Discovery of DS-6930, a potent selective PPARγ modulator. Part I: Lead identification.

The lead identification of a novel potent selective PPARγ agonist, DS-6930 is reported. To avoid PPARγ-related adverse effects, a partial agonist was designed to prevent the direct interaction with helix 12 of PPARγ-LBD. Because the TZD group is known to interact with helix 12, the TZD in efatutazone (CS-7017) was replaced to discover novel PPARγ intermediate partial agonist 8i. The optimization of 8i yielded 13ac with high potency in vitro. Compound 13ac exhibited robust plasma glucose lowering effects comparable to those of rosiglitazone (3 mg/kg) in Zucker diabetic fatty rats. Upon toxicological evaluation, compound 13ac (300 mg/kg) induced hemodilution to a lower extent than rosiglitazone; however, 13ac elevated liver enzyme activities. X-ray crystallography revealed no direct interaction of 13ac with helix 12, and the additional lipophilic interactions are also suggested to be related to the maximum transcriptional activity of 13ac.

Discovery of DS-6930, a potent selective PPARγ modulator. Part II: Lead optimization.

Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine resulted in the substantial loss of potency. Because poor membrane permeability was responsible for poor potency in vitro, the adjustment of lipophilicity was examined, which resulted in the discovery of dimethyl pyridine derivative (I, DS-6930). In preclinical studies, DS-6930 demonstrated high PPARγ agonist potency with robust plasma glucose reduction. DS-6930 maintained diminished PPARγ-related adverse effects upon toxicological evaluation in vivo, and demonstrated no hepatotoxicity. Cofactor recruitment assay showed that several cofactors, such as RIP140 and PGC1, were significantly recruited, whereas several canonical factors was not affected. This selective cofactor recruitment was caused due to the distinct binding mode of DS-6930. The calcium salt, DS-6930b, which is expected to be an effective inducer of insulin sensitization without edema, could be evaluated clinically in T2DM patients.

Usefulness of Mirogabalin in Central Neuropathic Pain After Stroke: Post Hoc Analysis of a Phase 3 Study by Stroke Type and Location.

INTRODUCTION: Central post-stroke pain (CPSP) is a common type of central neuropathic pain (CNeP) that can occur following the onset of stroke. The oral gabapentinoid mirogabalin besylate (mirogabalin) is a selective α2δ ligand that is effective for the treatment of CNeP, including CPSP. However, it is unknown whether the analgesic effect of mirogabalin on CPSP varies in patients with different background factors. METHODS: This was a post hoc subgroup analysis of a multinational, open-label, long-term phase 3 study of mirogabalin for the treatment of CNeP conducted between March 2019 and December 2020. Data from patients with CPSP were stratified by type of stroke (ischemic or hemorrhagic), stroke location (thalamus, putamen, brainstem, or other), presence/absence of motor weakness, median time since stroke (≥ 59 or < 59 months), and median duration of CPSP (≥ 55.5 or < 55.5 months). Efficacy was assessed with the short-form McGill Pain Questionnaire (SF-MPQ), and treatment-emergent adverse events (TEAEs) and adverse drug reactions (ADRs) were recorded. RESULTS: This subanalysis included all 94 patients with CPSP from the phase 3 study; all were Japanese, and the mean age was 65.3 years. The least squares mean change [95% confidence interval] in SF-MPQ visual analog scale (VAS) score from baseline at week 52 (last observation carried forward) was - 17.0 [- 22.1, - 11.9] mm. Among the subgroups, least squares mean changes in SF-MPQ VAS scores were not different. Most TEAEs were mild or moderate; severe TEAEs occurred in six patients (6.4%). Somnolence (25.5%), peripheral edema (13.8%), dizziness (11.7%), and weight gain (6.4%) were the most common ADRs, and the types and frequencies of ADRs were similar among subgroups. CONCLUSION: Mirogabalin was generally effective and well tolerated in patients with CPSP, regardless of background factors such as stroke type or location, presence/absence of motor weakness, time since stroke, and duration of CPSP. TRIAL REGISTRATION: Trial registration number NCT03901352.

