Kisspeptins to predict and treat delayed puberty

Abstract

To provide a method for identifying a subject as having, being at risk for, or having an attenuated form of pathologic hypogonadotropic hypogonadism.SOLUTION: The method comprises measuring a baseline level of LH in a subject; administering to the subject a stimulating dose of kisspeptin or a kisspeptin analog, for example, a dose comprising 0.08 to 15 nmol / kg kisspeptin-10 administered via intravenous bolus, or a dose comprising 0.8 to 500 nmol / kg kisspeptin-10 administered via subcutaneous (SC) injection; measuring at least one LH level after administration of the stimulating dose; comparing the baseline level of LH in the subject to the level after administration of the stimulating dose; and identifying a subject with delayed puberty who has a level of LH after administration of the stimulating dose that is not significantly different from the baseline level of LH as having pathologic hypogonadotropic hypogonadism.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claim This application is a continuation of U.S. Provisional Patent Application No. 62 / 858,479, filed June 7, 2019. The entire contents of the foregoing are incorporated herein by reference. .

[0002] Federally sponsored research or development This invention was made in part with funding from Grant No. HD043341 awarded by the National Institutes of Health; and with government support under Grant No. FD005712 awarded by the Food and Drug Administration. The Government has certain rights in this invention.

[0003] Technical Field Described herein are methods for diagnosing and treating delayed puberty using kisspeptin. It is listed. [Background technology]

[0004] Idiopathic hypogonadotropic hypogonadism (IHH) is a condition in which GnRH receptors are released from the hypothalamus. Rare disorders caused by the absence of neurons or regulatory neural circuits This is due to the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This results in a loss of smell and failure to achieve normal reproductive function by age 18. In cases where IHH is present, it is treated as Kallmann syndrome (KS). Untreated patients may present with infantile sexual It remains the same.

[0005] Children who exhibit delayed puberty pose a formidable challenge to clinicians, which is the question of which children ultimately This is because it is difficult to predict which children will progress during adolescence and which will not (1, 2) Some causes of delayed puberty are readily recognizable, such as primary hypogonadism. , anatomical lesions in the hypothalamic / pituitary region, and functional hypogonadotropic hypogonadism Disorders (i.e., reproductive-endocrine axis disorders due to chronic illness, stress, or negative energy balance) However, these symptoms are indicative of pediatric endocrinology treatment, and are not considered to be a symptom of a 3-year-old girl. It explains only 6–47% of delayed puberty in women and 11–29% of delayed puberty in boys (3–5). For the majority of girls and boys with delayed puberty, providers have noted markedly different outcomes, including constitutional delay. Two possible diagnoses remain: constitutional delay of puberty and IHH (1). starts late (or starts and then pauses briefly), but eventually starts and reaches full maturity. It is a self-limited condition that progresses to the achievement of human reproductive endocrine function (6). In contrast, IHH It is a pathological disorder that requires treatment (7). Currently, constitutional delay is not considered to be a pathological disorder that is a progressive disorder from IHH. There is no reliable way to distinguish between these two. Summary of the Invention

[0006] As used herein, reproductive endocrine dysfunction, optionally pathological hypogonadotropic gonadism A method of treating a subject having hypolipidemic syndrome is provided. The method comprises administering a therapeutically effective amount of (i) multiple Several doses (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 2, 3 , 4, 5, or 6 times a day) kisspeptin or kisspeptin analogues, and / or (ii) administering an opioid antagonist or mixed agonist-antagonist to the subject; As used herein, multiple doses of kisspeptin or kisspeptin analogs are administered. and / or (ii) an opioid antagonist or mixed agonist-agonist. Treatment of patients with pathological hypogonadotropic hypogonadism, including administering a steroid antagonist (i) kisspeptin or kisspeptin analogues for use in a method of treating an elephant and / or (ii) an opioid antagonist or mixed agonist-agonist Compositions comprising the antagonist are provided.

[0007] In some embodiments, multiple doses are administered at intervals of 1 to 6 hours, preferably 2 to 3 hours. at intervals of at least 2-3 days, preferably at least 1, 2, 3, 4, 6, or 12 It is administered over a period of months.

[0008] In some embodiments, the multiple doses are each administered by intravenous bolus (IVB), subcutaneous 0.08–2.4 nmol / kg q.s. administered by injection (SC) or pump Contains a dose equivalent to Peptin-10.

[0009] In some embodiments, each of the multiple doses is 0.2-0.3 nmol / kg. Kisspeptin-10, preferably equivalent to 0.24 nmol / kg kisspeptin-10 Includes dosage.

[0010] In some embodiments, the multiple doses include 2.4 to 24 nmol / kg kisspeptide. In some embodiments, the method comprises administering to a subject a dose of at least one compound equivalent to 100 mg of benzophenone-10. , at least one supraphysiological dose is the first dose of a multiple dose regimen, or the first two or Three or more doses are administered, optionally all in multiple doses.

[0011] In some embodiments, the method further comprises administering a therapeutically effective amount of an opioid antagonist or or a mixed agonist-antagonist to a subject. wherein the opioid antagonist is naloxone or narotrexone, or or a mixed agonist-antagonist is buprenorphine.

[0012] In some embodiments, the method further comprises administering to a subject a subject a dose of one or more gonadotropins (e.g., luteinizing agents). Furthermore, administration of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) Included.

[0013] As used herein, also refers to reproductive endocrine dysfunction (RED), such as pathological hypogonadotropin. Identifying subjects with, at risk for, or with milder forms of gonadotropin-dependent hypogonadism The method includes measuring a baseline level of LH in a subject. administering a stimulating dose of kisspeptin or a kisspeptin analogue, e.g., an intravenous bolus; Kisspeptin-10 at 0.08–15 nmol / kg administered by intravenous venipuncture (IBV) or administered by subcutaneous (SC) injection, 0.8–500 nmol / kg administering to the subject a dose comprising kisspeptin-10 of at least one L within about 10, 15, 20, 30, 45, or 60 minutes after administration of the dose measuring LH levels, determining baseline levels of LH in the subject after administration of a stimulating dose; Comparison of LH levels and LH after administration of a stimulatory dose equivalent to baseline LH levels (e.g., less than 3.5, 3, 2.5, or 2 times baseline), Subjects with delayed puberty are considered to have RED, e.g., pathological hypogonadotropic hypogonadism. This includes identifying something as something.

[0014] Alternatively, the method may involve administering a priming dose, e.g., an intravenous bolus (IVB). , intravenous doses containing 0.08–15 nmol / kg kisspeptin-10, or subcutaneous ( 0.8 to 500 nmol / kg kisspeptin-10 administered by SC injection administering a stimulating dose of kisspeptin or a kisspeptin analog to a subject; at least one dose within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose. At least one LH level should be measured, and the LH level after the administration of the stimulating dose should be used as the reference level. Comparison and stimulation doses below (or not significantly different from) reference levels of LH A subject with delayed puberty is considered to have a RED, e.g., pathological hypogonadism, having an LH level after administration of This may include identifying the patient as having tropic hypogonadism.

[0015] In some embodiments, the method is directed to treating reproductive endocrine dysfunction, such as pathological hypogonadotropin. The present invention includes administering to the identified subject a treatment for hypogonadism.

[0016] In some embodiments, the treatment comprises multiple doses of kisspeptin, or kisspeptin In some embodiments, the multiple doses include administering the analog over a 1-6 hour period. at intervals of at least 2 to 3 days, preferably at intervals of at least 1 In some embodiments, the multiple doses are each administered over a period of 1 to 12 months. Administered by intravenous bolus (IVB), subcutaneous injection (SC) or pump, 0.0 Contains doses equivalent to 8 to 2.4 nmol / kg kisspeptin-10. In this case, multiple doses are administered at 0.2 to 0.3 nmol / kg kisspeptin-1. 0, preferably 0.24 nmol / kg kisspeptin-10 equivalent dose. In some embodiments, the multiple doses include 2.4 to 24 nmol / kg kisspeptin-1. In some embodiments, at least one supraphysiological dose is equal to or greater than 0. At least one supraphysiological dose may be the first dose of multiple doses or the first two or more The above doses, optionally all of the multiple doses, are administered.

[0017] In some embodiments, the method further comprises administering a therapeutically effective amount of an opioid antagonist or or a mixed agonist-antagonist to a subject. wherein the opioid antagonist is naloxone or naltrexone, or A mixed agonist-antagonist is buprenorphine.

[0018] In some embodiments, the treatment is gonadal steroid replacement therapy, e.g., in men testosterone in men and estrogen and / or progesterone / progesterone in women Further comprising administering a gestin.

[0019] In some embodiments, the method further comprises administering to a subject a therapeutically effective amount of one or more gonadotropins (e.g., administration of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) Further includes:

[0020] In some embodiments, multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours. It is administered for at least 2 to 3 days, preferably for at least 1 to 12 months.

[0021] Unless otherwise specified, all technical and scientific terms used herein refer to the The term "compound" has the same meaning as commonly understood by a person skilled in the art to which the term pertains. Methods and materials for doing so are described herein and are similar to those of others known in the art. Any suitable methods and materials may also be used. Materials, methods and examples are for illustrative purposes only. All publications mentioned herein are for illustrative purposes only and are not intended to be limiting. All products, patent applications, patents, sequences, database entries, and other references are hereby incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0022] Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as the claims. It will be clear from the surrounding area. [Brief explanation of the drawings]

