Trilostane, a 3ß-hydroxysteroid dehydrogenase inhibitor, suppresses growth of hepatocellular carcinoma and enhances anti-cancer effects of sorafenib.

Background Human 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) is an enzyme associated with steroidogenesis, however its' role in hepatocellular carcinoma (HCC) biology is unknown. Trilostane is an inhibitor of HSD3B1 and has been tested as a treatment for patients with breast cancer but has not been studied in patients with HCC. Methods and Results The expression of HSD3B1 in HCC tumors in 57 patients were examined. A total of 44 out of 57 tumors (77.2%) showed increased HSD3B1 expression. The increased HSD3B1 in tumors was significantly associated with advanced HCC. In vitro, the knockdown of HSD3B1 expression in Mahlavu HCC cells by a short hairpin RNA (shRNA) led to significant decreases in colony formation and cell migration. The suppression of clonogenicity in the HSD3B1-knockdown HCC cells was reversed by testosterone and 17ß-estradiol. Trilostane-mediated inhibition of HSD3B1 in different HCC cells also caused significant inhibition of clonogenicity and cell migration. In subcutaneous HCC Mahlavu xenografts, trilostane (30 or 60 mg/kg, intraperitoneal injection) significantly inhibited tumor growth in a dose-dependent manner. Furthermore, the combination of trilostane and sorafenib significantly enhanced the inhibition of clonogenicity and xenograft growth, surpassing the effects of each drug used alone, with no documented additional toxicity to animals. HSD3B1 blockade was found to suppress the phosphorylation of extracellular signal-regulated kinase (ERK). The decreased ERK phosphorylation was reversed by testosterone or 17b-estradiol. Conclusions Trilostane significantly inhibited the growth of HCC by inhibiting HSD3B1 function and augmenting the efficacy of sorafenib.

Successful management of a patient with active Cushing's disease complicated with coronavirus disease 2019 (COVID-19) pneumonia.

We provide the details of the successful management of a patient with active Cushing's disease complicated with coronavirus disease 2019 (COVID-19) pneumonia. The patient was a 27-year-old Japanese female healthcare worker who was scheduled to undergo pituitary surgery for Cushing's disease. She had been in close contact with an undiagnosed patient infected with COVID-19 and then developed COVID-19 pneumonia. Despite a lack of known risk factors associated with severe COVID-19 infection, the patient's dyspnea worsened and her respiratory condition deteriorated, as indicated by the need for 7 L/min oxygen supply by mask to maintain her oxygen saturation at >90%. Medical treatment was initiated to control hypercortisolism by the 'block and replace' regimen using steroidogenesis inhibitors and hydrocortisone. The COVID-19 pneumonia improved with multi-modal treatment including antiviral therapy. One month later, after a negative severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) test result and with appropriate protection against virus transmission to medical staff in the operating room and daily medical care nurses, trans-sphenoidal surgery was performed by our highly experienced pituitary surgeon. One month after the surgery, the patient's basal ACTH and cortisol levels and urinary free cortisol were all under the detection limit. Surgical remission was expected. Since hypercortisolism due to active Cushing's disease may worsen a COVID-19 infection, multi-disciplinary management that includes appropriate and prompt treatment strategies is mandatory in such cases.

Evaluation of Iatrogenic Hypocortisolemia Following Trilostane Therapy in 48 Dogs with Pituitary-Dependent Hyperadrenocorticism.

This study aimed to retrospectively describe the clinical progression following diagnosis of iatrogenic hypocortisolemia (iHC) in 48 dogs receiving trilostane for pituitary-dependent hyperadrenocorticism. Cortisol concentrations were ≥1.5 mg/dL within 6 mo following diagnosis of iHC in 76.3% of dogs (95% confidence interval [CI] 59.8-88.6%). At the time of study completion, 25% of dogs (95% CI 13.6-39.6%) were receiving either glucocorticoids or mineralocorticoids or both; 42% of dogs (95% CI 27.6-56.8%) were on no adrenal-related medications; and the remaining 33% of dogs (95% CI 20.4-48.4%) were receiving trilostane. No patient-, clinicopathologic-, or trilostane-associated factors were identified to influence adrenal recovery following diagnosis of iHC, and it remains difficult to predict the clinical progression in this population of dogs.

