Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice

Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.

Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in echinococcosis patients

An assay for the simultaneous determination of the enantiomers of hydroxymebendazole (OH-MBZ) and hydroxyaminomebendazole (OH-AMBZ) together with aminomebendazole (AMBZ) in human plasma is described for the first time. It is based upon liquid-liquid extraction at alkaline pH from 0.5 mL plasma followed by analysis of the reconstituted extract by CE with reversed polarity in the presence of a 50 mM, pH 4.2 acetate buffer containing 15 mg/mL sulfated beta-CD as chiral selector. For all compounds, detection limits are between 0.01 and 0.04 microg/mL, and intraday and interday precisions evaluated from peak area ratios are <6.9 and <8.5%, respectively. Analysis of 39 samples of echinoccocosis patients undergoing pharmacotherapy with mebendazole (MBZ) revealed that the ketoreduction of MBZ and AMBZ is highly stereoselective. One enantiomer of each metabolite (firstly detected peak in both cases) could only be detected. The CE data revealed that OH-MBZ (mean: 0.715 microg/mL) is the major metabolite followed by AMBZ (mean: 0.165 microg/mL) and OH-AMBZ (mean: 0.055 microg/mL) whereas the MBZ plasma levels (mean: 0.096 microg/mL, levels determined by HPLC) were between those of AMBZ and OH-AMBZ.

Stereoselective biotransformation of ketamine in equine liver and lung microsomes

Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and, in a second step, into R/S-downstream metabolites. In liver and lung microsomes, levels of R-ketamine exceeded those of S-ketamine at all time points and S-norketamine exceeded R-norketamine at time points below the maximum concentration. In liver and lung microsomes, significant differences in the enzyme velocity (V(max)) were observed between S- and R-norketamine formation and between V(max) of S-norketamine formation when S-ketamine was compared to S-ketamine of the racemate. Our investigations in microsomal reactions in vitro suggest that stereoselective ketamine biotransformation in horses occurs in the liver and the lung with a slower elimination of S-ketamine in the presence of R-ketamine. Scaling of the in vitro parameters to liver and lung organ clearances provided an excellent fit with previously published in vivo data and confirmed a lung first-pass effect.

CE provides evidence of the stereoselective hydroxylation of norketamine in equines

CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of standard compounds, urinary extracts were fractionated by HPLC followed by characterization of the collected fractions with CE and LC-MS(n) with 0.7 mmu mass discrimination. Comparison of LC-MS(n) data obtained with the fractions, an in vitro microsomal sample, and both pony urine and hydrolyzed pony urine led to the identification of four hydroxylated norketamine metabolites with hydroxylation at the cyclohexanone ring, two with hydroxylation at the aromatic ring and four hydroxylated metabolites of ketamine. Due to the lower detection sensitivity, only the four hydroxynorketamine metabolites with hydroxylation at the cyclohexanone ring were observed by CE. The data suggest that demethylation of ketamine followed by hydroxylation of norketamine at the cyclohexanone ring is the major metabolic pathway in equine species and that the ketamine metabolism is highly stereoselective.

Pharmacokinetics and excretion of gamma-hydroxybutyrate (GHB) in healthy subjects

In Europe and the United States, the recreational use of gamma-hydroxy butyric acid (GHB) at dance clubs and "rave" parties has increased substantially. In addition, GHB is used to assist in the commission of sexual assaults. The aim of this controlled clinical study was to acquire pharmacokinetic profiles, detection times, and excretion rates in human subjects. Eight GHB-naïve volunteers were administered a single 25-mg/kg body weight oral dose of GHB, and plasma, urine, and oral fluid specimens were analyzed by using gas chromatography-mass spectrometry (GC-MS). Liquid-liquid extraction was performed after acid conversion of GHB to gamma-butyrolactone. Limits of quantitation of 0.1 (oral fluid), 0.2 (urine), and 0.5 microg/mL (plasma) could be achieved in the selected ion monitoring mode. GHB plasma peaks of 39.4 +/- 25.2 microg/mL (mean +/- SEM) occurred 20-45 min after administration. The terminal plasma elimination half-life was 30.4 +/- 2.45 min, the distribution volume 52.7 +/- 15.0 L, and the total clearance 1228 +/- 233 microL/min. In oral fluid, GHB could be detected up to 360 min, with peak concentrations of 203 +/- 92.4 microg/mL in the 10-min samples. In urine, 200 +/- 71.8 and 230 +/- 86.3 microg/mL, were the highest GHB levels measured at 30 and 60 min, respectively. Only 1.2 +/- 0.2% of the dose was excreted, resulting in a detection window of 720 min. Common side-effects were confusion, sleepiness, and dizziness; euphoria and change of vital functions were not observed. GHB is extensively metabolized and rapidly eliminated in urine and oral fluid. Consequently, samples should be collected as soon as possible after ingestion.

