A phase I, randomized, double-blind study to assess the safety, tolerability and efficacy of the topical RORC2 inverse agonist PF-06763809 in participants with mild-to-moderate plaque psoriasis.

BACKGROUND: Transcription factor retinoic acid-related orphan receptor 2 (RORC2/RORγT) mediates interleukin (IL)-17A and IL-17F expression. IL-17A plays a central role in the pathogenesis of several inflammatory disorders, including psoriasis. The RORC2 inhibitor PF-06763809 has been hypothesized to inhibit IL-17A production in T-helper 17 (Th17) cells, thereby reducing psoriasis symptoms. AIM: To assess the safety, tolerability and effect on skin infiltrate thickness of PF-06763809 in participants with mild/moderate chronic plaque psoriasis. METHODS: This was a randomized, double-blind, first-in-human study (trial registration: ClinicalTrials.gov NCT03469336). Participants received each of the following six treatments once daily for 18 days: three topical doses (2.3%, 0.8%, 0.23%) of PF-06763809, a vehicle and two active comparators (betamethasone and calcipotriol). Primary endpoints included change from baseline in psoriatic skin infiltrate thickness [echo-poor band (EPB) on ultrasonography] at Day 19, and safety. Change in psoriasis-associated gene expression (Day 19), evaluated by real-time reverse transcription PCR of skin biopsies, was an exploratory endpoint. RESULTS: In total, 17 participants completed the study. Change from baseline in the EPB on Day 19 for all three doses of PF-06763809 was not significantly different from that of vehicle (P > 0.05). A significant reduction in EPB from baseline was observed with betamethasone on Day 19 relative to all other treatments (P < 0.0001). Treatment-related adverse events were mild/moderate. There were no significant differences in gene expression on Day 19 between vehicle and PF-06763809-treated skin lesions. CONCLUSION: Using a psoriasis plaque test design, PF-06763809 was found to be well tolerated with an acceptable safety profile in participants with psoriasis, but without reduction in skin infiltrate thickness or disease biomarkers.

The CONSTANCES Cohort Biobank: An Open Tool for Research in Epidemiology and Prevention of Diseases.

"General-purpose cohorts" in epidemiology and public health are designed to cover a broad scope of determinants and outcomes, in order to answer several research questions, including those not defined at study inception. In this context, the general objective of the CONSTANCES project is to set up a large population-based cohort that will contribute to the development of epidemiological research by hosting ancillary projects on a wide range of scientific domains, and to provide public health information. CONSTANCES was designed as a randomly selected sample of French adults aged 18-69 years at study inception; 202,045 subjects were included over an 8-year period. At inclusion, the selected participants are invited to attend one of the 24 participating Health Prevention Centers (HPCs) for a comprehensive health examination. The follow-up includes a yearly self-administered questionnaire, and a periodic visit to an HPC. Procedures have been developed to use the national healthcare databases to allow identification and validation of diseases over the follow-up. The biological collection (serum, lithium heparinized plasma, EDTA plasma, urine and buffy coat) began gradually in June 2018. At the end of the inclusions, specimens from 83,000 donors will have been collected. Specimens are collected according to a standardized protocol, identical in all recruitment centers. All operations relating to bio-banking have been entrusted by Inserm to the Integrated Biobank of Luxembourg (IBBL). A quality management system has been put in place. Particular attention has been paid to the traceability of all operations. The nature of the biological samples stored has been deliberately limited due to the economic and organizational constraints of the inclusion centers. Some research works may require specific collection conditions, and can be developed on request for a limited number of subjects and in specially trained centers. The biological specimens that are collected will allow for a large spectrum of biomarkers studies and genetic and epigenetic markers through candidate or agnostic approaches. By linking the extensive data on personal, lifestyle, environmental, occupational and social factors with the biomarker data, the CONSTANCES cohort offers the opportunity to study the interplays between these factors using an integrative approach and state-of-the-art methods.

Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches-microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses-to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Diagnostic value of an ELISA using a recombinant 54-kDa species-specific protein from Chlamydia pneumoniae.