Augmentation of endogenous neurosteroid synthesis alters experimental status epilepticus dynamics.

Neurosteroids can modulate γ-aminobutyric acid type A receptor-mediated inhibitory currents. Recently, we discovered that the neurosteroids progesterone, 5α-dihydroprogesterone, allopregnanolone, and pregnanolone are reduced in the cerebrospinal fluid of patients with status epilepticus (SE). However, it is undetermined whether neurosteroids influence SE. For this reason, first we evaluated whether the inhibitor of adrenocortical steroid production trilostane (50 mg/kg) could modify the levels of neurosteroids in the hippocampus and neocortex, and we found a remarkable increase in pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone levels using liquid chromatography tandem mass spectrometry. Second, we characterized the dynamics of SE in the presence of the varied neurosteroidal milieu by a single intraperitoneal kainic acid (KA; 15 mg/kg) injection in trilostane-treated rats and their controls. Convulsions started in advance in the trilostane group, already appearing 90 minutes after the KA injection. In contrast to controls, convulsions prevalently developed as generalized seizures with loss of posture in the trilostane group. However, this effect was transient, and convulsions waned 2 hours before the control group. Moreover, electrocorticographic traces of convulsions were shorter in trilostane-treated rats, especially at the 180-minute (P < .001) and 210-minute (P < .01) time points. These findings indicate that endogenous neurosteroids remarkably modulate SE dynamics.

Ectopic Cushing's syndrome associated with a pheochromocytoma in a dog: a case report.

BACKGROUND: Ectopic Cushing's syndrome (ECS) associated with malignant tumors, such as small cell lung carcinoma, bronchial carcinoids, and pheochromocytoma, has been reported in human medicine. However, ECS related to pheochromocytoma has not been reported in dogs. CASE PRESENTATION: An 11-year-old castrated, male Scottish terrier was diagnosed with a left adrenal mass. Cushing's syndrome was suspected based on clinical signs, including pot belly, polyuria, polydipsia, bilateral alopecia, recurrent pyoderma, and calcinosis cutis. Cushing's syndrome was diagnosed on the basis of consistent clinical signs and repeated adrenocorticotropic hormone (ACTH) stimulation tests. In addition, tests for fractionated plasma metanephrine/normetanephrine suggested a pheochromocytoma. Unilateral adrenalectomy was performed after medical management with trilostane and phenoxybenzamine. Histopathology confirmed a diagnosis of pheochromocytoma without cortical lesions. After surgery, fractionated metanephrine/normetanephrine and the findings of low-dose dexamethasone suppression and ACTH stimulation tests were within the normal ranges without any medication. There were no clinical signs or evidence of recurrence and metastasis on thoracic and abdominal X-rays and ultrasonography up to 8 months after surgery. CONCLUSIONS: Pheochromocytoma should be considered a differential diagnosis for dogs with Cushing's syndrome with an adrenal tumor. A good prognosis can be expected with prompt diagnosis and surgical intervention.

Favorable outcome of pheochromocytoma in a dog with atypical Cushing's syndrome and diabetes mellitus following medical treatment: a case report.