[0023] [Figure 1] Recruitment and participation overview. [Figure 2-1] Neuroendocrine characteristics in children presenting with delayed or arrested puberty. A schematic of the protocol is shown in Panel A. Further details of the protocol are provided in Reference (20). At the first visit, participants had serum LH measured, overnight spontaneous pulsatility assessed, and responses to kisspeptin and GnRH charted. Participants then received exogenous pulsatile GnRH to enhance pituitary responsiveness to GnRH. They then returned for a second visit to measure LH secretion in response to kisspeptin and GnRH after this pituitary "priming." Results are shown for Participant A (B), a "kisspeptin non-responder," and Participant B (C), a "kisspeptin responder." [Figure 2-2]Neuroendocrine characteristics in children presenting with delayed or arrested puberty. A schematic of the protocol is shown in Panel A. Further details of the protocol are provided in Reference (20). At the first visit, participants had serum LH measured, overnight spontaneous pulsatility assessed, and responses to kisspeptin and GnRH charted. Participants then received exogenous pulsatile GnRH to enhance pituitary responsiveness to GnRH. They then returned for a second visit to measure LH secretion in response to kisspeptin and GnRH after this pituitary "priming." Results are shown for Participant A (B), a "kisspeptin non-responder," and Participant B (C), a "kisspeptin responder." [Figure 3] Differential response to kisspeptin in children who did and did not progress through puberty. Girls (open circles) and boys (closed circles) presenting with delayed or arrested puberty underwent a kisspeptin stimulation test to assess changes in luteinizing hormone in response to exogenous kisspeptin (ΔLH kisspeptin). Participants were then followed until age 18 to determine whether they had spontaneously progressed through puberty. [Figure 4-1] Further hormonal evaluation of children who did or did not progress through puberty. Girls (open circles) and boys (closed circles) showing delayed and arrested puberty were evaluated for serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at presentation (A and B, respectively) and the change in LH in response to exogenous gonadotropin-releasing hormone (GnRH, panel C). Boys were further evaluated for serum inhibin B (D). [Figure 4-2] Further hormonal evaluation of children who did or did not progress through puberty. Girls (open circles) and boys (closed circles) showing delayed and arrested puberty were evaluated for serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at presentation (A and B, respectively) and the change in LH in response to exogenous gonadotropin-releasing hormone (GnRH, panel C). Boys were further evaluated for serum inhibin B (D). [Figure 5-1]LH response to kisspeptin. Dotted lines represent administration time points. (Downward-pointing filled triangle) = LH pulse determined by the modified Santen & Bardin criteria. A: Healthy male. Genetics not assessed. Kisspeptin 0.313 μg / kg IVB at 6 hours. (+) Response. B: KS male. PROKR2 c.518T>G p.L173R heterozygous, DMXL2 c.4016T>G p.V1339G heterozygous. Kisspeptin 0.313 μg / kg IVB at 6 hours. (-) Response. C: KS female. Genetics not assessed. LH response graphed as a function of kisspeptin dose. (+) LH response across a range of doses. D: KS male. CHD7 c.2417T>Cp.M806T heterozygous; PROKR2 c.254G>Ap.R85H heterozygous. Same dose as panel C. (+) LH response. All data are graphed on the same Y-axis. [Figure 5-2] LH response to kisspeptin. Dotted lines represent administration time points. (Downward-pointing filled triangle) = LH pulse determined by the modified Santen & Bardin criteria. A: Healthy male. Genetics not assessed. Kisspeptin 0.313 μg / kg IVB at 6 hours. (+) Response. B: KS male. PROKR2 c.518T>G p.L173R heterozygous, DMXL2 c.4016T>G p.V1339G heterozygous. Kisspeptin 0.313 μg / kg IVB at 6 hours. (-) Response. C: KS female. Genetics not assessed. LH response graphed as a function of kisspeptin dose. (+) LH response across a range of doses. D: KS male. CHD7 c.2417T>Cp.M806T heterozygous; PROKR2 c.254G>Ap.R85H heterozygous. Same dose as panel C. (+) LH response. All data are graphed on the same Y-axis. [Figure 5-3]LH response to kisspeptin. Dotted lines represent administration time points. (Downward-pointing filled triangle) = LH pulse determined by the modified Santen & Bardin criteria. A: Healthy male. Genetics not assessed. Kisspeptin 0.313 μg / kg IVB at 6 hours. (+) Response. B: KS male. PROKR2 c.518T>G p.L173R heterozygous, DMXL2 c.4016T>G p.V1339G heterozygous. Kisspeptin 0.313 μg / kg IVB at 6 hours. (-) Response. C: KS female. Genetics not assessed. LH response graphed as a function of kisspeptin dose. (+) LH response across a range of doses. D: KS male. CHD7 c.2417T>Cp.M806T heterozygous; PROKR2 c.254G>Ap.R85H heterozygous. Same dose as panel C. (+) LH response. All data are graphed on the same Y-axis. [Figure 6] Re-graphed data from FIG. 5C showing the response of KS females to kisspeptin at variable doses at 2-hour frequency. [Figure 7-1] Baseline neuroendocrine profiling. A. Study schematic. B. Study subject with IHH undergoing 8-hour sampling in 2010–2011. C. Healthy sister in early follicular phase (EFP). E2 = estradiol. P = progesterone. LH = luteinizing hormone. [Figure 7-2] Baseline neuroendocrine profiling. A. Study schematic. B. Study subject with IHH undergoing 8-hour sampling in 2010–2011. C. Healthy sister in early follicular phase (EFP). E2 = estradiol. P = progesterone. LH = luteinizing hormone. [Figure 8-1]Baseline study of response to kisspeptin and GnRH. A. Study schematic, B. Study subjects. Arrows indicate luteinizing hormone pulses detected by the algorithm. K = kisspeptin-10 intravenous bolus; subscripts indicate dose: 1 = 0.24 nmol / kg, 2 = 0.72 nmol / kg, 3 = 2.4 nmol / kg. G = GnRH IVB 75 ng / kg. E2 = estradiol, FSH = follicle-stimulating hormone, LH = luteinizing hormone. [Figure 8-2] Baseline study of response to kisspeptin and GnRH. A. Study schematic, B. Study subjects. Arrows indicate luteinizing hormone pulses detected by the algorithm. K = kisspeptin-10 intravenous bolus; subscripts indicate dose: 1 = 0.24 nmol / kg, 2 = 0.72 nmol / kg, 3 = 2.4 nmol / kg. G = GnRH IVB 75 ng / kg. E2 = estradiol, FSH = follicle-stimulating hormone, LH = luteinizing hormone. [Figure 9] Response to kisspeptin infusion and GnRH. A. Study schematic; B. Study subject. Arrows indicate luteinizing hormone pulses detected by the algorithm. G = GnRH IVB 75 ng / kg; E2 = estradiol; FSH = follicle-stimulating hormone; LH = luteinizing hormone. [Figure 10-1] Responses to kisspeptin and GnRH and neuropeptide administration. A. Study schematic; B. Study subjects. Arrows indicate luteinizing hormone pulses detected by the algorithm. K = kisspeptin-10 intravenous bolus; subscripts indicate dose: 1 = 0.24 nmol / kg, 2 = 0.72 nmol / kg, 3 = 2.4 nmol / kg. G = GnRH IVB 75 ng / kg. E2 = estradiol, FSH = follicle-stimulating hormone, LH = luteinizing hormone. [Figure 10-2]Responses to kisspeptin and GnRH and neuropeptide administration. A. Study schematic; B. Study subjects. Arrows indicate luteinizing hormone pulses detected by the algorithm. K = kisspeptin-10 intravenous bolus; subscripts indicate dose: 1 = 0.24 nmol / kg, 2 = 0.72 nmol / kg, 3 = 2.4 nmol / kg. G = GnRH IVB 75 ng / kg. E2 = estradiol, FSH = follicle-stimulating hormone, LH = luteinizing hormone. [Figure 11-1] Kisspeptin administration to Tac2 knockout mice and littermates for control during sexual development. Dotted line = kisspeptin administration. Luteinizing hormone values ​​are mean ± SEM for each time point. [Figure 11-2] Kisspeptin administration to Tac2 knockout mice and littermates for control during sexual development. Dotted line = kisspeptin administration. Luteinizing hormone values ​​are mean ± SEM for each time point. [Figure 12-1] LH pulse profiles (A and D) and the effects of naloxone (NLX) (B, C, E, and F) in adult OVX WT and Tac2 knockout mice. A and D: LH pulses 120 min before and 180 min after NLX injection. NLX injection is indicated by arrows. Arrowheads indicate LH pulses. B and E: Changes in LH secretion (mean ± SEM) 60 min before and 120 min after NLX in WT and OVX Tac2KO mice, respectively. C and F: The effects of NLX treatment on LH release are also shown as mean ± SEM 20 min before (pre-NLX) and 20 min after (post-NLX) NLX injection. *P<0.05, Student's t-test. [Figure 12-2]LH pulse profiles (A and D) and the effects of naloxone (NLX) (B, C, E, and F) in adult OVX WT and Tac2 knockout mice. A and D: LH pulses 120 min before and 180 min after NLX injection. NLX injection is indicated by arrows. Arrowheads indicate LH pulses. B and E: Changes in LH secretion (mean ± SEM) 60 min before and 120 min after NLX in WT and OVX Tac2KO mice, respectively. C and F: The effects of NLX treatment on LH release are also shown as mean ± SEM 20 min before (pre-NLX) and 20 min after (post-NLX) NLX injection. *P<0.05, Student's t-test. [Figure 12-3] LH pulse profiles (A and D) and the effects of naloxone (NLX) (B, C, E, and F) in adult OVX WT and Tac2 knockout mice. A and D: LH pulses 120 min before and 180 min after NLX injection. NLX injection is indicated by arrows. Arrowheads indicate LH pulses. B and E: Changes in LH secretion (mean ± SEM) 60 min before and 120 min after NLX in WT and OVX Tac2KO mice, respectively. C and F: The effects of NLX treatment on LH release are also shown as mean ± SEM 20 min before (pre-NLX) and 20 min after (post-NLX) NLX injection. *P<0.05, Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0024] Although nearly 50 years have passed since the discovery of GnRH (31), the number of women who have not yet begun to develop sexual maturity has increased. Understanding the factors that maintain reproductive function subsequently complicates diagnosis and treatment. Idiopathic hypogonadotropic hypogonadism (IHH) Patients with these signals have abnormal GnRH secretion / action (32, 33). Most IHH patients have delayed puberty. Presenting as a teenager with developmental and, if left untreated, lifelong sexual infantilism and infertility ( 32 , 33 ).

[0025] Described herein are methods for diagnosing and treating delayed puberty using kisspeptin. It is listed.

[0026] Diagnosis of pathological hypogonadotropic hypogonadism For decades, clinicians and researchers have believed that children with delayed puberty eventually progress to adolescence. (8) These studies were conducted in a baseline setting. In (unstimulated) serum gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone) , FSH), gonadotropins after stimulation with GnRH or GnRH analogs, and Unfortunately, all of these tests are sensitive and For example, the test may show that constitutional delay is showed a significant overlap in serum inhibin B concentrations between boys with and without IHH, 10–29% of boys with constitutional delay and 22–45% of boys with IHH The overlapping range of inhibin B (9-11) is present. Despite increasing understanding, genetic testing is currently ineffective in predicting pubertal outcomes. Currently, fewer than half of IHH patients have an identifiable mutation in the IHH gene. (7) Furthermore, even when a mutation is found, its predictive power depends on the variable penetrance and The incidence of IHH gene mutations is limited by their severity and expressivity. Leah may have IHH and the other may have constitutional delay ( 24 ).

[0027] Children with delayed puberty, which progresses later into adolescence, often have persistent puberty at first presentation. It is indistinguishable from children with persistent hypogonadism, and the outcome of a given child is unknown. It is known that waiting times of several years may be required for treatment. As a result, The donor considered two common management approaches for delayed puberty: treatment with sex steroids. Alternatively, they may face challenges in deciding between wait-and-see treatment and no intervention.

[0028] Kisspeptin inhibits GnRH in all mammalian species tested to date, including humans. It is a neuropeptide secreted in the hypothalamus that potently stimulates the secretion of steroid hormones (12). In studies by this and other groups, reproductively intact adults received a single bolus of In response to kisspeptin, LH rises strongly, whereas adults with IHH do not respond significantly. (13-19) Therefore, provocation tests using kisspeptin are not suitable for individual The method can be used to assess the GnRH secretion capacity of the

[0029] Children with delayed or arrested puberty may have kissing reactions ranging from no response to a strong response. (20) Herein, the response of children to kisspeptin is Prospective study results show that response ability can predict a child's subsequent progression through adolescence. The results are listed.

[0030] The kisspeptin stimulation test described herein can be used to predict pubertal outcomes in children with delayed puberty. This overcomes two fundamental challenges in predicting puberty. Physiological hypogonadotropic hypogonadism in children with IHH is considered pathological hypogonadotropic hypogonadism. The problem is that there is no method to distinguish it from hypogonadism. Therefore, the kisspeptin stimulation test measures the child's potential future GnRH secretion capacity. Indeed, we have demonstrated that kisspeptin is a potent anti-inflammatory drug that acts both at the time of physical examination and daily. Children who were considered prepubertal at the time of their laboratory evaluation but ultimately progressed to puberty We found that it can induce an LH response in children.

[0031] The second challenge is to identify children with a temporary pubertal arrest and those with persistent pubertal arrest. The aim of this study is to distinguish children with IHH from those with partial pubertal development. and adults with partial reproductive endocrine activity are unable to respond to kisspeptin. (19) Similarly, in the current study, 1 showed partial reproductive endocrine activity. One participant (Participant 8) had a decreased response to kisspeptin, which persisted until the age of 18. accurately predicted the absence of pubertal development, whereas inhibin B and GnR H-induced LH was suggested to progress later into puberty.

