Cleavage of Escherichia coli tryptophan indole-lyase by trypsin at Lys406 affects the transmission of conformational changes associated with monovalent cation activation.

Escherichia coli tryptophan indole-lyase (Trpase) is a pyridoxal 5'-phosphate(pyridoxal-P)-dependent enzyme which catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal-P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20-100-fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal-P, and a shift in the lambda(max) from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal-P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50-fold for native enzyme, but the K(A) for K+ is unaffected. Both the native and trypsin-nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid-scanning stopped-flow experiments. However, the rate constant for quinonoid intermediate formation from L-tryptophan is reduced by at least 400-fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of L-alanine and S-ethyl-L-cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray-ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active-site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate alpha-carboxylate-binding site, and His463, are located in the sequence between Lys406 and the C-terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.

Pharmacokinetics and metabolism of 3'-azido-2',3'-dideoxy-5-methylcytidine in rhesus monkeys.

3'-Azido-2',3'-dideoxy-5-methylcytidine (AzddMeC, CS-92), a nucleoside analog with structural similarities to both 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), has been shown to be a potent and selective inhibitor of human immunodeficiency virus type 1 in vitro. The pharmacokinetics of AzddMeC were characterized following intravenous and oral administration of 60 mg/kg of the compound to male rhesus monkeys. AZT was found to be a major metabolite of AzddMeC in monkeys. AzddMeC concentrations in serum declined rapidly in a biexponential fashion with the terminal half-life ranging from 0.5 to 1.3 hr. Total clearance of AzddMeC was 2.00 +/- 0.41 liters/hr/kg (mean +/- SD) with the fraction of AzddMeC metabolized to AZT of 0.32 +/- 0.05. Renal excretion of unchanged nucleoside and metabolic deamination yielding AZT were the primary routes of AzddMeC clearance. No glucuronide metabolite of AzddMeC was detected in urine samples, although, AZT-glucuronide was found in urine. The volume of the central compartment of AzddMeC was 0.53 +/- 0.28 liters/kg, the volume of the tissue compartment was 0.37 +/- 0.29 liters/kg, and the steady-state volume of distribution was 0.90 +/- 0.55 liters/kg. Volume of distribution values indicate that AzddMeC distributes extravascularly. The first-order oral absorption rate constant was 0.53 +/- 0.56 hr-1, and oral bioavailability was 26 +/- 13%. Thus, the rate of absorption of AzddMeC after oral administration was variable, and oral bioavailability was incomplete.

Comparative bioavailability of slow release diclofenac (Voveran SR) with enteric coated tablet and internationally used Voltaren Retard.

The objective of the study was to compare the enteric coated diclofenac sodium (Voveran), the slow release formulation developed in India (Voveran SR) and the internationally marketed formulation Voltaren Retard. Ten healthy volunteers were administered 100 mg each of the three formulations in a three-way crossover fashion. Blood samples were collected over 24 hours following administration of the drug; plasma levels of unchanged drug were determined by gas chromatography. Pharmacokinetic parameters for the three formulations were compared. The extent of the drug available from the three formulations was the same as the mean AUC values were not significantly different. Cmax and MRT values for the two slow release formulations were comparable but were significantly different from the values obtained with the enteric coated formulation. Tmax values for the two slow release formulations were similar while the enteric coated tablet had faster time to peak. Voveran SR is comparable to Voltaren Retard and has the distinct advantage of a slow release formulation in that its Cmax is much lower and levels are maintained over 12 hours and detectable upto 24 hours. This slow release formulation will offer clinical advantages of better compliance, relief of early morning symptoms and better tolerability over long term usage.

Hybrid pharmacokinetic models to describe anti-HIV nucleoside brain disposition following parent and prodrug administration in mice.

Brain delivery of active anti-HIV compounds is important for successful treatment of the AIDS patient. As an initial step in predicting human brain drug concentrations, hybrid pharmacokinetic models were developed to characterize the disposition of anti-HIV nucleosides following parent and prodrug administrations in mice. Mouse data were obtained following intravenous administration of 3'-azido-2',3'-dideoxyuridine (AZddU or AZDU), 3'-azido-3'-deoxythymidine (AZT), and their dihydropyridine prodrugs (AZddU-DHP and AZT-DHP). Exponential equations were fitted to the serum concentration-time data for each species, including the pyridinium ion moieties, and subsequently used in differential mass balance equations describing the brain dynamics of each compound. Model parameters for the mass balance equations were estimated by various techniques, including the utilization of in vitro data. In general, model-predicted brain concentrations agreed with the observed data. Similar data in larger animals will permit scale-up of the current model to predict human brain drug concentrations.

