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Patients with acromegaly not cured by surgery are often initially treated with injected peptide long-acting somatostatin receptor ligands (SRLs). Non-peptide small molecules can also activate the somatostatin receptor and do so with a high degree of precision for the target therapeutic receptor subtype. Paltusotine (formerly {"type":"entrez-protein","attrs":{"text":"CRN00808","term_id":"1048626668","term_text":"CRN00808"}}CRN00808) is a small molecule somatostatin type 2 (SST2) receptor agonist with high oral bioavailability (70%) and pharmacokinetic profile suitable for once daily dosing. In healthy volunteers, paltusotine has been shown to lower growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. We hypothesized that patients with acromegaly could switch from injected SRLs to once daily oral paltusotine while maintaining baseline IGF-1 levels. ACROBAT Edge ({"type":"clinical-trial","attrs":{"text":"NCT03789656","term_id":"NCT03789656"}}NCT03789656) was a single-arm study designed to evaluate the safety and efficacy of switching from injected SRLs to paltusotine in patients with acromegaly. The primary analysis population consisted of those who had not achieved normal IGF-1 levels despite stable therapy with long-acting octreotide or lanreotide. Eligible patients received their last injection of SRL 4 weeks prior to switching to once daily oral paltusotine monotherapy for a 13-week treatment period. The starting dose of 10 mg per day was uptitrated in 10 mg increments at specified study visits to a maximal dose of 40 mg per day based on protocol specified study drug toleration and IGF-1 criteria. The primary endpoint was change in IGF-1 from baseline to the completion of the 13-week treatment period. Statistical testing was based on non-parametric Wilcoxon Sign Rank test of whether the median change is different from zero. In addition, the rise in IGF-1 during a 4-week washout period was used to provide supportive evidence of efficacy. Twenty-five patients were enrolled in the primary analysis group, three patients discontinued from the study for non-study drug related reasons, two during the treatment period and one during the washout period after completing treatment. The primary endpoint was achieved as paltusotine treatment resulted in no significant change in IGF-1 levels at week 13 compared to baseline [change in IGF-1 =-0.034 (-0.107, 0.107), median (IQR), p>0.6]. Of the 23 patients who completed the dosing period, 20 (87%) achieved IGF-1 levels at the end of treatment that were within 20% of baseline or lower. Median IGF-1 values rose significantly after paltusotine washout (p 10%) included: headache, arthralgia, fatigue, peripheral swelling, paresthesia and hyperhidrosis. There were no discontinuations due to adverse events and no treatment related serious adverse events. These results suggest that patients with acromegaly treated with injected SRLs can switch to oral paltusotine while maintaining IGF-1 and that paltusotine appeared to be well tolerated.

Adrenocorticotropic hormone (ACTH) is an important modulator of steroidal hormone synthesis and secretion from the adrenal gland and its selective activity at the melanocortin type 2 receptor (MC2) dictates the synthesis and secretion of cortisol (corticosterone in rats). Excess ACTH action contribute to the pathophysiology of Cushing’s disease (CD), ectopic ACTH secreting tumors (EAS), and Congenital Adrenal Hyperplasia (CAH). Cushing’s disease results from a microadenoma derived from pituitary corticotrophic cells that secretes excess ACTH, whereas EAS arises from nonpituitary ACTH secreting tumors. Excess ACTH action at the adrenal gland and resulting hypercortisolemia presents in a myriad of symptoms that result in high morbidity. CAH results from inactivating mutations in steroid synthesis pathways, resulting in lack of cortisol and aldosterone production. Lack of negative feedback by cortisol at the level of the pituitary causes the over-secretion of ACTH, and overproduction of adrenal androgens, causing significant virilization and reduction in quality of life. We hypothesize that blocking ACTH action directly via a selective MC2 receptor antagonist may provide an important new therapeutic mechanism for these patients. To test this hypothesis, Crinetics launched an iterative medicinal chemistry program to identify potent and selective nonpeptide ACTH antagonists with pharmaceutical and safety characteristics suitable for evaluation in human clinical trials. Unlike most other G protein coupled receptors, MC2 requires the presence of an accessory protein (MRAP) for cell surface expression and recognition of ACTH. Using CHO-K cells stably expressing this MC2-MRAP complex, iterative optimization led to the discovery of multiple chemical classes of highly potent, nonpeptide MC2 receptor selective antagonist leads, which were then further optimized for drug-like characteristics. We identified multiple compounds that exhibit high potency for human and rat MC2 receptors (hMC2 Kb