BACKGROUND: Pheochromocytoma (PCC) has poor prognosis and adrenalectomy is hard to be performed, in case of caudal vena cava invasion. The long-term administration of phenoxybenzamine in PCC has not been reported in dogs. CASE PRESENTATION: A 14-year-old castrated male Poodle dog presented with an abdominal mass. On physical examination, hypertension, increased lens opacity, calcinosis cutis, generalized alopecia, and systolic murmur were observed. Serum chemistry and urinalysis profiles revealed hyperglycemia, hypercholesterolemia, elevated liver enzymes, and glucosuria. Abdominal ultrasonography showed a right adrenal mass with invasion of the caudal vena cava, which was cytologically diagnosed as suspected PCC. An adrenal mass (width × height × length, 28 × 26 × 48 mm3) was found on computed tomography and diagnosed as PCC with increased plasma metanephrines and normetanephrines. An adrenocorticotropin hormone stimulation test showed elevated adrenal hormones (androstenedione, estradiol, progesterone, and 17-OH progesterone) with normal cortisol, compatible with atypical Cushing's syndrome. The dog was managed with trilostane, phenoxybenzamine, and insulin therapy. Glycosylated hemoglobin and fructosamine levels gradually decreased, and hypertension resolved. In the 10-month follow-up period, the liver enzymes levels gradually decreased, and the clinical signs of the dog were well-controlled without deterioration. CONCLUSIONS: This case report describes long-term medical management without adrenalectomy of PCC complicated with atypical Cushing's syndrome and DM.

The gut microbiota is a major regulator of androgen metabolism in intestinal contents.

Androgens exert important effects both in androgen-responsive tissues and in the intestinal tract. To determine the impact of the gut microbiota (GM) on intestinal androgen metabolism, we measured unconjugated (free) and glucuronidated androgen levels in intestinal contents from the small intestine, with a low bacterial density, and from cecum and colon, with a high bacterial density. Using a specific, sensitive gas chromatography-tandem mass spectrometry method, we detected high levels of glucuronidated testosterone (T) and dihydrotestosterone (DHT) in small intestinal content of mice of both sexes, whereas in the distal intestine we observed remarkably high levels of free DHT, exceeding serum levels by >20-fold. Similarly, in young adult men high levels of unconjugated DHT, >70-fold higher than in serum, were detected in feces. In contrast to mice with a normal GM composition, germ-free mice had high levels of glucuronidated T and DHT, but very low free DHT levels, in the distal intestine. These findings demonstrate that the GM is involved in intestinal metabolism and deglucuronidation of DHT and T, resulting in extremely high free levels of the most potent androgen, DHT, in the colonic content of young and healthy mice and men.

Differential effects of androgens, estrogens and socio-sexual context on sexual behaviors in the castrated male goat.

The behavioral and endocrine activation of sexual behaviors exhibited by male goats, especially self-enurination (SE), is poorly understood. In the first experiment, to assess the influence of socio-sexual context on SE in bucks, the effects of distance from does, the presence of estrous versus non-estrous does and the presence of another buck on SE and courtship frequencies of intact male goats (bucks; n = 12) were tested using a unique behavior test apparatus. For experiments 2 and 3, to test the relative contributions of sex steroid hormones and socio-sexual context on SE, castrated male goats (wethers; n = 20) were randomly divided into five groups and injected for seven weeks with one of the following: 25 mg testosterone propionate (T), 25 mg dihydrotestosterone propionate (DHT), 100 µg estradiol benzoate (E), 100 µg E and 25 mg DHT (E + DHT), or oil (CON). The effects of these treatments on frequency of SE and courtship were assessed using the behavior test apparatus (social scenarios) adapted from the findings in experiment 1. In one scenario, a wether could observe (from 4.6 m) a buck and estrous female (doe) together in a wire mesh holding pen. In a different scenario, the wether could observe (from the same distance) a buck that could only court the estrous doe through a wire mesh barrier. Finally, to observe the effects of steroid treatment on mounting and ejaculation frequencies, in addition to SE and courtship, each wether was placed in a pen with an estrous doe for 10 min. After a five-week, treatment-washout period, wethers were randomly assigned to different treatment groups and retested. In experiment 1, bucks that were distanced from females displayed more SEs than those with fence-line contact, while those with fence-line contact displayed more bouts of courtship (P < 0.05). In experiments 2 and 3, courtship frequencies displayed in all three scenarios were greater than CON only for groups exposed to estrogen directly or via aromatization (T, E + DHT, E; P < 0.05). Frequencies of SE exhibited during behavior tests in which the wether was watching were greater than CON only for androgen-treated groups (T, E + DHT, DHT; P < 0.05). In contrast, when the wether was free to interact with the female, only the DHT group displayed SE at a higher frequency than CON (P < 0.05). Treatment had no effect on mount frequencies in this test scenario, however ejaculation frequencies were highest for T and E + DHT (P < 0.05). These studies suggest that the courtship behaviors of the male goat are estrogen-dependent. However, SE appears to be activated by androgens. It was also demonstrated that social context contributes as much to behavior expression as steroid treatment, as in social scenario 2 some sexual behaviors were displayed in similar frequencies across groups, despite differing sex steroid treatments.

