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Jasper Dingemanse, Martine Gehin, Hans Gabriel Cruz, Petra HoeverDepartment of Clinical Pharmacology, Actelion Pharmaceuticals Ltd, Allschwil, SwitzerlandAbstract: Almorexant, a dual orexin receptor antagonist, was investigated for the treatment of insomnia. The following observations initiated further formulation development: the active pharmaceutical ingredient (API) was sticking to the apparatus used during tablet compression; almorexant has an absolute bioavailability of 11.2%; and almorexant modestly decreased the latency to persistent sleep by 10.4 minutes in patients. Two randomized crossover studies were performed to investigate the pharmacokinetics of several new formulations in healthy subjects. In study I, the old “sticky” tablet was compared to two new formulations developed to prevent sticking: a qualitatively similar tablet but with a larger API crystal size and a tablet with 30% more excipients as well as a larger API crystal size. This latter formulation was available in two strengths. The geometric mean ratios and 90% confidence interval of the area under the curve (AUC) were within the bioequivalence range of 0.80–1.25 for the different comparisons between formulations. In study II, 100 mg of the reference tablet was compared to 25 and 50 mg of a liquid-filled hard gelatin capsule developed to increase the bioavailability of almorexant. The geometric mean ratios of the maximum concentration and AUC comparing the new 25 and 50 mg capsule formulations to the reference tablet did not exceed 0.25 and 0.50, respectively, indicating that the new capsule formulation did not increase the maximum concentration of or the total exposure to almorexant. In conclusion, a new tablet was developed but formulation development aimed at increasing the bioavailability of almorexant failed.Keywords: almorexant, orexin receptor antagonist, pharmacokinetics, formulation development, healthy subjects

Researchers and clinicians have long used meaningful intransitive (i.e., not tool-related; MFI) gestures to assess apraxia-a complex and frequent motor-cognitive disorder. Nevertheless, the neurocognitive bases of these gestures remain incompletely understood. Models of apraxia have assumed that meaningful intransitive gestures depend on either long-term memory (i.e., semantic memory and action lexicons) stored in the left hemisphere, or social cognition and the right hemisphere. This meta-analysis of 42 studies reports the performance of 2659 patients with either left or right hemisphere damage in tests of meaningful intransitive gestures, as compared to other gestures (i.e., MFT or meaningful transitive and MLI or meaningless intransitive) and cognitive tests. The key findings are as follows: (1) deficits of meaningful intransitive gestures are more frequent and severe after left than right hemisphere lesions, but they have been reported in both groups; (2) we found a transitivity effect in patients with lesions of the left hemisphere (i.e., meaningful transitive gestures more difficult than meaningful intransitive gestures) but a "reverse" transitivity effect in patients with lesions of the right hemisphere (i.e., meaningful transitive gestures easier than meaningful intransitive gestures); (3) there is a strong association between meaningful intransitive and transitive (but not meaningless) gestures; (4) isolated deficits of meaningful intransitive gestures are more frequent in cases with right than left hemisphere lesions; (5) these deficits may occur in the absence of language and semantic memory impairments; (6) meaningful intransitive gesture performance seems to vary according to the emotional content of gestures (i.e., body-centered gestures and emotional valence-intensity). These findings are partially consistent with the social cognition hypothesis. Methodological recommendations are given for future studies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Objectives: Daridorexant, a dual orexin receptor antagonist was recently approved for the treatment of insomnia at doses up to 50 mg once per night. This study investigated the effect of single-dose and multiple-dose daridorexant 50 mg at steady state on the pharmacokinetics (PK) of the cytochrome P450 (CYP) 3A4-sensitive substrate midazolam, and the effect of single-dose daridorexant 50 mg on the PK and pharmacodynamics (PD) of the CYP2C9-sensitive substrate warfarin., Methods: In this prospective, single-center, open-label, fixed-sequence, phase I, drug-drug interaction study, 18 healthy male subjects sequentially received Treatment A, B, and C in three periods. Treatment A consisted of a single oral concomitant administration of midazolam 2 mg and warfarin 25 mg on day 1 of the first period. Treatment B consisted of one oral administration of daridorexant 50 mg followed 1 h later by a single oral dose of midazolam 2 mg concomitantly with a single oral dose of warfarin 25 mg on day 1 and a once-daily oral administration of daridorexant 50 mg for 6 days of the second period. Treatment C consisted of a single oral administration of daridorexant 50 mg at steady state followed 1 h later by a single oral administration of midazolam 2 mg on day 1 of the third period. Blood samples were assessed for midazolam and S-warfarin PK, and PD (international normalized ratio and factor VII). Noncompartmental  PK parameters and PD variables were evaluated with geometric mean ratios and 90% confidence intervals of Treatment B/A versus C/A for midazolam, and treatment B/A for warfarin. Safety and tolerability of each treatment were also assessed., Results: Midazolam maximum plasma concentration (C max ) and area under the plasma concentration-time curve from 0 to 24 h (AUC 0-24 ) were 1.13- and 1.42-fold higher, respectively, after single-dose administration of daridorexant 50 mg compared to administration of midazolam alone, while C max and AUC 0-24 were 1.12- and 1.35-fold higher, respectively, after administration of daridorexant 50 mg once daily at steady state. Terminal half-life and time to maximum plasma concentration were comparable between treatments. Daridorexant had no influence on the PK and PD of warfarin. All treatments were safe and well tolerated., Conclusions: Daridorexant at 50 mg is classified as a weak CYP3A4 inhibitor after single- and multiple-dose administration once daily at steady state. Daridorexant 50 mg did not induce CYP3A4 activity or inhibit CYP2C9 activity., Clinical Trial Registration: This trial (NCT05480488) was registered on 29 July, 2022., (© 2024. The Author(s).)