[0032] As described in Example 1, two participants (Participants B and 11) were given L The absence of an H pulse did not accurately predict participants' eventual progression to puberty. Kisspeptin stimulation testing accurately predicted outcomes in these participants. were receiving sex steroid treatment at the time of the study visit, which suppressed endogenous LH secretion. In contrast, their response to kisspeptin was strong and therefore The steroid stimulation test is a method for assessing the effects of exogenous sex steroid treatment (e.g., testosterone in men) on the development of steroid-related hormones. in men, and estrogen and / or progesterone / progestin status in women It reliably predicted final pubertal progression, even when performed in a controlled setting.

[0033] Some patients with IHH may have an intact response to kisspeptin. It is possible that the neurokinin B or its receptor genes, TAC3 and TACR3, or kisspeptin itself (25, 26) However, this is not the case in patients with IH. It is likely that only a small subset of H patients have mutations in TACR3. , present in only approximately 5% of patients with normal olfactory IHH, and mutations in TAC3 and KISS1 This is because it is a rare cause of HH (27-30).

[0034] Thus, the methods of the present invention provide for the treatment of pathological hypoglycemia in a subject, e.g., a mammal, e.g., a human subject. It can be used to diagnose gonadotropic hypogonadism, such as IHH. In some embodiments, the subject has at least 10, 11, 12, 13, 14, or 15 and / or under the age of 18, 19, or 20, e.g., 10-20 years old, 10 ~19, 10~18, 11~18, 11~19, 11~20, 12~18, 12~19, or 12 to 20 years of age. The method also includes, for example, administering intrareproductive endocrine therapy to a subject of any age. Dysfunction of secretory function or partial or late-onset forms of pathological hypogonadotropic gonads In some embodiments, the method may be used to detect hypofunction. , in children of any age, e.g., newborns, a congenital form of pathological hypogonadotropic gonads It is used to diagnose hypofunction.

[0035] In some embodiments, the current official clinical definition of IHH is when a patient is 18 years of age or older. However, in subjects under 18 years of age, the method may be used to determine whether the subject has IHH. and that this diagnosis can be made earlier than the traditional age cutoff. In some embodiments, the method comprises determining whether the subject is at risk of developing IHH. to determine whether a patient has or is likely to develop IHH.

[0036] The method includes administering at least one steroid hormone, preferably administered as an intravenous bolus (IVB). A stimulating dose of kisspeptin or kisspeptin analogue, e.g., at least 0.08, 0. 1, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22 or 0.24 nm ol / kg kisspeptin-10, up to approximately 8, 10, 12, 14, or 15 nmol / kg kisspeptin-10, e.g., 0.08 to 15 nmol / kg kisspeptin-10 , e.g., 0.1 to 12 nmol / kg kisspeptin-10, e.g., 0.2 to 10.11 nmol / kg kisspeptin-10, e.g., 0.2-0.5 nmol / kg kisspeptin kisspeptin-10, e.g., 0.22 to 0.25 nmol / kg kisspeptin-10, e.g., 0 LH levels in response to administration of a dose equivalent to 0.24 nmol / kg kisspeptin-10 Doses may also be administered, for example, subcutaneously (SC) or as an intranasal bolus. When administered SC, the dose may be higher than the IV dose range, e.g., within the ranges listed above. 10, 20 or about 30 times higher, e.g., at least 0.8 nmol / kg, up to The intranasal dose is typically about 500, 600, or 750 nmol / kg. Approximately 10 times the lower dose, i.e., 24 to 5000, 6000, or 7500 nmol / kg is.

[0037] The method includes obtaining at least one, e.g., one or more, samples from a subject; and and assessing the level of LH in the sample and comparing the level with one or more reference, For example, if you have normal LH levels, e.g., if you do not have pathological hypogonadotropic hypogonadism Subject references representing levels in subjects with low and / or pathological hypogonadotropic gonadism This may include comparing the LH levels in subjects with hypothyroidism to a disease reference. Suitable reference values ​​may include those shown in the Examples below. In a preferred embodiment, The method involves obtaining multiple samples from a subject, e.g., before, during, and after administration of a stimulating dose. and determining the LH level in the sample and the sample before administration of the stimulation dose. The LH level in the sample (e.g., baseline level) is compared to the level after administration of the stimulating dose. Thus, the method involves comparing absolute LH levels after kisspeptin administration to: Reference (e.g., a reference representative of a cohort of subjects, or a baseline level in the subjects) ) and / or changes in LH levels from baseline to post-stimulation a) unit increase (difference); b) relative change (ratio) In some embodiments, 3.5, 4, 4.5, or more, e.g. Subjects with an LH ratio of 4.85 or greater were identified as having constitutional delay and a subject having an LH ratio of less than 3.5, e.g., less than 3, e.g., 2.33 or less. identify as having pathological HH.

[0038] As used herein, the term "sample" refers to a sample obtained by the methods described herein. When referring to materials to be tested for the presence of LH using Contains feces or urine.

[0039] Various methods are known in the art for quantifying LH from a sample. The method may include isolation and / or purification of LH from the sample prior to quantification. An "isolated" or "purified" biological marker is one that is identical to the cell from which the biological marker was derived. Substantially free from cellular material or other contaminants derived from cells or tissues, i.e., During introduction, the product is partially or completely changed or removed from its natural state. The nucleic acids contained in the sample are first purified according to standard methods, e.g., by lytic enzymes, chemical isolated using a specific solution or by nucleic acid binding resin according to the manufacturer's instructions. It is isolated.

[0040] The presence and / or levels of LH can be determined by methods known in the art, e.g., standard methods. Various electrophoretic and quantitative immunoassay methods for proteins, including but not limited to: , Western blot, enzyme-linked immunosorbent assay (ELISA), biotin / avidin Isotopic assay, protein array detection, radioimmunoassay, immunohistochemistry (IHC), immunoassay It can be assessed using immunoprecipitation assays, FACS (fluorescence activated cell sorting), and mass spectrometry (Ki m (2010) Am J Clin Pathol 134:157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8):1013-1022; Philips (2014) PLOS One 9(3): e90226; Pfaffe (2011) Clin Chem 57(5): 675-687). This method is typically performed by direct or Labels, e.g., fluorescent, chemiluminescent, radioactive, that provide a signal either directly or indirectly , and revealing enzymes or dye molecules. A "label" is a detectable substance, such as a radioactive agent or a fluorophore (e.g., a phyco Coupling of erythrin (PE) or indocyanine (Cy5) to antibodies or probes a ring (i.e., physical bond), and a probe or refers to indirect labeling of antibodies (e.g., horseradish peroxidase, HRP).

[0041] In some embodiments, an ELISA method may be used, where microtiter plates are The wells of the plate are coated with an antibody against the protein to be tested. A sample containing or suspected of containing the target marker is then applied to the wells. After allowing sufficient time for antigen complex formation, the plate is washed to remove unbound material and the assay is performed. The elutably labeled molecule is added. Again, after a sufficient incubation period, the plate is The pellet is washed to remove excess unbound molecules, and the presence of the labeled molecule is detected using methods known in the art. Variations of the ELISA method, such as competitive ELISA or competitive assays, are used to determine the ELISA, and sandwich ELISA may also be used, as these are well known to those skilled in the art. Immunometric assays can be used.

[0042] Mass spectrometry, and specifically matrix-assisted laser desorption / ionization mass spectrometry (MALS) LDI-MS), and surface-enhanced laser desorption / ionization mass spectrometry (SELDI-MS) is useful for detecting LH (U.S. Pat. Nos. 5,118,937 and 5,045,694 (See Nos. 5,719,060 and 6,225,047).

[0043] Specific assays for LH are described, for example, in Wheeler, Methods Mol Biol. 2006;324: 109-24; Beastall et al., Ann Clin Biochem. 1987 May;24 (Pt 3):246-62; Pappa et a I., Theriogenology. 1999 Apr 1;51(5):911-26.

[0044] In some embodiments, the LH level after kisspeptin stimulation is , the presence and / or level of protein, and the subject is The subject has one or more symptoms associated with pathological hypogonadism, in which case the subject is In some embodiments, the subject has pathological hypogonadism. No overt signs or symptoms of gonadotropic hypogonadism but were evaluated The presence and / or level of one or more of the proteins is related to the protein in the disease reference the presence and / or equivalent of a condition in which the subject is diagnosed with a mild form of pathological hypogonadotropin have hypogonadism and / or develop pathological hypogonadotropic hypogonadism In some embodiments, the subject has a high likelihood of pathological hypogonadotropinemia. have signs or symptoms of hypogonadism but a normal LH response, in which case the subject In some embodiments, the individual is diagnosed with pathological hypogonadotropinemia and is sent for further testing. Have hypogonadism or develop pathological hypogonadotropic hypogonadism Once a high risk is determined, treatments, e.g., those known in the art or described herein, may be used. Treatments described herein may be administered.

[0045] Suitable reference values ​​can be determined by methods known in the art, such as standard clinical testing methods and It may be determined using statistical analysis. The reference value may have any relevant form. In some cases, the reference represents a meaningful LH level, e.g., a normal LH level. a subject reference level, e.g., a subject who is afflicted or who develops a disease as described herein levels in subjects not at risk for pathological hypogonadotropic hypogonadism Disease references representing LH levels associated with hypoglycemia, e.g., in subjects with IHH or KS The value includes a predetermined value for the level at which the

[0046] The predetermined level may be a single cutoff (threshold) value, e.g., a median or mean value, or Statistically different from the upper or lower quartile, tertile, or other segment It may be at a level that defines the boundaries of other segments of the clinical trial population being determined. can be a range of cut-off (or threshold) values, e.g., a confidence interval. For example, where the risk of disease occurrence in a defined group is established based on the risk of disease occurrence in a defined group. The association with risk or presence of disease is higher than the risk or presence of disease in other defined groups. may be several times higher or lower (e.g., about 2, 4, 8, 16, or more times higher). may be a particular range, for example, where a subject population (e.g., control subjects) Groups, such as low-risk, intermediate-risk, and high-risk groups, or the lowest quartile, are used to determine risk. the quartile where n is the lowest risk subject and the top quartile where n is the highest risk subject, or The bottom of the n-quantile is the subject with the lowest risk, and the top of the n-quantile is the subject with the highest risk. The subjects are divided equally (or not) into n-quantiles (i.e., n regularly affected intervals). )

[0047] In some embodiments, the predetermined level can be determined by comparing different levels of the same subject, e.g., different time points. time point, e.g., an earlier time point (e.g., kisspeptin or kisspeptin The level or appearance of the signal transduction pathway (before stimulation with the steroid analogue) is therefore unclear. In an embodiment, the method includes calculating a ratio of levels or a difference in levels, and Detects the presence of changes in LH levels following stimulation with kisspeptin or kisspeptin analogs This may include:

[0048] An object that is associated with a given value is typically referred to as a reference object. For example, in some embodiments In this embodiment, the control reference subject is a subject with a disorder described herein (e.g., pathological hypogonadotropinemia). If the control subject is preferably male, It may be desirable for the subject to be female, and the reference level used for subjects of the same sex may be A baseline is established. Control subjects are found to have pathological hypogonadotropic hypogonadism. Where desired, control subjects should also be free of pathological hypogonadotropic hypogonadism (e.g., For example, subjects with constitutional delayed puberty who ultimately undergo puberty independently without intervention. In some cases, it may be desirable to

[0049] Disease referent subjects are subjects with pathological hypogonadotropic hypogonadism.

