Construction and characterization of a humanized SLCO1B1 rat model with its application in evaluating the uptake of different statins.

Organic anion-transporting polypeptides 1B1 (OATP1B1) plays a crucial role in the transport of statins. However, there are too few animal models related to OATP1B1, especially humanized animal models. In this study, the human SLCO1B1 cDNA was inserted into the second exon of the rat Slco1b2 gene using CRISPR/Cas9 technology. Pharmacokinetic characteristics of statins were conducted in wild-type (WT), humanized OATP1B1 (hOATP1B1), and OATP1B2 knockout (OATP1B2 KO) rats, respectively. The results showed that human OATP1B1 was successfully expressed in rat liver and exhibited transport function. Furthermore, the pharmacokinetic results revealed that OATP1B1 exhibited varying uptake levels of pivastatin, rosuvastatin, and fluvastatin, leading to different levels of exposure within the body. These results were consistent with those obtained from in vitro experiments using overexpressed cell lines. In conclusion, we established a novel humanized SLCO1B1 transgenic rat model to assess the role of human OATP1B1 in the uptake of different statins. The different uptake mediated by OATP1B1 may be an important reason for the different efficacy of statins. The hOATP1B1 rat is a promising model for improving the prediction of human drug transport.

Improving antifouling performance of FO membrane by surface immobilization of silver nanoparticles based on a tannic acid: diethylenetriamine precursor layer for municipal wastewater treatment.

In this study, a facile method for multifunctional surface modification on forward osmosis (FO) membrane was constructed by surface immobilization of AgNPs based on tannic acid (TA)/diethylenetriamine (DETA) precursor layer. The cellulose triacetate (CTA) FO membranes modified by TA and DETA with different co-deposition time (6 h, 12 h, 24 h) were investigated. Results indicated that the TA/DETA (24)-Ag CTA membrane with a TA/DETA co-deposition time of 24 h was identified to be optimal, which attained more hydrophilic. And it had the bacterial mortality of Escherichia coli and Staphylococcus aureus reaching 98.23% and 99.83% respectively and possessed excellent physical and chemical binding stability. Meanwhile, the coating layer resulted in the antifouling ability without damaging the membrane intrinsic transport characteristics. As for synthetic municipal wastewater treatment, the water flux of CTA FO membrane decreased approximately 49% of the initial flux after running for 14 days. In contrast, the flux decline rate of TA/DETA (24)-Ag CTA membrane was about 37%. Furthermore, less foulant deposition and higher recovery rate of water flux was observed for TA/DETA (24)-Ag CTA membrane, implying that the modified membrane effectively alleviated membrane fouling and processed a lower flux decline during municipal wastewater treatment. It was attributed to the enhanced surface hydrophilicity and antibacterial property of the coating layer, which improved antifouling property.

Sorafenib reduces the production of epoxyeicosatrienoic acids and leads to cardiac injury by inhibiting CYP2J in rats.

Sorafenib, an important cancer drug in clinical practice, has caused heart problems such as hypertension, myocardial infarction, and thrombosis. Although some mechanisms of sorafenib-induced cardiotoxicity have been proposed, there is still more research needed to reach a well-established definition of the causes of cardiotoxicity of sorafenib. In this report, we demonstrate that sorafenib is a potent inhibitor of the CYP2J enzyme. Sorafenib significantly inhibited the production of epoxyeicosatrienoic acids (EETs) in rat cardiac microsomes. The in vivo experimental results also showed that after the administration of sorafenib, the levels of 11,12-EET and 14,15-EET in rat plasma were significantly reduced, which was similar to the results of CYP2J gene knockout. Sorafenib decreased the levels of EETs, leading to abnormal expression of mitochondrial fusion and fission factors in heart tissue. In addition, the expression of mitochondrial energy metabolism factors (Pgc-1α, Pgc-1ß, Ampk, and Sirt1) and cardiac mechanism factors (Scn5a and Prkag2) was significantly reduced, increasing the risk of arrhythmia and heart failure. Meanwhile, the increase in injury markers Anp, CK, and CK-MB further confirmed the cardiotoxicity of sorafenib. This study is of great significance for understanding the cardiotoxicity of sorafenib, and is also a model for studying the cardiotoxicity of other drugs that inhibit CYP2J activity.

