Modifications of microvascular filtration capacity in human limbs by training and electrical stimulation.

This study investigated whether an increase in microvascular surface area as a result of endurance training, which increases human skeletal muscle capillarity, would translate to greater capacity for fluid filtration compared with strength training, which does not affect capillary supply. Values for filtration capacity, Kf, derived from the slope of calf volume change, Jv, measured by venous occlusion plethysmography, against cuff pressure during a protocol of small cumulative pressure steps, were significantly higher in endurance athletes (5.78 +/- 0.88 mL min(-1) 100 mL(-1) mmHg(-1) x 10(-3), P < 0.05) than controls (3.38 +/- 0.32 mL min(-1) 100 mL(-1) mmHg(-1) x 10(-3) whereas strength-trained athletes had values similar to control (4.08 +/- 0.56 mL min(-1) 100 mL(-1) mmHg(-1) x 10(-3), ns), suggesting that surface area is important. However, when sedentary subjects underwent either a 4-week unilateral dynamic plantarflexion training programme (70% peak power, 20 min day(-1), 5 days week(-1) or a calf muscle electrical stimulation programme (8 Hz, 3 x 20 min day(-1), 5 days week(-1), neither of which caused limb blood flow to alter after training nor would be expected to increase capillarity, only the stimulation group showed a significant increase in Kf (6.68 +/- 0.62 mL min(-1) 100 mL(-1) mmHg(-1) x 10(-3) post-training vs. 3.38 +/- 0.38 pre-training, P < 0.05). This may be because stimulation enhances perfusion preferentially to glycolytic fibres, or maintains high levels of vascular endothelial growth factor (VEGF) or changes lymph clearance.

Pharmacokinetic, pharmacodynamic and clinical effects of a humanized IgG1 anti-CD4 monoclonal antibody in the peripheral blood and synovial fluid of rheumatoid arthritis patients.

BACKGROUND: CD4(+) T cells are important mediators in the pathogenesis of rheumatoid arthritis (RA). In this open-label, dose-escalating study, we examined the pharmacokinetic (PK), clinical, biological and immunological effects of a humanized IgG1 anti-CD4 monoclonal antibody (mAb), 4162W94, in the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHOD: Twenty-four patients in four cohorts (six patients in each cohort) were allocated to be treated with five consecutive daily doses of 4162W94 (10, 30, 100 or 300 mg i.v.). Disease activity was measured by the American College of Rheumatology (ACR) criteria and disease activity score (DAS). We also measured 4162W94 concentration, the percentage of 4162W94-coated CD4(+) lymphocytes, percentage down-modulation of CD4, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha) levels in the PB and SF. RESULTS: A direct relationship between 4162W94 dose, biological response and clinical outcome was seen. Treatment with 10 and 30 mg of 4162W94 for 5 consecutive days resulted in transient coating and down-modulation of CD4(+) lymphocytes, with little effect observed beyond the final dose. However, treatment with 100 and 300 mg resulted in sustained coating and/or down-modulation for 3 weeks and 4 weeks, respectively, in PB and >4 weeks in SF in one patient from the 300 mg cohort. There was a dose-related moderate but transient depression in the CD4(+) lymphocyte count in most patients, with all but three returning to >0.40 x 10(9)/l or >75% baseline by the end of the study period. Significant clinical improvement (ACR 20%) was seen in only 1/6 patients in each of the 10- and 30-mg cohorts; however, 3/6 and 5/5 patients in the 100 and 300-mg cohorts, respectively, were ACR 20% responders. In addition, there were significant reductions in PB acute phase reactants as well as SF IL-6 and TNFalpha concentrations in parallel to clinical improvement. CONCLUSION: Data from this pilot study suggest that 4162W94 is a clinically active novel immunotherapeutic agent that may suppress inflammation in RA.

Identification of a protein product of the myotonic dystrophy gene using peptide specific antibodies.

In order to identify the protein product of the recently characterised myotonic dystrophy gene, we have raised an antibody (DMAP1) to a peptide sequence of the predicted gene product. This antibody identifies a novel 52 kDa protein in a range of mouse tissues, and in addition a related 42 kDa protein in brain and heart. A second antibody raised to a different peptide from the same predicted sequence, also identifies the 52 kDa protein, which strongly implies that this 52 kDa protein is a major translation product of the myotonic dystrophy gene.

Differential assay of zidovudine and its glucuronide metabolite in serum and urine with a radioimmunoassay kit.

We developed an ancillary procedure for the ZDV-Trac RIA (Incstar) to allow simultaneous determination of both zidovudine (3'-azido-3'-deoxythymidine, ZDV, AZT, Retrovir) and its metabolite, the glucuronide of ZDV (3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine, ZDVG, GAZT), in human serum and urine. Using the ZDV-Trac RIA, we measured ZDV concentrations before and after ZDVG in samples was hydrolyzed to ZDV by beta-glucuronidase (EC 3.2.1.31); ZDVG concentration was calculated as the difference between the two results. This method enables rapid evaluation of a large number of samples with a total turn-around time of 6 h. The lower detection limit of the RIA was 0.27 micrograms/L; the measurements varied linearly with ZDV concentrations from 0.27 to 217 micrograms/L, with the 50% inhibitory concentration being approximately 10 micrograms/L. Analytical recoveries of inhouse serum and urine controls for both ZDV and ZDVG exceeded 90%. Coefficients of variation (CVs) of serum controls were less than 6% for ZDV and less than 11% for ZDVG; for urine controls, CVs for both ZDV and ZDVG were less than 6%. Results for ZDVG concentrations obtained by HPLC and by the ZDV-Trac RIA system compared well: r = 0.978, slope 1.0, for serum samples, and r = 0.993, slope 1.09, for urine samples.