[0050] Thus, in some cases, the LH level in a subject may be determined based on a reference level of LH. (or change in LH after stimulation) is indicative of a clinical condition (e.g., In other cases, the LH level in the subject is indicative of pathological hypogonadotropic hypogonadism. A level of LH above or equal to the reference level indicates the absence of disease. In some embodiments, the present invention indicates that the patient is at normal risk for the disease. The amount by which the level in a subject falls below the reference level is sufficient to distinguish the subject from control subjects. minutes, and optionally statistically significantly below the levels in control subjects. If the LH level in a woman is equivalent to a reference level of LH, then "equivalent" is almost It means that the results are nearly equivalent (e.g., not statistically different).

[0051] In prepubertal children with hypogonadism (insufficiency at the brain or pituitary level) (i.e., in contrast to hypogonadotropic hypogonadism), The answer is that they may be larger than in the general population. They may still have pathological hypogonadotropic Have a "normal risk" for hypogonadism.

[0052] The given value may depend on the particular subject (e.g., human subject) population selected. For example, a population that appears to be healthy may have pathological hypogonadotropic hypogonadism. LH levels in the "normal" range that differs from those in the target population who are likely to have, or are at high risk for, Thus, the selected predetermined value will be determined based on the subject's (e.g., human) Categories of interest (e.g., gender, age, presence of other diseases) may be considered. The categories and types can be selected by one of ordinary skill in the art using only routine experimentation. do.

[0053] In characterizing likelihood or risk, a number of predetermined values ​​may be established.

[0054] Treatment method The method may include treatment for pathological hypogonadotropic hypogonadism. In some embodiments, treatment is administered to a subject identified by the methods described herein. .

[0055] The methods described herein include the treatment of disorders associated with pathological hypogonadotropic hypogonadism. In some embodiments, the disorder is IHH, or KS ( For example, if the subject has anosmia. Generally, the method comprises: A therapeutically effective amount of kisspeptin or a kisspeptin analog is administered to a patient in need of such treatment. The method also includes administering to a subject who has received or is determined to be in need of treatment. For example, negative energy balance states (e.g., malnutrition, anorexia, or athletes), Hyperprolactinemia, adult-onset hypogonadotropic hypogonadism, drug therapy effects (e.g. hypothalamic amenorrhea due to glucocorticoids, opioids, or milder forms of pathological hypothalamic amenorrhea Subjects with gonadotropic hypogonadism and / or at risk of developing pathological HH Subjects with a risk of infertility, e.g., idiopathic infertility, irregular menstrual cycles, or abnormal semen analysis The present invention can be used to treat subjects with

[0056] As used in this context, "treating" refers to treating a pathological hypogonadotropic gonadal system. Pathological hypogonadism means the amelioration of at least one symptom of a disorder associated with pathological hypogonadism. Nadotropic hypogonadism is often manifested by, for example, absence of puberty until age 18, and is characterized by poor or absent development of secondary sexual characteristics or infertility Therefore, treatment may prevent the onset of puberty, the development of secondary sexual characteristics, and / or the restoration of fertility. The compounds described herein for the treatment of pathological hypogonadotropic hypogonadism can result in The administration of a therapeutically effective amount of a compound containing the compound can increase LH levels, sex steroids (e.g. For example, testosterone in men and estrogen and / or progesterone in women. Increased levels of steroids / progestins, and gametogenesis, e.g., in some cases In this study, LH secretion in response to the administration of stimulatory doses of kisspeptin or kisspeptin analogues was This results in one or more of the following increased levels:

[0057] Thus, in some embodiments, pathological hypogonadotropic hypogonadism, For example, methods for treating IHH or KS are provided herein. or multiple doses, e.g., supraphysiological and / or physiological or near-physiological doses The method may include administering a kisspeptin or kisspeptin analogue of In some embodiments, the doses are administered, e.g., periodically, e.g., 1-6, 1-4, 2-6, or or every 2-4 hours for days, weeks, months or years, e.g., 2-4 days, e.g., 4 Over an 8-hour period, optionally once a month, once every two months, or once every three months Once every four months, once every six months, once every eight months, once every ten months, or once a year, optionally over a longer period, e.g., days, weeks, months or years. Treatment may be administered daily for a period of time. It may continue until sexual development is achieved, at which point it may be optionally stopped. In some embodiments, at least some of the The amount is at physiological or near-physiological levels, e.g., 0.08 to 2.4, e.g., 0.1 ~5, 0.2-0.4, e.g., 0.24 nmol / kg IVB. In some embodiments, the method includes administering at least one supraphysiological dose, e.g., 2 to 25, e.g., , 2.4 to 24 nmol / kg kisspeptin-10, e.g., at least 2, 2.4, 2 .5, 2.6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 6, 17, 18, 19, or 20 nmol / kg kisspeptin-10, up to 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , or an equivalent dose of 25 nmol / kg kisspeptin-10. In some embodiments, the dose is a physiological dose of 0.8 to 60 nmol / kg, and and is administered subcutaneously at supraphysiological doses of 60–1200 nmol / kg.

[0058] In some embodiments, administration is, for example, intravenous, subcutaneous, or intranasal. In some embodiments, the method includes using a pump to administer the dose. Thus, administration can be, for example, in the form of a device or system carried by the subject or patient. A reservoir containing a liquid composition comprising an active agent and delivery of the composition to a subject or patient. / Infusion pumps containing an infusion pump for administration or periodic boluses of the active agent in a corresponding form a miniaturized device suitable for implantation within the body of a subject or patient that can deliver a This can occur using devices similar to insulin pumps, for example.

[0059] In some embodiments, the method includes administering a kisspeptide, as described above, for example, after 2-3 days. In some embodiments, treatment includes repeating the chin stimulation test. For example, for ovulation induction in women, In this case, the process may take anywhere from 1 month to 6 months. The process can take anywhere from 3 to 24 months. For maintenance of endocrine function, treatment may extend over several years.

[0060] The method may additionally or alternatively comprise administering an opioid antagonist, and / or a pathological hypoglycemic agent. This may include administering conventional treatment for gonadotropic hypogonadism.

[0061] Figures 5C and D are in patients with KS, so kisspeptin resistance is It can occur in the usual olfactory or anosmia forms of hypogonadism. does not predict a person's ability to respond to kisspeptin. (See Figure 5D), which indicates that the two genes have variants. Patients were still shown to respond. Data from this study support the various underlying genetic Despite the clinical diagnosis, there is no comprehensive pathomechanism for congenital hypogonadism. Supporting the role of speptin resistance, this may be due to, for example, negative energy balance states (e.g. (e.g., malnutrition, anorexia, or athletes), hyperprolactinemia, adult-onset hypogonadism Tropic hypogonadism, medication effects (e.g., glucocorticoids, opioids) hypothalamic amenorrhea due to steroids or milder forms of pathological hypogonadotropic hypogonadism Subjects at risk of developing reproductive endocrine dysfunction and / or pathological HH, e.g., Other causes of infertility, irregular menstrual cycles, or abnormal semen analysis in subjects Sensitivity to kisspeptin increases with repeated exposure, so In some embodiments, the method further comprises determining whether endogenous kisspeptin secretion is in the form of kisspeptin. To induce reversal of tolerance, to increase sensitivity to a point that produces a meaningful response. , may involve repeated administration of kisspeptin.

[0062] Kisspeptin and Kisspeptin Analogs As used herein, kisspeptin is a protein encoded by the KISS1 gene. A family of neuropeptides derived by cleavage of a 145 amino acid precursor peptide (Lee et al. 1996, Ohtaki et al. 2001). In humans, the active form of kisspeptide It is believed to be a 54 amino acid peptide (Ohtaki et al. 2001, Terao et al. 2 004). This method is applicable to full-length kisspeptin (NP_002247.3), a 54 amino acid peptide. tide (kisspeptin-54 (KP54)), or the sequence YNWNSFGLRF (SEQ ID NO: 1) and 2) may include administration of kisspeptin-10, a 10 amino acid peptide containing Analogs of Kiss-10 are known in the art and are described in Curtis et al., Am J Physiol Endocrinol Metab. 2010 Feb; 298(2): E296-E303; Gutierrez-Pascual et al., Mol Pharmacol. 2009 Jul; 76(1):58-67; Niida et al., Bioorg Med Chem Lett. 2006 Jan 1; 16(1):134-7; Orsini et al., J Med Chem. 2007 Feb 8; 50(3):462-71; Rosewei r et al., J Neurosci. 2009 Mar 25; 29(12):3920-9. In some embodiments, the analog is [dY] 1 KP-10. Analog is saved Substitutions, e.g., 1, 2, or 3 amino acid substitutions, may be made in the sequences described herein. wherein the analog is a nucleotide sequence for KISS1R. In some embodiments, the analogs possess, inter alia, For example, as described in International Publication No. 2014118318, The peptide bond between the glycine residues located in the adjacent region and the adjacent residue is disubstituted. Peptide compounds substituted with 1,2,3-triazole rings, U.S. Patent Application Publication Peptide compounds described in U.S. Patent Application Publication No. 20200172575, No. 200046797, U.S. Patent Nos. 7,960,348 and 8,404,643 Compound 1, described in Parker et al., Theriogenology. 2019 May;130:111-119 C6, KISS1-305, TAK-448 (Ac-D-Tyr-DT rp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH 2 (SEQ ID NO: 2), Decourt et al., Sci Rep. 2016; 6: 26908; MacLean et al., J Cli n Endocrinol Metab. 2014 Aug;99(8):E1445-53; Skorupskaite et al., Hum Reprod Upd ate. 2014 Jul; 20(4): 485-500) (e.g., TAK-448 acetate), or TA K-683 (N-acetyl-YWNTFGL{Met-R}W-NH2 (SEQ ID NO: 3), See Kanai et al., J Reprod Dev. 2017 Jun; 63(3): 305-310), and U.S. Patent Application Publication No. International Publication No. 20160074320, International Publication No. 200285399, International Publication No. 2004 060264, International Publication No. 2004101747, International Publication No. 2004063221 No. 1604682, WO 2005117939, and The compound is described in European Patent Application Publication No. 1464652. See also, Neuroendocrinology 2014;99:49-60. Kisspeptin or kisspeptin analogue and pharmaceutically acceptable salts thereof, pharmaceutical compositions used in the methods herein. possible.

[0063] [ka]

[0064] In some embodiments, when a peptide agonist is used, the peptide is modified. A peptide containing a reverse sequence, e.g., FRLGFSNWNY (SEQ ID NO: 4), Analogues may also be used.

[0065] The peptide analogs described herein are known in the art as peptidomimetics. For example, the method described in Qvit et al., D rug Discov Today. 2017 Feb; 22(2): 454-462; Farhadi and Hashemian, Drug Des Deve l Ther. 2018; 12: 1239-1254; Avan et al., Chem. Soc. Rev., 2014,43, 3575-3594; P athak, et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmi erski, WM, ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodm an et al., eds., Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003), and Mayo et al., J. Biol. Chem., 278:45746 (2003). In some cases, the compounds described herein These modified peptidomimetic versions of peptides and fragments are referred to as non-peptidomimetic peptides. shows enhanced stability in vivo compared to

[0066] Methods for making peptidomimetics include substituting one or more of the amino acids in a peptide sequence. Such sequences include, for example, all substitutions with D amino acid enantiomers. In other methods, the amino acid sequence from the N-terminus to the C-terminus is referred to as a "retro" sequence. The order is reversed, and the order of amino acid residues from the N-terminus to the C-terminus of the original peptide is The order of amino acid residues from the C-terminus to the N-terminus of the mimetic is: , which may be referred to as an "inverso" arrangement.

