Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3.

The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies. The protein can be purified on a nickel affinity resin due to a hexa-His tag that is incorporated onto the amino terminus of the domain. PBD binds to Cdc42Hs in a guanine nucleotide-dependent manner as demonstrated by a novel fluorescence assay that takes advantage of the spectroscopic properties of N-methylanthraniloyl (Mant)-guanine nucleotides. Ionic strength has little effect on the affinity of PBD for Cdc42Hs, but alkaline pH values tend to weaken the interaction. We have shown that the inhibition of the GTPase activity of Cdc42Hs, as well as a previously undescribed inhibition of guanine nucleotide dissociation, is mediated by the PBD portion of the mPAK-3 molecule. These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleotide domain. We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotide binding site, had any effect on PBD binding. Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold decrease in affinity. Finally, the catalytic domain of the GTPase activating protein, Cdc42-GAP, was shown to inhibit PBD binding in a competitive manner, indicating that this target molecule and the negative regulator (GAP) bind to overlapping sites on the Cdc42Hs molecule.

Antipyrine and doxycycline pharmacokinetics in patients with thyroid disorders.

Pathological conditions are known to affect pharmacokinetics of many drugs. Antipyrine half-life is used as a marker of liver microsomal enzyme function. Antipyrine pharmacokinetics, therefore, was investigated in 23 thyrotoxic and 11 euthyroid goitre patients. Of these, 11 thyrotoxic and 9 euthyroid goitre patients also participated in doxycycline bioavailability studies. In thyrotoxic patients, antipyrine half-life and AUCo infinity and doxycycline Cpmax and AUCo infinity were found to be reduced as compared to those of healthy euthyroid normal subjects. Following treatment of thyrotoxicosis, the antipyrine half-life and AUCo infinity returned to normal. Doxycycline AUCo infinity returned to near normal range but Cpmax did not.

Suitability of laboratory animals for screening anti-hyperlipidemic agents.

The present study was undertaken to evaluate and compare the lipid profiles of various laboratory animals to that of human beings. The human subjects and animals included in the study were from three age groups based on key physiological states. A record of the usual dietary constituents and their daily consumption was maintained. The results indicated that the lipid profile of pigs and dogs bears similarity to that of human beings. Results also revealed that lipid profile was labile in the second group of these animals indicating that this age is suitable to bring about the required changes to produce a hyperlipidemic animal.

Centbutindole vs trifluoperazine: a double-blind controlled clinical study in acute schizophrenia.

Twenty-nine acute schizophrenic patients were treated under double-blind conditions for six weeks with either centbutindole in a dose range of 3 mg/day to 4.5 mg/day or trifluoperazine in the dose range of 15 mg/day to 22.5 mg/day. Both drugs produced a significant improvement in initial psychopathology. No significant differences were demonstrated between the two treatment conditions.

Influence of menstrual cycle on antipyrine pharmacokinetics in healthy Indian female volunteers.

The effect of the menstrual cycle on antipyrine pharmacokinetics was studied in 11 normal, healthy Indian female volunteers. Antipyrine half-life, apparent volume of distribution, clearance and AUC were calculated by standard methods. Results indicated that in females, antipyrine half-life was significantly longer on day 5 as compared with that on days 15 and 21 of the menstrual cycle. It appears that hormonal changes during the menstrual cycle affect the pharmacokinetics of drugs in normal healthy females.

Effect of iron deficiency anaemia and its treatment on the absorption and elimination of phenformin.

1. The extent of phenformin absorption and its rate of urinary excretion have been assessed in adult patients with iron deficiency anaemia, a condition which compromises gastrointestinal function. 2. Phenformin (100 mg) was administered orally to patients before treatment, three days after the start of a course of iron treatment (oral 300 mg b.d. or total intravenous iron) and at the end of 28 days, when haemoglobin was over 10 gm%. 3. No significant difference was found between mean total amounts of phenformin and 4-hydroxyphenformin excreted in urine, before treatment or after 3 or 28 days replacement therapy. It is concluded that phenformin absorption is not affected by iron deficiency. 4. In addition, iron deficiency had no significant effect on phenformin elimination half-life.

Plasma rifampicin levels during oral contraception.