Pharmacokinetics of nitroxazepine in depressed patients.

A pharmacokinetic study was done on 10 depressed patients (DSM-III-R 296.3). The patients were treated with Sintamil (R) (nitroxazepine HCl) with titrated dose from 75 mg to 225 mg for 6 weeks. Plasma levels of nitroxazepine (Sintamil (R)) and its metabolites desmethyl (D), N-oxide (N-O) and carboxylic acid (c) were estimated. Anti-depressant efficacy was judged by reduction in Hamilton Rating Depression Scale (HDRS) scores, and tolerability was monitored by reports of unwanted effects.The overall reduction in HDRS score was about 50% by 6 weeks. The plasma levels of nitroxazepin (ng/ral) showed a rise from a mean ( +SEM) level. 47.0 4-7.3 on day 1 (dose 75 mg) to 129.84-24.6 on day 7 (dose 150 mg) (p< 0.01) and remained steady till day 21. There were large interindividual variations. The metabolites followed a similar pattern. The HDRS score showed a steady reduction between day 14 and 42 when the levels of nitroxizepine and des-methyl metabolites were maintained between 176.5 ng/ml to 251 ng/ml.

Brain targeting of anti-HIV nucleosides: synthesis and in vitro and in vivo studies of dihydropyridine derivatives of 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine.

A significant number of patients with AIDS and AIDS-related complex develop neurological complications. Therefore, it is critical that anti-HIV agents penetrate the blood-brain barrier and suppress viral replication in the brain. In an effort to increase the brain delivery of anti-HIV nucleosides, in vitro and in vivo pharmacokinetics of dihydropyridine derivatives of 3'-azido-2',3'-dideoxyuridine (AzddU, AZDU, or CS-87) and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) have been studied. In vitro studies of the prodrugs (AzddU-DHP and AZT-DHP) in human serum, mouse serum, and mouse brain homogenate indicated that the rates of serum conversion from prodrugs to parent drugs are species dependent: mouse brain homogenate greater than mouse serum greater than human serum. Half-lives in human serum, mouse serum, and mouse brain homogenate are 4.33, 0.56, 0.17 h, respectively, for AzddU and 7.70, 1.40, and 0.18 h, respectively, for AZT. In vivo studies of AzddU-DHP and AZT-DHP showed that the prodrugs have areas under the serum concentration-time curves (AUC) similar to those of the parent drugs. The AUC in serum for AzddU following prodrug administration is 25.79 micrograms h/mL, which is similar to the value of 25.83 micrograms h/mL when AzddU was administered. Analogously, the serum AUCs for AZT when AZT-DHP and AZT were administered are 25.38 and 26.64 micrograms h/mL, respectively. However, the brain AUCs for both AzddU and AZT derived from prodrugs, being 11.43 and 11.28 micrograms h/mL, respectively, are greater than the brain AUCs for AzddU (2.09 micrograms h/mL) and AZT (1.21 micrograms h/mL) when the parent drugs were administered. Thus, the relative brain exposure (re) for AzddU (5.47) and AZT (9.32) indicate a significant increase in exposure to the anti-HIV nucleosides following prodrug administrations. The results of extended half-lives of the synthesized prodrugs in human serum along with the higher re values in vivo warrant studies in larger animals to determine the potential usefulness of the prodrugs in humans.

Pharmacokinetics of 3'-fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine in rhesus monkeys.