Background: The P2Y 12 receptor antagonist selatogrel is being developed for subcutaneous self-administration with a ready-to-use autoinjector at the onset of acute myocardial infarction (AMI) symptoms. The unique pharmacological profile of selatogrel (fast, potent, and short-acting) can bridge the time gap between the onset of AMI and first medical care. A clinical Phase 1 study showed a time-dependent pharmacodynamic interaction between selatogrel and loading doses of clopidogrel and prasugrel. As treatment switching is a common clinical practice, the assessment of subsequent switching from a clopidogrel loading dose to the first maintenance dose of oral P2Y 12 receptor antagonists is highly relevant. Objectives: Model-based predictions of inhibition of platelet aggregation (IPA) for the drugs triggering pharmacodynamic interactions were to be derived to support clinical guidance on the transition from selatogrel to oral P2Y 12 receptor antagonists. Methods: Scenarios with selatogrel 16 mg administration or placebo followed by a clopidogrel loading dose and, in turn, prasugrel or ticagrelor maintenance doses at different times of administration were studied. Population pharmacokinetic/pharmacodynamic modeling and simulations of different treatment scenarios were used to derive quantitative estimates for IPA over time. Results: Following selatogrel/placebo and a clopidogrel loading dose, maintenance treatment with ticagrelor or a prasugrel loading dose followed by maintenance treatment quickly achieved sustained IPA levels above 80%. Prior to maintenance treatment, a short time span from 18 to 24 h was identified where IPA levels were predicted to be lower with selatogrel than with placebo if clopidogrel was administered 12 h after selatogrel or placebo. Predicted IPA levels reached with placebo alone and a clopidogrel loading dose at 4 h were consistently lower than with selatogrel administration, followed by a clopidogrel loading dose at 12 h. If a clopidogrel loading dose is administered at 12 h, selatogrel maintains higher IPA levels up to 16 h. IPA levels are subsequently lower than on the placebo until the administration of the first maintenance dose. Conclusions: Model-based predictions informed the transition from selatogrel subcutaneous administration to oral P2Y 12 therapy. The application of modeling techniques illustrates the value of employing pharmacokinetic and pharmacodynamic modeling for the simulation of various clinical scenarios of switching therapies.

