Methods of using kisspeptin or kisspeptin analogs or pharmaceutical compositions

Abstract

The invention features methods of treating a subject having a disorder by administering to the subject a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. Disorders that may be treated by the kisspeptin analogs or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition include reproductive disorders, hypoactive sexual arousal disorder, respiratory diseases, metabolic disorders, liver disorders, bone disorders, VMS, neurodegenerative disorders, and sequelae of neurotropic viruses. Also provided herein are methods of identifying a subject as having congenital hypogonadotropic hypogonadism (HH) and methods of determining whether a patient having a disorder is likely to respond to a therapy comprising a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

Description

[0001] PATENT

[0002] Attorney Docket No.: 51796-005WO3

[0003] BWH Ref.: 2024-010

[0004] METHODS OF USING KISSPEPTIN OR KISSPEPTIN ANALOGS OR PHARMACEUTICAL COMPOSITIONS

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 28, 2026, is named “51796-005WO3_Sequence_Listing_1_28_26” and is 8,432 bytes in size.

[0007] STATEMENT AS TO FEDERALLY FUNDED RESEARCH

[0008] This invention was made with government support under 1R01FD007843-01 awarded by the FDA and under 5R37HD043341 -21, HD090151, HD099084, and DK133760 awarded by the NIH. The government has certain rights in the invention.

[0009] CROSS-REFERENCE TO RELATED APPLICATIONS

[0010] This application claims the benefit of U.S. Provisional Application No. 63 / 751,582, filed January 30, 2025, and U.S. Provisional Application No. 63 / 814,773, filed May 30, 2025, the contents of each of which are incorporated herein by reference in their entireties.

[0011] BACKGROUND OF THE INVENTION

[0012] This application relates to treating and diagnosing disorders related to kisspeptin signaling. The kisspeptin receptor (KISS1R) is a member of the G protein-coupled receptor family.

[0013] Kisspeptins, the native ligands of KISS1 R, are encoded as a 145 amino acid precursor, which is proteolyzed to fragments of between 10-54 amino acids in length. The mRNA of human KISS1R is expressed abundantly in placenta, pituitary, spinal cord and pancreas, and is expressed at a low level in other tissues including different parts of brain (thalamus, caudate nucleus, substantia nigra, hippocampus, amygdala, prefrontal cortex, hypothalamus, and cerebellum), stomach, small intestine, thymus, spleen, lung, testis, kidney and fetal liver.

[0014] KISS1R signaling results in gonadotropin-releasing hormone (GnRH) release, which is required for the activation of gonadotropes at the pituitary level, and downstream release of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin in males and females, estradiol and anti-mullerian hormone (AMH) in females, and testosterone in males. There is a need for therapeutic modulators of KISS1R signaling.

[0015] SUMMARY OF THE INVENTION

[0016] In one aspect, the invention features a method of treating a subject having a disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein the administration is pulsatile.

[0017] In some embodiments, the therapeutically effective amount is between about 0.05 pg / kg to about 90 ng / kg. In some embodiments, the therapeutically effective amount is between about 10 pg / kg to about 1 ng / kg.PATENT

[0018] Attorney Docket No.: 51796-005WO3

[0019] BWH Ref.: 2024-010

[0020] In some embodiments, the therapeutically effective amount is between about 90 ng / kg to about 5 pg / kg. In some embodiments, the therapeutically effective amount is between about 90 ng / kg to about 0.5 pg / kg. In some embodiments, the therapeutically effective amount is between about 0.5 pg / kg to about 1 pg / kg. In some embodiments, the therapeutically effective amount is between about 1 pg / kg to about 5 pg / kg.

[0021] In some embodiments, the therapeutically effective amount is about 5 pg / kg or greater. In some embodiments, the therapeutically effective amount is between about 5 pg / kg to about 30 pg / kg. In some embodiments, the therapeutically effective amount is between about 5 pg / kg to about 10 pg / kg. In some embodiments, the therapeutically effective amount is between about 10 pg / kg to about 20 pg / kg. In some embodiments, the therapeutically effective amount is between about 20 pg / kg to about 30 pg / kg.

[0022] In some embodiments, the pulsatile administration is at a frequency of between about 20 minutes to about 4 days. In some embodiments, the pulsatile administration is at a frequency of between about 45 minutes to about 2 hours.

