Kisspeptin for predicting and treating delayed puberty

Abstract

Methods for diagnosing and treating pathological hypogonadotropic hypogonadism and reproductive endocrine dysfunction using kisspeptin and kisspeptin analogs.

Description

[Technical Field]

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

[0002] Federally sponsored research or development This invention was made with government support under Grant No. HD043341 awarded by the National Institutes of Health and 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. [Background technology]

[0004] Idiopathic hypogonadotropic hypogonadism (IHH) is a rare disorder caused by the absence of GnRH neurons or regulatory neural circuits in the hypothalamus. This results in defective secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and failure to achieve normal reproductive function by age 18. When accompanied by anosmia, IHH is treated as Kallmann syndrome (KS). Untreated patients remain sexually infantile.

[0005] Children who exhibit delayed puberty pose a formidable challenge to clinicians because it is difficult to predict which children will ultimately progress through puberty 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 (i.e., physiological suppression of the reproductive endocrine axis due to chronic illness, stress, or negative energy balance). However, these conditions explain only 36–47% of girls and 11–29% of boys with delayed puberty who present for pediatric endocrinology treatment (3–5). For the majority of girls and boys with delayed puberty, providers are left with two possible diagnoses with markedly different outcomes: constitutional delay and IHH (1). Constitutional delay is a self-limited condition in which puberty begins late (or is temporarily halted after initiation) but eventually begins and progresses to the achievement of full adult reproductive endocrine function (6). In contrast, IHH is a pathological disorder that requires treatment (7). Currently, there are no reliable methods to prospectively distinguish constitutional delay from IHH. Summary of the Invention

[0006] Provided herein are methods for treating a subject with reproductive endocrine dysfunction, optionally pathological hypogonadotropic hypogonadism, comprising administering to the subject a therapeutically effective amount of (i) multiple doses (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 2, 3, 4, 5, or 6 per day) of kisspeptin or a kisspeptin analog, and / or (ii) an opioid antagonist or mixed agonist-antagonist. Provided herein are compositions comprising (i) kisspeptin or a kisspeptin analog, and / or (ii) an opioid antagonist or mixed agonist-antagonist, for use in a method of treating a subject having pathological hypogonadotropic hypogonadism, comprising administering multiple doses of kisspeptin or a kisspeptin analog, and / or (ii) an opioid antagonist or mixed agonist-antagonist.

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

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

[0009] In some embodiments, each of the multiple doses comprises a dose equivalent to 0.2-0.3 nmol / kg kisspeptin-10, preferably 0.24 nmol / kg kisspeptin-10.

[0010] In some embodiments, the multiple doses include at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10. In some embodiments, the at least one supraphysiological dose is administered as the first dose of the multiple doses, or as the first two or more doses of the multiple doses, optionally all of the multiple doses.

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

[0012] In some embodiments, the method further comprises administering one or more gonadotropins (eg, luteinizing hormone (LH) and / or follicle stimulating hormone (FSH)).

[0013] Also provided herein are methods for identifying subjects having, at risk of, or having a mild form of reproductive endocrine dysfunction (RED), such as pathological hypogonadotropic hypogonadism. The method includes measuring a baseline level of LH in the subject; administering to the subject a stimulatory dose of kisspeptin or a kisspeptin analog, e.g., a dose comprising 0.08 to 15 nmol / kg kisspeptin-10 administered by intravenous bolus (IBV) or a dose comprising 0.8 to 500 nmol / kg kisspeptin-10 administered by subcutaneous (SC) injection; measuring at least one LH level after administration of the stimulatory dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulatory dose; comparing the baseline level of LH in the subject with the LH level after administration of the stimulatory dose; and identifying a subject with delayed puberty having an LH level after administration of the stimulatory dose that is equivalent to the baseline LH (e.g., less than 3.5, 3, 2.5, or 2 times baseline) as having RED, e.g., pathological hypogonadotropic hypogonadism.

[0014] Alternatively, the method may include administering to the subject a stimulatory dose of kisspeptin or a kisspeptin analog, e.g., an intravenous dose containing 0.08 to 15 nmol / kg kisspeptin-10 administered by intravenous bolus (IVB) or a dose containing 0.8 to 500 nmol / kg kisspeptin-10 administered by subcutaneous (SC) injection; measuring at least one LH level after administration of the stimulatory dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulatory dose; comparing the LH level after administration of the stimulatory dose with a reference level; and identifying a subject with delayed puberty having an LH level after administration of the stimulatory dose that is below (or not significantly different from) the reference level of LH as having RED, e.g., pathological hypogonadotropic hypogonadism.

[0015] In some embodiments, the methods include administering to the identified subject a treatment for a reproductive endocrine dysfunction, such as pathological hypogonadotropic hypogonadism.

[0016] In some embodiments, treatment involves administering multiple doses of kisspeptin or a kisspeptin analog. In some embodiments, the multiple doses are administered 1 to 6 hours apart, preferably 2 to 3 hours apart, over a period of at least 2 to 3 days, preferably at least 1 to 12 months. In some embodiments, the multiple doses each contain a dose equivalent to 0.08 to 2.4 nmol / kg kisspeptin-10 administered by intravenous bolus (IVB), subcutaneous injection (SC), or pump. In some embodiments, the multiple doses each contain a dose equivalent to 0.2 to 0.3 nmol / kg kisspeptin-10, preferably 0.24 nmol / kg kisspeptin-10. In some embodiments, the multiple doses include at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10. In some embodiments, the at least one supraphysiological dose is administered as the first dose of the multiple doses, or as the first two or more doses of the multiple doses, or optionally as all of the multiple doses.

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

[0018] In some embodiments, the treatment further comprises administering gonadal steroid replacement therapy, eg, testosterone in men, or estrogen and / or progesterone / progestin in women.

[0019] In some embodiments, the method further comprises administering one or more gonadotropins (eg, luteinizing hormone (LH) and / or follicle stimulating hormone (FSH)).

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

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods and examples are for illustrative purposes only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, shall prevail.

[0022] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [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] Regraphed 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] Despite nearly 50 years since the discovery of GnRH (31), understanding the factors that initiate sexual maturation and subsequently maintain reproductive function remains a challenge, complicating diagnosis and treatment. Patients with idiopathic hypogonadotropic hypogonadism (IHH) represent an important population for elucidating these signals because they have abnormal GnRH secretion / action (32, 33). Most IHH patients present as teenagers with delayed pubertal development and, if left untreated, suffer from lifelong sexual infantility and infertility (32, 33).

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

[0026] Diagnosis of pathological hypogonadotropic hypogonadism For decades, clinicians and researchers have attempted to develop tests to predict whether children with delayed puberty will ultimately progress to puberty (8). These tests include measurements of baseline (unstimulated) serum gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone, FSH), gonadotropins after stimulation with GnRH or GnRH analogs, and baseline inhibin B. Unfortunately, all of these tests have significant deficiencies in sensitivity, specificity, or both (8). For example, tests have shown significant overlap in serum inhibin B concentrations between boys with constitutional delay and those with IHH, with 10–29% of boys with constitutional delay and 22–45% of boys with IHH having inhibin B levels within the overlapping range (9–11). Despite an increasingly better understanding of the genetics of IHH and constitutional delay, genetic testing is currently inadequate for predicting pubertal outcome. Currently, fewer than half of patients with IHH have an identifiable mutation in the IHH gene. (7) Furthermore, even when a mutation is found, predictive power is limited by variable penetrance and expressivity; within the same family, some carriers of IHH gene mutations may have IHH and others may have constitutional delayed onset. (24)

[0027] It is known that children with delayed puberty who progress later into puberty are often indistinguishable from those with persistent hypogonadism at initial presentation, and several years of waiting may be required before a given child's outcome is known. As a result, patients, families, and providers may face a challenge in deciding between the two common management approaches for delayed puberty: treatment with sex steroids or watchful waiting without intervention.

[0028] Kisspeptin is a neuropeptide secreted in the hypothalamus that potently stimulates GnRH secretion in all mammalian species tested to date, including humans (12). In studies by us and others, reproductively intact adults responded to a single bolus of kisspeptin with a robust LH rise, whereas adults with IHH did not respond significantly (13-19). Therefore, provocative testing using kisspeptin can be used to assess an individual's GnRH secretory capacity.

[0029] Children with delayed or arrested puberty have responses to kisspeptin ranging from no response to a strong response. (20) Herein, we describe the results of a prospective study showing that a child's ability to respond to kisspeptin may predict whether they will subsequently progress through puberty.

[0030] The kisspeptin stimulation test described herein overcomes two fundamental challenges in predicting pubertal outcomes in children with delayed puberty. The first challenge is the lack of a method for distinguishing physiological hypogonadotropic hypogonadism in normal pubertal children from pathological hypogonadotropic hypogonadism in children with IHH. As a provocative test, the kisspeptin stimulation test provides the first method for measuring a child's potential for future GnRH secretion. Indeed, we found that kisspeptin can induce an LH response in children who have progressed to puberty at a time considered prepubertal based on physical examination and daytime laboratory evaluation.