6-formylindolo[3, 2-b]carbazole alters gut microbiota and prevents the progression of ankylosing spondylitis in mice.

Ankylosing spondylitis (AS), is known as a chronic inflammatory autoimmune disease, there is evidence to suggest that gut microbiota disorders may be related to the occurrence and development of AS. Studies have shown that 6-formylindolo[3, 2-b]carbazole (FICZ) has the ability to modulate intestinal homeostasis and inhibit inflammatory responses. The purpose of this work is to evaluate the protective role of FICZ in treating AS and elucidate potential mechanisms. FICZ was administered to the proteoglycan (PG)-induced AS mice for 7 consecutive weeks. The effects of FICZ on AS mice were evaluated by the disease severity, intestinal histopathology, proinflammatory cytokine levels, and intestinal mucosal barrier function. The gut microbiota compositions were profiled through 16S rDNA high-throughput sequencing. We found that FICZ significantly reduced the severity of AS and resulted in the downregulating of TNF-α and IL-17A inflammatory cytokines. Moreover, FICZ ameliorated pathological changes in the ileal and improved intestinal mucosal barrier function. Furthermore, FICZ altered the composition of the gut microbiota by increasing the Bacteroidetes/Firmicutes phylum ratio and enriched the genes related to "glycan biosynthesis and metabolism", thus reversing the process of AS. In conclusion, FICZ suppressed the progression of AS and altered gut microbiota in AS mice, which provided new insight into AS therapy strategy.

GRP78 promotes bone metastasis of prostate cancer by regulating bone microenvironment through Sonic hedgehog signaling.

Bone metastasis is the leading cause of tumor-related deaths in patients with prostate cancer (PCa). The interactions between PCa and the bone microenvironment form a vicious cycle. However, the complex molecular mechanism by which PCa regulates the bone microenvironment remains unclear. To determine the role of glucose-regulated protein (GRP78) in bone metastasis and growth, we established intracardiac injection and tibial injection models, and performed their histological staining. To assess the effect of GRP78 on the differentiation of osteoblasts and osteoclasts, we performed cell co-culture, enzyme-linked immunosorbent assay, alizarin red staining, and tartrate-resistant acid phosphatase staining. We found that GRP78 is upregulated in PCa tissues and that its upregulation is associated with PCa progression in patients. Functional experiments showed that GRP78 overexpression in PCa cells considerably promotes bone metastasis and induces bone microstructure changes. Silencing GRP78 substantially inhibits the migration and invasion of PCa cells in vitro and bone metastasis and tumor growth in vivo. Mechanistically, GRP78 promotes the migration and invasion of PCa cells via the Sonic hedgehog (Shh) signaling pathway. Cell co-culture showed that GRP78 promotes the differentiation of osteoblasts and osteoclasts through Shh signaling. Our findings suggest that tumor-bone matrix interactions owing to GRP78-activated paracrine Shh signaling by PCa cells regulate the differentiation of osteoblasts and osteoclasts. This process promotes bone metastasis and the proliferation of PCa cells in the bone microenvironment. Targeting the GRP78/Shh axis can serve as a therapeutic strategy to prevent bone metastasis and improve the quality of life of patients with PCa.

Photocatalytic membrane coated with α-Fe2O3/Fe3O4 for enhanced filtration performance and antifouling property in surface water treatment.

A photocatalytic membrane coated with α-Fe2O3/Fe3O4 nanoparticles has been developed to address the challenges of membrane fouling and organic removal in the treatment of natural surface water. The photocatalytic and filtration properties of the membranes were investigated through a variety of methods. The successful preparation of iron oxide was confirmed by UV-vis diffuse reflectance spectra and X-ray diffractometry, with α-Fe2O3 identified as the primary photocatalytic agent. A commercial ultrafiltration (UF) membrane was employed as a comparison to evaluate the photocatalytic performance and filtration properties of the modified membrane. Results showed that the photocatalytic membrane achieved better removal rates for UV254 (22.0%) and specific fluorescent organic compounds, such as component 2 (19.38%) and component 3 (16.89%), compared to the control group. Furthermore, both irreversible and reversible fouling resistances of the prepared membranes were significantly lower than that of the control group, with reductions of 39.4% and 50.2%, respectively. The membrane coated with α-Fe2O3/Fe3O4 nanoparticles exhibited moderate removal of protein-like and terrestrially derived humic-like fluorescent organics while controlling membrane fouling. Although the α-Fe2O3/Fe3O4 nanoparticles-coated photocatalytic membrane demonstrated good anti-fouling properties, the removals of organic matters were not as effective as anticipated due to the shorter hydraulic retention time. This study provides valuable insights for enhancing pollutant degradation and anti-fouling properties of membranes through the utilization of solar photocatalytic α-Fe2O3/Fe3O4 surface-modified membranes in the treatment of natural surface water.