[0023] In some embodiments, the pulsatile administration is over a period of at least about 1 day. in some embodiments, the pulsatile administration is over a period of at least about 14 days.

[0024] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes a sequence of:

[0025] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or

[0026] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2). In some embodiments, the disorder is a reproductive disorder. In some embodiments, the reproductive disorder is associated with a suppressed reproductive axis. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof activates the reproductive axis. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt or kisspeptin-10 or a pharmaceutically acceptable salt thereof thereof enables fertility. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof increases the level of a reproductive health biomarker.

[0027] In some embodiments, the subject is a female. In some embodiments, the reproductive health biomarker is luteinizing hormone (LH), follicle-stimulating hormone (FSH), inhibin, estradiol, anti-mullerian hormone (AMH), external signs of puberty onset (Tanner scale), sexual desire, follicle size, bone density, uterine lining (e.g., endometrial lining), cervical mucus, vaginal moisture, basal body temperature, a marker of egg quality, or a marker of embryo quality. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof results in follicle growth. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof improves the manner in which eggs grow, the number of eggs that grow, the quality of the eggs that grow, or the ability of eggs to ovulate.

[0028] In some embodiments, the subject is a male. In some embodiments, the reproductive health biomarker is LH, FSH, inhibin, testosterone, external signs of puberty onset (Tanner scale), sexual desire, semen analysis, bone density, or testicular size. In some embodiments, administration of the kisspeptinPATENT

[0029] Attorney Docket No.: 51796-005WO3

[0030] BWH Ref.: 2024-010

[0031] analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof results in spermatogenesis.

[0032] In some embodiments, the reproductive disorder is hypogonadotropic hypogonadism (HH). In some embodiments, the HH is congenital HH. In some embodiments, the congenital HH is congenital idiopathic HH (normosmic). In some embodiments, the congenital HH is congenital Kallmann syndrome (anosmic / hyposmic). In some embodiments, the HH is acquired HH. In some embodiments, the acquired HH is hypothalamic amenorrhea. In some embodiments, the acquired HH is hyperprolactinemia. In some embodiments, the acquired HH is associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse.

[0033] In some embodiments, the reproductive disorder is abnormal pregnancy; the reproductive disorder is infertility; or the reproductive disorder is a reproductive disorder treated with an IVF protocol.

[0034] In some embodiments, the disorder is hypoactive sexual arousal disorder.

[0035] In some embodiments, the disorder is vasomotor symptoms (VMS).

[0036] In some embodiments, the disorder is a respiratory disease. In some embodiments, the respiratory disease is asthma.

[0037] In some embodiments, the disorder is a metabolic disorder. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the metabolic disorder is obesity. In some embodiments, the metabolic disorder is metabolic dysfunction-associated steatotic liver disease (MASLD). In some embodiments, the metabolic disorder is metabolic dysfunction-associated steatohepatitis (MASH).

[0038] In some embodiments, the disorder is a liver disorder. In some embodiments, the liver disorder is MASLD. In some embodiments, the liver disorder is MASH.

[0039] In some embodiments, the disorder is a bone disorder. In some embodiments, the bone disorder is osteoporosis.

[0040] In some embodiments, the disorder is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Alzheimer’s disease.

[0041] In some embodiments, the disorder is a sequela of a neurotropic virus. In some embodiments, the neurotropic virus is SARS CoV-2.

[0042] In another aspect, the invention provides a method of treating a subject having a disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein the administration is continuous.

[0043] In some embodiments, the therapeutically effective amount is between about 0.05 pg / kg / h to about 250 ng / kg / h. In some embodiments, the therapeutically effective amount is between about 10 pg / kg / h to about 1 ng / kg / h.

[0044] In some embodiments, the continuous administration is over a period of at least about 2 hours. In some embodiments, the continuous administration is over a period of at least about 16 hours.

[0045] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes a sequence of:

[0046] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); orPATENT

[0047] Attorney Docket No.: 51796-005WO3

[0048] BWH Ref.: 2024-010

[0049] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2). In some embodiments, the disorder is a reproductive disorder. In some embodiments, the reproductive disorder is associated with a hyperactive reproductive axis. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof suppresses the reproductive axis. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof decreases the level of a reproductive health biomarker.