Inhibition of 3ß-Hydroxysteroid Dehydrogenase Type 1 Suppresses Interleukin-6 in Breast Cancer.

BACKGROUND: Recently, we demonstrated that the expression of 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) in breast cancer is associated with shorter recurrence-free survival, and genetic or pharmacologic inhibition of HSD3B1 reduced colony formation and xenograft growth. However, the mechanisms are unclear. METHODS: Triple-negative MDA-MB-231 and BT-20 breast cancer cells underwent HSD3B1 silencing. Microarray and bioinformatic analysis were performed. The interleukin-6 (IL-6) expression and secretion were evaluated using real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Clonogenic ability and cell viability were determined in the absence or presence of recombinant IL-6. RESULTS: Functional and pathway enrichment analyses showed that HSD3B1 silencing modulates the expression of several growth factors and cytokines. Cells transfected with HSD3B1-targeting small interfering RNA or treated with an HSD3B1 inhibitor (trilostane) had decreased IL-6 expression and secretion. HSD3B1 inhibition reduced colony formation, which was partially rescued by IL-6 supplementation. The HSD3B1 knockdown enhanced paclitaxel sensitivity, and IL-6 treatment partially reversed the augmented cytotoxicity. CONCLUSIONS: Our findings suggest that the therapeutic potential of targeting HSD3B1 is in part mediated by IL-6 suppression.

Infradian Rhythms of Resistance to a Dissociative Anesthetic in Wistar Male Rats under Normal Conditions and After Surgical Removal of the Adrenal Glands and Testes.

Daily dynamics of changes in the latency of a response to dissociative anesthetic tiletamine (time from injection to ataxia) was studied in mature Wistar rats. Both intramuscular and intravenous administration of the anesthetic was associated with 4-day oscillations of the latent period synchronous with the dynamics of changes in the concentration of glucocorticoid hormones. The period and phases of the infradian rhythm of resistance to the anesthetic remained unchanged after removal of both adrenal glands and testes and administration of corticosterone synthesis blocker trilostane diminishing the 4-day cycle of changes in corticosterone level. Therefore, hormones of the adrenal glands and testes do not play the key role in the mechanisms of formation of the 4-day infradian rhythm.

Comparison of two prepill cortisol concentrations in dogs with hypercortisolism treated with trilostane.