Long-Term Safety and Efficacy of Mirogabalin for Central Neuropathic Pain: A Multinational, Phase 3, 52-Week, Open-Label Study in Asia.

INTRODUCTION: Central neuropathic pain (CNeP) is difficult to treat and has diverse etiology, including spinal cord injury (CNePSCI), Parkinson's disease (CNePPD), and central post-stroke pain (CPSP). The safety and efficacy of mirogabalin have been demonstrated in short-term trials, including patients with CNePSCI. The objective of our study was to confirm the safety/efficacy of mirogabalin in patients with CNePPD and CPSP, and obtain long-term data for CNePSCI. METHODS: This 52-week, open-label extension of a previous randomized controlled study was conducted across Japan, Korea, and Taiwan. Patients with CNePSCI, CNePPD, or CPSP received twice daily (BID) 5-10 mg mirogabalin for a 4-week titration period, after which the dosage was maintained for 47 weeks at a maximum of 15 mg BID, followed by a 1-week taper period receiving the same dose but only administered once daily. The primary endpoint was safety, assessed primarily by incidence and severity of treatment-emergent adverse events (TEAEs). Efficacy was assessed in a post hoc analysis of data obtained by the short-form McGill Pain Questionnaire (SF-MPQ). RESULTS: Of the 210 patients enrolled, 106, 94, and 10 had CNePSCI, CPSP, and CNePPD, respectively. The mean overall age of patients was 62.9 years, and most patients were male and of Japanese ethnicity. TEAEs occurred in 84.8% of patients, the most common being somnolence (16.7%), peripheral edema (12.4%), edema (11.4%), nasopharyngitis (11.0%), and dizziness (7.6%). Most TEAEs were mild. Severe and serious TEAEs occurred in 6.2% and 13.3% of patients, respectively. All patient groups experienced reductions in SF-MPQ visual analog scores for pain: mean ± standard deviation changes from baseline at week 52 were -2.3 ± 21.13 mm (CNePSCI), -17.0 ± 24.99 mm (CPSP), and -17.1 ± 35.32 mm (CNePPD). CONCLUSION: Mirogabalin was generally safe, well tolerated, and effective for treatment of CNeP in this long-term study. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT03901352.

Mirogabalin for Central Neuropathic Pain After Spinal Cord Injury: A Randomized, Double-Blind, Placebo-Controlled, Phase 3 Study in Asia.

BACKGROUND AND OBJECTIVES: Patients with spinal cord injury (SCI) commonly experience central neuropathic pain (CNeP), which is challenging to treat. Mirogabalin is effective for peripheral neuropathic pain, but evidence for CNeP is lacking. METHODS: This randomized, double-blind, placebo-controlled, phase 3 study investigated mirogabalin efficacy and safety for the treatment of CNeP in patients with traumatic SCI. Adult patients from 120 sites throughout Japan, Korea, and Taiwan were randomized (1:1) to receive placebo or mirogabalin (5 mg twice daily [BID] for 1 week, 10 mg BID for 1 week, and 10 or 15 mg BID for 12 weeks). Patients with moderate renal impairment received half the dosage. The primary efficacy endpoint was change from baseline in the weekly average daily pain score (ADPS) at week 14. The secondary endpoints included ADPS responder rates, the Short-Form McGill Pain Questionnaire (SF-MPQ), average daily sleep interference score (ADSIS), and Neuropathic Pain Symptom Inventory (NPSI). Adverse events were monitored for safety. RESULTS: Each treatment group comprised 150 patients. Mirogabalin elicited a statistical and clinically relevant improvement in change from baseline in the weekly ADPS at week 14 (least-squares mean difference [95% CI] vs placebo -0.71 [-1.08 to -0.34], p = 0.0001). Responder rates at week 14 were higher for mirogabalin than those for placebo (odds ratio [95% CI] 1.91 [1.11-3.27] for the ≥30% responder rate; 2.52 [1.11-5.71] for the ≥50% responder rate). Statistical improvements (i.e., least-squares mean difference [95% CI] vs placebo) were also observed in the SF-MPQ (-2.4 [-3.8 to -1.1]), ADSIS -0.71 (-1.04 to -0.38), and NPSI -7.7 (-11.1 to -4.4) scores. Most treatment-emergent adverse events were mild; no serious adverse drug reactions were reported. DISCUSSION: Mirogabalin elicited clinically relevant decreases in pain and was well tolerated, suggesting that mirogabalin is a promising treatment for patients with CNeP due to SCI. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov (NCT03901352); first submitted April 3, 2019; first patient enrolled March 14, 2019; available at clinicaltrials.gov/ct2/show/NCT03901352. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that in adult patients with CNeP due to traumatic SCI, mirogabalin, 10 or 15 mg BID, effectively improves weekly ADPS at week 14.