The aim of this study was to evaluate a 54-kDa recombinant protein encoded by the CPn0980 gene for use in a Chlamydia pneumoniae-specific ELISA. The ability of this affinity-purified protein to detect C. pneumoniae-specific antibodies was evaluated with a panel of 105 serum samples from 62 patients with community-acquired pneumonia. The results of this assay were compared with those obtained with a direct PCR-based detection assay and an outer-membrane complex-based immunoassay. The 54-kDa protein induced specific antibodies following infection of humans, and the recombinant 54-kDa ELISA detected anti-C. pneumoniae IgG and/or IgM antibodies with a sensitivity of 66.7% and a specificity of 79.2% compared with the direct PCR-based detection assay.

Pharmacokinetics and metabolism of a selective PDE5 inhibitor (UK-343,664) in rat and dog.

1. UK-343,664 is a novel potent and selective PDE5 inhibitor. Plasma clearances in the male and female rat were high (120 and 54 ml min(-1) kg(-1)), giving rise to short elimination half-lives (0.2 and 0.3h respectively). Lower clearance in dog (14 ml min(-1) kg(-1)) was the primary factor resulting in a longer elimination half-life (3.7 h). The higher clearance in rat than dog was in agreement with in vitro metabolism rates in hepatic microsomes. 2. The volume of distribution was lower in rat (1.3-2.11 kg(-1)) compared with dog (4.61 kg(-1)) probably due to increased plasma protein binding in rat (96 versus 81% in dog). 3. Oral bioavailabilities were 2, 12 and 70% in the male and female rat and dog respectively. Tmax < or = 0.5 h in all animals. 4. In multiple oral dose studies, increased systemic exposure was seen with increasing dose up to doses of 200 mg kg(-1) in rat and 150 mg kg(-1) in dog. A marked super-proportional increase in the male rat indicated a capacity-limited clearance at high doses. 5. At the maximal dose of 200 mg kg(-1) in the female rat, no clinical signs were observed after 14 days of treatment. Only minimal signs were recorded in the male rat and dog at the highest dose levels investigated. 6. After single oral or intravenous doses of [14C]-UK-343,664, the majority of radioactivity was excreted in the faeces of both species. 7. UK-343,664 was extensively metabolized in both rat and dog. The major primary pathways in dog involved piperazine N-deethylation and loss of a two carbon fragment from the piperazine ring (N,N'-de-ethylation). More extensive metabolism in the rat included additional notable metalbolites arising from hydroxylation and lactamization of the piperazine ring, which were only minor metabolites in the dog.

Potential role for P-glycoprotein in the non-proportional pharmacokinetics of UK-343,664 in man.

1. UK-343,664 is a potent and specific PDE5 inhibitor. Following single oral doses to human volunteers, it exhibited non-proportional pharmacokinetics over the dose range 30-800 mg. Over this 27-fold dose range, Cmax and AUCt increased 247- and 287-fold respectively. The half-life (4-6 h) was similar at all doses. No systemic exposure was quantifiable at doses <10 mg. 2. UK-343,664 is a lipophilic molecule (log D7.4 = 3.1) and as such is expected to be cleared mainly by metabolism. Based on studies with expressed human P450 enzymes it was concluded that the metabolism of UK-343,664 was predominantly mediated by CYP3A4. With a moderate Km = 76 microM for this enzyme, saturation of first-pass metabolism alone was considered unlikely to account for the non-proportional pharmacokinetics. 3. UK-343,664 showed high affinity for P-glycoprotein in vitro, with a Km = 7.3 microM. In transport studies in LLC-PK1 cell monolayers transfected with P-glycoprotein, UK343,664 showed marked polarized transport which was concentration dependent. 4. The high affinity of UK-343,664 for P-glycoprotein is considered to be the primary source of the non-proportional pharmacokinetic profile observed in man.

Use of cloned and expressed human UDP-glucuronosyltransferases for the assessment of human drug conjugation and identification of potential drug interactions.