BACKGROUND: The ideal method for monitoring trilostane therapy in dogs with hypercortisolism is still open to debate. Recently, determination of the pre-trilostane (prepill) cortisol concentration has been proposed to be more repeatable than either post-trilostane or post-ACTH cortisol. The aim of this study was to compare two prepill cortisol concentrations in dogs with hypercortisolism during trilostane therapy. Sixteen client-owned dogs with naturally occurring hypercortisolism were prospectively included and cortisol concentrations were measured twice, 1 h apart, before the morning trilostane dose (prepill 1 and 2 cortisol). RESULTS: A total of 47 prepill cortisol measurement pairs were included. Compared to prepill 1, prepill 2 cortisol was higher in 15, equal in 8 and lower in 24 pairs. Group agreement between prepill 1 and 2 cortisol was 70% (moderate agreement - weighted kappa 0.55). In 30% of the pairs, group assignment was discrepant, implying a different therapeutic decision. In some dogs certain circumstances (e.g. excessive barking, difficulties during blood collection, excitement at arrival) were identified as potential factors explaining the discrepancy between prepill 1 and 2 cortisol measurements. CONCLUSIONS: In a substantial number of dogs treated with trilostane, the two prepill cortisol concentrations differed. Part of this difference might be ascribable to stressful events during test performance. When using prepill cortisol measurements to monitor trilostane therapy, recording of any incident during handling that might affect cortisol release might be helpful to make a reliable decision about a trilostane dose adaptation.

Urinary calculi in a shih tzu dog with hyperadrenocorticism.

An 11-year-old spayed female shih tzu dog was presented with pollakiuria, stranguria, and hematuria. Radiographs revealed a large number of radiodense urinary calculi within the bladder. Physical examination, complete blood cell count, biochemistry and ACTH stimulation test suggested possible hyperadrenocorticism. A cystotomy was performed and the patient was treated for hyperadrenocorticism.

Calculs urinaires chez une chienne Shih Tzu atteinte d'hyperadrénocorticisme. Une chienne Shih Tzu stérilisée âgée de 11 ans a été présentée avec de la pollakiurie, de la strangurie et de l'hématurie. Les radiographies ont révélé un grand nombre de calculs urinaires radio-opaques dans la vessie. L'examen physique, une formule sanguine complète et la biochimie ont suggéré la possibilité d'hyperadrénocorticisme. Une cystotomie a été réalisée et la patiente a été traitée pour l'hyperadrénocorticisme.(Traduit par Isabelle Vallières).

Update on the use of trilostane in dogs.

Many articles published in the past few years have contributed to a better understanding of the use of trilostane in dogs. Trilostane is a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase, the enzyme essential for synthesis of cortisol and all other steroids. Trilostane is reported to be safe and effective in the treatment of pituitary-dependent hyperadrenocorticism (HAC), adrenal-dependent HAC, and alopecia X. While trilostane controls most of the clinical signs associated with HAC, abnormalities such as hypertension, hypercoagulability, and proteinuria may persist despite therapy. Because the duration of cortisol suppression after a dose of trilostane is often less than 12 hours, many dogs with HAC could benefit from low dose trilostane treatment every 12 hours. Many controversies regarding trilostane still exist. This review provides a comprehensive commentary on trilostane's indications, mode of action, dose, monitoring, efficacy, and adverse effects.

Mise à jour sur l'utilisation du trilostane chez les chiens. De nombreux articles publiés au cours des dernières années ont contribué à une meilleure compréhension de l'utilisation du trilostane chez les chiens. Le trilostane est un inhibiteur compétitif de la 3ß-hydroxystéroïde déshydrogénase, l'enzyme essentiel pour la synthèse du cortisol et de tous les autres stéroïdes. On signale que le trilostane est sûr et efficace pour le traitement de l'hyperadrénocorticisme pituitaire (HAC), le HAC adrénal et l'alopécie X. Bien que le trilostane maîtrise la plupart des signes cliniques associés au HAC, des anomalies comme l'hypertension, l'hypercoagulabilité et la protéinurie peuvent persister malgré la thérapie. Parce que la durée de la suppression du cortisol après une dose de trilostane est souvent de moins de 12 heures, plusieurs chiens atteints de HAC pourraient bénéficier d'un traitement à faible dose de trilostane toutes les 12 heures. Il subsiste encore beaucoup de controverse concernant le trilostane. Cet examen fournit un commentaire exhaustif sur les indications, le mode d'action, la dose, la surveillance, l'efficacité et les effets secondaires du trilostane.(Traduit par Isabelle Vallières).

