Maternal and Fetal Pharmacokinetic Analysis of Cannabidiol during Pregnancy in Mice.

Cannabidiol (CBD), a major component of cannabis, has various effects, such as antiemetic and anxiolytic activities, and has recently been marketed as a supplement. The number of people using CBD during pregnancy is increasing, and there are concerns about its effects on the fetus. In addition, the scientific evidence supporting the fetal safety of CBD use during pregnancy is insufficient. To investigate CBD transfer from the mother to the fetus, a single intravenous dose of CBD was administered to pregnant mice in this study, and fetal pharmacokinetics (distribution and elimination) was analyzed. The transfer of CBD from the maternal blood to the fetus was rapid, and the compound accumulated in the fetal brain, liver, and gastrointestinal tract. Conversely, little CBD was transferred from the mother to the amniotic fluid. We analyzed the pharmacokinetics of CBD using a two-compartment model and found that the maternal and fetal half-lives of CBD were approximately 5 and 2 hours, respectively. Furthermore, we performed a moment analysis of the pharmacokinetics of CBD, observing a mean residence time of less than 2 hours in both the mother and fetus. These results suggest that once-daily CBD intake during pregnancy is unlikely to result in CBD accumulation in the mother or fetus. SIGNIFICANCE STATEMENT: CBD is currently marketed as a supplement, and despite its increasing use during pregnancy, little information concerning its fetal effects has been reported. In the present study, CBD was administered to pregnant mice, and the pharmacokinetics in the fetus was investigated using a two-compartment model and moment analysis. The results of these analyses provide important information for estimating the risk to the fetus if CBD is mistakenly consumed during pregnancy.

Changes in the Expression of Aquaporin-3 in the Gastrointestinal Tract Affect Drug Absorption.

Aquaporin-3 (AQP3) plays an important role in water transport in the gastrointestinal (GI) tract. In this study, we conducted a Caco-2 cell permeability assay to examine how changes in the expression and function of AQP3 affect the rate at which a drug is absorbed via passive transport in the GI tract. When the function of AQP3 was inhibited by mercuric chloride or phloretin, there was no change in warfarin permeability. In contrast, when the expression of AQP3 protein was decreased by prostaglandin E2 (PGE2) treatment, warfarin permeability increased to approximately twice the control level, and membrane fluidity increased by 15%. In addition, warfarin permeability increased to an extent comparable to that after PGE2 treatment when cell membrane fluidity was increased by 10% via boric acid/EDTA treatment. These findings suggest the possibility that the increased drug absorption under decreased AQP3 expression was attributable to increased membrane fluidity. The results of this study demonstrate that the rate of water transport has little effect on drug absorption. However, our findings also indicate that although AQP3 and other similar transmembrane proteins do not themselves transport drugs, changes in their expression levels can cause changes in cell membrane fluidity, thus affecting drug absorption rates.

Anti-Hypertensive Effects of Acacia Polyphenol in Spontaneously Hypertensive Rats.

We have previously demonstrated that acacia polyphenol (AP) exerts strong anti-obesity, anti-diabetic, and anti-atopic dermatitis effects. In the present study, we investigated the anti-hypertensive effects of AP. Spontaneously hypertensive rats (SHR) with hypertension and control Wistar Kyoto rats (WKY) were used. WKY and SHR were fed AP-containing food or AP-free food (control group) ad libitum for 4 weeks, and their blood pressures were measured. After AP administration, both systolic and diastolic blood pressures were significantly lower in the SHR group than in the control group. There were no differences in the systolic or diastolic blood pressure of WKY between the AP group and the control group. Angiotensin-converting enzyme (ACE) activity, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression, and superoxide dismutase (SOD) activity in SHR kidneys were not altered by AP administration. Blood SOD activity in SHR was significantly higher in the AP group than in the control group. AP exerts anti-hypertensive effects on hypertension but has almost no effect on normal blood pressure. The anti-hypertensive effects of AP may be related to the anti-oxidative effects of increased blood SOD activity.

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon.

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea.

Relationship between Aging-Related Skin Dryness and Aquaporins.

Skin function deteriorates with aging, and the dermal water content decreases. In this study, we have analyzed the mechanism of aging-related skin dryness focusing on aquaporins (AQPs), which are the water channels. Mice aged 3 and 20 months were designated as young and aged mice, respectively, to be used in the experiments. No differences were observed in transepidermal water loss between the young mice and aged mice. However, the dermal water content in aged mice was significantly lower than that in young mice, thus showing skin dryness. The expression of AQP1, AQP3, AQP4, AQP7, and AQP9 was observed in the skin. All the mRNA expression levels of these AQPs were significantly lower in aged mice. For AQP3, which was expressed dominantly in the skin, the protein level was lower in aged mice than in young mice. The results of the study showed that the expression level of AQPs in the skin decreased with aging, suggesting the possibility that this was one of the causes of skin dryness. New targets for the prevention and treatment of aging-related skin dryness are expected to be proposed when the substance that increases the expression of AQP3 is found.

