Detection of lymphocyte subpopulations in EDTA whole blood samples using flow cytometry. Stability and normal values

Series of T-cell-specific monoclonal antibodies (OKT 11 for peripheral T-lymphocytes, OKT 4 for helper/inducer T-cells, OKT 8 for suppressor/cytotoxic T-cells), one B-cell-specific antibody (anti-LEU-10) and the antibody anti-LEU-7 for LGL/NK-cells were used to detect the distribution of different lymphocyte subpopulations in the peripheral blood of 60 healthy, clinically examined test persons. The flow cytometry was performed by means of direct immunofluorescence method. The statistical evaluation showed that the results in all three age groups studied did not essentially differ with the exception of the so-called natural killer cells which increase significantly with advancing years. The described method allows the dispatch of lymphocytes samples even from places far away and the analysis of the specimen within 36 to 48 h.     

Characterization of beta-adrenoceptor subtype in bladder smooth muscle in cynomolgus monkey

We first investigated the relaxations of the urinary bladder induced by beta-adrenoceptor agonists in anesthetized cynomolgus monkeys and then employed a variety of beta-adrenoceptor agonists and antagonists in vitro to identify the beta-adrenoceptor subtype responsible for the relaxation (using isolated monkey detrusors). Isoprenaline reduced bladder pressure in a dose-dependent manner. Isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of isolated detrusor strips, the rank order of relaxing potencies being isoprenaline > noradrenaline > adrenaline. Subtype-selective beta-adrenoceptor agonists also relaxed isolated detrusor strips, the rank order of potencies being CGP-12177 > BRL 37344 > dobutamine, salbutamol, procaterol > xamoterol. In the antagonist experiment, bupranolol (beta-antagonist, 10(-6) to 10(-5) M) and SR 58894A (beta3-antagonist, 10(-7) to 10(-5) M) caused a rightward shift of the concentration-relaxation curve for isoprenaline, but CGP-20712A (beta1-antagonist, 10(-9) to 10(-7) M) and ICI-118551 (beta2-antagonist, 10(-9) to 10(-7) M) did not. The present functional study provides the first evidence that relaxation of the monkey detrusor by beta-adrenoceptor activation is mediated via the beta3-subtype.     

Dalcetrapib pharmacokinetics and metabolism in the cynomolgus monkey

The thioester dalcetrapib is undergoing Phase III clinical evaluation for the prevention and regression of atherosclerosis and the prevention of cardiovascular events through targeting cholesteryl ester transfer protein and increasing high-density lipoprotein cholesterol levels. Dalcetrapib undergoes rapid hydrolysis to generate the pharmacologically active form (dalcetrapib-thiol), which undergoes extensive metabolism via glucuronic acid conjugation, methylation, and hydroxylation, predominately forming the pharmacologically inactive S-methyl (dalcetrapib-S-Me) and S-glucuronide (dalcetrapib-S-Glu) metabolites. The purpose of this study was to characterize the absorption and disposition of dalcetrapib-thiol and its primary metabolites in cynomolgus monkeys following first pass through the intestines and liver using an in vivo dual portal and peripheral vein cannulation. Results showed the high influence of glucuronidation of dalcetrapib-thiol on the first-pass effect. Following passage through the primate intestinal wall, area under the plasma concentration-time curve indicated a marked loss (by ~85%) of active compound and formation of dalcetrapib-S-Glu and dalcetrapib-S-Me. Based on time to maximum drug concentrations (T(max)) values in the portal vein, metabolism of dalcetrapib-thiol to dalcetrapib-S-Glu appears to occur almost instantly (median T(max) 6.0 and 5.5 h, respectively), whereas methylation to dalcetrapib-S-Me occurs much more slowly (median T(max), 24 h). A relatively modest impact on systemic exposure followed hepatic first pass, with a further decrease in dalcetrapib-thiol exposure of 58% (AUC), a 3-fold reduction in exposure levels of dalcetrapib-S-Me and near-complete decrease in exposure of dalcetrapib-S-Glu. Passage of drug-related material through the intestinal wall and the liver results in an overall decrease of exposure to dalcetrapib-thiol of >90%.     

Dalcetrapib pharmacokinetics and metabolism in the cynomolgus monkey

1. The thioester dalcetrapib is undergoing Phase III clinical evaluation for the prevention and regression of atherosclerosis and the prevention of cardiovascular events through targeting cholesteryl ester transfer protein and increasing high-density lipoprotein cholesterol levels. Dalcetrapib undergoes rapid hydrolysis to generate the pharmacologically active form (dalcetrapib-thiol), which undergoes extensive metabolism via glucuronic acid conjugation, methylation, and hydroxylation, predominately forming the pharmacologically inactive S-methyl (dalcetrapib-S-Me) and S-glucuronide (dalcetrapib-S-Glu) metabolites.

