The dosing regimen for 17-hydroxyprogesterone caproate was suboptimal: lessons for future pharmacotherapy for pregnant women.

BACKGROUND: Makena (17-hydroxyprogesterone caproate) was approved by the United States Food and Drug Administration for the prevention of recurrent spontaneous preterm birth in 2011 under the accelerated approval pathway, but fundamental pharmacokinetic or pharmacodynamic (Phase 1 and Phase 2) studies were not performed. At the time, there were no dose-response or concentration-response data. The therapeutic concentration was not known. The lack of such data brings into question the dosing regimen for 17-hydroxyprogesterone caproate and if it was optimized. OBJECTIVE: The purpose of this study was to evaluate the dosing regimen for 17-hydroxyprogesterone by analyzing 3 data sets in which the 17-hydroxyprogesterone caproate pharmacology was evaluated, namely the Maternal-Fetal Medicine Omega 3 study, the Obstetric-Fetal Pharmacology Research Units study, and the Obstetrical-Fetal Pharmacology Research Centers study. If an inappropriate dosing regimen could be identified, such information could inform future studies of pharmacotherapy in pregnancy. STUDY DESIGN: Data from the Omega 3 study were used to determine if plasma concentration was related to spontaneous preterm birth risk and if a threshold concentration could be identified. Data from the Obstetric-Fetal Pharmacology Research Units study were used to determine the half-life of 17-hydroxyprogesterone caproate and to develop a model to simulate drug concentrations with various dosing regimens. Data from the Obstetrical-Fetal Pharmacology Research Centers study were used to determine the relationship between dose and safety outcomes. RESULTS: Analysis of the Omega 3 data set indicated that the risk for spontaneous preterm birth decreased as the log concentration of 17-hydroxyprogesterone caproate increased (odds ratio, 0.04; 95% confidence interval, 0.00-0.90). A steady state concentration of >9 ng/mL (equivalent to >8 ng/mL at 25-28 weeks) was associated with the lowest risk for spontaneous preterm birth (hazard ratio, 0.52; 95% confidence interval, 0.27-0.98; P=.04); this concentration was not achieved in 25% of subjects who received the 250 mg weekly dose. In the Obstetrical-Fetal Pharmacology Research Units study, the adjusted half-life (median and interquartile range) of 17-hydroxyprogesterone caproate was 14.0 (11.5-17.2) days. Simulations indicated that with the 250 mg weekly dose, >5 weekly injections were required to reach the 9 ng/mL target; however, those with the shortest half-life (corresponding to higher clearance), never reached the targeted 9 ng/mL concentration. In 75% of subjects, a loading dose of 500 mg weekly for 2 weeks followed by 250 mg weekly achieved and maintained the 9 ng/mL concentration within 2 weeks but in those 25% with the shortest half-life, concentrations exceeded the 9 ng/mL target for only 3 weeks. In the Obstetrical-Fetal Pharmacology Research Centers study, all 65 subjects who received a weekly dose of 500 mg exceeded the 9 ng/mL steady state. CONCLUSION: The dosing regimen for 17-hydroxyprogesterone caproate was inadequate. There is a significant inverse relationship between drug concentration and spontaneous preterm birth. The risk was lowest when the concentration exceeded 9 ng/mL, but 25% of women who received the 250 mg weekly dose never reached or maintained this concentration. The drug's long half-life necessitates a loading dose to achieve therapeutic concentrations rapidly. The omission of basic pharmacologic studies to determine the proper dosing may have compromised the effectiveness of 17-hydroxyprogesterone caproate. Future pharmacotherapy trials in pregnancy must first complete fundamental pharmacology studies.

Assessment of glomerular filtration and tubular secretion in baboons with life-supporting pig kidney grafts.

With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculated were similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.

Effect of formulation and route of administration on the distribution of 17-hydroxyprogesterone caproate in rats.

