epithelial cells. The in vivo vaginal tolerance, absorption, and toxicity of a 2% WHI-05 gel-microemulsion was tested in the rabbit. Whereas ZDV displayed potent anti-HIV activity but lacked SIA, WHI-05 elicited both potent anti-HIV activity and SIA. WHI-05 inhibited the replication of ZDV-sensitive as well as ZDV-resistant strains of HIV in PBMC. CASA combined with HR-LVSEM demonstrated that WHI-05-induced SIA was not associated with membrane damage. Unlike, N-9, the spermicidal activity of WHI-05 was not associated with cytotoxicity to reproductive tract epithelial cells. Repetitive intravaginal application of a 2% WHI-05 gel-microemulsion did not damage the vaginal epithelium or cause local inflammation in the rabbit model. As a potent anti-HIV agent that has spermicidal activity and is devoid of mucosal toxicity, WHI-05 shows a unique clinical potential to become the active ingredient for a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Animals↗

PubMed1999

Evaluation of the pharmacokinetic features and tissue distribution of the potent nonnucleoside inhibitor of HIV-1 reverse transcriptase, N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240) with an analytical HPLC method.

PURPOSE: The purpose of the present study was to examine the pharmacokinetic features and tissue distribution of N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240), a novel non-nucleoside inhibitor of HIV reverse transcriptase with potent anti-viral activity against AZT-sensitive as well as multidrug-resistant HIV-1 strains. METHODS: A sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method was established to measure concentrations of HI-240 in pharmacokinetic studies. The plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameter values. RESULTS: HI-240 had an elimination half-life of 78.3 +/- 2.0 min after i.v. administration and 196.8 +/- 3.1 min after i.p. administration. The systemic clearance of HI-240 was 2194 +/- 61 ml/h/kg after i.v. administration and 9339 +/- 1160 ml/h/kg after i.p. administration. Following i.v. injection, HI-240 rapidly distributed to and accumulated in multiple tissues with particularly high accumulation in adipose tissue, adrenal gland, and uterus+ovary. The concentration of HI-240 in brain tissue was comparable to that in the plasma, indicating that HI-240 easily crosses the blood-brain-barrier. Following i.p. injection, HI-240 was rapidly absorbed with a t1/2ka and a tmax values of less than 10 min. Following oral administration, HI-240 was absorbed with a t1/2ka of 4.2 +/- 1.1 min and a tmax of 95.1 +/- 25.1 min. The intraperitoneal bioavailability was estimated at 23.5%, while the oral bioavailability was only 1%. CONCLUSIONS: The HPLC-based accurate and precise analytical detection method and pilot pharmacokinetic studies described herein provide the basis for advanced preclinical pharmacodynamic studies of HI-240. The ability of HI-240 to distribute rapidly and extensively into extravascular compartments and easily cross the blood-brain barrier represent significant pharmacokinetic advantages over AZT.

Administration, Oral↗