Cushing’s disease (CD) and Ectopic ACTH syndrome (EAS) stem from excess circulating adrenocorticotropic hormone (ACTH) and resulting hypercortisolemia. In CD, excess ACTH is secreted from pituitary tumors, whereas excess ACTH in EAS arises from nonpituitary tumors. ACTH acts on the adrenal melanocortin type 2 (MC2) receptor to control the synthesis and secretion of adrenal hormones, including the stress hormone cortisol (corticosterone in rats) which accounts for the comorbidities of CD and EAS. Availability of a potent ACTH antagonist that can normalize cortisol in patients with diseases of excess ACTH will be a major advance in endocrinology. Additionally, an ACTH antagonist will have utility in congenital adrenal hyperplasia (CAH) because of its ability to block production of excess adrenal androgens. Crinetics is evaluating and developing ACTH antagonists for the treatment of diseases of excess ACTH. To our knowledge, these compounds represent the first potent nonpeptide ACTH antagonists to demonstrate in vitro potency and in vivo efficacy. As a result, the direct effects of sustained MC2 receptor blockade on the structure and function of the adrenal gland have never been able to be assessed. We examined the effects of several orally bioavailable ACTH antagonists across a range of doses on Sprague-Dawley rat adrenal gland weight, histology, and hormone levels in repeat dosing (7-14 days) studies. Sustained MC2 receptor antagonism dose dependently blocked activity of ACTH at the level of the adrenal gland and reduced plasma corticosterone levels. In the normal rat, this resulted in dose-dependent atrophy of the adrenal gland as assessed by organ weights and microscopically. The atrophy was primarily observed in the cortisol producing zona fasciculata, as well as in the zona reticularis, with smaller reductions noted in the aldosterone producing zona glomerulosa. Additionally, hypertrophy of the adrenal glands caused by continuous subcutaneous administration of exogenous ACTH was reversed by treatment with an ACTH antagonist. The adrenal effects were accompanied by expected changes in corticosterone levels. These preclinical findings demonstrate the therapeutic potential of ACTH antagonism and provide a strong rationale for development of an orally bioavailable drug that can be used to combat CD, EAS, and CAH.

Congenital hyperinsulinism (CHI) results from mutations within the insulin secretion pathway and is characterized by excessive and/or inappropriate insulin secretion by pancreatic islet β-cells. CHI is the most common cause of persistent hypoglycemia in newborns and infants and is estimated to affect 1:2500 to 1:50,000 live births. Prompt recognition and treatment are vital to prevent coma, long-term neurological complications, and even death. If medical control of CHI is unsuccessful, a near-total pancreatectomy may be required, but hypoglycemia often persists. The neuropeptide somatostatin is an important modulator of pancreatic hormonal signaling and activity at different somatostatin receptor (sst) subtypes dictates the suppression of insulin and/or glucagon. The injectable peptide drugs octreotide and lanreotide are potent sst2 agonists used to treat CHI, but in addition to suppressing insulin, the sst2 activity of these peptides may also inhibit glucagon secretion, potentially reducing effectiveness and compromising a key defense against hypoglycemia. Glucagon secretion from α-cells is inhibited through activation of sst2 receptors, while insulin secretion from β-cells is inhibited through activation of sst2 and sst5. We therefore hypothesize that agonists selectively targeting sst5 and lacking sst2 activity will offer an improved efficacy/safety profile for patients with hyperinsulinemic hypoglycemia. Using iterative medicinal chemistry and pharmacology, Crinetics has discovered several classes of highly potent, orally bioavailable, small molecule sst-subtype selective agonists with drug-like pharmaceutical properties. Our discovery efforts aimed at finding a compound to treat CHI have yielded potent and selective nonpeptide sst5 agonists with sub-nanomolar EC50s in cell-based assays of receptor activation. Insulin secretion from isolated human and rat islets was suppressed upon exposure to sst5 agonists. Potent and selective sst5 agonists were then evaluated in a number acute and repeat dose in vivo models (e.g., oGTT, fed/fasted conditions, sulfonylurea-induced hypoglycemia) to assess physiological effects and to gain mechanistic insights. As predicted by the in vitro pharmacology, selective nonpeptide sst5 agonists suppressed insulin secretion and raised blood glucose levels in each model, while having minimal effects on glucagon secretion. Leading sst5 agonists were also evaluated for drug like characteristics, including stability in liver microsomes, lack of inhibition of cytochromes P450 and the hERG ion channel, and were shown to exhibit good exposure upon oral dosing in both rats and dogs. The culmination of these studies has led to a subset of candidate molecules that are being evaluated in genotoxicity, safety pharmacology, and general toxicity studies to determine the molecule most suitable for evaluation in human clinical trials.

Injected depot formulations of somatostatin peptide analogs are routinely used to treat acromegaly and neuroendocrine tumors (NETs). CRN00808, a small molecule nonpeptide selective somatostatin receptor 2 (sst2) agonist, is being evaluated for efficacy and safety in patients with acromegaly. The current Phase 1 study was conducted in two Parts: In Part A, the absorption, metabolism, excretion, and mass balance of a single oral dose of 20 mg [14C]-CRN00808 (3.0 MBq) oral solution was characterized in six healthy male subjects. Plasma, blood, urine, and feces were collected for up to 432 hours, and were analyzed for total radioactivity and CRN00808 concentrations (plasma only). Metabolite profiling was conducted on the plasma, urine, and feces samples. In Part B, the absolute bioavailability of CRN00808 was determined by administering a single oral dose of 20 mg CRN00808 compared with a single micro-tracer intravenous (IV) bolus injection of 50 µg [14C]-CRN00808 (0.0185 MBq) in five healthy male subjects. The IV dose was administered approximately 90 minutes after the oral dose. Plasma samples were collected for up to 144 hours and were analyzed for total radioactivity and CRN00808 concentrations (plasma only). Key data from Part A and Part B will be presented. Available data from Part A of the study show that >90% of radioactivity was recovered within 7 days of dosing. The primary route of excretion was the feces (>90%) with minimal excretion in the urine (

