Respective Plasma Concentrations Research Articles

Immunogenetic Profile Associated with Patients Living with HIV-1 and Epstein-Barr Virus (EBV) in the Brazilian Amazon Region.

Viral coinfection among HIV-positive patients, coupled with the development of AIDS, remains a major public health problem. The synergism between the presence of HIV and other viruses has consequences in relation to changes in the severity of the infection, as well as changes in the natural course of both infections. Several polymorphisms present in genes that encode cytokines have a relevant influence on their transcription and consequently on the production of such immunological molecules. The present study evaluated the influence of SNPs located in the promoter regions of genes encoding the cytokines INF-ɣ, TNF, IL-6, IL-4, and IL-2, as well as their respective plasma concentrations, in patients infected with HIV and/or EBV in the state of Pará. Additionally, this study described the epidemiological profile and compared CD4+ and CD8+ T lymphocyte counts among the groups studied. The associative analysis between the SNPs and plasma cytokine concentrations in different groups showed statistical relevance for three polymorphisms: rs2069762 (IL2), where the GG genotype demonstrated higher IL-2 levels in HIV mono-infected individuals; rs2243250 (IL4), where the CT genotype showed higher IL-4 levels in the control group; and rs2069705 (IFNG), where the TT genotype showed higher IFN-γ levels in the coinfected group. Regarding SNP associations with CD4+/CD8+ counts, significant findings were observed in HIV mono-infected individuals: the rs2069705 (IFNG) polymorphism was linked to higher CD4+ counts with the CT genotype, and rs1799964 (TNF) was associated with higher CD8+ counts with the CC genotype. Therefore, this study provides evidence that the rs2069705 (IFNG) SNP is associated with elevated IFN-γ levels, which may have pathogenic consequences, as depletion of this cytokine is concerning for people living with HIV due to its antiviral properties.

Effects of nickel supplementation on nutrient utilization, mineral balance, haematology and antioxidant status of crossbred dairy calves

BackgroundNickel has been identified as an important newer trace mineral playing essential role in animals however, its precise mechanism of action in the body is yet to be determined. Interaction of Ni with other essential minerals is suggested in reports limited to lab animals and needs to be explored further in large animals. AimThis study was conducted to study the influence of Ni supplementation at different levels, on minerals and health status of crossbred dairy calves. MethodTwenty-four Karan Fries crossbred (Tharparkar × Holstein Friesian) male dairy calves were selected based on body weight (137.09 ± 5.68) and age (10.78 ± 0.61) and were divided into 4 treatment groups (n = 6) given basal diet supplemented with 0 (Ni0), 5 (Ni5), 7.5 (Ni7.5) and 10 (Ni10) ppm Ni/kg of DM. Nickel was supplemented in the form of nickel sulphate hexahydrate (NiSO4.6 H2O) solution. To ensure the intake of the required amount of nickel by each animal, the calculated quantity of solution was mixed with 250 g concentrate mixture and offered individually to the calves. The calves were fed total mixed ration (TMR) consisting of green fodder, wheat straw and concentrate mixture in the ratio of 40:20:40 and the nutritional requirements were met according to NRC (2001) guidelines. Growth performance was recorded at fortnightly interval whereas, plasma minerals, haematology, antioxidant and immunity parameters were studied at monthly interval during the 150-day experimental period. Nutrient utilization and mineral balances were estimated with the help of a metabolism trial conducted at the end of feeding trial. ResultsSupplementation of Ni exhibited no influence on dry matter intake (DMI), body weight, average daily gain (ADG) and nutrient digestibility of dairy calves. However, the absorption and balance of minerals such as Ni, Fe, Cu, Zn and their respective plasma concentration increased (P < 0.05) with Ni supplementation and highest values were observed in calves fed 10 mg Ni/kg DM. The red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (HCT) and activity of superoxide dismutase (SOD) and catalase antioxidant enzymes showed highest increase (P < 0.05) in calves supplemented with Ni at level of 10 mg/kg DM as compared to other treatment groups. However, white blood cell (WBC) count, glutathione peroxidase (GPx), total antioxidant status (TAS), total immunoglobulins and IgG plasma concentration remained unaltered with addition of different levels of Ni in the diet of calves. ConclusionsThe supplementation of Ni at level of 10 mg/kg DM shows a positive effect on status of trace minerals such as Fe, Cu, Zn and improves the physiological conditions and health status of crossbred dairy calves indicated by improved haematology and antioxidant parameters.