Introduction: P2Y12 receptor antagonists (P2Y12 inhibitors) are well established for the treatment of coronary artery disease. The P2Y12 inhibitors currently commercially available present either pharmacokinetic limitations (due to delayed absorption, bioactivation requirement via CYP enzymes, or need of intravenous administration), pharmacodynamic (PD) limitations (limited % inhibition of platelet aggregation (IPA) or relevant PD interactions) or safety limitations (major bleeding in specific populations)., Areas Covered: Selatogrel, a 2-phenylpyrimidine-4-carboxamide analog, is a potent, reversible, and selective P2Y12 inhibitor administered subcutaneously that is under development for the treatment of acute myocardial infarction (AMI) in patients with a recent history of AMI. In this review, the authors summarize the results from preclinical, phase 1, and phase 2 trials which showed that selatogrel provides rapid, pronounced, and reversible P2Y12 receptor inhibition with a favorable safety profile., Expert Opinion: These unique characteristics added to the limited potential to interact with co-medications and manageable PD interactions with other P2Y12 inhibitors provide a clear rationale for investigating the benefit of selatogrel as an emergency treatment to improve clinical outcomes in patients with AMI.

Aims: Daridorexant is a dual orexin receptor antagonist developed for the treatment of insomnia. The solubility of daridorexant is pH-dependent and daridorexant has been shown to be a sensitive CYP3A4 substrate when co-administered with moderate CYP3A4 inhibitors. The purpose of this study was to assess the effect of an increased gastric pH on daridorexant pharmacokinetics (PK) and the extent of interaction when daridorexant is co-administered with a moderate CYP3A4 inducer., Methods: In this prospective, single-centre, randomized, open-label study, 24 male subjects consecutively received four treatments, i.e., daridorexant 50 mg single dose; famotidine 40 mg single dose + daridorexant 50 mg single dose; efavirenz 600 mg once a day (o.d.) for 10 days; and daridorexant 50 mg single dose + efavirenz 600 mg o.d. for 2 days. Plasma PK parameters of daridorexant were derived by noncompartmental analysis. Standard safety and tolerability evaluations were analysed descriptively., Results: When daridorexant administration was preceded by administration of famotidine, daridorexant C max decreased by 39%, geometric means ratio (GMR) (90% confidence interval [90% CI]): 0.61 (0.50, 0.73). AUC 0-∞ remained unchanged. In the presence of steady-state efavirenz, daridorexant C max , AUC 0-∞ and t ½ decreased by approximately 35% (GMR [90% CI]): 0.65 (0.54, 0.78), 61% (0.39 (0.348, 0.44), and 35% (0.65 (0.58, 0.73), respectively. t max remained unaffected. All treatments containing daridorexant were well tolerated., Conclusion: Daridorexant 50 mg can be administered concomitantly with gastric pH modifiers or with moderate CYP3A4 inducers without dose adaptation based on efficacy observed at lower doses in Phase 3 studies., (© 2021 British Pharmacological Society.)

In this first-in-human study, the tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of sinbaglustat, a dual inhibitor of glucosylceramide synthase (GCS) and non-lysosomal glucosyl ceramidase (GBA2), were investigated in healthy subjects. The single-ascending dose (SAD) and multiple-ascending dose (MAD) studies were randomized, double-blind, and placebo-controlled. Single doses from 10 to 2,000 mg in men and multiple doses from 30 to 1,000 mg twice daily for 7 days in male and female subjects were investigated. Tolerability, PK, and PD data were collected up to 3 days after (last) treatment administration and analyzed descriptively. Sinbaglustat was well-tolerated in the SAD and MAD studies, however, at the highest dose of the MAD, three of the four female subjects presented a similar pattern of general symptoms. In all cohorts, sinbaglustat was rapidly absorbed. Thereafter, plasma concentrations decreased biphasically. In the MAD study, steady-state conditions were reached on Day 2 without accumulation. During sinbaglustat treatment, plasma concentrations of glucosylceramide (GlcCer), lactosylceramide, and globotriaosylceramide decreased in a dose-dependent manner, reflecting GCS inhibition. The more complex the glycosphingolipid, the more time was required to elicit PD changes. After treatment stop, GlcCer levels returned to baseline and increased above baseline at lowest doses, probably due to the higher potency of sinbaglustat on GBA2 compared to GCS. Overall, sinbaglustat was welltolerated up to the highest tested doses. The PK profile is compatible with b.i.d. dosing. Sinbaglustat demonstrated target engagement in the periphery for GCS and GBA2., (© 2020 Idorsia Pharmaceuticals Ltd. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Serotonin (5-HT) is synthesized from dietary tryptophan (Trp) and plays an important role in numerous diseases of the central nervous system and periphery. Stable isotope tracers enable safe monitoring of metabolic rates. Here we demonstrate measurement of peripheral 5-HT synthesis in healthy subjects by monitoring the produced [ 13 C 10 ]-5-HT (h-5-HT) in EDTA-whole blood from three doses of orally administered [ 13 C 11 ]-Trp (h-Trp) tracer. h-Trp was rapidly absorbed and distributed in a multiphasic manner, followed by a slower terminal elimination phase. The h-5-HT synthesis rate was dependent on h-Trp dose, appeared linear up to 12 hours postdose, and could be reliably assessed for the two highest doses. The human data was compared to similar studies in rats and dogs, finding larger interspecies differences in the h-5-HT synthesis rate than in 5-HT levels. In future studies, the h-5-HT synthesis rate can be used to assess disease-dysregulated 5-HT synthesis or quantify the pharmacodynamics of 5-HT synthesis inhibitors., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