Depot injection formulations of peptide somatostatin receptor ligands (SRLs) are routinely used to treat acromegaly and neuroendocrine tumors (NETs). Paltusotine (CRN00808), an orally administered small molecule nonpeptide selective somatostatin receptor 2 (SST2) agonist has been shown to maintain GH and IGF-1 levels in acromegaly patients previously on depot SRLs (ACROBAT Edge NCT03789656). In this study, a capsule formulation was used, which did not exhibit dose proportional pharmacokinetics (PK) at doses >40 mg, required a 2-hour post-dose fast in overnight fasted patients, and had the potential for reduced bioavailability when taken with proton-pump inhibitors (PPI). A spray-dried dispersion (SDD) tablet formulation was developed with improved solubility in the physiological pH range and its performance was evaluated in healthy volunteers. Male and female healthy volunteers who met inclusion/exclusion criteria were enrolled in a single-center Phase 1 study (ANZCTR registration ACTRN12619001562167). A Cohort of 12 subjects was administered a single dose of paltusotine in a four-period cross-over design. Periods 1 and 2 assessed the effect of lansoprazole (a PPI) on PK of 20 mg dose of paltusotine SDD tablets. In Period 3, 20 mg dose of paltusotine SDD tablets was co-administered with a high fat, high-calorie meal. In Period 4, a 60 mg dose of paltusotine SDD tablets was administered to assess dose proportionality. In a separate cohort of subjects (n=12; also, a 4-period cross-over design), the relative bioavailability of capsules and SDD tablets was assessed, and the effect of food administration 0.5, 1, and 2 hour post-dose was evaluated. Subsequently, in another cohort of 12 subjects (a 3-period cross-over design), dose proportionality of the SDD tablets was evaluated at 40 mg and 80 mg dose with a 1-hour post-dose fast. A 4 hours post-dose fast was also assessed for the 80 mg dose. Pharmacokinetics and safety of paltusotine were evaluated. Paltusotine was generally well tolerated in this study. SDD tablets exhibited dose proportional increase in total systemic exposure (AUC) up to a dose of 80 mg. Healthy volunteers pretreated with the PPI, lansoprazole (15 mg bid for 3 days), and co-administered with paltusotine SDD tablets exhibited a small decrease (approximately 25%) in systemic exposure to paltusotine compared with the same subjects that had washed out from the PPI-pretreatment. SDD tablets exhibited significant reduction in systemic exposure when co-administered with a high-fat, high-calorie meal. However, the SDD formulation was less sensitive to timing of post-dose food administration compared to the capsule formulation. These data suggest that the SDD tablet formulation of paltusotine improves flexibility in dose administration, can be co-administered with PPIs and other agents that increase stomach pH, and reduces the post-dose fasting requirement.

CRN04894 is an orally administered nonpeptide that is a potent and selective antagonist for adrenocorticotropic hormone (ACTH) acting at the melanocortin 2 receptor (MC2R) and is currently under development for the treatment of diseases of ACTH excess such as Cushing’s disease, congenital adrenal hyperplasia, and ectopic ACTH-secreting tumors. Cushing’s disease results from an adenoma derived from pituitary corticotropic cells that secrete excess ACTH, whereas ectopic ACTH syndrome arises from nonpituitary ACTH secreting tumors. Congenital adrenal hyperplasia is a genetic disease that results in cortisol deficiency leading to high levels of ACTH and adrenal androgens. Each of these indications is characterized by high ACTH levels that act on MC2R expressed in the adrenal cortex to drive pathological elevations of adrenally derived steroid hormones. CRN04894 blocks the action of ACTH at MC2R, providing a potential novel treatment for these diseases. Preclinical models of chronic hypercortisolemia include implantation of ACTH-secreting pituitary tumor cells in mice and continuous administration of ACTH via subcutaneously implanted osmotic pumps in rats. These models induce features consistent with human diseases of ACTH excess including hypercortisolemia and hypertrophy of the adrenal glands. We employed both rodent models to examine the pharmacodynamic effects of CRN04894 on corticosterone levels and adrenal gland morphology. In the mouse pituitary tumor model, subcutaneous inoculation of the ACTH-secreting mouse pituitary tumor cell line, AtT-20, into immunodeficient mice resulted in formation of tumors and increased plasma ACTH and corticosterone levels. Repeated daily oral administration of CRN04894 for 14 days dose-dependently and robustly suppressed plasma corticosterone levels in mice with AtT-20 tumors. In the rat model, subcutaneous implantation of osmotic pumps delivering ACTH resulted in increased corticosterone levels, reduction in body weight, and hypertrophy of the adrenal glands after 7 days. Daily oral administration of CRN04894 over 7 days dose-dependently suppressed corticosterone levels, mitigated the effect of ACTH excess on body weight, and rescued the adrenal gland hypertrophy. These findings provide evidence that CRN04894 functions as an effective ACTH antagonist at MC2R to suppress adrenal corticosterone secretion in both mouse and rat models of ACTH excess and hypercortisolemia, thus providing a strong rationale for its potential therapeutic utility in diseases of ACTH excess. This work was supported in part by an SBIR grant from the NIH awarded to Dr. Struthers (R43- DK115245)

