Hyaluronic acid capacitation induces intracellular signals modulated by membrane-associated adenylate cyclase and tyrosine kinase involved in bovine in vitro fertilization.

Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.

Exposure-disease continuum for 2-chloro-2'-deoxyadenosine, a prototype ocular teratogen. 3. Intervention with PK11195.

BACKGROUND: Treatment of pregnant mice with 2-chloro-2'-deoxyadenosine (2CdA) on Day 8 of gestation induces microphthalmia through a mechanism linked to the p53 tumor suppressor pathway. The present study defines the response of Day 8 mouse embryos through time with respect to pharmacologic intervention with PK11195, a ligand of the mitochondrial peripheral benzodiazepine receptor (Bzrp). METHODS: Pregnant CD-1 mice dosed with 2CdA with or without PK11195 on gestation Day 8 provided fetuses for teratologic evaluation on Day 14 and Day 17; HPLC measured pyridine nucleotides (NADH/NAD+) at 1.5 hr, RT-PCR measured mitochondrial 16S rRNA abundance at 3.0 hr, and p53 protein induction was assessed with immunostaining at 4.5 hr postexposure. RESULTS: The mean incidences of malformed fetuses were significantly higher in the 7.5 mg/kg 2CdA treatment group (50.2% malformed) vs. the 2CdA + 4.0 mg/kg PK11195 co-treatment group (4.4% malformed). Malformed fetuses displayed a range of ocular defects that included microphthalmia and keratolenticular dysgenesis (Peters anomaly). No malformations were observed in the control or PK11195 alone groups. PK11195 also protected litters from increased resorption rates and fetal weight reduction. It did not rescue early effects on NADH balance (1.5 hr) or 16S rRNA expression (3.0 hr); however, the p53 response (4.5 hr) was downgraded in 2CdA + PK11195 embryos vs. 2CdA alone. By delaying the administration of PK11195 in 1.5 hr intervals it was determined that the window for protection closed between 4.5 to 6.0 hr after 2CdA. CONCLUSIONS: The capacity of PK11195 to suppress the pathogenesis of microphthalmia implies a critical role for mitochondrial peripheral benzodiazepine receptors in the p53-dependent mode of action of 2CdA on ocular development.

cycloSal-Pronucleotides of 2'-fluoro-ara- and 2'-fluoro-ribo-2',3'- dideoxyadenosine as a strategy to bypass a metabolic blockade.

Novel, lipophilic cycloSal triesters 4a-c and 5a-c were synthesized, respectively, from the ara- and ribo-configurated 2'-fluorinated-2', 3'-dideoxyadenosines 2 and 3. The cycloSal phosphotriesters were used as tools to study the effects of the two different sugar pucker conformations induced by two opposite configurations of the fluorine substituent at C2' of the dideoxyribose moiety. F-ara-ddA (2) is known to be an active anti-HIV agent, whereas the ribo-analogue 3 is inactive. Hydrolysis studies with the triester precursors 4a-c and 5a-c showed selective formation of the monophosphates of 2 and 3. The lipophilicity of the triester prodrugs was considerably increased by the cycloSal mask with respect to ddA (1), F-ara-ddA (2), and F-ribo-ddA (3). Phosphotriesters 4 and 5 proved to be completely resistant to ADA and AMPDA deamination. In parallel experiments, ribo-nucleoside 3 showed a 50-fold faster deamination rate relative to the ara-analogue 2. Against HIV in CEM cells, the phosphotriesters 4 proved to be 10-fold more potent than the parent nucleoside 2. Furthermore, the prodrugs 4 were active against MSV-induced transformation of C3H/3T3 fibroblasts, while 2 was inactive. More interestingly, the ribo-configurated phosphotriesters 5, prepared from the inactive F-ribo-ddA (3), showed a level of anti-HIV activity that was even higher than that of F-ara-ddA (2). Our findings clearly prove that the application of the cycloSal-pronucleotide concept to F-ribo-ddA (3) overcomes a metabolic blockade in the formation of the corresponding monophosphate.

Single dose and multiple dose pharmacokinetics of 2'-fluoro-2',3'-dideoxyadenosine and 2'-fluoro-2',3'-dideoxyinosine, anti-HIV agents, in rats.

