Discovery of Potent, Selective, and Short-Acting Peptidic V2 Receptor Agonists.

The vasopressin analogue desmopressin (desamino-d-arginine8 vasopressin, dDAVP, 1) is a potent vasopressin 2 (V2) receptor (V2R) agonist approved in many countries for the treatment of diabetes insipidus, primary nocturnal enuresis, nocturia, and coagulation disorders. Since 1 is primarily excreted via the kidneys, an age-related decline in kidney function leads to slower elimination, prolonged antidiuresis, and hyponatremia. In search of novel, potent, selective, and short-acting peptidic V2R agonists, we synthesized a series of C-terminally truncated analogues of [Val4]dDAVP, 2, modified in positions 2, 3, and 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency at the human V2 receptor, selectivity versus the related receptors (human vasopressin 1a receptor, human vasopressin 1b receptor, and human oxytocin receptor), and pharmacokinetic profiles in rodents and other higher species. The truncated analogues show excellent potency at the V2R, increased systemic clearance, and shorter half-life in rats. Two compounds 19 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-Agm) and 38 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-d-Arg-NEt2) have been selected for clinical development for nocturia.

Preclinical Efficacy of [V4 Q5 ]dDAVP, a Second Generation Vasopressin Analog, on Metastatic Spread and Tumor-Associated Angiogenesis in Colorectal Cancer.

PURPOSE: Control of metastatic spread of colorectal cancer (CRC) remains as a major therapeutic challenge. [V4 Q5 ]dDAVP is a vasopressin peptide analog with previously reported anticancer activity against carcinoma tumors. By acting as a selective agonist of arginine vasopressin type 2 membrane receptor (AVPR2) present in endothelial and tumor cells, [V4Q5]dDAVP is able to impair tumor aggressiveness and distant spread. Our aim was to evaluate the potential therapeutic benefits of [V4Q5]dDAVP on highly aggressive CRC disease using experimental models with translational relevance. MATERIALS AND METHODS: Murine CT-26 and human Colo-205 AVPR2-expressing CRC cell lines were used to test the preclinical efficacy of [V4Q5]dDAVP, both in vitro and in vivo. RESULTS: In syngeneic mice surgically implanted with CT-26 cells in the spleen, sustained intravenous treatment with [V4Q5]dDAVP (0.3 µg/kg) dramatically impaired metastatic progression to liver without overt signs of toxicity, and also reduced experimental lung colonization. The compound inhibited in vivo angiogenesis driven by Colo-205 cells in athymic mice, as well as in vitro endothelial cell migration and capillary tube formation. [V4Q5]dDAVP exerted AVPR2-dependent cytostatic activity in vitro (IC50 1.08 µM) and addition to 5-fluorouracil resulted in synergistic antiproliferative effects both in CT-26 and Colo-205 cells. CONCLUSION: The present preclinical study establishes for the first time the efficacy of [V4Q5]dDAVP on CRC. These encouraging. RESULTS: suggest that the novel second generation vasopressin analog could be used for the management of aggressive CRC as an adjuvant agent during surgery or to complement standard chemotherapy, limiting tumor angiogenesis and metastasis and thus protecting the patient from CRC recurrence.

Addition of vasopressin synthetic analogue [V(4)Q(5)]dDAVP to standard chemotherapy enhances tumour growth inhibition and impairs metastatic spread in aggressive breast tumour models.

