Morphine diesters. II. Blood metabolism and analgesic activity in the rat

The kinetics of hydrolysis of dipropanoylmorphine (DPM) and dibutanoylmorphine (DBM) in human blood fractions and for diacetylmorphine (DAM) and DBM in rat blood fractions were investigated. In each case the hydrolysis of morphine diesters terminated with the production of the corresponding 6-monoester derivative. Generally, decreases in Km and Vmax were observed for the plasma, red blood cell (RBC) cytosol, and RBC membrane esterases responsible for morphine diester hydrolysis as the alkyl chain length of the ester moiety increased. This resulted in an overall decrease in the rate of hydrolysis of morphine diesters by human or rat blood with longer chain homologs of DAM. The analgesic potency and duration of morphine, DAM, and DBM were assessed at various i.v. dosages in the rat by means of the tail-flick latency test. A comparison of equianalgesic doses of morphine, DAM, and DBM indicated that DAM and DBM were 11.5 and 6 times as potent and 0.8 and 1.2 times as long acting, respectively, as morphine.     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.     

A maternal-fetal comparison of ovine plasma esterases.

The esterases of maternal and fetal ovine plasma were characterized and compared early in the third trimester of gestation. Various techniques were used including statistical evaluation of kinetic parameters, gel-filtration and sensitivity to various activators and inhibitors. Maternal esterase activity, expressed in terms of p-nitrophenyl acetate hydrolysis, was found to be 8-fold higher than fetal activity and although both arylesterase and aliesterase appeared to be present in fetal plasma, their contribution to the total activity was relatively small.     

Distribution tissulaire des estérases chez l'etourneau sansonnet (Sturnus Vulgaris)

Tissue distribution of esterase was examined by electrophoresis in the blood, liver and heart tissues of the starling Sternus vulgaris. Esterases have been found in all tissues examined but the profile varies from tissue to tissue, depending on the absence or presence of particular isozymes, as well as the relative activity of these that are present. Overall, liver has the most esterase activity; red cells and plasma, liver tissue and heart tissue have distinctly different esterases.     

The response of trout red cells to adrenaline during seasonal acclimation and changes in temperature

Adult rainbow trout were acclimated to three different temperature and photoperiod regimes: 17°C, 14 h light: 10 h dark (summer); 7° C, 14 h light: 10 h dark; and 5° C, 8 h light: 16 h dark (winter). Blood was collected from these fish after 40 days acclimation, and the response of red blood cells to in vitro adrenergic stimulation was assessed. To examine potential seasonal variations in endogenous levels of circulating catecholamines, plasma levels of adrenaline (Ad) and noradrenaline (NAd) were measured at rest and after exercise. At rest, there were no differences between groups in plasma levels of either Ad or NAd, but, after exercise, the pattern of catecholamine elevation differed. In fish acclimated to 17 and 7° C in summer, Ad and NAd increased by about the same amount (10–15 times). In fish acclimated to 5° C in winter, NAd increased about three-fold, compared to the near 50-fold increase in Ad levels. Whether this difference between groups can be attributed to seasonal influences is unclear. At both low (0·5%) and high (2%) PCO ₂, adrenergic stimulation (2 × 10^-7 M Ad) of trout red cells in vitro led to a significant reduction in MCHC (mean cell [haemoglobin]), compared to non-stimulated cells. However, only at the high PCO ₂ were pHe and red cell pHi significantly different from those in the non-stimulated cells: the latter was higher and the former lower in the stimulated cells. There were no differences in the response of red cells to adrenergic stimulation between groups of fish. Under the conditions of the present study no influence of season and/or temperature on the in vitro response of trout red cells to adrenergic stimulation was shown.     

Determination of tripamide and its metabolites in plasma,red blood cells and urine by high-performance liquid chromatography

A procedure for the determination of tripamide and its hydroxylated metabolites in plasma, red blood cells and urine by reversed-phase high-performance liquid chromatography is described.The concentrations in red blood cells showed a monophasic decline and the half-life was 9.5 h. The concentration in red blood cells was markedly higher than that in plasma, showing that 95–98% of the drug is present in whole blood, after a dose of tripamide (90 mg) in man. The specificity and sensitivity of this procedure appear to be satisfactory for pharmacokinetic studies.     

Acute effects of nitrite on ion regulation in two neotropical fish species

To broaden the understanding of physiological responses of tropical fish to environmental stressors, the effects of nitrite on haematological parameters and plasma and red blood cell ion regulation were studied in two neotropical fish species, Astyanax altiparanae and Prochilodus lineatus. Both fish species were exposed to NaNO2 (30 mg l(-1)) over a 96-h period and blood samples were taken for ion and haematological analyses. The results revealed that nitrite leads to a decrease in P. lineatus blood haematocrit and haemoglobin content and an increase in blood methaemoglobin. A. altiparanae did not exhibit any significant difference in these haematological parameters. During the exposure to NO2- both fish species had significantly reduced plasma Na+ concentration and red blood cell (RBC) K+ concentration, but only P. lineatus showed an increase in extracellular K+ concentration. When RBC volume was analyzed in vitro, after 2 min of exposure to NaNO2, a 36% shrinkage was observed in P. lineatus cells, while only a 10% shrinkage was observed in A. altiparanae cells. These results suggest that for P. lineatus, nitrite entrance into the cell leads to methaemoglobin formation and K+ efflux, causing red cell shrinkage and increased plasma K+. However, A. altiparanae proved to be a species more resistant to nitrite, exhibiting fewer responses to this compound.     