Dual Targeting of MDM2 and BCL2/Bclxl Demonstrates Potent Synergistic Activity in High-Risk Adult Acute Lymphoblastic Leukemia

Despite the use of both intensive multi-modal cytotoxic therapeutic regimens and novel immune-based therapies in adult acute lymphoblastic leukemia (ALL) outcomes remain dismal, with five-year survival rates less than 45%. As such, there is an urgent need to identify novel therapies to improve patient outcomes. While TP53 mutations are infrequent in ALL, alternative mechanisms such as MDM2 overexpression or CDKN2A/B deletion may otherwise phenotypically disrupt and circumvent normal p53 function. We previously demonstrated encouraging pre-clinical activity of p53-MDM2 antagonist idasanutlin (RG7388) in diverse high-risk primary and patient-derived xenograft (PDX) ALL samples at concentrations well-below clinically attainable plasma levels [Bell et al., ASH 2020]. However, clinical studies reveal only modest responses to idasanutlin monotherapy in various hematological malignancies, emphasizing the need to identify effective drug combinations. To systematically identify combination treatments which enhance cytotoxic idasanutlin activity, we performed comprehensive high-throughput phenotypic combination screening against 1971 FDA-approved and clinically advanced drugs. Extensive use of an ex vivo coculture system of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSCs) was employed to support growth and survival of the ALL blasts, complemented by high-content image analysis to discriminate and enumerate the respective cell populations. Using the relapsed NALM6 B-ALL cell line and a panel of 6 PDX ALL samples - including relapsed and high-risk cytogenetic subtypes - we iteratively identified drugs (n=10) exhibiting significant synergistic and potent interaction with idasanutlin at concentrations well-below respective drug peak plasma concentrations reported in patients (Bliss synergy δ > 10). These included clinical ALL drugs dexamethasone and daunorubicin, proteasome inhibitor carfilzomib, BCL2/BCLxL inhibitor navitoclax, and histone deacetylase inhibitors romidepsin and pracinostat. Counter-screening of each drug candidate in a NALM6 TP53-isogenic cell model confirmed on-target p53-dependent combination activity (p<0.05). Further interrogation of each drug candidate was performed across a broad landscape of drug combination ratios with idasanutlin in primary (n=5) and PDX samples (n=6), including high-risk KMT2A-rearranged, ABL class T-ALL, and ETP-ALL. Combination of idasanutlin with navitoclax exhibited both the greatest and most consistent synergistic interaction (δ = 24.7±8.7, n=11) of the candidate combinations (n=10) across an extensive landscape of dose combinations; additionally alluding to synergy indiscriminate of ALL subtype. Further characterizing the cytotoxic antileukemic activity of navitoclax, single-agent exposure achieved low nanomolar range, dose-dependent ex vivo sensitivity in a panel of B- (n=10) and T-ALL (n=4) primary and PDX samples, with half maximal inhibitory concentrations (IC50) in the range of 3 to 11 nM (mean IC50 = 6.4 nM) - almost 1000-fold lower than clinically attainable concentrations [Roberts et al., BJH2015]. While both idasanutlin and navitoclax effectively induced apoptosis in their own right, combination exposure markedly enhanced cell death. In an exceptionally high-risk TCF3-HLF PDX sample, 24 hours exposure to the drug combination increased annexin-V positive cells by 37.3±5.0% and 33.3±3.5% as compared to either drug (IC50s, p<0.0001). In the NALM6 and RS4;11 relapsed ALL cell lines, combination exposure significantly increased annexin-V positive cells by at least two-fold (p<0.001) and enhanced apoptotic hallmark products cleaved poly(ADP-ribose) polymerase (PARP) (p<0.01) and cleaved caspase-3 as compared to either drug alone (IC50s). Mechanistically, we found protein levels of MCL-1-binding pro-apoptotic sensitizer NOXA to be up to 3-fold increased in combination-treated blasts (p<0.05), potentially inhibiting compensatory anti-apoptotic MCL-1 activity - a known clinical resistance factor to BH3 mimetics. Together, we provide strong evidence that concurrent targeting of MDM2-p53 binding and BCL2/BCLxL leads to potent and synergistic enhancement of apoptotic cell death in a range of high-risk ALL subtypes. The proposed combination of two clinical-stage compounds could have considerable positive clinical impact for the treatment of adult ALL.

Quantification of serotonin and eight of its metabolites in plasma of healthy volunteers by mass spectrometry

Serotonin is transformed into melatonin under the control of the light/dark cycle, representing a cornerstone of circadian rhythmicity. Serotonin also undergoes extensive metabolism to produce 5-hydroxyindoleacetic acid (5-HIAA), a biomarker for the diagnosis and monitoring of serotonin secreting neuroendocrine tumors (NETs). While serotonin, melatonin and their metabolites are part of an integrated comprehensive system, human observations about their respective plasma concentrations are still limited.We report here for the first time a multiplex UHPLC-MS/MS assay for the quantification of serotonin, 5-HIAA, 5-hydroxytryptophol (5-HTPL), N-acetyl-serotonin (NAS), Mel, 6-OH-Mel, 5-methoxytryptamine (5-MT), 5-methoxytryptophol (5-MTPL), and 5-methoxyindoleacetic acid (5-MIAA) in human plasma. Analytes were extracted by protein precipitation and solid phase extraction. Plasma concentrations for these analytes were determined in 102 healthy volunteers.The LLOQ of the assay ranges from 2.2 nM for serotonin to 1.0 pM for 6-OH-Mel. This sensitivity enables the quantification of circulating serotonin, 5-HIAA, NAS, Mel, and 5-MIAA, even at their lowest diurnal concentrations.This assay will enable specific, precise and accurate measurement of serotonin, Mel and their metabolites to draw a detailed picture of this complex pineal metabolism, allowing a dynamic understanding of these pathways and providing promising biomarkers and a metabolic signature for serotonin-secreting NETs.