[0031] A second challenge is distinguishing children with temporarily arrested pubertal development from those with IHH and persistently arrested partial pubertal development. We have previously shown that adults with IHH and partial reproductive endocrine activity fail to respond to kisspeptin (19). Similarly, in the current study, one participant with partial reproductive endocrine activity (Participant 8) had a reduced response to kisspeptin, which accurately predicted the absence of pubertal development until age 18, whereas inhibin B and GnRH-induced LH suggested later pubertal progression.

[0032] As described in Example 1, the absence of LH pulses during overnight measurements in two participants (Participants B and 11) did not accurately predict their eventual pubertal progression, whereas kisspeptin stimulation testing accurately predicted their outcome. These participants were receiving sex steroid treatment at the time of their study visit, which may have suppressed endogenous LH secretion. In contrast, their response to kisspeptin was robust, and thus kisspeptin stimulation testing reliably predicted eventual pubertal progression, even when performed in the context of exogenous sex steroid treatment (e.g., testosterone in males, estrogen and / or progesterone / progestin in females).

[0033] Some patients with IHH may have an intact response to kisspeptin (e.g., patients with mutations in TAC3 and TACR3, genes for neurokinin B or its receptor, which are thought to function upstream of kisspeptin, or KISS1, which encodes kisspeptin itself) (25,26). However, this is likely to represent only a small subset of IHH patients, because mutations in TACR3 are present in only approximately 5% of patients with normal olfactory IHH, and mutations in TAC3 and KISS1 are rare causes of IHH (27-30).

[0034] Thus, the methods of the present invention can be used to diagnose pathological hypogonadotropic hypogonadism, e.g., IHH, in a subject, e.g., a mammal, e.g., a human subject. In some embodiments, the subject is at least 10, 11, 12, 13, 14, or 15 years of age and / or less than 18, 19, or 20 years of age, e.g., 10-20 years, 10-19 years, 10-18 years, 11-18 years, 11-19 years, 11-20 years, 12-18 years, 12-19 years, or 12-20 years of age. The methods can also be used to detect reproductive endocrine dysfunction, or partial or late-onset forms of pathological hypogonadotropic hypogonadism, e.g., in subjects of any age. In some embodiments, the methods are used to diagnose congenital forms of pathological hypogonadotropic hypogonadism, e.g., in children of any age, e.g., newborns.

[0035] In some embodiments, the current official clinical definition of IHH requires that patients be 18 years of age or older, but in subjects under 18 years of age, the methods determine that the subject has IHH, allowing this diagnosis to be made at an earlier age than the traditional age cutoff. In some embodiments, the methods determine that the subject is at risk for or likely to develop IHH.

[0036] The method includes administering at least one stimulant 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 nmol / kg kisspeptin-10, up to about 8, 10, 12, 14, or 15 nmol / kg kisspeptin-10, e.g., 0.08 to 15 nmol / kg kisspeptin-10, preferably administered as an intravenous bolus (IVB). The administration of kisspeptin-10 is dependent upon detection of LH levels in response to administration of a dose equivalent to 0.1-12 nmol / kg kisspeptin-10, e.g., 0.2-10.11 nmol / kg kisspeptin-10, e.g., 0.2-0.5 nmol / kg kisspeptin-10, e.g., 0.22-0.25 nmol / kg kisspeptin-10, e.g., 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 is higher than the IV dose range, e.g., 10, 20, or about 30-fold higher than the ranges described above, e.g., at least 0.8 nmol / kg and up to about 500, 600, or 750 nmol / kg. Intranasal doses are typically about 10 times the subcutaneous dose, i.e., 24 to 5000, 6000, or 7500 nmol / kg.

[0037] The method may include obtaining at least one, for example, one or more samples from a subject, assessing the LH level in the sample, and comparing the level with one or more references, for example, a subject reference representing a normal LH level, for example, a level in a subject without pathological hypogonadotropic hypogonadism, and / or a disease reference representing the LH level in a subject with pathological hypogonadotropic hypogonadism.Suitable reference values ​​may include those shown in the examples below.In a preferred embodiment, the method includes obtaining multiple samples from a subject, for example, before, during, and after administration of a stimulating dose, determining the LH level in the samples, and comparing the LH level in the samples before administration of the stimulating dose (for example, baseline level) with the level after administration of the stimulating dose. Thus, the method may include comparing the absolute LH level after kisspeptin administration to a reference (e.g., a reference representative of a cohort of subjects, or a baseline level in the subject) and / or determining the change in LH level from baseline to post-stimulation as either a) a unit increase (difference), or b) a relative change (ratio). In some embodiments, subjects with an LH ratio of 3.5, 4, 4.5, or more, e.g., 4.85 or more, are identified as having constitutional delay, and subjects with an LH ratio less than 3.5, e.g., less than 3, e.g., 2.33 or less, are identified as having pathological LH.

[0038] As used herein, the term "sample" when referring to a material to be tested for the presence of LH using the methods described herein includes, inter alia, whole blood, plasma, serum or urine.

[0039] Various methods for quantifying LH from a sample are well known in the art.This method may include isolating and / or purifying LH from the sample before quantification.An "isolated" or "purified" biological marker is substantially free from cellular material or other contaminants from the cell or tissue from which the biological marker is derived, i.e., partially or completely changed or removed from its natural state during human intervention.For example, the nucleic acid contained in the sample is first isolated according to standard methods, for example, by using lytic enzymes, chemical solutions, or by nucleic acid binding resin according to manufacturer's instructions.

[0040] The presence and / or level of LH can be assessed using methods known in the art, such as standard electrophoresis and quantitative immunoassay methods for proteins, including but not limited to Western blot, enzyme-linked immunosorbent assay (ELISA), biotin / avidin-type assay, protein array detection, radioimmunoassay, immunohistochemistry (IHC), immunoprecipitation assay, FACS (fluorescence activated cell sorting), mass spectrometry (Kim (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). The methods typically involve revealing labels, such as fluorescent, chemiluminescent, radioactive, and enzyme or dye molecules, that provide a signal either directly or indirectly. As used herein, the term "label" refers to the coupling (i.e., physical binding) of a detectable substance, such as a radioactive agent or fluorophore (e.g., phycoerythrin (PE) or indocyanine (Cy5)), to an antibody or probe, as well as the indirect labeling of a probe or antibody by reaction with a detectable substance (e.g., horseradish peroxidase, HRP).

[0041] In some embodiments, ELISA may be used, in which the wells of a microtiter plate are coated with an antibody against the protein to be tested. A sample containing or suspected to contain a biological marker is then applied to the well. After a sufficient time for the antibody-antigen complex to form, the plate is washed to remove unbound molecules, and a detectably labeled molecule is added. Again, after a sufficient incubation period, the plate is washed to remove excess unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of ELISA, such as competitive ELISA or competitive assays and sandwich ELISA, may also be used, as they are well known to those skilled in the art. Immunometric assays may be used.

[0042] Mass spectrometry, and specifically matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and surface-enhanced laser desorption / ionization mass spectrometry (SELDI-MS), are useful for detecting LH (see U.S. Pat. Nos. 5,118,937, 5,045,694, 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 al., Theriogenology. 1999 Apr 1;51(5):911-26.

[0044] In some embodiments, the LH level after kisspeptin stimulation is equivalent to the presence and / or level of the protein in the disease reference, and the subject has one or more symptoms associated with pathological hypogonadotropic hypogonadism, in which case the subject has pathological hypogonadotropic hypogonadism. In some embodiments, the subject does not have any overt signs or symptoms of pathological hypogonadotropic hypogonadism, but the presence and / or level of one or more of the assessed proteins is equivalent to the presence and / or level of the protein in the disease reference, in which case the subject has a mild form of pathological hypogonadotropic hypogonadism and / or has a high likelihood of developing pathological hypogonadotropic hypogonadism. In some embodiments, the subject has signs or symptoms of pathological hypogonadotropic hypogonadism but has a normal LH response, in which case the subject is sent for further testing. In some embodiments, once an individual is determined to have pathological hypogonadotropic hypogonadism or to be at high risk for developing pathological hypogonadotropic hypogonadism, they may be administered a treatment, e.g., a treatment known in the art or described herein.

[0045] Appropriate reference value can be determined by methods known in the art, for example, by standard clinical test method and statistical analysis.Reference value can have any relevant form.In some cases, reference comprises the predetermined value of meaningful LH level, for example, the subject reference level that represents normal LH level, for example, the level in the subject that is not affected or does not have the risk of developing the diseases described herein, and / or the disease reference that represents the LH level related to pathological hypogonadotropic hypogonadism, for example, the level in the subject that has IHH or KS.

[0046] The predetermined level can be a single cutoff (threshold) value, such as a median or mean value, or an upper or lower quartile, tertile, or level that defines the boundary of another segment of a clinical trial population that is determined to be statistically different from other segments. It can also be a range of cutoff (or threshold) values, such as a confidence interval. It can also be established based on comparison groups, for example, where the association between the risk of developing or the presence of disease in one defined group is several times higher or lower (e.g., about 2-fold, 4-fold, 8-fold, 16-fold, or more) than the risk or presence of disease in another defined group. This may be a particular range, for example, where a subject population (e.g., control subjects) is divided evenly (or unequally) into groups, such as a low-risk group, an intermediate-risk group, and a high-risk group, or quartiles in which the lowest quartile is the subject at lowest risk and the top quartile is the subject at highest risk, or n-quantiles (i.e., n regularly affected intervals) in which the bottom n-quantile is the subject at lowest risk and the top n-quantile is the subject at highest risk.

