Association of gestational anemia with pregnancy conditions and outcomes: A nested case-control study.

BACKGROUND: Gestational anemia is a serious public health problem that affects pregnant women worldwide. Pregnancy conditions and outcomes might be associated with the presence of gestational anemia. This study investigated the association of pregnancy characteristics with anemia, exploring the potential etiology of the disease. AIM: To assess the association of pregnancy parameters with gestational anemia. METHODS: A nested case-control study was conducted based on the Chinese Pregnant Women Cohort Study-Peking Union Medical College Project (CPWCS-PUMC). A total of 3172 women were included. Patient characteristics and gestational anemia occurrence were extracted, and univariable and multivariable logistic regression models were used to analyze the association of pregnancy parameters with gestational anemia. RESULTS: Among the 3172 women, 14.0% were anemic, 46.4% were 25-30 years of age, 21.9% resided in eastern, 15.7% in middle, 12.4% in western 18.0% in southern and 32.0% in northern regions of China. Most women (65.0%) had a normal prepregnancy body mass index. Multivariable analysis found that the occurrence of gestational anemia was lower in the middle and western regions than that in the eastern region [odds ratio (OR) = 0.406, 95% confidence interval (CI): 0.309-0.533, P < 0.001)], higher in the northern than in the southern region (OR = 7.169, 95%CI: 5.139-10.003, P < 0.001), lower in full-term than in premature births (OR = 0.491, 95%CI: 0.316-0.763, P = 0.002), and higher in cases with premature membrane rupture (OR=1.404, 95%CI: 1.051-1.876, P = 0.02). CONCLUSION: Gestational anemia continues to be a health problem in China, and geographical factors may contribute to the situation. Premature birth and premature membrane rupture may be associated with gestational anemia. Therefore, we should vigorously promote local policy reformation to adapt to the demographic characteristics of at-risk pregnant women, which would potentially reduce the occurrence of gestational anemia.

Investigation of optimal gestational weight gain based on the occurrence of adverse pregnancy outcomes for Chinese women: a prospective cohort study.

OBJECTIVE: To investigate recommendations for appropriate gestational weight gain (GWG) of Chinese females. METHODS: In total of 3,172 eligible women in the first trimester were recruited into the Chinese Pregnant Women Cohort Study (CPWCS) project. Pregnancy complications and outcomes were collated using the hospital medical records system. The method of occurrence of participants with adverse pregnancy outcomes (Occurrence Method) was conducted to calculate the recommended total GWG for each participant's pre-pregnancy BMI. Occurrence Method data were judged against the Institute of Medicine (IOM) and Japanese recommended criteria in terms of the total occurrence of adverse pregnancy outcomes of pregnant women with appropriate weight gain. RESULTS: The most frequent GWG was ≥ 14 kg and < 16 kg (19.4%), followed by ≥ 10 kg and < 12 kg (15.5%) and ≥ 12 kg and < 14 kg (15.2%). The most frequently occurring adverse pregnancy outcomes were cesarean sections for underweight (30.0%), normal weight (40.4%), overweight (53.6%) and obese (53.7%) women. A large for gestational age (LGA) accounted for 18.0% of the overweight and 20.9% of the obesity group. Gestational diabetes mellitus (GDM) occurred in 16.9% of overweight and 23.1% of obese women. The recommended total GWG in a Chinese women population is ≥ 8 and < 12 kg if underweight, ≥ 12 and < 14 kg for normal weight, ≥ 8.0 and < 10.0 kg if overweight, and < 8 kg for women with obesity. CONCLUSIONS: Current Chinese recommendations provide the optimal ranges of GWG to minimize the occurrence of undesirable pregnancy outcomes for each group of pre-pregnancy BMIs in a Chinese population. TRIAL REGISTRATION: Registered with ClinicalTrials ( NCT03403543 ).

Safety, Tolerability, and Pharmacokinetics of Multiple Repeated Oral Doses of the α2/3/5-Subtype Selective GABAA -Positive Allosteric Modulator PF-06372865 in Healthy Volunteers.