Combined biotin, folic acid, and vitamin B12 supplementation given during the transition period to dairy cows: Part I. Effects on lactation performance, energy and protein metabolism, and hormones

Biotin (B8), folates (B9), and vitamin B12 (B12) are involved and interrelated in several metabolic reactions related to energy and protein metabolism. We hypothesized that a low supply of one of the latter vitamins during the transition period would impair metabolic status. The purpose of this study was to evaluate the effect of B8 supplementation on the response of lactation performance and selected energy and protein metabolites and hormones to a combined supplementation of B9 and B12 given to periparturient dairy cows, from d -21 to 21 relative to calving. A total of 32 multiparous Holstein cows housed in tie stalls were randomly assigned, according to their previous 305-d milk yield, to 8 incomplete blocks of 4 treatments: (1) a 2-mL weekly i.m. injection of saline (0.9% NaCl; B8-/B9B12-); (2) 20 mg/d of dietary B8 (unprotected from ruminal degradation) and 2-mL weekly i.m. injection of 0.9% NaCl (B8+/B9B12-); (3) 2.6 g/d of dietary B9 (unprotected) and 2-mL weekly i.m. injection of 10 mg of B12 (B8-/B9B12+); and (4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9, and weekly i.m. injection of 10 mg of B12 (B8+/B9B12+) in a 2 × 2 factorial arrangement. Milk yield and dry matter intake were obtained daily and milk components weekly. Blood samples were taken weekly from d -21 to calving and 3 times per week from calving to 21 d following parturition. Prepartum plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHB), and adiponectin were unaffected by treatments. Biotin, B9, and B12 supplements increased their respective concentrations in plasma and milk. Cows fed the B8 supplement tended to have lower dry matter intake, but only cows in B8+/B9B12- had greater plasma concentrations of NEFA compared with B8-/B9B12-. Milk and total solid yields were greater by 13.5 and 13.9%, respectively, for B8-/B9B12+ [45.5 (standard error, SE: 1.8) and 5.81 (0.22) kg/d, respectively] compared with B8-/B9B12- [40.1 (1.9) and 5.10 (0.23) kg/d, respectively], but these effects were suppressed when combined with the B8 supplement. Cows in the B8-/B9B12+ group had decreased plasma insulin and tended to have increased NEFA concentrations, but postpartum plasma concentrations of glucose, BHB, leptin, and adiponectin were not affected. These cows also mobilized more body fat reserves, as suggested by a tendency to increased plasma NEFA and more milk total solids compared with B8-/B9B12- cows. However, plasma concentrations of BHB and adiponectin were similar among treatments. This suggests that the B9 and B12 supplements enhanced efficiency of energy metabolism in early lactation cows. Folic acid and B12 supplementation increased postpartum plasma Cys and homocysteine concentrations but did not affect plasma Met concentration, suggesting an upregulation of the transsulfuration pathway. In summary, our results showed that, under the current experimental conditions, increasing B8 supply did not improve responses to the B9 and B12 supplementation.

Infant Dextromethorphan and Dextrorphan Exposure via Breast Milk From Mothers Who Are CYP2D6 Extensive Metabolizers.

The risk of infant exposure to dextromethorphan (DM) and its active metabolite, dextrorphan (DX), through breast milk has not been evaluated. In this study, bound and unbound DM and DX concentrations in breast milk and plasma at 2hours post-dose were measured in 20 lactating women (n=20) following a single 30 mg oral dose of DM. The DM and DX concentrations in breast milk were positively correlated with their respective plasma concentrations. The breast milk-to-plasma (M/P) ratios of 1.0 and 1.6 and the unbound M/P ratios of 1.1 and 2.0 for DM and DX, respectively, suggested that DM and DX are extensively distributed into breast milk. The infant exposure following a single dose of 30 mg DM was estimated using breast milk concentrations of 0.33±0.32 and 1.8±1.0μg/kg/day for DM and DX, respectively. The steady-state infant exposure was estimated using the M/P ratios and previously reported area under the concentration-time curve (AUC) of DM and DX following repeated dosing of DM 60mg orally, twice daily, to be 0.64±0.22 and 1.23±0.38μg/kg/day, respectively. Based on these estimated infant doses, the relative infant doses (RIDs) were estimated to be <1%, suggesting the infant is only exposed to a minor fraction of adult dose through breast milk; however, one nursing infant developed an erythematous rash during this study, which warrants additional research to fully elucidate the risks of infant exposure to DM and DX through breast milk.

