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Ke Y.,Endoceutics Inc. | Gonthier R.,Endoceutics Inc. | Labrie F.,Endoceutics Inc.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2017

In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315 > 97 & 315 > 109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315 > 97–315 > 109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315 > 109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1 × 3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70% ∼ 80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1 × 3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5–2000 pg/mL, a good linearity is observed with R > 0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74 ∼ 91.7 pg/mL, which is well below the reported concentrations of 314 pg/mL ∼ 942 pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mL ∼ 18.6 ng/mL. © 2017 Elsevier B.V.


Bertin J.,EndoCeutics Inc | Bertin J.,Laval University
American journal of obstetrics and gynecology | Year: 2014

OBJECTIVE: Estrogens are well recognized to have beneficial effects on vulvovaginal atrophy because of menopause. The distribution of estrogen receptors and enzymes responsible for estradiol (E2) formation within the vagina may provide insight into how dehydroepiandrosterone, a precursor of both estrogens and androgens, improves vulvovaginal atrophy.CONCLUSION: The enzymes responsible for E2 formation as well as ERs are expressed mainly in the superficial layer of the stratified epithelium as well as the muscle layer of the vagina. The present data provide morphologic and biochemical support for the role of local dehydroepiandrosterone transformation into estrogens in regulating epithelial cell maturation, pH, fluid secretion, smooth muscle activity, and blood flow regulation in the primate vagina.STUDY DESIGN: The purpose of the study was to determine where the steroidogenic enzymes responsible for E2 formation as well as estrogen receptors are localized in vaginal specimens collected from cynomolgus monkeys (Macaca fascicularis), the closest model to the human. HSD3B1, HSD17B1, HSD17B5, HSD17B12, aromatase (CYP19A1), estrogen receptor (ER)-α, and ER-β were measured or localized by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence. Estrogens were quantified by liquid chromatography/tandem mass spectrometry.RESULTS: All steroidogenic enzymes and estrogen receptors are localized mainly in the superficial layer of the stratified squamous epithelium, blood vessel walls, and muscle fibers of the vagina. Immunolabeling of HSD17B5 and HSD17B12 shows that these enzymes are uniformly distributed from the basal membrane to the superficial keratinized cells, whereas HSD3B1 and aromatase are particularly localized in the outer (external) portion of the epithelial layer. ER-α and ER-β are also distributed within the vaginal epithelium, with expression especially elevated at the basal membrane level. Copyright © 2014 Elsevier Inc. All rights reserved.


Dury A.Y.,EndoCeutics Inc. | Ke Y.,EndoCeutics Inc. | Gonthier R.,EndoCeutics Inc. | Isabelle M.,EndoCeutics Inc. | And 2 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2015

Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno-based assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1-S), androsterone sulfate (ADT-S), pregnenolone sulfate (Preg-S) and allopregnanolone sulfate (Allopreg-S). E1-S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1-S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40 ng/mL, 40 pg/mL, 5 ng/mL, 1.5 ng/mL and 0.25 ng/mL for DHEA-S, E1-S, ADT-S, Preg-S and Allopreg-S, respectively. A good linearity is obtained with R > 0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1-S, ADT-S and Preg-S while for Allopreg-S, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. © 2015 Elsevier Ltd.


Labrie F.,EndoCeutics Inc.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2015