Analysis of CYP2R1 and CYP26A1 Expression Patterns in Regeneration in Mice with Liver Injury.

Cytochrome P450 enzymes (CYPs) are involved in the metabolism of various substances in the liver and small intestine and show markedly higher expression levels in the liver compared to other organs. The liver exhibits a remarkable capacity to regenerate. After excision of 70% of the liver, the organ can regenerate to its original size in approximately 1 week. Unlike the normal liver, in the injured liver, hepatic stem cells known as oval cells are considered to play an important role in regeneration. However, the role of CYPs in liver regeneration remains unclear. In the present study, we investigated the role of CYPs in the regeneration of injured liver. Liver injury was induced by 4-week repeated doses of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in the diet. Next, both DDC-fed mice and control diet (containing no DDC)-fed mice were subjected to 70% hepatectomy, and the hepatic gene expression patterns measured during regeneration were analyzed. Mice with DDC-induced liver injury expressed the oval cell markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM), and partial hepatectomy increased the expression levels of CYP2R1 and CYP26A1 as well as the hepatoblast marker alpha-fetoprotein (AFP) in these mice. The results of this study suggest that CYP2R1 and CYP26A1 are important in the differentiation of oval cells into hepatoblast-like cells in the injured liver.

Role of the Drug-Metabolizing Enzyme CYP during Mouse Liver Development.

The drug-metabolizing enzyme CYP is mainly involved in the metabolism of various substances in the liver, such as drugs, endogenous substances, and carcinogens. Recent reports have also revealed that CYP1B1 plays a major role in the developmental process. Because the level of CYP expression is markedly high in the liver, we hypothesize that CYP plays a role in the developmental process of the liver. To verify this hypothesis, we analyzed the expression patterns of various CYP molecular species and their functions during the differentiation of embryonic stem cells (ES cells) into hepatocytes and the developmental process in mice. The results demonstrated that CYP2R1 and CYP26A1 are expressed at an earlier stage of the differentiation of ES cells into hepatocytes than hepatoblast-specific markers. Additionally, during the development of the mouse liver, CYP2R1 and CYP26A1 were mostly up-regulated during the stage when hepatoblasts appeared. In addition, when CYP2R1 and CYP26A1 expressions were forced in ES cells and liver of adult mice, they differentiated into hepatoblast marker positive cells. These results suggest that CYP2R1 and CYP26A1 may play a major role in hepatoblast cell differentiation during the development of the liver.

Mechanism for Increased Expression of UGT2B in the Liver of Mice with Neuropathic Pain.

Approximately 30% of patients with cancer pain experience concurrent neuropathic pain. Since these patients are not sufficiently responsive to morphine, the development of an effective method of pain relief is urgently needed. Decreased function of the µ opioid receptor, which binds to the active metabolite of morphine M-6-G in the brain, has been proposed as a mechanism for morphine resistance. Previously, we pharmacokinetically examined morphine resistance in mice with neuropathic pain, and demonstrated that the brain morphine concentration was decreased, expression level of P-glycoprotein (P-gp) in the small intestine was increased, and expression level and activity of uridine diphosphate glucuronosyltransferase (UGT)2B in the liver were increased. In order to clarify the mechanism of the increased expression of UGT2B, we examined the phase of neuropathic pain during which UGT2B expression in the liver begins to increase, and whether this increased expression is nuclear receptor-mediated. The results of this study revealed that the increased expression of UGT2B in the liver occurred during the maintenance phase of neuropathic pain, suggesting that it may be caused by transcriptional regulation which was not accompanied by increased nuclear import of pregnane X receptor (PXR).

Aquaporins in the Colon as a New Therapeutic Target in Diarrhea and Constipation.

Aquaporins (AQPs) play important roles in the water transport system in the human body. There are currently 13 types of AQP, AQP0 through AQP12, which are expressed in various organs. Many members of the AQP family are expressed in the intestinal tract. AQP3 is predominantly expressed in the colon, ultimately controlling the water transport. Recently, it was clarified that several laxatives exhibit a laxative effect by changing the AQP3 expression level in the colon. In addition, it was revealed that morphine causes severe constipation by increasing the AQP3 expression level in the colon. These findings have shown that AQP3 is one of the most important functional molecules in water transport in the colon. This review will focus on the physiological and pathological roles of AQP3 in the colon, and discuss clinical applications of colon AQP3.