[0067] Peptide mimetics are both inverso versions of the peptides described herein, i.e. The novel peptidomimetics may be "retro-inverso" versions of the peptides. The order of amino acid residues from N-terminus to C-terminus in the peptide mimetic is the same as that from C-terminus to N-terminus in the original peptide. It may be composed of D amino acids arranged in a manner that corresponds to the order of the amino acid residues at the ends. stomach.

[0068] Another method for producing a peptidomimetic is to replace one or more amino acid residues in a peptide with , chemically distinct but functionally recognized analogs of amino acids, i.e., artificial amino acids Artificial amino acid analogs include β-amino acids, β-substituted β- Amino acids (β 3 -amino acids"), phosphorous analogs of amino acids, e.g., ∀-aminophos These include α-aminophosphinic acids and ∀-aminophosphinic acids, and amino acids with non-peptide bonds. Artificial amino acids can be peptide mimetics, such as peptide oligomers (e.g., peptoid amino acids). peptides, cyclic peptides, oligoureas or oligocaprolactones), β-peptides, cyclic peptides, oligoureas or oligocaprolactones It can be used to create carbamate peptides, or heterocyclic ring molecules. Verso peptidomimetics include FRLGFSNWNY, where the sequence is all D -Contains amino acids.

[0069] The sequences may also be modified, for example, by biotinylation or PEGylation of the amino terminus, and / or by calcitonination. It may be modified by amidation of the carboxy terminus.

[0070] Opioid antagonists In some embodiments, the methods described herein comprise administering an effective amount of an opioid antagonist. agonists, such as naloxone or naltrexone, or mixed agonist-antagonists This includes the administration of antidepressants such as buprenorphine. Others, such as nalmefene, may also be used. The opioid antagonist may be a kisspeptin or kisspeptin analogue, or as an alternative to, or administered before, after, or simultaneously with, other treatments described herein. Therefore, as used herein, (i) kisspeptin or kisspeptin analyte and (ii) an opioid antagonist, such as naloxone or naltrexone. Compositions containing agonists, or mixed agonists-antagonists, such as buprenorphine, are provided. It is served.

[0071] Conventional treatment of pathological hypogonadotropic hypogonadism In some embodiments, the method includes gonadal steroid replacement therapy, e.g., in men. Testosterone (e.g., testosterone esters, e.g., enanthate esters, cypionate, undecanoate) and estrogen and / or or progestins (e.g., conjugated estrogens (Premarin), ethinyl estrogen estradiol, or estradiol, and / or medroxyprogesterone, particulate progesterone), and / or hormonal infertility medications (e.g., ethinyl estradiol) The method also includes administering a gonadotropin, e.g., thiazolinone / norethindrone. For example, administering follicle-stimulating hormone (FSH) or human chorionic gonadotropin (hCG) Optionally, clomiphene and / or letrozole may be administered to the patient. It may also be used to stimulate ovulation in a subset of individuals. [Example]

[0072] The invention is further described in the following examples, which are set forth in the claims. It is not intended to limit the scope of the invention.

[0073] [Example 1] Use of kisspeptin to predict pubertal outcomes in youth with delayed puberty material and method The following materials and methods were used in this example.

[0074] Test Approval Physiological testing during kisspeptin stimulation testing was performed at the Massachusetts General Hospital. al Hospital (MGH) / Partners IRB approved and Registered with ClinicalTrials.gov (NCT01438034). Putin is used under IND 113,591 and GnRH is used under IND 93,353. The genetic studies of delayed puberty and IHH were conducted at the Boston Scientific Center for Genetics and Genetics, respectively. All participants were approved by the Children's Hospital and MGH. The parent must provide a written informed consent and at least one parent must provide a written informed consent. The two participants who underwent retesting were included in the repeat test. The study was approved by the Partners IRB, and participants and their parents were informed of the possibility of retesting. and provided written informed consent and consent.

[0075] Test Protocol Details of the study protocol have been described previously (20). Briefly, the inclusion criteria were: For girls, age 12 years or older, and for boys, age 13.5 years or older, with delayed puberty Breast development or cessation is defined based on breast development in girls and testicular volume in boys. Children with identifiable causes of delayed puberty were excluded. Participants were asked to provide a baseline LH secretion, LH secretion in response to 0.313 mcg / kg kisspeptin-10 (ΔL H キスペプチン ), and LH secretion in response to 75 ng / kg GnRH (ΔLH GnR H ) every 10 minutes for blood sampling, tional and Clinical Research Center (TCRC) He had been hospitalized twice. キスペプチン and ΔLH GnRH 6 days, 2 "Priming" of the pituitary gland with hourly pulsatile 75 ng / kg subcutaneous GnRH The pituitary response to GnRH was assessed both before and after the initiation of ovulation (8).

[0076] Participants then returned for follow-up visits every six months for physical examinations and FSH, LH, and and estradiol or testosterone levels were measured to assess reproductive endocrine activity. Upon reaching age 18, participants were monitored for physical and laboratory signs of reproductive endocrine activity. For the six steroid-treated participants, the treatment was Before laboratory testing, sex steroid measurements were performed to determine whether the exogenous administration (e.g., estradiol) 4 weeks for injectable testosterone, 6 weeks for transdermal testosterone This was done to ensure that the results reflected endogenous production more reliably than the control (over a 2-week period).

[0077] Laboratory Assays For TCRC visits, LH, FSH, testosterone (T), and estrogen levels As previously described (20), the MGH Clinical Laboratories tory Research Core, Brigham and Women's H Hospital Research Assay & Analysis Core and For example, for serum LH and FSH, automated Abbo AxSYM system (Abbott Laboratories, Chicago) , IL), and serum T concentrations were measured using the DPC Coat-A-Count RIA kit (D iagnostics Products Corporation, Los Ange LH levels were measured using a microparticle enzyme immunoassay (ELISA) using a 1000-μL LH antibody (60). The accuracy of the assay was 4.3–6.4%. Inhibin B was measured by the University of f Immunoassay by Virginia Ligand Assay Core The intra-assay coefficient of variation was 2.5%. For diagnostics, laboratory testing is available from Labcorp and Quest Diagnostics. For participants outside the Boston area, follow-up data were were obtained through routine clinical care from local endocrinologists and clinical laboratories. .

[0078] Genetic Testing Whole-exome analysis was performed at the Broad Institute (Cambridge, MA). Exome sequencing data showed association with IHH / KS, delayed puberty, or both. We screened for variants in 30 genes associated with genomic DNA damage (listed in Table 1 (21)). Variants are listed by the American College of Medical Genetics The cases were classified according to the diagnostic criteria of tics and Genomics ( 22 ).

[0079] statistics Fisher's exact test was used to compare the relationship between kisspeptin stimulation test and pubertal outcomes. Correlations were assessed using p values ​​less than 0.05. Jeffreys intervals were used. Confidence intervals were calculated for sensitivity and specificity.

[0080] [Table 1]

[0081] [Table 2]

[0082] [Table 3]

[0083] [Table 4]

[0084] [Table 5]

[0085] result Response to kisspeptin Seventeen participants with delayed or arrested puberty participated in the study (four girls and and 13 boys (Figure 1). 15 of these participants (Participants 1–15) The results of the kisspeptin- and GnRH-stimulation studies were previously reported in reference (20). Participant A was newly enrolled in this study and showed a "kisspeptin non-responder" pattern. Meanwhile, participant B, who was also new to the study, showed a "kisspeptin responder" pattern. The characteristics of the participants and their neuroendocrine phenotypes are shown in Table 4 and Table 2 (21 ) shown.

[0086] Long-term follow-up After this initial neuroendocrine characterization, participants returned for follow-up visits every 6 months. Eight participants (two girls and six boys) progressed to puberty during the follow-up period. (Table 4, Table 3(21)). In the absence of exogenous treatment, boys showed a progressive increase in testicular volume. The girls showed an increase in breast size and progressive breast development. Kisspeptin is a hormone that responds to LH and increases by 0.8mIU / mL or more. Responders (Figure 3, Table 4).

[0087] In contrast, eight participants (one girl and seven boys) had progressed to puberty by age 18. These participants were unable to carry out the procedure (Table 4, Table 3(21)). The boys showed persistent sexual immaturity, with testicular volumes of <4 mL and the girls with exogenous estrogen. There was no breast development until treatment with diol was initiated. At the time of assessment of endocrine reproductive activity, all of these participants had serum concentrations below the adult reference range. Sex steroids (estradiol for girls and testosterone for boys) and in situations of low or low sex steroids, serum glutamate levels that are inappropriately normal. These individuals who failed to enter puberty had high nadtropin levels. "Kisspeptin non-responders" (1 person) who showed little to no response to kisspeptin 1 girl and 6 boys) (an eighth "non-responder" was lost to follow-up) and 1 "Intermediate responders" in men who showed an LH response to 0.4mIU / mL kisspeptin " (Figure 3, Table 4).

[0088] Therefore, participants' responses to kisspeptin were more likely to be related to participants who progressed to puberty later in life. This significantly differentiated participants from those who did not progress (p=0.0002). The sensitivity and specificity for the putin stimulation test were both 100% (95% CI 7.5-9.5%). 4~100%).

[0089] LH measurements under three conditions: daytime, overnight, and after GnRH stimulation Unstimulated LH concentrations (measured during the day or overnight) and after stimulation with exogenous GnRH Determined LH has been tested as a way to predict whether a child will eventually enter puberty. In this study, two boys had unstimulated serum LH levels in the pubertal range at enrollment. One patient had a range of puberty at enrollment, and one patient later showed signs of progression during puberty. Of the remaining 14 participants with LH in the first trimester, 7 progressed to puberty and 7 did not. Therefore, unstimulated LH is not a precise predictor of pubertal outcomes. It wasn't.

[0090] During early puberty, diurnal gonadotropin measurements accurately reflect the activity of the reproductive endocrine axis. This may not be the case because the GnRH and LH secretion pulses are not constant during the night. This occurs only during the deep stages of sleep (23). Sampling was performed to detect sleep-related LH secretion. All six children who had had the disease at least once later showed signs of progression during adolescence (Table 4). In contrast, 10 children who did not experience an LH pulse overnight had a variety of outcomes. Eight patients failed to progress to puberty, while two showed pubertal progression. These two included one girl and one boy who were being treated with exogenous sex steroids at the time of the neuroendocrine evaluation. and one boy (Participant B and 11), which may have suppressed endogenous LH secretion. be.

[0091] GnRH-stimulated LH secretion has also been studied as a test to predict pubertal outcome. In our cohort, the LH response to GnRH overlapped and was later observed during puberty. In subjects who progressed to rheumatoid arthritis, the range was 1.2 to 15.4 mIU / mL, and in subjects who did not progress, the range was 1.2 to 15.4 mIU / mL. In the present study, the range was 0.2 to 7.5 mIU / mL (Figure 4, Table 4).

[0092] Therefore, these tests were conducted during the day and at night. Neither unstimulated nor GnRH-stimulated LH was associated with pubertal mortality in this study cohort. The kisspeptin stimulation test was not accurate for predicting outcome.

[0093] Inhibin B Measurement of serum inhibin B predicts pubertal outcome in children with delayed puberty. Serum inhibin B levels have been proposed as a way to prevent puberty (8-11). In boys who progressed, the range was 39-209 pg / mL, and in boys who did not progress, The range of values ​​for α-glucan was <17 pg / mL to 48 pg / mL (Figure 4, Table 4). Inhibin B positively differentiates between children who will progress to puberty later in life and those who will not. There was no clear distinction.