The fluorometric microculture cytotoxicity assay

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.     

Development of a Physiologically Based Model to Describe the Pharmacokinetics of Methylphenidate in Juvenile and Adult Humans and Nonhuman Primates

The widespread usage of methylphenidate (MPH) in the pediatric population has received considerable attention due to its potential effect on child development. For the first time a physiologically based pharmacokinetic (PBPK) model has been developed in juvenile and adult humans and nonhuman primates to quantitatively evaluate species- and age-dependent enantiomer specific pharmacokinetics of MPH and its primary metabolite ritalinic acid. The PBPK model was first calibrated in adult humans using in vitro enzyme kinetic data of MPH enantiomers, together with plasma and urine pharmacokinetic data with MPH in adult humans. Metabolism of MPH in the small intestine was assumed to account for the low oral bioavailability of MPH. Due to lack of information, model development for children and juvenile and adult nonhuman primates primarily relied on intra- and interspecies extrapolation using allometric scaling. The juvenile monkeys appear to metabolize MPH more rapidly than adult monkeys and humans, both adults and children. Model prediction performance is comparable between juvenile monkeys and children, with average root mean squared error values of 4.1 and 2.1, providing scientific basis for interspecies extrapolation of toxicity findings. Model estimated human equivalent doses in children that achieve similar internal dose metrics to those associated with pubertal delays in juvenile monkeys were found to be close to the therapeutic doses of MPH used in pediatric patients. This computational analysis suggests that continued pharmacovigilance assessment is prudent for the safe use of MPH.     

Transfer of excess cholesterol from human red blood cells to plasma in vitro

This study examined the ability of plasma and plasma fractions from normolipidaemic subjects and plasma from a patient with homozygous familial high density lipoprotein deficiency (Tangier disease) to promote loss of excess cholesterol from red blood cells in vitro. Isolated high density lipoproteins were the most potent plasma fraction for removing excess cellular cholesterol. Lipoprotein-deficient plasma and human serum albumin, but not very low density lipoproteins and low density lipoproteins, also removed excess cholesterol from the red blood cells. The near absence of high density lipoproteins in plasma from the patient with Tangier disease did not result in an abnormally low rate of cholesterol loss from the enriched red blood cells. These results suggest that normal levels of high density lipoproteins are not vital for the removal of excess cholesterol from red blood cells by plasma.     

Aluminum transfer as glutamate complex through blood-brain barrier

In vitro distribution of aluminium between plasma and erythrocytes has been studied in the presence of variable amounts of sodium L-glutamate. With a red blood cell suspension in isotonic sodium chloride, aluminium remains confined in erythrocytes even when the sodium L-glutamate concentration increases in the medium. Aluminium initially present in plasma penetrates red blood cells when sodium L-glutamate increases in whole blood, showing that this metal is able in vitro to cross the erythrocyte membrane as glutamate complex. In vivo experiments with male Wistar rats prove that aluminium is also able to pass the blood--brain barrier as glutamate complex and deposit in the brain cortex.     

Erythrocyte adenosine triphosphate depletion during voluntary hyperventilation

Chronic hypophosphatemia in humans is associated with a slow depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, combined with shape alteration, impaired deformability, and viability of the cells. Likewise, incubation of erythrocytes in alkaline solution is associated with ATP depletion. Since in hyperventilation both hypophosphatemia and alkalosis are present, we have investigated red cell organic phosphates, shape, deformability, and osmotic fragility before, during, and after 20 min of voluntary hyperventilation. On the average, red cell ATP decreased by 42%, the blood pH increased by 0.2 units, and plasma inorganic phosphorus decreased by 46% compared with the initial values. Red cell 2,3-DPG, shape, deformability, and osmotic fragility remained unchanged. After the end of hyperventilation ATP increased rapidly to control values in parallel with the normalization of the blood pH, whereas inorganic plasma phosphorus remained at the low level observed during hyperventilation. It is concluded that the combined effects of hypophosphatemia and alkalosis in acute hyperventilation lead to an isolated fall of red cell ATP, which occurs as rapid as after total inhibition of red cell glycolysis in vitro.     