Aim: The aim of the present study was to investigate whether increasing the bosentan dosing frequency from 2 mg kg -1 twice daily (b.i.d.) to 2 mg kg -1 three times daily (t.i.d.) in children with pulmonary arterial hypertension (PAH) (from ≥3 months to <12 years of age) would increase exposure., Methods: An open-label, prospective, randomized, multicentre, multiple-dose, phase III study was conducted. Patients (n = 64) were randomized 1:1 to receive oral doses of bosentan of 2 mg kg -1 b.i.d. or t.i.d. The main pharmacokinetic endpoint was the daily exposure to bosentan over 24 h corrected to the 2 mg kg -1 dose (AUC 0-24C ). The maximum plasma concentration corrected to the 2 mg kg -1 dose (C maxC ), the time to reach the maximum plasma concentration (t max ) and safety endpoints were also assessed., Results: The geometric mean [95% confidence interval (CI)] for AUC 0-24C was 8535 h.ng ml -1 (6936, 10 504) and 7275 h.ng ml -1 (5468, 9679) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.85 (0.61, 1.20)]. The geometric mean (95% CI) for C maxC was 743 ng ml -1 (573, 963) and 528 ng ml -1 (386, 722) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.71 (0.48, 1.05)]. The median (range) for t max was 3.0 h (0.0-7.5) and 3.0 h (1.0-8.0) for 2 mg kg -1 b.i.d. and t.i.d., respectively. The proportions of patients who experienced ≥1 adverse event were similar in the b.i.d. (66.7%) and t.i.d. (67.7%) groups., Conclusions: There appeared to be no clinically relevant difference in exposure to bosentan, or in safety, when increasing the frequency of bosentan dosing from b.i.d. to t.i.d. Therefore, the present study provides no indication that the dosing recommendation should be changed, and 2 mg kg -1 b.i.d. remains the recommended dosing regimen for bosentan in paediatric PAH patients., (© 2017 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Objective: To evaluate the efficacy, safety, and pharmacokinetics of the endothelin receptor antagonist bosentan as adjunctive therapy for neonates with persistent pulmonary hypertension of the newborn (PPHN)., Study Design: This was a phase 3, multicenter, randomized, placebo-controlled exploratory trial (FUTURE-4). Eligible patients were >34 weeks gestation, <7 days old, receiving inhaled nitric oxide (iNO) treatment (≥4 hours), and had persistent respiratory failure (oxygenation index [OI] ≥12). After 2:1 randomization, bosentan 2 mg/kg or placebo was given by nasogastric tube twice daily for ≥48 hours and up to 1 day after iNO weaning., Results: Twenty-one neonates received a study drug (13 bosentan, 8 placebo). Compared with the placebo group, the group treated with bosentan had a higher median baseline OI and greater need for vasoactive agents. One treatment failure (need for extracorporeal membrane oxygenation) occurred in the group treated with bosentan. The time to weaning from iNO or mechanical ventilation was not different between the groups. Bosentan was well tolerated and did not adversely affect systemic blood pressure or hepatic transaminase levels. Anemia and edema were more frequent in patients receiving bosentan. Blood concentrations of bosentan were low and variable on day 1, and achieved steady state on day 5., Conclusion: Adjunctive bosentan was well tolerated, but did not improve oxygenation or other outcomes in our patients with PPHN. This effect may be related to delayed absorption of bosentan on treatment initiation in critically ill neonates or to more severe illness of the neonates who received bosentan., Trial Registration: ClinicalTrials.gov:NCT01389856., (Copyright © 2016. Published by Elsevier Inc.)