Pharmacology and in vitro profiling of a novel peroxisome proliferator-activated receptor γ ligand, Cerco-A.

Peroxisome proliferator-activated receptor γ (PPARγ; NR1C3) is known as a key regulator of adipocytogenesis and the molecular target of thiazolidinediones (TZDs), also known as antidiabetic agents. Despite the clinical benefits of TZDs, their use is often associated with adverse effects including peripheral edema, congestive heart failure, and weight gain. Here we report the identification and characterization of a non-thiazolidinedione PPARγ partial agonist, Cerco-A, which is a derivative of the natural product, (-)-cercosporamide. Cerco-A was found to be a binder of the PPARγ ligand-binding domain in a ligand competitive binding assay and showed a unique cofactor recruitment profile compared to rosiglitazone. A crystal structure analysis revealed that Cerco-A binds to PPARγ without direct hydrogen bonding to helix12. In PPARγ transcriptional activation assay and an adipocyte differentiation assay, Cerco-A was a potent partial agonist of PPARγ. After a 14-day oral administration, once per day of Cerco-A in Zucker diabetic fatty (ZDF) rats, an apparent decrease of plasma glucose and triglyceride was observed, as with pioglitazone. To evaluate drug safety, Cerco-A was administered for 13 days orally in non-diabetic Zucker fatty (ZF) rats. Each of the hemodilution parameters (hematocrit, red blood cells number, and hemoglobin), which are considered as undesirable effects of TZDs, was improved significantly compared to pioglitazone. While Cerco-A showed body weight gain, as with pioglitazone, Cerco-A had significantly lower effects on heart and white adipose tissues weight gain. The results suggest that Cerco-A offers beneficial effects on glycemic control with attenuated undesirable side effects.

A review of a new voltage-gated Ca2+ channel α2δ ligand, mirogabalin, for the treatment of peripheral neuropathic pain.

INTRODUCTION: Neuropathic pain (NeP) is a chronic and refractory condition in many patients, and its treatment is a challenge for physicians. A new voltage-gated Ca2+ channel α2δ ligand, mirogabalin, has a high specific binding affinity for the α2δ subunit, with a slower dissociation rate for α2δ-1 than α2δ-2 compared to that of pregabalin. Mirogabalin was shown to be effective in NeP animal models, with a margin of safety between central nervous system side effects and the analgesic effect of the dose. It exerted a favorable analgesic effect, was well tolerated in patients with peripheral NeP (P-NeP), and was first approved in Japan in 2019 and subsequently in Korea and Taiwan in 2020. AREAS COVERED: The purpose of this article is to review the pharmacological characteristics, pharmacokinetics, and efficacy and safety of mirogabalin for NeP based on the results of non-clinical and clinical studies. EXPERT OPINION: Although there are several first-line therapies for NeP, insufficient efficacy and adverse drug reactions of NeP drugs often cause patient dissatisfaction. Mirogabalin was effective and well tolerated with a step-wise dose increase in clinical studies on P-NeP patients. Thus, mirogabalin is expected to be a useful treatment option for patients with P-NeP.