2. The purpose of this study was to characterize the absorption and disposition of dalcetrapib-thiol and its primary metabolites in cynomolgus monkeys following first pass through the intestines and liver using an in vivo dual portal and peripheral vein cannulation.

3. Results showed the high influence of glucuronidation of dalcetrapib-thiol on the first-pass effect. Following passage through the primate intestinal wall, area under the plasma concentration–time curve indicated a marked loss (by ~85%) of active compound and formation of dalcetrapib-S-Glu and dalcetrapib-S-Me.

4. Based on time to maximum drug concentrations (T_max) values in the portal vein, metabolism of dalcetrapib-thiol to dalcetrapib-S-Glu appears to occur almost instantly (median T_max 6.0 and 5.5?h, respectively), whereas methylation to dalcetrapib-S-Me occurs much more slowly (median T_max, 24?h). A relatively modest impact on systemic exposure followed hepatic first pass, with a further decrease in dalcetrapib-thiol exposure of 58% (AUC), a 3-fold reduction in exposure levels of dalcetrapib-S-Me and near-complete decrease in exposure of dalcetrapib-S-Glu. Passage of drug-related material through the intestinal wall and the liver results in an overall decrease of exposure to dalcetrapib-thiol of >90%.

     

Cell-based screening using high-throughput flow cytometry

This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC? Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt?, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12?min, respectively. Embedded in the system is HyperView?, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.     

A toxicity profile of osteoprotegerin in the cynomolgus monkey

Osteoprotegerin (OPG) is a novel secreted glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that acts as an antiresorptive agent inhibiting osteoclast maturation. OPG acts by competitively inhibiting the association of the OPG ligand with the RANK receptor on osteoclasts and osteoclast precursors. This inhibition of osteoclasts can lead to excess accumulation of newly synthesized bone and cartilage in vivo. The purpose of this study was to investigate the potential toxicity of a human recombinant form of OPG in the young cynomolgus monkey. OPG was administered by intravenous (i.v.) or subcutaneous (s.c.) injection three times per week for either 4 or 13 weeks. There were no deaths during the study, no clinical signs related to treatment, no effect on body weight, appetence, or ophthalmology. No toxicologically relevant changes in routine laboratory investigations, organ weights, or gross or histopathological findings were observed. Serum ionized calcium and phosphorus were decreased at all dose levels. Evaluations were performed to monitor biochemical markers of bone resorption (N-telopeptide [NTx], deoxypyridinoline [DPD]), bone formation (skeletal alkaline phosphatase [sALP], osteocalcin [OC]), parathyroid hormone [PTH], and bone density of the proximal tibia and distal radius in vivo. Dose-related decreases in NTx and/or DPD were observed at each dose level, with up to a 90% decrease in NTx noted for animals treated i.v. or s.c. at 15 mg/kg. Similar decreases were observed for sALP and OC. PTH was increased for animals treated at 5 and 15 mg/kg (i.v. or s.c.). Trabecular bone density was increased for the majority of males and females treated i.v. or s.c. at 15 mg/kg and males treated i.v. at 5 mg/kg. Microscopic examination of the sternebrae revealed corresponding increases in bone. Decreases in markers of bone turnover, and corresponding increases in bone density, were consistent with the pharmacological action of OPG as an osteoclast inhibitor. The no-observable-adverse-effect level (NOAEL) of OPG was 15 mg/kg.     

Pharmacokinetics of proterguride in rat and cynomolgus monkey

1. The pharmacokinetics of proterguride were studied in rat and cynomolgus monkey using 3H- and 14C-labelled drug and a radioimmunoassay for concentration measurements of unchanged drug. 2. Proterguride was rapidly and completely absorbed at low doses but not completely at higher dose levels, especially in rat. 3. Bioavailability was 18% in the monkey (low and high doses) and 79% (low doses) and 38% (high dose), respectively, in the rat. 4. Proterguride was able to pass the blood-brain barrier achieving concentrations in brain similar to those in plasma. 5. Excretion of labelled compounds was mainly in the faeces in rat, but in monkey elimination was equal in faeces and urine.     

A 12-week study of BHA in the cynomolgus monkey

Butylated hydroxyanisole (BHA) given by gavage to female cynomolgus monkeys on 5 days/wk for 84 days produced transient changes in selected serum chemistry and haematology parameters. Terminal observations revealed increased liver size, decreased hepatic monooxygenase activity and an increase in the mitotic index of the oesophageal epithelium. Several of these observations are similar to those reported for rodents also given BHA at or near the maximum tolerated dose. Gastroscopic evaluation of the stomach and oesophagus at monthly intervals and extensive gross and histopathological examination failed to reveal the proliferative effects seen in the forestomach of rats fed diets containing BHA.     

