Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

Participation of beta-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release.

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.

Retention of 14C label is lower in old than in young Wistar rats after oral dosing with [14C]pyridoxine.

Ten young (6-mo-old) and ten old (31-mo-old) male Wistar rats fed a purified diet (250 g casein and 6 mg pyridoxine.HCl per kg) from weaning were given either a single oral dose or five repeated oral doses of 14C-labeled pyridoxine. At various times after dosing animals of each age group were killed. Absorption of orally dosed [14C]pyridoxine.HCl was not found to be different between young and old rats. Total body retention of 14C label administered was modestly but significantly lower in old than in young rats. However, distribution of 14C label over various tissues and among the various B-6 vitamers was similar. No significant age-related differences were observed in the biokinetic parameters derived from urinary excretion data. Contrary to the findings for 14C label distribution, age-related differences were observed for absolute level of tissue 14C-labeled vitamers. The lower [14C]pyridoxal-5'-phosphate content in liver and muscle, and [14C]pyridoxamine-5'-phosphate content in liver, of old animals indicated an age-related difference in liver and muscle vitamin B-6 disposition. In both young and old rats, and in both liver and muscle tissue, pyridoxamine-5'-phosphate was observed to be a faster-exchanging tissue vitamin B-6 pool than pyridoxal-5'-phosphate.

Influence of age and sex on vitamin B-6 vitamer distribution and on vitamin B-6 metabolizing enzymes in Wistar rats.

Vitamin B-6 vitamer distribution and activities of vitamin B-6 metabolizing enzymes were evaluated in aging male and female Wistar rats fed a purified diet (containing 250 g of casein and 6 mg of pyridoxine hydrochloride per kg) from weaning until 31 mo of age. Plasma pyridoxal 5'-phosphate (PLP) concentration became lower with increasing age, with the largest decrease in the 1st yr of life. An age-related change in vitamin B-6 distribution between the various tissues examined was observed: B-6 vitamer content increased in heart and brain, whereas PLP content decreased in gastrocnemius muscle, kidney and liver. The decrease in muscle PLP content occurred in concert with a decrease in muscle glycogen phosphorylase activity. Urinary 4-pyridoxic acid (4-PA) excretion increased with age, especially in female rats, in parallel with an increase in liver pyridoxal oxidase and pyridoxal dehydrogenase activities. Age-related changes in vitamin B-6 distribution were probably not causally related to changes in activity of vitamin B-6 metabolizing enzymes; they were regarded as consequences of changes in protein metabolism. The higher urinary 4-PA excretion in older rats may reflect a lower vitamin B-6 requirement; however, the lower PLP content of gastrocnemius muscle may indicate an age-related decrease in vitamin B-6 body stores.

The effect of apolipoprotein A on in vitro apolipoprotein C binding and in vivo removal of artificial triacylglycerol-rich particles.

Although the presence of apolipoprotein A on the surface of chylomicrons and its exchange for apolipoprotein C in the mesenteric lymph is known, the functional role of apolipoprotein A in triacylglycerol transport has not been elucidated. Mimicking the in vivo situation, artificial triacylglycerol rich particles (TGRP) with which apolipoprotein AI had been associated, were incubated with high-density lipoproteins (HDL). It was found that apo AI-TGRP bound approximately twice as much apolipoprotein C from HDL as nonprotein-containing TGRP, losing 75% of the apolipoprotein AI originally present. To test whether an increased apolipoprotein C binding in vitro implicated an increased removal rate in vivo, boluses of apolipoprotein AI preincubated and control TGRP were given intravenously (IV) to six hypertriacylglycerolemic patients. The fractional catabolic rate was 35% (range 6% to 65%) higher for apolipoprotein AI preincubated TGRP than for control TGRP. In accordance with the in vitro incubations, the molar ratio of apolipoprotein C to phospholipid on TGRP reisolated 30 minutes after injection was 39% (range 12% to 115%) higher for apo AI triacylglycerol rich particles (TRP) than for control TGRP. The maximal removal capacity of apo AI-TRP, tested in one patient by constant infusion, was increased 53% as compared to control TGRP. Thus, the function of apolipoprotein AI in triacylglycerol transport may be to enhance apolipoprotein C binding to chylomicrons, which are comparable to TRP, and in doing so to enhance their removal from the plasma compartment.

The CII/CIII ratio of transferable apolipoprotein in primary and secondary hypertriglyceridemia.

Since the apolipoproteins C transfer to triglyceride-rich particles when they enter the lymph-blood compartment, and since the apo CII/CIII ratio may influence the removal of triglyceride-rich particles from that compartment, the apo CII/CIII ratio of proteins that transfer from plasma to an artificial fat emulsion was studied in hypertriglyceridemic and control patients. It appeared (1) that the apo CII/CIII ratio was lower in 10 patients with primary hypertriglyceridemia (0.06 +/- 0.20) than in 13 normals (1.55 +/- 0.73, p less than 0.001) and (2) that it was lower in 16 patients who previously had secondary hypertriglyceridemia, but who were normotriglyceridemic when tested (0.58 +/- 0.24), than in 15 patients with comparable disease but without secondary hypertriglyceridemia (0.91 +/- 0.47, p less than 0.02). These findings suggest that a low CII/CIII ratio of transferable apolipoprotein contributes to the potential development of secondary hypertriglyceridemia.