[0047] In some embodiments, the predetermined level is the level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point (e.g., before stimulation with kisspeptin or a kisspeptin analog). Thus, in some embodiments, the method can include calculating a ratio of the levels or a difference in the levels, and detecting the presence of a change in the level of LH after stimulation with kisspeptin or a kisspeptin analog.

[0048] The subject that is related to a predetermined value is typically referred to as a reference subject.For example, in some embodiments, the control reference subject does not have the disorder described herein (for example, pathological hypogonadotropic hypogonadism).The control subject may be preferably male, or the control subject may be preferably female, and the reference level used for the subject of the same gender is established.The control subject may be preferably with pathological hypogonadotropic hypogonadism, or the control subject may be preferably not with pathological hypogonadotropic hypogonadism (for example, the subject with constitutional delayed puberty, but eventually undergoes puberty on its own without intervention).

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

[0050] Therefore, in some cases, the LH level in a subject that is less than or equal to the reference level of LH (or the change in LH after stimulation) indicates a clinical condition (for example, pathological hypogonadotropic hypogonadism).In other cases, the LH level in a subject that is greater than or equal to the reference level of LH indicates the absence of disease or normal risk of disease.In some embodiments, the amount by which the level in a subject is below the reference level is sufficient to distinguish the subject from control subjects, and optionally is statistically significantly lower than the level in control subjects.When the LH level in a subject is equivalent to the reference level of LH, "equivalent" refers to being nearly equivalent (for example, not statistically different).

[0051] In prepubertal children with hypogonadism (as opposed to insufficient brain or pituitary levels, i.e., hypogonadotropic hypogonadism), the response to kisspeptin may be greater than in the general population, although they are still at "normal risk" for pathological hypogonadotropic hypogonadism.

[0052] The predetermined value may depend on the specific subject (e.g., human subject) population that is selected.For example, a population that is considered healthy will have a "normal" range of LH level that is different from the subject population that has, is likely to have, or is at high risk of pathological hypogonadotropic hypogonadism.Therefore, the predetermined value selected may take into account the category (e.g., gender, age, presence of other diseases) that the subject (e.g., human subject) belongs to.Appropriate ranges and categories can be selected by those skilled in the art through simple experimentation.

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

[0054] Treatment method The methods can 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 methods for the treatment of disorders associated with pathological hypogonadotropic hypogonadism. In some embodiments, the disorder is IHH or KS (e.g., when the subject has anosmia). Generally, the methods involve administering a therapeutically effective amount of kisspeptin or a kisspeptin analog described herein to a subject in need of, or determined to be in need of, such treatment. The methods may also be used to treat subjects with, for example, a negative energy balance state (e.g., malnutrition, anorexia, or athletes), hyperprolactinemia, adult-onset hypogonadotropic hypogonadism, hypothalamic amenorrhea due to medication effects (e.g., glucocorticoids, opioids), or mild forms of pathological hypogonadotropic hypogonadism, and / or subjects at risk of developing pathological HH, such as subjects with idiopathic infertility, irregular menstrual cycles, or abnormal semen analysis.

[0056] In this context, "treating" refers to alleviating at least one symptom of the disorder associated with pathological hypogonadotropic hypogonadism.Pathological hypogonadotropic hypogonadism often results in, for example, the absence of puberty by age 18, or the poor or non-development of secondary sexual characteristics, or infertility, and thus treatment can result in the onset of puberty, the development of secondary sexual characteristics, and / or the restoration of fertility.Administering a therapeutically effective amount of the compounds described herein for the treatment of pathological hypogonadotropic hypogonadism can result in one or more of the following: increased LH levels, increased levels of sex steroids (e.g., testosterone in men, estrogen and / or progesterone / progestin in women), and increased gametogenesis, for example, in some cases, increased levels of LH in response to the administration of a stimulating dose of kisspeptin or kisspeptin analog.

[0057] Thus, in some embodiments, methods for treating pathological hypogonadotropic hypogonadism, such as IHH or KS, are provided herein. The methods may include administering one or more doses, e.g., supraphysiological and / or physiological or near-physiological doses, of kisspeptin or a kisspeptin analog. In some embodiments, doses may be administered periodically, e.g., every 1-6, 1-4, 2-6, or 2-4 hours, for days, weeks, months, or years, e.g., over a 2-4 day, e.g., 48-hour period, optionally once a month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 10 months, or once a year, or, optionally, chronically, e.g., daily over a period of days, weeks, months, or years. Treatment may continue until a desired outcome, e.g., fertility or development of secondary sexual characteristics, is achieved, at which time it may optionally be stopped or reduced. In some embodiments, at least some doses are at physiological or near-physiological levels, e.g., 0.08-2.4, e.g., 0.1-5, 0.2-0.4, e.g., 0.24 nmol / kg IVB. In some embodiments, the methods include administering at least one supraphysiological dose, e.g., 2-25, e.g., 2.4-24 nmol / kg kisspeptin-10, e.g., a dose equivalent to at least 2, 2.4, 2.5, 2.6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nmol / kg kisspeptin-10, up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nmol / kg kisspeptin-10. In some embodiments, the dose is administered subcutaneously at physiological doses of 0.8 to 60 nmol / kg and supraphysiological doses of 60 to 1200 nmol / kg.

[0058] In some embodiments, administration is, for example, intravenous, subcutaneous or nasal.In some embodiments, the method comprises using a pump to administer dosage.Therefore, administration can be, for example, by using an infusion pump, which comprises a reservoir containing a liquid composition comprising active agent and an infusion pump for delivering / administering the composition to subject or patient, in the form of a device or system that subject or patient has, or a miniaturized device that can deliver a periodic bolus of corresponding active agent, suitable for implantation in the body of subject or patient, for example, a device similar to an insulin pump.

[0059] In some embodiments, the method includes repeating the kisspeptin stimulation test, as described above, for example, after 2-3 days. In some embodiments, treatment continues for months and years until all desired effects are seen. For example, for ovulation induction in females, the course may be anywhere from 1 to 6 months. For spermatogenesis induction in males, the course may be anywhere from 3 to 24 months. For puberty induction and / or maintenance of reproductive endocrine function, treatment may extend over several years.

[0060] The method may additionally or alternatively include administering an opioid antagonist and / or conventional treatment for pathological hypogonadotropic hypogonadism.

[0061] Figures 5C and 5D are from patients with KS, so kisspeptin resistance can occur in either the normal or anosmia form of hypogonadism. A patient's genetic characteristics do not predict their ability to respond to kisspeptin. This is supported by Example 3 (see Figure 5D), which showed that patients with variants in two genes still responded. The data from this study support the role of kisspeptin resistance as an overarching pathomechanism for congenital hypogonadism, despite various underlying genetic diagnoses, and may also extend to other causes of reproductive endocrine dysfunction, including, for example, negative energy balance states (e.g., malnutrition, anorexia, or athletes), hyperprolactinemia, adult-onset hypogonadotropic hypogonadism, hypothalamic amenorrhea due to medication effects (e.g., glucocorticoids, opioids), or mild forms of pathological hypogonadotropic hypogonadism, and / or pathological HH in subjects at risk of developing it, for example, subjects with idiopathic infertility, irregular menstrual cycles, or abnormal semen analysis. Sensitivity to kisspeptin increases with repeated exposure, and therefore in some embodiments the method may involve repeated administration of kisspeptin to increase sensitivity to the point where endogenous kisspeptin secretion results in a meaningful response to induce reversal of kisspeptin tolerance.

