Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells

Background Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. Methods We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. Results We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. Conclusion This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

Development of Saccharomyces cerevisiae Y486–based biosensor carrying both CPR-CYP3A4 and DIN7-GFP constructs for mutagenicity testing of procarcinogens

In the world and Vietnam, a great number of toxic substances from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. Many exogenous harmful substances are procarcinogens, but become carcinogens by the bioactivation of human cytochrome P450 enzymes (CYPs). Thus, development of analytical testing for rapid detection of procarcinogens plays a crucial role in food safety and environmental monitoring. This study aims to establish a biosensor basing on Saccharomyces cerevisiae Y486 cotransformed with two promoter–gene constructs, CYP3A4–CPR and DIN7–GFP. The results showed that all recombinant proteins were coexpressed in Y486 cells. The molecular weight of recombinant CPR and CYP3A4 were 75 kDa and 56 kDa, respectively. CYP3A4 enzyme only showed its catalytic activity in biotransformation of the specific substance as coexpressed with CPR. Kinetic constants, Km, Vmax, and Vmax/Km, of this CPR–CYP3A4 enzyme complex were 3.2 µM, 3.5 pmol/pmol CYP/min, and 1.1 μL/pmol CYP/min, respectively. Coexpressing constructs of CPR–CYP3A4 and DIN7-GFP in Y486 strain was able to identify aflatoxin B1 in the range of 0.1 - 0.4 µM; benzo(c)pyrene in the range of 10 - 40 µM. However, this system could not detect other procacinogens, such as, N-Nitrosodimethylamine, at any investigated concentrations. These findings were the first trial for further development of other biosensors to determine diverse procarcinogens in the enviroment by redesign of coexpressing constructs or replacement of the specific CYPs and inducible promoters.

Changes in the Pharmacokinetics and Pharmacodynamics of Sildenafil in Cigarette and Cannabis Smokers

Sildenafil citrate, a widely-used oral therapy for erectile dysfunction, is a cytochrome P3A4 (CYP3A4) enzyme substrate. Studies have reported that this substrate has an inhibitory effect on CYP3A4 enzymes in long-term cigarette and cannabis smokers, which predominantly mediate the hepatic elimination of sildenafil. Cigarette and/or cannabis smoking could therefore alter the exposure of sildenafil. The aim of this study was to examine the effect of smoking cigarettes and/or cannabis on the pharmacokinetics, pharmacodynamics, safety and tolerability of sildenafil. Thirty-six healthy human subjects were equally divided into three groups: non-smokers, cigarette smokers and cannabis smokers. Each group was administered a single dose of sildenafil (50 mg tablets). The primary outcome measures included the maximum concentration of sildenafil in plasma (Cmax), the elimination half-life (t1/2) and the area under the plasma concentration time curve from zero to time (AUC0–t). The pharmacodynamics were assessed by the International Index of Erectile Function (IIEF-5). The exposure of sildenafil (AUC0–t) showed a statistically significant increase in cigarette smokers (1156 ± 542 ng·h/mL) of 61% (p < 0.05) while in cannabis smokers (967 ± 262 ng·h/mL), a non-significant increase in AUC0–t of 35% (p > 0.05) was observed relative to non-smokers (717 ± 311 ng·h/mL). Moreover, the Cmax of sildenafil increased by 63% (p < 0.05) and 22% (p > 0.05) in cigarette smokers and cannabis smokers, respectively. Cigarette smoking increases the exposure of sildenafil to a statistically significant level with no effect on its pharmacodynamics, safety and tolerability.

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min−1, kinact/Ki = 0.03 min−1 µM−1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.

In Silico Prediction and Pharmacokinetic Comparison of Ursodeoxycholic Acid and Obeticholic Acid in the Management of Primary Biliary Cholangitis

Background: Primary Biliary Cholangitis (PBC) is a persistent liver disease. Ursodeoxycholic acid is used as a first-line treatment for the past two decades. However, concurrent use of Ursodeoxycholic acid reported with a severe adverse drug reaction. Obeticholic acid has been started utilizing as monotherapy and also with Ursodeoxycholic acid in a patient who is intolerant to Ursodeoxycholic acid therapy. We primarily aimed to compare the pharmacokinetic & toxicity profiles of Ursodeoxycholic acid and Obeticholic acid using in silico methods. Method: The pharmacokinetic profile of UDCA & OCA was observed from PKCSM server online database, OSIRIS® property Explorer, T.E.S.T. (Toxicity estimation software tool) & Molinspiration® is used to estimate the drug toxicity profiles. Result: This computer-aided response provides a great understanding and creates a gap between the theoretical and clinical evidence for UDCA & OCA's preference in PBC management. Conclusion: Co-administration of Obeticholic acid with Ursodeoxycholic acid will be an effective treatment for PBC in patients with UDCA intolerants. However, both medications are well-recognized substrates of the CYP3A4 enzyme and may lead to unintended drug interactions and side effects. Keywords: Primary Biliary Cholangitis, Obeticholic acid, Ursodeoxycholic acid, CYP3A4, Drug Interactions, Pharmacokinetics.