Method validation in the bioanalytical laboratory.

Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to engender confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of a bioanalytical method are: the evaluation of drug and analyte stability, selectivity, limits of quantification and detection, accuracy, precision, linearity and recovery. The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which the data are put. Specific analytical techniques may require additional validation such as antibody-binding characteristics, peak purity determination, evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross-validated with a method utilizing a highly specific detector such as a mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of that assay should be monitored with quality control standards sent from the originating laboratory.

Validation of a radioimmunoassay for the determination of human plasma concentrations of lamotrigine.

A precise and sensitive radioimmunoassay to determine human plasma lamotrigine (430C78), Lamictal) is described. The method is a direct double antibody procedure employing a rabbit polyclonal first antibody raised to a BSA conjugate of lamotrigine, and an iodinated tyrosine methyl ester of lamotrigine as the tracer. Both reagents are added simultaneously to samples containing lamotrigine prior to an overnight incubation at 4 degrees C. The method has a sensitivity of 20 ng ml-1, when plasma samples are initially diluted 1:20 with phosphate buffer and sample volumes of 20 microliters are used. The intra-assay precision at 40, 80 and 160 ng ml-1 was 6.2, 2.1 and 4.8%, respectively, and the inter-assay precision at 500, 2000, 5000 and 10,000 ng ml-1 was 4.6, 5.7, 4.6 and 5.9%, respectively. The method was specific and showed reasonable correlation with an HPLC method [1].

A preliminary study of the pharmacodynamics and pharmacokinetics of a novel enkephalin analogue [Tyr-D.Arg-Gly-Phe (4NO2).Pro.NH2 (BW443C)] in healthy volunteers.

We have studied 16 healthy men to evaluate preliminary pharmacodynamics and kinetics of BW443C given by i.v. infusions. Four volunteers received escalating doses at weekly intervals, starting at 0.1 microgram.kg-1 for 60 min and increasing to a maximum of 2.0 micrograms.kg-1.min-1 for 180 min. Subsequently 12 different subjects received single i.v. infusions of 10 micrograms.kg-1.min-1 for 20 min. Subjective effects were reported and objective measurements made of central nervous and cardiovascular effects. Blood was sampled at intervals on all occasions, plasma concentrations were determined by radioimmunoassay and pharmacokinetic profiles were analysed using NONLIN. Dry mouth and some nasal stuffiness were reported and postural hypotension occurred in 5/16 subjects at plasma concentrations greater than 0.8 microgram.ml-1. Supine blood pressure was well maintained in all subjects and hypotension resolved within 60-90 min of discontinuing the infusion. There was no evidence of sedation, mood change, nausea, vomiting, miosis, change in accommodation or respiratory depression. Rapid infusions produced transient feelings of warmth, heavy eyelids, heavy legs, and increased bowel sounds, which resolved despite increasing plasma concentrations. The disposition of the peptide was adequately described by a 2-compartment model with a mean +/- SD plasma clearance of 123 +/- 18 ml.min-1 and a half-life of 2.0 +/- 0.4 h.

Alcohol and bupropion pharmacokinetics in healthy male volunteers.

A study was performed to determine whether there is a pharmacokinetic interaction between alcohol and the novel antidepressant bupropion. In the first part 8 healthy male volunteers received single doses of 100 mg bupropion hydrochloride orally on 2 occasions accompanied by either ethanol in orange or plain orange drink according to a balanced cross over design. Plasma bupropion concentrations were determined by radioimmunoassay and kinetics analysed with the aid of NONLIN. Blood alcohol levels were assessed by breathalyser. The disposition of bupropion was adequately described by a 2 compartment model and kinetic parameters were not significantly altered by the presence of alcohol. In the second part of the study the same subjects received 40 ml ethanol in orange drink 3.5 h after ingestion of 100 mg bupropion or dummy tablet in a double blind cross over fashion. Bupropion did not affect alcohol kinetics. In contrast to many other psychotropic drugs there is no evidence for a kinetic interaction between bupropion and alcohol.

Tolerance and pharmacokinetics of A515U, an acyclovir analogue, in healthy volunteers.

A515U (6-deoxyacyclovir) is an analogue of acyclovir devoid of antiviral activity in vitro but which is well absorbed and undergoes conversion to acyclovir after oral administration to rats. The tolerance and pharmacokinetics of various doses of A515U have been studied in 8 healthy volunteers. Single oral doses of 25, 50, 100, 200 and 400 mg A515U and 400 mg acyclovir for comparison were administered to the volunteers at weekly intervals. Concentrations of the parent drug and acyclovir were determined in plasma and urine. The prodrug was well tolerated and did not cause adverse reactions or changes in haematological or biochemical variables. It was well absorbed and conversion to acyclovir was rapid and extensive at all doses. Plasma concentrations of acyclovir achieved with 50 mg A515U orally were comparable to and less variable than those produced by 400 mg acyclovir. A515U was rapidly cleared with a short plasma elimination half life of approximately 0.5 h. The attainment of high plasma concentrations of acyclovir by oral administration of a prodrug may represent an important advance in antiviral chemotherapy.