A single and multiple dose pharmacokinetic (PK) study was conducted in rats following oral administration of 2'-fluoro-2',3'-dideoxyadenosine (FddA) and 2'-fluoro-2',3'-dideoxyinosine (FddI) at three dose levels. Six rats/gender were assigned to one of the three FddA or FddI dose levels: 40, 250, and 1000 mg/kg/day. Three rats/gender were assigned to the PK study on day 1, while the remaining 3 rats/gender were assigned to the PK study on day 14. The rats received the appropriate doses of either FddA or FddI orally by gavage once a day for 14 days. Serial blood samples up to 24 h and cumulative urine samples (0-24 h) were collected on both days 1 and 14. Plasma and urine samples were analyzed for the concentrations of intact FddA and/or FddI using a validated assay. The data were subjected to non-compartmental PK analyses. Over the dose range of 40-1000 mg/kg. both FddA and FddI exhibited dose dependent pharmacokinetics in rats. Following FddA administration, there was a rapid and extensive in vivo conversion of FddA to FddI; FddI was the major circulating moiety as reflected by Cmax and AUC values (generally 2-3-fold greater than those of FddA at each dose level) as well as the amount excreted (%UR) in the urine. In contrast, following FddI administration, Cmax, AUC, and %UR values were 2-5-fold lower as compared to the FddI generated from FddA administration at each dose level, which also suggested that FddI was not absorbed as extensively as FddA. Based on the findings of this study, FddA is an excellent prodrug of FddI.

Role of brain tissue localized purine metabolizing enzymes in the central nervous system delivery of anti-HIV agents 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.

PURPOSE: This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes. METHODS: Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions. RESULTS: The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x). CONCLUSIONS: PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.

Enhancement of the anti-HIV-1 activity of ddAdo by coformycin, EHNA and deaza-EHNA derivatives.

2',3'-dideoxyadenosine (ddAdo) and 2',3'-dideoxyinosine (ddIno) are potent and selective inhibitors of the replication of the human immunodeficiency virus type 1 (HIV1) in several cell culture systems. Equipotent in terms of antiviral activity, both compounds selectively inhibit the reverse transcription of HIV-1 by virtue of their conversion into ddATP. In human lymphoid cells ddAdo is converted to the active metabolite, ddATP, but it also undergoes rapid deamination, via adenosine deaminase, to form ddIno. ddIno, like ddAdo, gives rise to dideoxynucleotides of the dideoxy-adenylate series (ddAMP, ddADP and ddATP), as well as to IMP and to adenylate ribonucleotides. With the main object of blocking the deamination of ddAdo, we studied its anti-HIV-1 activity in the presence of different adenosine deaminase inhibitors, namely Coformycin (CF), 9-(erythro-2-hydroxy-3-nonyl) adenine (EHNA) and some deaza-EHNA derivatives. In contrast with reports on 2'-deoxycoformycin (Cooney et al., 1987), the adenosine deaminase inhibitors tested by us showed a significant increase in the antiviral activity of ddAdo, but not of ddIno. Enhancement was obtained with EHNA and CF concentrations up to 250 and > 12,500 times lower than their respective maximum non toxic doses. In combination with EHNA or CF, ddAdo could be used at concentrations up to ten times lower than those required to obtain the same degree of inhibition when ddAdo (or ddIno) was used alone. The use of EHNA or CF in combination with ddAdo at concentrations that inhibit the multiplication of HIV-1, allowed uninfected cells to maintain their normal multiplication rates. In fact, in combination experiments, cytotoxic effects were evident only with doses of EHNA, or CF and ddAdo 10 to 100 or more times higher than those required to inhibit HIV-1 significantly. The in vivo implications of these results for anti-HIV chemotherapy are discussed.

Pharmacokinetics of 2',3'-dideoxyadenosine in dogs.