Expression of hepatic cytochrome P450 in a mouse model of ulcerative colitis changes with pathological conditions.

BACKGROUND AND AIM: The expression levels of cytochrome P450 (CYP) in the liver were analyzed over time in dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model, from the initial active stage to the remission stage, to investigate the relationship between the changes in pathological conditions and CYP expression levels. METHODS: DSS solution was given to mice for 10 days, after which water without DSS was provided for 40 days. Pathological conditions and CYP expression levels were examined over time. The mechanism for variation in CYP expression was also analyzed. RESULTS: The mRNA expression levels of CYP (CYP3A11, CYP1A2, CYP2C29, CYP2D9, and CYP2E1) decreased as pathological conditions worsened and reached their lowest levels on day 10 of DSS treatment. Pathological conditions improved following the discontinuation of DSS, and CYP expression levels normalized by day 50. Blood lipopolysaccharide levels, the hepatic expression of inflammatory cytokines, and the nuclear translocation of pregnane X receptor and constitutive androstane receptor in the liver exhibited patterns similar to the observed variations in CYP expression levels. CONCLUSION: The capacity for metabolizing drugs that are substrates of CYP decreases during the active stage of ulcerative colitis but subsequently improves during the remission stage. This decrease in CYP expression was likely caused by the observed reduction in the levels of nuclearly localized pregnane X receptor and constitutive androstane receptor, and the increase in the production of inflammatory cytokines triggered by lipopolysaccharides.

Effects of menthol on the pharmacokinetics of triazolam and phenytoin.

We have previously shown that menthol attenuates the anticoagulant effect of warfarin by increasing the expression levels of CYP3A and CYP2C in the liver. This study evaluated the effects of menthol on the pharmacokinetics of the CYP3A substrate triazolam and the CYP2C substrate phenytoin. Menthol was orally administered to mice for 7 d. Twenty-four hours after the administration of menthol, triazolam was orally administered, and the plasma concentration was measured. In addition, the CYP3A metabolic activity for triazolam and the CYP3A expression level in the liver were determined. The effects of menthol on the pharmacokinetics of phenytoin were assessed in the same manner. In the menthol-treated group, the area under the blood concentration-time curve (AUC) of triazolam was lower and its clearance was higher compared with the control group. The CYP3A metabolic activity and CYP3A expression level in the liver were significantly increased in the menthol-treated group compared with the control group. Similarly, the AUC of phenytoin was lower and the hepatic CYP2C expression level was higher in the menthol-treated group. Thus, menthol lowered the plasma concentrations of triazolam and phenytoin when concurrently administered. These effects may be attributed to an increased metabolic activity for these drugs due to the increased expression of CYP3A and CYP2C in the liver.

Transdermal administration of aqueous pregabalin solution as a potential treatment option for patients with neuropathic pain to avoid central nervous system-mediated side effects.

Pregabalin, (S)-3-isobutyl-γ-aminobutyric acid (GABA), is a widely used adjuvant therapy for patients with neuropathic pain, which is defined as chronic pain caused by lesions or diseases of the somatosensory nervous system. However, dizziness and somnolence (sleepiness) are common dose-limiting side effects, probably due to excessive sedative effects on higher centers of the central nervous system (CNS) which are involved in the anticonvulsant and analgesic actions of pregabalin. We speculated that transdermal delivery would minimize centrally mediated side effects. To test this idea, we evaluated the analgesic effects of pregabalin delivered through the transdermal route in animal models of neuropathic pain. Transdermally administered pregabalin increased the pain thresholds in response to mechanical stimuli in a partial sciatic nerve ligation model in rats and a spinal nerve ligation model in mice, and surprisingly also in normal animals. It is noteworthy that simple transdermal application of an aqueous solution of pregabalin is effective. This could be a useful treatment option to avoid or minimize the CNS-mediated side effects of orally administered pregabalin.

Gastrectomy increases the expression of hepatic cytochrome P450 3A by increasing lithocholic acid-producing enteric bacteria in mice.