Expression of 3ß-Hydroxysteroid Dehydrogenase Type 1 in Breast Cancer is Associated with Poor Prognosis Independent of Estrogen Receptor Status.

BACKGROUND: Human 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) plays a vital role in steroidogenesis in breast tumors and may therefore be a suitable target for treatment of breast cancer. This study investigated the role of HSD3B1 in the pathogenesis of breast cancer in clinical and experimental settings. METHODS: Expression of HSD3B1 in primary tumors of 258 breast cancer patients was evaluated by immunohistochemistry. Screening of breast cancer cell lines indicated that triple-negative MDA-MB-231 cells expressed HSD3B1. The effects from genetic and pharmacologic inhibition of HSD3B1 were assessed in vitro and in vivo. RESULTS: The findings showed that 44% of the 258 breast cancers were HSD3B1-positive. The HSD3B1-positivity was associated with advanced-stage disease (p = 0.009) and reduced recurrence-free survival (p = 0.048) but not with tumor subtype or estrogen receptor status. Silencing of HSD3B1 or treatment with an HSD3B1 inhibitor (trilostane) reduced colony formation in breast cancer cells. Knockdown of HSD3B1 inhibited cell proliferation and migration. Analysis of a murine xenograft tumor model indicated that trilostane significantly slowed tumor growth. CONCLUSIONS: Expression of HSD3B1 in breast cancer is negatively associated with prognosis. The study found HSD3B1 to be a potential therapeutic target for breast cancer independent of estrogen receptor status.

Effects of Trilostane on urinary Catecholamines and their metabolites in dogs with Hypercortisolism.

BACKGROUND: Glucocorticoids influence the synthesis and metabolism of catecholamines (epinephrine and norepinephrine) and metanephrines (metanephrine and normetanephrine). The aim of this study was to measure urinary catecholamines and metanephrines in dogs with hypercortisolism before and during trilostane therapy. Urine samples were collected during initial work up and during therapy with trilostane in 14 dogs with hypercortisolism and in 25 healthy dogs. Epinephrine, norepinephrine, metanephrine and normetanephrine were measured using high-pressure liquid chromatography and expressed as ratios to urinary creatinine concentration. RESULTS: Untreated dogs with hypercortisolism had significantly higher epinephrine, norepinephrine, and normetanephrine:creatinine ratios compared to healthy dogs. During trilostane therapy, urinary catecholamines and their metabolites did not decrease significantly. However, dogs with low post-ACTH cortisol concentrations during trilostane therapy had less increased epinephrine, norepinephrine and normetanephrine:creatinine ratios compared to healthy dogs. There was no correlation of urinary catecholamines and their metabolites with baseline or post-ACTH cortisol or endogenous ACTH concentrations during trilostane therapy. CONCLUSION: Influences between steroid hormones and catecholamines seem to occur, as dogs with hypercortisolism have significantly higher urinary epinephrine, norepinephrine, and normetanephrine:creatinine ratios. Once-daily trilostane therapy does not lead to a significant decrease in catecholamines and their metabolites. Trilostane-treated dogs still have increased urinary epinephrine, norepinephrine and normetanephrine:creatinine ratios during trilostane therapy.

Aromatized testosterone attenuates contextual generalization of fear in male rats.

Generalization is a common symptom of many anxiety disorders, and females are 60% more likely to suffer from an anxiety disorder than males. We have previously demonstrated that female rats display significantly accelerated rates of contextual fear generalization compared to male rats; a process driven, in part, by activation of ERß. The current study was designed to determine the impact of estrogens on contextual fear generalization in male rats. For experiment 1, adult male rats were gonadectomized (GDX) and implanted with a capsule containing testosterone proprionate, estradiol, dihydrotestosterone proprionate (DHT), or an empty capsule. Treatment with testosterone or estradiol maintained memory precision when rats were tested in a different (neutral) context 1day after training. However, male rats treated with DHT or empty capsules displayed significant levels of fear generalization, exhibiting high levels of fear in the neutral context. In Experiment 2, we used acute injections of gonadal hormones at a time known to elicit fear generalization in female rats (e.g. 24h before testing). Injection treatment followed the same pattern of results seen in Experiment 1. Finally, animals given daily injections of the aromatase inhibitor, Fadrozole, displayed significant fear generalization. These data suggest that testosterone attenuates fear generalization likely through the aromatization testosterone into estradiol as animals treated with the non-aromatizable androgen, DHT, or animals treated with Fadrozole, displayed significant generalized fear. Overall, these results demonstrate a sex-dependent effect of estradiol on the generalization of contextual fear.