[0050] In some embodiments, the subject is a female. In some embodiments, the reproductive health biomarker is LH, FSH, inhibin, estradiol, AMH, external signs of puberty onset (Tanner scale), sexual desire, follicle size, bone density, uterine lining (e.g., endometrial lining), cervical mucus, vaginal moisture, basal body temperature, a marker of egg quality, or a marker of embryo quality. In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof decreases follicle growth.

[0051] In some embodiments, the subject is a male. In some embodiments, the reproductive health biomarker is LH, FSH, inhibin, testosterone, external signs of puberty onset (Tanner scale), sexual desire, semen analysis, bone density, or testicular size.

[0052] In some embodiments, administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof modifies spermatogenesis or the potential for spermatogenesis (for example, which is applicable for boys with precocious pubertal development requiring suppression of the reproductive axis). Administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof may be used to treat precocious puberty in males, for example, such treatments would help to abort abnormally rapid testicular growth, and by extension, spermatogenesis.

[0053] In some embodiments, the reproductive disorder is precocious puberty, sex hormone-dependent cancer, or gender dysphoria.

[0054] In some embodiments, the reproductive disorder is polycystic ovary syndrome (PCOS), endometriosis, uterine fibroid, or abnormal pregnancy.

[0055] In some embodiments, the disorder is a metabolic disorder. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the metabolic disorder is obesity.

[0056] In some embodiments, the metabolic disorder is MASLD. In some embodiments, the metabolic disorder is MASH.

[0057] In some embodiments, the disorder is a liver disorder. In some embodiments, the liver disorder is MASLD. In some embodiments, the liver disorder is MASH.

[0058] In some embodiments, the disorder is a bone disorder. In some embodiments, the bone disorder is osteoporosis.

[0059] In some embodiments, the disorder is vasomotor symptoms (VMS).

[0060] In some embodiments, the disorder is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Alzheimer's disease.

[0061] In some embodiments, the disorder is a sequela of a neurotropic virus. In some embodiments, the neurotropic virus is SARS CoV-2.PATENT

[0062] Attorney Docket No.: 51796-005WO3

[0063] BWH Ref.: 2024-010

[0064] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intravenously, subcutaneously, or intramuscularly.

[0065] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered subcutaneously at a dose of about 5 pg / kg.

[0066] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intranasally.

[0067] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered by inhalation.

[0068] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intrathecally.

[0069] In any of the preceding aspects, the method further includes administering to the subject a gonadotropin-releasing hormone receptor (GnRHR) agonist or antagonist, neurokinin receptor agonist or antagonist, or opioid agonist, antagonist, or agonist-antagonist. In some embodiments, the GnRHR agonist is leuprolide, gonadorelin, goserelin, nafarelin, histrelin, triptorelin, or buserelin. In some embodiments, the GnRHR antagonist is abarelix, cetrorelix, degarelix, elagolix, ganirelix, or relugolix. In some embodiments, the neurokinin receptor antagonist is fezolinetant, aprepitant, rolapitant, casopitant, fosaprepitant, netupitant, or maropitant. In some embodiments, the opioid agonist is morphine, methadone, or alfentanil. In some embodiments, the opioid antagonist is naloxone or naltrexone. In some embodiments, the opioid agonist-antagonist is buprenorphine.

[0070] In another aspect, the disclosure provides for a method of identifying a subject as having congenital HH, the method including:

[0071] (a) providing a reference level of LH;

[0072] (b) administering to the subject a stimulating dose of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein:

[0073] (i) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg; or

[0074] (ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg;

[0075] (c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose;

[0076] (d) comparing the reference level of LH to the level of LH in the subject after administration of the stimulating dose; and

[0077] (e) identifying a subject who has a level of LH after administration of the stimulating dose that is not different from the reference level of LH as having congenital HH.