Injected depot formulations of somatostatin peptide analogs are routinely used to treat acromegaly and neuroendocrine tumors (NETs). CRN00808 is a small molecule nonpeptide selective somatostatin receptor 2 agonist whose safety, pharmacokinetics (PK), and pharmacodynamics (PD) has been characterized in preclinical studies. This study describes the final results from a first-in-human, single and multiple ascending dose Phase 1 study in healthy volunteers to measure the safety, PK, PD, and midazolam drug interaction potential of CRN00808 (NCT03276858; preliminary results with blinded safety data presented at ENDO 2018). In the single dose arm of the study, cohorts of 8 subjects (6 active: 2 placebo) received CRN00808 as an oral solution or capsules (1.25 mg to 60 mg, or placebo). The effect of food on CRN00808 PK was also evaluated. In the multiple dose arm, cohorts of 9 subjects (6 active: 3 placebo) received CRN00808 capsules once daily (5 mg to 30 mg, or placebo) for 7-10 days. In the drug-interaction arm, a single cohort of 8 subjects received 20 mg of CRN00808 for 7 days; midazolam PK was assessed before (Day -2) and after (Day 7) administration of CRN00808. Safety and PK were assessed in all phases of the study. Suppression of GHRH-induced GH secretion and suppression of serum IGF-1 were measured as PD endpoints in the single and multiple dose phases of the study, respectively. Once daily administration of 5-30 mg CRN00808 capsules exhibited dose-dependent increases in peak (Cmax) and total (AUC) plasma exposures. The apparent terminal elimination half-life was determined to be of 42-50 hours and steady state was achieved in 3-5 days. Capsules taken with a standard high fat, high calorie meal resulted in a markedly lower plasma CRN00808 AUC (83%). Oral administration of CRN00808 resulted in dose-dependent suppression of both GHRH stimulated GH and IGF-1 secretion; a single 10 mg dose was found to cause 91% suppression of GHRH-stimulated GH and 10 mg once per day for 10 days resulted in maximal suppression of serum IGF-1. Midazolam PK was unaffected by co-administration of 20 mg CRN00808, suggesting little or no risk of drug interaction with CYP3A4/5 substrates. Treatment emergent adverse events associated with CRN00808 were generally mild and transient, and consistent with those reported with other somatostatin agonists. In conclusion, results from this Phase I clinical trial in healthy volunteers support further clinical development of CRN00808 as a once-daily oral treatment of patients with acromegaly.

Cushing’s disease is most commonly the result of a microadenoma derived from pituitary corticotrophic cells that secretes excess adrenocorticotropic hormone (ACTH). ACTH is an important modulator of steroidal hormone synthesis and secretion from the adrenal gland and its selective activity at the melanocortin type 2 receptor (MC2) dictates the synthesis and secretion of cortisol (corticosterone in rats). The resulting hypercortisolemia in Cushing’s patients presents in a myriad of symptoms that include growth of fat pads, excessive sweating, dilation of capillaries, thinning of the skin, muscle weakness, hirsutism, depression/anxiety, hypertension, osteoporosis, insulin resistance, hyperglycemia, and heart disease, among others that result in high morbidity. We hypothesize that blocking ACTH action directly via a selective MC2 receptor antagonist may provide an important new therapeutic mechanism to help better manage Cushing’s disease in patients. To test this hypothesis, we launched an iterative medicinal chemistry program to identify potent and selective nonpeptide MC2 receptor antagonists with pharmaceutical and safety characteristics suitable for evaluation in human clinical trials. Unlike most other G protein coupled receptors, MC2 requires the presence of an accessory protein (MRAP) for cell surface expression and recognition of ACTH and our effort led to small molecule nonpeptides with antagonist activity in CHO-K cells stably expressing the MC2-MRAP complex. Iterative optimization led rapidly to the discovery of multiple chemical classes of highly potent, nonpeptide MC2 selective antagonist leads, which were then further optimized for drug-like characteristics. We have identified multiple compounds that exhibit high potency for human and rat MC2 receptors (hMC2 Kb