Following the arrest of estradiol secretion by the ovaries at menopause, all estrogens and all androgens in postmenopausal women are made locally in peripheral target tissues according to the physiological mechanisms of intracrinology. The locally made sex steroids exert their action and are inactivated intracellularly without biologically significant release of the active sex steroids in the circulation. The level of expression of the steroid-forming and steroid-inactivating enzymes is specific to each cell type in each tissue, thus permitting to each cell/tissue to synthesize a small amount of androgens and/or estrogens in order to meet the local physiological needs without affecting the other tissues of the organism. Achieved after 500 million years of evolution, combination of the arrest of ovarian estrogen secretion, the availability of high circulating levels of DHEA and the expression of the peripheral sex steroid-forming enzymes have permitted the appearance of menopause with a continuing access to intratissular sex steroids for the individual cells/tissues without systemic exposure to circulating estradiol. In fact, one essential condition of menopause is to maintain serum estradiol at biologically inactive (substhreshold) concentrations, thus avoiding stimulation of the endometrium and risk of endometrial cancer. Measurement of the low levels of serum estrogens and androgens in postmenopausal women absolutely requires the use of MS/MS-based technology in order to obtain reliable accurate, specific and precise assays. While the activity of the series of steroidogenic enzymes can vary, the serum levels of DHEA show large individual variations going from barely detectable to practically normal "premenopausal" values, thus explaining the absence of menopausal symptoms in about 25% of women. It should be added that the intracrine system has no feedback elements to adjust the serum levels of DHEA, thus meaning that women with low DHEA activity will not be improved without external supplementation. Exogenous DHEA, however, follows the same intracrine rules as described for endogenous DHEA, thus maintaining serum estrogen levels at substhreshold or biologically inactive concentrations. Such blood concentrations are not different from those observed in normal postmenopausal women having high serum DHEA concentrations. Androgens, on the other hand, are practically all made intracellularly from DHEA by the mechanisms of intracrinology and are always maintained at very low levels in the blood in both pre- and postmenopausal women. Proof of the importance of intracrinology is also provided, among others, by the well-recognized benefits of aromatase inhibitors and antiestrogens used successfully for the treatment of breast cancer in postmenopausal women where all estrogens are made locally. Each medical indication for the use of DHEA, however, requires clinical trials performed according to the FDA guidelines and the best rules of clinical medicine. © 2014 Elsevier Ltd.


Pelletier G.,Laval University | Ouellet J.,Laval University | Martel C.,EndoCeutics Inc. | Labrie F.,Laval University | Labrie F.,EndoCeutics Inc.
Journal of Sexual Medicine | Year: 2013

Introduction: We have recently reported that dehydroepiandrosterone (DHEA) increases the density of nerve fibers in the ovariectomized (OVX) rat vagina. Aim: To better define the mechanism of action of DHEA, we have examined the effect of DHEA, conjugated estrogens (premarin) and the potent blocker of estrogen action acolbifene on the innervation in the lamina propria in the OVX rat vagina. Methods: Female Sprague-Dawley rats (10-12 weeks old) were used. Innervation of the vagina was examined 9 months after OVX and was compared to that of OVX animals treated daily with DHEA (80mg/kg) by topical application on the skin, premarin (0.5mg/kg) orally as well as acolbifene (2.5mg/kg) orally administrated alone or in combination with DHEA or premarin. Main Outcome Measures: Four histological sections from each vagina (5 animals/group) were immunostained using antibodies to the panneuronal marker protein gene product 9.5 (PGP 9.5). The areas were measured by stereological analysis. Results: OVX reduced the area of the lamina propria to 44% of the intact value, an effect which was reversed to 69% and 84% of the intact value by DHEA and premarin, respectively, at the doses used. When acolbifene was used, no inhibition of the stimulatory effect of DHEA was observed, while the action of premarin was completely blocked. Evaluation of the PGP 9.5 fiber density revealed that DHEA treatment increased the density of fibers by 60% compared to OVX animals, while a further 27% increase was observed when acolbifene was combined with DHEA. Premarin, on the other hand, had no effect on the density of PGP 9.5 fibers. Conclusions: Considering that the antiestrogen acolbifene had no inhibitory effect on the effect of DHEA in rat vagina while blocking the stimulatory effect of premarin, the present data indicate that DHEA exerts its stimulatory effect on the fiber density through an androgenic action. © 2013 International Society for Sexual Medicine.