Safety and Efficacy of Mirogabalin for Peripheral Neuropathic Pain: Pooled Analysis of Two Pivotal Phase III Studies.

PURPOSE: Mirogabalin besylate has been approved in several countries to treat peripheral neuropathic pain. This pooled analysis, using data from the two pivotal Phase III studies in Asian patients with diabetic peripheral neuropathic pain and post-herpetic neuralgia, aimed to provide clinicians with more detailed and precise information relating to mirogabalin's safety and efficacy. METHODS: Data were pooled from 2 multicenter, double-blind, placebo-controlled, parallel-group, 14-week treatment studies of mirogabalin conducted at ∼350 study sites (Japan, South Korea, Taiwan, Singapore, Malaysia, and Thailand). Eligible patients in both studies were randomized in a 2:1:1:1 ratio, stratified according to a baseline average daily pain score (ADPS) of <6 or ≥6, to placebo, mirogabalin 15-mg once daily (QD), mirogabalin 10-mg twice daily (BID), or mirogabalin 15-mg BID treatment groups. Safety was assessed based on treatment-emergent adverse events identified from the adverse events collected throughout both studies. The primary efficacy end point of both studies was the change from baseline in ADPS at week 14. FINDINGS: In total, 1587 patients (824 with diabetic peripheral neuropathic pain; 763 with post-herpetic neuralgia) who received at least 1 dose of study drug were analyzed (633 received placebo, 954 treated with mirogabalin). Treatment-emergent adverse events included somnolence (3.8%, 10.8%, 14.5%, and 19.1%) and dizziness (2.7%, 5.7%, 9.1%, and 13.1%) in patients receiving placebo, mirogabalin 15 mg QD, mirogabalin 10 mg BID, and mirogabalin 15 mg BID, respectively. In patients treated with mirogabalin 15 mg QD, 2 (0.6%) of 316 patients discontinued due to somnolence. In the mirogabalin 10-mg BID group, somnolence, edema, and peripheral edema each resulted in 3 (0.9%) of 318 patient discontinuations. In the mirogabalin 15-mg BID group, 6 (1.9%) of 320 patients discontinued due to dizziness and 3 (0.9%) due to somnolence. At week 14, mirogabalin 10 mg BID and 15 mg BID statistically significantly improved ADPS versus placebo, with least squares mean changes (95% CI) of -0.31 (-0.55, -0.08) and -0.63 (-0.86, -0.40). Post hoc analysis showed a statistically significant difference 2 days after administration in the mirogabalin 10-mg and 15-mg BID groups compared with placebo. Female sex, age ≥65 years, and baseline weight <60 kg may influence the safety of mirogabalin, particularly regarding the incidence of somnolence and dizziness, but had no notable impact on efficacy. ClinicalTrials.gov identifiers: NCT02318706 and NCT02318719. IMPLICATIONS: This pooled analysis showed that mirogabalin was efficacious and well-tolerated by Asian patients with peripheral neuropathic pain.

Discovery of a novel selective PPARgamma modulator from (-)-Cercosporamide derivatives.

In an investigation of (-)-Cercosporamide derivatives with a plasma glucose-lowering effect, we found that N-benzylcarboxamide derivative 4 was a partial agonist of PPARgamma. A SAR study of the substituents on carboxamide nitrogen afforded the N-(1-naphthyl)methylcarboxamide derivative 23 as the most potent selective PPARgamma modulator. An X-ray crystallography study revealed that compound 23 bounded to the PPARgamma ligand binding domain in a unique way without any interaction with helix12. Compound 23 displayed a potent plasma glucose-lowering effect in db/db mice without the undesirable increase in body fluid and heart weight that is typically observed when PPARgamma full agonists are administrated.