Weekly intramuscular (250 mg/week) or subcutaneous (275 mg/week) injections of 17-hydroxyprogesterone caproate (17-OHPC) is the only treatment option for the prevention of preterm birth in women with a prior history of preterm delivery.The objective of the current study was to determine the relative distribution of 17-OHPC in selected tissues in adult female SD rats after IM (oily formulation or solution), IV (solution), PO (solution), or intravaginal (suppository) administration.Plasma, uterus, adipose, and liver samples were collected at various times and analysed by LC-MS-MS.The highest concentrations of 17-OHPC were observed in the adipose tissue, after IM (oily formulation), and intravaginal administration.Substantial concentrations of 17-OHPC were also observed in the uterus after IM, intravaginal and IV administration.17-OHPC was not detected in the liver and in any of the tissues tested after PO administration.17-OHPC levels in plasma after intravaginal suppository administration were low despite substantial concentrations in the adipose and the uterus.The distribution of 17-OHPC depends on the formulation, the route of administration, and the sampling time.Low systemic concentrations and substantial distribution in the tissues of interest after intravaginal administration warrants future studies to evaluate the potential of the daily intravaginal route of administration of 17-OHPC.

Pregnancy Alters CYP- and UGT-Mediated Metabolism of Buprenorphine.

BACKGROUND: In the United States, drug addiction has become a nationwide health crisis. Recently, buprenorphine (BUP), a maintenance therapy approved by the Food and Drug Administration, has been increasingly used in pregnant women for the treatment of opioid use disorder. Pregnancy is associated with various anatomic and physiological changes, which may result in altered drug pharmacokinetics (PKs). Previously, we reported that dose-adjusted plasma concentrations of BUP are lower during pregnancy than after pregnancy. The mechanism(s) responsible for this difference has not yet been defined. Our study aimed to evaluate alterations in cytochromes P450 (CYP)- and uridine diphosphate glucunosyltransferases (UGT)-mediated metabolism of BUP during pregnancy to determine the mechanism(s) responsible for this observation. METHODS: Data from 2 clinical studies were included in the current analysis. Study 1 was a prospective, open-labeled, nonrandomized longitudinal BUP PK study in pregnant women with a singleton gestation, stabilized on twice-daily sublingual BUP opioid substitution therapy. Each subject participated in up to 3 studies during and after pregnancy (the second, third trimester, and postpartum). The design of study 2 was similar to study 1, with patients evaluated at different time points during the pregnancy (first, second-half of pregnancy), as well as during the postpartum period. In addition, the dosing frequency of BUP study 2 participants was not restricted to twice-daily dosing. At each study visit, blood samples were collected before a BUP dose, followed by multiple collection times (10-12) after the dose, for up to 12 hours or till the end of the dosing interval. Plasma concentrations of BUP and 3 metabolites were quantified using validated ultraperformance liquid chromatography-tandem mass spectrometric assays. RESULTS: In total, 19, 18, and 14 subjects completed the PK study during 1/2 trimester, third trimester, and postpartum, respectively. The AUC ratios of norbuprenorphine and norbuprenorphine glucuronide to buprenorphine, a measure of CYP3A mediated N-demethylation, were 1.89, 1.84, and 1.33 during the first and second, third trimesters, and postpartum, respectively. The AUC ratios of buprenorphine glucuronide to BUP, indicative of UGT activity, were 0.71, 2.07, and 0.3 at first/second trimesters, third trimester, and postpartum, respectively. Linear mixed-effect modeling analysis indicated that the AUC ratios of CYP- and UGT-mediated metabolism of BUP were significantly higher during pregnancy compared with postpartum. CONCLUSIONS: The CYP and UGT activities were significantly increased as determined by the metabolic ratios of BUP during pregnancy compared with the postpartum period. The increased UGT activity appeared to account for a substantial part of the observed change in metabolic activity during pregnancy. This is in agreement with the need for BUP dose increment in pregnant women to reach similar BUP exposure and therapeutic effect as in nonpregnant subjects.