Multiple-dose pharmacokinetics (PK) and safety were investigated in this phase 1 study of PF-06372865, a positive allosteric modulator of α2/3/5 subunit-containing γ-aminobutyric acid A receptors (NCT03351751). In 2 cohorts (7-8 PF-06372865 and 2 placebo in each cohort), healthy adult subjects received twice-daily oral doses of PF-06372865 for 21 days, which included titration in the first 7 days, followed by a maintenance dose of 25 mg twice daily (Cohort 1) and 42.5 mg twice daily (Cohort 2) for 14 days. Serial PK samples were collected on days 1 and 21. Nineteen subjects were assigned to study treatments; 18 completed the study. Approximate dose-proportional increases in maximum plasma concentratin and area under the plasma concentration-time curve over the dosing interval were observed. PF-06372865 was rapidly absorbed with a median time to maximum concentration of 1 to 2 hours following both single- and multiple-dose administration. Mean terminal elimination half-life on day 21 was approximately 11 hours in both cohorts. All adverse events were mild; the most frequently reported was dizziness. After titration, there were no reports of somnolence. There were no clinically significant safety findings, including a lack of withdrawal symptoms on discontinuation of treatment. These results demonstrate that PF-06372865 is safe and well tolerated at doses estimated to achieve high receptor occupancy (>80%), a profile differentiated from nonselective benzodiazepines.

Effects of pre-pregnancy body mass index and gestational weight gain on maternal and infant complications.

BACKGROUND: The potential effects of pre-pregnancy body mass (BMI) and gestational weight gain (GWG) on pregnancy outcomes remain unclear. Thus, we investigated socio-demographic characteristics that affect pre-pregnancy BMIs and GWG and the effects of pre-pregnancy BMI and GWG on Chinese maternal and infant complications. METHODS: 3172 women were enrolled in the Chinese Pregnant Women Cohort Study-Peking Union Medical College from July 25, 2017 to July 24, 2018, whose babies were delivered before December 31, 2018. Regression analysis was employed to evaluate the socio-demographic characteristics affecting pre-pregnancy BMI and GWG values and their effects on adverse maternal and infant complications. RESULTS: Multivariate logistic regression analysis revealed that age groups < 20 years (OR: 1.97), 25-30 years (OR: 1.66), 30-35 years (OR: 2.24), 35-40 years (OR: 3.90) and ≥ 40 years (OR: 3.33) as well as elementary school or education below (OR: 3.53), middle school (OR: 1.53), high school (OR: 1.40), and living in the north (OR: 1.37) were risk factors in maintaining a normal pre-pregnancy BMI. An age range of 30-35 years (OR: 0.76), living in the north (OR: 1.32) and race of ethnic minorities (OR: 1.51) were factors affecting GWG. Overweight (OR: 2.01) and inadequate GWG (OR: 1.60) were risk factors for gestational diabetes mellitus (GDM). Overweight (OR: 2.80) and obesity (OR: 5.42) were risk factors for gestational hypertension (GHp). Overweight (OR: 1.92), obesity (OR: 2.48) and excessive GWG (OR: 1.95) were risk factors for macrosomia. Overweight and excessive GWG were risk factors for a large gestational age (LGA) and inadequate GWG was a risk factor for low birth weights. CONCLUSIONS: Overweight and obesity before pregnancy and an excessive GWG are associated with a greater risk of developing GDM, GHp, macrosomia and LGA. The control of body weight before and during the course of pregnancy is recommended to decrease adverse pregnancy outcomes, especially in pregnant women aged < 20 or > 25 years old educated below university and college levels, for ethnic minorities and those women who live in the north of China. TRIAL REGISTRATION: Registered at Clinical Trials ( NCT03403543 ), September 29, 2017.

Comparison of autoregressive integrated moving average model and generalised regression neural network model for prediction of haemorrhagic fever with renal syndrome in China: a time-series study.

