High-performance liquid chromatographic analysis of the anti-tumor agent SCH 66336 in cynomolgus monkey plasma and evaluation of its chiral inversion in animals

SCH 66336 is a novel non-cytotoxic anti-tumor agent that is in phase I/II clinical trials for the treatment of solid tumors. This compound is a single enantiomer with one chiral center. Prior to evaluation of this drug candidate in man, it was necessary to evaluate its pharmacokinetics and possible chiral inversion in animals. Thus, high-performance liquid chromatographic (HPLC) methods have been developed for its determination in cynomolgus monkey plasma and for the evaluation of its chiral inversion in rats and cynomolgus monkeys. The achiral HPLC analysis involved extraction with 30% methylene chloride in hexane followed by separation on a CN column and quantitation by UV absorbance at 280 nm. The method was linear over a concentration range of 0.1 to 20 μg/ml in monkey plasma. The chiral HPLC analysis involved the use of a Chiralpak AD column set at 39°C with a mobile phase of hexane–ethanol–diethylamine mixture and a UV detector set at 280 nm. Plasma samples were subjected to solid-phase extraction on a C₂ cartridge prior to HPLC analysis. The method was linear over a concentration range of 0.25 to 10 μg/ml in rat and cynomolgus monkey plasma for both enantiomers. Both methods showed good linearity (r²>0.99), accuracy (bias<13%) and precision (CV<12%). Chiral HPLC analysis indicated that SCH 66336 was not subjected to chiral inversion in rats and cynomolgus monkeys     

Pharmacokinetics of the active antifungal enantiomer, SCH 42427 (RR), and evaluation of its chiral inversion in animals following its oral administration and the oral administration of its racemate genaconazole (RR/SS)

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower C(max) and a longer t(1/2) than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration-time profiles and C(max), AUC(I), and t(1/2) values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer.     

Comparative morphology and morphometry of alveolar macrophages from six species

Pulmonary alveolar macrophages (PAM) were collected from normal, healthy mice, rats, dogs, cynomolgus monkeys, chimpanzees, and humans and evaluated for morphologic and morphometric characteristics. The PAM of mice, rats, and dogs were morphologically similar and had statistically similar frequency distributions for size. The cell size distribution for these three species was relatively homogeneous. The PAM of nonhuman primates and humans were morphologically heterogenous with sometimes prominent cytoplasmic vacuolation, irregular cell outlines, and increased numbers of multinucleated cells as compared to the PAM of rodents and dogs. The mean size of human PAM was statistically greater than that for all other species evaluated, including nonhuman primates. These data indicate that significant differences in PAM morphology and size exist among species.     

Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans

The prochiral carbonyl group of fenofibrate (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propionate) is reduced during its metabolism giving rise to a chiral secondary alcohol, "reduced fenofibric acid." Chiral and diastereomeric HPLC methods have been developed for the determination of its enantiomeric composition and these have been applied to the measurement of the "reduced fenofibric acid" enantiomers in urine of rats, guinea pigs, dogs, and human volunteers given [14C]fenofibrate. In the three animal species, the reduction is markedly enantioselective for the (-)-isomer, the enantiomeric ratios (-/+) being 95:5. This was not due to differences in the excretion of the enantiomers, since when racemic "reduced fenofibric acid" was given to rats it was recovered in the urine with the same enantiomeric composition as the dose form. In humans the ratio was 52:48 showing the lack of stereoselectivity of reduction in this species.     

Relationship between body weight and hematological and serum biochemical parameters in female cynomolgus monkeys (Macaca fascicularis).

Obesity is a risk factor triggering a variety of metabolic diseases. Cynomolgus monkeys (Macaca fascicularis) exhibit spontaneous onset of obesity in adulthood, similar to such onset exhibited by humans. To clarify the characteristics accompanying obesity in female cynomolgus monkeys, we used simple and multiple regression analyses to determine the relationship between body weight and hematological and serum biochemical parameters as well as obesity-related hormones, namely, leptin and insulin. Simple regression analysis showed that body weight was significantly (P < 0.05) correlated with leptin level, insulin level, hemoglobin concentration, hematocrit values, mean corpuscular volume, glucose concentration, and triglyceride concentration. In addition, a multiple regression model containing leptin level, insulin level, mean corpuscular volume, and red blood cell count explained 66.9% of the variance in body weight. Therefore, female cynomolgus monkeys show similar obesity characteristics to humans, i.e., obesity is associated with enhanced synthesis and excretion of leptin in adipocytes, high risk of diabetes mellitus, and high levels of hematocytes. Our results indicate that female cynomolgus monkeys are good models for studying obesity in humans.     

