Influence of human p16(INK4) and p21(CIP1) on the in vitro activity of recombinant Plasmodium falciparum cyclin-dependent protein kinases.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.

Design and synthesis of Pfmrk inhibitors as potential antimalarial agents.

The synthesis and inhibitory activities of 10 potential inhibitors of Pfmrk, a Plasmodium falciparum cyclin-dependent protein kinase, are described. The most potent inhibitor is a 3-phenyl-quinolinone compound with an IC(50) value of 18 microM. It is the first compound reported to inhibit Pfmrk at the micro molar range.

Cyclin H activation and drug susceptibility of the Pfmrk cyclin dependent protein kinase from Plasmodium falciparum.

The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs). Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully. To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity. Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity. Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro. Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity. This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs. CDKs are attractive drug targets due to their role in cellular proliferation. Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target. Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds. A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk.

Topical anesthetic-induced methemoglobinemia and sulfhemoglobinemia in macaques: a comparison of benzocaine and lidocaine.

Benzocaine (BNZ) and lidocaine (LC) are commonly used topical (spray) anesthetics approved for use in humans. Benzocaine has structural similarities to methemoglobin (MHb)-forming drugs that are current candidates for cyanide prophylaxis, while LC has been reported to increase MHb in man. In this study, we compared MHb and sulfhemoglobin (SHb) production in three groups of Macaques (Chinese rhesus and Indian rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina)) after exposure to BNZ and LC. Formation of SHb, unlike MHb, is not thought to be reversible and therefore is considered to be of greater toxic significance. Both MHb and SHb levels were measured periodically on a CO-Oximeter. All rhesus macaques (n = 8) were administered an intratracheal/intranasal) dose of 56 mg (low dose) or 280 mg (high dose) of BNZ or 40 mg of LC in a randomized cross-over design (all animals received all three treatments). Pig-tailed macaques (n = 6) were given an intranasal dose of 56 mg of BNZ and 40 mg of LC. As no differences in the peak MHb or time to peak (mean +/- SD) were observed among the three macaque subspecies, the data were pooled. Lidocaine did not cause MHb or SHb formation above baseline in any monkey. In contrast, all monkeys (n = 14) had a significant elevation in peak MHb formation after 56 mg of BNZ, which ranged from 4.0% to 19.4% with an average of 8.6 +/- 4.0% (mean +/- SD), with peak MHb levels reached at 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte and plasma cholinesterase activity in male and female rhesus monkeys before and after exposure to sarin.

The rhesus monkey (Macaca mulatta) has a menstrual cycle similar to the human. Differences in hormone levels have been demonstrated between the sexes and in females during the menstrual cycle but these differences in terms of organophosphorus toxicity have not been explored. Plasma cholinesterase (ChE/BuChE) and erythrocyte (RBC) acetylcholinesterase (AChE) activity were measured before and after exposure to the organophosphorus compound sarin (11 micrograms/kg, i.v.; 0.75 LD50) in six male and six female rhesus monkeys. After baseline measurements were obtained, sarin was administered to atropinized monkeys to determine in vivo differences between the sexes in their response to sarin. With the baseline values, the intraanimal and intragroup BuChE/AChE variations were found to be minimal. Following sarin intoxication and 2-PAM treatment no significant differences were seen between the sexes in the rate of reactivation of BuChE or AChE by 2-PAM. The rate of aging of sarin phosphonylated RBC AChE between the sexes was also similar. De novo regeneration of RBC AChE and plasma BuChE after sarin intoxication was different between the male and female monkeys. The female plasma BuChE recovery rate was 48% slower than the male recovery rate, while the early (first 63 days) RBC AChE recovery rate was 24.5% faster in the females. In conclusion, there probably are not any clinically significant differences between male and female rhesus monkeys acutely intoxicated with sarin. However, on subsequent exposure clinical differences may be observed due to substantial differences in the rate of de novo synthesis of both plasma BuChE and RBC AChE.

HI-6 pharmacokinetics in rabbits after intravenous and intramuscular administration.