[V(4)Q(5)]dDAVP is a novel 2nd generation vasopressin analogue with robust antitumour activity against metastatic breast cancer. We recently reported that, by acting on vasopressin V2r membrane receptor present in tumour cells and microvascular endothelium, [V(4)Q(5)]dDAVP inhibits angiogenesis and metastatic progression of the disease without overt toxicity. Despite chemotherapy remaining as a primary therapeutic option for aggressive breast cancer, its use is limited by low selectivity and associated adverse effects. In this regard, we evaluated potential combinational benefits by adding [V(4)Q(5)]dDAVP to standard-of-care chemotherapy. In vitro, combination of [V(4)Q(5)]dDAVP with sub-IC50 concentrations of paclitaxel or carmustine resulted in a cooperative inhibition of breast cancer cell growth in comparison to single-agent therapy. In vivo antitumour efficacy of [V(4)Q(5)]dDAVP addition to chemotherapy was first evaluated using the triple-negative MDA-MB-231 breast cancer xenograft model. Tumour-bearing mice were treated with i.v. injections of [V(4)Q(5)]dDAVP (0.3 µg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10 mg/kg i.p.). After 6 weeks of treatment, combination regimen resulted in greater tumour growth inhibition compared to monotherapy. [V(4)Q(5)]dDAVP addition was also associated with reduction of local aggressiveness, and impairment of tumour invasion and infiltration of the skin. Benefits of combined therapy were confirmed in the hormone-independent and metastatic F3II breast cancer model by combining [V(4)Q(5)]dDAVP with carmustine (25 mg/kg i.p.). Interestingly, [V(4)Q(5)]dDAVP plus cytotoxic agents severely impaired colony forming ability of tumour cells and inhibited breast cancer metastasis to lung. The present study shows that [V(4)Q(5)]dDAVP may complement conventional chemotherapy by modulating metastatic progression and early stages of microtumour establishment, and thus supports further preclinical testing of the compound for the management of aggressive breast cancer.

Feeding response following central administration of mesotocin and arginine-vasotocin receptor agonists in chicks (Gallus gallus).

Mesotocin (MT) and arginine-vasotocin (AVT) are posterior pituitary derived hormones in birds and are homologous to mammalian oxytocin (OT) and vasopressin (VP), respectively. We previously reported that intracerebroventricular (ICV) injection of both MT and AVT inhibit feeding and induce wing-flapping in chicks (Gallus gallus). Because both peptides cause similar effects suggests that they might act via common receptors. However, the specific receptors of MT and AVT which mediate their anorexigenic effect have not been clarified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in MT- and AVT-induced anorexia and behavioral patterns by using several agonists. ICV injection of vasopressin-1 receptor agonist (V1R) (homologous to chicken AVT receptor-2 and -4 [VT2R and VT4R, respectively]), significantly decreased food intake while agonists of vasopressin-2 receptor (V2R) and OT receptor (OTR) (homologues of chicken AVT receptor-1 and MT receptor respectively) had no effect. In addition, V1R agonist induced wing-flapping although this was not affected by V2R or OTR agonists. Since VT2R has not been found in the brain of chicks, the present study suggested that VT4R might be related to the anorexigenic effect and wing-flapping induced by MT and AVT in chicks.

The novel desmopressin analogue [V4Q5]dDAVP inhibits angiogenesis, tumour growth and metastases in vasopressin type 2 receptor-expressing breast cancer models.

Desmopressin (dDAVP) is a safe haemostatic agent with previously reported antitumour activity. It acts as a selective agonist for the V2 vasopressin membrane receptor (V2r) present on tumour cells and microvasculature. The purpose of this study was to evaluate the novel peptide derivative [V4Q5]dDAVP in V2r-expressing preclinical mouse models of breast cancer. We assessed antitumour effects of [V4Q5]dDAVP using human MCF-7 and MDA-MB-231 breast carcinoma cells, as well as the highly metastatic mouse F3II cell line. Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. In order to study the effect of intravenously administered [V4Q5]dDAVP on tumour growth and angiogenesis, breast cancer xenografts were generated in athymic mice. F3II cells were injected into syngeneic mice to evaluate the effect of [V4Q5]dDAVP on spontaneous and experimental metastatic spread. In vitro cytostatic effects of [V4Q5]dDAVP against breast cancer cells were greater than those of dDAVP, and associated with V2r-activated signal transduction and partial cell cycle arrest. In MDA-MB-231 xenografts, [V4Q5]dDAVP (0.3 µg/kg, thrice a week) reduced tumour growth and angiogenesis. Treatment of F3II mammary tumour-bearing immunocompetent mice resulted in complete inhibition of metastatic progression. [V4Q5]dDAVP also displayed greater antimetastatic efficacy than dDAVP on experimental lung colonisation by F3II cells. The novel analogue was well tolerated in preliminary acute toxicology studies, at doses ≥ 300-fold above that required for anti-angiogenic/antimetastatic effects. Our data establish the preclinical activity of [V4Q5]dDAVP in aggressive breast cancer, providing the rationale for further clinical trials.