Background and Objective: The chemoattractant receptor-homologous molecule expressed on T helper-2 cells (CRTH2) is a G-protein-coupled receptor for prostaglandin D2 (PGD2), a key mediator in inflammatory disorders. Two selective and potent CRTH2 antagonists currently in clinical development, ACT-453859 and setipiprant, were compared with respect to their (predicted) clinical efficacy., Methods: Population pharmacokinetic (PK) and pharmacodynamic (PD) models were developed to characterize how plasma concentrations (PK) of ACT-453859, its active metabolite ACT-463036 and setipiprant related to their effect on blocking PGD2-induced internalization of CRTH2 on eosinophils (PD). Simulations were used to identify doses and dosing regimens leading to 90 % of maximum blockade of CRTH2 internalization at trough., Results: A combined concentration of ACT-453859 and its metabolite ACT-463036, with weights proportional to potency (based on an eosinophil shape change assay), enabled good characterization of the PD effect. The modelling and simulation results facilitated decision making by suggesting an ACT-453859 dose of 400 mg once daily (or 100 mg twice daily) for clinically relevant CRTH2 antagonism., Conclusion: Pharmacometric quantification demonstrated that CRTH2 internalization is a useful new biomarker to study CRTH2 antagonism. Ninety percent of maximum blockade of CRTH2 internalization at trough is suggested as a quantitative PD target in clinical studies.

Cadazolid is under development as an oral treatment for Clostridium difficile infection (CDI), which is the most common infectious cause of antibiotic-associated diarrhoea. Low systemic cadazolid exposures were previously reported in healthy subjects following both single and multiple oral dosing. The main objective of this study was to investigate systemic cadazolid exposure in patients with severe CDI with potential disrupted lining of the gastrointestinal tract. A single 3000 mg oral dose of cadazolid was administered to six patients with microbiologically-confirmed severe CDI. Plasma and faeces were collected up to 144 h post-dose for determination of cadazolid concentrations. Safety assessments were conducted over the 144-h investigational period. Cadazolid was well tolerated in patients with severe CDI, with no reported drug-related adverse events. Cadazolid systemic exposure following a single 3000 mg oral dose was very low, with a peak plasma concentration (C(max)) of 2.64 ng/mL and an area under the concentration-time curve (AUC(0-144)) of 125 ng×h/mL. The median peak daily faecal cadazolid concentration was 5675 times the C. difficile MIC(90) of 0.25 mg/L. In subjects with severe CDI, cadazolid systemic exposure was very low following a single high oral dose. Cadazolid plasma concentrations were similar in magnitude to those previously reported for healthy subjects, whereas total systemic exposure was ca. 5-6 times higher, but was still low. Peak daily faecal cadazolid concentrations were 5675 times the 0.25 mg/L C. difficile MIC(90), and on Day 4 five of the six patients presented a daily faecal cadazolid concentration ≥1651 times the MIC(90) [ClinicalTrial.gov ID: NCT02053181]., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Purpose: Setipiprant, a selective oral CRTH2 antagonist, has been investigated for the treatment of allergic rhinitis and asthma. In vitro data showed that setipiprant has a weak induction potential on CYP3A4. An interaction at the hepatic level between setipiprant and CYP3A4 substrates was not expected even at the dosing regimen of 1,000 mg setipiprant b.i.d. due to the high plasma protein binding. However, at this dosing regimen, interactions at the gut level could not be excluded., Methods: In this single-center, open-label study, 40 mg of simvastatin was administered orally on Day 1, and then concomitantly with setipiprant on Day 10 following 9 days of setipiprant 1,000 mg b.i.d. to 22 healthy male subjects., Results: In the presence of setipiprant, the simvastatin concentration-time profile was similar to that of simvastatin alone. The concentrations of simvastatin were, however, slightly lower, resulting in a 9 % decrease in C max (geometric mean ratio (GMR) 0.91, 90 % confidence interval (CI) (0.73, 1.13)) and in a 16 % lower AUC0-∞ (GMR 0.84, 90 % CI (0.72, 0.99)). Exposure to simvastatin acid was similar when comparing simvastatin with or without setipiprant. The GMR and 90 % CI for AUC0-∞ were within the 0.8 to 1.25 limits, whereas those for C max were outside (GMR 2.73, 90 % CI (2.11, 3.53)). Moreover, the median t max of simvastatin acid occurred earlier (1.8 h) when combined compared to 3.0 h when administered alone., Conclusions: As setipiprant has little impact on simvastatin pharmacokinetics, it does not modulate CYP3A4 in a clinically relevant manner.