Long-term safety and efficacy of mirogabalin in Asian patients with diabetic peripheral neuropathic pain.

AIMS/INTRODUCTION: Diabetic peripheral neuropathic pain (DPNP) affects the functionality, mood and sleep patterns of patients with diabetes. Mirogabalin, an α2 δ ligand with a slower dissociation for α2 δ-1 versus α2 δ-2 subunits, showed efficacy and safety in a randomized, double-blind, placebo-controlled, 14-week study in Asian patients with DPNP. This open-label extension study evaluated the long-term safety and efficacy of mirogabalin in Asian patients with DPNP. MATERIAL AND METHODS: This 52-week open-label extension study was carried out in Japan, Korea and Taiwan in patients with DPNP. Patients received mirogabalin, initiated at 5 mg twice daily and increased to a flexible maintenance dosage of 10 or 15 mg twice daily. Adverse events were monitored throughout the study. Patients provided a self-assessment of pain using the Short-Form McGill Pain Questionnaire. RESULTS: Of the 214 patients who entered the study, 172 (80.4%) completed the extension study. Of 172 patients who completed the study, 149 received the highest dosage of mirogabalin (15 mg twice daily). The most common treatment-emergent adverse events were nasopharyngitis, diabetic retinopathy, peripheral edema, somnolence, diarrhea, increased weight and dizziness. Most treatment-emergent adverse events were mild or moderate in severity. The incidence of treatment-emergent adverse events leading to treatment discontinuation was 13.1%. The visual analog scale and all other Short-Form McGill Pain Questionnaire subscales (sensory score, affective score, total score and present pain intensity) generally decreased over time from baseline until week 52. CONCLUSIONS: This extension study showed the safety and efficacy of a long-term flexible dosing regimen of mirogabalin 10 or 15 mg twice daily in patients with DPNP.

Results of Mirogabalin Treatment for Diabetic Peripheral Neuropathic Pain in Asian Subjects: A Phase 2, Double-Blind, Randomized, Placebo-Controlled, Study.

INTRODUCTION: Almost one-quarter of Asian patients with diabetes experience diabetic peripheral neuropathic pain (DPNP), which may be associated with moderate or severe levels of pain, insomnia, mood disorders, and worsened quality of life. Current treatments are generally ineffective and may be poorly tolerated. We evaluated mirogabalin as a treatment for DPNP in Asian subjects. METHODS: This phase 2, randomized, double-blind, controlled study was conducted in Japan, South Korea, and Taiwan. Subjects (n = 450) with DPNP were randomized (1:1:1:1:1) to treatment with 5, 10, or 15 mg twice-daily (BID) mirogabalin, 150 mg BID pregabalin, or placebo. The primary endpoint was change from baseline in average daily pain score (ADPS) at week 7; secondary endpoints included responder rates, Short-Form McGill Pain Questionnaire (SF-MPQ), Patient Global Impression of Change (PGIC), average daily sleep-interference score (ADSIS), and incidence of treatment-emergent adverse events (TEAEs). RESULTS: A greater improvement was noted for each mirogabalin treatment group for change from baseline in ADPS at week 7 compared with both placebo and with pregabalin, although these improvements were not statistically significant. The percentage of 30, 50, and 75% responders and subjects with PGIC improvements was greater in each mirogabalin group versus placebo. Mirogabalin 15 mg BID significantly improved the SF-MPQ sensory (p = 0.0313) and visual analog scale scores (p = 0.0093), and ADSIS (p = 0.0002), versus placebo. Treatment was generally well tolerated; the most frequently reported TEAEs in the mirogabalin groups were somnolence (14.7%) and dizziness (11.0%), and most AEs were mild or moderate even at the highest dose. CONCLUSIONS: In Asian subjects with DPNP, mirogabalin (5, 10, and 15 mg BID) was well tolerated. Although no significant differences were observed in the primary endpoint, there was a tendency toward improvement of pain with mirogabalin treatment, and this trend was also observed in the secondary endpoints. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01504412.

