Michiel J. B. Kemme

Influence of a microemulsion vehicle on cutaneous bioequivalence of a lipophilic model drug assessed by microdialysis and pharmacodynamics.

AbstractPurpose. The aim of the study was to investigate the cutaneous bioequivalence of a lipophilic model drug (lidocaine) applied in a novel topical microemulsion vehicle, compared to a conventional oil–in–water (O/W) emulsion, assessed by a pharmacokinetics microdialysis model and a pharmacodynamic method. Methods. Dermal delivery of lidocaine was estimated by microdialysis in 8 volunteers. Absorption coefficients and lag times were determined by pharmacokinetic modelling of the microdialysis data. Subsequently, the anaesthetic effect of the treatments was assessed by mechanical stimuli using von Frey hairs in 12 volunteers. Results. The microemulsion formulation increased the cutaneous absorption coefficient of lidocaine 2.9 times (95% confidence interval: 1.9/4.6) compared with the O/W emulsion–based cream. Also, lag time decreased from 110 ± 43 min to 87 ± 32 min (P = 0.02). The compartmental pharmacokinetic model provided an excellent fit of the concentration–time curves with reliable estimation of absorption coefficient and lag time. A significant anaesthetic effect was found for both active treatments compared to placebo (P < 0.02), but the effect did not diverge significantly between the two formulations. Conclusions. The microemulsion vehicle can be applied to increase dermal drug delivery of lipophilic drugs in humans. The microdialysis technique combined with an appropriate pharmacokinetic model provides a high sensitivity in bioequivalence studies of topically applied substances.

The influence of reduced liver blood flow on the pharmacokinetics and pharmacodynamics of recombinant tissue factor pathway inhibitor

Recombinant tissue factor pathway inhibitor (rTFPI) has been shown to be an effective treatment in animal models of sepsis and is under investigation for human use. Reduced liver blood flow during septic shock may substantially alter the pharmacokinetics of rTFPI because clearance of rTFPI approaches liver blood flow. The aim of this study was to examine the effect of exercise‐induced reduction in liver blood flow on the pharmacokinetics and pharmacodynamics of rTFPI.

Modulation of vasoactivity and platelet aggregation by selective 5-HT receptor antagonism in humans.

Background Distinct serotonin [5-hydroxytryptamine (5-HT)] receptors are involved in platelet aggregation and vasoconstriction. Compounds that simultaneously and selectively inhibit the pertaining 5-HT receptors may therefore represent a therapeutic strategy for arterial thrombosis, observed frequently after atherosclerotic plaque rupture. The vasoactive and antiplatelet effects of the combined 5-HT1B and 5-HT2A receptor blocker SL65.0472-00 were investigated in humans to elucidate the functional involvement of these receptors. Methods Twenty-four volunteers, divided into 2 groups of 12, received an oral dose of 20 mg of SL65.0472-00 or placebo in a randomized, double-blind, crossover study. Pre dose and at 2, 4, and 6 hours after dosing, intra-arterial infusions of 5-HT1B agonist sumatriptan (n = 12) or 5-HT (n = 12) were administered. Forearm blood flow (FBF) was measured using plethysmography, and platelet aggregation was measured using whole blood aggregometry induced by a combination of a threshold collagen concentration and an excess of 5-HT. Treatments were compared using analysis of variance. Results After placebo treatment, infusion of 1 ng·kg−1·min−1 5-HT induced vasodilatation (FBF +80% change from baseline), whereas infusion of 30 and 80 ng·kg−1·min−1 5-HT resulted in vasoconstriction (FBF −25% and −50%). After SL65.0472-00 treatment, all 5-HT doses induced vasodilatation (FBF +25%–60%). Sumatriptan dose dependently decreased FBF (maximally −35%), but this effect was not altered by SL65.0472-00 treatment. 5-HT–induced platelet aggregation was effectively inhibited by 90% after SL65.0472-00 treatment for at least 6 hours. Conclusions SL65.0472-00 has potent antagonistic effect on 5-HT–induced vasoconstriction and platelet aggregation but not on sumatriptan-induced vasoconstriction. This suggests that in humans, SL65.0472-00 is a 5-HT2A blocker without clear 5-HT1B antagonistic activity.