[0062] Kisspeptin and Kisspeptin Analogs As used herein, kisspeptin refers to a family of neuropeptides resulting from cleavage of a 145-amino acid precursor peptide encoded by the KISS1 gene (Lee et al. 1996, Ohtaki et al. 2001). In humans, the active form of kisspeptin is thought to be a 54-amino acid peptide (Ohtaki et al. 2001, Terao et al. 2004). The method may involve administration of full-length kisspeptin (NP_002247.3), the 54-amino acid peptide (kisspeptin-54 (KP54)), or kisspeptin-10, a 10-amino acid peptide containing the sequence YNWNSFGLRF (SEQ ID NO: 1). Analogs of Kiss-10 are known in the art and include those 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; Roseweir et al., J Neurosci. 2009 Mar 25; 29(12):3920-9. In some embodiments, the analog is [dY] 1KP-10. Analogs may include conservative substitutions, e.g., one, two, or three amino acid substitutions, made in the sequences described herein, wherein the analog maintains agonist activity for KISS1R. In some embodiments, the analog is, among others, a peptide compound in which the peptide bond between a glycine residue located in a region proximal to the C-terminus of the compound and an adjacent residue is replaced by a disubstituted 1,2,3-triazole ring, as described, for example, in WO 2014118318; a peptide compound described in U.S. Patent Application Publication No. 20200172575; Compound 1 described in U.S. Patent Application Publication No. 20200046797; U.S. Patent Nos. 7,960,348 and 8,404,643; C6 described in Parker et al., Theriogenology. 2019 May;130:111-119; KISS1-305; TAK-448 (Ac-D-Tyr-D-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2); Decourt et al., Sci Rep. 2016;6: 26908; MacLean et al., J Clin Endocrinol Metab. 2014 Aug;99(8):E1445-53; Skorupskaite et al., Hum Reprod Update. 2014 Jul;20(4):485-500) (e.g., TAK-448 acetate), or TAK-683 (N-acetyl-YWNTFGL{Met-R}W-NH2 (SEQ ID NO: 3), Kanai et al., J Reprod Dev. 2017 Jun;63(3): 305-310), and compounds described in U.S. Patent Application Publication No. 20160074320, WO 200285399, WO 2004060264, WO 2004101747, WO 2004063221, EP 1604682, WO 2005117939, and EP 1464652.See also Matsui and Asami, Neuroendocrinology 2014;99:49-60. Pharmaceutical compositions containing kisspeptin or kisspeptin analogs, including pharmaceutically acceptable salts thereof, may be used in the methods herein.

[0063] [ka]

[0064] In some embodiments, when a peptide agonist is used, the peptide is modified. Peptide analogs containing the reverse sequence, for example, FRLGFSNWNY (SEQ ID NO: 4), can also be used.

[0065] Peptide analogs described herein may include those modified according to methods known in the art for making peptidomimetics. For example, Qvit et al., Drug Discov Today. 2017 Feb; 22(2): 454-462; Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12: 1239-1254; Avan et al., Chem. Soc. Rev., 2014,43, 3575-3594; Pathak, et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmierski, WM, ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman 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, these modified peptidomimetic versions of the peptides and fragments described herein exhibit enhanced stability in vivo compared to non-peptidomimetic peptides.

[0066] A method for producing a peptidomimetic involves substituting one or more, for example all, of the amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as "retro" sequences. In other methods, the N- to C-terminal order of amino acid residues is reversed, so that the order of amino acid residues from the N- to C-terminus of the original peptide is the order of amino acid residues from the C- to N-terminus of the modified peptidomimetic. Such sequences may be referred to as "inverso" sequences.

[0067] Peptide mimetics may be both inverso versions, or "retro-inverso" versions, of the peptides described herein. The new peptidomimetics may be composed of D-amino acids arranged such that the order of amino acid residues from N- to C-terminus in the peptidomimetic corresponds to the order of amino acid residues from C- to N-terminus in the original peptide.

[0068] Another method for creating peptidomimetics involves replacing one or more amino acid residues in a peptide with a chemically distinct but functionally recognized analog of an amino acid, i.e., an artificial amino acid analog. Artificial amino acid analogs include β-amino acids, β-substituted β-amino acids ("β"). 3 Artificial amino acids include α-amino acids (∀-amino acids), phosphorous analogs of amino acids such as ∀-aminophosphonic acids and ∀-aminophosphinic acids, and amino acids with non-peptide bonds. Artificial amino acids can be used to create peptidomimetics, such as peptide oligomers (e.g., peptoid amide or ester analogs), β-peptides, cyclic peptides, oligourea or oligocarbamate peptides, or heterocyclic ring molecules. An exemplary retro-inverso peptidomimetic includes FRLGFSNWNY, where the sequence contains all D-amino acids.

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

[0070] Opioid antagonists In some embodiments, the methods described herein include administering an effective amount of an opioid antagonist, such as naloxone or naltrexone, or a mixed agonist-antagonist, such as buprenorphine.Others, such as nalmefene, can also be used.The opioid antagonist can be administered instead of, before, after, or simultaneously with kisspeptin or kisspeptin analogs, or other treatments described herein.Therefore, provided herein is a composition comprising (i) kisspeptin or kisspeptin analogs, and (ii) an opioid antagonist, such as naloxone or naltrexone, or a mixed agonist-antagonist, such as buprenorphine.

[0071] Conventional treatment of pathological hypogonadotropic hypogonadism In some embodiments, the methods include administering gonadal steroid replacement therapy, such as testosterone in men (e.g., testosterone esters, (e.g., enanthate, cypionate, undecanoate) and estrogen and / or progestin in women (e.g., conjugated estrogens (Premarin), ethinyl estradiol, or estradiol, and / or medroxyprogesterone, micronized progesterone), and / or hormonal infertility medications (e.g., ethinyl estradiol / norethindrone). The methods may also include administering a gonadotropin, such as follicle-stimulating hormone (FSH) or human chorionic gonadotropin (hCG). Optionally, clomiphene and / or letrozole may be used to stimulate ovulation in a subset of patients. [Example]

[0072] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[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 The physiological kisspeptin stimulation study was approved by the Massachusetts General Hospital (MGH) / Partners IRB and registered with ClinicalTrials.gov (NCT01438034). Kisspeptin was administered under IND 113,591, and GnRH was administered under IND 93,353. Studies of the genetics of delayed puberty and IHH were approved by Boston Children's Hospital and MGH, respectively. All participants provided written informed consent, and at least one parent provided written informed consent. For the two participants who underwent retesting, a repeat study was approved by the Partners IRB, and the participants and their parents provided written informed consent for retesting.

[0075] Test Protocol Details of the study protocol have been described previously (20). Briefly, inclusion criteria were age 12 years or older for girls and age 13.5 years or older for boys, with delayed or arrested puberty defined based on breast development for girls and testicular volume for boys. Children with identifiable causes of delayed puberty were excluded. Participants were assessed for overnight baseline LH secretion, LH secretion in response to 0.313 mcg / kg kisspeptin-10 (ΔLH キスペプチン ), and LH secretion in response to 75 ng / kg GnRH (ΔLH GnRH The patient underwent two admissions to the MGH Translational and Clinical Research Center (TCRC) for blood sampling every 10 minutes to measure ΔLH. キスペプチン and ΔLH GnRHevaluated both before and after pituitary “priming” with pulsatile 75 ng / kg subcutaneous GnRH every 2 hours for 6 days to ensure a robust pituitary response to GnRH ( 8 ).

[0076] Participants then returned for follow-up visits every 6 months for physical examinations and measurements of FSH, LH, and estradiol or testosterone to assess reproductive endocrine activity. Upon reaching age 18, participants underwent a final evaluation of physical and laboratory signs of reproductive endocrine activity. For participants receiving the six steroid treatments, treatment preceded laboratory testing to ensure that sex steroid measurements reflected endogenous production rather than exogenous administration (over 4 weeks for estradiol, 6 weeks for injectable testosterone, and 2 weeks for transdermal testosterone).

[0077] Laboratory Assays For TCRC visits, LH, FSH, testosterone (T), and estradiol were measured by the MGH Clinical Laboratory Research Core, the Brigham and Women's Hospital Research Assay & Analysis Core, and Labcorp as previously described (20). For example, serum LH and FSH were measured using a microparticle enzyme immunoassay with an automated Abbott AxSYM system (Abbott Laboratories, Chicago, IL), and serum T concentrations were measured using a DPC Coat-A-Count RIA kit (Diagnostics Products Corporation, Los Angeles, CA) (60). The precision of the LH assay ranged from 4.3 to 6.4%. Inhibin B was measured in a single batch by immunoassay at the University of Virginia Ligand Assay Core, with an intra-assay coefficient of variation of 2.5%. For follow-up visits, laboratory testing was performed by Labcorp and Quest Diagnostics. For participants outside the Boston area, follow-up data were obtained through routine clinical care from local endocrinologists and clinical laboratories.

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

[0079] statistics The Fisher's exact test was used to assess the correlation between kisspeptin stimulation testing and pubertal outcomes. A p value of less than 0.05 was considered significant. Jeffreys intervals were used to calculate confidence intervals 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 this study (four girls and 13 boys; Figure 1). The results of kisspeptin- and GnRH-stimulation tests for 15 of these participants (Participants 1–15) have been previously reported in reference (20). Participant A, newly enrolled in this study, demonstrated a "kisspeptin non-responder" pattern, whereas Participant B, also newly enrolled in this study, demonstrated a "kisspeptin responder" pattern (Figure 2). The characteristics of the participants and their neuroendocrine phenotypes are shown in Tables 4 and 2 (21).

[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, the boys showed a progressive increase in testicular volume, and the girls showed progressive breast development. All of these participants were "kisspeptin responders," responding to exogenous kisspeptin with an LH rise of 0.8 mIU / mL or more (Figure 3, Table 4).