(-)-Cercosporamide derivatives as novel antihyperglycemic agents.

In our exploratory campaign for an antihyperglycemic agent with a novel mechanism of action, (-)-Cercosporamide 1, which is known as an antifungal agent, showed a potent plasma glucose lowering effect in hyperglycemic KK/Ta mice. The trouble was that it was accompanied by a decrease in food intake and a loss of body weight. We synthesized some (-)-Cercosporamide derivatives and succeeded to separate these actions. N,O-ketal type derivatives, especially compound 10, had the most potent plasma glucose lowering effect without affecting the food consumption or body weight.

[Pharmacological, pharmacodynamics, and clinical profile of mirogabalin besylate (Tarlige® tablets 2.5†mg∙5†mg∙10†mg∙15†mg)].

Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, has been approved for the treatment of peripheral neuropathic pain including painful diabetic peripheral neuropathy (DPNP) and postherpetic neuralgia (PHN) in Japan. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than for the α2δ-2 subunit. It also showed potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for the central nervous system side effects. A pharmacological study using mutant mice demonstrated that the analgesic effects of mirogabalin were mediated by binding of the drug to the α2δ-1 subunit, not the α2δ-2 subunit. The pharmacological properties of mirogabalin can be associated with its unique binding characteristics. The bioavailability of mirogabalin is high and its plasma exposure increases dose-proportionally. Mirogabalin is mainly excreted via the kidneys in an unchanged form, thus, mirogabalin has a low possibility of undergoing drug-drug interaction, while dose adjustment based on the creatinine clearance level is specified in patients with renal impairment. In double-blind, placebo-controlled phase 3 studies in Asian patients with DPNP and PHN, mirogabalin showed significant and dose-dependent pain relief, and all tested doses of mirogabalin were well tolerated. In summary, mirogabalin has a balanced efficacy versus safety profile, and can provide an alternative therapeutic option for the treatment of peripheral neuropathic pain.

Mirogabalin for the treatment of diabetic peripheral neuropathic pain: A randomized, double-blind, placebo-controlled phase III study in Asian patients.

AIMS/INTRODUCTION: This study evaluated the efficacy and safety of mirogabalin, a novel, potent, selective ligand of the α2 δ subunit of voltage-dependent Ca2+ channels, for the treatment of diabetic peripheral neuropathic pain (DPNP). MATERIALS AND METHODS: During this double-blind, multisite, placebo-controlled phase III study, Asian patients aged ≥20 years with type 1 or 2 diabetes and DPNP were randomized 2:1:1:1 to a placebo, mirogabalin 15, 20 or 30 mg/day for up to 14 weeks, with a 1- to 2-week titration (NCT02318706). The primary endpoint was the change from baseline in average daily pain score (ADPS) at week 14, defined as a weekly average of daily pain (0 = no pain to 10 = worst possible pain, for the past 24 h). RESULTS: Of 834 randomized patients, 330, 164, 165 and 165 received placebo, mirogabalin 15, 20 or 30 mg/day, respectively, and were included in analyses (modified intention-to-treat population, n = 824); 755 (90.5%) completed the study. At week 14, the least squares mean average daily pain score change from baseline was -1.31, -1.34, -1.47 and -1.81, respectively, showing statistical significance for mirogabalin 30 mg/day versus placebo (P = 0.0027). The treatment-emergent adverse events observed were mostly mild-to-moderate in all mirogabalin doses, and the most frequent treatment-emergent adverse events were nasopharyngitis, somnolence, dizziness, peripheral edema and weight increase. CONCLUSIONS: Mirogabalin relieved DPNP in a dose-dependent manner; mirogabalin 30 mg/day showed statistically significant pain relief (vs placebo) in Asian DPNP patients. All doses of mirogabalin tested were well tolerated.