No evidence for functional involvement of 5-HT2B receptors in serotonin-induced vasodilatation in the human forearm.

The receptor involved in the serotonin (5-hydroxytryptamine [5-HT])-induced vasodilatation in the human forearm has not yet been identified. Experimental data point to the 5-HT2B receptor located on the endothelium. RS-127445 (2-amino-4-(4-fluoronaphthyl-1-yl)-6-isopropylpyrimidine) is a novel potent and selective 5-HT2B receptor antagonist. The effect of oral RS-127445 (500 mg) on 5-HT-induced vasodilatation was studied in a double-blind, randomized, placebo-controlled, crossover study in six healthy volunteers. On each study day 5-HT (0.5 ng/kg/min) was infused into the brachial artery for 8 min, before drug administration and at intervals of 20, 65, 110, 230, and 470 min after oral ingestion. At each infusion, plasma samples for study drug assay were taken and forearm blood flow was assessed using venous occlusion plethysmography. Although (log) drug concentrations exceeded pKi, there was no correlation between RS-127445 concentrations and 5-HT-induced vasodilatation. 5-HT-induced vasodilatation did not differ between treatments and time points. It appears that there is no functional involvement of 5-HT2B receptors in 5-HT-mediated vasodilatation in the human forearm.

Endothelial binding of recombinant tissue plasminogen activator: quantification in vivo using a recirculatory model.

Binding of tissue plasminogen activator (t-PA) to the endothelium may be important in the prevention of thrombus formation. The aim was to develop a method to quantify endothelial binding in vivo. Nine healthy male volunteers received a 40 min continuous infusion with low dose recombinant t-PA (3.75 μg/min) and an indocycanine green infusion (0.5 mg/min) as control. A three-compartment recirculatory model was developed to account for non-specific circulatory delay effects. t-PA antigen, activity and t-PA/PAI-1 complex profiles showed a marked delay in increase at the beginning of the infusion. A reversible and concentration-dependent binding component was incorporated in the model which resulted in an accurate description of the t-PA concentration profile. t-PA binding was characterized by a dissociation constant of 5.9 ng/ml (SEM 1.8, CV 0%; fixed) and a binding capacity of 70 μg t-PA (SEM 10, CV 48%). This model can be used as a tool to quantify the ability of the endothelium to bind t-PA.

Clinically Important Interaction Between Tedisamil and Verapamil

Tedisamil, a class III antiarrhythmic drug, is a P‐glycoprotein substrate. Tedisamil treatment may implicate coadministration with class IV antiarrhythmics such as verapamil, a P‐glycoprotein inhibitor. Pharmacokinetic and pharmacodynamic interactions between tedisamil and verapamil were evaluated in a double‐blind, crossover study. Twelve healthy volunteers received a 3‐day treatment of tedisamil (100 mg bid), verapamil (180 mg bid), a combination of these drugs, or placebo. Blood pressure and electrocardiograms were assessed daily and cardiac output and pharmacokinetics on day 3. Combination of tedisamil and verapamil increased tedisamil plasma concentrations (AUC0−12 h: +77%, CI90%: +51% to +108%; Cmax: +78%, CI90%: +57% to +102%) compared to tedisamil monotreatment but decreased plasma concentrations of verapamil (AUC0−12 h: −21%, CI90%: −32% to −8%; Cmax: −28%, CI90%: −39% to −14%) and norverapamil (AUC0−12 h: −17%, CI90%: −28% to −6%; Cmax: −20%, CI90%: −29% to −10%) compared to verapamil monotreatment. Compared to placebo, verapamil and the combination treatment increased PR by 23.5 (CI95%: 17.9 to 29.2) ms and 12.2 (5.7 to 17.0) ms, respectively. Compared to placebo, tedisamil and the combination treatment increased QTc by 27.8 (15.8 to 39.8) ms and 45.7 (33.7 to 57.7) ms, respectively. Thus, concomitant use of tedisamil with P‐glycoprotein inhibitors likely results in clinically significant drug interactions.