3'-Fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine are nucleoside analogs which inhibit human and simian immunodeficiency virus in vitro. The pharmacokinetic properties of these compounds in rhesus monkeys after intravenous, oral, and subcutaneous administration of the drug were compared. Half-lives, total clearances, and steady-state volumes of distribution of the two drugs were determined. The half-lives for the drugs by the different routes were between 0.58 and 1.4 h. Oral bioavailability of 3'-deoxy-2',3'-didehydrothymidine was incomplete, with an average of 42% +/- 15% of the dose reaching the systemic circulation. Absorption of 3'-fluoro-3'-deoxythymidine after oral administration was variable, with bioavailability ranging from 21 to 95%. Bioavailability after subcutaneous administration ranged from 59 to 77% for 3'-deoxy-2',3'-didehydrothymidine and from 52 to 59% for 3'-fluoro-3'-deoxythymidine. The ratio of concentrations in cerebrospinal fluid and serum for the drugs was about 0.15 at 1 h after drug administration and was independent of the route of administration, suggesting that a nucleoside carrier-mediated process is involved in the transport of these compounds to the central nervous system. Because of the similar metabolism of nucleoside analogs in monkeys and humans, the potential glucuronide formation was assessed. Whereas the glucuronide of 3'-fluoro-3'-deoxythymidine was readily detected in urine, the amount of 3'-deoxy-2',3'-didehydrothymidine glucuronidated was small or not detectable in one-half of the urine samples. Pharmacokinetic parameters for the two drugs were similar to each other and analogous to those for 3'-azido-3'-deoxythymidine in monkeys, suggesting that the same dose and scheduling of the drug can be used for all three compounds in prophylactic and therapeutic efficacy drug studies in rhesus monkeys.

3'-Azido-2',3'-dideoxyuridine (AzddU): comparative pharmacokinetics with 3'-azido-3'-deoxythymidine (AZT) in monkeys.

The pharmacokinetics of the anti-human immunodeficiency virus type 1 nucleosides, 3'-azido-2',3'-dideoxyuridine (AzddU) and 3'-azido-3'-deoxythymidine (AZT) were characterized in rhesus monkeys. Half-life, total clearance, and steady-state volume of distribution were similar for both compounds. The observed pharmacokinetic parameters for AZT were comparable to those previously reported in humans. Oral absorption of AzddU and AZT was virtually complete after 60 mg/kg. However, bioavailability of both nucleosides was markedly lower (less than 50%) after 200 mg/kg, possibly indicating the involvement of a saturable absorption mechanism. The nucleosides were also well absorbed after subcutaneous administration. AzddU and AZT penetrated the cerebrospinal fluid (CSF) with concentration ratios in CSF:serum ranging from 0.05 to 0.25 one hour after drug administration. The glucuronides of AZT and AzddU were readily detected in urine. Hemogram and blood chemistry values for animals receiving short-term treatment (3 doses) with either AZT or AzddU did not exhibit any significant changes when compared with untreated control monkeys. The similar pharmacokinetic characteristics of AzddU compared with AZT suggest that clinical trials of AzddU are warranted.

Antiretroviral activity, biochemistry, and pharmacokinetics of 3'-azido-2',3'-dideoxy-5-methylcytidine.

3'-Azido-2',3'-dideoxy-5-methylcytidine (CS-92, AzddMeC) is an antiviral nucleoside analogue structurally related to 3'-azido-3'-deoxythymidine (AZT). CS-92 is a potent and selective inhibitor of HIV-1 reverse transcriptase and HIV-1 replication in human lymphocytes and macrophages. The EC50 for CS-92 in HIV-1-infected human PBM cells was 0.09 microM. In HIV-1-infected human macrophages, the EC50 was 0.006 microM. This compound was also effective against human immunodeficiency virus type 2 in lymphocytes. The replication of Friend murine virus was only weakly inhibited, and no effect was observed against herpes simplex virus type 1 and type 2 and coxsackievirus B4. CS-92 was not toxic to PBM or Vero cells when tested up to 200 microM and was, furthermore, at least 40 times less toxic to granulocyte-macrophage and erythroid precursor cells in vitro than was AZT. The interaction of the 5'-triphosphate of CS-92 with HIV-1 reverse transcriptase indicated competitive inhibition (the inhibition constant, Kis, was 0.0093 microM) with a 30-fold greater affinity for CS-92-TP than for ddCTP. CS-92-TP inhibited HIV-1 reverse transcriptase by 50% at a concentration 6,000-fold lower than that which was required for a similar inhibition of DNA polymerase alpha. Pharmacokinetic studies showed that CS-92 was not deaminated to AZT in rats, but this compound was found to have a half-life of 2.7 hours. In rhesus monkeys, however, a compound with a retention time and ultraviolet spectra characteristics similar to AZT was detected. The mean half-life in rhesus monkeys for CS-92 was 1.52 and 1.74 h after intravenous and oral administration, respectively, and the oral bioavailability was about 21 percent. Additional preclinical studies with CS-92 will determine the ultimate utility of this antiviral agent for the treatment of HIV-1 infections.