Glucuronidation is an important pathway for human drug metabolism. Four cloned and expressed human UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B15) were used to screen a series of three potential drug substrates differing only in position of the phenol moiety. The meta and para phenols, UK-156,037 and UK-157,147, were found to be substrates for UGT1A1 with K(m) values of 256 and 105 microM, respectively. The ortho phenol UK-157,261 was glucuronidated predominantly by UGT1A9 with a K(m) of 45 microM. The latter K(m) compares favorably with the known UGT1A9 substrate propofol (K(m) = 200 microM). In a series of competition experiments, UK-157,261 was shown to inhibit the glucuronidation of propofol by UGT1A9 with a K(i) value of 65 microM. This result indicates that even the most potent of these compounds is extremely unlikely to interact in the clinic with the glucuronidation of propofol. This study shows the utility of the expressed human UDP-glucuronosyltransferases in determining substrate structure-activity relationships and potential drug-drug interactions.

The role of P-glycoprotein in determining the oral absorption and clearance of the NK2 antagonist, UK-224,671.

UK-224,671 has been shown to exhibit low oral bioavailability in vivo due to poor absorption from the GI tract. The purpose of this study was to investigate the underlying reason for this observation. In Caco-2 cell flux experiments, the absorptive (A to B) flux of UK-224,671 was low, consistent with poor in vivo absorption. However, flux in the B to A direction was significantly greater, suggesting that UK-224,671 can permeate the membrane of the gut wall cell. Such a Caco-2 cell flux is indicative of transporter mediated efflux, possibly by P-glycoprotein. In P-glycoprotein knockout mice, the oral bioavailability of UK-224,671 was 22%, representing a significant increase over the P-glycoprotein expressing wild type mice (<2%). However, in the knockout mice absorption was still incomplete, suggesting that both P-glycoprotein mediated efflux and poor membrane permeation combine to limit the oral absorption of UK-224,671 in wild type mice. Lack of P-glycoprotein expression had no effect on the clearance of UK-224,671 in mice, which suggests that uptake from the blood into the excretory cell is mediated by a transporter other than P-glycoprotein. Bile duct cannulated rat experiments show that approximately 20% of the clearance of UK-224,671 occurs by direct secretion across the gut wall into the faeces. This clearance pathway requires UK-224,671 to cross both the basolateral and apical membranes of the gut wall cell. P-glycoprotein is likely to be involved in the passage of the compound across the apical membrane as has been observed for other P-glycoprotein substrates.

Pharmacokinetics and metabolism of a sulphamide NK2 antagonist in rat, dog and human.

1. UK-224,671 is a sulphamide-containing NK2 antagonist with moderate lipophilicity and basicity. 2. The physicochemical properties of UK-224,671 are reflected in its pharmacokinetics following intravenous (i.v.) administration. The compound partitioned extensively into red blood cells in all species examined and the blood clearance was moderate to low with respect to liver blood flow and distribution into tissues was extensive. 3. UK-224,671 exhibited species differences in oral bioavailability. In dog, the compound exhibited moderate bioavailability (55%), whereas in rat and man oral bioavailability was < 10%. 4. In rat and dog, the major excreted form after i.v. administration was unchanged UK-224,671 in both urine and faeces. In addition, of three metabolites observed, the most abundant was the N-descyclopropylmethyl (UK-280,045). 5. The profile of radioactivity in rat following oral administration of [14C]-UK-224,671 was not consistent with a 10% absorbed compound with 40% of the dose present as metabolites. This suggests that the low bioavailability of UK-224,671 in rat is due to a combination of moderate intestinal permeability and extensive first-pass metabolism by the gut and does not result from poor gastrointestinal absorption per se.

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures.

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.

Pharmacokinetics and metabolism of darifenacin in the mouse, rat, dog and man.

