Simultaneous determination of the novel tyrosine kinase inhibitor meditinib and its active metabolite demethylation meditinib in monkey plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

Meditinib (ME) is a novel tyrosine kinase inhibitor used as an antichronic myeloid leukemia drug. A simple, sensitive and specific LC/MS/MS method was developed and validated for the analysis of ME and its metabolite demethylation meditinib (PI) in monkey plasma using naltrexone as the internal standard. Sample preparation involved protein precipitation with methanol. The analysis was carried out on an Agilent C8 column (3.5 µm, 2.1 × 50 mm). Elution was achieved with a mobile phase gradient varying the proportion of a water solution containing 0.1% formic acid (solvent A) and a 0.1% formic acid in methanol solution (solvent B) at a flow rate of 300 µL/min. The method had a linear calibration curve over the concentration range of 2-1000 ng/mL for ME and 2-1000 ng/mL for PI. The lower limits of quantification of ME and PI were 2 and 2 ng/mL, respectively. The intra- and inter-day precision values were <15% and accuracy values were within ±10.0%. The mean recoveries of ME and PI from plasma were >85%. The assay has been successfully used for pharmacokinetic evaluation of ME and PI using the monkey as an animal model, and those data are reported for the first time.

Blood meal analysis of Anopheles vectors of simian malaria based on laboratory and field studies.

Blood feeding and host-seeking behaviors of a mosquito play an imperative role in determining its vectorial capacity in transmitting pathogens. Unfortunately, limited information is available regarding blood feeding behavior of Anopheles species in Malaysia. Collection of resting Anopheles mosquitoes for blood meal analysis poses a great challenge especially for forest dwelling mosquitoes. Therefore, a laboratory-based study was conducted to evaluate the potential use of mosquitoes caught using human landing catch (HLC) for blood meal analysis, and subsequently to document blood feeding behavior of local Anopheles mosquitoes in Peninsular Malaysia. The laboratory-based experiment from this study revealed that mosquitoes caught using HLC had the potential to be used for blood meal analysis. Besides HLC, mosquitoes were also collected using manual aspirator and Mosquito Magnet. Overall, 47.4% of 321 field-caught Anopheles mosquitoes belonging to six species were positive for vertebrate host DNA in their blood meal. The most frequent blood meal source was human (45.9%) followed by wild boar (27.4%), dog (15.3%) and monkey (7.5%). Interestingly, only Anopheles cracens and Anopheles introlatus (Leucosphyrus Group) fed on monkey. This study further confirmed that members of the Leucosphyrus Group are the predominant vectors for knowlesi malaria transmission in Peninsular Malaysia mainly due to their simio-anthropophagic feeding behavior.

Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry.

A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC-MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC-MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.

Promiscuity and the primate immune system.

The behavioral and ecological factors involved in immune system evolution remain poorly explored. We present a phylogenetic analysis of white blood cell counts in primates to test three hypotheses related to disease risk: increases in risk are expected with group size or population density, exposure to soil-borne pathogens, and mating promiscuity. White blood cell counts were significantly greater in species where females have more mating partners, indicating that the risk of sexually transmitted disease is likely to be a major factor leading to systematic differences in the primate immune system.

A simple and reliable method to blood type monkeys using serum samples.

Monkeys are frequently used in experimental transplantation research because of their physical traits and availability. As ABO incompatibility may result in humoral injury, it is important to identify the ABO blood typing of monkeys before transplantation. However, monkeys lack expression of ABH antigens on red blood cells, which makes accurate determination of the blood type difficult. The gel agglutination assay has been widely used as a routine blood grouping test clinically for more than 10 years. In this study, we evaluated the efficacy and the interference factors of using the gel system (including the direct gel system and the reverse gel system) for ABO typing in rhesus monkeys (n = 38) and cynomolgus monkeys (n = 26). Immunohistochemistry assay was used to obtain the accurate blood type data of monkeys. The results revealed that the direct gel system was ineffective in blood typing of monkeys, whereas the reverse gel system assay, which is based on preabsorbed serum, provided reproducible results that were confirmed by histologic analysis. We conclude that the reverse gel system assay with use of preabsorbed serum is a simple and reliable method for ABO typing of monkeys.

Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC-MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum.

PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC-MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k'), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of logk' versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC-MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.

Per- and polyfluoroalkyl substances (PFASs) in the blood of two colobine monkey species from China: Occurrence and exposure pathways.

Per-/polyfluoroalkyl substances (PFASs), which are widely used in industrial and commercial products, have been identified as global and ubiquitous pollutants. Despite this, limited data are available regarding the impacts of PFAS exposure and intake in non-human primates. Here, we report for the first time on the occurrence of PFASs in the blood and dietary sources of two rare and endangered primate species, namely, the golden snub-nosed monkey (Rhinopithecus roxellana) and Francois' leaf monkey (Trachypithecus francoisi). Results showed that perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) were dominant and found at the highest proportions in the blood of both species at the four study sites. The ∑PFAS levels in blood samples from captive golden snub-nosed monkeys in Tongling Zoo (mean: 2.51 ng/mL) and Shanghai Wild Zoo (3.52 ng/mL) near urbanized areas were one order of magnitude higher than the levels in wild monkeys from Shennongjia Nature Reserve (0.27 ng/mL). Furthermore, significant age positive relationships for perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS), and 6:2 chlorinated polyfluorinated ether sulfonates (6:2 Cl-PFESA) were observed in both golden snub-nosed monkeys at Shanghai Wild Zoo and Francois' leaf monkeys at Wuzhou Breeding Center. In addition, PFAS levels in frequently consumed food and drinking water were analyzed for Francois' leaf monkeys. Results showed that tree leaves accounted for the highest percentage of total daily intake of PFASs, especially PFOA, thus highlighting tree leaf consumption as a primary PFAS exposure route for this species. Overall, however, dietary exposure to PFASs was of relatively low risk to Francois' leaf monkey health.

Radioimmunoassay of human plasma lecithin-cholesterol acyltransferase.

A sensitive and precise competitive-displacement double-antibody radioimmunoassay was developed for the human plasma enzyme lecithin-cholesterol acyltransferase (LCAT; Ec 2.3 1.43). The ability of plasma from various animal species to displace labeled human LCAT from goat anti-human LCAT could be ranked in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat. Normolipidemic subjects had levels of LCAT of 6.14 +/- 0.98 micrograms/ml (mean +/- SD, n = 66). Subjects with dysbeta-lipoproteinemia had the highest plasma LCAT levels (7.88 +/- 0.39 micrograms/ml, n = 7, P < 0.05), followed by hypercholesterolemic subjects (7.00 +/- 1.30, n = 41) and hypertriglyceridemic subjects (6.96 +/- 1.3, n = 10). LCAT-deficient subjects had the lowest enzyme levels (0.89, 0.83, and 0.05 micrograms/ml, respectively, and two subjects with no detectable enzyme). Males had lower LCAT levels (6.42 +/- 1.05 micrograms/ml, n = 90, for all subjects; 5.99 +/- 1.03, n = 44, for normolipidemics) than females (7.01 +/- 1.14, n = 34, for all subjects P < 0.01; 6.44 +/- 0.79, n = 22, for normolipidemics, P < 0.01). LCAT levels correlated significantly with total cholesterol (males, r = 0.384, P < 0.001; females, r = 0.519, P < 0.002); and total triglyceride (only in females, r = 0.512, P < 0.002). LCAT levels in females correlated inversely with HDL cholesterol (r = 0.341, P < 0.05) and apoprotein D (r = 0.443, P < 0.02), but no such relationship existed in males.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. SIGNIFICANCE: An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for Homo sapiens. The great similarity found between the primate's molecules and those for humans supported the use of the monkeys or their cells for continuing assays involved in studying malaria. Integral membrane receptors used by Plasmodium for invading cells were also found; this required timely characterisation for evaluating their therapeutic role. The list of erythrocyte protein composition reported here represents a useful source of basic knowledge for advancing biomedical investigation in this field.

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

AIM: Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). RESULTS: In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. CONCLUSION: The validated method was robust and implemented in support of a preclinical PK/PD study.