Structure-activity relationship of 1-desamino-8-D-arginine vasopressin as an antiproliferative agent on human vasopressin V2 receptor-expressing cancer cells.

The synthetic nonapeptide 1­desamino­8­D­arginine vasopressin (dDAVP) can reduce tumor cell growth through agonist action on the vasopressin V2 receptor. A structure­antiproliferative activity relationship analysis of dDAVP was performed using the alanine scanning technique on the aggressive MDA­MB­231 human breast carcinoma cell line. The results from this analysis demonstrated that the amino acids located at the loop of dDAVP are important for the antiproliferative activity of dDAVP, highlighting the key role of the N­terminal region of the peptide in the interaction with the tumor cell surface receptor. The findings from this study present novel strategies for designing improved compounds with enhanced stability for cancer therapy.

Adaptation and content validation of the Brazilian version of the Posttraumatic Cognitions Inventory.

OBJECTIVE: To translate, adapt and validate the contents of the Brazilian version of the Posttraumatic Cognitions Inventory. METHODS: The process of translation and adaptation of the initial instructions and items involved five steps: (1) translation; (2) back translation; (3) correction and semantic adaptation; (4) content validation by professional experts (judges); and (5) test of final version through a verbal-numbered scale. As performance indicators for understanding, the scores of central tendency (mean) and dispersion (standard deviation) were calculated for each item in step 5. Satisfactory comprehension was defined as a mean score ≥ 3. RESULTS The 36 questions and the initial instructions were translated and adapted to create the Brazilian version of the Posttraumatic Cognitions Inventory. In the final stage of testing, 45 adults answered the items and demonstrated an adequate understanding of the instrument in the verbal-numbered scale (M = 4.13; dp = 0.11). CONCLUSIONS The Posttraumatic Cognitions Inventory is an easily understood and semantically valid instrument. Further studies are needed to verify and evaluate the appropriateness of its psychometric properties for the Brazilian population.

Adaptação e validação de conteúdo da versão brasileira do Posttraumatic Cognitions Inventory / Adaptación y validación de contenido de la versión brasileña del Posttraumatic Cognitions Inventory / Adaptation and content validation of the Brazilian version of the Posttraumatic Cognitions Inventory

OBJETIVO: Realizar a tradução, adaptação e validação de conteúdo da versão brasileira do Posttraumatic Cognitions Inventory . MÉTODOS: O processo de tradução e adaptação das instruções iniciais e dos itens do PTCI envolveu cinco etapas: (1) tradução; (2) retradução; (3) correção e adaptação semântica; (4) validação do conteúdo por profissionais da área (juízes); e (5) teste da versão final, por meio de uma escala verbal-numérica. Como indicadores de desempenho para a compreensão, foram computados os escores de tendência central (média) e dispersão (desvio padrão) para cada item na etapa 5. Definiu-se escore médio ≥ 3 para compreensão satisfatória. RESULTADOS: As 36 questões e as instruções iniciais foram traduzidas e adaptadas para compor a versão brasileira do Posttraumatic Cognitions Inventory . Quarenta e cinco adultos responderam aos itens do Posttraumatic Cognitions Inventory na etapa teste da versão final, mostrando compreensão adequada do instrumento na escala verbal-numérica (M = 4,13; dp = 0,11). CONCLUSÕES: O Posttraumatic Cognitions Inventory é um instrumento de fácil compreensão e semanticamente válido. Estudos posteriores são necessários para a verificação e adequação da avaliação de suas propriedades psicométricas na população brasileira. .