Almorexant, a tetrahydroisoquinoline orexin receptor antagonist and first representative of a new class of compounds for the treatment of insomnia, is a substrate of the cytochrome P450 3A4 isoenzyme (CYP3A4). Two randomized two-way crossover studies were performed in healthy subjects investigating the pharmacokinetic interaction between almorexant and the CYP3A4 inhibitors ketoconazole and diltiazem. When administered as a single dose of 100 mg almorexant during steady state of ketoconazole (400 mg once daily for 14 days) or diltiazem treatment (300 mg once daily for 11 days), the exposure to almorexant was 10.5- and 3.5-fold, respectively, greater when compared with almorexant alone. Exposure to the phenol metabolites M3 and M8 increased in the presence of the CYP3A4 inhibitors, whereas that to M6 (dealkylated metabolite) decreased. Concomitant ketoconazole decreased formation of the dehydrogenated metabolite M5 and diltiazem increased concentrations of this metabolite. Higher almorexant exposure was associated with an increased incidence of typical almorexant-related adverse events such as fatigue (both studies) and somnolence (ketoconazole study only). The present results indicate that dose adaptation must be considered when almorexant would be coadministered with inhibitors of CYP3A4., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The transcription-intermediary-factor-2 (TIF-2) is a coactivator of the glucocorticoid receptor (GR), and its disruption would be expected to influence glucocorticoid-mediated control of the hypothalamo-pituitary-adrenal (HPA) axis. Here, we show that its targeted deletion in mice is associated with altered expression of several glucocorticoid-dependent components of HPA regulation (e.g., corticotropin-releasing hormone, vasopressin, ACTH, glucocorticoid receptors), suggestive of hyperactivity under basal conditions. At the same time, TIF-2(-/-) mice display significantly lower basal corticosterone levels and a sluggish and blunted initial secretory response to brief emotional and prolonged physical stress. Subsequent analysis revealed this discrepancy to result from pronounced aberrations in the structure and function of the adrenal gland, including the cytoarchitectural organization of the zona fasciculata and basal and stress-induced expression of key elements of steroid hormone synthesis, such as the steroidogenic acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). In addition, altered expression levels of two nuclear receptors, DAX-1 and steroidogenic factor 1 (SF-1), in the adrenal cortex strengthen the view that TIF-2 deletion disrupts adrenocortical development and steroid biosynthesis. Thus, hyperactivity of the hypothalamo-pituitary unit is ascribed to insidious adrenal insufficiency and impaired glucocorticoid feedback.

Although the essential involvement of the progesterone receptor (PR) in female reproductive tissues is firmly established, the coregulators preferentially enlisted by PR to mediate its physiological effects have yet to be fully delineated. To further dissect the roles of members of the steroid receptor coactivator (SRC)/p160 family in PR-mediated reproductive processes in vivo, state-of-the-art cre-loxP engineering strategies were employed to generate a mouse model (PR(Cre/+) SRC-2(flox/flox)) in which SRC-2 function was abrogated only in cell lineages that express the PR. Fertility tests revealed that while ovarian activity was normal, PR(Cre/+) SRC-2(flox/flox) mouse uterine function was severely compromised. Absence of SRC-2 in PR-positive uterine cells was shown to contribute to an early block in embryo implantation, a phenotype not shared by SRC-1 or -3 knockout mice. In addition, histological and molecular analyses revealed an inability of the PR(Cre/+) SRC-2(flox/flox) mouse uterus to undergo the necessary cellular and molecular changes that precede complete P-induced decidual progression. Moreover, removal of SRC-1 in the PR(Cre/+) SRC-2(flox/flox) mouse uterus resulted in the absence of a decidual response, confirming that uterine SRC-2 and -1 cooperate in P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analysis disclosed the absence of significant ductal side branching and alveologenesis in the hormone-treated PR(Cre/+) SRC-2(flox/flox) mammary gland, reinforcing an important role for SRC-2 in cellular proliferative changes that require PR. We conclude that SRC-2 is appropriated by PR in a subset of transcriptional cascades obligate for normal uterine and mammary morphogenesis and function.