Comparative pharmacokinetics of 3'-azido-3'-deoxythymidine (AZT) and 3'-azido-2',3'-dideoxyuridine (AZddU) in mice.

The pharmacokinetics of 3'-azido-3'-deoxythymidine (AZT) and 3'-azido-2',3'-dideoxyuridine (AZddU, CS-87), active anti-HIV compounds, were characterized in uninfected mice. Sensitive and specific HPLC techniques were used to quantitate AZT and AZddU concentrations in serum and brain homogenates following iv doses of 50 mg/kg and 250 mg/kg. The pharmacokinetic parameters of t1/2, CIt, and Vss were similar for both compounds at each dose; however, CIt and Vss decreased at the higher dose, indicating a dose dependency. At the 50 mg/kg doses, the CIt of AZddU and AZT was 1.27 liters/hr/kg and 1.38 liters/hr/kg, respectively, which is analogous to the clearance value of AZT observed in humans. Brain/serum concentration ratios for AZddU tended to be greater than those obtained for AZT and were significantly different at the 50 mg/kg dose, being 0.234 +/- 0.282 for AZddU and 0.064 +/- 0.025 for AZT.

Pharmacokinetics of primaquine in patients with P. vivax malaria.

The pharmacokinetics of primaquine (PQ) and its major carboxylic acid metabolite (PQC) have been studied in seven Indian patients with P. vivax malaria following PQ 15 mg/day p.o. for 14 days. After a single oral dose on Day 1, a mean peak blood concentration of 50.7 ng/ml PQ was attained after 2.3 h, which declined monoexponentially with a half-life of 5.6 h. The mean total body clearance was 37.6 l/h and the volume of distribution was 292 l. The mean renal excretion (0-24 h) of the drug was only 0.54% of the dose and renal clearance was 0.189 l/h. Following chronic administration, none of the pharmacokinetic parameters was affected, and a steady state blood concentration of 2.5-4.2 ng/ml PQ was attained. After the first dose of PQ, PQC had a mean area under the blood concentration - time curve 11-fold higher than that of the parent drug. In contrast to the rapid distribution and elimination of PQ, the metabolite showed a longer mean residence time and accumulation in the body. The mean Cmax and AUC of the metabolite on Day 14 were 48 and 40% higher than the corresponding Day 1 values. The metabolite could not be detected in urine at any time in any patient. PQ and its metabolite did not show any accumulation in blood cells.

Kinetic studies of the interaction between isoniazid and reducing sugars.

The interaction between isoniazid and reducing sugars is acid-catalyzed and reversible. Kinetic studies of hydrazone formation from isoniazid and glucose, lactose, maltose, and galactose have been carried out in simulated gastric juice at 37 degrees C. The forward reaction was found to follow second-order kinetics, while the reverse reaction, the hydrolysis of the sugar isonicotinoyl hydrazone, is pseudo-first-order. The effects of the concentration of reactants, pH, and temperature on the rate have been studied, and the rate constants and the energy of activation were determined.

Spectrophotometric determination of chloramphenicol and its esters in complex drug mixtures.

When aromatic nitro compounds are reduced with zinc and calcium chloride and reacted with trisodium pentacyanoaminoferrate they give a purple product having an absorbance maximum between 480 and 540 nm. Applying this reaction, a quantitative method has been developed for the determination of chloramphenicol and its esters. Various reaction conditions have been standardized. Beer's law is obeyed in the concentration range of 4 to 32 micrograms/mL reaction mixture. Average recoveries and standard deviations were 99.78 +/- 0.627 and 99.90 +/- 0.660; 101.06 +/- 0.702; and 99.90 +/- 0.880% for chloramphenicol, chloramphenicol sodium succinate, and chloramphenicol palmitate, respectively. The method has also been applied to determine chloramphenicol and its esters as well as chloramphenical in the presence of combination drugs in dosage forms. The presence of benzocaine, lignocaine, sulfadiazine, nitrofurantoin, ascorbic acid, hydrocortisone, prednisolone, streptomycin, and tetracycline does not interfere with the proposed spectrophotometric procedure. The method does not require prior separation of chloramphenicol from combination drugs.