OBJETIVO: Realizar la traducción, adaptación y validación de contenido de la versión brasileña del Posttraumatic Cognitions Inventory (PTCI). MÉTODOS: El proceso de traducción y adaptación de las instrucciones iníciales y de los ítems del PTCI involucró cinco etapas: (1) traducción; (2) re-traducción; (3) corrección y adaptación semántica; (4) validación del contenido por profesionales del área (jueces) y (5) prueba de la versión final, por medio de una escala verbal-numérica. Como indicadores de desempeño para la comprensión, se computaron los escores de tendencia central (promedio) y dispersión (desvío estándar) para cada ítem en la etapa 5. Se definió, escores promedio ≥ 3 para comprensión satisfactoria. RESULTADOS: Las 36 preguntas y las instrucciones iníciales fueron traducidas y adaptadas para componer la versión brasileña del PTCI. Cuarenta y cinco adultos respondieron a los ítems del PTCI en la etapa de prueba de la versión final, mostrando comprensión adecuada del instrumento en la escala verbal-numérica (M=4,13; ds=0,11). CONCLUSIONES: el PTCI es un instrumento de fácil comprensión y semánticamente válido. Estudios posteriores son necesarios para la verificación y adecuación de la evaluación de sus propiedades psicométricas en la población brasileña. .

OBJECTIVE To translate, adapt and validate the contents of the Brazilian version of the Posttraumatic Cognitions Inventory. METHODS: The process of translation and adaptation of the initial instructions and items involved five steps: (1) translation; (2) back translation; (3) correction and semantic adaptation; (4) content validation by professional experts (judges); and (5) test of final version through a verbal-numbered scale. As performance indicators for understanding, the scores of central tendency (mean) and dispersion (standard deviation) were calculated for each item in step 5. Satisfactory comprehension was defined as a mean score ≥ 3. RESULTS The 36 questions and the initial instructions were translated and adapted to create the Brazilian version of the Posttraumatic Cognitions Inventory. In the final stage of testing, 45 adults answered the items and demonstrated an adequate understanding of the instrument in the verbal-numbered scale (M = 4.13; dp = 0.11). CONCLUSIONS The Posttraumatic Cognitions Inventory is an easily understood and semantically valid instrument. Further studies are needed to verify and evaluate the appropriateness of its psychometric properties for the Brazilian population. .

Polycystic ovary syndrome in Salvador, Brazil: a prevalence study in primary healthcare.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common condition in women of reproductive age. It is characterized by hyperandrogenism, oligomenorrhea/amenorrhea and polycystic ovaries. It is associated with obesity, diabetes, dyslipidemia and cardiovascular disease. No studies have been conducted on the prevalence of PCOS in Brazilian or South American women. Few studies using the Rotterdam criteria have been published. The objective of the present study was to calculate the prevalence of PCOS at primary healthcare level in Salvador, Brazil based on these criteria. METHODS: This was a cross-sectional, two-phase study conducted in a probability sample of women of 18-45 years of age screened for cervical cancer in the primary healthcare network of the city of Salvador, Brazil. In the first phase, interviews were conducted, weight, height, waist circumference, blood pressure and random blood sugar levels were measured, and the presence of acne and hirsutism was investigated. Women with at least one diagnostic criterion were referred for the second phase, which consisted of specialist consultation, pelvic ultrasonography and hormone measurements for differential diagnosis and/or investigation of a second criterion. RESULTS: Of the 859 women interviewed, 88.5% were black and 58.7% had 11 years of schooling or less. A diagnosis of PCOS was excluded in 84.4%, undetermined in 7.1% and confirmed in 8.5% (95% CI: 6.80-10.56). There were no statistically significant differences between these three groups with respect to weight, body mass index, waist circumference, blood sugar levels or arterial blood pressure. Women with PCOS were younger (p = 0.00), taller (p = 0.04), had fewer children (p = 0.00), were better educated (p = 0.01), and had higher total testosterone levels (p = 0.01) and a higher LH/FSH ratio (p = 0.01). CONCLUSION: According to the Rotterdam criteria, the prevalence of PCOS in women seeking primary healthcare in Salvador, Brazil, was 8.5%.

Recent patenting activities in the discovery and development of vasopressin V2 receptor agonists.