Triacylglycerol and very low density lipoprotein secretion into plasma of squirrel monkeys.

We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d less than 1.006) lipoproteins, but there was an increase in the ratio of traicylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted Triton WR-1339-treated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylglycerol during fasting, the rate of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipoproteins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins. There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

Heterogenetiy of glucocorticoid binders: a high affinity triamcinolone acetonide binder in bovine serum.

While investigating glucocorticoid-binding proteins in bovine tissues, a new binder was found in fresh bovine serum which exhibited high affinity for certain synthetic glucocorticoids. This serum binder was characterized using [3H]triamcinolone acetonide (TA) as the ligand. On sucrose gradients, the [3H]TA peak sedimented at 8S, which was easily distinguishable from the [3H]cortisol-transcortin peak at 4S. Unlike the tissue receptor, which showed ionically dependent transformation from 8S in equilibrium or formed from 4S, the serum binder sedimented at 8S in both hypo- and hypertonic gradients. Binding properties were evaluated employing sephadex G-50 chromatography to separate bound from free steroid. Scatchard analysis of specific [3H]TA binding data revealed a straight line. The apparent equilibrium dissociation constant (Kd) was 7.8 +/- 0.7 X 10(-8) M, and the binding capacity was 772 +/- 70 fmol/mg serum protein. Hormonal specificity was determined by a competitive binding assay and revealed the following sequence: TA (100%) > betamethasone (47%) > triamcinolone (33%) > dexamethasone (2%) = cortisol = progesterone; aldosterone, estradiol, and testosterone exhibited negligible competitive activity. The serum binder was very stable, withstanding heating to 37 C for 60 min and long term storage in the frozen state. However, binding was significantly destroyed by trypsin. The binder was absent from fresh samples of chicken, mouse, rat, rabbit, dog, monkey, and human sera and frozen horse and porcine sera, but was clearly present in commercially available frozen calf, fetal calf, and lamb sera. At this time, we are unable to define the function of this binder, although its existence in both ovine and bovine sera suggests a possible role in ruminants. However, since bovine serum is routinely employed in tissue culture studies, the presence of this glucocorticoid binder might significantly influence many experiments.

Dietary saturated fatty acids (12:0, 14:0, 16:0) differ in their impact on plasma cholesterol and lipoproteins in nonhuman primates.

Three species of monkey (rhesus, cebus, and squirrel) were rotated through five purified diets containing 31% energy as various fat blends (P:S between 0.1 and 1.0) for 12-wk periods to compare the impact of specific dietary fatty acids on plasma lipids and lipoproteins. As 12:0 + 14:0 was replaced by 16:0, a significant decrease occurred in total and LDL cholesterol, whereas slight increases in total cholesterol and the LDL-HDL ratio occurred when 16:0 replaced 18:2. Hegsted and Keys regression equations provided a good fit for the observed data, but the predicted total cholesterol response was perfect (r = 0.995) for both equations when 16:0 was considered neutral. Thus, under these conditions 16:0 was less cholesterolemic than 12:0 + 14:0 and only slightly cholesterolemic compared with 18:2.

Activity and attention in zinc-deprived adolescent monkeys.

Lethargy is characteristic of malnourished populations, but little is known about the biologic mechanism or consequences for cognitive performance. In the current experiment, 24-h activity patterns and performance of an attention task were studied in adolescent female monkeys (18-33 mo of age, n = 10/group) under conditions of moderate dietary zinc deprivation (2 micrograms Zn/g diet) and adequate dietary zinc (50 micrograms Zn/g). There were progressive decreases in daytime activity levels in the zinc-deprived group followed by slowing of growth around the time of the growth spurt. Attention performance was also impaired before the onset of growth retardation. Zinc-deprived monkeys failed to show the shift to later initiation of the rest phase of the diurnal cycle seen in controls in late adolescence. These data support previous findings that activity and attention can be affected during early stages of zinc deprivation before the onset of growth retardation.

Fibrinopeptides A and B of Japanese monkey (Macaca fuscata) and patas monkey (Erythrocebus patas): their amino acid sequences, restricted mutations, and a molecular phylogeny for macaques, guenons, and baboons.