[0087] In contrast, eight participants (one girl and seven boys) failed to progress through puberty by age 18 years (Table 4, Table 3(21)). These participants demonstrated persistent sexual immaturity on physical examination, with the boy exhibiting a testicular volume of <4 mL and the girl experiencing no breast development until treatment with exogenous estradiol was initiated. Upon evaluation of endogenous reproductive endocrine activity after discontinuation of sex steroid treatment, all of these participants had serum concentrations of sex steroids (estradiol for girls and testosterone for boys) below the adult reference range and serum gonadotropin concentrations that were low or, in the context of low sex steroids, inappropriately normal. Those individuals who failed to enter puberty were "kisspeptin non-responders" (one girl and six boys) who showed little to no response to kisspeptin (the eighth "non-responder" was lost to follow-up) and one boy, an "intermediate responder," who showed an LH response to 0.4 mIU / mL kisspeptin (Figure 3, Table 4).

[0088] Thus, participants' response to kisspeptin significantly distinguished those who progressed to later puberty from those who did not (p=0.0002). Both the sensitivity and specificity for the kisspeptin stimulation test were 100% (95% CI 74-100%).

[0089] LH measurements under three conditions: daytime, overnight, and after GnRH stimulation Unstimulated LH concentrations (measured during the day or overnight) and LH measured after stimulation with exogenous GnRH have been tested as methods for predicting whether a child will ultimately enter puberty (8). In this study, two boys had unstimulated serum LH concentrations in the pubertal range at enrollment, and one later progressed to puberty. Of the remaining 14 participants with LH levels in the pubertal range at enrollment, seven progressed to puberty and seven did not (Figure 4, Table 4). Thus, unstimulated LH was not a reliable predictor of pubertal outcome.

[0090] During early puberty, daytime gonadotropin measurements may not accurately reflect the activity of the reproductive endocrine axis because GnRH and LH secretion pulses occur only during deep stages of nocturnal sleep (23). Therefore, we performed overnight blood sampling to detect sleep-related LH secretion. All six children who had at least one overnight LH secretion pulse were subsequently observed to progress to puberty (Table 4). In contrast, the 10 children who did not have an overnight LH pulse had variable outcomes. Eight children failed to progress to puberty, while two showed pubertal progression. These two children were one girl and one boy (Participants B and 11) who were being treated with exogenous sex steroids at the time of neuroendocrine evaluation, which may have suppressed endogenous LH secretion.

[0091] GnRH-stimulated LH secretion has also been studied as a test for predicting pubertal outcome (8). In our cohort, LH responses to GnRH overlapped, ranging from 1.2 to 15.4 mIU / mL in subjects who later progressed to puberty and from 0.2 to 7.5 mIU / mL in subjects who did not (Figure 4, Table 4).

[0092] Thus, neither of these tests, unstimulated LH measured during the day or at night, nor GnRH-stimulated LH, was an accurate kisspeptin stimulation test for predicting pubertal outcome in this study cohort.

[0093] Inhibin B Measurement of serum inhibin B has been proposed as a method for predicting pubertal outcome in children with delayed puberty (8-11). Serum inhibin B ranged from 39 to 209 pg / mL in boys who progressed later in puberty and from <17 pg / mL to 48 pg / mL in boys who did not progress (Figure 4, Table 4). Therefore, inhibin B did not accurately distinguish between children who would progress later in puberty and those who would not progress.

[0094] Genetic Testing A subset of patients consented to exome analysis. Pathogenic and likely pathogenic variants in the IHH gene (Table 1 (21)) were identified in two of the five participants who later progressed to puberty and in two of the six participants who did not (Table 5). No pathogenic or likely pathogenic variants were identified in IGSF10, a gene that may be associated with constitutional delay. Therefore, genetic testing could not predict pubertal outcome for children with delayed puberty.

[0095] [Table 6]

[0096] [Table 7]

[0097] [Example 2] Supraphysiological kisspeptin for improving pubertal outcomes for youth with delayed puberty One subject with delayed puberty in the study described in Example 1 received 0.313 μg / kg IVB (physiological dose) kisspeptin-10 at age 15.3 years and failed to mount an LH response. Shortly thereafter, during another hospitalization, the subject received a supraphysiological dose of kisspeptin (18 μg / kg) and, surprisingly, responded. The subject's final diagnosis was IHH, as evidenced by her failure to show physical signs of puberty by age 18 years. This indicates 1) that GnRH deficiency may be due to kisspeptin resistance and 2) that this resistance can be overcome by high-dose kisspeptin.

[0098] Based on this observation, we hypothesized that high-dose and / or repeated kisspeptin administration could stimulate GnRH-induced LH secretion in patients with IHH / KS. To address this hypothesis, we initiated the following protocol: 1) q10-min blood sampling × 6 h, 2) q2-h kisspeptin administration × 40 h (five sets of four doses each: 0.313, 0.939, 3.13, and 13.19 μg / kg). For comparison, Figure 5A shows a healthy male. He had normal LH pulses, and physiological doses of kisspeptin clearly produced GnRH-induced LH pulses. Figure 5B shows a KS male with no endogenous LH pulses and no LH response to kisspeptin (i.e., kisspeptin resistance). The male has rare variants in the genes encoding prokinectin receptors, PROKR2 and DMXL2. Figure 5C shows a KS woman who did not respond to physiological kisspeptin doses but responded to supraphysiological doses, producing clear kisspeptin-induced GnRH-induced LH pulses. Furthermore, the amplitude of the pulses increased over time, suggesting a "priming" effect of repeated exposure. The genetic characteristics of the woman are unknown. Figure 5D shows a KS man who also carries a genetic variant; his response to supraphysiological kisspeptin was not as pronounced. Figure 6 shows the data from Figure 5C re-graphed, clearly demonstrating the response to each dose over time. Thus, although patients with persistent hypogonadism were thought to be kisspeptin-resistant to a single IV bolus dose, these patients showed significantly different responses to repeated kisspeptin administration, especially at higher doses. The data indicate that GnRH neurons in patients with IHH / KS can be evoked from their resting state by repeated kisspeptin stimulation. Furthermore, this repeated kisspeptin stimulation resulted in GnRH-induced LH pulses.

[0099] [Example 3] Hypothalamic reproductive endocrine pulse generator activity is independent of neurokinin B and dynorphin signaling Identification of the afferent pathways through which endogenous factors (e.g., gonadal steroids, stress hormones, and nutritional signals) and external cues (e.g., social cues and photoperiod) regulate GnRH release has recently focused on the kisspeptin / neurokinin B / dynorphin system (34). Inactivating mutations in kisspeptin, neurokinin B (NKB), and their respective receptors cause IHH in humans and mice, implicating these neuropeptides in the generation of GnRH pulses (35-42). Dynorphin is thought to oppose this stimulatory activity by providing a significant delay in GnRH pulse-generating activity in response to progesterone during the luteal phase of the menstrual cycle (43-45). These three neuropeptides fuse in a neuronal population of the arcuate nucleus, the KNDy (kisspeptin-neurokinin B-dynorphin) dyuron, and act in a coordinated manner to synchronize the secretory activity of GnRH neurons and generate the pulse of GnRH secretion necessary to drive reproductive endocrine function ( 46 – 48 ).

[0100] Because biallelic loss-of-function mutations disrupt both copies of a gene, patients with such mutations (i.e., "human knockouts") provide novel insights into the phenotypic consequences of gene disruption or loss. In this study, four sisters with biallelic complete loss-of-function mutations in the gene encoding NKB (a key neuropeptide in KNDy neurons) underwent genotype-driven phenotyping. Despite their initial diagnosis of IHH, some sisters spontaneously recovered reproductive endocrine function in adulthood. Studies were performed in both normal and neurokinin B-deficient family members and normal and neurokinin B-deficient mice to examine the role of NKB in GnRH pulse generation and analyze the interactions between NKB, kisspeptin, and dynorphin. The use of a combination of specific neuroendocrine probes revealed that the hypothalamus was able to generate GnRH-induced LH pulses despite genetic and pharmacological antagonism of two of the three KNDy components, NKB and dynorphin.

[0101] The results demonstrate elevated LH levels during NLX infusion in subjects with IHH. To date, the ability to stimulate endogenous GnRH-induced LH pulsations that mimic normal physiology in patients with IHH has not existed.

[0102] Considerations regarding LH pulses include the observation that Subject 4 appeared to have a more pronounced response to NLX than Subject 5. Subject 4 received pituitary priming with exogenous GnRH, whereas Subject 5 did not, which may have amplified the effect of NLX on the LH response in Subject 4. Subject 4 was also receiving intermittent hormone replacement therapy, which may have enhanced endogenous kisspeptin action during GnRH release.