OBJECTIVES: Haemorrhagic fever with renal syndrome (HFRS) is a serious threat to public health in China, accounting for almost 90% cases reported globally. Infectious disease prediction may help in disease prevention despite some uncontrollable influence factors. This study conducted a comparison between a hybrid model and two single models in forecasting the monthly incidence of HFRS in China. DESIGN: Time-series study. SETTING: The People's Republic of China. METHODS: Autoregressive integrated moving average (ARIMA) model, generalised regression neural network (GRNN) model and hybrid ARIMA-GRNN model were constructed by R V.3.4.3 software. The monthly reported incidence of HFRS from January 2011 to May 2018 were adopted to evaluate models' performance. Root mean square error (RMSE), mean absolute error (MAE) and mean absolute percentage error (MAPE) were adopted to evaluate these models' effectiveness. Spatial stratified heterogeneity of the time series was tested by month and another GRNN model was built with a new series. RESULTS: The monthly incidence of HFRS in the past several years showed a slight downtrend and obvious seasonal variation. A total of four plausible ARIMA models were built and ARIMA(2,1,1) (2,1,1)12 model was selected as the optimal model in HFRS fitting. The smooth factors of the basic GRNN model and the hybrid model were 0.027 and 0.043, respectively. The single ARIMA model was the best in fitting part (MAPE=9.1154, MAE=89.0302, RMSE=138.8356) while the hybrid model was the best in prediction (MAPE=17.8335, MAE=152.3013, RMSE=196.4682). GRNN model was revised by building model with new series and the forecasting performance of revised model (MAPE=17.6095, MAE=163.8000, RMSE=169.4751) was better than original GRNN model (MAPE=19.2029, MAE=177.0356, RMSE=202.1684). CONCLUSIONS: The hybrid ARIMA-GRNN model was better than single ARIMA and basic GRNN model in forecasting monthly incidence of HFRS in China. It could be considered as a decision-making tool in HFRS prevention and control.

Analyzing maternal mortality rate in rural China by Grey-Markov model.

Maternal mortality rate (MMR) in China has reduced during a decade but still higher than many countries around the world. Rural China is the key region which affects over all maternal death. This study aims to develop a suitable model in forecasting rural MMR and offer some suggestions for rural MMR intervention. Data in this study were collected through the Health Statistical Yearbook (2017) which included the overall MMR in China and urban and rural mortality rate. A basic grey model (GM(1,1)), 3 metabolic grey models (MGM), and a hybrid GM(1,1)-Markov model were presented to estimate rural MMR tendency. Average relative error (ARE), the post-test ratio (C), and small error probability (P) were adopted to evaluate models' fitting performance while forecasting effectiveness was compared by relative error.The MMR in rural China reduced obviously from 63.0 per 100,000 live births in 2005 to 21.1 per 100,000 live births in 2017. One basic GM(1,1) model was built to fit the rural MMR and the expression was X^((1)) (k + 1) = 553.80e^0.0947k - 550.00 (C = 0.0456, P > .99). Three MGM models expressions were X^((1)) (k + 1)  = 548.67e^0.0923k - 503.17 (C = 0.0540, P > .99), X^((1)) (k + 1) = 449.39e^0.0887k - 408.09 (C = 0.0560, P > .99), X^((1)) (k + 1) = 461.33e^0.0893k - 425.23(C = 0.0660, P > .99). Hybrid GM(1,1)-Markov model showed the best fitting performance (C = 0.0804, P > .99). The relative errors of basic GM(1,1) model and hybrid model in fitting part were 2.42% and 2.03%, respectively, while 5.35% and 2.08%, respectively, in forecasting part. The average relative errors of MGM were 2.07% in fitting part and 17.37% in forecasting part.Data update was crucial in maintain model's effectiveness. The hybrid GM(1,1)-Markov model was better than basic GM(1,1) model in rural MMR prediction. It could be considered as a decision-making tool in rural MMR intervention.

Comparison of ARIMA and GM(1,1) models for prediction of hepatitis B in China.