HDL from an Alzheimer's disease perspective.

PURPOSE OF REVIEW: We review current knowledge regarding HDL and Alzheimer's disease, focusing on HDL's vasoprotective functions and potential as a biomarker and therapeutic target for the vascular contributions of Alzheimer's disease. RECENT FINDINGS: Many epidemiological studies have observed that circulating HDL levels associate with decreased Alzheimer's disease risk. However, it is now understood that the functions of HDL may be more informative than levels of HDL cholesterol (HDL-C). Animal model studies demonstrate that HDL protects against memory deficits, neuroinflammation, and cerebral amyloid angiopathy (CAA). In-vitro studies using state-of-the-art 3D models of the human blood-brain barrier (BBB) confirm that HDL reduces vascular Aß accumulation and attenuates Aß-induced endothelial inflammation. Although HDL-based therapeutics have not been tested in clinical trials for Alzheimer's disease , several HDL formulations are in advanced phase clinical trials for coronary artery disease and atherosclerosis and could be leveraged toward Alzheimer's disease . SUMMARY: Evidence from human studies, animal models, and bioengineered arteries supports the hypothesis that HDL protects against cerebrovascular dysfunction in Alzheimer's disease. Assays of HDL functions relevant to Alzheimer's disease may be desirable biomarkers of cerebrovascular health. HDL-based therapeutics may also be of interest for Alzheimer's disease, using stand-alone or combination therapy approaches.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway.

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

Pharmacokinetics of Intravenous Isavuconazole in Solid-Organ Transplant Recipients.

Isavuconazole may be useful in treating and preventing fungal infections in solid-organ transplant (SOT) recipients due to its safety profile and activity against Aspergillus and some Mucorales Isavuconazole has favorable pharmacokinetics based on clinical trials in various patient populations, but data are limited in SOT recipients. We evaluated the steady-state pharmacokinetics of isavuconazole in 26 SOT recipients receiving the drug intravenously for prophylaxis. There was moderate interpatient variability in isavuconazole pharmacokinetic parameters (coefficients of variation of 51% for the area under the plasma concentration-versus-time curve [AUC] and 59% for the trough plasma concentration [Ctrough]). AUC and steady-state Ctrough were significantly lower in women, patients with a body mass index of ≥18.5 kg/m2, and those receiving hemodialysis. Trough plasma concentrations were highly correlated with AUCs (R2 = 0.94) and can serve as a suitable measure of isavuconazole exposure in patients. In conclusion, moderate interpatient variability in isavuconazole exposure, the identification of factors associated with lower exposure, the recognition that Ctrough is a surrogate marker for AUC, and the availability of a simple analytical method suggest that therapeutic drug monitoring (TDM) may be useful for guiding treatment in at least some SOT recipients. Future studies are needed to correlate isavuconazole exposure with patients' clinical outcomes and to determine the clinical role of TDM.

Reduced-Concentration Clavulanate for Young Children with Acute Otitis Media.

Amoxicillin-clavulanate (A/C) is currently the most effective oral antimicrobial in treating children with acute otitis media (AOM), but the standard dosage of 90 mg amoxicillin/6.4 mg clavulanate/kg of body weight/day commonly causes diarrhea. We examined whether an A/C formulation containing lower concentrations of clavulanate would result in less diarrhea while maintaining plasma levels of amoxicillin and clavulanate adequate to eradicate middle-ear pathogens and to achieve clinical success. We conducted an open-label study in children with AOM who were 6 to 23 months of age. In phase 1, we treated 40 children with a reduced-clavulanate A/C formulation providing 90 mg amoxicillin/3.2 mg clavulanate/kg/day for 10 days. In phase 2, we treated 72 children with the same formulation at a dosage of 80 mg amoxicillin/2.85 mg clavulanate/kg/day for 10 days. We compared the rates of protocol-defined diarrhea (PDD), diaper dermatitis, and AOM clinical response in these children with rates we had reported in children who received the standard A/C regimen, and we obtained plasma levels of amoxicillin and clavulanate at various time points. Outcomes in phase 1 children and in children who had received the standard regimen did not differ significantly. Rates of PDD in children receiving phase 2 and standard regimens were 17% and 26%, respectively (P = 0.10). The corresponding rates of diaper dermatitis were 21% and 33% (P = 0.04) and of AOM treatment failure were 12% and 16% (P = 0.44). Symptomatic responses did not differ significantly between regimens; both gave clavulanate levels sufficient to inhibit ß-lactamase activity. In young children with AOM, clavulanate dosages lower than those currently used may be associated with fewer side effects without reducing clinical efficacy. (This study has been registered at ClinicalTrials.gov under registration no. NCT02630992.).