Enhanced elimination of betamethasone in dichorionic twin pregnancies

Aims: No study has evaluated the BET pharmacokinetics in twin pregnancies separated by chorionicity. The aim this study is to describe and compare the BET pharmacokinetic parameters in singleton, dichorionic (DC) and monochorionic (MC) twin pregnancies in the third trimester of pregnancy. Methods: Twenty-six pregnant women received an intramuscular dose of 6 mg of BET sodium phosphate plus 6 mg BET acetate. Serial blood samples were collected for 24 hours after the first intramuscular BET esters dose. BET plasma concentrations were quantified using a validated HPLC analytical method. BET pharmacokinetic parameters were obtained employing a non-compartment model, and were compared using ANOVA’s test with Tukey’s multiple comparisons test. Correlations between clinical features and pharmacokinetic parameters were analyzed using Pearson’s correlation. Preliminary data on the BET placental transfer were also presented. Results: The geometric mean (IC 95%) of AUC0-∞ 670.0 (504.3-805.2) vs 434.9 (311.2-539.6) ng.h/mL and the CL/F 18.38 (13.84-22.65) vs 29.40 (21.17-36.69) were significantly lower and higher, respectively, in DC twin pregnancies compared to singleton. Others pharmacokinetic parameters did not differ among the groups. Conclusions: Data from this study suggest that the presence of two fetoplacental units may increase the BET metabolism by CYP3A4 enzyme and increase its elimination. Pharmacokinetic-pharmacodynamic clinical studies are needed to investigate whether this BET pharmacokinetic changes have clinical repercussions for the newborns and require dose adjustment in DC twin pregnancies.

Iatrogenic Cushing syndrome following lumbar medial branch block in a patient with HIV on ritonavir and darunavir

We present a case report of a 62-year old female with HIV and chronic facetogenic back pain who underwent bilateral L3–L4 and L4–L5 medial branch nerve blocks using triamcinolone acetonide 80 mg. 2 weeks later she presented to the emergency department with acute anxiety/depression and was discharged with psychiatric follow-up. 2 weeks after this she presented to the outpatient HIV clinic with persistent uncontrolled depression alongside classic cushingoid features (e.g., buffalo hump, moon facies). She was diagnosed with iatrogenic Cushing syndrome caused by a drug–drug interaction between triamcinolone and ritonavir, a protease inhibitor and a CYP3A4 enzyme inhibitor. While the literature describes the interaction of ritonavir with intra-articular/intranasal/epidural triamcinolone, this is the first documented occurrence following a nerve block procedure. Symptoms resolved within 6 months alongside discontinuation of protease inhibitor therapy.

Gilteritinib administration in a hemodialysis patient

Introduction Gilteritinib is a multitargeted tyrosine kinase inhibitor (TKI) approved by the Food and Drug Administration (FDA) for acute myeloid leukemia (AML) with a FMS-related tyrosine kinase 3 (FLT3) mutation. The pharmacokinetics of gilteritinib in the setting of severe renal impairment (creatinine clearance [CrCl] 15-29 mL/min utilizing Cockcroft-Gault method) and end-stage renal disease are unknown. Gilteritinib is primarily metabolized by the liver through the CYP3A4 enzyme and is eliminated in both the feces and urine. Its excretion is primarily through the fecal route, accounting for 64.5% of the recovered dose. Only about 16.4% of the recovered dose has been detected in the urine of human subjects. Case Report We describe our patient case documenting the administration of gilteritinib in the setting of end-stage renal disease (ESRD) and hemodialysis (HD). Management and Outcomes: Our patient was initiated on single agent gilteritinib 120 mg by mouth once daily for relapse FLT3-TDK positive AML. Treatment course was complicated by pancytopenia, neutropenic fever, and staphylococcus lugdunensis bacteremia requiring temporary interruption of therapy. Discussion Given that gilteritinib is metabolized by the liver and eliminated primarily in the feces, one does not expect an increase in toxicity related to impaired renal function. Although this report describes the successful utilization of gilteritinib, caution should be exercised when administering in patient populations with end organ disease, and patient comorbidities should be taken into account.

Physiological Responses of Earthworm Under Acid Rain Stress

Acid rain has become one of the major global environmental problems, and some researches reported that acid rain may have a certain inhibition on soil biodiversity. Besides this, it is well known that earthworm (Eisenia fetida) plays an important role in the functioning of soil ecosystems. For this point, we conducted a series of experiments to investigate whether acid rain would take effects on earthworms. In the present study, the earthworms were incubated on filter paper and in soil under acid rain stress. The mortality and behavior of earthworms were recorded, and epidermal damage and the activity of the CYP3A4 enzyme were measured for the tested earthworms. Our experimental results showed that the earthworms could not survive in the acid rain stress of pH below 2.5, and acid rain with weak acidity (i.e., 4.0 ≤ pH ≤ 5.5) promoted the activity of the CYP3A4 enzyme in the earthworms, while acid rain with strong acidity (i.e., 3.0 ≤ pH ≤ 3.5) inhibited it. Moreover, the degree of damage in sensitive parts of the earthworms increased with the decrease of pH value. This study suggests that acid rain can cause discomfort response and the direct epidermal damage of earthworms, and even kill them.