INTRODUCTION: Vasopressin V(2) agonists are well known as effective therapies in the treatment of central diabetes insipidus and nocturnal enuresis. Furthermore, given their mode of action, these particular agonists have more recently been considered, in both the pharmaceutical industry and in academia, as viable therapies for urological conditions such as nocturia. AREAS COVERED: For the past 10 years, significant progress has been made in the discovery and development of vasopressin V(2) agonists. This article provides the reader with information on the recent progress in the discovery and development of these compounds based on patents published from 2002 onward. Specifically, the article looks at the discovery of new non-peptide agonists as well as well as novel formulations of the vasopressin V(2) agonist desmopressin. EXPERT OPINION: The V(2) receptor is currently one of the hottest therapeutic targets investigated for the treatment of urinary disorders such as nocturia and central diabetes. Over the past 10 years, significant progress has been made in the discovery and development of vasopressin V(2) receptor agonists, for the treatment of these disorders. The author anticipates that these agonists will be launched to market in the not-too-distant future.

Central vasopressin V1a receptors modulate neural processing in mothers facing intruder threat to pups.

Vasopressin V1a receptors in the rat brain have been studied for their role in modulating aggression and anxiety. In the current study blood-oxygen-level-dependent (BOLD) functional MRI was used to test whether V1a receptors modulate neural processing in the maternal brain when dams are exposed to a male intruder. Primiparous females were given an intracerebroventricular (ICV) injection of vehicle or V1a receptor antagonist ([beta-Mercapto-beta,beta-cyclopentamethylenepropionyl(1), O-me-Tyr(2),Arg(8)]-Vasopressin, [corrected] 125 ng/10 microL) 90-120 min before imaging. During fMRI, awake dams were presented with a male intruder threat to pups using a specialized chamber that contained separate compartments for pups and a male intruder. Our results indicate that the number of activated voxels was reduced in the cortical amygdala with V1a receptor blockade, while an increase was observed in the anterior olfactory nucleus and other areas. Dams treated with V1a antagonist showed significantly greater BOLD responses in the anterior olfactory nucleus, infralimbic prefrontal cortex, gustatory cortex, somatosensory cortex, and substantia innominata when presented with a novel male intruder. BOLD responses were reduced in the cortical amygdala and ventromedial hypothalamus. The V1a receptor sensitive areas play roles in the processing of smell, taste and touch and emotional reactivity. Thus one interpretation of the present fMRI data is that vasopressin, acting through V1a receptors, may modulate sensory processing and perhaps coordinate this effect with changes in visceromotor activity during the initial stages of maternal aggressive motivation and/or anxiogenic responses.

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct.

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n=22) or immunoblotting (n=6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.

Induction of uPA release in human peripheral blood lymphocytes by [deamino-Cysl,D-Arg8]-vasopressin (dDAVP).

[deamino-Cys(l),d-Arg(8)]-vasopressin (dDAVP), known to be an arginine vasopressin (AVP) V(2) receptor agonist, is an agent that increases fibrinolytic activity levels in plasma after its infusion into the human body. However, mechanisms underlying an increase and exact localization of the extrarenal dDAVP-responsive V(2) receptor remain unclarified. Two AVP receptors, V(1a) and V(2), and a related oxytocin (OT) receptor were found to be expressed in human lymphocytes. Furthermore, we found an increase of fibrinolytic activity in the medium of peripheral lymphocytes obtained from human volunteers less than 20 min after dDAVP infusion. The increased activity was also detected in the medium after incubating the lymphocytes in the presence of dDAVP in vitro, being highest at 20 min after the incubation. In accord with the increased fibrinolytic activity, the levels of urokinase-type plasminogen activator (uPA) in the medium were also increased. However, there was no significant difference of plasminogen activator inhibitor-1 (PAI-1), pro-uPA, and tissue-type plasminogen activator (tPA) concentrations in the medium between dDAVP treatment and control. When lymphocytes were preincubated with a V(2) receptor antagonist [Adamantaneacetyl(1),O-Et-d-Tyr(2),Val(4),Aminobutyryl(6),Arg(8,9)]-vasopressin, the dDAVP-induced uPA increase was diminished. In contrast, preincubation with a V(1) receptor antagonist, [beta-Mercapto-beta,beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2),Arg(8)]-vasopressin, prior to dDAVP treatment resulted in a greater increase of the uPA concentration in the medium than with the dDAVP treatment alone. Thus it was suggested that dDAVP may induce uPA release from human lymphocytes via V(2) receptor-mediated reaction, and also via cross-talk between V(1) and V(2) receptors.

Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4.

The glutamine(4) residue in [deamino-Cys(1)]arginine vasopressin (dAVP) was replaced by a broad series of aliphatic, aromatic, polar, and charged amino acids to give the following peptides: d[Gly(4)]AVP (1), d[Ala(4)]AVP (2), d[Abu(4)]AVP (3), d[Nva(4)]AVP (4), d[Nle(4)]AVP (5), d[Leu(4)]AVP (6), d[Ile(4)]AVP (7), d[Thi(4)]AVP (8), d[Phe(4)]AVP (9), d[Tyr(4)]AVP (10), d[Trp(4)]AVP (11), d[Asn(4)]AVP (12), d[Ser(4)]AVP (13), d[Thr(4)]AVP (14), d[Dap(4)]AVP (15), d[Dab(4)]AVP (16), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), d[Arg(4)]AVP (19), d[Har(4)]AVP (20), and d[Glu(4)]AVP (21). All peptides were synthesized by solid-phase methods using BOC chemistry for all but one peptide (8), which required the use of Fmoc chemistry. The binding and functional properties of these position 4 substituted analogues of dAVP (d[X(4)]AVP) and the previously reported d[Cha(4)]AVP (Derick et al. Endocrinology 2002, 143, 4655-4664) were evaluated on human arginine vasopressin (AVP) V(1a), V(1b), and V(2) receptors and on the human oxytocin (OT) receptor expressed in living Chinese hamster ovary (CHO) cells. Binding studies revealed that broad modifications of the fourth residue of dAVP do not significantly alter affinity for the human V(1b) receptor. Only aromatic (Phe, Tyr, Trp) or negatively charged (Glu) residues reduce V(1b) affinity. By contrast, the human V(1a) and more particularly the human V(2) and the OT receptors are more sensitive to many of these modifications. Thus, the replacement of the Gln(4) residue of dAVP by aliphatic (Leu, Cha) or positively charged (Orn, Lys, Arg, Har) amino acids led to analogues exhibiting drastic reductions of their affinity for the human V(1a), V(2), and OT receptors. Consequently, in addition to the previously reported d[Cha(4)]AVP, peptides 6 and 17-20 display excellent selectivities for the human V(1b) receptor. The key structural requirement responsible for optimal V(1b) selectivity appears to be the length and branching of the aliphatic side chain of the fourth residue of dAVP. Functional studies performed on CHO cells expressing the different human AVP/OT receptors confirm the V(1b) selectivity of peptides 6, 17, 18, 20, and d[Cha(4)]AVP. However, d[Arg(4)]AVP (19), which triggers an excellent coupling between the human V(2) receptor and adenylyl cyclase, was found to exhibit both V(1b) and V(2) agonism in functional tests. More interestingly, these functional experiments revealed that, depending on the AVP/OT receptor, a given d[X(4)]AVP analogue may behave as a full agonist or as a partial agonist. This strongly suggests that the fourth residue of dAVP plays an important role in the coupling between the hormone-receptor complex, the heterotrimeric G protein, and the effectors. In conclusion, the synthesis of these d[X(4)]AVP analogues led to the discovery of new V(1b) agonists with high affinity and greatly enhanced selectivities. Thus, in addition to d[Cha(4)]AVP, d[Leu(4)]AVP (6), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), and d[Har(4)]AVP (20) are useful new tools for studying the structure and the function of the human V(1b) receptor.

Effect of rosiglitazone on spontaneous and clomiphene citrate-induced ovulation in women with polycystic ovary syndrome.