[0078] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes a sequence of:

[0079] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 1); orPATENT

[0080] Attorney Docket No.: 51796-005WO3

[0081] BWH Ref.: 2024-010

[0082] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2). In some embodiments, the congenital HH is congenital Kallmann syndrome (anosmic / hyposmic). In another aspect, the disclosure provides for a method of determining whether a patient having a disorder is likely to respond to a therapy including a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof, the method including:

[0083] (a) providing a reference level of LH;

[0084] (b) administering to the subject a provocative dose of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof;

[0085] (c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose;

[0086] (d) comparing the reference level of LH to the level of LH in the subject after administration of the provocative dose; and

[0087] (e) identifying a subject who has a level of LH after administration of the provocative dose that is greater than the reference level of LH as likely to respond to the therapy.

[0088] in some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes a sequence of:

[0089] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or

[0090] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2). In some embodiments, the provocative dose is administered intravenously at a dose of between about 1 pg / kg to about 1 pg / kg. In some embodiments, the provocative dose is administered subcutaneously at a dose of between about 10 pg / kg to about 10 pg / kg.

[0091] In some embodiments, the disorder is a reproductive disorder, hypoactive sexual arousal disorder, respiratory disease, metabolic disorder, liver disorder, bone disorder, VMS, neurodegenerative disorder, or a sequela of a neurotropic virus. In some embodiments, the reproductive disorder is HH, abnormal pregnancy, infertility, precocious puberty, sex hormone-dependent cancer, gender dysphoria, PCOS, endometriosis, uterine fibroid, or a reproductive disorder treated with an IVF protocol. In some embodiments, the HH is congenital HH or acquired HH. In some embodiments, the congenital HH is congenital idiopathic HH (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic).

[0092] In another aspect, the invention provides a pharmaceutical composition comprising a kisspeptin analog or a pharmaceutically acceptable salt thereof, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:

[0093] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 1); or

[0094] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 2). In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 1. In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 2.

[0095] In some embodiments, the pharmaceutical composition is an aqueous formulation having a pH of between about 3.5 to about 8.0. In some embodiments, the formulation has a pH of about 4.0 to aboutPATENT

[0096] Attorney Docket No.: 51796-005WO3

[0097] BWH Ref.: 2024-010

[0098] In some embodiments, the pharmaceutical composition comprises a buffer. In some embodiments, the buffer is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the buffer is present at a concentration of about 5 mM. In some embodiments, the buffer is an acetate buffer, such as a sodium acetate buffer.

[0099] In some embodiments, the pharmaceutical composition comprises a tonicity agent. In some embodiments, the tonicity agent is present at a concentration of about 1 mg / mL to about 100 mg / mL. In some embodiments, the tonicity agent is present at a concentration of about 40 mg / mL. In some embodiments, the tonicity agent is mannitol.

[0100] In some embodiments, the pharmaceutical composition comprises one or more surfactants. In some embodiments, each of the one or more surfactants is present at a concentration of about 0.01% to about 20%.

[0101] In some embodiments, the one or more surfactants is polysorbate 80 (PS80). In some embodiments, the pharmaceutical composition comprises about 0.05% to about 1% PS80. In some embodiments, the pharmaceutical composition comprises about 0.1% PS80.

[0102] In some embodiments, the one or more surfactants is polysorbate 20 (PS20). In some embodiments, the pharmaceutical composition comprises about 0.05% to about 1% PS20. In some embodiments, the pharmaceutical composition comprises about 0.1% PS20.

[0103] In some embodiments, the one or more surfactants is propylene glycol (PG). In some embodiments, the pharmaceutical composition comprises about 1% to about 10% PG. In some embodiments, the pharmaceutical composition comprises about 5% PG.

[0104] In some embodiments, the pharmaceutical composition comprises about 5 mM sodium-acetate buffer, about 40 mg / mL mannitol, about 0.1% PS80, and about 5% PG, wherein the pH is about 5.0.

[0105] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof is present at a concentration of about 0.01 ng / mL to about 100 mg / mL.

[0106] In another aspect, the invention features method of treating a subject having a disorder, the method comprising administering to the subject a pharmaceutical composition disclosed herein.

[0107] In some embodiments, the subject is a male or a female.

[0108] In some embodiments, the administration is pulsatile.

[0109] In some embodiments, the disorder is a reproductive disorder, hypoactive sexual arousal disorder, VMS, respiratory disease, metabolic disorder, liver disorder, bone disorder, neurodegenerative disorder, or a sequela of a neurotropic virus. In some embodiments, the disorder is a reproductive disorder and administration of the pharmaceutical composition activates the reproductive axis, enables fertility, increases the level of a reproductive health biomarker, results in follicle growth, improves the manner in which eggs grow, improves the number of eggs that grow, improves the quality of the eggs that grow, improves the ability of eggs to ovulate, or results in spermatogenesis.