Ankylosing spondylitis PET imaging and quantifications via P2X7 receptor-targeting radioligand [18F]GSK1482160.

PURPOSE: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial spine; however, the quantitative detection of inflammation in AS remains a challenge in clinical settings. We aimed to investigate the feasibility of using a specific P2X7R-targeting 18F-labeled tracer [18F]GSK1482160 for positron emission tomography (PET) imaging and the quantification of AS. METHODS: The radioligand [18F]GSK1482160 was obtained based on nucleophilic aliphatic substitution. Dynamic [18F]GSK1482160 and [18F]FDG micro-PET/CT imaging were performed on AS mice (n = 8) and age-matched controls (n = 8). Tracer kinetics modeling was performed using Logan's graphical arterial input function analysis to quantify the in vivo expression of P2X7R. The post-PET tissues were collected for hematoxylin-eosin (H&E), immunohistochemical (IHC), and immunofluorescence (IF) staining. RESULTS: [18F]GSK1482160 PET/CT imaging revealed that the specific binding in the ankle joint and sacroiliac joint (SIJ) of the AS at 8 weeks group (BPNDankle-AS-8W (non-displaceable binding potential of the ankle) 3.931 ± 0.74; BPND SIJ-AS-8W (BPBD of the SIJ) 4.225 ± 0.84) were significantly higher than the controls at 8 weeks group (BPNDankle-Ctr-8W 0.325 ± 0.15, BPNDSJJ-Ctr-8W 0.319 ± 0.17) respectively, and the AS at 14 weeks group (BPNDankle-AS-14W 12.212 ± 2.25; BPNDSJJ-AS-14W 13.389 ± 3.60) were significantly higher than the controls at 14 weeks group (BPNDankle-Ctr-14W 0.204 ± 0.16, BPNDSJJ-Ctr-14W 0.655 ± 0.35) respectively. The four groups had no significant difference in the [18F]FDG uptake of ankle and SIJ. IHC and IF staining revealed that the overexpression of P2X7R was colocalized with activated macrophages from the ankle synovium and spinal endplate in mice with AS, indicating that quantification of P2X7R may contribute to the understanding of the pathogenesis of inflammation in human AS. CONCLUSION: This study developed a novel P2X7R-targeting PET tracer [18F]GSK1482160 to detect the expression of P2X7R in AS mouse models and provided powerful non-invasive PET imaging and quantification for AS.

Comparative Effectiveness of MRI, 4D-CT and Ultrasonography in Patients with Secondary Hyperparathyroidism.

Objective: Accurate preoperative localization of abnormal parathyroid glands is crucial for successful surgical management of secondary hyperparathyroidism (SHPT). This study was conducted to compare the effectiveness of preoperative MRI, 4D-CT, and ultrasonography (US) in localizing parathyroid lesions in patients with SHPT. Methods: We performed a retrospective review of prospectively collected data from a tertiary-care hospital and identified 52 patients who received preoperative MRI and/or 4D-CT and/or US and/or 99mTc-MIBI and subsequently underwent surgery for SHPT between May 2013 and March 2020. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each imaging modality to accurately detect enlarged parathyroid glands were determined using histopathology as the criterion standard with confirmation using the postoperative biochemical response. Results: A total of 198 lesions were identified intraoperatively among the 52 patients included in this investigation. MRI outperformed 4D-CT and US in terms of sensitivity (P < 0.01), specificity (P = 0.455), PPV (P = 0.753), and NPV (P = 0.185). The sensitivity and specificity for MRI, 4D-CT, and US were 90.91%, 88.95%, and 66.23% and 58.33%, 63.64%, and 50.00%, respectively. The PPV of combined MRI and 4D-CT (96.52%) was the highest among the combined 2 modalities. The smallest diameter of the parathyroid gland precisely localized by MRI was 8×3 mm, 5×5 mm by 4D-CT, and 5×3 mm by US. Conclusion: MRI has superior diagnostic performance compared with other modalities as a first-line imaging study for patients undergoing renal hyperparathyroidism, especially for ectopic or small parathyroid lesions. We suggest performing US first for diagnosis and then MRI to make a precise localization, and MRI proved to be very helpful in achieving a high success rate in the surgical treatment of renal hyperparathyroidism in our own experience.