Fully automated synthesis of [(18) F]fluoro-dihydrotestosterone ([(18) F]FDHT) using the FlexLab module.

Imaging of androgen receptor expression in prostate cancer using F-18 FDHT is becoming increasingly popular. With the radiolabelling precursor now commercially available, developing a fully automated synthesis of [(18) F] FDHT is important. We have fully automated the synthesis of F-18 FDHT using the iPhase FlexLab module using only commercially available components. Total synthesis time was 90 min, radiochemical yields were 25-33% (n = 11). Radiochemical purity of the final formulation was > 99% and specific activity was > 18.5 GBq/µmol for all batches. This method can be up-scaled as desired, thus making it possible to study multiple patients in a day. Furthermore, our procedure uses 4 mg of precursor only and is therefore cost-effective. The synthesis has now been validated at Austin Health and is currently used for [(18) F]FDHT studies in patients. We believe that this method can easily adapted by other modules to further widen the availability of [(18) F]FDHT.

Lack of association between clinical signs and laboratory parameters in dogs with hyperadrenocorticism before and during trilostane treatment.

INTRODUCTION: Trilostane therapy, the treatment of choice for pituitary- dependent hyperadrenocorticism (HAC) in dogs, is monitored by assessing resolution of clinical signs and measuring adrenocortical reserve capacity with an ACTH-stimulation test. The aim of this prospective study was to evaluate agreement between clinical signs reported by owners and cortisol or ACTH concentrations before and during trilostane therapy (starting dose 1-2 mg/kg once daily). A questionnaire on signs of HAC was used and a clinical score calculated as the sum of the 9 questions. Eighteen questionnaires at diagnosis and 97 during therapy were filled out by owners of 32 dogs. An ACTH-stimulation test was performed at each reevaluation. There were weak correlations between abdominal girth, appetite or weight gain and cortisol concentrations during therapy. However, the clinical score did not correlate with cortisol or cACTH values. In 50% of dogs, trilostane application had to be changed from once daily to twice daily during the study. Clinical signs reported by owners matched poorly with cortisol or cACTH concentrations at any time point. If low-dose trilostane is used, treatment frequency often has to be increased.

INTRODUCTION: Le traitement au trilostane, médicament de choix dans les cas d'hyperadrénocorticisme hypophyso-dépendant chez le chien, est évalué sur la base de la disparition des symptômes cliniques et des résultats des tests de stimulation à l'ACTH. Le but de la présente étude prospective était de comparer les symptômes cliniques (évalués par les propriétaires) avec les concentrations de cortisol et d'ACTH endogène avant et durant un traitement au trilostane (dose initiale 1­2 mg/kg, 1× par jour). On a utilisé un questionnaire composé de 9 questions relatives aux symptômes cliniques sur la base desquels on a calculé un score clinique total. Dix-huit questionnaires ont été remplis au moment du diagnostic et 97 durant le traitement par les propriétaires de 32 chiens. Un test de stimulation à l'ACTH a été réalisé lors de chaque contrôle. Il existait de faibles corrélations entre le périmètre abdominal, l'appétit et la prise de poids et les taux de cortisol durant le traitement. Le score clinique total n'était toutefois pas corrélé avec les concentrations de cortisol ou d'ACTH. Chez la moitié des chiens, la dose de trilostane a du être répartie en deux prises journalières. Les symptômes cliniques jugés par les propriétaires montraient une mauvaise corrélation avec les taux de cortisol et d'ACTH durant le traitement au trilostane. Si on dose ce médicament de façon faible, il y a souvent lieu d'augmenter la fréquence des prises.