Effects of aging on bone mineral content and bone biomarkers in female cynomolgus monkeys.

Age-related changes in bone mineral content and bone biomarkers were assessed over the complete lifespan of female cynomolgus monkeys. The bone mass of the lumbar spine increased linearly from birth to about 2.5 years of age, and this increase gradually slowed thereafter until a peak bone mass was achieved at 9 years of age. The bone mass stabilized after 9 years of age, showing no sign of further reduction with age. In contrast with the significant increase in bone mass before 2.5 years of age, significant decreases occurred in the serum concentrations of the following bone formation markers: intact osteocalcin, bone-specific alkaline phosphatase and amino-terminal propeptide of type I procollagen, but the serum concentration of carboxy-terminal propeptide of type I procollagen did not change significantly throughout the entire lifespan. Concerning the bone resorption markers, the levels of tartrate-resistant acid phosphatase fluctuated throughout the entire lifespan. The skeleton of an aging female monkey undergoes changes similar to those observed in senescent humans, but did not undergo the menopausal changes seen in women. The use of female cynomolgus monkeys to model human skeletal interventions should therefore be undertaken with consideration of the similarities and differences between cynomolgus monkeys and humans.     

Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)‐ and R(−)‐enantiomers of tiaprofenic acid (S‐TIA, R‐TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S‐TIA and R‐TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R‐TIA it was found that 1) there was a time delay of peak S‐TIA plasma concentrations, 2) S‐TIA concentrations exceeded R‐TIA concentrations from ∼2 hr after dosing, 3) C_max and AUC_(0‐∞) for S‐TIA were greater than for R‐TIA following administration of S‐TIA, and 4) inversion was bidirectional but favored inversion of R‐TIA to S‐TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R‐enantiomer to the S‐enantiomer. In conclusion, stereoselective pharmacokinetics of R‐ and S‐TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2‐arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley‐Liss, Inc.     

Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.     

Novel hyperprolactinemia and hyperprolactinemic anovulation model using the cynomolgus monkey (Macaca fascicularis)

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.     

Ratio of leptin to adiponectin as an obesity index of cynomolgus monkeys (Macaca fascicularis).

Obesity is responsible for inducing various metabolic diseases. Laboratory-bred cynomolgus monkeys exhibit spontaneous onset of obesity. However, to date, no blood chemistry index to identify the state of obesity in cynomolgus monkeys has been determined. In the present study, to determine such an index, we measured the serum levels of two adipocyte-derived hormones, leptin and adiponectin, and evaluated the relationship between these hormones and other serum energy metabolic factors (i.e. insulin, total protein, glucose, total cholesterol and triglyceride) as well as the percentage of body fat (%Fat) in mature cynomolgus monkeys. Both in females and males, leptin was positively correlated with insulin and %Fat, and adiponectin was negatively correlated with insulin and %Fat. In female cynomolgus monkeys, leptin, adiponectin, and glucose were selected as the most important determinants for %Fat in multiple regression analysis, and in male cynomolgus monkeys, leptin was selected. The ratio of leptin to adiponectin (L/A ratio) was significantly elevated in the animals with %Fat over 40 (P < 0.01). The results indicate that L/A ratio is a potential index for comprehensively identifying obesity in cynomolgus monkeys.     

No regional differences of cytochrome P450 expression in the liver of Cynomolgus monkeys (Macaca fascicularis).

Non-human primates are frequently used in toxicological studies the result of which are extrapolated to humans, but background data on drug metabolism ability among monkeys derived from different countries has not been published, especially on the key enzyme, cytochrome P450 (CYP450). We assessed the amounts of hepatic CYP450 obtained from cynomolgus monkeys of different ages and from different countries in this study. There were no regional differences of total P450 content, as well as major CYP450 isozymes (CYP 1A, 2A, 2B, 2C, 2D, 2E1 and 3A4) in cynomolgus monkeys by westernblot analysis. Similarly, there were no significant differences with hybrid cynomolgus monkeys, but variations in individual values were large. As for aging, total P450 contents declined in old cynomolgus monkeys (12-32 years of age). These results indicate the usefulness of basic data of hepatic CYP450 obtained from cynomolgus monkeys of different ages and from different countries.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Sensitive high-performance liquid chromatographic method for the determination of a benzonaphthazepine antipsychotic agent, SCH 39166, and its active metabolite, SCH 40853, in human plasma and its cross-validation with a gas chromatographic method

A sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of a benzonaphthazepine antipsychotic agent, SCH 39166, and its active metabolite, SCH 40853. The HPLC method required a single-step organic extraction at alkali pH followed by HPLC analysis utilizing a CN column with UV detection at 205 nm. The limit of quantitation was 1 ng/ml for SCH 39166 and 0.5 ng/ml for SCH 40853. The HPLC method was cross-validated with a previously reported GC method by the analysis of 73 plasma samples spiked with various concentrations of SCH 39166 and SCH 40853. The correlation coefficient was 0.9969 for SCH 39166 and 0.9984 for SCH 40853. Both GC and HPLC methods were used for the determination of plasma concentrations and yielded similar pharmacokinetic parameters for SCH 39166 and SCH 40853 in man following oral administration of SCH 39166 (100 mg).     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Chiral separation and modeling of baclofen,bupropion, and etodolac profens on amylose reversed phase chiral column

Chiral resolution of baclofen, bupropion, and etodolac profens was obtained with amylose derivatized chiral reversed stationary phase (carbamate groups). The eluent used for bupropion and etodolac was MeOH–water (20:80, v /v) and for baclofen was water–methanol (95:5, v /v). The eluent run rates, finding wavelength and temperature, were 1.0 mL/min, 220 nm and 27 ± 1 °C for all the eluents. The magnitude of the retardation factors for S‐ and R‐enantiomers of baclofen, bupropion, and etodolac were 1.37, 2.62, 2.25, 3.25, 1.8, and 3.0. The magnitudes of separation and resolution factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and 2.04. Limits of detection and quantitation were 1.0–2.0 and 5.1–10.0 μg/mL. Chiral recognition mechanisms were recognized by simulation and high‐performance liquid chromatography (HPLC) experiments. It was seen that hydrogen interactions, hydrophobic interactions, and π–π exchanges were the chief interactions for chiral recognition mechanisms. The described methods may be exploited for the chiral separation of baclofen, bupropion, and etodolac profens in any unknown sample.     

Cynomolgus and rhesus monkey visual pigments. Application of Fourier transform smoothing and statistical techniques to the determination of spectral parameters

Microspectrophotometric measurements were performed on 217 photoreceptors from cynomolgus, Macaca fascicularis, and rhesus, M. mulatta, monkeys. The distributions of cell types, for rods and blue, green, and red cones were: 52, 12, 47, and 44, respectively, for the cynomolgus, and 22, 4, 13, and 13 for the rhesus. Visual cells were obtained fresh (unfixed), mounted in various media (some containing 11-cis-retinal), and then located visually under dim red (650 nm) illumination. Absolute absorbance (A), linear dichroism (LD), and bleaching difference (BD) absorbance spectra were recorded through the sides of outer segments. The spectra were subjected to rigorous selection criteria, followed by digital averaging and Fourier transform filtering. Statistical methods were also applied to the accepted samples in the estimation of population means and variances. The wavelength of mean peak absorbance (lambda max) and the standard error at 95% certainty of the rod and blue, green, and red cone pigments in cynomolgus were 499.7 +/- 2.5, 431.4 +/- 4.2, 533.9 +/- 2.4, and 565.9 +/- 2.8 nm, respectively. The rhesus pigments were statistically indistinguishable from the cynomolgus, having lambda max of approximately 500, 431, 534, and 566 nm. Statistical tests did not reveal the presence of a lambda max subpopulation (i.e., anomalous pigments). The bandwidth of each alpha-band was determined in two segments, giving rise to the longwave half-density (LWHDBW), shortwave half-density (SWHDBW), and total half-density (THDBW) bandwidths. The LWHDBW was found to have the smallest variance. Both the LWHDBW and the THDBW showed linear dependence on the peak wavenumber (lambda max)-1 for the four macaque pigments.     

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>)

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2–10), and average heterozygosity was 0.63 (range 0.10–0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.     

Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys

Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PL < S-PL) was observed in the formation of 5-OH-PL and/or NDP. Kinetic experiments revealed that the formation of all metabolites was biphasic in cynomolgus monkeys, whereas only the formation of NDP was biphasic in marmosets. Inhibition experiments employing human CYP antibodies and chemical inhibitors suggested that mainly CYP2D enzymes and partially CYP1A and 2C enzymes are involved in the oxidation of PL in both monkey liver microsomes. In small intestinal microsomes, activity levels were much higher in cynomolgus monkeys than in marmosets and humans and reversed substrate enantioselectivity (R-PL > S-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.     

Discovery of a vorapaxar analog with increased aqueous solubility

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.