OBJECTIVE: In women suffering from polycystic ovary syndrome (PCOS), correction of hyperinsulinemia results enhances spontaneous ovulation or alternatively, the responsiveness to ovulation induction agents such as clomiphene citrate (CC). We investigated the effect of rosiglitazone maleate on ovulation induction in overweight and obese, CC-resistant women with PCOS. DESIGN: Double-blind, randomized, placebo-controlled trial. SETTING: Academic reproductive endocrinology clinic. PATIENT(S): Overweight and obese women with clinical and laboratory manifestations of PCOS who desired pregnancy and were resistant to CC. INTERVENTION(S): Twenty-five women were randomized into two treatment groups. Subjects in Group I (n = 12) were randomized to receive rosiglitazone 4 mg b.i.d. with a placebo on cycle days 5-9. Group II (n = 13) was randomized to receive rosiglitazone 4 mg b.i.d. with CC on cycle days 5-9. The duration of the study was 2 months. MAIN OUTCOME MEASURE(S): The primary outcome was ovulation as defined by luteal serum progesterone greater than 5 ng/dL assessed on days 21, 24, and 28 of the cycle. Secondary outcomes were pregnancy and changes in insulin sensitivity, serum lipoproteins, and androgens. RESULT(S): Overall, 14 of 25 (56%) women, who were previously resistant to CC, successfully ovulated. In subjects taking rosiglitazone alone (Group I), 4 of 12 (33%) subjects ovulated compared with 10 of 13 (77%) women randomized to rosiglitazone with CC (Group II) (P=.04, Fisher's exact). One subject in Group I became pregnant, resulting in one uncomplicated live birth; two subjects in Group II conceived, with one successful live birth and one first trimester, spontaneous abortion. For all subjects, fasting insulin declined from 29.4 +/- 13.8 microU/mL to 17.3 +/- 7.8 microU/mL after rosiglitazone (P=.003, paired t-test). Although mean levels of total testosterone (T) and dehydroepiandrosterone sulfate (DHEAS) did not decline significantly, sex hormone-binding globulin (SHBG) did increase from 0.7 +/- 0.3 microg/dL to 1.0 +/- 0.3 microg/dL after rosiglitazone therapy (P=.001, paired t test). There was also a decrease in luteinizing hormone (LH) from 9.4 +/- 6.3 mU/mL to 7.2 +/- 3.7 mU/mL (P=.01). Lipoproteins including total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides did not change. CONCLUSIONS: Short-term rosiglitazone therapy enhances both spontaneous and clomiphene-induced ovulation in overweight and obese women with PCOS. Rosiglitazone therapy improves insulin sensitivity and decreases hyperandrogenemia primarily through increases in SHBG.

Conformations and receptor activity of desmopressin analogues, which contain gamma-turn mimetics or a psi[CH(2)O] isostere.

Three analogues of the antidiuretic drug desmopressin ([1-desamino,8-D-arginine]vasopressin) have been prepared. In two of these, gamma-turn mimetics based on a morpholin-3-one framework have been inserted instead of residues Phe3-Asn5, whereas the third analogue has a methylene ether isostere in place of the amide bond between residues 3 and 4. The three analogues were used to probe if the structure determined for desmopressin in aqueous solution, which contains an inverse gamma-turn centered around Gln4, is important in interactions with the vasopressin V(2) receptor. Conformational studies revealed that the analogues that contain either an inverse gamma-turn mimetic or a methylene ether isostere mimicked the conformation of desmopressin fairly well and very well, respectively. Despite this, the analogues displayed only very low agonistic activities at the vasopressin V(2) receptor. Consequently, an inverse gamma-turn involving residues Phe3-Asn5 does not appear to be important when desmopressin is bound to the V(2) receptor. In addition, it was concluded that the amide bond between Phe3 and Gln4 in desmopressin is crucial for interactions with the antidiuretic V(2) receptor.

Stereoselective synthesis of Psi[CH(2)O] pseudodipeptides and conformational analysis of a PhePsi[CH(2)O]Ala containing analogue of the drug desmopressin.