Establishment of LC-MS/MS method for quantifying chlorpromazine metabolites with application to its metabolism in liver and placenta microsomes.

Chlorpromazine has sedative and antiemetic pharmacological effects and is widely used in clinic. Its main metabolites include 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide, which affect the therapeutic efficacy. To support metabolism research, the quantitative analysis method of 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide in microsomal enzymes was established for the first time by LC-MS/MS. This method has been fully validated in rat liver microsomes, and partially verified in human liver microsomes and human placenta microsomes. The intra-day and inter-day accuracy and precision of the analytes were all within ± 15%. The extraction recovery was good, and no matrix effect was detected. This accurate and sensitive method was successfully applied to chlorpromazine metabolism in different microsomal enzymes. In particular, the biotransformation of chlorpromazine in human placenta microsomes was detected for the first time. The metabolites detected in human liver and placenta microsomes presented different formation rates, indicating the wide distribution and different activities of drug-metabolizing enzymes.

Design and Construction of Carboxylesterase 2c Gene Knockout Rats by CRISPR/Cas9.

BACKGROUND: Carboxylesterase 2 (CES2) is mainly distributed in the human liver and gut, and plays an active role in the metabolic activation of many prodrugs and lipid metabolism. Although CES2 is of great significance, there are still few animal models related to CES2. OBJECTIVES: This research aims to construct Ces2c gene knockout (KO) rats and further study the function of CES2. METHODS: CRISPR/Cas9 gene editing technology was used to target and cleave the rat Ces2c gene. Compensatory effects of major CES subtypes both in the liver and small intestine of KO rats were detected at mRNA levels. Meanwhile, diltiazem and aspirin were used as substrates to test the metabolic capacity of Ces2c in KO rats. RESULTS: This Ces2c KO rat model showed normal growth and breeding without off-target effects. The metabolic function of Ces2c KO rats was verified by the metabolic study of CES2 substrates in vitro. The results showed that the metabolic capacity of diltiazem in KO rats was weakened, while the metabolic ability of aspirin did not change significantly. In addition, the serum physiological indexes showed that the Ces2c deletion did not affect the liver function of rats.. CONCLUSION: The Ces2c KO rat model was successfully constructed by CRISPR/Cas9 system. This rat model can not only be used as an important tool to study the drug metabolism mediated by CES2, but also as an important animal model to study the physiological function of CES2.

[Prenatal screening and diagnosis for a fetus with mosaic sex chromosome abnormality].

OBJECTIVE: To carry out prenatal screening and diagnosis for a woman with advanced maternal age. METHODS: Non-invasive prenatal testing (NIPT) was carried out to determine the risk of fetal chromosome aneuploidy. Aminiocentesis was proceeded for fetal chromosomal karyotyping and copy number variation sequencing (CNV-seq). The fetus was subjected to systematic ultrasound screening in the second trimester. RESULTS: NIPT has indicated there was a loss of fetal sex chromosome. Karotyping of the amniocyte showed a mosaic sex chromosome abnormality 45,X[53]/46,X,+mar[7]. The result of fetal DNA CNV-seq was seq[GRCh37]del(Yq11.1q12) chrY: g.13 104 553-28 819 361del, seq[GRCh37]del(Yp11.32p11.2) chrY: g.10 001-9 873 915del (mosaic ratio: 30%). Ultrasonography discovered that the fetus had renal dysplasia and male external genitalia. The karyotypes of the couple were both normal. CONCLUSION: Multiple genetic tests should be carried out for fetus with a high risk for chromosome aneuploidies signaled by NIPT. It is difficult to predict the post-natal phenotype for fetuses with mosaic sex chromosomal aneuploidies. The couple should be carefully counseled upon genetic counseling.