BACKGROUND: Hepatitis B virus (HBV) infection is a major public health threat in China for China has a hepatitis B prevalence of more than one million people in 2017 year. Disease incidence prediction may help hepatitis B prevention and control. This study intends to build and compare 2 forecasting models for hepatitis B incidence in China. METHODS: Autoregressive integrated moving average (ARIMA) model and grey model GM(1,1) were adopted to fit the monthly incidence of hepatitis B in China from March 2010 to October 2017. The fitting and forecasting performances of the 2 models were evaluated. The better one was adopted to predict the incidence from November 2017 to March 2018. Database was built by Excel 2016 and statistical analysis was completed using R 3.4.3 software. RESULTS: Descriptive analysis showed that the incidence of hepatitis B in China has seasonal variation and has shown a downward trend from 2010 to 2017. We selected the ARIMA (3,1,1) (0,1,2)12 model among all the ARIMA models for it has the lowest AIC value. Model expression of GM (1,1) was X(1) (k + 1) = 3386876.7478e0.0249k - 3289206.7428. The root mean square error (RMSE), mean absolute error (MAE) and mean absolute percentage error (MAPE) of ARIMA(3,1,1)(0,1,2)12 model were lower than GM(1,1) model on fitting part and forecasting part. According to the forecast results, the incidence may have a slight fluctuation during the following months. CONCLUSIONS: The ARIMA model showed better hepatitis B fitting and forecasting performance than GM(1,1) model. It is a potential decision supportive tool for controlling hepatitis B in China before a predictive hepatitis B outbreak.

[Effect of cadmium chloride on immigration of mouse neural stem cell].

OBJECTIVE: To investigate the effects of cadmium chloride on cytoactive and immigration of mouse neural stem cell (mNSC). METHODS: MTT assay was used to detect cytoactive at 24 hours. The immigration of mNSC was determined by immunofluorescence staining. RESULTS: Compared with control, CdCl2 treatment at 10.0 µmol/L for 24 h resulted in a decrease in cellular viability (70.08 ± 6.21)% (P < 0.05). Compared with control, Aa/Ab and Dm/Db display decreasing tendency in a dose-dependent manner (r(s Aa/Ab) = - 0.90, γ(s Dm/Db) = - 0.90, P < 0.05) after CdCl2 treatment at 0.1 - 10.0 µmol/L for 24 h. CONCLUSION: Cadmium chloride treatment inhibits immigration of mNSC, and shows negative effect on cell viability. Meanwhile, the effect of cadmium chloride on immigration is more obvious than cell viability at the same concentration for same treatment time.

Metabolism, excretion and pharmacokinetics of [14C]crizotinib following oral administration to healthy subjects.

1. Crizotinib (XALKORI®), an oral inhibitor of anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor kinase (c-Met), is currently approved for the treatment of patients with non-small cell lung cancer that is ALK-positive. 2. The metabolism, excretion and pharmacokinetics of crizotinib were investigated following administration of a single oral dose of 250 mg/100 µCi [(14)C]crizotinib to six healthy male subjects. 3. Mean recovery of [(14)C]crizotinib-related radioactivity in excreta samples was 85% of the dose (63% in feces and 22% in urine). 4. Crizotinib and its metabolite, crizotinib lactam, were the major components circulating in plasma, accounting for 33% and 10%, respectively, of the 0-96 h plasma radioactivity. Unchanged crizotinib was the major excreted component in feces (∼ 53% of the dose). In urine, crizotinib and O-desalkyl crizotinib lactam accounted for ∼ 2% and 5% of the dose, respectively. Collectively, these data indicate that the primary clearance pathway for crizotinib in humans is oxidative metabolism/hepatic elimination. 5. Based on plasma exposure in healthy subjects following a single dose of crizotinib and in vitro potency against ALK and c-Met, the crizotinib lactam diastereomers are not anticipated to contribute significantly to in vivo activity; however, additional assessment in cancer patients is warranted.

Application of target-mediated drug disposition model to small molecule heat shock protein 90 inhibitors.