Congenital hyperinsulinism (CHI) results from mutations within the insulin secretion pathway and is characterized by excessive and/or inappropriate insulin secretion by pancreatic islet β-cells. CHI is the most common cause of persistent hypoglycemia in newborns and infants and is estimated to affect 1/30,000 to 1/50,000 live births. Prompt recognition and treatment are vital to prevent coma, long-term neurological complications, and even death. If medical control of CHI is unsuccessful, near-total pancreatectomy may be required. The neuropeptide somatostatin is an important modulator of pancreatic hormonal signaling and activity at different somatostatin receptor (sst) subtypes dictates the suppression of insulin and/or glucagon. Glucagon secretion from α-cells is inhibited through sst2 receptors and insulin secretion from β-cells is inhibited through activation of sst2, sst3, and sst5. The injectable peptide drugs octreotide and lanreotide are potent agonists at sst2 and are often deployed as the last medical intervention to prevent or delay pancreatectomy. These peptides’ sst2 activity leads to inhibition of glucagon secretion, potentially reducing their effectiveness and compromising a key defense mechanism against hypoglycemia. We hypothesize that agonists targeting sst5 but lacking sst2 activity will possess an optimal efficacy/safety profile for patients with hyperinsulinemic hypoglycemia. Using iterative medicinal chemistry, Crinetics has discovered several classes of highly potent, orally bioavailable, small molecule sst-subtype selective agonists with drug-like pharmaceutical properties. Our discovery efforts aimed at finding a compound to treat CHI have yielded potent and selective nonpeptide sst5 agonists with sub-nanomolar EC50s in cell-based assays of receptor activation. These compounds also typically possess similar potency for the rat sst5 receptor. To probe their physiological consequences and to gain mechanistic insights, we compared the acute and chronic effects of these agonists to the peptide pasireotide, a pan-sst agonist that is most potent at sst5, on glycemic control in several rat models, which generally demonstrate a high degree of translation to humans. These preclinical studies evaluated the effects of the sst5 agonists during oGTT, ipGTT, sulfonylurea-induced hypoglycemia, and on blood glucose levels in both the fed and fasted states. In each model, selective nonpeptide sst5 agonists suppressed insulin secretion and raised blood glucose levels while having minimal effects on glucagon secretion, as predicted by their in vitro pharmacology. These results support our efforts to develop potent nonpeptide selective sst5 agonists with pharmaceutical and safety profiles suitable for evaluation in human clinical trials.

Introduction. Odontogenic cysts are distinct entities and quite a common occurrence in the jaw bones. These are individual lesions which arise from the same odontogenic apparatus but with varying pathogenesis. Cytokeratins are integral components in tooth development and are expressed across the odontogenic tissues in physiological and pathological states. Aim. To elucidate the role of cytokeratin-7 in the pathogenesis of odontogenic cysts by immunohistochemistry Method. Cytokeratin-7 (CK-7) was assessed in 39 cases of odontogenic lesions retrieved from the archival files which included 15 cases of Dentigerous cysts (DC), 12 cases of Odontogenic keratocysts (OKC) and 12 cases of Radicular cysts (RC) and also 8 cases of control specimens. Statistical analysis. Results obtained were statistically analyzed using chi-square test to assess the association between different odontogenic cysts used in this study and Cytokeration-7 staining. The difference was considered to be of statistical significance if the p value was ≤ 0.05. Results. CK7 expression was maximum in dentigerous cycts (66.66%) followed by radicular cysts (41.66%) and odontogenic keratocysts (16.6%). On evaluation of staining and expression pattern, highest positivity is shown in Dentigerous cysts and the positivity is seen in suprabasal (60%) and superficial layers (40%) whereas radicular cysts and odontogenic keratocysts showed positivity in superficial and spinous layers. Conclusion. Cytokeratin-7 expression correlates with the degree of differentiation of the epithelium. So the cysts with a well-differentiated epithelium (RC and DC) express CK-7, while the cysts with a less well-differentiated epithelium (OKC) show slight positivity. Thus it can be useful to differentiate OKC from DC and RC.

The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2, is approved for the treatment of tardive dyskinesia. Valbenazine is converted to two significant circulating metabolites in vivo, namely, (+)-α-dihydrotetrabenazine (R,R,R-HTBZ) and a mono-oxy metabolite, NBI-136110. Radioligand-binding studies were conducted to assess and compare valbenazine, tetrabenazine, and their respective metabolites in their abilities to selectively and potently inhibit [3H]-HTBZ binding to VMAT2 in rat striatal, rat forebrain, and human platelet homogenates. A broad panel screen was conducted to evaluate possible off-target interactions of valbenazine, R,R,R-HTBZ, and NBI-136110 at >80 receptor, transporter, and ion channel sites. Radioligand binding showed R,R,R-HTBZ to be a potent VMAT2 inhibitor in homogenates of rat striatum (Ki = 1.0-2.8 nM), rat forebrain (Ki = 4.2 nM), and human platelets (Ki = 2.6-3.3 nM). Valbenazine (Ki = 110-190 nM) and NBI-136110 (Ki = 160-220 nM) also exhibited inhibitory effects on VMAT2, but with lower potency than R,R,R-HTBZ. Neither valbenazine, R,R,R-HTBZ, nor NBI-136110 had significant off-target interactions at serotonin (5-HT1A, 5-HT2A, 5-HT2B) or dopamine (D1 or D2) receptor sites. In vivo studies measuring ptosis and prolactin secretion in the rat confirmed the specific and dose-dependent interactions of tetrabenazine and R,R,R-HTBZ with VMAT2. Evaluations of potency and selectivity of tetrabenazine and its pharmacologically active metabolites were also performed. Overall, the pharmacologic characteristics of valbenazine appear consistent with the favorable efficacy and tolerability findings of recent clinical studies [KINECT 2 (NCT01733121), KINECT 3 (NCT02274558)].