The pharmacokinetics of HI-6 ((4-carboxamidopyridinium (1) methyl)-(2'-hydroxyiminomethyl-pyridinium (1') methyl) ether dichloride) have been studied in rabbits receiving an intramuscular (50 micrograms kg-1) or intravenous (12.5 micrograms kg-1) dose. The plasma concentration-time profile for the intramuscular dose (n = 8) fits a one-compartment open model with first-order absorption and elimination. The absorption half-life was 2 min and maximum concentration (51 micrograms mL-1) was reached in 9 min. The pharmacokinetics for the intravenous dose (n = 8) was described by a two-compartment open model with first-order distribution and elimination. The apparent volume of distribution was 0.1 L kg-1. Half-lives of distribution and elimination were 5 and 38 min, respectively. The results indicate HI-6 is rapidly absorbed, distributed and eliminated in rabbits receiving an intramuscular dose.

The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman or VX in rats.

This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase (AChE). In all experiments, rats were given PYR (131 micrograms/kg, im; I70 dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agent the accumulation in the brain or estimating blood AChE activity, sufficient soman (47 micrograms/kg, iv) or VX (21.3 micrograms/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI-6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigmine failed to alter covalent binding of labelled VX or soman in the brain. The 24-hr survival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p > .05), whereas with atropine + HI-6 they decreased significantly from 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (p < .05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivated AChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine+oxime efficacy against VX-induced lethality.

Evaluation of several oximes as reactivators of unaged soman-inhibited whole blood acetylcholinesterase in rabbits.

The antidotal benefit of oximes against organophosphorus (OP) anticholinesterase intoxication is thought to be due to reactivation of the OP-inhibited acetylcholinesterase (AChE). This study was conducted to determine whether the antidotal efficacy against soman by the oximes 2-hydroxyiminomethyl-3-methyl-1-[2-(3-methyl-3-nitrobutyl oxymethyl)]-imidazolium Cl (ICD 467) and 1,1'-methylenebis[4-(hydroxyiminomethyl) pyridinium] di-Cl (MMB-4) resulted, in part, from reactivation of the inhibited AChE. These oximes were tested in parallel with pralidoxime Cl (2-PAM) and 1-(2-hydroxyiminomethyl-1-pyridinio-3-(4-carbamoyl-1-pyridinio+ ++)-2-oxapropane di-Cl (HI-6). Rabbits were atropinized (8 mg/kg, i.m.) and intoxicated with soman (13 micrograms/kg, i.v.; 1.2 x LD50) 5 min later. Three minutes after soman, animals were treated with oxime (50, 100 or 150 mumol/kg, i.m.). Whole blood was collected from a catheter in the central artery of the ear just before soman, at 2 min after soman and at 2, 5, 10, 15, 30, and 60 min after oxime or vehicle for determination of AChE activity. Shortly thereafter, animals were anesthetized and exsanguinated with immediate flushing using heparinized saline. AChE activity was also determined on the cortex, medulla-pons and diaphragm to assess central and peripheral reactivation. Treatment with HI-6 or MMB-4 (50 mumol/kg, i.m.) resulted in significant (P less than 0.05) reactivation of soman-inhibited whole blood AChE and diaphragm cholinesterase (ChE), but not brain AChE. In contrast, 2-PAM was completely ineffective in reactivating soman-inhibited AChE. HI-6 was significantly better than MMB-4 in reactivating blood AChE; they were essentially equal against soman-inhibited diaphragm ChE. Three animals exposed to soman and treated with ICD 467 died within 15 min. When animals not exposed to soman were treated with ICD 467 (25 mumol/kg, i.m.), whole blood AChE activity was depressed by 60% within 5-10 min after treatment. Furthermore, ICD 467 failed to reactivate significantly unaged soman-inhibited erythrocyte AChE, in vitro. These observations indicate that ICD 467 would be contraindicated as a therapy for anti-ChE intoxication and that the efficacy of HI-6 or MMB-4 can be explained, in part, by reactivation of soman-inhibited AChE.