Replacement of hydrogen with fluorine within three pairs of structurally similar small molecule inhibitors of heat shock protein 90 (HSP90) resulted in differences in inhibition constants (K(i)) in vitro as well as marked differences in rat intravenous pharmacokinetic profiles. The difference in pharmacokinetic profiles between lower and higher affinity inhibitors (LAIs and HAIs, respectively) was characterized by remarkably different estimates for steady-state volumes of distribution (V(ss): 1.8-2.0 versus 10-13 l/kg) with comparable clearance estimates (3.2-3.5 l/h per kilogram). When the observed V(ss) estimates were compared with the values predicted with the tissue-composition-based model, the observed V(ss) estimates for HAIs were 4- to 8-fold larger than the predicted values, whereas the V(ss) values for LAIs were comparable. Accordingly, a negative relationship between in vitro HSP90 K(i) versus in vivo V(ss) estimates was observed among these inhibitors. We therefore hypothesized that pharmacokinetic profiles of these inhibitors could be characterized by a target-mediated drug disposition (TMDD) model. In vivo equilibrium dissociation constant (K(D)) estimates for HAIs due to target binding by TMDD model with rapid binding approximation were 1-6 nM (equivalent to 0.3-2 nM free drug), which appeared comparable to the in vitro K(i) estimates (2-3 nM). In vivo KD values of LAIs were not accurately determined by the TMDD model, likely due to nonspecific binding-dependent tissue distribution obscuring TMDD profiles. Overall, these results suggest that the observed large Vss estimates for potent HSP90 inhibitors are likely due to pharmacological target binding.

Prediction of crizotinib-midazolam interaction using the Simcyp population-based simulator: comparison of CYP3A time-dependent inhibition between human liver microsomes versus hepatocytes.

Crizotinib (Xalkori) is an orally available potent inhibitor of multiple tyrosine kinases, including anaplastic lymphoma kinase and mesenchymal-epithelial transition factor. Objectives of the present study were as follows: 1) to characterize crizotinib time-dependent inhibition (TDI) potency for CYP3A in human liver microsomes (HLM) and cryopreserved human hepatocytes suspended in human plasma (HSP); 2) to characterize crizotinib enzyme induction potency on CYP3A4 in cryopreserved human hepatocytes; 3) to predict crizotinib steady-state plasma concentrations in patients (e.g., autoinhibition and autoinduction) using the mechanistic dynamic model, Simcyp population-based simulator; and 4) to predict a clinical crizotinib-midazolam interaction using the dynamic model as well as the static mathematical model. Crizotinib inactivation constant (K(I)) and maximum inactivation rate constant (k(inact)) for TDI were estimated as, respectively, 0.37 µM and 6.9 h(-1) in HLM and 0.89 µM and 0.78 h(-1) in HSP. Thus, crizotinib inactivation efficiency (k(inact)/K(I)) was ∼20-fold lower in HSP relative to HLM. Crizotinib E(max) and EC(50) for CYP3A4 induction (measured as mRNA expression) were estimated as 6.4- to 29-fold and 0.47 to 3.1 µM, respectively. Based on these in vitro parameters, the predicted crizotinib steady-state area under plasma concentration-time curve (AUC) with HLM-TDI was 2.1-fold higher than the observed AUC, whereas that with HSP-TDI was consistent with the observed result (≤1.1-fold). The increase in midazolam AUC with coadministration of crizotinib (21-fold) was significantly overpredicted using HLM-TDI, whereas the prediction using HSP-TDI (3.6-fold) was consistent with the observed result (3.7-fold). Collectively, the present study demonstrated the value of HSP to predict in vivo CYP3A-mediated drug-drug interaction.

Discovery pharmacokinetic studies in mice using serial microsampling, dried blood spots and microbore LC-MS/MS.

BACKGROUND: Initial pharmacokinetic (PK) studies of discovery compounds are conducted in mice to demonstrate exposure prior to conducting efficacy studies. PK information obtained from a single mouse by serial blood microsampling, dried blood spot collection and analyses using microbore (1 mm internal diameter column) LC-MS/MS is presented. Ex vivo blood to plasma concentration ratios (BPRs) from mouse PK studies were compared with in vitro BPRs for 15 compounds. RESULTS: Two compounds were orally dosed and blood was collected at time points via serial blood sampling. The calculated PK parameters (AUC, T(max) and C(max)) were comparable across liquid blood, dried blood spot and plasma matrices. The BPR results from both methods were comparable. CONCLUSION: Serial blood microsampling has led to reduced animal and compound usage with improved PK data. Ex vivo BPR is suitable in a discovery setting. Microbore LC-MS/MS is well suited in instances where sample volume is limited, and enables faster analyses, reduced solvent use, and less frequent MS source cleaning.

Metabolite profiling of dasatinib dosed to Wistar Han rats using automated dried blood spot collection.