[0103] Previous studies have shown that the same dose of kp-10, which produces a strong GnRH-induced LH response in healthy men and luteal-phase women, fails to produce any effect in IHH patients with a range of genotypes, indicating that the functional capacity of the GnRH neural network is fundamentally impaired in patients with IHH (58). In contrast to previous observations in IHH patients with genotypes other than TAC3 or TACR3, subjects 3, 4, and 5 responded to kp-10 IVB (58). Here, the ability to respond to low-frequency pulses and exogenous kp-10 administration indicates that the GnRH neural circuitry required for pulse generation remains intact in patients lacking NKB. However, the ability to respond to kp-10 with LH pulses was observed only in the context of IVB administration, not with continuous infusion, as previously reported by others (59). Differences in the kp-10 dose, LH assay, and LH pulse algorithm 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). Subjects were either reproductively normal (Subject 1; genotype TAC3 c.61_61ΔG p.A21LfsX44 heterozygous) or had a diagnosis of hypogonadotropic hypogonadism (Subjects 2–5; genotype TAC3 c.61_61ΔG p.A21LfsX44 homozygous). Siblings and parents were unavailable for study participation. IHH was defined as hypogonadal steroid levels (estradiol <20 pg / mL in women) in the setting of low or normal gonadotropin levels, in the absence of age 18 years or older and identifiable medical conditions that could cause hypogonadotropic hypogonadism. As previously reported (19), recovery of IHH in women was defined as 1) fertility without exogenous GnRH or gonadotropin therapy, 2) at least 3 months of spontaneous menstrual cycles in the absence of treatment, and / or 3) LH pulse frequency and amplitude within the normal range for women. Relapse after recovery was defined as the return of hypogonadal steroid levels (in women, serum estradiol <20 pg / mL) and / or amenorrhea.

[0106] Subjects also participated in genetic testing. Patient DNA was screened for rare sequence variants (RSVs), defined as those with a minority allele frequency of <1% in the Genome Aggregation Database (gnomAD), in genes known to cause IHH, as previously described ( 20 , 21 ). The genes screened were CHD7 (MIM608892), FGF8 (MIM600483), FGFR1 (MIM136350), GNRH1 (MIM152760), GNRHR (MIM138850), HS6ST1 (MIM604846), ANOS1 (formerly KAL1, MIM300836), KISS1 (MIM603286), KISS1R (MIM604161), NSMF (formerly NELF, MIM60813), PROK2 (MIM607002), PROKR2 (MIM607123), TAC3 (MIM162330), and TACR3 (MIM162332), and were identified by PCR amplification of exons followed by Sanger sequencing. RSVs were reported if they were predicted to be damaging in at least two of four in silico prediction programs: PolyPhen-2 ( 22 ), SIFT ( 23 ), Mutation Taster ( 24 ), or Panther ( 25 ). The University of Pennsylvania Smell Identification Test (UPSIT) score from a 12-item olfactory test was used to classify 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) underwent detailed neuroendocrine phenotyping, mapping endogenous LH pulsations, with blood sampling every 10 minutes (q10 min) for 6–8 hours, under the direction of Professor I. Sadaf Farooqi at the Wellcome Trust Clinical Research Facility, Cambridge, UK (Figure 7A).

[0110] In 2016, subjects 1, 3, 4, and 5 were invited to participate in a second series of daytime studies at the Massachusetts General Hospital (MGH) Clinical Research Center (CRC) to determine whether endogenous LH pulse patterns could be altered by administration of naloxone (NLX), a nonspecific opioid antagonist that blocks GnRH, kisspeptin 112-121 (kp-10), and dynorphin (Figures 8A, 9A, and 10A). To ensure that pituitary gonadotropes were in a primed state, subjects 3 and 4 received exogenous pulsatile GnRH at 25 ng / kg every 2 hours (q2h) via a Crono F portable infusion pump (Cane SpA, Turin, Italy) 3 days before admission to the MGH CRC (28). Subject 5 did not undergo pulsatile GnRH priming because she had recent evidence of some neuroendocrine activity (years of spontaneous bleeding) (Table 6).

[0111] Baseline testing: All subjects underwent 10-minute blood sampling for at least 6 hours to assess endogenous GnRH-induced LH secretion during the day of their MGH CRC visit (Figures 7A, 8A).

[0112] Kisspeptin Bolus: After assessment of endogenous GnRH-induced LH secretion, subjects 3, 4, and 5 received an intravenous bolus (IVB) of 0.24 nmol / kg kp-10, a dose previously shown by our group to consistently induce GnRH-induced LH pulses of physiological amplitude in healthy men and healthy luteal-phase women (29, 30) (Figure 8A). Subjects 4 and 5 then received 0.72 and 2.4 nmol / kg kp-10 IVB. Subjects 3, 4, and 5 then received 75 ng / kg IVB GnRH at the end of these studies, as our group has previously shown this dose to produce robust GnRH-induced LH responses in individuals with intact gonadotrophic cell function (31).

[0113] Kisspeptin infusion: In contrast to the IVB study, subject 3 returned to CRC and participated in a second admission in which kp-10 was administered as a 12-hour continuous infusion (9.5 nmol / kg / hour) to determine its effect on endogenous GnRH-induced LH pulsations. As in the IVB study, blood samples were drawn q10 min, 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 CRC and received a 13-hour NLX infusion (0.8 mg / hour infusion after 10 mg IVB NLX) to determine the effect of opioid antagonism on endogenous LH pulses in the absence of NKB signaling. Midway through the infusion, boluses of kp-10 and GnRH (kp-10 dose range: 0.24–2.4 nmol / kg, GnRH: 75 ng / kg) were administered to determine whether NLX administration could enhance the response to these peptides (Figure 10A). 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, a 10-amino acid isoform of kisspeptin (corresponding to amino acids 112-121 of the preprohormone), and GnRH were synthesized according to good manufacturing practices by NeoMPS (PolyPeptide Laboratories, San Diego, CA). NeoMPS supplied kisspeptin 112-121 under contract to the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Naloxone was ordered from Hospira (Lake Forest, IL).

[0116] Human laboratory assays LH and 2-hour pool estradiol for each sample were measured by direct immunoassay using the automated Abbott ARCHITECT system (Abbott Laboratories, Inc., Abbott Park, IL) as previously described (28). Estradiol was measured by a second-generation immunoassay traceable to a mass spectrometry-based assay for the 2010–2011 study and by Elecsys (Roche Diagnostics, Indianapolis, IN) for the 2016 study (32, 33).

[0117] Assessment of pulsatile LH release in adolescent and adult Tac2 knockout mice Tac2+ / - breeding pairs were generated and genotyped by the Texas A&M Institute for Genomic Medicine (College Station, TX) (34). All mice were generated and maintained on an Sv129 / C57BL / 6 hybrid background and group-housed (3–5 mice per cage) in a temperature- and light-controlled environment at Brigham and Women's Hospital, with light on from 06:00 to 18:00 h and free access to food and water. Mice were handled daily for 2–6 weeks prior to the experiment to allow for acclimation to the sampling conditions.

[0118] Changes in LH secretion were measured in sexually mature (6 weeks old) and adult (16 weeks old) intact and ovariectomized (OVX) Tac2 knockout (KO) female mice and control (wild-type, WT) littermates (n = 4–5 per group). These mice lack NKB, as Tac2 in mice encodes NKB in humans. Pulsatile LH secretion was assessed by repeated blood collection via a single incision at the tip of the tail. After the tail was washed with saline, 4 μl of blood was collected from the cut tail with a pipette at each time point. Whole blood was immediately diluted with 116 μl of 0.05% PBST, vortexed, and frozen on dry ice. Samples were stored at -80°C for subsequent LH ELISA. For the KP-10 administration study, 36 consecutive blood samples were collected over a 6-hour sampling period. At 170 min of sampling (or 180 min for adolescent Tac2 knockout mice), mice were intraperitoneally injected with mouse kp-10 (7.5 nmol / 100 μl saline, Phoenix Pharmaceuticals). For NLX administration studies, 30 consecutive blood samples were collected over a 5-h sampling period from WT and Tac2 KO mice. WT and Tac2 KO mice were ovariectomized to increase the frequency and amplitude of LH pulses to better determine the effect of dynorphin removal on LH pulse generation. At 120 min of sampling, mice were intraperitoneally injected with NLX (5 mg / kg / 100 μl saline, Sigma-Aldrich).

[0119] Data analysis Human pulse analysis: LH pulses were identified using a modified version of the validated Santen and Bardin method (35, 36) and augmented by a deconvolution algorithm (29). Pulse amplitudes of kp-10- or GnRH-induced LH pulses were calculated as the difference between time 0 of kp-10 or GnRH administration and the peak of the pulse.

[0120] Mouse pulse analysis: LH pulses were identified using custom-written MATLAB code that reads LH pulse data collected by LH sandwich ELISA. The code contains a loop that determines a pulse based on whether a) the height of the LH value exceeds the height of either of the two previous values ​​by 20% and the height of the following value by 10%; or b) the peak in the second time interval (i=2) exceeds the previous single value by 20% to be considered a pulse.

[0121] Statistics: Paired two-tailed t-tests were used to assess mean LH, LH amplitude (nadir to peak of LH pulse) and FSH at baseline compared with the response to neuropeptide intervention as defined in the methods described above. All values ​​are reported as mean ± standard error unless otherwise stated.

[0122] Test Approval All human studies were approved by the Institutional Review Board of MGH / Partners Healthcare or the Local Regional Ethics Committee of Cambridge, United Kingdom. All subjects provided written informed consent before participating in the studies. For mouse studies, the Brigham and Women's Hospital Institutional Animal Care and Use Committee approved all procedures.