[0110] In some embodiments, the administration is continuous.

[0111] In some embodiments, the disorder is a reproductive disorder, a metabolic disorder, a liver disorder, a bone disorder, a neurodegenerative disorder, abnormal pregnancy, VMS, or a sequela of a neurotropic virus. In some embodiments, the disorder is a reproductive disorder and administration of the pharmaceutical composition suppresses the reproductive axis, decreases the level of a reproductivePATENT

[0112] Attorney Docket No.: 51796-005WO3

[0113] BWH Ref.: 2024-010

[0114] health biomarker, decreases follicle growth, or modifies spermatogenesis or the potential for spermatogenesis.

[0115] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a therapeutically effective amount.

[0116] In another aspect, the invention provides a method of identifying a subject as having congenital HH, the method comprising:

[0117] (a) providing a reference level of LH;

[0118] (b) administering to the subject a pharmaceutical composition disclosed herein, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a stimulating dose, wherein:

[0119] (i) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg; or

[0120] (ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg;

[0121] (c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose;

[0122] (d) comparing the reference level of LH to the level of LH in the subject after administration of the stimulating dose; and

[0123] (e) identifying a subject who has a level of LH after administration of the stimulating dose that is not different from the reference level of LH as having congenital HH.

[0124] In another aspect, the invention provides a method of determining whether a patient having a disorder is likely to respond to a therapy comprising a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, the method comprising:

[0125] (a) providing a reference level of LH;

[0126] (b) administering to the subject a pharmaceutical composition disclosed herein, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a provocative dose;

[0127] (c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose;

[0128] (d) comparing the reference level of LH to the level of LH in the subject after administration of the provocative dose; and

[0129] (e) identifying a subject who has a level of LH after administration of the provocative dose that is greater than the reference level of LH as likely to respond to the therapy.

[0130] In any of the preceding embodiments, the kisspeptin analog or pharmaceutically acceptable salt thereof includes a sequence of SEQ ID NO: 1. In other embodiments, the kisspeptin analog or pharmaceutically acceptable salt thereof is a sequence of SEQ ID NO: 1

[0131] In any of the preceding embodiments, the kisspeptin analog or pharmaceutically acceptable salt thereof includes a sequence of SEQ ID NO: 2. In other embodiments, the kisspeptin analog or pharmaceutically acceptable salt thereof is a sequence of SEQ ID NO: 2.PATENT

[0132] Attorney Docket No.: 51796-005WO3

[0133] BWH Ref.: 2024-010

[0134] In any of the preceding embodiments, the kisspeptin analog or pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein is delivered to a human using a programmable pump.

[0135] In any of the preceding embodiments, a kisspeptin including the sequence of SEQ ID NO: 3 may be used. In other embodiments, the kisspeptin is kisspeptin-10, which has a sequence of SEQ ID NO: 3, may be used.

[0136] In any of the preceding embodiments, kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered intravenously, subcutaneously, intramuscularly, intranasally, by inhalation, or intrathecally.

[0137] In some embodiments, the invention involves administration of a single bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, which is useful for treating any disorder described herein, identifying a subject as having congenital HH, or determining whether a patient having a disorder is likely to respond to a therapy.

[0138] Advantageously, any of the kisspeptin analogs or a pharmaceutically acceptable salt thereof described herein or a pharmaceutical composition described herein have an enhanced function in the methods described herein (e.g., potency or stability) relative to kisspeptin.