5'-Esters of 2'-deoxyadenosine and 2-chloro-2'-deoxyadenosine with cell differentiation-provoking agents

Phenylacetic and retinoic acids are carboxyacidic cell differentiating agents displaying anticancer activities. We report on a new class of compounds including the 5'-esters of 2'-deoxyadenosine (dA) or 2-chloro-2'-deoxyadenosine (cladribine, 2CdA) and the aforementioned acids. The rationale behind the synthesis of these esters was that if they are hydrolyzed inside the lymphoid cells, either dA will be removed from the intracellular environment by deamination, or 2CdA will be phosphorylated and accumulated. In either case targetted delivery of the differentiating agent to the lymphoid cells may be envisaged. The said compounds were synthesized by the Mitsunobu procedure employing triphenylphosphine and azadicarboxylic acid esters, and their stability was tested against various esterases. Esters of dA and 2CdA with phenylacetic acids were found to be resistant to enzymatic hydrolysis, whereas those with retinoic acids were efficiently hydrolyzed by commercially available hepatic esterase as well as by esterases present in the blood plasma and in diluted human lymphocyte lysate. Susceptibility to enzymatic hydrolysis was found to be a prerequisite of cytotoxic and/or differentiating activity of these esters in leukemic cell lines.     

Alteration of gangliosides in plasma and red cells of humans bearing melanoma tumors

In melanoma tumor-bearing humans, the levels of lipid-bound sialic acid were significantly elevated in both plasma and erythrocytes. Disialosyllactosylceramide in the plasma and sialosyllactosylceramide in red cells were the gangliosides mainly concerned by the increase, as compared to their concentration in the blood of healthy humans. In surgically treated patients, the plasma gangliosides remained higher than the controls, whereas a downward trend was noticeable in red cells. It is suggested that the occurrence of increased amounts of disialosyllactosylceramide in patients' plasma reflects the previously shown presence of this ganglioside as a major component in the sialic acid-containing glycolipid fraction of malignant melanocytes.     

Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administration

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.     

The effect of host blood in the in vitrotransformation of bloodstream trypanosomes by tsetse midgut homogenates

Abstract. Midgut homogenates prepared from Glossina morsitans morsitans , that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.     

A new acivicin prodrug designed for tumor-targeted delivery

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.     

Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels

Background

Acute stress drives a ‘high-alert’ response in the immune system. Psychoactive drugs induce distinct stress hormone profiles, offering a sought-after opportunity to dissect the in vivo immunological effects of acute stress in humans.

Methods

3,4-methylenedioxymethamphetamine (MDMA), methylphenidate (MPH), or both, were administered to healthy volunteers in a randomized, double-blind, placebo-controlled crossover-study. Lymphocyte subset frequencies, natural killer (NK) cell immune-phenotypes, and changes in effector function were assessed, and linked to stress hormone levels and expression of CD62L, CX3CR1, CD18, and stress hormone receptors on NK cells.

Results

MDMA/MPH > MDMA > MPH robustly induced an epinephrine-dominant stress response. Immunologically, rapid redistribution of peripheral blood lymphocyte-subsets towards phenotypically mature NK cells occurred. NK cytotoxicity was unaltered, but they expressed slightly reduced levels of the activating receptor NKG2D. Preferential circulation of mature NK cells was associated with high epinephrine receptor expression among this subset, as well as expression of integrin ligands previously linked to epinephrine-induced endothelial detachment.

Conclusion

The acute epinephrine-induced stress response was characterized by rapid accumulation of mature and functional NK cells in the peripheral circulation. This is in line with studies using other acute stressors and supports the role of the acute stress response in rapidly mobilizing the innate immune system to counteract incoming threats.     

Influence of prolonged physical exercise on the erythropoietin concentration in blood

Erythropoietin (EPO) and red blood cells were studied in 15 well-trained men before and several times after a marathon run. Changes in red blood cells reflected changes of plasma volume. Immediately after the run, red blood cells were increased due to haemoconcentration, whereas 31 h later the values were decreased due to haemodilution. The EPO concentration was increased 3 h, and more impressive 31 h, after the run. This long-lasting increase in EPO concentration after the marathon run would seem to be responsible for the increased red blood cell mass in long distance runners.     

An Evaluation of Erythrocytes as Plasma Glutamate Scavengers for Enhanced Brain-to-Blood Glutamate Efflux

Several acute brain pathological conditions are characterized by the presence of excess glutamate in brain interstitial fluid. We have previously shown that decreasing blood glutamate levels increases the driving force for an enhanced brain-to-blood efflux of glutamate. The present study investigated the glutamate pumping ability of glutamate-depleted erythrocytes both in vitro and in vivo to determine whether the latter could potentially be used in a blood exchange procedure for neuroprotection. We have observed that glutamate is taken up in red blood cells only via a passive diffusive process with a diffusion constant of 0.144/h. When glutamate-depleted blood cells resuspended in 6% hetastarch were injected into recipient rats, using a blood exchange protocol, a decrease of blood glutamate was observed but attributed to plasma dilution. These observations are discussed in light of a novel neuroprotective strategy based on blood glutamate scavenging.