Screening of new antioxidant molecules using flow cytometry

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.     

Pharmacokinetics and anti-inflammatory pharmacodynamics of prednisolone in cynomolgus monkey

1. The purpose was to investigate whether the pharmacokinetics and pharmacodynamics of prednisolone in the non-human primate was an appropriate surrogate for man.

2. After single intravenous doses of 0.03, 0.3, and 3?mg kg^?1, prednisolone demonstrated a dose-dependent clearance and volume of distribution. When corrected for concentration-dependent protein binding, the free clearance was linear at the tested dose levels. The protein binding-corrected volume of distribution was similar across doses. The serum half-life was estimated as being between 2 and 4?h. Prednisolone exhibits near complete inhibition of the cytokines TNF-α, IL-1β, IL-6 and IL-8 with very similar IC₅₀ estimates from 0.09 to 0.16 μg ml^?1 (from 0.24 to 0.44 μM).

3. The monkey demonstrated a similar pharmacokinetics–pharmacodynamics profile of prednisolone when compared with man (from the literature).

     

Disposition of the enantiomers of cromakalim in rat and cynomolgus monkey

1. Disposition of the 3R,4S(+) and 3S,4R(-) enantiomers of the racemic antihypertensive drug cromakalim has been studied in rats and cynomolgus monkeys using the ¹⁴C-drug labelled in either the 3R,4S(+) or the 3S,4R(-) enantiomer.

2. After oral administration to rat, blood concentrations of the 3R,4S(+) enantiomer were up to fourfold higher than those of the 3S,4R(-) enantiomer. Metabolism of the former was not as extensive as that of the latter and consequently plasma and urinary radio-metabolite patterns were quantitatively different.

3. In contrast to rat, there were much greater differences in the disposition of the two enantiomers following oral administration of cromakalim to the cynomolgus monkey. Plasma concentrations of the 3R,4S(+) enantiomer were approximately 100 x those of the 3S,4R(-) enantiomer and the rate of urinary ¹⁴C elimination for the 3R,4S(+) enantiomer was much faster than that for the 3S,4R(-) enantiomer. Plasma and urinary radio-metabolite patterns were very different for the two isomers. Metabolic end products of the 3R,4S(+) enantiomer were predominantly phase I metabolites whereas the 3S,4R(-) enantiomer was almost entirely metabolized by glucuronidation.

4. A study of the racemic drug alone would have led to a misunderstanding of the fate of the compound in these species.     

Assessment of the renal toxicity of novel anti-inflammatory compounds using cynomolgus monkey and human kidney cells

PF1, an anti-inflammatory drug candidate, was nephrotoxic in cynomolgus monkeys in a manner that was qualitatively comparable to that observed with the two previous exploratory drug candidates (PF2 and PF3). Based on the severity of nephrotoxicity, PF1 ranked between the other two compounds, with PF2 inducing mortality at all doses and PF3 eliciting only mild nephrotoxicity. To further characterize nephrotoxicity in monkeys and enable direct comparisons with humans, primary cultures of proximal tubular (PT) cells from monkey and human kidneys were used as in vitro tools, using lactate dehydrogenase release as the biomarker of cytotoxicity. In both human and monkey PT cells, PF2 was by far the most cytotoxic compound of the three drugs. PF1 exhibited modest cytotoxicity at the highest concentration tested in human PT cells but none in monkey kidney cells whereas PF3 exhibited the reverse pattern. Because these drugs are organic anions, mechanistic studies using human organic anion transporters 1 and 3 (hOAT1 and hOAT3) transfected cell lines were pursued to evaluate the potential of these compounds to interact with these transporters. All three drugs exhibited high affinity for hOAT3 (PF1 exhibited the lowest IC₅₀ of 6 μM) but only weakly interacted with hOAT1 (with no interaction found for PF2). PF2 was a strong hOAT3 (not hOAT1) substrate, whereas PF1 and PF3 were substrates for both hOAT1 and hOAT3. Upon pretreatment of monkeys with the OAT substrate probenecid, PF3 systemic exposure (AUC) and half-life (t_1/2) increased ∼2-fold whereas clearance (CL) and volume of distribution (Vd_ss) decreased, as compared to naïve monkeys. This indicated that PF3 competed with probenecid for hOAT1 and/or hOAT3 mediated elimination of PF3. Thus, hOAT1 and/or hOAT3 may be responsible for the uptake of this series of drugs in renal PT cells, which may directly or indirectly lead to the observed nephrotoxicity in vivo.     