Hypercholesterolemia reduces the expression and function of hepatic drug metabolizing enzymes and transporters in rats.

Hypercholesterolemia, one of the most common lipid metabolic diseases, may cause severe complications and even death. However, the effect of hypercholesterolemia on drug-metabolizing enzymes and transporters remains unclear. In this report, we established a rat model of diet-induced hypercholesterolemia. Quantitative real-time PCR and Western blot analysis were used to study the mRNA and protein expression of drug-metabolizing enzymes and transporters. The functions of these enzymes and transporters were evaluated by the cocktail assay. In hypercholesterolemic rats, the expression of phase I enzymes (CYP1A2, CYP2C11, CYP2E1, CYP3A1/2, CYP4A1 and FMO1/3) and phase II enzymes (UGT1A1/3, PROG, AZTG, SULT1A1, NAT1 and GSTT1) decreased. In addition, the mRNA levels of drug transporter Slco1a1/2, Slco1b2, Slc22a5, Abcc2, Abcb1a and Abcg2 decreased in rats with hypercholesterolemia, while Abcb1b and Abcc3 increased. The decreased expression of hepatic phase I and II enzymes and transporters may be caused by the changes of CAR, FXR, PXR, and Hnf4α levels. In conclusion, diet-induced hypercholesterolemia changes the expression and function of hepatic drug-metabolizing enzymes and transporters in rats, thereby possibly affecting drug metabolism and pharmacokinetics. In clinical hyperlipidemia, patients should strengthen drug monitoring to avoid possible drug exposure mediated risks.

Relationship between Gut Microbiota and Bone Health.

Gut Microbiota (GM) are microorganisms that live in the host gastrointestinal tract, and their abundance varies throughout the host's life. With the development of sequencing technology, the role of GM in various diseases has been increasingly elucidated. Unlike earlier studies on orthopedic diseases, this review elucidates the correlation between GM health and bone health and discusses the potential mechanism of GM effects on host metabolism, inflammation, and ability to induce or aggravate some common orthopedic diseases, such as osteoarthritis, osteoporosis, rheumatoid arthritis, etc. Finally, the prospective methods of GM manipulation and evaluation of potential GM-targeting strategies in the diagnosis and treatment of orthopedic diseases are reviewed.

LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS.

INTRODUCTION: The diagnosis and treatment of osteoporosis, a frequent age-related metabolic bone disorder, remain incomprehensive and challenging. The potential regulatory role of lncRNA XIST and sphingosine kinase 1 (SPHK1) pathway need experimental investigations. MATERIALS AND METHODS: RAW264.7 cells and BMMs were obtained for in vitro studies and 30 ng/mL RANKL was implemented for induction of osteoclast differentiation. The suppressing of lncRNA XIST, SPHK1 and fused in sarcoma (FUS) was achieved using small hairpin RNA, while overexpression of XIST and FUS was constructed by pcDNA3.1 vector system. Tartrate-resistant acid phosphatase (TRAP) staining was used for observation of formation of osteoclasts. RNA-pulldown analysis and RNA binding protein immunoprecipitation (RIP) was implemented for measuring mRNA and protein interactions. RT-qPCR was conducted to determining mRNA expression, whereas ELISA and Western blotting assay was performed for monitoring protein expression. RESULTS: RANKL induced osteoclast differentiation and upregulated expression of osteoclastogenesis-related genes that included NFATc1, CTSK, TRAP and SPHK1 and the level of lncRNA XIST in both RAW264.7 cells and BMMs. However, knockdown of lncRNA XIST or suppressing SPHK1 significantly reserved the effects of RANKL. LncRNA XIST was further demonstrated to be interacted with FUS and increased the stability of SPHK1, indicating its ability in promoting osteoclast differentiation through SPHK1/S1P/ERK signaling pathway. CONCLUSION: LncRNA XIST promoted osteoclast differentiation via interacting with FUS and upregulating SPHK1/S1P/ERK pathway.

Dual role of boron-doped diamond (BDD) anode in effluent organic matter degradation and ultrafiltration membrane fouling mitigation.