[0139] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0140] Definitions

[0141] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0142] The terms “polypeptide” and “peptide” are used interchangeably herein to mean a compound that contains a sequence of amino acids bonded to each other through peptidic bonds. Polypeptides may contain both natural and unnatural amino acids. Commonly accepted abbreviations that are used in the art as well as herein to represent unnatural amino acids include the following:

[0143] D-Phe(2,4-DiCI): 2,4-dichloro-D-phenylalanine

[0144] Arg(Me): N-ω-methyl-arginine

[0145] HoPro: homoproline, S-homoproline, S-Pip, S-high Proline, or (S)-piperidine-2-formic acid A6c: 1-amino-cyclohexyl formic acid

[0146] azaGly: azaglycine

[0147] Ac: acetyl

[0148] As used herein, the term “kisspeptin” refers to a family of neuropeptides that result from the cleavage of a 145-amino-acid precursor peptide that is encoded by the KISS1 gene. In humans, the active form of kisspeptin is a 54-amino-acid peptide. The present methods can include administration of the full-length kisspeptin, the 54-amino-acid peptide (kisspeptin-54 (KP54)), or kisspeptin-10, a 10-amino-acid peptide including the sequence Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe (SEQ ID NO: 3).PATENT

[0149] Attorney Docket No.: 51796-005WO3

[0150] BWH Ref.: 2024-010

[0151] As used herein, the term “kisspeptin analog” refers to a peptide that is not identical, but has analogous functional or structural features to kisspeptin. For example, a kisspeptin analog retains the biological activity of kisspeptin, while having certain biochemical modifications that enhance the analog's function (for example, potency or stability) relative to kisspeptin. In some embodiments, a kisspeptin analog may include an unnatural amino acid. In some embodiments, a kisspeptin analog may be a peptidomimetic. In some embodiments, the kisspeptin analog includes the sequence of any one of:

[0152] (a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or

[0153] (b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2). The term “pharmaceutically acceptable salt,” as used herein, represents salts of kisspeptin-10 or the kisspeptin analogs described herein that are suitable for use in methods described herein.

[0154] Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Pharmaceutical Salts: Properties, Selection, and Use (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of kisspeptin-10 or the kisspeptin analogs or separately by reacting the free base group with a suitable organic acid.

[0155] As used herein, “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms condition, disease, or illness, unless otherwise indicated.

[0156] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a disorder. The disorder may be a reproductive disorder (e.g., hypogonadotropic hypogonadism (e.g., congenital hypogonadotropic hypogonadism (e.g., congenital idiopathic hypogonadotropic hypogonadism (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic)) or acquired hypogonadotropic hypogonadism (e.g., hypothalamic amenorrhea, hyperprolactinemia, or acquired hypogonadotropic hypogonadism associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse)), abnormal pregnancy, infertility, precocious puberty, sex hormone-dependent cancer, gender dysphoria, polycystic ovary syndrome, endometriosis, uterine fibroid, or a reproductive disorder treated with an in vitro fertilization protocol), hypoactive sexual arousal disorder, a respiratory disease (e.g., asthma), a metabolic disorder (e.g., obesity, diabetes, metabolic dysfunction-associated steatotic liver disease (MASLD), or metabolic dysfunction-associated steatohepatitis (MASH)), a liver disorder (e.g., MASLD or MASH), a bone disorder (e.g., osteoporosis), vasomotor symptoms, a neurodegenerative disorder (e.g., Alzheimer’s disease), or a sequela of a neurotropic virus. Treatment may be administered to a subject who does not exhibit signs of the disease and / or to a subject who exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0157] As used herein, the terms “subject” and “patient” are interchangeable and refer to a subject (e.g., a mammalian subject, e.g., a human subject) that receives treatment or diagnosis for a disorder as described herein.

[0158] As used herein, “therapeutically effective amount” refers to an amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereofPATENT

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[0161] described herein sufficient to treat a disorder as described herein in a subject (e.g., a human), in some embodiments, the administration is pulsatile and the therapeutically effective amount is between about 0.05 pg / kg to about 90 ng / kg. In some embodiments, the administration is pulsatile and the therapeutically effective amount is between about 90 ng / kg to about 5 pg / kg. In some embodiments, the administration is pulsatile and the therapeutically effective amount is between about 5 pg / kg to about 30 pg / kg. In some embodiments, the administration is continuous and the therapeutically effective amount is between about 0.05 pg / kg / h to about 250 ng / kg / h.

[0162] As used herein, the term “pulsatile administration” refers to repeated administration of a single bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof followed by a period of little / no release of the kisspeptin analog or kisspeptin-10 (lag phase) in a defined frequency. The boluses may be released at a frequency of between about 20 minutes to about 4 days. The pulsatile administration may occur over a period of at least about 1 day (for example, the boluses may be released every 1.5 hours for 2 weeks or the boluses may be released every 2 hours for 2 days). Pulsatile administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof sensitizes KISS1R to activation. In some embodiments, pulsatile administration refers to administration of a kisspeptin-10 or a pharmaceutically acceptable salt thereof.