PIP: Researchers conducted a study on the effect of oral contraceptives (OCs) on rifampicin plasma levels in 6 healthy women between 19-38 years old. These women's weight ranged between 40-60 kg an their height between 150-160 cms. All hemoglobin levels were 10 g. They had not used OCs for 3 months before the research began. As a control, the volunteers 1st did not receive an OC and had a menstrual cycle. Blood samples were collected between days 15-28 of the menstrual cycle. They took a daily low dose OC containing 1 mg norethisterone acetate and 30 mcg ethinyl estradiol in the next cycle. Before and after the 2nd OC cycle, they took 450 mg of rifampicin while the stomach was void and laboratory personnel estimated plasma levels of rifampicin by microbiological assay at 0, 1, 2, 4, 6 and 8 hours. When the women were taking no OC, peak plasma levels of rifampicin ranged between 8.2-36 mcg/ml, while they varied between 11.25-29 mcg/dl during the 2nd OC cycle. Further, the time of peak concentration of rifampicin when the women did not use an OC occurred between 2-4 hours in 5 women and at 6 hours for the other woman. During OC administration, all the women's peak concentration was 2 or 4 hours. The area under curve (AUC) of rifampicin while no OC was used extended from 29.85-176 + or - 22.1 mcg/ml/hour and 61.9-157.7 + or - 14.9 mcg/ml/hour while the women took the OC. No significant difference existed between before and during OC use plasma levels of rifampicin and AUC. Even though studies show that rifampicin treatment reduces plasma levels of OCs, these results demonstrate that a low dose OC does not change rifampicin levels.^ieng

Effect of ascariasis and its treatment on drug absorption.

Ascariasis has been reported to impair the absorption of nutrients, vitamin A, and D-xylose, which is corrected on treatment. The effect of ascariasis and its treatment on the absorption of sulphadimidine and isoniazid has been investigated. There was no difference between drug absorption before and after the treatment or in comparison with a normal population.

Effect of pectin and kaolin on bioavailability of co-trimoxazole suspension.

Bioavailability of co-trimoxazole suspension was determined with and without concurrent administration of pectin and kaolin in 8 volunteers. Twenty ml suspension of co-trimoxazole containing 160 mg trimethoprim (TMP) and 800 mg sulphamethoxazole (SMX) and co-trimoxazole suspension along with 20 ml of pectin-kaolin suspension were administered in a random order with 7 days interval. Plasma estimation of trimethoprim and sulphonamide was carried out at serial intervals. Area under curve (AUC) and Cmax of TMP were significantly higher when co-trimoxazole suspension alone was used. No statistically significant changes were observed in case of sulphamethoxazole. Clinical study is necessary to verify whether concurrent administration of co-trimoxazole and pectin-kaolin leads to loss of antibacterial efficacy.

Effect of iron deficiency anaemia and its treatment on sulphadimidine absorption.

The effect of iron deficiency anaemia and its treatment on the absorption of sulphadimidine has been investigated in adult patients. The absorption judged by total % of the dose excreted in urine and Cmax, tmax, AUC and Kabs in plasma, was not significantly different before and after iron therapy or correction of anaemia. However, sulphadimidine absorption by the anaemic patients was significantly greater than in normals.

Clonidine and enphenamic acid interaction study in dogs.

The drug interaction between clonidine (Cl) and enphenamic acid (En.A.), a newer non-steroidal anti-inflammatory drug (NSAID) was studied in anaesthetized dogs. Prior administration of En.A. in animals modified the blood pressure response to clonidine. Thus En.A., administered 1 hr before clonidine, potentiated the hypertensive response and blocked the subsequent hypotensive response to clonidine. Intracerebroventricular administration of En.A. did not affect the usual blood pressure response to clonidine. Similarly, this interaction was not observed in reserpinized animals and it was only partially blocked in normal dogs, treated with tolazoline. It is concluded that En.A. interferes with blood pressure responses to clonidine and that this interaction is probably of a peripheral, vascular, nature.

Drug interaction between guanfacine and nonsteroidal antiinflammatory drugs in dogs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to attenuate the hypotensive actions of various antihypertensive agents. This experimental study in dogs was undertaken to find out whether NSAIDs modified the pharmacological actions of an antihypertensive drug, guanfacine. Indomethacin and enphenamic acid significantly prolonged the initial hypertensive response and blunted the subsequent hypotension produced by intravenous guanfacine. Ibuprofen and acetyl salicylic acid also interacted in a similar manner, but to a lesser extent. Phenylbutazone, on the other hand, caused a blunting of the pressor response and potentiated the hypotension following guanfacine. When indomethacin was given intracerebroventricularly, there was no interaction. These results suggest the necessity of monitoring hypertensive subjects taking guanfacine when NSAIDs are co-administered.

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.