Ultrafiltration (UF) is effective in retaining macromolecules during tertiary treatment, but the membrane fouling caused by the effluent organic matter (EfOM) limits its application. This study employed electrochemical oxidation (EO) as a pretreatment method for UF in tertiary treatment to investigate the effects of anode materials on membrane fouling alleviation and EfOM degradation. Compared with the dimensionally stable (DSA) and platinum (Pt) anodes, EO with a boron-doped diamond (BDD) anode exhibited better performances for membrane fouling mitigation due to the higher hydroxyl radical production activity of the BDD anode. It was observed that the current density and electrolysis time were closely related to membrane fouling when using a BDD anode, where increasing the current density or electrolysis time led to a significant improvement of specific flux. The BDD-based pre-oxidation efficiently removed 64% DOC, 76% UV254, and 95% fluorescence organic matter in EfOM, among which the concentrations of DOC and UV254 were positively correlated with the total fouling index (TFI). Meanwhile, 70% SMX in the secondary effluent was removed by the BDD anode. Furthermore, the BDD anode also mitigated membrane fouling by decomposing high molecular weight organic matter into smaller fractions and enhancing the electrostatic repulsion between membrane and EfOM. Therefore, the BDD-based EO process is a promising pretreatment strategy for UF to alleviate membrane fouling and improve the permeate quality.

The nitrogen-doped multi-walled carbon nanotubes modified membrane activated peroxymonosulfate for enhanced degradation of organics and membrane fouling mitigation in natural waters treatment.

The synthesized catalyst nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) were introduced into membrane technology for peroxymonosulfate (PMS) activation. The enhanced permeability of the N-MWCNTs-modified membrane might be attributed to the increase in hydrophilicity and membrane porosity. The catalytic degradation and membrane filtration performance for the N-MWCNTs-modified membrane/PMS system in treating different types of natural waters were evaluated. The removal of phenol by the N-MWCNTs-modified membrane was 83.67% in 2 min, which was greater than the phenol removal by the virgin membrane (3.39%) and N-MWCNT powder (41.42%), respectively. Moreover, the resultant membrane coupled with PMS activation exhibited outstanding removal effects on the fluorescent organics in the secondary effluent and Songhua River water. The combination effectively reduced the total membrane fouling caused by the secondary effluent, Songhua River water, and three typical model organics by 28.19-61.98%. Electron paramagnetic resonance and classical quenching tests presented that the active species (SO4·-, ·OH, and 1O2) and other non-radical processes generated by N-MWCNTs activated PMS decreased the foulants deposition on the membrane surface. Meanwhile, the membrane interception accelerated the aggregation of pollutants and PMS towards the membrane surface through applied pressure, facilitating their mass transfer to the N-MWCNTs surface for the catalysis exerted more effectively. This study demonstrated the potential application of the coupling of N-MWCNTs catalytic oxidation and the UF, which offers a promising prospect to improve the permeate quality and simultaneously overcome the membrane fouling barriers.

CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics.

The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as Cyp, Abcb1, Oatp1b2 gene knockout rats. These new rat models are not only widely used in the study of drug metabolism, chemical toxicity, and carcinogenicity, but also promote the study of DMPK related mechanism, and further strengthen the relationship between drug metabolism and pharmacology/toxicology. This review systematically introduces the advantages and disadvantages of CRISPR-Cas9, summarizes the methods of establishing DMPK rat models, discusses the main challenges in this field, and proposes strategies to overcome these problems.

P-glycoprotein mediates the pharmacokinetic interaction of olanzapine with fluoxetine in rats.