AIMS: To evaluate the pharmacokinetics (PK) of five H1 receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg). METHODS: Five H1 receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy. RESULTS: The median clearance (CL), apparent volume of distribution (V d) and apparent terminal elimination half-life (t1/2) of diphenhydramine after an i.v. microdose were 24.7 l h-1, 302 l and 9.3 h, and the oral Cmax and AUC0-� were 0.195 ng ml-1 and 1.52 ng h ml-1, respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2. CONCLUSIONS: Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection. © 2009 Neurocrine Biosciences.

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 13b displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-pyrrolidine 13b-1 possessed a Ki of 1.0 nM and an EC50 of 3.8 nM, while its 3R,4S-isomer 13b-2 exhibited a Ki of 4.7 and an IC50 of 64 nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 13b-1 also demonstrated efficacy in a diet-induced obesity model in rats.

Benzylamine and pyridinemethylamine derivatives were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor (MC4R). These compounds were also profiled in rodents for their pharmacokinetic properties. Two compounds with diversified profiles in chemical structure, pharmacological activities, and pharmacokinetics, 10 and 12b, showed efficacy in an established murine cachexia model. For example, 12b had a K(i) value of 3.4 nM at MC4R, was more than 200-fold selective over MC3R, and had a good pharmacokinetic profile in mice, including high brain penetration. Moreover, 12b was able to stimulate food intake in the tumor-bearing mice and reverse their lean body mass loss. Our results provided further evidence that a potent and selective MC4R antagonist with appropriate pharmacokinetic properties might potentially be useful for the treatment of cancer cachexia.

A series of alpha-benzylpropionylpiperazines were synthesized and tested as antagonists of the melanocortin-4 receptor. In addition to its high potency and selectivity, R-11a had desirable pharmacokinetic properties including high brain penetration in mice.

SU5416, 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2). It was the first VEGFR2 inhibitor to enter clinical trials for the treatment of colorectal and non-small cell lung cancers. Pre-clinical evaluation of SU5416 included studies related to the distribution, metabolism and excretion of this compound. These studies have provided information useful in understanding the disposition and metabolism of the indolinone class of chemicals, which has not been studied previously with therapeutic intent. The lessons we learned from SU5416 have been successfully applied in developing next generation indolinone compounds targeting tumor angiogenesis.

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of energy homeostasis. Recent studies have shown that blockade of the MC4R reverses tumor-induced weight loss in mice. Herein, we describe the synthesis and identification of potent and selective non-peptide antagonists of the human MC4R from a series of 2-ethoxycarbonylcyclohexyl-piperazines. Compound 12i was found to possess low nanomolar affinity for the MC4R, and exhibit oral bioavailability in rats. More importantly, when administered orally to mice (10 mg/kg), it led to statistically significant increases in food intake over a 24-h period.

Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), beta-naphthoflavone (33 microM), phenobarbital (100 or 250 microM), isoniazid (100 microM) and/or rifampin (20 or 50 microM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by beta-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital (n = 7) and rifampin (n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital (n = 5) or rifampin (n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by beta-naphthoflavone. Based on these observations, we generalize that beta-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.

Objective: There are five situations in a women’s life during which hormone fluctuations make them more susceptible to oral health problems – during puberty, at certain points in the monthly menstrual cycle, when using birth control pills, during pregnancy, and at menopause. The present study aimed at evaluating the effect of menstruation, pregnancy and menopause on salivary flow rate, pH and gustatory function. Materials and Methods: The study was carried out on 120 patients including 30 controls (with normal menstrual cycle of 28 to 30 d) and 90 cases (30 patients within three days of menstruation, 30 pregnant and 30 postmenopausal). Paraffin-stimulated saliva samples were obtained by expectoration to calculate salivary flow rate, pH was measured electrometically and patients were prospectively evaluated for gustatory function. Then, whole mouth taste test was performed in which the quality identification and intensity ratings of taste solutions were measured. Results: No statistically significant difference was found between the groups with respect to salivary flow rate but pH values were significantly lower in post menopausal women (p

Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Castleman's disease (CD) is characterized by lymph node enlargement due to hyperplasia of abnormal lymphoid follicles and paracortical lymphocytic hyaline vascular (HV) stroma or plasmacytosis (PC). The lymphoid follicles in CD show involuted germinal centers and prominent mantle zone lymphocytes. Ninety-seven cases clinically suspected to be CD were analyzed according to conventional histologic criteria established by Castleman and Keller for diagnosis. Twenty-two cases were excluded as nonspecific hyperplasia (12); Hodgkin's and non-Hodgkin's lymphoma (9); and multiple myeloma involving lymph node paracortex (1). The 75 remaining cases, consisting of 51 cases of CD and 24 with altered follicles or paracortex suggestive of CD, were further analyzed immunohistologically for changes in follicular dendritic reticulum cells (FDRC) using the monoclonal antibody Ki-M4p, for germinal center proliferation with Ki-S5, for mantle zone immunophenotype with Ki-B3 and Ki-B5, for paracortical plasmacytoid monocytes with Ki-M1p, and for plasma cell clonality by applying antibodies to kappa and lambda immunoglobulin light chains. Lymph nodes showing nonspecific follicular and paracortical hyperplasia were included as controls. Hyaline vascular CD and plasma cell CD showed enlarged, polyploid FDRC with prominent nucleoli, decreased germinal center proliferation, and mantle zone populations of immunophenotypically aberrant, Ki-B3-negative B lymphocytes. Thirty-seven percent of hyaline vascular CD and plasma cell CD contained plasmacytoid monocytes, and 15% showed interstitial areas of lambda predominant plasma cells. Plasmacytoid monocytes were common in hyaline vascular CD but rare in plasma cell CD. Cases suspected to be CD that demonstrated a mantle zone population of Ki-B3-negative B lymphocytes had clinical finding of POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal paraprotein, and skin changes or sclerotic bone lesions) syndrome and were reclassified as hyaline vascular CD, plasma cell CD, and mantle zone CD with an aberrant mantle zone immunophenotype only (lacking follicular center and paracortical histologic or immunohistologic abnormalities). Immunohistochemistry was valuable for identification of dysplastic FDRC, decreased germinal center proliferation, and plasmacytoid monocytes. In addition, immunohistochemistry was essential for detection of plasma cell clonality, an aberrant mantle zone immunophenotype, and mantle-zone-restricted CD that was devoid of diagnostic alterations of germinal center or paracortex.

Analogues of the known H(1)-antihistamine R-dimethindene were profiled as potential agents for the treatment of insomnia. Several highly selective compounds were efficacious in rodent sleep models. On the basis of overall profile, indene 1d and benzothiophene 2a had pharmacokinetic properties suitable for evaluation in night time dosing. Compound 2a did not show an in vivo cardiovascular effect from weak hERG channel inhibition.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5-2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within +/-10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.

Thien-2-yl 1S,2R-milnacipran analogues were synthesized and characterized as norepinephrine/serotonin transporter inhibitors. These compounds possessed higher potencies than 1S,2R-milnacipran (2R-1) while maintaining low molecular weight and moderate lipophilicity, which are the important features for the pharmacological and pharmacokinetic characteristics of milnacipran (1). Thus, compound 5c exhibited IC50 values of 2.3 and 32 nM, respectively, at NET and SERT, which were more than 10-fold better than those of 1 (NET IC50 = 77 nM, SERT IC50 = 420 nM). Moreover, 5c achieved the same efficacy as 1, but with much lower doses, in a rodent spinal nerve ligation pain model. In addition, 5c displayed desirable pharmacokinetic properties in several species, including high oral availability and significant brain penetration.

This study was designed to study the in vitro metabolism of indiplon, a novel hypnotic agent, and to assess its potential to cause drug interactions. In incubations with pooled human liver microsomes, indiplon was converted to two major, pharmacologically inactive metabolites, N-desmethyl-indiplon and N-desacetyl-indiplon. The N-deacetylation reaction did not require NADPH, and appeared to be catalyzed by organophosphate-sensitive microsomal carboxylesterases. The N-demethylation of indiplon was catalyzed by CYP3A4/5 based on the following observations: (1) the sample-to-sample variation in N-demethylation of indiplon ([S] = 100 microM) in a bank of human liver microsomes was strongly correlated with testosterone 6beta-hydroxylase (CYP3A4/5) activity (r(2) = 0.98), but not with any other CYP enzyme; (2) recombinant CYP1A1, CYP1A2, CYP3A4, CYP3A5 and CYP3A7 had the ability to catalyze this reaction; (3) the N-demethylation of indiplon was inhibited by CYP3A4/5 inhibitors (ketoconazole and troleandomycin), but not by a CYP1A2 inhibitor (furafylline). In pooled human liver microsomes, indiplon exhibited a weak capacity to inhibit CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4/5 and carboxylesterase (p-nitrophenylacetate hydrolysis) activities (IC50 >/= 20 microM). Clinical data available on indiplon support the conclusions of this paper that the in vitro metabolism of indiplon is catalyzed by multiple enzymes, and indiplon is a weak inhibitor of human CYP enzymes.

A series of pyrrolidinones derived from phenylalanines were synthesized as potent antagonists of the human melanocortin-4 receptor. These compounds showed high potencies and selectivities, and several of them had good oral bioavailabilities. In addition, 12e demonstrated in vivo efficacy in a murine cachexia model.

