Pharmacokinetics of ethylenediaminemalonatoplatinum(II) (JM-40) during phase I trial

Pharmacokinetics of the cis-platin analog ethylenediaminemalonatoplatinum(II) (JM-410) was studied in 28 cycles of 19 patients during the phase I study of this drug. The drug was administered intravenously by short-term (10-60 min) infusion. Doses ranged from 20 to 1,200mg m-2. JM-40 was determined in plasma ultrafiltrate and urine by HPLC. Platinum (Pt) concentrations were determined in plasma, plasma ultrafiltrate, urine and red blood cells by atomic absorption spectrometry up to 5 days after administration of the drug. Ultrafilterable Pt could be determined up to 45 days after the infusion in one patient sampled over such a long period. Pharmacokinetics of JM-40 showed a linear behaviour. The final half-life of total Pt in plasma was 4.1 +/- 0.9 days. The disposition of JM-40 was similar to that of ultrafilterable Pt in respect to t1/2 alpha (10 and 13 min), t1/2 beta (44 and 57 min), volumes of distribution Vc (11 and 121) and Vss (17 and 201), systemic clearance (256 and 223 ml min-1), renal clearance (69 and 73 ml min-1) and metabolic clearance (183 and 154 ml min-1). During the first 6 h 27 +/- 9% of the administered dose was excreted as JM-40. Cumulative platinum excretion in the urine amounted to 29 +/- 13% and 60 +/- 13% over the first 6 h, 24 h and 5 days, respectively. The uptake of platinum in red blood cells was limited, comprising only 0.24 +/- 0.12% of the administered dose. Although JM-40 and carboplatin are structurally closely related, pharmocokinetics and toxicity of JM-40 were more similar to cis-platin than to carboplatin.     

Class III P-glycoproteins mediate the formation of lipoprotein X in the mouse.

Cholestasis is associated with hypercholesterolemia and appearance of the abnormal lipoprotein X (LpX) in plasma. Using mice with a disrupted Mdr2 gene, we tested the hypothesis that LpX originates as a biliary lipid vesicle. Mdr2-deficient mice lack Mdr2 P-glycoprotein, the canalicular translocator for phosphatidylcholine, and secrete virtually no phospholipid and cholesterol in bile. Bile duct ligation of Mdr2(+)/+ mice induced a dramatic increase in the plasma cholesterol and phospholipid concentration. Agarose electrophoresis, density gradient ultracentrifugation, gel permeation, and electron microscopy revealed that the majority of phospholipid and cholesterol was present as LpX, a 40-100 nm vesicle with an aqueous lumen. In contrast, the plasma cholesterol and phospholipid concentration in Mdr2(-)/- mice decreased upon bile duct ligation, and plasma fractionation revealed a complete absence of LpX. In mice with various expression levels of Mdr2 or MDR3, the human homolog of Mdr2, we observed that the plasma level of cholesterol and phospholipid during cholestasis correlated very closely with the expression level of these canalicular P-glycoproteins. These data demonstrate that during cholestasis there is a quantitative shift of lipid secretion from bile to the plasma compartment in the form of LpX. The concentration of this lipoprotein is determined by the activity of the canalicular phospholipid translocator.     

Hepatic,intestinal and renal transport of 1-naphthol-β-d-glucuronide in mutant rats with hereditary-conjugated hyperbilirubinemia

Summary Recently, a mutant rat strain was described with a genetic defect for the biliary excretion of organic anions (TR^– rats). To determine the possible heterogeneity of the transport systems in liver, intestine and kidney we investigated the transport of the anion 1-naphthol--d-glucuronide (1-NG) in isolated vascularly perfused organ preparations of the rat liver, intestine and kidney of both Wistar rats and TR^– rats. 1-NG was administered as such (liver and kidney experiments) or formed intracellularly from 1-naphthol (1-N) (liver and gut experiments). Independent of the type of exposure to 1-NG, the biliary excretion was considerably impaired in TR^– rats. In the intestine the total appearance and the vascular/luminal distribution pattern of 1-NG were not significantly different from the values in control rats. Furthermore, no significant disturbance was found with respect to the renal clearance of 1-NG in the TR^– rat when compared with the Wistar rat. Thus, the genetic defect in the TR^– rat is restricted to an impaired hepatobiliary excretion of 1-NG and does not affect the excretory systems of the intestine and kidney. These results suggest that the excretion of 1-NG by the liver, intestine and kidney involves distinct organ-specific transport systems.     