Labrie F.,Endoceutics Inc. | Labrie F.,Laval University | Martel C.,Endoceutics Inc. | Balser J.,Veristat Inc.
Menopause | Year: 2011

Objective: Because the exclusive source of sex steroids (at least estrogens) after menopause is recognized to be dehydroepiandrosterone (DHEA), this study examines the interindividual variability of serum DHEA and its metabolites as well as the contribution of the ovary to global sex steroid physiology in postmenopausal women. Methods: Serum levels of DHEA and 11 of its metabolites were measured by gas or liquid chromatography/mass spectrometry in 442 intact and 71 ovariectomized postmenopausal women aged 42 to 74 years. Results: With a mean ± SD concentration of 2.03 ± 1.33 ng/mL, serum DHEA in intact postmenopausal women is highly variable with 5th and 95th centiles at 0.55 and 4.34 ng/mL, respectively, for a 7.9-fold difference. A comparable variability is observed for the 11 metabolites of DHEA. The 22.3% higher serum DHEA in intact compared with ovariectomized women is accompanied by parallel differences for all the other steroids, thus indicating that all sex steroids originate from circulating DHEA in postmenopausal women with no direct secretion of active estrogens or androgens by the postmenopausal ovary. Conclusions: The 7.9-fold difference between low and high serum DHEA levels provides an explanation for the lack of signs of hormone deficiency in some women, whereas most of them have symptoms or signs. The approximately 20% contribution of the ovary to the total pool of DHEA with no direct secretion of estrogens or androgens in the circulation could possibly explain the reported negative effect of oophorectomy on longevity, especially from coronary heart disease events. © 2011 by The North American Menopause Society.


Trademark
Endoceutics Inc. | Date: 2016-12-23

pharmaceutical preparations for the prevention and treatment of breast and uterine cancer, Alzheimers disease, medical conditions related to menopause, bone loss, muscle loss, type 2 diabetes, fat accumulation, osteoporosis, hot flushes, skin atrophy, memory loss, and cognition loss.


Trademark
Endoceutics Inc. | Date: 2013-06-14

pharmaceutical preparations for the prevention and treatment of breast and uterine cancer, Alzheimers disease, medical conditions related to menopause, bone loss, muscle loss, type 2 diabetes, fat accumulation, osteoporosis, hot flushes, skin atrophy, memory loss, and cognition loss.


Trademark
Endoceutics Inc. | Date: 2015-09-21

Pharmaceutical preparations for the prevention and treatment of vaginal atrophy, decreased libido and sexual dysfunction.


Trademark
Endoceutics Inc. | Date: 2016-11-22

Pharmaceutical preparations and substances for the prevention and treatment of menopause, sexual dysfunction, vaginal atrophy, decreased libido, gynaecological disorders affecting menstruation, premenstrual syndrome, sterility, menstrual syndromes, dysmenorrhea, infertility, genital tract infections, pelvic inflammatory diseases, endometriosis, vaginal discomfort due to menopausal atrophic changes, anatomical abnormalities; pharmaceutical preparations and substances for the prevention and treatment of menopausal problems, neoplasia, breast neoplasm, breast cancer, mastopathy, mastodynia, breast pain; pharmaceutical preparations and substances for the prevention and treatment of the endocrine system; pharmaceutical preparations and substances for the prevention and treatment of oily skin, acne, male baldness; pharmaceutical preparations and substances for the prevention and treatment of prostate cancer and benign prostate enlargement; pharmaceutical preparations and substances for the prevention and treatment of obesity, high cholesterol, diabetes, loss of muscle mass and strength, bone loss. Wholesale distributorship of pharmaceuticals for others. Custom manufacture of pharmaceutical products to the order and specification of others. Pharmaceutical educational services and patient educational services, namely, providing classes, workshops and seminars in the fields of treatment and prevention of cancer and endocrine-related disorders and diseases. Research and development in the field of pharmaceuticals; conducting early evaluations in the field of new pharmaceuticals; medical laboratory services; laboratory research in the fields of treatment and prevention of cancer and endocrine-related disorders and diseases.

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