Comparative pharmacokinetics of antipyrine (phenazone) in the baboon,cynomolgus monkey and rhesus monkey

The pharmacokinetics of antipyrine (phenazone) in 3 species of non-human primate have been evaluated following its intravenous administration at a dose level of 92 mg/kg.Mean peak plasma concentrations of antipyrine of 132, 137 and 155 μg/ ml in the rhesus monkey, the cynomolgus monkey and the baboon respectively were not observed until 5 min after intravenous injection. Thereafter, concentrations declined with an apparent half-life of elimination of 1.5–2 h. The time-course of plasma antipyrine concentrations was adequately described by a one-compartment open model and no notable differences in pharmacokinetic parameters utilising a 2-compartment open model were observed.Antipyrine was mainly distributed in total body water. The mean volume of distribution was equivalent to 88, 73 and 66% of body weight in the rhesus monkey, the cynomolgus monkey and the baboon, respectively.An analysis of variance of volumes of distribution, apparent half-lives of elimination and systemic clearances showed that there was a statistically significant species-related difference in systemic clearance (P < 0.05) and volumes of distribution (P < 0.01) which were lower in the cynomolgus monkey than in the other 2 species.The pharmacokinetics of antipyrine in the non-human primate are more similar to those of other laboratory animal species than to those of humans.     

褪黑素对急性脑梗死患者周围血T淋巴细胞亚群的干预作用

目的探讨褪黑素对急性脑梗死患者免疫受损恢复的干预作用。方法将60例入选的急性脑梗死患者随机分为褪黑素(Melatonin,MT)干预组和维生素C对照组,另在健康查体者中选30人作正常对照组。急性脑梗死患者于发病后第3天开始分别给与MT(9mg/d,每晚临睡前口服1次)片和维生素C片(0.3/d,每晚口服1次),用流式细胞仪(FCM)直接荧光染色法检测发病后第3天和给药后第14天的外周血CD3+、CD4+、CD8+T淋巴细胞数量,并统计两组的感染率。结果维生素C对照组第14天的CD3+、CD4+和CD4+/CD8+均较正常对照组明显降低(P<0.01),而MT干预组第14天的CD3+、CD4+和CD4+/CD8+与正常对照组比较无显著差异(P=0.25),但与维生素C对照组比较有显著性差异(P<0.01)。与维生素C对照组相比,MT组的感染并发症较少(P<0.01)且较轻。结论适当补充MT能扭转或改善急性脑梗死受损的T细胞免疫功能,减少感染并发症的发生。     

Identification of contact and respiratory sensitizers using flow cytometry

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analyses were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-gamma-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.     

用流式细胞仪检测血小板P—选择素和血小板—中性粒细胞复合物

用流式细胞仪方法测定血小板的 ＣＤ６２Ｐ和 ＰＮＣ．选 ２５名健康不吸烟自愿者（男 １１名,女 １４名,年龄 ２２－６８岁,平均４２岁）．在用血小板激活物ＡＤＰ前后来评估流式细胞仪检测血小板的方法．ＣＤ６２Ｐ和ＰＮＣ在ＡＤＰ刺激后明显增高［分别为（８．０±２．７）％到（１８．８±６．４）％,Ｐ＜０．０１,（９．４±３．４）％到（１４．５±７．２）％Ｐ＜０．０１）］．论证,流式细胞仪测定血小板的方法是可行的,该方法简便,快速,敏感性高．     

Disposition of the enantiomers of cromakalim in rat and cynomolgus monkey.

1. Disposition of the 3R,4S(+) and 3S,4R(-) enantiomers of the racemic antihypertensive drug cromakalim has been studied in rats and cynomolgus monkeys using the 14C-drug labelled in either the 3R,4S(+) or the 3S,4R(-) enantiomer. 2. After oral administration to rat, blood concentrations of the 3R,4S(+) enantiomer were up to fourfold higher than those of the 3S,4R(-) enantiomer. Metabolism of the former was not as extensive as that of the latter and consequently plasma and urinary radiometabolite patterns were quantitatively different. 3. In contrast to rat, there were much greater differences in the disposition of the two enantiomers following oral administration of cromakalim to the cynomolgus monkey. Plasma concentrations of the 3R,4S(+) enantiomer were approximately 100 x those of the 3S,4R(-) enantiomer and the rate of urinary 14C elimination for the 3R,4S(+) enantiomer was much faster than that for the 3S,4R(-) enantiomer. Plasma and urinary radiometabolite patterns were very different for the two isomers. Metabolic end products of the 3R,4S(+) enantiomer were predominantly phase I metabolites whereas the 3S,4R(-) enantiomer was almost entirely metabolized by glucuronidation. 4. A study of the racemic drug alone would have led to a misunderstanding of the fate of the compound in these species.