Clinical oral doses of dexamethasone decreases intrinsic clearance of quinidine, a cytochrome P450 3A substrate in dogs.

We investigated the effect of dexamethasone (DEX) at clinical doses on the pharmacokinetics of quinidine (QN) in dogs. Dogs (5 healthy 1-year-old male beagles) were orally administered DEX once daily for 5 days at 2.5 or 7.5 mg/day. QN (2 mg/kg) was intravenously injected 3 weeks before and one day after the DEX treatment. The plasma concentration of QN was determined by high-performance liquid chromatography with fluorometric detection. Plasma concentrations of albumin and alpha(1)-acid glycoprotein (AGP) were determined by a bromocresol green method and a single immunodiffusion method, respectively. In order to calculate unbound concentrations of QN in plasma, the binding kinetics of QN in plasma was examined by an ultrafiltration method using pooled plasma from the 5 dogs when they were drug-free. Total body clearance of QN was decreased dose-dependently By the DEX treatment, although the decrease was not statistically significant. Elimination half-lives significantly increased (more than twice at 7.5 mg), and intrinsic clearance significantly decreased (about 50%). The volume of distribution increased significantly (about two-fold). Plasma levels of AGP significantly decreased, and the unbound fraction of QN in plasma significantly increased. Our results demonstrate that clinical doses of DEX significantly affect the pharmacokinetics of QN, a CYP3A substrate in dogs, by decreasing CYP3A activity and plasma AGP levels. There is a possibility that adverse drug-drug interaction occurs during DEX therapy through its effects on CYP3A activity and plasma AGP levels.

Effect of oral administration of clinically relevant doses of dexamethasone on regulation of cytochrome P450 subfamilies in hepatic microsomes from dogs and rats.

OBJECTIVE: To evaluate the effect of oral administration of dexamethasone (DEX) at clinically relevant doses on metabolic activities of cytochrome P450 (CYP) isoenzymes in dogs and rats. ANIMALS: 15 healthy 1-year-old male Beagles and 20 healthy 10-week-old male Wistar rats. PROCEDURE: Hepatic microsomes were harvested from dogs treated orally with DEX at 2.5 and 7.5 mg for 5 days and from rats treated orally with DEX at 0.75, 6, and 48 mg/kg for 5 days. 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam were used as CYP1A, CYP2C, CYP2D, and CYP3A substrates, respectively. Concentrations of metabolites formed by CYPs were measured by use of high-performance liquid chromatography, except for the resorufin concentrations measured by use of a fluorometric method. Reaction velocity-substrate concentration data were analyzed to obtain maximum reaction velocity (Vmax) and Michaelis-Menten constant (Km). RESULTS: Values of Vmax for midazolam 4-hydroxylation were significantly decreased by treatment with DEX at 2.5 and 7.5 mg in dogs, although values of Km were not affected. Values of Vmax for bufuralol 1'-hydroxylation were also decreased by treatment with DEX. In rats, values of Vmax for midazolam 4- hydroxylation were significantly decreased by treatment with DEX at 0.75 and 6 mg/kg but significantly increased at 48 mg/kg. Other reactions were not affected by treatment with DEX. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that DEX downregulates the CYP3A subfamily when administered at clinically relevant doses to dogs. The effect of downregulation of CYP3A in dogs treated with DEX should be considered to avoid adverse effects from coadministration of drugs.

Effect of oral ketoconazole on first-pass effect of nifedipine after oral administration in dogs.