Higher habitual intake of dietary dicarbonyls is associated with higher corresponding plasma dicarbonyl concentrations and skin autofluorescence: the Maastricht Study

Dicarbonyls are highly reactive compounds and major precursors of advanced glycation end products (AGEs). Both dicarbonyls and AGEs are associated with development of age-related diseases. Dicarbonyls are formed endogenously but also during food processing. To what extent dicarbonyls from the diet contribute to circulating dicarbonyls and AGEs in tissues is unknown. To examine cross-sectional associations of dietary dicarbonyl intake with plasma dicarbonyl concentrations and skin AGEs. In 2566 individuals of the population-based Maastricht Study (age: 60±8 y, 50% males, 26% with type 2 diabetes), we estimated habitual intake of the dicarbonyls methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG) by combining FFQs with our dietary dicarbonyl database of MGO, GO, and 3-DG concentrations in>200 commonly consumed food products. Fasting plasma concentrations of MGO, GO, and 3-DG were measured by ultra-performance liquid chromatography-tandem mass spectrometry. Skin AGEs were measured as skin autofluorescence (SAF), using the AGE Reader. Associations of dietary dicarbonyl intake with their respective plasma concentrations and SAF (all standardized) were examined using linear regression models, adjusted for age, sex, potential confounders related to cardiometabolic risk factors, and lifestyle. Median intake of MGO, GO, and 3-DG was 3.6, 3.5, and 17mg/d, respectively. Coffee was the main dietary source of MGO, whereas this was bread for GO and 3-DG. In the fully adjusted models, dietary MGO was associated with plasma MGO (β: 0.08; 95% CI: 0.02, 0.13) and SAF (β: 0.12; 95% CI: 0.07, 0.17). Dietary GO was associated with plasma GO (β: 0.10; 95% CI: 0.04, 0.16) but not with SAF. 3-DG was not significantly associated with either plasma 3-DG or SAF. Higher habitual intake of dietary MGO and GO, but not 3-DG, was associated with higher corresponding plasma concentrations. Higher intake of MGO was also associated with higher SAF. These results suggest dietary absorption of MGO and GO. Biological implications of dietary absorption of MGO and GO need to be determined. The study has been approved by the institutional medical ethical committee (NL31329.068.10) and the Minister of Health, Welfare and Sports of the Netherlands (Permit 131088-105234-PG).

Pilot study of oral fluid and plasma meropenem and piperacillin concentrations in the intensive care unit

BackgroundTherapeutic drug monitoring (TDM) of β-lactam antibiotics may be used to optimize dosing for patients in the intensive care unit (ICU). A noninvasive matrix such as oral fluid may be interesting in selected patient groups. We compared the oral fluid concentrations of piperacillin and meropenem with the respective unbound and total concentrations in plasma. A secondary objective was to evaluate feasibility of the collection of oral fluid samples in this specific patient population. MethodsThe study included 20 non-intubated ICU patients, age 22 to 77 y, receiving piperacillin or meropenem via continuous intravenous infusion. The standard protocol consisted of collecting a paired plasma-oral fluid sample for 3 consecutive days. Oral fluid was obtained from the patients using a standardized procedure by spitting in a plastic container after 2 min of gathering oral fluid in the mouth. ResultsAntibiotic concentrations of piperacillin and meropenem are measurable, albeit very low, in unstimulated oral fluid of ICU patients. For piperacillin, a poor correlation was found between oral fluid and both total and unbound plasma concentrations (Spearman’s correlation coefficients (Rs) 0.46 and 0.48 respectively). For meropenem this correlation was better (Rs for oral fluid versus total and unbound plasma meropenem concentration 0.92 and 0.93 respectively). Dispersion of antibiotic concentrations was greater in oral fluid than in blood. Collecting oral fluid samples was difficult in non-intubated ICU patients. ConclusionsOral fluid from non-intubated ICU patients, obtained through a standardized procedure, cannot be recommended as an alternative matrix for quantitative meropenem or piperacillin TDM.

Drug–drug interactions and pharmacodynamics of concomitant clobazam and cannabidiol or stiripentol in refractory seizures