Programmable co-delivery of the immune checkpoint inhibitor NLG919 and chemotherapeutic doxorubicin via a redox-responsive immunostimulatory polymeric prodrug carrier.

To achieve synergistic therapeutic efficacy and prevent cancer relapse, chemotherapy and immunotherapy have been combined as a new modality for tumor treatment. In this work, we designed a redox-responsive immunostimulatory polymeric prodrug carrier, PSSN10, for programmable co-delivery of an immune checkpoint inhibitor NLG919 (NLG) and a chemotherapeutic doxorubicin (DOX). NLG-containing PSSN10 prodrug polymers were self-assembled into nano-sized micelles that served as a carrier to load DOX (DOX/PSSN10 micelles). DOX/PSSN10 micelles displayed spherical morphology with a size of ∼170 nm. DOX was effectively loaded into PSSN10 micelles with a loading efficiency of 84.0%. In vitro DOX release studies showed that rapid drug release could be achieved in the highly redox environment after intracellular uptake by tumor cells. In 4T1.2 tumor-bearing mice, DOX/PSSN10 micelles exhibited greater accumulation of DOX and NLG in the tumor tissues compared with other organs. The PSSN10 carrier dose-dependently enhanced T-cell immune responses in the lymphocyte-Panc02 co-culture experiments, and significantly inhibited tumor growth in vivo. DOX/PSSN10 micelles showed potent cytotoxicity in vitro against 4T1.2 mouse breast cancer cells and PC-3 human prostate cancer cells comparable to that of DOX. In 4T1.2 tumor-bearing mice, DOX/PSSN10 mixed micelles (5 mg DOX/kg, iv) was more effective than DOXIL (a clinical formulation of liposomal DOX) or free DOX in inhibiting the tumor growth and prolonging the survival of the treated mice. In addition, a more immunoactive tumor microenvironment was observed in the mice treated with PSSN10 or DOX/PSSN10 micelles compared with the other treatment groups. In conclusion, systemic delivery of DOX via PSSN10 nanocarrier results in synergistic anti-tumor activity.

Pharmacokinetics of Posaconazole Suspension in Lung Transplant Patients with and without Cystic Fibrosis.

Invasive fungal infections (IFIs) are common among lung transplant recipients (LTRs). Posaconazole is an important antifungal agent for both prophylaxis and treatment of IFIs; however, detailed pharmacokinetic data are limited among LTRs, particularly those with cystic fibrosis (CF). Our objective was to conduct a pharmacokinetic study of posaconazole oral suspension among LTRs, with particular attention to patients with CF. We enrolled 20 LTRs, 7 with CF and 13 with other underlying lung diseases. Average daily doses in CF and non-CF patients were 829 and 862 mg, respectively. After ≥5 days of treatment, only 4 patients had average plasma concentrations of >0.7 µg/ml. Average steady-state plasma concentrations were 61% lower in CF patients (0.233 µg/ml) than in non-CF LTRs (0.594 µg/ml; P = 0.03). The average dose-normalized plasma area-under-the-curve (AUC) values were also lower in CF (0.007 h·µg/ml) than in non-CF LTRs (0.02 h·µg/ml; P = 0.02). The weight-normalized apparent oral clearance values were 2.51 and 0.74 liters/h/kg among CF and non-CF LTRs, respectively (P = 0.005). Despite significant interpatient variability, plasma trough concentrations were strongly correlated with posaconazole AUC across all LTRs (r(2) = 0.95, P < 0.0001). Taken together, our study highlights a critical need to incorporate new formulations of posaconazole into prophylaxis and treatment strategies for LTRs, particularly those with CF. Future pharmacokinetic studies of both tablet and intravenous formulations must consider LTR-specific factors and incorporate a therapeutic drug monitoring plan in this patient population.