1. Following intravenous administration to animals at 2.5 mg/kg, darifenacin exhibited terminal plasma half-lifes < 2 h due to high plasma clearance (with respect to blood flow) and volumes of distribution greater than total body water. 2. Following oral administration to animals at doses > 4 mg/kg there was evidence of saturation of clearance since oral AUCs exceeded those expected from the high plasma clearances. In addition, terminal plasma half-lifes were greater than those estimated from intravenous administration. 3. In man, oral clearance was high with respect to liver blood flow. 4. Following oral administration of the radiolabelled drug to animals and man, unchanged darifenacin was only a minor component of the faecal radioactivity indicating that darifenacin was well absorbed from the gut. 5. Darifenacin was metabolized by three main routes in all species: monohydroxylation, oxidative dihydrobenzfuran ring opening and N-dealkylation. There were no marked species differences in the metabolism of darifenacin.

A universal radiochemical high-performance liquid chromatographic assay for the determination of UDP-glucuronosyltransferase activity.

A new unified assay for the determination of UDP-glucuronosyltransferase (UGT) activities has been developed. The resolution of [14C]uridine diphosphate glucuronic acid from radiolabeled glucuronides formed by incorporation of this radiolabel can now be achieved by a sensitive and rapid-gradient HPLC method which utilizes a radioactivity endpoint as a universal detection method. One important application of this method is the determination of kinetic parameters for cloned and expressed UGT isoforms with greater speed and precision than can be afforded by TLC methodology. Moreover, assays with 14C-labeled substrates indicate that gradient HPLC can easily resolve the substrate from the glucuronide products and present an alternative to the time-consuming optimization of conditions for organic phase extraction assays.

Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors.

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.

Regulation of the third member of the uncoupling protein family, UCP3, by cold and thyroid hormone.

Uncoupling protein (UCP1) is a transmembrane proton transporter present in the mitochondria of brown adipose tissue (BAT), a specialized tissue which functions in temperature homeostasis and energy balance (Nicholls, D. G., and Locke, R. M. (1984) Physiol. Rev. 64, 2-40; Lowell, D. D., and Flier, J. S. (1997) Annu. Rev. Med.). UCP1 mediates the thermogenesis that is characteristic of BAT by uncoupling mitochondrial oxidation of substrates from ATP synthesis. Recently, two proteins related to UCP1 have been identified and designated UCP2 (Fleury, C., et al. (1997) Nature Genetics 15, 269-272) or UCP homolog (UCPH) (Gimeno, R. E., et al. (1997) Diabetes 46, 900-906) and UCP3 (Boss, O., et al. (1997) FEBS Lett. 408, 39-42; Vidal-Puig, A., et al. (1997) Biochem. Biophys. Res. Commun. 235, 79-82). We investigated the regulation in rats of UCP3, which is expressed primarily in skeletal muscle and BAT. Expression of rat UCP3 mRNA in BAT was upregulated by in vivo treatment with triiodothyronine (T3) and by exposure to cold, suggesting that UCP3 is active in thermogenesis and energy expenditure. In skeletal muscle, UCP3 mRNA was also upregulated by T3 but, surprisingly, not by cold exposure. A hypothesis is proposed to account for this differential regulation.

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats.

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRP alpha (Ki values 0.12 +/- 0.06, 0.017 +/- 0.007, 3.83 +/- 1.14 and 0.007 +/- 0.001 nM, respectively). In contrast Ki values for displacement of [125I]rat amylin from accumbens membranes (amylin receptors), and [125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRP alpha in these preparations (51 +/- 5, 34 +/- 2, 0.024 +/- 0.002, 0.31 +/- 0.07 nM, respectively, at amylin receptors; 33 +/- 5, 69 +/- 29, 2.7 +/- 1.5 and 13 +/- 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRP alpha. In contrast, for amylin or calcitonin agonist actions (inhibition of [14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Pharmacokinetics of a series of bis(methanesulphonamido-arylalkyl)amines in the beagle dog.