ObjectiveThe goal of this study was to characterize the drug–drug interactions between clobazam and 2 antiseizure drugs, cannabidiol and stiripentol, for treatment of refractory seizures through the use of pharmacokinetic modeling. MethodsA population pharmacokinetic/pharmacodynamic model was developed to characterize the combined effect of clobazam and its active metabolite, N-desmethylclobazam (i.e., N-clobazam), on seizure protection in patients with Lennox–Gastaut syndrome using data from the phase 3 CONTAIN trial. Drug–drug interactions between clobazam and cannabidiol were examined by comparing model-generated data to data from a study of 13 patients taking concomitant clobazam and cannabidiol. Modeling data were also descriptively compared with studies of patients administered both clobazam and stiripentol. Sedation-related adverse events from CONTAIN were analyzed to determine the exposure–somnolence relationship of clobazam. ResultsExposure-efficacy analysis from the pharmacokinetic/pharmacodynamic model using CONTAIN data indicated that clobazam (half-maximal effective concentration [EC50], 303 ng/mL) was 3 times more potent than N-clobazam (EC50, 899 ng/mL). After administration of clobazam, when both clobazam and N-clobazam concentrations were each 1 to 2 times the EC50 value (clobazam dose, 20 mg), 70.0%–74.9% seizure protection was predicted; when concentrations were >2 times the EC50 value (clobazam dose, 40 mg), 74.0%–96.9% seizure protection was predicted. Generalized additive model analyses demonstrated decreased seizure probability with higher plasma concentration of clobazam. Coadministration of stiripentol and clobazam resulted in increased respective median plasma concentrations of clobazam and N-clobazam (1.1–1.2 times and 5.2–8.2 times) compared with administration of placebo and clobazam. Probability of somnolence significantly increased with age and higher N-clobazam plasma concentration. SignificanceAwareness of drug–drug interactions between clobazam and cannabidiol is needed when adding cannabidiol or stiripentol to a regimen of clobazam or vice versa. Based upon our population pharmacokinetic/pharmacodynamic model, we predict that an increase in N-clobazam levels, which patient data show may enhance efficacy and/or make adverse events such as somnolence more likely.

Effect of maternal biotin, folic acid, and vitamin B12 supplementation before parturition on colostral and Holstein calf plasma concentrations in those vitamins

The newborn calf relies on placental and colostral transfers of B vitamins to cover its requirements. This study was conducted to evaluate biotin, folate and vitamin B12 concentrations in colostrum of cows receiving those vitamin supplements and in plasma of their calves fed with their colostrum. According to a 2 × 2 factorial arrangement, non-lactating pregnant cows (n = 34) were assigned to 2 levels of biotin supplement (0 or 20 mg/day of dietary biotin) and 2 levels of folic acid and vitamin B12 supplements (0 or 2.6 g/day of dietary folic acid and 10 mg of intramuscular injections of vitamin B12). Treatments were initiated 26 (SD: 8) days before calving until the parturition. Colostrum sample from the first milking was harvested. Offspring (n = 23) were fed 2.4 (SE: 0.3) kg of their dam colostrum within 3 (SD: 2) h of life (P > 0.24); calves born as twin, stillborn or not receiving their dam colostrum were removed. Calves were blood sampled and weighed 25 (SD: 4) h after birth. Supplementary biotin, folic acid, and vitamin B12 successfully increased their respective prepartum dam plasma and colostrum vitamin concentrations (P < 0.001). Colostral biotin, folate, and vitamin B12 concentrations were respectively increased from 26.8 (95% confidence interval (CI): 20.4–35.1) to 253.7 (95% CI: 193.8–332.3), 673 to 1094 (SE: 52), and 28.6 to 57.9 (SE: 3.3) ng/mL by their respective vitamin supplementation. Colostral IgG concentration did not differ among treatments (P > 0.14) and averaged 49.8 (SD: 23.5) g/L. Calves born to dams receiving folic acid and vitamin B12 supplements were heavier (50 vs. 44, SE: 1 kg; P = 0.003); biotin supplement did not affect calf weight (P = 0.66). Plasma biotin concentrations of calves were increased from 1.1 (95% CI: 0.9–1.4) to 7.5 (95% CI: 6.1–9.2) ng/mL by the maternal biotin supplementation (P < 0.001). Maternal folate and vitamin B12 supplementation during the prepartum period increased calf plasma concentration of folates from 15.9 (SE: 1.9) to 30.8 (SE: 1.8) ng/mL and tended to augment plasma vitamin B12 concentration of offspring, from 0.76 (95% CI: 0.51–1.12) to 1.25 (95% CI: 0.87–1.80) ng/mL (P < 0.07). No treatment effect was noted on calf plasma concentration of IgG (P > 0.54). Results suggest that biotin, folic acid and vitamin B12 supplementation given to the dam before calving increased supply of those vitamins for newborn calves who rely on external supply of the B vitamins to fulfill their requirements before the development of their functional rumen.

Abstract 3078: Tesetaxel, a novel, oral taxane, crosses intact blood-brain barrier (BBB) at therapeutically relevant concentrations