We had previously revealed that drug metabolism, as well as the expression level of hepatic CYP3A, a drug-metabolizing enzyme, increase 12 weeks after gastrectomy in mice. In this study, we elucidated the mechanism of the increased CYP3A expression. The levels of lithocholic acid (LCA)-producing bacteria (Bacteroides fragilis) and LCA in the colon did not show a significant increase up to 4 weeks after gastrectomy compared to the sham operation group. In contrast, at 12 and 24 weeks post-gastrectomy, the levels of Bacteroides fragilis and LCA were significantly higher in the gastrectomy group than in the sham operation group. At 12 and 24 weeks after gastrectomy, the hepatic nuclear translocation of pregnane X receptor (PXR) had also increased. The hepatic CYP3A11 mRNA expression and nuclear translocation of PXR after intraperitoneal administration of LCA to normal mice was significantly higher than those of the control group. The intraperitoneal administration of taurolithocholic acid (TLCA), a taurine conjugate of LCA, caused no change in the expression level of CYP3A11. We suggest that the increase in the expression level of CYP3A after gastrectomy is caused by an increase in the nuclear translocation of PXR, which is triggered by an increase in LCA-producing bacteria.

Consumption of a high-fat diet during pregnancy changes the expression of cytochrome P450 in the livers of infant male mice.

It has been recently reported that the consumption of a high-fat diet during pregnancy exerts various effects on fetuses and newborn mice. The purpose of this study was to determine the effects of a high-fat diet during pregnancy on the expression of cytochrome P450 (CYP) in the livers of offspring. Mouse dams were fed a high-fat diet during pregnancy from the time of conception. After their birth, the newborn mice were fed a normal diet until 12 weeks of age. In the livers of the infant male mice that consumed a high-fat diet, the protein expression of CYP3A and CYP2C was decreased, and the protein expression of CYP1A and CYP2E was increased at 6 and 12 weeks of age. However, almost no changes were observed in the CYP proteins at 6 and 12 weeks of age in the livers of the infant female mice that consumed a high-fat diet. The amount of pregnane X receptor (PXR) translocated into the nucleus was reduced in the livers of infant male mice that consumed a high-fat diet. However, there was neither an increase in tumor necrosis factor-α or interleukin-1ß nor a decrease in lithocholic acid. These data suggested that CYP3A and CYP2C might decrease as a result of the decrease in the amount of nuclear PXR in infant male mice that consumed a high-fat diet. The results of this study suggested that the consumption of a high-fat diet by pregnant mothers may be one explanation for individual differences in pharmacokinetics.

Different diets cause alterations in the enteric environment and trigger changes in the expression of hepatic cytochrome P450 3A, a drug-metabolizing enzyme.

Changes in the expression level and activity of cytochrome P450 (CYP) in the liver are caused by various factors and affect the pharmacokinetics of drugs. The purpose of this study was to determine whether the expression of CYP3A is affected by a high-fat diet. In addition, we examined whether the type of diet given to mice could produce changes in the expression level and activity of CYP3A. Mice were fed a purified diet containing 10 kcal% lard (control group) or 60 kcal% lard (HF group) or regular mouse chow containing 13 kcal% of fat (MF group) for 4 weeks. No significant differences were observed in the hepatic CYP3A protein expression level between the HF group and the control group. The CYP3A protein expression in the MF group was significantly higher than that observed in the control group. In the MF group, the area under the curve (AUC) of intraperitoneally administered triazolam was lower. Because lithocholic acid (LCA) is known to increase hepatic CYP3A expression, the levels of Clostridium sordellii and LCA in the feces were measured. In the MF group, the levels of Clostridium sordellii and LCA were higher. It has been demonstrated that a high-fat diet does not cause any changes in hepatic CYP3A expression. In addition, the different diets caused alterations in the enteric environment, which triggered changes in CYP3A expression. Therefore, it is necessary to carefully consider the type of feed while performing animal experiments to evaluate the pharmacokinetics of drugs.

Elucidating the mechanism by which Gypsum fibrosum, a traditional Chinese medicine, maintains cutaneous water content.

Aquaporin-3 (AQP3) plays an important role in maintaining the normal water content of the skin. Previously, we revealed that the expression of cutaneous AQP3 increased following oral administration of Gypsum fibrosum (main component: CaSO4) to mice. The purpose of this study is to elucidate the mechanism by which Gypsum fibrosum increases the expression of cutaneous AQP3 in a keratinocyte cell line. Gypsum fibrosum or CaSO4 was added to keratinocytes, and the expression level of AQP3, the Ca concentration, the activity of protein kinase C (PKC), and the degrees of phosphorylation of both extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were measured. The mRNA and protein expression levels of AQP3 increased significantly 6 h-post addition of Gypsum fibrosum. In keratinocytes treated with Gypsum fibrosum, increases in the concentration of intracellular Ca, PKC activity, and the phosphorylation of ERK and CREB were observed. Pre-treatment with GF109203X, a PKC inhibitor, suppressed the mRNA expression levels of AQP3. Similarly to treatment with Gypsum fibrosum, the addition of CaSO4 led to the same observations in keratinocytes. It is hypothesized that Gypsum fibrosum causes an increase in the intracellular Ca concentration, PKC activity, and the phosphorylation levels of ERK and CREB, resulting in increased AQP3 expression in keratinocytes. In addition, it is possible that the effect of Gypsum fibrosum is attributable to CaSO4, based on the results demonstrating that the mechanisms of action of Gypsum fibrosum and CaSO4 were nearly identical.