The pharmacokinetics of 2',3'-dideoxyadenosine (ddAdo) and 2'-3'-dideoxyinosine (ddIno) were determined after intravenous bolus administration and long-term intravenous infusion of ddAdo in dogs. ddAdo was rapidly deaminated to ddIno and ddAdo plasma concentrations were only a fraction of ddIno concentrations. The total body clearance of ddAdo exceeded the literature value for the cardiac output of the dog, indicating an extremely rapid metabolism, and the existence of extrahepatic metabolism. Urinary excretion of unchanged ddAdo was a minor route of elimination (approximately 1%). The pharmacokinetics of ddIno was determined assuming complete conversions of ddAdo to ddIno. ddIno elimination was dose-dependent with total body clearance ranging from 4 to 55 ml/min/kg in individual animals. The plasma half-life was approximately 30 min after most routes of administration, but increased to approximately 60 min in two animals receiving a large intravenous dose of 500 mg/kg. ddIno penetrated into the cerebrospinal fluid to a limited extent, reaching concentrations of 3-11% of those in plasma. Urinary excretion of unchanged ddIno accounted for approximately 20% of the administered dose of ddAdo, while uric acid and hypoxanthine were minor urinary metabolites. Concentrations exceeding the in vitro minimal viral inhibitory concentration (2.4 micrograms/mL) could be safely maintained in plasma for a 10-day period. Infusions which gave cerebrospinal fluid concentrations of 12 to 17 micrograms/mL resulted in dose limiting myelosuppression and intestinal toxicity, after less than 10 days of infusion. Orally administered ddAdo was absorbed as ddIno, with bioavailabilities ranging from 28 to 93% in experiments where no emesis occurred. These studies indicate the rapid in vivo conversion of ddAdo to ddIno, and support the selection of ddIno over ddAdo for further drug development.

Pharmacokinetics of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine in patients with severe human immunodeficiency virus infection.

This article describes the pharmacokinetics of 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) as determined during phase I clinical trials in patients with acquired immunodeficiency syndrome and acquired immunodeficiency syndrome-related complex. Drug levels were determined by HPLC in plasma, cerebrospinal fluid, and urine after administration of the drugs either intravenously or as an oral liquid given with antacid. ddA was metabolized rapidly and quantitatively to ddI to such an extent that ddA was undetectable in the plasma even during continuous intravenous administration of ddA. The plasma kinetics of ddI were generally monoexponential and were characterized by a half-life of 38 minutes. This probably does not accurately reflect the kinetics of the active species of ddI, which appears to be 2',3'-dideoxyadenosine triphosphate, formed intracellularly. Oral bioavailability was 38% for oral liquid given with antacid. The total body clearance averaged 1.00 L/kg/hr, with a volume of distribution of 1.01 L/kg. Approximately 36% of the intravenous dose could be recovered unchanged in the urine. The level of ddI in the cerebrospinal fluid 1 hour after drug infusion averaged 21% of that of the simultaneous plasma level. It is concluded that ddI has pharmacokinetic properties that are amenable to its clinical use.

Disposition of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine in mice.

Mice were dosed with [3H]2',3'-dideoxyadenosine ([3H]ddA) in three procedures: intravenously, intraperitoneally, and interperitoneally following a dose of 2'-deoxycoformycin (dCF). For mice dosed intravenously, the content of radioactivity in plasma and tissue samples were essentially constant after 30 min. Of the radioactivity in plasma and brain samples collected between 30 min and 24 hr, more than 94% was present as 3H2O, indicating that most of the tritium from [3H]ddA had exchanged with water. No intact ddA was detected, and the deamination product, 2',3'-dideoxyinosine (ddI), was present only transiently. In the urine, the major radioactive material was [3H]ddI. Also detected were 3H2O and small amounts of [3H]hypoxanthine and [3H]ddA. Following intraperitoneal doses to mice, levels of radioactivity in plasma, liver, and kidney increased to a maximum by 15-30 min after dosing but dropped to essentially constant levels thereafter, again indicating that the tritium had exchanged with water. At 5, 15, and 30 min after dosing, ddI was the major radioactive component in plasma. Only small amounts of ddA were present. When dCF was administered 24 hr prior to intraperitoneal [3H]ddA, levels of radioactivity in plasma, liver, and kidney reached a maximum of 30 to 60 min after dosing and decreased to essentially constant levels thereafter. The dCF transiently inhibited the deamination of ddA to ddI, since, in plasma, [3H]ddA was the main radioactive component at 5 and 15 min after dosing. Comparison of HPLC assays based on radioactivity detection and UV absorbance showed that they were equivalent for measuring ddA and ddI in samples derived from dosed mice.(ABSTRACT TRUNCATED AT 250 WORDS)