[0094] Genetic Testing A subset of patients consented to exome sequencing. and likely pathogenic variants (Table 1 (21)) that later progress during adolescence. Two of the five participants who had not entered puberty and two of the six participants who had not entered puberty Pathogenic or likely pathogenic variants were identified in IG It was not identified in SF10, which is a gene that may be associated with constitutional delay. Therefore, genetic testing does not provide information about pubertal outcomes for children with delayed puberty. It was not possible to predict.

[0095] [Table 6]

[0096] [Table 7]

[0097] [Example 2] Supraphysiological kissing techniques to improve pubertal outcomes for youth with delayed puberty hmm One subject with delayed puberty in the study described in Example 1 had a pubertal delay of 15.3 years of age. 0.313 μg / kg IVB (physiological dose) of kisspeptin-10 was administered. Shortly thereafter, during another hospitalization, the subject developed an ultrasonographic They received a physiological dose of kisspeptin (18 μg / kg) and, surprisingly, responded The subject's final diagnosis was failure to show physical signs of puberty by age 18. As shown by the above, it was IHH. This suggests that 1) GnRH deficiency is 1) this tolerance may be due to kisspeptin resistance, and 2) this tolerance may be due to high doses of kisspeptin. It has been shown that this can be overcome by peptin.

[0098] Based on this observation, high doses and / or repeated kisspeptin administration may be beneficial for IHH / It was hypothesized that GnRH-induced LH secretion in patients with KS could be stimulated. To address this theory, we initiated the following protocol: 1) 6 x 10-minute blood sampling. 2) Kisspeptin administration q2h x 40h (5 sets of 4 doses each: 0.31 3, 0.939, 3.13, and 13.19 μg / kg). For comparison, Figure 5A shows the normal This shows a normal male who had normal LH pulses and was receiving physiological doses of kisspeptin. The GnRH-induced LH pulse was clearly observed. Figure 5B shows that the LH pulse was not induced by the GnRH-induced LH pulse. KS men who do not respond to kisspeptin (i.e., kisspeptin resistant) Males have higher expression of the genes encoding the prokinectin receptors, PROKR2 and DMX Figure 5C shows that the IL-1 gene has a rare variant in L2, which is unresponsive to physiological doses of kisspeptin. However, they responded to supraphysiological doses and showed clear kisspeptin-induced GnRH-induced LH pulses. Furthermore, the pulse amplitude increased over time, which suggests that repeated This indicates a "priming" effect of exposure. The genetic characteristics of women are not yet known. Figure 5D shows a KS man who also has the genetic variant, and although his response was not as pronounced, Responses to each dose over time were also observed. The data shown are from Figure 5C, re-graphed, clearly demonstrating that persistent gonads Patients with hypothyroidism were found to be kisspeptin resistant after a single IV bolus dose. However, these patients were given kisspeptin repeatedly, especially at higher doses. The data showed significantly different responses in patients with IHH / KS. GnRH neurons can be evoked from their resting state by repeated kisspeptin stimulation. Furthermore, this repeated kisspeptin stimulation suppresses GnRH-induced LH pulsations. brought about.

[0099] [Example 3] Hypothalamic reproductive endocrine secretion independent of neurokinin B and dynorphin signaling Pulse generating activity Endogenous factors (e.g., gonadal steroids, stress hormones, and nutritional signals) and afferent neurons in which external cues (e.g., social cues and day length) regulate GnRH release Pathway identification has recently focused on the kisspeptin / neurokinin B / dynorphin system. (34) Kisspeptin, neurokinin B (NKB), and their respective receptors Inactivating mutations in the receptor cause IHH in humans and mice, and GnRH receptor agonists These neuropeptides have been implicated in the generation of dyslipidemia (35-42). The GnRH pulse-generating activity in response to progesterone during the luteal phase of the menstrual cycle was It is thought to oppose this stimulatory activity by providing a significant delay in These three neuropeptides are expressed in the arcuate nucleus, KNDy (kisspeptin-neuronal nucleus), and the nucleus basalis. (B-dynorphin) fusion and coordination in a population of neurons It acts in a gonadotropin-dependent manner to synchronize the secretory activity of GnRH neurons and drive reproductive endocrine functions. It generates the pulse of GnRH secretion necessary for the ovulation process (46-48).

[0100] Biallelic loss-of-function mutations disrupt both copies of a gene, making it difficult to differentiate between the two. Patients who undergo this procedure (i.e., "human knockouts") will not experience the phenotypic consequences of the gene disruption or loss. This study provides novel insights into the effects of NKB (KNDy) neurons. A patient with biallelic, complete loss-of-function mutation in a gene encoding a key neuropeptide Four sisters with IHH underwent genotype-driven phenotyping. Nevertheless, some sisters recovered their reproductive endocrine function spontaneously in adulthood. , normal and neurokinin B-deficient family members and normal and neurokinin B We performed this study in both deficiency and innate immune cells to investigate the role of NKB in GnRH pulse generation. The interactions between NKB, kisspeptin, and dynorphin were analyzed. By using a combination of secretory probes, the hypothalamus was able to detect the secretion of three KNDy components, NKB and Despite genetic and pharmacological antagonism of two of the dynorphins, It has been shown that GnRH-induced LH pulses can be generated.

[0101] Results show elevated LH levels during NLX infusion in subjects with IHH To date, endogenous GnRH induction mimics normal physiology in patients with IHH. The ability to stimulate LH pulsations was non-existent.

[0102] Consideration of the LH pulse indicates that subject 4 had a more pronounced response to NLX than subject 5. Subject 4 had pituitary priming with exogenous GnRH. Subject 4 received NLX and subject 5 did not, which may explain the effect of NLX on the LH response in subject 4. Subject 4 also showed increased endogenous kisspeptin activity during GnRH release, possibly amplifying the effect. She was receiving intermittent hormone replacement therapy, which may have augmented her hormone use.

[0103] In a preliminary study, a strong GnRH-induced LH response was observed in healthy men and women in the luteal phase. What effect does the same dose of kp-10 have on IHH patients with a range of genotypes? The functional capacity of the GnRH neuronal network is also impaired by IHH. These TAC3 and TAC4 genes have been shown to be fundamentally impaired in patients with glaucoma (58). In contrast to previous observations in IHH patients with genotypes other than TACR3 or TACR4, Elephants 3, 4, and 5 responded to kp-10 IVB (58). The ability to respond to pulses and exogenous kp-10 administration was related to the GnRH required for pulse generation. This indicates that neural circuits remain intact in patients lacking NKB. However, the ability to respond to kp-10 with an LH pulse was only observed in the setting of IVB administration. observed with continuous infusion, as previously reported by others (59). The dose of kp-10, LH assay and LH pulse algorithm were different. This may explain this discrepancy.

[0104] method The following materials and methods were used in Example 3.

[0105] Subjects and Eligibility Criteria Five women from a single consanguineous family were recruited based on their genotype (Table 6). Subject 1 was reproductively normal (genotype TAC3 c.61_61ΔG p.A2 1LfsX44 heterozygote) or with a diagnosis of hypogonadotropic hypogonadism (Subjects 2-5, genotype TAC3 c.61_61ΔG p.A21LfsX4 4 homozygous). Siblings and parents were unavailable for study participation. IHH were aged 18 years or older. Age and identifiable medical conditions that may cause hypogonadotropic hypogonadism Hypogonadism in the setting of low or normal gonadotropin levels in the absence of defined as low sex steroid levels (in women, estradiol <20 pg / mL) As previously reported by our team (19), the recovery of IHH in women was primarily due to: 1) external 1) Fertility without the use of gonadotropin therapy or primogonial GnRH; 2) Fertility in the absence of treatment 3) at least 3 months of spontaneous menstrual cycles, and / or 3) normal for women Relapse after recovery was defined as LH pulse frequency and amplitude within the normal range. Sex steroid levels (in women, serum estradiol <20 pg / mL) and and / or amenorrhea again.

[0106] Subjects also participated in genetic testing. Patient DNA was analyzed as previously described. (20, 21), in genes known to cause IHH, Genome Minority of less than 1% in the Aggregation Database (gnomAD) Rare sequence variants (RSVs), defined as those with an allele frequency of The genes screened were CHD7 (MIM608892), F GF8(MIM600483), FGFR1(MIM136350), GNRH1(MI M152760), GNRHR (MIM138850), HS6ST1 (MIM6048) 46), ANOS1 (formerly called KAL1, MIM300836), KISS1 (M IM603286), KISS1R (MIM604161), NSMF (formerly NELF These are called MIM60813), PROK2 (MIM607002), and PROKR2 (M IM607123), TAC3 (MIM162330) and TACR3 (MIM162 332), which was performed by PCR amplification of the exons and subsequent Sanger sequencing. RSV has been predicted by four in silico prediction programs, PolyPhen-2 (22) , SIFT (23), Mutation Taster (24) or Panther ( 25) were reported when they were predicted to be damaging in at least two of the following cases: 1 University of Pennsylvania Sm from two-item olfactory test The olfactory sensory perception test (UPSIT) score was used to assess olfactory ability. (26, 27)

[0107] [Table 8]

[0108] [Table 9]

[0109] Test Design In 2010, subjects with hypogonadotropic hypogonadism (subjects 2, 3, 4, and 5 ) blood samples were taken every 10 minutes (q10 min) for 6-8 hours and Wellco me Trust Clinical Research Facility, Camb ridge, UK, under the direction of Professor I. Sadaf Farooqi. Detailed neuroendocrine phenotyping was performed, mapping LH pulsations (Fig. 7A).

[0110] In 2016, subjects 1, 3, 4, and 5 had endogenous LH pulse patterns that were similar to GnRH. , nonspecific blocking of kisspeptin 112-121 (kp-10) and dynorphin To determine whether this could be altered by administration of the opioid antagonist, naloxone (NLX), Massachusetts General Hospital (MG) H) Secondary Clinical Research Center (CRC) Each rat was invited to participate in a series of daytime tests (Figures 8A, 9A, and 10A). To ensure that the stimulating hormone secreting cells are in a ready state, subject 3 and 4, 25 ng / kg exogenous pulsatile GnRH every 2 hours (q2h), Cr by the ono F portable infusion pump (Cane SpA, Turin, Italy) ly) was administered 3 days before admission to MGH CRC (28). Subject 5 had any neurological disorder. Because there was recent evidence of endocrine activity (years of spontaneous bleeding), pulsatile GnRH No priming was performed (Table 6).

[0111] Baseline testing: All subjects completed 10-minute blood sampling for at least 6 hours. The patients were then screened for endogenous GnRH-induced LH secretion during the day of their MGH CRC visit. (Figures 7A and 8A).

[0112] Kisspeptin bolus: After assessment of endogenous GnRH-induced LH secretion, subjects 3, 4, and 5 Previous work from our group has shown that intravenous administration of 0.24 nmol / kg of kp-10 This dose was administered to healthy men and healthy women in the luteal phase. It has been shown to consistently induce GnRH-induced LH pulses of physiological amplitude in (29, 30) (Figure 8A). Subjects 4 and 5 then had 0.72 and 2.4 nmol / kJ. Subjects 3, 4, and 5 were then administered 100 mg of kp-10IVB. Finally, 75 ng / kg IVB GnRH was administered, which is the same as the previous study by our group. This dose is potent in individuals with intact gonadotroph function. This is because it has been shown to result in a GnRH-induced LH response (31).

[0113] Kisspeptin infusion: In contrast to the IVB study, subject 3 returned to CRC and had a kp-10 of 1 Participate in the second hospitalization administered as a 2-hour continuous infusion (9.5 nmol / kg / hour) The effect on endogenous GnRH-induced LH pulsations was determined. Samples were collected over 10 minutes, and 75 ng / kg IVB GnRH was administered at the end of the study. (Figure 9A).