Pharmacokinetics and safety profile of the human anti-Pseudomonas aeruginosa serotype O11 immunoglobulin M monoclonal antibody KBPA-101 in healthy volunteers

KBPA-101 is a human monoclonal antibody of the immunoglobulin M isotype, which is directed against the O-polysaccharide moiety of Pseudomonas aeruginosa serotype O11. This double-blind, dose escalation study evaluated the safety and pharmacokinetics of KBPA-101 in 32 healthy volunteers aged 19 to 46 years. Each subject received a single intravenous infusion of KBPA-101 at a dose of 0.1, 0.4, 1.2, or 4 mg/kg of body weight or placebo infused over 2 h. Plasma samples for pharmacokinetic assessments were taken before infusion as well as 0.25, 0.5, 1, 2, 2.5, 4, 6, 8, 12, 24, 36, and 48 h and 4, 7, 10, and 14 days after start of dosing. Plasma concentrations of KBPA-101 were detected with mean maximum concentrations of drug in plasma of 1,877, 7,571, 24,923, and 83,197 ng/ml following doses of 0.1, 0.4, 1.2, and 4.0 mg/kg body weight, respectively. The mean elimination half-life was between 70 and 95 h. The mean volume of distribution was between 4.76 and 5.47 liters. Clearance ranged between 0.039 and 0.120 liters/h. At the highest dose of 4.0 mg/kg, plasma KBPA-101 levels were greater than 5,000 ng/ml for 14 days. KBPA-101 exhibited linear kinetics across all doses. No anti-KBPA-101 antibodies were detected after dosing in any subject. Overall, the human monoclonal antibody KBPA-101 was well tolerated over the entire dose range in healthy volunteers, and no serious adverse events have been reported.

Significant yield improvement in the stereoselective synthesis of allyl cyanides from Baylis-Hillman derivatives in aqueous medium under the influence of the Phase-Transfer catalyst

The nucleophilic reaction of NaCN with the acetyl derivative of Baylis-Hillman adducts in the presence of a phase-transfer catalyst in aqueous medium stereoselectively affords the corresponding allyl cyanides in a short period and excellent yields.

Stereoselective synthesis and structure determination of a bicyclo[3.3.2]decapeptide

By analogy to the structural diversity of covalent bond networks between atoms within organic molecules, one can design topologically diverse peptides from mathematical graphs by assigning amino acids to graph nodes and peptide bonds to graph edges. The key is to use diamino acids or amino diacids as equivalents of trivalent graph nodes, which enables a variety of graph topologies beyond the standard linear and monocyclic graphs in natural peptides. Here the bicyclic decapeptide A1FGk2VFPE1AG2 (1b) was prepared and crystallized to assign its bridge stereochemistry. The bridge configuration appears as planned by the chirality of the branching amino acids. Bicyclization furthermore depends on the presence of matched chiralities in the branching amino acids. The stereoselective formation of the second bridge opens the way for the synthesis of a large family of bicyclic peptides as promising new scaffolds for drug design.

Pharmacokinetics of GHB and detection window in Serum and Urine after single uptake of a low dose of GLB - an Experiment with two volunteers

During the last few years γ-hydroxybutyric acid (GHB) and γ-butyrolactone (GBL) have attracted much interest as recreational drugs and knock-out drops in drug-facilitated sexual assaults. This experiment aims at getting an insight into the pharmacokinetics of GHB after intake of GBL. Therefore Two volunteers took a single dose of 1.5 ml GBL, which had been spiked to a soft drink. Assuming that GBL was completely metabolized to GHB, the corresponding amount of GHB was 2.1 g. Blood and urine samples were collected 5 h and 24 h after ingestion, respectively. Additionally, hair samples (head hair and beard hair) were taken within four to five weeks after intake of GBL. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after protein precipitation with acetonitrile. The following observations were made: spiked to a soft drink, GBL, which tastes very bitter, formed a liquid layer at the bottom of the glass, only disappearing when stirring. Both volunteers reported weak central effects after approximately 15 min, which disappeared completely half an hour later. Maximum concentrations of GHB in serum were measured after 20 min (95 µg/ml and 106 µg/ml). Already after 4-5 h the GHB concentrations in serum decreased below 1 µg/ml. In urine maximum GHB concentrations (140 µg/ml and 120 µg/ml) were measured after 1-2 h, and decreased to less than 1 µg/ml within 8-10 h. The Ratio of GHB in serum versus blood was 1.2 and 1.6