To characterize and enable efficient rat pharmacokinetic (PK) screening in early drug discovery, automated sampling of blood time points are routinely employed. With the development of dried blood spot (DBS) technology for drug level quantitation, an opportunity exists for the automated collection of rat PK time points using DBS. DBS, as an alternative sample collection technique has led to the increased collection of PK study samples for the quantitative analyses of drug candidates in both pre-clinical and clinical studies. However, the feasibility of using DBS samples for drug metabolite profiling including both phase I and phase II metabolites has not been well established. This work reports the study of metabolite profiling of dasatinib dosed to Wistar Han rats using automated DBS collection. Automated DBS and plasma collection using a rat AccuSampler (VeruTech AB, Sweden) was employed using dasatinib as a model compound. The DBS and plasma samples were extracted by methanol and acetonitrile and both plasma and DBS extracts were analyzed using a Sciex API4000 Qtrap mass spectrometer coupled to a Shimazdzu HPLC system. Dasatinib and its metabolites were analyzed by multiple reaction monitoring (MRM) and MRM trigger enhanced product ion scan (MRM-EPI). Both phase I oxidative metabolites and phase II glucuronide conjugates and sulfate conjugates were detected from both rat plasma and DBS samples. Overall, comparable metabolite profiles including phase I oxidative and phase II glucuronide and sulfate conjugates were observed from both extracts of plasma and DBS samples when using the untreated DBS cards for dasatinib. Chemically treated DBS cards such as DMPK-A and DMPK-B cards may affect the dasatinib metabolites. Similar PK parameters were obtained for dasatinib from both plasma and DBS samples, after correcting for blood to plasma ratio. The results obtained from this study suggest that collection of study samples by DBS can be used for metabolite profiling, however, the availability of limited samples may be a concern for multiple injections.

Pharmacokinetic/pharmacodynamic modeling of crizotinib for anaplastic lymphoma kinase inhibition and antitumor efficacy in human tumor xenograft mouse models.

Crizotinib [Xalkori; PF02341066; (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine] is an orally available dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor. The objectives of the present studies were to characterize: 1) the pharmacokinetic/pharmacodynamic relationship of crizotinib plasma concentrations to the inhibition of ALK phosphorylation in tumors, and 2) the relationship of ALK inhibition to antitumor efficacy in human tumor xenograft models. Crizotinib was orally administered to athymic nu/nu mice implanted with H3122 non-small-cell lung carcinomas or severe combined immunodeficient/beige mice implanted with Karpas299 anaplastic large-cell lymphomas. Plasma concentration-time courses of crizotinib were adequately described by a one-compartment pharmacokinetic model. A pharmacodynamic link model reasonably fit the time courses of ALK inhibition in both H3122 and Karpas299 models with EC(50) values of 233 and 666 ng/ml, respectively. A tumor growth inhibition model also reasonably fit the time course of individual tumor growth curves with EC(50) values of 255 and 875 ng/ml, respectively. Thus, the EC(50) for ALK inhibition approximately corresponded to the EC(50) for tumor growth inhibition in both xenograft models, suggesting that >50% ALK inhibition would be required for significant antitumor efficacy (>50%). Furthermore, based on the observed clinical pharmacokinetic data coupled with the pharmacodynamic parameters obtained from the present nonclinical xenograft mouse model, >70% ALK inhibition was projected in patients with non-small-cell lung cancer who were administered the clinically recommended dosage of crizotinib, twice-daily doses of 250 mg (500 mg/day). The result suggests that crizotinib could sufficiently inhibit ALK phosphorylation for significant antitumor efficacy in patients.

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry.

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 µL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 µL out of a minimum sample volume of 15 µL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 µL plasma aliquot from 15 µL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 µm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 µL of plasma from 15 µL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.

Pharmacokinetic-pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available heat shock protein 90 inhibitor in a human tumor xenograft mouse model.