Amino acid sequences of fibrinopeptides A and B from the macaque, Macaca fuscata (Japanese monkey) and the guenon, Erythrocebus patas (patas monkey) were established. Fibrinopeptides A of the monkeys had a sequence identical with those of baboons: Ala-Asp-Thr-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Fibrinopeptides B were 9-residue, "short," peptides with the sequences Asn-Glu-Glu-Ser-Leu-Phe-Ser-Gly-Arg for M. fuscata and Asn-Glu-Glu-Val-Leu-Phe-Gly-Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from that of a close-related species, M. mulatta (rhesus monkey), at a single site, Leu (M.f.)----Pro (M.m.). A single replacement between the B peptides of E. patas and Cercocebus aethiops (green monkey), Val (E.p.)----Gly (C.a.), was detected. A phylogenic relationship of macaques, guenons, and baboons, named Cercopithecinae (Old World monkey), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old World monkeys, suggesting a restricted mutation of their fibrinopeptides during primate evolution.

NCA: a differentiation antigen of myelopoietic cells in humans and hominoid monkeys.

NCA, a normal colonic and granulocytic antigen, could be demonstrated in serum and in myelopoietic, but not lymphopoietic or erythropoietic, cells of Homo sapiens and other Primates. The levels of NCA in both serum and myelopoietic cells of Homo and hominoids were higher than those of more distant relatives of the same order. Thus, the classic phylogenetic differences are reflected also by the distribution of NCA. Hyperimmunization of Macaca irus, in which the NCA content of serum and cells is low, led to occurrence of anti-NCA IgG in all animals. The phylogenetic differences may accordingly have to do with slight antigenic NCA differences between Homo and other Primates rather than differences in amount only. Purified NCA did not affect growth and maturation of myelopoietic stem cells in vitro, whereas anti-NCA inhibited development of the majority of myelopoietic clusters and colonies.

Protamine sulfate neutralization of the anticoagulant activity of Aprosulate, a synthetic sulfated lactobionic acid amide.

Aprosulate or lactobionic acid is a highly sulfated analogue of heparin which is currently undergoing clinical trials in Europe as a potential antithrombotic drug. Aprosulate exerts a strong anticoagulant effect in plasma as a result of its interaction with heparin cofactor II. In this study, the ability of protamine sulfate to neutralize the anticoagulant activity of Aprosulate was investigated. In vitro, ex vivo, and in vivo coagulation studies were performed using various clotting assays such as the APTT, Heptest, and thrombin time as a measure of the anticoagulant activity of Aprosulate. In the first study, protamine sulfate when administered in vitro to plasma samples containing various concentrations of Aprosulate was found to effectively neutralize the anticoagulant activity of the Aprosulate in both normal human and normal monkey plasma systems. However, the relative index of neutralization of Aprosulate was assay dependent. Protamine sulfate was also found to antagonize the anticoagulant effects of Aprosulate in an ex vivo study. The ex vivo supplementation of protamine sulfate to plasma samples collected at various time intervals following the subcutaneous administration of Aprosulate to a group of primates completely neutralized the anticoagulant activity of the Aprosulate. In a third in vivo study, protamine sulfate when injected intravenously into the bloodstream of a group of primate was found to completely neutralize the anticoagulant effects of a previously administered dosage of Aprosulate. The results of these three studies clearly suggest that protamine sulfate can be used to effectively neutralize the anticoagulant activity of Aprosulate.

A non-haemagglutinating isolate of mink enteritis virus.

A virus was isolated from mink showing clinical and pathological signs of mink enteritis. This virus was identified as mink enteritis virus (MEV) from results of serological tests, determination of its density in CsCl (1.415 g cm-3), and morphology, including size (20 nm in diameter). The isolate was designated MEV-S. In contrast to other known MEV strains, the MEV-S isolate has no haemagglutinating (HA) activity with swine red blood cells (RBCs) at 4 degrees C and pH 6.8. Neither was there any HA at other pH values and temperatures, or when horse, bovine and rhesus monkey RBC's were used.