Mode of activation of the metabolic burst in polymorphonuclear leukocytes by calcium oxalate crystals

Microcrystals of calcium oxalate cause an activation of the metabolic burst in polymorphonuclear leukocytes, measured as NBT reduction. Crystal-induced NBT reduction, which was mainly due to superoxide release, was accompanied by enzyme release. Modulation of calcium oxalate-induced activation by several agents resembles the effect of these agents on activation by opsonized zymosan and by phorbol myristate acetate. Both the activation of the metabolic burst and concomitant enzyme release could be counteracted by certain anions, such as oxalate, pyruvate and citrate, indicating that positive charges on the crystals play an important role in crystal-cell interaction. Removal of the negatively charged sialic acid from the cell surface by neuraminidase did not affect the action of the crystals. The mechanism, by which calcium oxalate activates polymorphonuclear leukocytes, is discussed.     

Activation of neutrophil migration by dioctanoyl-sn-glycerol and fMet-Leu-Phe is controlled by different pathways

Migration activated by fMet-Leu-Phe is inhibited by GTP[S] and is little affected by protein kinase C inhibitors. We investigated the effects of GTP[S] and the protein kinase C inhibitor AMG-C₁₆ on dioctanoyl-sn-glycerol (DiC8)-activated migration of rabbit neutrophils and compared them with the effects on fMet-Leu-Phe-activated migration and random migration. GTP[S] did not inhibit DiC8-activated migration or random migration but inhibited fMet-Leu-Phe-activated migration. AMG-C₁₆ gave a strong inhibition of DiC8-activated migration but had only a small effect on fMet-Leu-Phe-activated migration and random migration. When fMet-Leu-Phe and DiC8 were added together in suboptimal concentrations an additive effect was found. Pretreatment with the diacylglycerol kinase inhibitor R59022 enhanced random migration. The enhancement was completely inhibited by AMG-C₁₆ and was unaffected by GTP[S]. These findings suggest that DiC8-activated migration and fMet-Leu-Phe-activated migration are controlled by different pathways.

     

Phenotypic and functional characterization of human suppressor T-cell clones: II. Activation by Mycobacterium leprae presented by HLA-DR molecules to alpha beta T-cell receptors.

We have been studying human T-cell clones that suppress anti-mycobacterial T-cell responses but not T-cell responses to an unrelated antigen or mitogen. In the present paper we report our studies on the activation requirements of these suppressor-T-cell clones. The suppressor-T-cell clones could proliferate and produce interferon-gamma upon stimulation with Mycobacterium leprae and other mycobacteria but not with unrelated antigens or autologous T cells. Both suppressor and nonsuppressor clones react to a 36-kDa antigen of M. leprae. Thus far, we have not been able to demonstrate whether they see the same or different epitopes. The antigen-driven proliferation of suppressor-T-cell clones was, however, significantly lower than that observed for T-cell clones that did not mediate suppression. The proliferation of suppressor-T-cell clones to M. leprae antigens could be blocked by monoclonal antibodies to HLA-DR, alpha beta T-cell receptor, interleukin-2 receptor, and, in the case of CD4-positive suppressor-T-cell clones, anti-CD4 monoclonal antibodies. DR restriction of the antigen presentation to these suppressor-T-cell clones was shown in mixing experiments using antigen-presenting cells as mononuclear cells from family members and unrelated individuals. These experiments also indicated that apart from regular DR-restriction a hitherto unknown factor may be required for presentation to or activation of suppressor-T-cell clones that is present in the family members and unrelated individuals with the same ethnic and geographic background but absent in DR/Dw-matched healthy Dutch individuals.     