PF04942847 [2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide] was identified as an orally available, ATP-competitive, small-molecule inhibitor of heat shock protein 90 (HSP90). The objectives of the present study were: 1) to characterize the pharmacokinetic-pharmacodynamic relationship of the plasma concentrations of PF04942847 to the inhibition of HSP90-dependent protein kinase, AKT, as a biomarker and 2) to characterize the relationship of AKT degradation to tumor growth inhibition as a pharmacological response (antitumor efficacy). Athymic mice implanted with MDA-MB-231 human breast cancer cells were treated with PF04942847 once daily at doses selected to encompass ED(50) values. Plasma concentrations of PF04942847 were adequately described by a two-compartment pharmacokinetic model. A time delay (hysteresis) was observed between the plasma concentrations of PF04942847 and AKT degradation; therefore, a link model was used to account for the hysteresis. The model reasonably fit the time courses of AKT degradation with the estimated EC(50) of 18 ng/ml. For tumor growth inhibition, the signal transduction model reasonably fit the inhibition of individual tumor growth curves with the estimated EC(50) of 7.3 ng/ml. Thus, the EC(50) for AKT degradation approximately corresponded to the EC(50) to EC(80) for tumor growth inhibition, suggesting that 50% AKT degradation was required for significant antitumor efficacy (50-80%). The consistent relationship between AKT degradation and antitumor efficacy was also demonstrated by applying an integrated signal transduction model for linking AKT degradation to tumor growth inhibition. The present results will be helpful in determining the appropriate dosing regimen and guiding dose escalation to achieve efficacious systemic exposure in the clinic.

Quantitative analysis of PD 0332991 in xenograft mouse tumor tissue by a 96-well supported liquid extraction format and liquid chromatography/mass spectrometry.

Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6h post-oral PD 0332991 administration at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.

Separation and quantification of two diastereomers of a Drug Candidate in rat plasma by ultra-high pressure liquid chromatography/mass spectrometry.

Ultra-high pressure liquid chromatography (UHPLC) is a relatively new technology which utilizes chromatographic media with a 1.7 microm particle size. This technology has the potential to offer significant advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometric detection. Drug Candidate A, under development at Merck Research Laboratories, contains two chiral centers which have the absolute configuration R, S. Under in vivo and ex vivo conditions, one of the chiral centers readily epimerizes to produce the R, R diastereomer. Initially, a traditional high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was developed to separate and quantify these two diastereomers in rat plasma. The lower limit of quantification (LOQ) of the two analytes was 2 ng/mL, and a chromatographic run time of approximately 11 min was needed to separate R, S-(A) and R, R-(A). In this study, we explored a simple and robust UHPLC-MS/MS method in order to increase sample throughput and productivity. We were able to achieve a two-fold reduction in the lower limit of quantification and a three-fold reduction in retention time utilizing the UHPLC method, while keeping the same sample extraction procedure and similar MS/MS methodology. The new method exhibited good intra- and inter-day accuracy and precision, and was linear over a dynamic range of 1-500 ng/mL for each diastereomer. The method was successfully applied for the determination of R, S-(A) and R, R-(A) concentrations for in vitro and in vivo studies of epimerization of A in Sprague-Dawley rats.

Enantiomeric separation and quantification of pindolol in human plasma by chiral liquid chromatography/tandem mass spectrometry using staggered injection with a CTC Trio Valve system.

Pindolol is a non-selective beta-adrenergic antagonist (beta-blocker) for the treatment of cardiovascular diseases such as hypertension and angina pectoris. It has one chiral center, and, therefore, two optical isomers. It was essential to develop an enantioselective assay to measure each enantiomer in human plasma. However, separation of enantiomers using chiral chromatography usually requires relatively long retention times. This can pose a problem for rapid turnaround of a large number of samples (i.e., clinical studies). In the present study, a simple and sensitive chiral liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the determination of S-(-)- and R-(+)-pindolol in human plasma. To increase throughput, staggered sample injection was employed using a CTC Trio Valve system on a CTC HTS PAL autosampler. The method exhibited good intra- and inter-day accuracy and precision, and was linear over a dynamic range of 250 pg/mL to 250 ng/mL for each pindolol enantiomer. Intra- and inter-day accuracy ranged between 90.0-106% and 91.6-104% for both quality control (QC) samples of S-(-)- and R-(+)-pindolol, respectively. The respective intra- and inter-day precision ranged between 4.24-7.86% and 4.98-10.4%.