Synthesis of Am80 (tamibarotene) prodrug candidates, congeners and metabolites.

Compound 1 (IT-M-07000) was previously reported as a candidate prodrug of Am80 (Tamibarotene; used to treat acute promyelocytic leukemia), and shown to be efficiently metabolized to Am80 via ß-oxidation. Here, we describe in detail the synthesis of 1, together with another tetradeuterated candidate prodrug, IT-YA-00616 (2), as well as two congeners, and several metabolic intermediates of 1 previously detected in mouse plasma.

(R)- and (S)-4-Amino-3-(trimethylsilyl)methylbutanoic acids ameliorate neuropathic pain without central nervous system-related side effects.

Neuropathic pain is a chronic pain condition resulting from neuronal damage, and is usually treated with pregabalin or gabapentin, which are structurally related to γ-aminobutyric acid (GABA) and are originally developed as anticonvulsant drugs. Here, we report the synthesis and pharmacology of (R)- and (S)-4-amino-3-(trimethylsilyl)methylbutanoic acids (1a and 1b), which showed analgesic activity as potent as that of pregabalin in the Chung spinal nerve ligation model. However, unlike pregabalin, 1a and 1b do not have antiepileptic effects, and they are therefore promising candidates for selective therapeutic agents to treat neuropathic pain without central nervous system-related side effects.

Effect of Conclevan on endurance capacity in mice.

The purpose of this study was to clarify the anti-fatigue effect of Conclevan, which is mainly composed of liver hydrolysate, via a forced swimming test using mice. Conclevan was administered to mice for 6 weeks, and a forced swimming test was conducted to measure swimming time. After six weeks, the blood ammonia and glutamine concentrations were measured. In the Conclevan administration group, swimming time increased significantly compared to the swimming control group. In the swimming control group, an increase in blood ammonia and a decrease in blood glutamine were observed, relative to the non-swimming control group. In the Conclevan administration group, the increased blood ammonia and decreased blood glutamine induced by swimming were significantly reduced, compared to the swimming control group. The mRNA expression levels of the hepatic enzymes of the urea cycle (carbamoyl-phosphate synthetase, argininosuccinate synthetase, and arginase) and glutamine synthesis (glutamate dehydrogenase and glutamine synthetase) were significantly increased in the Conclevan administration group, compared to the swimming control group. The results of this study demonstrated the anti-fatigue effects of Conclevan. This product may inhibit an increase in the fatigue-inducing ammonia concentration in the blood by increasing the expression of hepatic enzymes, which convert ammonia to urea, leading to increased swimming time. In addition, Conclevan may prolong swimming time by increasing the hepatic synthesis of glutamine, which is an important amino acid for supplying energy in muscles.

Inhibition of aquaporin-3 water channel in the colon induces diarrhea.

Aquaporin (AQP) 3, which is predominantly expressed in the colon, is considered to play an important role in regulating the fecal water content in the colon. In this study, the role of AQP3 in the colon was examined using HgCl(2) and CuSO(4), which are known to inhibit AQP3 function. The fecal water content was measured up to 1 h after the rectal administration of HgCl(2) or CuSO(4) to rats. The results showed that the fecal water content in the HgCl(2) administration group increased significantly to approximately 4 times that in the control group, and severe diarrhea was observed. However, no changes were observed in the mRNA expression level of the osmoregulatory genes (sodium myo-inositol transporter and taurine transporter) and the level and distribution of AQP3 protein expression, as determined 1 h after the administration of HgCl(2). Comparable results were observed in the CuSO(4) administration group. The results of this study indicated that the inhibition of AQP3 function in the colon caused diarrhea. Therefore, it has been revealed that the fecal water content in the colon is controlled by the transport of water from the luminal side to the vascular side, which is mediated by AQP3. Our findings suggest that a drug that modulates the function or expression of AQP3 in the colon may represent a new target for the development of laxatives.