[0163] As used herein, the term “continuous administration” refers to consistent administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof over an extended period. The continuous administration may be over a period of at least about 2 hours (for example, continuous administration for 16 hours). Continuous administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof desensitizes KISS1R to activation. In some embodiments, continuous administration refers to administration of a kisspeptin-10 or a pharmaceutically acceptable salt thereof.

[0164] As used herein, the term “reproductive disorder” refers to conditions or diseases involving the reproductive axis, including congenital abnormalities, cancers of the reproductive system and sexual dysfunction. In some embodiments, a reproductive disorder may be hypogonadotropic hypogonadism (HH) (e.g., congenital HH (e.g., congenital idiopathic HH (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic)) or acquired HH (e.g., hypothalamic amenorrhea, hyperprolactinemia, or acquired HH associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse)), precocious puberty, sex hormone-dependent cancer, gender dysphoria, polycystic ovary syndrome (PCOS), uterine fibroids, endometriosis, infertility, abnormal pregnancy, or a reproductive disorder treated with an in vitro fertilization (IVF) protocol.

[0165] As used herein, the term “reproductive axis” is used interchangeably with “hypothalamic-pituitary-gonadal axis” and refers to the hypothalamus, pituitary gland, and gonads. In females, the reproductive axis is referred to as the hypothalamic-pituitary-ovarian axis, while in males, the reproductive axis is referred to as the hypothalamic-pituitary-testicular axis.

[0166] As used herein, the term “suppressed reproductive axis” refers to a condition where the subject has lowered levels of a reproductive health biomarker as compared to a normal individual.PATENT

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[0169] As used herein, the term “activates the reproductive axis” means increasing levels of a reproductive health biomarker.

[0170] As used herein, the term “hyperactive reproductive axis” refers to a condition where the subject has elevated levels of a reproductive health biomarker as compared to a normal individual.

[0171] As used herein, the term “suppresses the reproductive axis” means decreasing levels of a reproductive health biomarker.

[0172] As used herein, the term “reproductive health biomarker” refers to a characteristic that is objectively measured and evaluated as an indicator of normal reproductive health. In some embodiments, the reproductive health biomarker is a downstream marker of reproductive health, e.g., a marker of overall health, such as bone density or basal body temperature. In some embodiments, the reproductive health biomarker is a female reproductive health biomarker, which may include luteinizing hormone (LH), follicle- stimulating hormone (FSH), inhibin, estradiol, anti-mullerian hormone (AMH), a marker of egg quality, or a marker of embryo quality. In some embodiments, the reproductive health biomarker is phenotypic, such as external signs of puberty onset (Tanner scale), sexual desire, follicle size (as determined by ovarian imaging), uterine lining (e.g., endometrial lining), cervical mucus, or vaginal moisture in females. In some embodiments, the reproductive health biomarker is a male reproductive health biomarker, which may include luteinizing hormone (LH), follicle-stimulating hormone (FSH), inhibin, or testosterone. In some embodiments, the reproductive health biomarker is phenotypic, such as external signs of puberty onset (Tanner scale), sexual desire, semen analysis, or testicular size in males.

[0173] As used herein, the term "hypogonadotropic hypogonadism" (HH) refers to a condition where the subject produces little or no sex hormones due to a lack of signaling from the pituitary gland or hypothalamus. In some embodiments, the HH may be congenital HH or acquired HH.

[0174] The term "congenital hypogonadotropic hypogonadism" (congenital HH) refers to a condition where a patient was on a trajectory since birth to never go through puberty. Congenital HH may be congenital idiopathic HH (normosmic). Congenital HH may be associated with an inability to smell, which is referred to as congenital Kallmann syndrome (anosmic / hyposmic).

[0175] The term "acquired hypogonadotropic hypogonadism" (acquired HH) refers to a postnatal onset of disorders that damage or alter the function of gonadotropin-releasing hormone (GnRH) neurons and / or pituitary gonadotroph cells. In some embodiments, the acquired HH is hypothalamic amenorrhea. In some embodiments, the acquired HH is hyperprolactinemia. In some embodiments, the acquired HH may be associated with negative energy balance, malnutrition, obesity, stress, metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse.