Abstract Background: Central nervous system (CNS) metastases in cancers such as breast and lung cancer are common and associated with poor outcomes. The taxanes paclitaxel (P) and docetaxel (D) do not significantly cross the BBB (brain to plasma exposure of only 1% and 8%, respectively), making them ineffective in treating CNS metastases. Tesetaxel (T) is a novel, orally administered taxane currently in a Phase 3 study in patients with advanced breast cancer (aBC; NCT03326674). Several properties make it unique, including: oral administration with a low pill burden; a long (~8-day) terminal plasma half-life in humans, allowing infrequent dosing; no history of hypersensitivity reactions; significant activity against chemo-resistant tumors; and, unlike P and D, minimal efflux by the P-glycoprotein pump, a key element of the BBB. The in vivo tissue distribution of tesetaxel was previously investigated in mice treated with 14C-T at 4 mg/kg and sacrificed between 1-168 hours. We now report 2 additional studies in dogs and monkeys. Methods: Dogs and monkeys were dosed at 0.6 mg/kg and 1 mg/kg of 14C-T, respectively, and tissue distribution was assessed at 336 hours. These doses equate to ~44% of the 27 mg/m2 dose in the ongoing Phase 3 study. In vivo radioactivity concentrations in the cerebrum were compared to plasma concentrations and mean tumor GI50 (concentration resulting in 50% tumor growth inhibition) determined by MTT assay in 23 tumor cell lines. Results: The absolute brain concentrations of T in dogs and monkeys far exceeded both the respective plasma concentrations and the tumor GI50 (Table 1). Conclusions: In dogs and monkeys, T brain concentrations far exceed its tumor GI50 for a sustained period when administered at therapeutically relevant concentrations. The inclusion criteria for the ongoing Phase 3 study of T in patients with aBC allows for CNS metastases. Table 1:Tesetaxel CNS Levels Far Exceed Human GI50a in Dogs and Monkeys14C-T Radioactivity Concentration (ng eq./g or mL) bCerebrum Concentration/ Tumor GI50 cNCerebrum (Mean ± SD)Plasma (Mean ± SD)Cerebrum/ PlasmaDog d310.9 ± 4.00.9 ± 0.112x18xMonkey e36.5 ± 3.80.9 ± 0.27x11xaConcentration of drug required to inhibit growth by 50%b14 days after dosingcMean GI50 for T across 23 tumor cells lines = 0.6 ng/mLdSingle dose of 0.6 mg/kg (equivalent to 44% of a human dose of 27 mg/m2)eSingle dose of 1 mg/kg (equivalent to 44% of a human dose of 27 mg/m2) Citation Format: Joyce James, Kevin Tang, Thomas Wei. Tesetaxel, a novel, oral taxane, crosses intact blood-brain barrier (BBB) at therapeutically relevant concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3078.

Nintedanib plus letrozole in early breast cancer: a phase 0/I pharmacodynamic, pharmacokinetic, and safety clinical trial of combined FGFR1 and aromatase inhibition

BackgroundThe combined use of a FGFR1 blocker and aromatase inhibitors is appealing for treating breast cancer patients with FGFR1 amplification. However, no pharmacodynamic studies have addressed the effects of this combined target modulation. We conducted a phase 0/I clinical trial in an adjuvant setting, with the goal of obtaining pharmacodynamic proof of the effects of combined aromatase and FGFR1 inhibition and to establish the RP2D for nintedanib combined with letrozole.Patients and methodsWomen with early-stage luminal breast cancer were eligible for enrollment in the study. Dose level 1 was nintedanib (150 mg/bid) plus letrozole (2.5 mg/day) administered for a single 28-day cycle (DLT assessment period), followed by a classic 3 + 3 schedule. FGF23 and 17-B-estradiol levels were determined on days 0 and 15; pharmacokinetic parameters were assessed on days 1 and 28. Patients were allowed to continue treatment for 6 cycles. The primary study endpoint was a demonstration of FGFR1 modulation (defined as a 25% increase in the plasma FGF23 level).ResultsA total of 19 patients were enrolled in the study (10 in the expansion cohort following dose escalation). At the RP2D (nintedanib 200 mg/bid plus letrozole 2.5 mg/day), we observed a 55% mean increase in the plasma FGF23 level, and 81.2% of the patients had no detectable level of 17-B-estradiol in their plasma (87.5% of the patients treated with letrozole alone). Nintedanib and letrozole displayed a pharmacokinetic interaction that led to three- and twofold increases in their respective plasma concentrations. Most G3 toxic events (5 out of 6: 2 diarrhea and 3 hypertransaminasemia) occurred subsequent to the DLT assessment period.ConclusionCombined treatment with nintedanib (200 mg/bid) plus letrozole (2.5 mg/day) effectively suppressed FGFR1 and aromatase activity, and these respective doses can be used as starting doses in any subsequent trials. However, drug-drug interactions may produce tolerability issues when these drugs are co-administered for an extended time period (e.g., 6 months). Patients enrolled in future trials with these drugs should be carefully monitored for their FGF23 levels and signs of toxicity, and those findings should guide individualized treatment decisions.Trial registrationThis trial was registered at www.clinicaltrials.gov under reg. # NCT02619162, on December 2, 2015.

Multiplex analysis of genetic polymorphisms within UGT1A9, a gene involved in phase II of Δ9-THC metabolism.