1. The pharmacokinetics of three closely related analogues of dofetilide have been investigated in the beagle dog. These have been compared with those of dofetilide and related to physicochemical properties and structural features of the molecules. 2. Following intravenous administration, the four compounds exhibit elimination half-lives ranging from 4.6 to 19 h. This range is due to changes in both volume of distribution and plasma clearance across the series. 3. In vitro plasma protein shows a relationship to lipophilicity within this series. Protein binding increasing from 54% for dofetilide, the least lipophilic compound (log D7.4 = 0.73) to 92% for the most lipophilic analogue (log D7.4 = 2.07). There is a trend for a decrease in the volume of distribution with increased plasma protein binding. 4. Plasma clearance values range from 2.4 to 10.2 ml/min/kg and are comprised of renal and non-renal components. Renal clearance ranges fro 0.11 to 2.9 ml/min/kg and shows an inverse correlation with and lipophilicity of the compounds. Values for the renal clearance of unbound drug suggest that only the most lipophilic derivative (III), has sufficient membrane affinity to undergo tubular reabsorption. 5. Non-renal clearance of either total or free drug shows no relationship with lipophilicity. Highest values are observed for the two compounds with a methyl substituent on the tertiary amine and lowest values for the two compounds in which the tertiary amine is incorporated into a 7-membered ring. In vitro metabolism in dog liver microsomes also shows increased lability for the two N-methyl compounds. The N-desmethyl metabolite is the major product in both cases.

Pharmacokinetics and metabolism of zamifenacin in mouse, rat, dog and man.

1. Zamifenacin was rapidly metabolized in vitro by liver microsomes from rat, dog, and man. 2. Zamifenacin exhibited extensive plasma protein binding with human plasma showing 20 and 10-fold higher binding that that in rat and dog respectively. 3. Following oral administration to animals, metabolic clearance resulted in decreased bioavailability due to first-pass metabolism in rat and mouse. Oral clearance in man was low as a result of increased metabolic stability and increased plasma protein binding compared with animals. 4. Metabolism was the major route of clearance of zamifenacin with the primary metabolic step resulting in opening of the methylenedioxy ring to yield the catechol. In man, this metabolite was excreted as the glucuronide conjugate, whereas in the animal species it was further metabolized by mono-methylation of the catechol.

Different pharmacological characteristics in L6 and C2C12 muscle cells and intact rat skeletal muscle for amylin, CGRP and calcitonin.

1. We compared the ability of rat amylin, rat calcitonin gene-related peptide (CGRP) and rat and salmon calcitonins to elevate cyclic AMP levels and to inhibit [U-14C]-glucose incorporation into glycogen in insulin-stimulated intact rat soleus muscle and in two cell lines derived from rodent skeletal muscle, L6 and C2C12. 2. In intact soleus muscle, both amylin (EC50S of 0.7-6.1 nM) and salmon calcitonin (EC50S of 0.5-1.4 nM) were more potent than CGRP (EC50S of 5.6-15.8 nM) and were much more potent than rat calcitonin (EC50S of 50-137 nM) at stimulating cyclic AMP production, activating glycogen phosphorylase and inhibiting insulin-stimulated [14C]-glycogen formation. 3. In contrast, in both L6 and C2C12 cells, CGRP (EC50S of 0.042-0.12 nM) stimulated cyclic AMP formation and inhibited insulin-stimulated [U-14C]-glucose incorporation into glycogen approximately 1000 times more potently than amylin (EC50S 34-240 nM), while salmon calcitonin was without measurable effect. 4. There was a correlation between elevation of cyclic AMP and inhibition of insulin-stimulated [U-14C]-glucose incorporation into glycogen evoked by these peptides in both intact muscle (r2 = 0.69, P < 0.0004) and muscle cell lines (r2 = 0.96, P < 0.0001). 5. In conclusion, the effects of amylin, CGRP, and calcitonin on soleus muscle glycogen metabolism appear to be mediated by adenylyl cyclase-coupled receptors which show a pharmacological profile similar to high affinity amylin binding sites that have been previously reported in rat brain. In contrast, the effects of amylin and CGRP in L6 and C2C12 rodent muscle cell lines appear to be mediated by adenylyl cyclase-coupled receptors that behave like CGRP receptors.