PEG-farnesyl thiosalicylic acid telodendrimer micelles as an improved formulation for targeted delivery of paclitaxel.

We have recently designed and developed a dual-functional drug carrier that is based on poly(ethylene glycol) (PEG)-derivatized farnesylthiosalicylate (FTS, a nontoxic Ras antagonist). PEG5K-FTS2 readily form micelles (20-30 nm) and hydrophobic drugs such as paclitaxel (PTX) could be effectively loaded into these micelles. PTX formulated in PEG5K-FTS2 micelles showed an antitumor activity that was more efficacious than Taxol in a syngeneic mouse model of breast cancer (4T1.2). In order to further improve our PEG-FTS micellar system, four PEG-FTS conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/FTS (1/2 vs 1/4) in the conjugates. These conjugates were characterized including CMC, drug loading capacity, stability, and their efficacy in delivery of anticancer drug PTX to tumor cells in vitro and in vivo. Our data showed that the conjugates with four FTS molecules were more effective than the conjugates with two molecules of FTS and that FTS conjugates with PEG5K were more effective than the counterparts with PEG2K in forming stable mixed micelles. PTX formulated in PEG5K-FTS4 micelles was the most effective formulation in inhibiting the tumor growth in vivo.

Targeted delivery of Doxorubicin by folic acid-decorated dual functional nanocarrier.

Doxorubicin (DOX) is one of the most commonly used antineoplastic agents, but its clinical application is oftentimes coupled with severe side effects. Selective delivery of DOX to tumors via nanosized drug carrier represents an attractive approach to this problem. Previously, we developed a dual functional nanomicellar carrier, PEG5K-embelin2 (PEG5K-EB2), which was able to deliver paclitaxel (PTX) selectively to tumors and to achieve an enhanced therapeutic effect. In the present study, we examined the utility of PEG5K-EB2 to deliver DOX to tumors. In addition, folic acid (FA) was coupled to the surface of the PEG5K-EB2 micelles (FA-PEG5K-EB2) to further improve the selective targetability of the system. DOX-loaded PEG5K-EB2 micelles were uniformly spherical particles with a diameter of approximately 20 nm. Incorporation of FA had minimal effect on the size of the particles. The DOX loading efficiency was as high as 91.7% and 93.5% for PEG5K-EB2 and FA-PEG5K-EB2, respectively. DOX formulated in PEG5K-EB2 micelles (with or without FA decoration) demonstrated sustained kinetics of DOX release compared to free DOX. FA-PEG5K-EB2 significantly facilitated the intracellular uptake of DOX over free DOX and PEGylated liposomal DOX (Doxil) in breast cancer cells, 4T1.2, and drug resistant cells, NCI/ADR-RES. P-gp ATPase assay showed that PEG5K-EB2 significantly inhibited the function of the P-gp efflux pump. The maximum tolerated dose of DOX-loaded PEG5K-EB2 micelles was 15 mg/kg in mice, which was 1.5-fold greater than that for free DOX. Pharmacokinetics (PK) and biodistribution studies showed that both types of DOX-loaded micelles, especially FA-PEG5K-EB2, were able to significantly prolong the blood circulation time of DOX and facilitate its preferential accumulation at the tumor tissue. Finally, DOX/PEG5K-EB2 mixed micelles demonstrated significantly enhanced tumor growth inhibitory effect with minimal toxicity in comparison to free DOX and Doxil and the antitumor activity was further enhanced after the decoration by folic acid. Our data suggest that FA-PEG5K-EB2 micelles represent a promising DOX delivery system that warrants more study in the future.