We present a novel multiplex assay for the simultaneous detection of 12 polymorphisms within the UGT1A9 sequence, which codes for enzymes involved in phase II biotransformation. The assay combines a multiplexed amplification step with single-base extension sequencing. The method described here is fast, cost-effective, and easy-to-use, combining the relevant features of screening methods for research and diagnostics in pharmacogenetics. To validate the assay, we tested reproducibility and sensitivity and analysed allele frequencies of 110 Caucasian individuals. Furthermore, we describe combining genetic information of individuals consuming Cannabis sativa products with respective plasma concentrations of a metabolite.

Urinary renin-angiotensin markers in polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD), activation of the renin-angiotensin aldosterone system (RAAS) may contribute to hypertension and disease progression. Although previous studies have focused on circulating RAAS components, preliminary evidence suggests that APDKD may increase urinary RAAS components. Therefore, our aim was to analyze circulating and urinary RAAS components in ADPKD. We cross-sectionally compared 60 patients with ADPKD with 57 patients with non-ADPKD chronic kidney disease (CKD). The two groups were matched by sex, estimated glomerular filtration rate (eGFR), blood pressure, and RAAS inhibitor use. Despite similar plasma levels of angiotensinogen and renin, urinary angiotensinogen and renin excretion were five- to sixfold higher in ADPKD (P < 0.001). These differences persisted when adjusting for group differences and were present regardless of RAAS inhibitor use. In multivariable analyses, ADPKD, albuminuria, and the respective plasma concentrations were independent predictors for urinary angiotensinogen and renin excretion. In ADPKD, both plasma and urinary renin correlated negatively with eGFR. Total kidney volume correlated with plasma renin and albuminuria but not with urinary renin or angiotensinogen excretions. Albuminuria correlated positively with urinary angiotensinogen and renin excretions in ADPKD and CKD. In three ADPKD patients who underwent nephrectomy, the concentrations of albumin and angiotensinogen were highest in plasma, followed by cyst fluid and urine; urinary renin concentrations were higher than cyst fluid. In conclusion, this study shows that, despite similar circulating RAAS component levels, higher urinary excretions of angiotensinogen and renin are a unique feature of ADPKD. Future studies should address the underlying mechanism and whether this may contribute to hypertension or disease progression in ADPKD.

Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice

Elevated circulating fatty acids (FAs) contribute to the development of obesity-associated metabolic complications such as insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). Hence, reducing adipose tissue lipolysis to diminish the mobilization of FAs and lower their respective plasma concentrations represents a potential treatment strategy to counteract obesity-associated disorders. Here we show that specific inhibition of adipose triglyceride lipase (Atgl) with the chemical inhibitor, Atglistatin, effectively reduces adipose tissue lipolysis, weight gain, IR and NAFLD in mice fed a high-fat diet. Importantly, even long-term treatment does not lead to lipid accumulation in ectopic tissues such as the skeletal muscle or heart. Thus, the severe cardiac steatosis and cardiomyopathy that is observed in genetic models of Atgl deficiency does not occur in Atglistatin-treated mice. Our data validate the pharmacological inhibition of Atgl as a potentially powerful therapeutic strategy to treat obesity and associated metabolic disorders.

Shortening of the electromechanical window in the ketamine/xylazine-anesthetized guinea pig model to assess pro-arrhythmic risk in early drug development

BackgroundA negative electromechanical window (EMw) was recently proposed as a better preclinical tool than QTc interval to predict clinical pro-arrhythmic potential. As such, we utilized the ketamine/xylazine anesthetized guinea pig to characterize the EMw and QTc interval for a diverse set of reference agents with known clinical pro-arrhythmic potential. Then we determined the clinical proarrhythmia predictive capacity of EMw shortening compared to hERG inhibition or QTc interval prolongation alone. MethodsChanges in EMw and QTc interval by 26 reference agents were evaluated in the ketamine/xylazine-anesthetized guinea pig. Confusion matrix analysis using the hERG, QTc and EMw indexes (hERG IC50, QTc EC5 or the EMw EC−10 divided by their respective free therapeutic maximal plasma concentration) at various folds the therapeutic concentrations was conducted to assess the concordance of each index to predict clinical pro-arrhythmic risk. ResultsShortening of the EMw concomitant to an increase in QTc interval was observed in the GP with known pro-arrhythmic drugs. Non-torsadogenic compounds did not cause EMw shortening, although some prolonged the QTc interval. The preclinical:clinical concordance of the EMw index (88%) was similar (p>0.05) to using QTc interval prolongation alone (85%) but significantly greater (p<0.05) than using hERG inhibition alone (69%). In addition, the specificity when using the EMw (87%) was largely greater (p<0.05) than using QTc interval (73%) or hERG inhibition (60%) alone. When the components of the response (duration of left ventricular pressure (LVP) cycle (QLVPend) or QT interval) that caused EMw shortening were considered, the concordance is further improved (>95%). ConclusionEMw shortening improves QTc interval prolongation recording in early drug development and increases the translatability over existing preclinical tools in predicting clinical arrhythmias.

Bioavailability Studies and in vitro Profiling of the Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitor UCPH-102.

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood-brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg(-1) ) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH-102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20 mg kg(-1) ) did not induce acute effects or any visible changes in behavior.