[0114] Naloxone infusion, dynorphin blockade: Subjects 4 and 5 returned to the CRC and underwent NLX infusion ( 10 mg IVB followed by NLX followed by 0.8 mg / hour infusion for 13 hours, and opioid Blockade of dynorphin signaling by antagonism results in the absence of NKB signaling. The effects of the infusion on the endogenous LH pulse were determined. and GnRH bolus (kp-10 dose range: 0.24-2.4 nmol / kg, GnR H: 75ng / kg), and NLX administration enhanced the response to these peptides. Again, blood samples were collected q10 min for hormone measurements. Due to a nursing error, subject 5 received an NLX infusion that was terminated prematurely at 9 hours. .

[0115] Peptide source Kisspeptin 112-121 (pre-kisspeptin), a 10-amino acid isoform of kisspeptin The prohormone (corresponding to amino acids 112-121 of the prohormone) and GnRH are expressed in NeoMPS (Po (Peptide Laboratories, San Diego, CA) NeoMPS was synthesized using good manufacturing practices. hriver National Institute of Child Healt Kisspeptin 1 under contract to the U.S. Food and Drug Administration and Human Development Naloxone was provided by Hospira (Lake Forest, IL). It was ordered by L.

[0116] Human laboratory assays LH and 2-hour pool estradiol for each sample were analyzed using automated Abbo tt ARCHITECT system (Abbott Laboratories, In c., Abbott Park, IL) as previously described ( 28 ). estradiol was measured by direct immunoassay. Testing for this is performed using second-generation immunoassays that are traceable to mass spectrometry-based assays. For the 2016 study, Elecsys (Roche Diagnostic s, Indianapolis, IN) ( 32 , 33 ).

[0117] Assessment of pulsatile LH release in adolescent and adult Tac2 knockout mice Tac2+ / - breeding pairs were obtained from the Texas A&M Institute for Genome Research. omic Medicine (College Station, TX) All mice were genotyped (34). Created and maintained in the Brigham and Women's In the hospital, light is provided from 06:00 to 18:00 hours, and food and They were group-housed (per cage) in a temperature- and light-controlled environment with free access to cattle and water. Mice were kept for 2 days before the experiment to allow them to adapt to the sampling conditions. Handled daily for ~6 weeks.

[0118] Changes in LH secretion occur during sexual maturation (6 weeks of age) and in adulthood (16 weeks of age). Intact and ovariectomized (OVX) Tac2 knockout (KO) female mice and control (wild-type, WT) littermates (n = 4-5 per group). Tac2 in the mouse model encodes the NKB gene in humans, so these mice express the NKB gene. Measurement of pulsatile LH secretion is performed repeatedly via a single incision at the tip of the tail. Blood was collected from the tail and washed with saline. 4 μl of blood was then pipetted into the Whole blood was collected from the cut tail at each time point. The samples were diluted with 0.05% PBST, vortexed, and frozen on dry ice. The pools were stored at -80°C for the subsequent LH ELISA. Thirty-six consecutive blood samples were collected over a 6-hour sampling period. At 170 min after puberty (or for sampling of adolescent Tac2 knockout mice), At 180 minutes (in the case of the 1st generation), mice were intraperitoneally injected with mouse kp-10 (7.5 nmol / kg). l / 100 μl saline, Phoenix Pharmaceuticals). NL For the X-administration study, 30 consecutive blood samples were collected from WT and Tac2 KO mice. The cells were collected over a 5-hour sampling period from WT and Tac2 KO mice. The mice were ovariectomized to better understand the effect of dynorphin removal on the generation of LH pulses. To better determine the LH pulse frequency and amplitude were increased. At 1 min, mice were intraperitoneally injected with NLX (5 mg / kg / 100 μl saline). saline, Sigma Aldrich).

[0119] Data analysis Human pulse analysis: LH pulses were analyzed using a modified version of the validated Santen and Bardin method. The variance (35, 36) was used to identify the variance and augment it with a deconvolution algorithm (29 ) The pulse amplitude of the kp-10-induced or GnRH-induced LH pulse was measured using the It was calculated as the difference between the time point 0 of nRH administration and the peak of the pulse.

[0120] Mouse pulse analysis: LH pulses are collected by LH sandwich ELISA. This was determined using a custom-written MATLAB code that reads the LH pulse data. The code is: a) the height of the LH value is 20% greater than the height of either of the two previous values, and b) the peak in the second time interval (i=2) exceeds the height of A loop that determines a pulse based on whether it exceeds a single value by 20% before being considered a pulse. include.

[0121] Statistics: Paired two-tailed t-test was used to compare mean LH, LH amplitude (LH pulse) at baseline. The nadir to peak of FSH and FSH were measured by neuropeptide as defined in the above method. All values ​​are mean ± standard deviation unless otherwise stated. reported as an error.

[0122] Test Approval All human studies were approved by the Institutional Review Board of MGH / Partners Healthcare or Local Regional l Ethics Committee of Cambridge, United K All subjects were required to complete a written agreement prior to participating in the study. Written informed consent was provided. Mouse studies were performed as described in Brigham and W omen's Hospital Institutional Animal Car The Human Use Committee approved all methods.

[0123] result Initial clinical presentation and subsequent course of study subjects Subject 1 had normal menarche timing, normal menstrual cycles, and spontaneous conception ( (Table 6). Their sisters, Subjects 2, 3, 4, and 5, were 13–15 years old and had been diagnosed with HIV. She presented with primary amenorrhea and received estrogen therapy to induce secondary sexual characteristics. Spontaneous sexual maturation by age 18 years, absence of normal MRI, and low gonadotropins Subjects 2, 3, 4, and 5 all received a diagnosis of IHH due to their olfactory responses (Table 6). None of the four IHH sisters had loss of consciousness. Three of the four IHH sisters were receiving fertility medication. Unauthorized pregnancy (subjects 3 and 4) and regular spontaneous menstrual cycle (subject 5) ) showed reversal of hypogonadism between the ages of 22 and 28 years. However, the recovery was not sustained, and at the time of physiological testing, subjects 3, 4, and and 5 had reverted to a state of hypogonadotropic hypogonadism (Table 6).

[0124] Genetics Sequencing of candidate genes confirmed that subject 1 (with normal pubertal timing and normal menstrual cycle) The gene encoding NKB (TAC3) is involved in a single nucleotide deletion. It was revealed that the gene was heterozygous (c.61_61ΔG p.A21LfsX44) This base pair deletion was caused by a frameshift mutation and a prepro-formaldehyde mutation before the NKB sequence. This results in a premature stop codon in the mon, which triggers nonsense-mediated decay. Even if the transcript escapes nonsense-mediated decay, The REM shift mutation is processed to produce a decapeptide known as NKB. Subjects 2, 3, 4, and 5 all had low gonadotropin levels. have endotropic hypogonadism and are homozygous for this frameshift mutation This mutation is novel and was identified in 123,136 exomes and 15,496 genomes. It is not found in gnomAD, a normative database that includes (21). Notably, gnomAD contains a homozygous mutant for a protein-truncating mutation in TAC3. Some individuals are completely absent. This family has no other genes known to cause IHH. Does not carry a gene mutation.

[0125] Baseline test: A slow LH pulse frequency resulted in IHH individuals without neurokinin B. characterize a person At the time of these baseline examinations, the IHH sisters (Subjects 2, 3, 4, and 5) were hormonally Without medication, she had amenorrhea and low but detectable serum estradiol levels, and Progesterone levels were low (Table 6, Figure 7B). All subjects with IHH had low A weakened but organized GnRH pulse, evidenced by LH secretory events had a pathological basis (characterized by low estradiol and low progesterone) For comparison in the physiological early follicular phase, the LH frequency was 7.0±1.8 pulses / 12 hours. During this time, the LH amplitude was 2.3±1.0 IU / L [mean±2SD] (37, 38). At 5 and 6, one pulse was observed in the sampling interval (7-8 hours, average The mean LH amplitude was 1.5 ± 0.8 mIU / mL (Figure 7B). In subject 3, the pulse was Furthermore, LH levels in subjects 3, 4, and 5 were significantly higher than those in subjects 1, 2, and 3. It showed a gradual decay at the beginning of the interval, indicating that the LH secretory event occurred before the start of the test. Therefore, all subjects had an abnormally low frequency of LH secretion. During a repeat study in 2016, test subjects (subjects 3, 4, and 5) again Amenorrhea and low but detectable estradiol levels without hormone medication All studies reproduced the same endogenous LH pattern observed in 2010. However, the frequency of LH secretory events was low, with a mean LH amplitude of 1.3 ± 1.1 mIU / mL. (Figure 8B).

[0126] In contrast, subject 1, a healthy sister with a heterozygous truncating variant in TAC3 underwent blood sampling on day 4 of their menstrual cycle (early follicular phase; EFP). The subject had 11 LH pulses in 12 hours, with a mean LH pulse amplitude of 0.46 ± 0.2 5 mIU / mL (Fig. 7C) (Healthy early follicular phase women: frequency, 7.0 ± 1.8 pa rs / 12 h, amplitude 2.3 ± 1.0 IU / L [mean ± 2 SD]) (19, 20).

[0127] Kisspeptin bolus: IHH individuals without NKB respond to kisspeptin All subjects responded to kisspeptin with an LH pulse (Figure 8B). Elephants received three kisspeptin boluses, with kisspeptin in five of the six boluses. The one exception was the LH pulse after kisspeptin, which was released immediately after the endogenous LH peak. This occurred when α-glucan was administered, resulting in a single peak of prolongation (Figure 8B, subject 5). Consistent with gender, all subjects demonstrated appropriate pituitary priming, which was associated with kisspeptin This indicates that there is no pituitary defect that could impair responsiveness (LH pulse amplitude after GnRH administration). Width, subject 3: 1.6mIU / mL, subject 4: 5.1mIU / mL, subject 5: 3.0mIU / mL).

[0128] Kisspeptin infusion: no pulsatile LH secretion Subject 3 received a 12-hour kp-10 infusion (9.5 nmol / kg / hour) and an LH pulse. During the infusion, a mild increase in mean LH was observed (baseline: 0.05). .46±0.24mIU / mL;kp-10 injection: 0.63±0.08mIU / mL;p <0.0001) (Figures 8B & 9B). Mean FSH levels also increased significantly compared to baseline. elevated (baseline: 1.9±0.2mIU / mL; kp-10 infusion: 2.4±0. After the kp-10 injection, subject 3 had a IVB GnRH This resulted in L amplitudes comparable to those observed in the baseline test the previous day. H pulse was delivered (baseline, 1.6 mIU / mL, after kp-10 injection, 2. 5mIU / mL).