Both SRC-1 and TIF2 are members of the p160 steroid receptor coactivator family. Genetic analyses have shown that inactivation of TIF2, but not SRC-1, reduces postnatal survival, growth, and male reproductive function. Here, we demonstrate that, through analyses of SRC-1/TIF2 compound mutant mice, SRC-1 can partially compensate for the effects of a loss of TIF2 on mouse survival and growth, whereas SRC-1 and TIF2 are dispensable for primary organogenesis. The highly variable onset of defects observed in TIF2(-/-) testes due to the absence of TIF2 in Sertoli cells, including abnormal spermiogenesis, age-dependent degeneration of seminiferous epithelium, and disorder of cholesterol homeostasis, is uniformly accelerated upon inactivation of SRC-1 alleles in the TIF2 null genetic background, thus demonstrating that TIF2 and SRC-1 can perform redundant functions in Sertoli cells. Massive desquamation of immature germ cells together with an increase in germ cell apoptosis and a decrease in germ cell proliferation may be responsible for the early onset of the severe seminiferous epithelial degeneration observed in SRC-1(+/-)/TIF2(-/-) testes. Interestingly, the overall abnormal features displayed by the SRC-1(+/-)/TIF2(-/-) and SRC-1(-/-)/TIF2(-/-) mutant testes, including spermatid maturation defects, increase in Sertoli cell lipid stores, loss of immature germ cells, and formation of giant multinucleated spermatids, are commonly detected in testes of elderly men, suggesting that deficiencies in molecular pathways involving TIF2 and SRC-1 in Sertoli cells could participate in testicular senescence.

Human TIF2 (hTIF2) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 and p/CIP. Although the functions of hTIF2 and of its mouse homolog (GRIP1 or mTIF2) have been clearly established in vitro, their physiological role remains elusive. Here, we have generated mice lacking mTIF2/GRIP1 and examined their phenotype with a particular emphasis on reproductive functions. TIF2(-/-) mice are viable, but the fertility of both sexes is impaired. Male hypofertility is due to defects in both spermiogenesis (teratozoospermia) and age-dependent testicular degeneration, and TIF2 expression appears to be essential for adhesion of Sertoli cells to germ cells. Female hypofertility is due to a placental hypoplasia that most probably reflects a requirement for maternal TIF2 in decidua stromal cells that face the developing placenta. We conclude that TIF2 plays a critical role in mouse reproductive functions, whereas previous reports have not revealed serious fertility impairment in SRC-1(-/-) or p/CIP(-/-) mutants. Thus, even though the three p160 coactivators exhibit strong sequence homology and similar activity in assays in vitro, they play distinct physiological roles in vivo, as their genetic eliminations result in distinct pathologies.

Steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)-2 are homologous nuclear receptor coactivators. We have investigated their possible redundancy as thyroid hormone (TH) coactivators by measuring thyroid function in compound SRC-1 and TIF-2 knock out (KO) mice. Whereas SRC-1 KO (SRC-1(-/-)) mice are resistant to TH and SRC-1(+/-) are not, we now demonstrate that TIF-2 KO (TIF-2(-/-)) mice have normal thyroid function. Yet double heterozygous, SRC-1(+/-)/TIF-2(+/-) mice manifested resistance to TH of a similar degree as that in mice completely deficient in SRC-1. KO of both SRC-1 and TIF-2 resulted in marked increases of serum TH and thyrotropin concentrations. This work demonstrates gene dosage effect in nuclear coactivators manifesting as haploinsufficiency and functional redundancy of SRC-1 and TIF-2.