[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). Her sisters, subjects 2, 3, 4, and 5, presented with primary amenorrhea at ages 13–15 years and received estrogen therapy to induce secondary sexual characteristics. Because of spontaneous sexual maturation by age 18 years, lack of normal MRI findings, and low gonadotropins, subjects 2, 3, 4, and 5 all received a diagnosis of IHH (Table 6). None of the sisters had anosmia. Three of the four IHH sisters demonstrated reversal of hypogonadism between ages 22–28 years, as evidenced by conception without fertility medication (subjects 3 and 4) and regular spontaneous menstrual cycles (subject 5). However, the reversal was not sustained, and by the time of physiological testing, subjects 3, 4, and 5 had reverted to a state of hypogonadotropic hypogonadism (Table 6).

[0124] Genetics Sequencing of the candidate gene revealed that subject 1 (with normal pubertal timing and normal menstrual cycles) was heterozygous for a single-nucleotide deletion (c.61_61ΔG p.A21LfsX44) in the gene encoding NKB (TAC3). This base pair deletion results in a frameshift mutation and a premature stop codon for the preprohormone before the NKB sequence, which is predicted to result in nonsense-mediated decay. Even if the transcript escapes nonsense-mediated decay, the frameshift mutation would destroy the portion of the preprohormone that is processed to produce the decapeptide known as NKB. Subjects 2, 3, 4, and 5 all have hypogonadotropic hypogonadism and are homozygous for this frameshift mutation. This mutation is novel and not found in gnomAD, a normative database containing 123,136 exomes and 15,496 genomes (21). Notably, no individuals in gnomAD are homozygous for protein-truncating mutations in TAC3, and this family does not carry any other genetic mutations known to cause IHH.

[0125] Baseline testing: Slow LH pulse frequency characterizes IHH individuals without neurokinin B During these baseline examinations, the IHH sisters (subjects 2, 3, 4, and 5) were amenorrheic, had low but detectable serum estradiol levels, and low progesterone levels, without hormonal medication (Table 6, Figure 7B). All subjects with IHH had evidence of a weakened but organized GnRH pulse generator, evidenced by infrequent LH secretory events (for comparison, during the physiological early follicular phase, characterized by low estradiol and progesterone, LH frequency was 7.0 ± 1.8 pulses / 12 h, LH amplitude was 2.3 ± 1.0 IU / L [mean ± 2 SD]) (37, 38). In subjects 2, 4, and 5, one pulse was observed during the sampling interval (7–8 h, mean LH amplitude 1.5 ± 0.8 mIU / mL) (Figure 7B). In subject 3, no pulses were observed during the study. Furthermore, the LH levels of subjects 3, 4, and 5 showed a gradual decay at the beginning of the sampling interval, indicating that LH secretory events had occurred before the start of the study. Accordingly, all subjects exhibited an abnormally low frequency of LH secretory events. When the study was repeated in 2016, the study subjects (subjects 3, 4, and 5) were again amenorrheal, and without hormonal medication, estradiol levels were low but detectable. All studies reproduced the same endogenous LH pattern observed in 2010, with a low frequency of LH secretory events and 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 her menstrual cycle (early follicular phase; EFP). The subject exhibited 11 LH pulses over 12 hours, with a mean LH pulse amplitude of 0.46 ± 0.25 mIU / mL (Figure 7C). (Healthy early follicular phase women: frequency, 7.0 ± 1.8 pulses / 12 hours, 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 LH pulses (Figure 8B). Two test subjects received three kisspeptin boluses and showed LH pulses after kisspeptin in five of the six boluses. The one exception occurred when kisspeptin was administered immediately after the endogenous LH peak, resulting in a prolonged single peak (Figure 8B, subject 5). Consistent with this responsiveness, all subjects demonstrated adequate pituitary priming, indicating the absence of a pituitary defect that could impair kisspeptin responsiveness (LH pulse amplitude after GnRH administration: subject 3: 1.6 mIU / mL, subject 4: 5.1 mIU / mL, subject 5: 3.0 mIU / mL).

[0128] Kisspeptin infusion: no pulsatile LH secretion Subject 3 received a kp-10 infusion (9.5 nmol / kg / hr) for 12 hours, with no detectable LH pulses. A mild increase in mean LH was observed during the infusion (baseline: 0.46 ± 0.24 mIU / mL; kp-10 infusion: 0.63 ± 0.08 mIU / mL; p < 0.0001) (Figures 8B & 9B). Mean FSH levels also increased compared to baseline (baseline: 1.9 ± 0.2 mIU / mL; kp-10 infusion: 2.4 ± 0.1 mIU / mL; p < 0.001). After the kp-10 infusion, Subject 3 received an IVB dose of GnRH, which resulted in LH pulses of similar amplitude to those observed in the baseline test the previous day (baseline, 1.6 mIU / mL; post-kp-10 infusion, 2.5 mIU / mL).

[0129] Naloxone infusion: Blockade of dynorphin by naloxone increases LH and FSH secretion and LH pulse frequency but does not amplify kisspeptin-induced LH pulses. Subjects 4 and 5 received the nonselective opioid antagonist, NLX, and increasing boluses of kisspeptin (0.24, 0.72, 2.4 nmol / kg) to determine the effect of blockade of dynorphin signaling on endogenous and kisspeptin-stimulated LH secretion patterns. Both studies demonstrated an increase in mean LH levels during NLX infusion compared to baseline (Subject 4—Baseline: 1.44 ± 0.76 mIU / mL, NLX: 2.82 ± 0.54 mIU / mL, p < 0.00001; Subject 5—Baseline: 0.6 ± 0.25 mIU / mL, NLX: 1.1 ± 0.37 mIU / mL, p < 0.00001, across matched time points) (Figures 8B, 10B). For the study subject, Subject 4, for whom LH sampling on and off NLX infusion was completed and comparable, LH pulse frequency increased from one pulse per 6 hours (Figure 8B) to four pulses per 6 hours (Figure 10B). Mean FSH levels also increased compared to baseline (Subject 4—Baseline: 3.7 ± 0.3 mIU / mL, NLX: 5.0 ± 0.9 mIU / mL; p < 0.01; Subject 5—Baseline: 3.3 ± 0.3 mIU / mL, NLX: 5.1 ± 0.1 mIU / mL; p < 0.0001). No corresponding changes were observed in LH pulse amplitude (Subject 4—Baseline: 2.59 mIU / mL, NLX: 0.45 ± 0.29 mIU / mL; Subject 5—Baseline: 0.82 mIU / mL, NLX: 1.22 and 1.39 mIU / mL). NLX infusion blocks dynorphin by inhibiting opioid tone and, through the absence of NKB signaling, increases gonadotropin secretion and improves LH pulse frequency in individuals with IHH.

[0130] Subjects 4 and 5 also received increasing boluses of kp-10 (0.24, 0.72, and 2.4 nmol / kg), followed by LH pulses; a replicate result was seen with NLX off (Figs. 8B and 10B). There were no significant differences in the kisspeptin-induced LH response to NLX on or off, and there was no clear dose-response relationship, although the small number of boluses at each dose limited the ability to assess such a relationship.

[0131] Kisspeptin bolus stimulates LH release in adolescent and adult WT and NKB-deficient (Tac2 KO) mice To confirm our findings in IHH patients, we performed studies in Tac2 KO and WT control female mice. Peripheral administration of kp-10 induced a robust increase in LH in all groups, regardless of age or genotype. Interestingly, adolescent Tac2 KO female mice lacking NKB exhibited a higher amplitude of LH release (5.29 ± 0.43 ng / ml, n = 5) than control females (2.67 ± 0.48 ng / ml, n = 5; p < 0.01) (Figure ​(Figure11). 11). However, LH returned to baseline more quickly in Tac2 KO mice (52 ± 3.72 min postinjection, n = 5) than in WT controls (68 ± 3.72 min, n = 5; p < 0.01). Adult WT mice showed the expected LH pulse in response to kp-10, whereas Tac2 KO mice showed a biphasic response, exhibiting two overlapping peaks of LH (Figure 10). In both adult groups, the induction of LH release appeared more sustained than in adolescent mice (Adolescent WT: 68 ± 3.742 min post-injection, n = 5 vs. adult WT 142.5 ± 4.78 min, n = 4, p < 0.0001; Adolescent Tac2 KO: 52 ± 3.742 min, n = 5 vs. adult Tac2 KO 156.7 ± 3.33 min, n = 3, p = 0.07).

[0132] Naloxone increases pulsatile LH release in adult OVX WT and Tac2 KO mice To determine the role of opiate (dynorphin)-mediated kisspeptin signaling in the absence of NKB, we examined the effect of NLX, a dynorphin inhibitor, on LH secretion. Peripheral administration of 5 mg / kg of NLX induced a rise in LH secretion within 20 min of administration in both WT (Fig. S1A-S1C) and Tac2 KO female mice (Fig. S1D-E) (WT: 20 min before NLX, 2.37 ± 0.59, n = 4 vs. 20 min after NLX, 4.31 ± 0.32, n = 4; p < 0.05. Tac2 KO: 20 min before NLX, 0.31 ± 0.06, n = 4 vs. 20 min after NLX, 1.22 ± 0.29, n = 4; p < 0.05).