Clinical trials of olanzapine combined with fluoxetine (Olanzapine/Fluoxetine Combination, OFC) in the treatment of refractory depression have shown significant efficacy, but the drug-drug interaction (DDI) between them remains unclear. In this report, the pharmacokinetic interaction between olanzapine and fluoxetine was studied in wild-type (WT) and Mdr1a/b gene knockout (KO) rats. By analyzing the pharmacokinetics and tissue distribution of olanzapine in single dose and combination, the potential DDI mediated by P-gp was explored. The results showed that in WT rats, the combination of fluoxetine increased the peak concentration (Cmax, 44.1 ± 5.1 ng/mL in the combination group vs 9.0 ± 1.5 ng/mL in the monotherapy group) and the exposure (AUC0-t, 235.8 ± 22.7 h × ng/mL in the combination group vs 47.5 ± 8.4 h × ng/mL in monotherapy group) of olanzapine, and decreased the clearance (CL, 8119.0 ± 677.9 mL/h/kg in the combination group vs 49,469.0 ± 10,306.0 mL/h/kg in monotherapy group). At the same time, fluoxetine significantly increased the in vivo exposure of olanzapine in brain, liver, kidney and ileum of WT rats, indicating the occurrence of DDI. The same phenomenon was observed in Caco-2 cells in vitro as well. However, in KO rats, there was no significant difference in pharmacokinetic parameters between the monotherapy group and the combination group. In conclusion, P-gp plays an important role in the pharmacokinetic interaction between olanzapine and fluoxetine in rats. This study may provide a reference for the clinical safety of olanzapine combined with fluoxetine.

Characterization of a Novel CYP1A2 Knockout Rat Model Constructed by CRISPR/Cas9.

CYP1A2, as one of the most important cytochrome P450 isoforms, is involved in the biotransformation of many important endogenous and exogenous substances. CYP1A2 also plays an important role in the development of many diseases because it is involved in the biotransformation of precancerous substances and poisons. Although the generation of Cyp1a2 knockout (KO) mouse model has been reported, there are still no relevant rat models for the study of CYP1A2-mediated pharmacokinetics and diseases. In this report, CYP1A2 KO rat model was established successfully by CRISPR/Cas9 without any detectable off-target effect. Compared with wild-type rats, this model showed a loss of CYP1A2 protein expression in the liver. The results of pharmacokinetics in vivo and incubation in vitro of specific substrates of CYP1A2 confirmed the lack of function of CYP1A2 in KO rats. In further studies of potential compensatory effects, we found that CYP1A1 was significantly upregulated, and CYP2E1, CYP3A2, and liver X receptor ß were downregulated in KO rats. In addition, CYP1A2 KO rats exhibited a significant increase in serum cholesterol and free testosterone accompanied by mild liver damage and lipid deposition, suggesting that CYP1A2 deficiency affects lipid metabolism and liver function to a certain extent. In summary, we successfully constructed the CYP1A2 KO rat model, which provides a useful tool for studying the metabolic function and physiologic function of CYP1A2. SIGNIFICANCE STATEMENT: Human CYP1A2 not only metabolizes clinical drugs and pollutants but also mediates the biotransformation of endogenous substances and plays an important role in the development of many diseases. However, there are no relevant CYP1A2 rat models for the research of pharmacokinetics and diseases. This study successfully established CYP1A2 knockout rat model by using CRISPR/Cas9. This rat model provides a powerful tool to study the function of CYP1A2 in drug metabolism and diseases.

A non-linear dose-response relation of female body mass index and in vitro fertilization outcomes.

PURPOSE: Obesity, measured by body mass index (BMI), is implicated in adverse pregnancy outcomes for women seeking in vitro fertilization (IVF) care. However, the shape of the dose-response relationship between BMI and IVF outcomes remains unclear. METHODS: We therefore conducted a dose-response meta-analysis using a random effects model to estimate summary relative risk (RR) for clinical pregnancy (CPR), live birth (LBR), and miscarriage risk (MR) after IVF. RESULTS: A total of 18 cohort-based studies involving 975,889 cycles were included. For each 5-unit increase in BMI, the summary RR was 0.95 (95% CI: 0.94-0.97) for CPR, 0.93 (95% CI: 0.92-0.95) for LBR, and 1.09 (95% CI: 1.05-1.12) for MR. There was evidence of a non-linear association between BMI and CPR (Pnon-linearity < 10-5) with CPR decreasing sharply among obese women (BMI > 30). Non-linear dose-response meta-analysis showed a relatively flat curve over a broad range of BMI from 16 to 30 for LBR (Pnon-linearity = 0.0009). In addition, we observed a J-shaped association between BMI and MR (Pnon-linearity = 0.006) with the lowest miscarriage risk observed with a BMI of 22-25. CONCLUSIONS: In conclusion, obesity contributed to increased risk of adverse IVF outcomes in a non-linear dose-response manner. More prospective trials in evaluating the effect of body weight control are necessary.