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited considerable binding affinity (Ki = 1 nM) and functional activity at MCH-R1, acceptable CYP2D6 inhibition, and good rat brain exposure.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are key regulators of xenobiotic-inducible cytochrome P450 gene expression. Whereas much is known about their role in regulating drug metabolism, little is known regarding their role in regulating drug transport in vivo. Wild-type mice and mice lacking PXR (PXR-KO) were used to examine the inducible expression of two drug transporter genes, Oatp2 (Slc21a5) and Mrp3 (Abcc3), in liver following treatment with selective PXR and CAR activators. Selective activation of PXR or CAR induced Oatp2 and Mrp3 expression in wild-type mice but not in PXR-KO mice. Basal expression levels of Oatp2 and Mrp3 gene were significantly higher in PXR-KO mice when compared with wild-type mice. Additionally, phenobarbital (PB)-inducible Oatp2 and Mrp3 gene expression was significantly increased in the PXR-KO mice when compared with wild-type PB-treated mice. We also examined the effect of PXR ablation on PB-inducible hepatic CYP3A activity in vivo. Microsomes isolated from PB-treated PXR-KO mice exhibited a significantly elevated rate of testosterone 6 beta-hydroxylation when compared with microsomes isolated from wild-type PB-treated mice. PB treatment produced significantly increased levels of hepatomegaly in PXR-KO mice when compared with wild-type PB-treated mice. Taken together, these results suggest that nonliganded PXR plays a net negative role in coregulating shared PXR/CAR-target gene expression in vivo and extend the hypothesis that PXR and CAR coregulate not only drug metabolism but also drug transport.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.

Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 microM) by human liver microsomes. Dioxane and tetra-hydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (Vmax = 179 to 2470 pmol/ mg protein/min), the Km for coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 microM). The following chemicals caused little or no inhibition of CYP2A6 as defined by a Ki > 200 microM: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 microM, failed to inhibit CYP2A6 when the concentration of coumarin was equal to Km (0.50 microM). The following chemicals were classified as strong inhibitors of CYP2A6 (defined by Ki < 200 microM): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k(inactivation) 0.5 min-1). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (Ki = 0.04 microM). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors.

S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) is a potent inhibitor of rat liver mitochondrial low Km aldehyde dehydrogenase (ALDH2) both in vivo and in vitro, and has been proposed to be the metabolite responsible for ALDH2 inhibition by disulfiram. Diethyldithiocarbamate methyl ester (DDTC-Me), a key intermediate in the metabolism of disulfiram, has been shown to be bioactivated by microsomal monooxygenases to diethyldithiocarbamate methyl ester sulfoxide (DDTC-Me sulfoxide). Studies were conducted to determine if DDTC-Me sulfoxide was also an active metabolite of disulfiram and inhibitor of ALDH2. DDTC-Me sulfoxide inhibited ALDH2 in vitro with an IC50 of 10 microM, and in vivo with an ID50 of 31 mg/kg (170 mumol/kg). Maximal ALDH2 inhibition in vivo was observed 8 hr after the administration of 45.2 mg/kg DDTC-Me sulfoxide, with ALDH2 activity returning to control levels after 48 hr. Although DDTC-Me sulfoxide inhibited ALDH2 in vivo, DDTC-Me sulfoxide was not detected in plasma from rats treated with either disulfiram (75 mg/kg), DDTC-Me (122.25 mg/kg), or DDTC-Me sulfoxide (45.2 mg/kg). However, DDTC-Me and S-methyl N,N-diethylthiolcarbamate (DETC-Me) were detected in plasma from rats treated with DDTC-Me sulfoxide. In rats treated with DDTC-Me sulfoxide and challenged with ethanol, a small increase of approximately microM in blood acetaldhyde and an inconsistent drop in blood pressure was observed. In conclusion, DDTC-Me sulfoxide inhibited ALDH2 in vitro and in vivo, was less potent than DETC- MeSO, and was not detected after disulfiram administration.

The aim of this study was to test suitable culture conditions for maintaining normal cellular cytoarchitecture and inducibility of P450 enzymes in primary cultures of human hepatocytes by prototypical inducers. The selectivity and sensitivity of a sandwich culture system were determined by treating cultures with a number of clinically relevant drugs that are known to be inducers of either rodent and/or human P450 enzymes. The results showed that considerable induction of CYP3A4 activity is observed at DMSO concentrations greater than 0.1% (v/v). No differences in P450 induction response were observed between cultures maintained under different matrix conditions. However, the matrix condition considered to be optimal for maintaining cellular integrity, protein yields, and P450 enzyme induction was a sandwich configuration in combination with modified Chee's medium containing insulin (6.25 microg/mL) and dexamethasone (< or =0.1 microM). Under these conditions, induction of CYP3A4 occurred at clinically relevant drug concentrations, and maximal activities were achieved after 3 days of exposure. Overall, the response of human hepatocyte cultures to treatment with both positive and negative modulators was found to reflect that observed in vivo with respect to both enzyme specificity and potency of enzyme induction, although considerable sample-to-sample variability was observed in the magnitude of induction.