[0129] Naloxone infusion: Blockade of dynorphin by naloxone inhibits LH & FSH secretion and L Increases H-pulse frequency but does not amplify kisspeptin-induced LH pulses Subjects 4 and 5 received the nonselective opioid antagonist, NLX, and an escalating bolus Kisspeptin (0.24, 0.72, 2.4 nmol / kg) was administered to the mice. Effects of blockade of fin signaling on endogenous and kisspeptin-stimulated LH secretion patterns Both studies measured the effect of NLX on mean LH levels during infusion compared to baseline. Subject 4 showed elevated serum IL-1 levels (Baseline: 1.44 ± 0.76 mIU / mL, NLX :2.82±0.54mIU / mL, p<0.00001; Subject 5 - Baseline:0. 6±0.25mIU / mL, NLX:1.1±0.37mIU / mL, p<0.0000 1, across adapted time points) (Fig. 8B, 10B). For subject 4, whose off LH sampling was completed and comparison was possible, The H pulse frequency increased from one pulse per 6 hours (Figure 8B) to four pulses per 6 hours (Figure 10B) ) Mean FSH levels were also elevated compared to baseline (subjects 4- Baseline: 3.7 ± 0.3 mIU / mL, NLX: 5.0 ± 0.9 mIU / mL; p <0.01; Subject 5 - Baseline: 3.3±0.3mIU / mL, NLX: 5.1±0 0.1 mIU / mL; p<0.0001). No corresponding changes were observed in LH pulse amplitude. (Subject 4 - Baseline: 2.59mIU / L, NLX: 0.45±0.29mIU / L) mL, subject 5 - baseline: 0.82 mIU / mL, NLX: 1.22 and 1.39 NLX infusion increases dynorphin production by inhibiting opioid tone. Blocking NKB signaling and the absence of NKB signaling may contribute to the development of gonadal hyperplasia in individuals with IHH. Increases estrogen secretion and improves LH pulse frequency.

[0130] Subjects 4 and 5 also had escalating bolus kp-10 (0.24, 0.72, 2.4 nm ol / kg), followed by LH pulses, and the results were replicated with NLX off ( 8B, 10B). Kisspeptin-induced LH responses to on or off NLX. There was no significant difference in the dose-response relationship, but a small number of balls at each dose were Russ had limited ability to assess such relationships.

[0131] Kisspeptin bolus inhibits adolescent and adult WT and NKB-deficient (Tac2 KO) Stimulates LH release in mice To substantiate our findings in IHH patients, we performed Tac2 KO and WT control females. A study was conducted in mice. Peripheral administration of kp-10 resulted in a significant improvement in the survival of mice regardless of age and genotype. induced a strong rise in LH in all groups. Tac2 KO female mice had significantly higher blood cholesterol levels than control females (2.67 ± 0.48 ng / ml, n = 5; p < 0.05). showed a higher amplitude of LH release (5.29±0.43ng / ml, n=5) than the control group (0.01). ) (Figure 11). However, LH decreased in Tac2 KO mice (52±3.72 post-injection). min, n = 5), faster than WT controls (68 ± 3.72 min, n = 5; p < 0.01). Adult WT mice showed the expected LH pulse in response to kp-10. On the other hand, Tac2 KO mice in response to kp-10 showed a biphasic response, which was consistent with L In both adult groups, the induction of LH release was , adolescent mice (adolescent WT: 68 ± 3.742 min after injection, n = 5 vs. adult WT1 42.5±4.78 min, n=4, p<0.0001; Adolescent Tac2 KO:52±3. 742, n=5 vs adult Tac2 KO 156.7±3.33 min, n=3, p=0 It was thought that the effect was better maintained than the previous standard deviation (.07).

[0132] Naloxone increases pulsatile LH release in adult OVX WT and Tac2 KO mice. Add Opiate (dynorphin) in the absence of NKB To determine the role of dynorphine in the effects of dynorphine on kisspeptin signaling, we The effect of NLX on LH secretion was examined. Blind injection resulted in significant increases in WT (Figure 12A-C) and Tac2 KO female mice within 20 minutes of administration. induced a rise in LH levels in both WT and NLX (Fig. 12D-E). 7±0.59, n=4 vs 20 minutes after NLX, 4.31±0.32, n=4; p<0. 05.Tac2 KO: 20 minutes before NLX, 0.31±0.06, n=4 vs after NLX 20 minutes, 1.22±0.29, n=4, p<0.05).

[0133] After NLX administration, WT mice responded by increasing the duration of subsequent LH pulses after NLX administration. Before NLX: WT 25 ± 2.67 min, n = 3; Tac2 KO 23.33 ± 2.10 min min, n=3, p=0.13; After NLX: WT:83.33±12.02 min, n=3;Ta c2 KO: 30 ± 5.77 min, n = 3; p < 0.01) (Fig. 12A). The increase in the duration of the subsequent LH pulse was accompanied by a significantly longer interpulse interval in WT mice. (WT interpulse interval, 25.38 ± 1.83 min before NLX, WT interpulse interval, N 46.67±3.33 minutes after LX, p<0.0002).

[0134] Tac2 KO animals had significantly reduced LH baseline and pulse responses compared with OVX controls. The number of LH pulses was 0-1 at 120 minutes before NLX. In this case, a strong LH pulse was induced 20 minutes after treatment, compared with baseline. had a peak that reached a two-fold increase (before NLX: 0.31 ± 0.06 mIU / mL, After NLX: 1.2±0.28mIU / mL, p<0.02). A limited number of LH pulses The analysis of the interval between pulses has been eliminated, and the data show that the NLX rises during the LH pulse. (Tac2 KO 23.33±2.10 min before NLX, n = 3, after NLX: Tac2 KO: 30 ± 5.77 min, n = 3, p > 0.05) (Figure 1 2D~E).

[0135] [Table 10-1]

[0136] [Table 10-2]

[0137] [Table 10-3]

[0138] [Table 10-4]

[0139] [Table 10-5]

[0140] [Table 10-6]

[0141] [Table 10-7]

[0142] [Table 10-8]

[0143] [Table 10-9]

[0144] Other embodiments While the present invention has been described in connection with its detailed description, the foregoing description is intended to be illustrative. and shall not be construed as limiting the scope of the invention, which is defined by the appended claims. It should be understood that other aspects, advantages, and modifications are not intended to be limiting. Within the scope of the following claims.

Claims

1. Treating subjects with reproductive endocrine dysfunction, optionally pathological hypogonadotropic hypogonadism 1. A method of administering a therapeutically effective amount of (i) multiple doses of kisspeptin or kisspeptin derivatives to a subject, comprising administering to said subject a therapeutically effective amount of (i) multiple doses of kisspeptin or kisspeptin derivatives to a subject (ii) an opioid analog, and / or (ii) an opioid antagonist or mixed agonist - administering an antagonist to said subject.

2. Preferably, said multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, for at least 2 to 3 days.

10. The method of claim 1, wherein the method is administered for at least 1 to 12 months.

3. Each of the multiple doses is administered by intravenous bolus (IVB), 10. The method of claim 1, comprising a dose equivalent to 2.4 nmol / kg kisspeptin-10. 。

4. Each of the multiple doses is 0.2-0.3 nmol / kg kisspeptin-10, preferably or a dose equivalent to 0.24 nmol / kg kisspeptin-10. How to post.

5. The multiple doses are at least equivalent to 2.4 to 24 nmol / kg kisspeptin-10.

10. The method of claim 1, further comprising administering one or more supraphysiological doses.

6. The at least one supraphysiological dose is the first dose of the plurality of doses, or the first two doses.

6. The method of claim 5, wherein the compound is administered in one or more doses, optionally all of said multiple doses. How to do it.

7. A therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist is administered. The method of claim 1, further comprising administering to the subject.

8. The opioid antagonist is naloxone or naltrexone, or 8. The method of claim 7, wherein the mixed agonist-antagonist is buprenorphine.

9. The subject is a pathologically hypogonadotropic hypogonadism patient with pathological hypogonadotropic hypogonadism. at risk of hypogonadism or have a mild form of pathological hypogonadotropic hypogonadism A method of identifying a product as having: measuring a baseline level of LH in the subject; Stimulating doses of kisspeptin or kisspeptin analogs, e.g., intravenous bolus (IVB) ) a dose containing 0.08 to 15 nmol / kg kisspeptin-10 administered by or 0.8-500 nmol / kg Kispe administered by subcutaneous (SC) injection administering to said subject a dose comprising aptin-10; after administration of the stimulating dose, e.g., about 10, 15, 20, 30, 4 measuring at least one LH level within 5 or 60 minutes; The baseline level of LH in the subject is compared with the LH level after administration of the stimulatory dose. Compare with Having an LH level after administration of said stimulatory dose that is not significantly different from said baseline level of LH. Subjects with delayed puberty are classified as those with pathological hypogonadotropic hypogonadism. To identify as A method comprising:

10. and administering to said identified subject a treatment for pathological hypogonadotropic hypogonadism. The method of claim 9, comprising:

11. The treatment may include administering multiple doses of kisspeptin or a kisspeptin analog. The method of claim 10.

12. Preferably, said multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, for at least 2 to 3 days. The method of claim 11, wherein the method is administered for at least 1 to 12 months.

13. Each of the multiple doses is administered by intravenous bolus (IVB), 12. The method of claim 11, comprising a dose equivalent to 2.4 nmol / kg kisspeptin-10. Law.

14. Each of the multiple doses is 0.2-0.3 nmol / kg kisspeptin-10, preferably or a dose equivalent to 0.24 nmol / kg kisspeptin-10. The method described.

15. The multiple doses are at least equivalent to 2.4 to 24 nmol / kg kisspeptin-10.

12. The method of claim 11, further comprising one supraphysiological dose.

16. The at least one supraphysiological dose is the first dose of the plurality of doses, or the first two doses.

16. The method of claim 15, wherein the compound is administered in one or more doses, optionally all of said doses. How to post.

17. A therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist is administered.

11. The method of claim 10, further comprising administering to the subject.

18. The opioid antagonist is naloxone or naltrexone, or a mixture thereof.

18. The method of claim 17, wherein the combined agonist-antagonist is buprenorphine.

19. 11. The method of claim 1 or 10, wherein the treatment further comprises administering gonadal steroid replacement therapy. The method described.

20. The gonadal steroid replacement therapy provides testosterone in men and estrogen in women.

2. The method of claim 1, further comprising administering strogen and / or progesterone / progestin.

19. The method according to claim 19.

21. 11. The method of claim 1 or 10, further comprising administration of one or more gonadotropins.

22. and administering multiple doses of kisspeptin or a kisspeptin analog to treat pathological hypogonadism. (i) for use in a method of treating a subject having nadotropic hypogonadism; (ii) a kisspeptin or kisspeptin analog, and / or (iii) an opioid antagonist. Compositions containing agonists or mixed agonist-antagonists.

23. Preferably, said multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, for at least 2 to 3 days. or for at least 1 to 12 months. composition.

24. Each of the multiple doses is administered by intravenous bolus (IVB), 23. The use of claim 22, comprising a dose equivalent to 2.4 nmol / kg kisspeptin-10. Composition for use.

25. Each of the multiple doses is 0.2-0.3 nmol / kg kisspeptin-10, preferably or a dose equivalent to 0.24 nmol / kg kisspeptin-10. A composition for the described use.

26. The multiple doses are at least equivalent to 2.4 to 24 nmol / kg kisspeptin-10.

23. The composition for use according to claim 22, comprising one or more supraphysiological doses.

27. The at least one supraphysiological dose is the first dose of the plurality of doses, or the first two doses.

27. The method of claim 26, wherein the compound is administered in one or more doses, optionally all of said multiple doses. The composition for use as described above.

28. A therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist is administered.

23. The composition for use of claim 22, further comprising administering to said subject.

29. The opioid antagonist is naloxone or naltrexone, or 23. The use according to claim 22, wherein the mixed agonist-antagonist is buprenorphine. Composition for.

30. 23. The method of claim 22, wherein the method further comprises administering gonadal steroid replacement therapy. Composition for use.

31. The gonadal steroid replacement therapy provides testosterone in men and estrogen in women. further comprising administering strogen and / or progesterone / progestin; 31. A composition for use according to claim 30.

32. 23. The use of claim 22, wherein the method further comprises the administration of one or more gonadotropins. Composition for.

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