Dependence of anxiolytic effects of the dipeptide TSPO ligand GD-23 on neurosteroid biosynthesis.

The elevated plus maze test showed that GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide), an original dipeptide ligand of TSPO, exerted anxiolytic effect when injected intraperitoneally at a dose of 0.5 mg/kg. This effect was completely blocked by the selective neurosteroid synthesis inhibitors, enzymes trilostane and finasteride. The same inhibitors do not prevent the anxiolytic effects of the benzodiazepine tranquillizer diazepam. The results of the study indicate the selective neurosteroidogenic mechanism of the anxiolytic action of GD-23.

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro.

STUDY QUESTION: What are the direct effects of progesterone (P4) and estradiol (E2) on the development and function of primate follicles in vitro from the pre-antral to early antral stage? SUMMARY ANSWER: In a steroid-depleted milieu, E2 improved follicle survival, growth, antrum formation and oocyte health, whereas P4 exerted minimal beneficial effects on follicle survival and reduced oocyte health. WHAT IS KNOWN ALREADY: Effects of P4 and E2 on follicle development have been studied primarily in large antral and pre-ovulatory follicles. Chronic P4 exposure suppresses antral follicle growth, but acute P4 exposure promotes oocyte maturation in pre-ovulatory follicles. Effects of E2 can be stimulatory or inhibitory depending upon species, dose and duration of exposure. STUDY DESIGN, SIZE, DURATION: Non-human primate model, randomized, control versus treatment. Macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult rhesus macaque secondary follicles were encapsulated in 0.25% alginate and cultured individually in media containing follicle stimulating hormone plus (i) vehicle, (ii) a steroid-synthesis inhibitor, trilostane (TRL, 250 ng/ml), (iii) TRL + low E2 (100 pg/ml) or progestin (P, 10 ng/ml R5020) and (iv) TRL + high E2 (1 ng/ml E2) or P (100 ng/ml R5020). Follicles reaching the antral stage (≥750 µm) were treated with human chorionic gonadotrophin for 34 h. End-points included follicle survival, antrum formation, growth pattern, plus oocyte health and maturation status, as well as media concentrations of P4, E2 and anti-Müllerian hormone (AMH). MAIN RESULTS AND THE ROLE OF CHANCE: In a steroid-depleted milieu, low dose, but not high dose, P improved (P < 0.05) follicle survival, but had no effect (P > 0.05) on antrum formation and AMH production. Low-dose P increased (P < 0.05) P4 production in fast-grow follicles, and both doses of P elevated (P < 0.05) E2 production in slow-grow follicles. Additionally, low-dose P increased (P < 0.05) the percentage of no-grow follicles, and high-dose P promoted oocyte degeneration. In contrast, E2, in a steroid-depleted milieu, improved (P < 0.05) follicle survival, growth, antrum formation and oocyte health. E2 had no effect on P4 or E2 production. Follicles exposed to E2 yielded mature oocytes capable of fertilization and early cleavage, at a rate similar to untreated control follicles. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro effects of P and E2 during the interval from the secondary to small antral stage of macaque follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel information on the direct actions of P4 and E2 on primate pre-antral follicle development. Combined with our previous report on the actions of androgens, our findings suggest that androgens appear to be a survival factor but hinder antral follicle differentiation, E2 appears to be a survival and growth factor at the pre-antral and early antral stage, whereas P4 may not be essential during early folliculogenesis in primates. STUDY FUNDING/COMPETING INTERESTS: NIH P50 HD071836 (NCTRI), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), ONPRC 8P51OD011092. There are no conflicts of interest.