Quantitative and qualitative differences in HLA-DR molecules correlated with antigen-presentation capacity

The monoclonal antibodies 7.3.19.1 (anti-DRw52-like) and B8.11.2 (anti-DR framework) were used for the isolation and characterization of HLA class II molecules expressed by HLA-DR3 and DR5 homozygous B cell lines. Sequential immunoprecipitation studies demonstrated that from these cells class II molecules can be isolated which are characterized by the presence or absence of DR framework (DR) and DRw52-like (DRw62) determinants: (DR+, DRw52+), (DR+, DRw52-) and (DR-, DRw52+). The DR3 donor cells appeared to express only the (DR+, DRw52+) and (DR-, DRw52+) class II molecules whereas DR5-positive cells express only the (DR+, DRw52+) and (DR+, DRw52-) class II molecules. Besides qualitative differences some of the above-mentioned molecules appeared to differ in their levels of expression. To investigate whether this might have functional implications, cells with the HLA-DR3 and -5 haplotypes were used to present antigen purified protein derivative of tuberculin (PPD) to PPD-specific T cell lines and the blocking capacity of the two monoclonal antibodies 7.3.19.1 and B8.11.2 was determined. A remarkable correlation was observed between the type of class II molecule blocked by these monoclonal antibodies and its quantitative expression. However, (DR-, DRw52+) molecules, clearly expressed by DR3 cells, were not involved in the presentation of PPD. This indicates that not only quantitative but also qualitative aspects may play a role in the selection of the type of class II molecule that will be involved in antigen presentation.     

Major histocompatibility complex class II-restricted antigen presentation across a species barrier: conservation of restriction determinants in evolution.

The existence of at least three alleles of the HLA-DRB3 gene within the human population is evident. These alleles express DRw52 determinants and react with monoclonal antibody (mAb) 7.3.19.1. The polymorphic epitope recognized by 7.3.19.1 is not only present on human cells but is also expressed on chimpanzee (Pan troglodytes) class II-positive cells. The 7.3.19.1 determinant already existed before speciation of man and chimpanzee, and is at least 5,000,000 yr old. Two-dimensional gel electrophoresis demonstrated that the various HLA- and Patr-DRw52 molecules that are reactive with 7.3.19.1 exhibit isoelectric point differences due to primary amino acid heterogeneity, as was confirmed by sequencing data. Sequence comparison allowed us to map the binding site of mAb 7.3.19.1 to the alpha helix of the major histocompatibility complex (MHC) class II DRB1 domain surrounding the antigen-binding cleft. Despite MHC sequence variation, chimpanzee antigen-presenting cells can present antigen (purified protein derivative) to human T cell lines and vice versa. Only the HLA- and Patr-DRw52 molecules were shown to function as restriction elements for antigen presentation across this species barrier. It is concluded that these particular restriction determinants probably have been conserved in evolution. The HLA- and Patr-DRw52 molecules represent alleles displaying polymorphism that has been selected for in evolution. Such "biomutants" may thus be more useful to study the biological significance of MHC molecules than MHC variants that have been generated by in vitro mutagenesis experiments.     

Lack of enteral nutrition during critical illness is associated with profound decrements in biliary lipid concentrations.

BACKGROUND: Food in the intestine drives the enterohepatic circulation of bile components. OBJECTIVE: We investigated whether parenteral or enteral delivery of nutrients alters serum and biliary lipids in critically ill patients. DESIGN: Eight intensive care unit (ICU) patients who had received >/= 5 d of total parenteral nutrition (TPN) were compared with 8 ICU patients who had fasted for >/=5 d. Both groups were studied before and after 5 d of enteral nutrition (EN). Each patient served as his or her own control. Duodenal bile was analyzed for biliary lipid content and serum lipids were determined simultaneously. Duodenal bile samples from 18 healthy persons served as controls. RESULTS: Bile salt concentrations in all ICU patients were 17% of control values before EN (P < 0.005) and 34% of control values after 5 d of EN (P < 0.005). Phospholipid concentrations were 12% of control before EN (P < 0. 0005) but increased almost 4-fold after EN (P < 0.0005). Biliary cholesterol concentrations were 20% of control values before EN (P < 0.001) and did not improve afterward. No difference in bile composition was observed between fasted ICU patients and those who received TPN. The inverse correlation between the severity of illness and biliary lipid concentrations observed before EN disappeared with enteric stimulation. The low serum concentrations of HDL cholesterol and apolipoprotein A-I increased significantly with EN in all ICU patients. CONCLUSION: Lack of EN during critical illness was associated with profound decrements in biliary lipid concentrations that normalized partially after 5 d of EN. We hypothesize that loss of enteric stimulation in ICU patients impairs hepatic lipid metabolism.