[0133] After NLX administration, WT mice responded by increasing the duration of the subsequent LH pulse (pre-NLX: WT 25 ± 2.67 min, n = 3; Tac2 KO 23.33 ± 2.10 min, n = 3, p = 0.13; post-NLX: WT: 83.33 ± 12.02 min, n = 3; Tac2 KO: 30 ± 5.77 min, n = 3, p < 0.01) (Figure 12A). Furthermore, the increased duration of the LH pulse after NLX was accompanied by significantly longer interpulse intervals in WT mice (WT interpulse interval, pre-NLX 25.38 ± 1.83 min; WT interpulse interval, post-NLX 46.67 ± 3.33 min, p < 0.0002).

[0134] Tac2 KO animals showed significantly lower LH baselines and pulse rates than OVX controls (0-1 LH pulse 120 min before NLX). NLX administration induced strong LH pulses in all cases, occurring 20 min after treatment, with peaks reaching a two-fold increase compared to baseline (pre-NLX: 0.31 ± 0.06 mIU / mL, post-NLX: 1.2 ± 0.28 mIU / mL, p < 0.02). The limited number of LH pulses precluded analysis of the interpulse interval, and the data show that NLX did not increase the duration of LH pulses (pre-NLX Tac2 KO: 23.33 ± 2.10 min, n = 3; post-NLX Tac2 KO: 30 ± 5.77 min, n = 3, p > 0.05) (Figure 12D-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, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are set forth below. [1] A method of treating a subject having reproductive endocrine dysfunction, optionally pathological hypogonadotropic hypogonadism, comprising administering to the subject a therapeutically effective amount of (i) multiple doses of kisspeptin or a kisspeptin analog, and / or (ii) an opioid antagonist or mixed agonist-antagonist. [2] The method of [1], wherein the multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, over a period of at least 2 to 3 days, preferably at least 1 to 12 months. [3] The method of [1], wherein each of the multiple doses comprises a dose equivalent to 0.08 to 2.4 nmol / kg of kisspeptin-10 administered by intravenous bolus (IVB). [4] The method of [3], wherein each of the multiple doses contains a dose equivalent to 0.2 to 0.3 nmol / kg kisspeptin-10, preferably 0.24 nmol / kg kisspeptin-10. [5] The method of [1], wherein the multiple doses include at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10. [6] The method of [5], wherein the at least one supraphysiological dose is administered as the first dose of the multiple doses, or as the first two or more doses of the multiple doses, optionally as all of the multiple doses. [7] The method of [1], further comprising administering to the subject a therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist. [8] The method of [7], wherein the opioid antagonist is naloxone or naltrexone, or the mixed agonist-antagonist is buprenorphine. [9] A method for identifying a subject as having, at risk for, or having a mild form of pathological hypogonadotropic hypogonadism, comprising: measuring a baseline level of LH in the subject; administering to the subject a priming dose of kisspeptin or kisspeptin analog, e.g., a dose containing 0.08 to 15 nmol / kg kisspeptin-10 administered by intravenous bolus (IVB) or a dose containing 0.8 to 500 nmol / kg kisspeptin-10 administered by subcutaneous (SC) injection; measuring at least one LH level after administration of the stimulatory dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulatory dose; comparing the baseline level of LH in the subject with the level of LH after administration of the stimulatory dose; identifying subjects with delayed puberty who have an LH level after administration of said stimulatory dose that is not significantly different from the baseline level of said LH as having pathological hypogonadotropic hypogonadism; A method comprising:

[10] The method of [9], further comprising administering treatment for pathological hypogonadotropic hypogonadism to the identified subject.

[11] The method of

[10] , wherein the treatment comprises administering multiple doses of kisspeptin or a kisspeptin analog.

[12] The method of

[11] , wherein the multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, over a period of at least 2 to 3 days, preferably at least 1 to 12 months.

[13] The method of

[11] , wherein each of the multiple doses comprises a dose equivalent to 0.08 to 2.4 nmol / kg kisspeptin-10 administered by intravenous bolus (IVB).

[14] The method of

[13] , wherein each of the multiple doses contains a dose equivalent to 0.2 to 0.3 nmol / kg kisspeptin-10, preferably 0.24 nmol / kg kisspeptin-10.

[15] The method of

[11] , wherein the multiple doses include at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10.

[16] The method of

[15] , wherein the at least one supraphysiological dose is administered as the first dose, or the first two or more doses of the multiple doses, optionally all of the multiple doses.

[17] The method of

[10] , further comprising administering to the subject a therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist.

[18] The method of

[17] , wherein the opioid antagonist is naloxone or naltrexone, or the mixed agonist-antagonist is buprenorphine.

[19] The method according to [1] or

[10] , wherein the treatment further comprises administering gonadal steroid replacement therapy.

[20] The method according to

[19] , wherein the gonadal steroid replacement therapy comprises administering testosterone to men and estrogen and / or progesterone / progestin to women.

[21] The method of [1] or

[10] , further comprising administration of one or more gonadotropins.

[22] A composition comprising (i) a kisspeptin or kisspeptin analog, and / or (ii) an opioid antagonist or mixed agonist-antagonist, for use in a method of treating a subject having pathological hypogonadotropic hypogonadism, comprising administering multiple doses of kisspeptin or a kisspeptin analog.

[23] The composition for use according to

[22] , wherein the multiple doses are administered at intervals of 1 to 6 hours, preferably 2 hours, over a period of at least 2 to 3 days, preferably at least 1 to 12 months.

[24] The composition for use according to

[22] , wherein each of the multiple doses comprises a dose equivalent to 0.08 to 2.4 nmol / kg of kisspeptin-10 administered by intravenous bolus (IVB).

[25] The composition for use according to

[24] , wherein each of the multiple doses contains a dose equivalent to 0.2 to 0.3 nmol / kg kisspeptin-10, preferably 0.24 nmol / kg kisspeptin-10.

[26] The composition for use according to

[22] , wherein the multiple doses include at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10.

[27] The composition for use of

[26] , wherein the at least one supraphysiological dose is administered as the first dose, or the first two or more doses of the multiple doses, optionally all of the multiple doses.

[28] The composition for use according to

[22] , further comprising administering to the subject a therapeutically effective amount of an opioid antagonist or mixed agonist-antagonist.

[29] The composition for use according to

[22] , wherein the opioid antagonist is naloxone or naltrexone, or the mixed agonist-antagonist is buprenorphine.

[30] The composition for use described in

[22] , wherein the method further comprises administering gonadal steroid replacement therapy.

[31] The composition for use described in

[30] , wherein the gonadal steroid replacement therapy further comprises administering testosterone in men and estrogen and / or progesterone / progestin in women.

[32] The composition for use according to

[22] , wherein the method further comprises administering one or more gonadotropins.

Claims

1. 1. A pharmaceutical composition comprising kisspeptin for treating a human subject having pathological hypogonadotropic hypogonadism, wherein said kisspeptin is administered in multiple doses; said multiple doses comprising at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10; The at least one supraphysiological dose is administered as the first dose of the multiple doses, or as the first two or more doses of the multiple doses, or as all of the multiple doses. Pharmaceutical compositions.

2. 10. The pharmaceutical composition of claim 1, wherein the multiple doses are administered at intervals of 1 to 6 hours over a period of at least 2 to 3 days.

3. 3. The pharmaceutical composition of claim 2, wherein the multiple doses are administered at intervals of 2 hours.

4. 3. The pharmaceutical composition of claim 2, wherein the multiple doses are administered over a period of at least 1 to 12 months.

5. 2. The pharmaceutical composition of claim 1, wherein the pathological hypogonadotropic hypogonadism is idiopathic hypogonadotropic hypogonadism (IHH) or Kallmann syndrome (KS).

6. 10. The pharmaceutical composition of claim 1, wherein each of the multiple doses comprises a dose equivalent to 0.08 to 2.4 nmol / kg kisspeptin-10 administered by intravenous bolus (IVB).

7. 7. The pharmaceutical composition of claim 6, wherein each of said multiple doses comprises a dose equivalent to 0.2-0.3 nmol / kg kisspeptin-10.

8. 8. The pharmaceutical composition of claim 7, wherein each of the multiple doses comprises a dose equivalent to 0.24 nmol / kg kisspeptin-10.

9. 10. The pharmaceutical composition of claim 1, administered in combination with a therapeutically effective amount of an opioid antagonist that is naloxone or naltrexone, or a mixed agonist-antagonist that is buprenorphine.

10. 10. The pharmaceutical composition of claim 1, administered in combination with a gonadal steroid.

11. 11. The pharmaceutical composition of claim 10, wherein the gonadal steroid is testosterone in men and estrogen and / or progesterone / progestin in women.

12. 10. The pharmaceutical composition of claim 1, administered in combination with one or more gonadotropins.

13. 1. A combination of (i) kisspeptin and (ii) an opioid antagonist that is naloxone or naltrexone or a mixed agonist-antagonist that is buprenorphine for treating a human subject having pathological hypogonadotropic hypogonadism, wherein said kisspeptin is administered in multiple doses; the multiple doses comprising at least one supraphysiological dose equivalent to 2.4 to 24 nmol / kg kisspeptin-10; Combination.

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