The long-term oral ketoconazole (KTZ) treatment extensively inhibits hepatic CYP3A activity. We investigated the effect of the KTZ treatment on hepatic and intestinal extraction of nifedipine (NIF) using beagle dogs. Four dogs were given orally KTZ for 20 days (200 mg, bid). NIF was administered either intravenously (0.5 mg/kg) or orally (20 mg) 10 and 20 days before the KTZ treatment and 10 and 20 days after start of KTZ treatment. CLtot of NIF after intravenous administration decreased to about 50% during the KTZ treatment. C(max) and AUC after oral administration increased to 2.5-fold and fourfold, respectively, by the KTZ treatment. The hepatic extraction ratio of NIF decreased to about a half by KTZ. A significant decrease in intestinal extraction ratio was not observed. In conclusion, the KTZ treatment inhibits hepatic extraction more profoundly than intestinal extraction of NIF. Therefore, inhibition of hepatic extraction of NIF by the KTZ treatment mainly results in substantial increase in systemic bioavailability in dogs. Because KTZ inhibits human CYP3A activities similar to canine CYP3A activities, the long-term oral KTZ treatment may dramatically increase bioavailability of NIF or other CYP3A substrates in humans.

In vitro characterization of the inhibitory effects of ketoconazole on metabolic activities of cytochrome P-450 in canine hepatic microsomes.

OBJECTIVE: To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs. ANIMALS: 4 healthy 1-year-old male Beagles. PROCEDURE: Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki). RESULTS: KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6+/-6.0, 170+/-2.5, and 0.180+/-0.131 microM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12.

Synthesis and biological evaluation of novel (-)-Cercosporamide derivatives as potent selective PPARγ modulators.

Selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators are expected to be a novel class of drugs improving plasma glucose levels without PPARγ-related adverse effects. As a continuation of our studies for (-)-Cercosporamide derivatives as selective PPARγ modulators, we synthesized substituted naphthalene type compounds and identified the most potent compound 15 (EC(50) = 0.94 nM, E(max) = 38%). Compound 15 selectively activated PPARγ transcription and did not activate PPARα and PPARδ. The potassium salt of compound 15 showed a high solubility and a good oral bioavailability (58%). Oral administration of the potassium salt remarkably improved the plasma glucose levels of female Zucker diabetic fatty rats at 1 mg/kg. Moreover, it did not cause a plasma volume increase or a cardiac enlargement in Wistar-Imamichi rats, even at 100 mg/kg.

Effect of multiple dosing of ketoconazole on pharmacokinetics of midazolam, a cytochrome P-450 3A substrate in beagle dogs.

To evaluate effects of multiple dosing of ketoconazole (KTZ) on hepatic CYP3A, the pharmacokinetics of intravenous midazolam (MDZ, 0.5 mg/kg) before and during multiple dosing of KTZ were investigated in beagle dogs. KTZ tablets were given orally to dogs (n = 4) for 30 days (200 mg b.i.d.). With coadministration of KTZ, t(1/2beta) of MDZ were significantly increased both on day 1 (2-fold) and on day 30 (3-fold). Total body clearance (CL(tot)) of MDZ declined gradually during the first 5 days after the start of KTZ treatment, and thereafter CL(tot) appeared to reach a plateau phase (one-fourth), depending on plasma KTZ concentrations. The effects of KTZ on the biotransformation of MDZ were also investigated using dog liver microsomes (n = 5). The K(i) values of KTZ for MDZ 1'-hydroxylation and 4-hydroxylation were 0.0237 and 0.111 microM, respectively, indicating that KTZ extensively inhibits hepatic CYP3A activity in dogs. CL(tot) values estimated from in vitro K(i) values corrected by unbound fraction of KTZ and unbound concentrations of the drug in plasma were consistent with in vivo CL(tot) of MDZ. The results in this study suggest that KTZ treatment is necessary until plasma concentrations of the drug reach a steady state to evaluate the effect of multiple dosing of the drug on hepatic CYP3A in vivo. In addition, it is suggested that K(i) values corrected by unbound fraction of KTZ and unbound concentrations of the drug in plasma enable precise in vitro-in vivo scaling.