No Influence of Acute Hypertriglyceridemia on Plasma t-PA in Healthy Male Volunteers

Dietary effects on liver blood flow may have biased the previously observed effects of hypertriglyceridemia on systemic tissue-type plasminogen activator (t-PA) concentrations. Therefore, in this study the effects of hypertriglyceridemia on plasma t-PA were determined by inducing hypertriglyceridemia with an intravenous fat emulsion (Intralipid) infusion. In a randomised crossover fashion, eight healthy male volunteers received Intralipid 10% (1.5 ml/min) or 0.9% saline for 2 h and 45 min. After 2 h of infusion, t-PA antigen, t-PA activity, t-PA/plasminogen activator inhibitor (PAI-1) complex, and PAI-1 activity were determined. Concomitantly, the effects of Intralipid t-PA clearance were determined from steady-state t-PA antigen concentrations of a 45-min recombinant tissue-type plasminogen activator (rt-PA) infusion (31.25 microg/min). Liver blood flow was assessed from steady-state concentrations of a continuous sorbitol infusion. Differences between treatments were calculated using the prevalue as the covariate. No significant differences were observed in mean+/-S.D. endogenous concentrations of t-PA antigen, 4.5+/-0.9/4.1+/-0.9 ng/ml (Intralipid vs. saline infusion; difference of 0.3 ng/ml, 95% confidence interval, CI: -0.2, 0.8); t-PA activity, 0.69+/-0.21/0.68+/-0.21 U/ml (difference of 0.04 U/ml, CI: -0.17, 0.25); t-PA/PAI-1 complex, 2.0+/-1.3/1.6+/-1.0 ng/ml (difference of 0.1 ng/ml, CI: -0.8, 0.6); and PAI-1 activity, 7.3+/-5.1/7.1+/-5.1 U/ml (difference of 0.26 U/ml, CI: -3.7, 4.3). Mean t-PA clearance and liver blood flow were unaffected by the Intralipid infusion. These results indicate that acute hypertriglyceridemia does not alter plasma fibrinolytic parameters in healthy male volunteers.

Measurement of collagen- and serotonin-induced platelet aggregation in whole blood

Upon erosion and rupture of an atherosclerotic plaque, collagen and serotonin (5-hydroxytyramine [5-HT]) induce a process of simultaneous platelet aggregation and vasoconstriction. Simultaneous inhibition of these pathophysiological processes, attainable by 5-HT inhibition, is a potential drug target and could offer an attractive treatment modality. The availability of a reliable and accurate test to measure inhibition of 5-HT-induced platelet aggregation would facilitate the rational development of such new compounds. Therefore, we developed a validated method to measure the additive effect of 5-HT on platelet aggregation in human whole blood after an initial induction by a low-concentration collagen, using impedance aggregometry. This method is feasible to measure 5-HT-induced platelet aggregation in whole blood for the evaluation of promising platelet aggregation inhibitors possessing 5-HT antagonistic activity. The availability of this method will support and stimulate selective 5-HT antagonism as effective management of thrombosis.

Evaluation of Skeletal Muscle and Adipose Tissue Biopsy Procedures for Microarray-Based Gene-Expression Profiling

Since the publication of the first microarray experiment in 1995 [1], this technology has rapidly evolved and has now become one of the major tools for global geneexpression profiling. The results of recent microarray studies have greatly added to our understanding of e.g., disease pathways [2-5], mechanisms of drug action [6], and tumor classification [7,8] underlining the increasingly important role of geneexpression profiling in biomedicine.

Tissue biopsy techniques for gene‐expression profiling in clinical pharmacology

Gene‐expression profiling studies require sufficient quality/quantity mRNA to obtain valid results. In animal studies, obtaining relevant tissue material is not a problem, but in humans there are obvious constraints. Therefore, we evalauted tissue biopsy techniques, perceived burden and the RNA extraction methods in 12 healthy subjects.