Retinol and Alpha-tocopherol in the Colostrum of Lactating Tunisian Women Delivering Prematurely: Associations with Maternal Characteristics

This study aims to assess vitamin A and E concentrations in the premature colostrum of lactating Tunisian women and to identify maternal characteristics that may affect these concentrations. Human colostrum was obtained from 105 mothers who gave birth prematurely in the Centre for Maternity and Neonatology of Tunis (Tunisia). Retinol and alpha-tocopherol were analyzed in the colostrum and in plasma by high-performance liquid chromatography. Retinol and alpha-tocopherol concentrations were 57.5 ± 50.1 μg/dL and 1222 ± 772 μg/dL in the colostrum, respectively, and 51.7 ± 20.0 μg/dL and 1351 ± 772 μg/dL in plasma, respectively. Concentrations of each vitamin in the colostrum were positively correlated with their respective concentrations in plasma (r = 0.415, p = 0.001 for retinol and r = 0.392, p = 0.003 for alpha-tocopherol). In multivariate analysis, colostrum vitamin A was associated with plasma vitamin A and preeclampsia, while colostrum vitamin E was associated with plasma vitamin E, gestational age, and preeclampsia. In Tunisian women, colostrum vitamin A and E levels are close to the average values reported in the literature. The levels are too low to cover the needs of very low birth weight (VLBW) infants, particularly in women with plasma vitamin deficiencies, preeclampsia, or very premature delivery. Given the undeniable beneficial effects of human colostrum, whenever feasible, VLBW infants should be fed colostrum. Infant vitamin A and E requirements should be met by milk fortification or supplementation.

Effect of the Urinary Tryptin Inhibitor Ulinastatin on Cardiopulmonary Bypass–Related Inflammatory Response and Clinical Outcomes: A Meta-Analysis of Randomized Controlled Trials

PurposeCardiopulmonary bypass (CPB) can cause systemic inflammatory responses and a series of subsequent complications that may harm patients. The aim of this study was to explore the effects of ulinastatin on inflammatory responses and clinical outcomes of CPB via a meta-analysis of published randomized controlled trials. MethodsA literature search was conducted, both manually and by using the PubMed, EMBASE, Cochrane Library, and Web of Knowledge databases from inception to February 2013, to identify randomized controlled trials. The abstracted efficacy measures included changes in the plasma levels of cytokines (interleukin-6 [IL-6], IL-8, and tumor necrosis factor-α [TNF-α]) measured during the perioperative period and clinical indicators of efficacy, including the duration of mechanical ventilation and the length of intensive care unit stay. Ten ulinastatin-related randomized controlled trials related to cardiac surgeries involving CPB were selected. FindingsIn terms of cytokine concentrations, there were no significant differences between patients who received ulinastatin and those who received placebo before CPB. However, as the surgeries progressed, cytokine concentrations were all significantly lower in the ulinastatin group (P < 0.05 at 1 hour; P < 0.0001 at 6 hours), and the respective plasma concentrations returned to baseline values 24 hours after CPB. In terms of the clinical outcome indices, the length of intensive care unit stay was not significantly different, but the duration of mechanical ventilation (95% CI, –6.75 to –0.39; P = 0.03) was significantly shorter in the ulinastatin group. ImplicationsThis meta-analysis found that changes in inflammatory cytokines occurred in a time-dependent manner and that the use of ulinastatin resulted in decreased duration of mechanical ventilation with CPB compared with placebo.

Opioid Concentrations in Oral Fluid and Plasma in Cancer Patients With Pain

ContextMeasuring opioid concentrations in pain treatment is warranted in situations where optimal opioid analgesia is difficult to reach. ObjectivesTo assess the usefulness of oral fluid (OFL) as an alternative to plasma in opioid concentration monitoring in cancer patients on chronic opioid therapy. MethodsWe collected OFL and plasma samples from 64 cancer patients on controlled-release (CR) oral morphine, CR oral oxycodone, or transdermal (TD) fentanyl for pain. Samples were obtained on up to five separate days. ResultsA total of 213 OFL and plasma samples were evaluable. All patients had detectable amounts of the CR or TD opioid in both plasma and OFL samples. The plasma concentrations of oxycodone and fentanyl (determination coefficient R2 = 0.628 and 0.700, respectively), but not morphine (R2 = 0.292), were moderately well correlated to the daily opioid doses. In contrast to morphine and fentanyl (mean OFL/plasma ratio 2.0 and 3.0, respectively), the OFL oxycodone concentrations were significantly higher than the respective plasma concentrations (mean OFL/plasma ratio 14.9). An active transporter could explain the much higher OFL vs. plasma concentrations of oxycodone compared with morphine and fentanyl. ConclusionOFL analysis is well suited for detecting the studied opioids. For morphine and fentanyl, an approximation of the plasma opioid concentrations is obtainable, whereas for oxycodone, the OFL/plasma concentration relationship is too variable for reliable approximation results.