A method for synthesis of XaaPsi[CH(2)O]Ala/Gly pseudodipeptides in good yields and excellent diastereoselectivity from azido alcohols and (R)-2-chloropropionic acid or tert-butyl bromoacetate has been developed. Insertion of one of the pseudodipeptide building blocks in the peptide drug desmopressin revealed that methylene ether isosteres may have only a minor influence on the secondary structure of peptides.

Changes in plasma arginine vasotocin (AVT) concentration and dorsal aortic blood pressure following AVT injection in the teleost Platichthys flesus.

Dorsal aortic blood pressure and plasma arginine vasotocin (AVT) concentrations have been assessed in free swimming, chronically cannulated flounder following AVT injection. Intraarterial AVT at doses of 4.76 x 10(-12) mol.kg-1 and greater caused a biphasic change in blood pressure (an initial fall followed by a sustained pressor response). Doses above 4.76 x 10(-12) mol.kg-1 were associated with plasma AVT concentrations (20 min after injection) 2-3 orders of magnitude greater than the physiological range and must be considered pharmacological. Injection of the lowest pressor AVT dose (4.76 x 10(-12) mol.kg-1), 20 min after injection, increased plasma AVT concentrations to 23.4 +/- 6.1 fmol x ml-1. This increase is close to plasma AVT concentrations recently reported in untreated fish; however, in the initial period after injection plasma levels were calculated to be considerably higher than the physiological range. These results confirm that AVT is pressor in flounder but suggest that the pressor response may occur only at circulating AVT levels above the normal physiological range. The biphasic response to AVT and the differing responses to mammalian V1 and V2 type receptor agonists in the current work suggests that AVT may contribute to regional blood flow distribution in teleosts rather than blood pressure regulation.

A V1-type receptor for mediating the neurohypophysial hormone-induced ACTH release in trout pituitary.

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss. Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D50) showed that these peptides affected ACTH release in the following order of potency: CRF (8 x 10(-13) M) > AVT (2 x 10(-10) M) > IT (10(-7) M). Maximal responses (Dmax) were obtained for hormonal concentrations of 10(-10) M, 10(-8) M and 10(-6) M respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides. Specific V1 or V2 agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V1 agonist, [Phe2, Orn8]-oxytocin, stimulated ACTH release while the V2 agonist, [deamino1, Val4, D-Arg8]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V1 agonist with 10(-7) M and 7 x 10(-9) M respectively, and were in the range of values obtained with natural peptides. The V1 antagonist, [d(CH2)5(1), O-Me-Tyr2, Arg8]-vasopressin, and the V2 antagonist, [d(CH2)5(1), D-Ile2, Ile4, Arg8, Ala9]-vasopressin, maximally reversed the 10(-9) M AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 x 10(-10) M concentration of the analogues and a D50 approximately 2 x 10(-11) M. These results demonstrated the presence of only one V1-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V1b-type.

Glycosylated peptide hormones: pharmacological properties and conformational studies of analogues of [1-desamino,8-D-arginine]vasopressin.

Two analogues of the antidiuretic drug [1-desamino,8-D-arginine]vasopressin (DDAVP), which have a glycosylated serine at position 4, have been prepared by Fmoc solid phase peptide synthesis. The glycosylated analogues had significantly higher bioavailabilities than the nonglycosylated [D-Tyr2,Ser4]DDAVP and DDAVP on intraintestinal administration in rat. The improved bioavailability resulted from an increased absorption from the small intestine and most likely from an increased stability toward enzymatic degradation, whereas plasma clearance was either unaffected or slightly increased by the glycosylation. The glycosylated analogues displayed only very low agonistic and antagonistic activities at the vasopressin V2-receptor. Conformational studies performed by 1H NMR spectroscopy did not reveal any major influence from glycosylation on the conformation of the peptide backbone. The lack of receptor binding displayed by the analogues is therefore most likely explained by steric repulsion between the carbohydrate moiety and the vasopressin receptor which prevents receptor binding.