Pharmacokinetics of meprobamate in overdose treated with continuous venovenous hemodiafiltration (CVVHDF)

We report a case of massive suicidal overdose of meprobamate leading to cardiovascular collapse, respiratory failure, and severe central nervous system depression. We observed first-order elimination kinetics despite significant overdose, and demonstrated effectiveness of continuous venovenous hemodiafiltration (CVVHDF) for extracorporeal removal of meprobamate in this patient. Total body clearance was calculated to be 87 mL/minute, with 64 mL/minute (74%) due to CVVHDF. CVVHDF was stopped after 36 hours, and the patient made an uneventful recovery.

CISPLATIN NEPHROTOXICITY AND ITS EFFECTS ON GENTAMICIN PHARMACOKINETICS

Myelosuppression is a common side effect of anticancer agents such as cisplatin. This makes patients more susceptible to infections. Gentamicin is an aminoglycoside antibiotic that is very effective in the treatment of gram negative infections. Both these drugs are excreted by the kidney, and are also nephrotoxic. Thus, each may affect the disposition of the other. This project deals with the nature and duration of the effects of cisplatin on gentamicin pharmacokinetics in F-344 rats.^ The appropriate cisplatin dose was determined by comparing the nephrotoxicity of four intravenous doses--3, 4, 5, and 6 mg/kg. The 6 mg/kg dose gave the most consistent nephrotoxic effect, with peak plasma urea nitrogen and creatinine levels on the 7th day. Plasma and tissue gentamicin levels were compared between rats given gentamicin alone (30 mg/kg, intraperitoneally, twice a day for four days), and those given cisplatin (6 mg/kg, intraperitoneally) with the first gentamicin dose. Cisplatin caused a significant elevation of gentamicin levels in plasma, liver, and spleen. However, cisplatin given in three weekly doses of 2 mg/kg each, had no effect on plasma or tissue gentamicin levels.^ In order to determine the duration of cisplatin effects, a single dose of gentamicin (30 mg/kg, intravenously) was given to different groups of rats either alone, or on day 1, 4, 7, 15, or 29 following cisplatin (6 mg/kg, intravenously on day 1). Plasma samples were collected through a cannula placed on the external jugular vein at 0.5, 1, 2, 3, 4, 5, and 6 hours after gentamicin; the rats were sacrificed at 24 hours. Cisplatin caused a significant decrease in gentamicin excretion and an elevation of gentamicin levels in plasma, kidneys, liver, and spleen at all the time points that were tested, except with concomitant administration. Plasma urea nitrogen was elevated, and creatinine clearance decreased by the 4th day after cisplatin and these continued to be significantly different even on the 29th day after cisplatin.^ These results demonstrate that cisplatin nephrotoxicity reduced gentamicin excretion for at least a month in F-344 rats. This could increase the risk of toxicity from the second drug by elevating its levels in plasma and tissue. Thus, caution should be exercised when renally excreted drugs are given after cisplatin. ^

The pharmacokinetics of dexmedetomidine administered as a constant rate infusion in horses.

Dexmedetomidine, the most selective α2 -adrenoceptor agonist in clinical use, is increasingly being used in both conscious and anaesthetized horses; however, the pharmacokinetics and sedative effects of this drug administered alone as an infusion are not previously described in horses. Seven horses received an infusion of 8 μg dexmedetomidine/kg/h for 150 min, venous blood samples were collected, and dexmedetomidine concentrations were assayed using liquid chromatography-mass spectrometry (LC/MS) and analyzed using noncompartmental pharmacokinetic analysis. Sedation was scored as the distance from the lower lip of the horse to the ground measured in centimetre. The harmonic mean (SD) plasma elimination half-life (Lambda z half-life) for dexmedetomidine was 20.9 (5.1) min, clearance (Cl) was 0.3 (0.20) L/min/kg, and volume of distribution at steady-state (Vdss ) was 13.7 (7.9) L/kg. There was a considerable individual variation in the concentration of dexmedetomidine vs. time profile. The level of sedation covaried with the plasma concentration of dexmedetomidine. This implies that for clinical use of dexmedetomidine constant rate infusion in conscious horses, infusion rates can be easily adjusted to effect, and this is preferable to an infusion at a predetermined value.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging

UNLABELLED Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.