Quality assessment of compounded 17-hydroxyprogesterone caproate.

OBJECTIVE: The purpose of this study was to evaluate the quality of compounded 17-hydroxyprogesterone caproate (17-OHPC). STUDY DESIGN: Compounded 17-OHPC that was obtained from 15 compounding pharmacies throughout the United States was analyzed for potency, impurities, sterility, and pyrogen status. RESULTS: Eighteen samples were supplied by 15 compounding pharmacies. The concentration of 17-OHPC in all samples was within the specification limits, and all tested samples passed sterility and pyrogen testing. Only 1 of 18 samples was out of specification limits for impurities. CONCLUSION: Compounded 17-OHPC that was obtained from 15 pharmacies throughout the United States did not raise safety concerns when assessed for potency, sterility, pyrogen status, or impurities.

Targeted delivery of anticancer agents via a dual function nanocarrier with an interfacial drug-interactive motif.

We have developed a dual-function drug carrier, polyethylene glycol (PEG)-derivatized farnesylthiosalicylate (FTS). Here we report that incorporation of a drug-interactive motif (Fmoc) into PEG5k-FTS2 led to further improvement in both drug loading capacity and formulation stability. Doxorubicin (DOX) formulated in PEG5k-Fmoc-FTS2 showed sustained release kinetics slower than those of DOX loaded in PEG5k-FTS2. The maximum tolerated dose of DOX- or paclitaxel (PTX)-loaded PEG5k-Fmoc-FTS2 was significantly higher than that of the free drug. Pharmacokinetics and biodistribution studies showed that DOX/PEG5k-Fmoc-FTS2 mixed micelles were able to retain DOX in the bloodstream for a significant amount of time and efficiently deliver the drug to tumor sites. More importantly, drug (DOX or PTX)-loaded PEG5k-Fmoc-FTS2 led to superior antitumor activity over other treatments including drugs formulated in PEG5k-FTS2 in breast cancer and prostate cancer models. Our improved dual function carrier with a built-in drug-interactive motif represents a simple and effective system for targeted delivery of anticancer agents.

Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Metabolomic and biochemical changes in the plasma and liver of toxic milk mice model of Wilson disease.

Wilson disease (WD) is an inherited disorder characterized by abnormal copper metabolism with complex pathological features. Currently, this mechanism of copper overload-induced hepatic injury remains unclear. In this study, male toxic milk (TX) mice were selected as experimental subjects. Copper levels and biochemical indices were measured by atomic absorption spectroscopy (AAS) and kits. Liver tissue ultrastructure was observed by hematoxylin-eosin (H&E), sirius red staining and transmission electron microscopy. Plasma and liver metabolic profiles of TX mice were characterized by untargeted metabolomics. In addition, the expression of enzymes related to arachidonic acid metabolism in liver tissue was detected by Western blotting. The results showed the excessive copper content, concomitant oxidative stress, and hepatic tissue structural damage in TX mice. Seventy-eight metabolites were significantly different in WD, mainly involved in the metabolism of arachidonic acid, glycerophospholipids, sphingolipids, niacin and nicotinamide, and phenylalanine. Furthermore, the arachidonic acid metabolic pathway is an important pathway involved in WD metabolism. The level of arachidonic acid in the liver of TX mice was significantly lower (p < 0.01) compared to the control group. The expression of cytoplasmic phospholipase A2 (cPLA2) and arachidonic acid 12-lipoxygenase (ALOX12), related to the arachidonic acid metabolic pathway, was significantly different in the liver of TX mice (p < 0.01). Modulation of the arachidonic acid metabolic pathway could be a potential therapeutic strategy to alleviate WD symptoms.

Deregulated hepatic metabolism exacerbates impaired testosterone production in Mrp4-deficient mice.

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.