[0176] As used herein, the term “hypoactive sexual desire disorder” refers to a condition in which desire for sexual activity is persistently or recurrently diminished or absent, causing marked distress or interpersonal difficulties. Sexual arousal disorder can be caused by psychological / emotional factors, relationship factors, illness, medications / drugs (e.g., analgesics, such as opioids) and hormone changes.

[0177] As used herein, the term “respiratory disease” refers to any disease of the nasal cavity, pharynx, throat, trachea, bronchioles, alveoli, or lungs that affects human respiration. For example, a respiratory disease may be asthma.PATENT

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[0180] As used herein, the term “metabolic disorder" refers to any disease that affects the body’s metabolism. Exemplary metabolic disorders include obesity, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is a condition in which fat (e.g., lipids) builds up in the liver due to causes other than excessive alcohol consumption. In some embodiments, the MASLD may progress to metabolic dysfunction-associated steatohepatitis (MASH).

[0181] As used herein, the term “liver disorder" refers to any disease that affects the liver. An exemplary liver disorder is metabolic dysfunction-associated steatotic liver disease (MASLD), which is a condition in which fat (e.g., lipids) builds up in the liver due to causes other than excessive alcohol consumption. In some embodiments, the MASLD may progress to metabolic dysfunction-associated steatohepatitis (MASH).

[0182] As used herein, the term “bone disorder” refers to any disease that affects the bone. An exemplary bone disorder is osteoporosis, in which bone mineral density decreases, which may lead to fractures.

[0183] As used herein, the term “vasomotor symptoms” (VMS) refers to a condition characterized by hot flashes, night sweats, palpitation of the heart rhythm joint pain, and muscular weakness. In a particular embodiment, the subject is female and the vasomotor symptoms are hot flashes and night sweats. The hot flashes or night sweats may be associated with menopause or hormonal dependent cancers such as breast cancer (Tamoxifen) in females or prostate cancer (androgen deprivation therapy) in males.

[0184] As used herein, the term “neurodegenerative disorder” refers to any disorder that affects the neurons. An exemplary neurodegenerative disorder is Alzheimer’s disease.

[0185] As used herein, the term “neurotropic virus” refers to a virus that affects the nervous system. In some embodiments, the neurotropic virus is SARS CoV-2.

[0186] As used herein, the term “sequela” refers to a condition that results from a prior disease. In some embodiments, the sequela is brain fog, resulting from a neurotropic virus (e.g., SARS CoV-2).

[0187] As used herein, the term “gonadotropin-releasing hormone receptor (GnRHR) agonist or antagonist” refers to a molecule that binds to GnRHR and acts as an agonist (activates GnRHR) or antagonist (does not activate GnRHR). GnRHR agonists may include leuprolide (LUPRON®), gonadorelin, goserelin (ZOLADEX®), nafarelin, histrelin (SUPPRELIN®, VANTAS®), triptorelin (TRELSTAR®), and buserelin. GnRHR antagonists may include abarelix (PLENAXIS®), cetrorelix (CETROTIDE®), degarelix (FIRMAGON®), elagolix (ORLISSA®), ganirelix (ORGALUTRAN®), and relugolix (RELUMINA®, ORGOVYK®).

[0188] As used herein, the term “neurokinin receptor agonist or antagonist” refers to a molecule that binds to a neurokinin receptor and acts as an agonist (activates the neurokinin receptor) or antagonist (does not activate the neurokinin receptor). Neurokinin receptor antagonists may include fezolinetant (VEOZAH®), aprepitant, rolapitant, casopitant, fosaprepitant, netupitant, and maropitant.

[0189] As used herein, the term “opioid agonist, antagonist, or agonist-antagonist” refers to a molecule that binds to an opioid receptor and acts as an agonist (activates the opioid receptor), antagonist (does not activate the opioid receptor) or agonist-antagonist (agonistic activity at some opioid receptors and antagonistic activity at other opioid receptors). Opioid agonists may include morphine, methadone, andPATENT

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[0192] alfentanil. Opioid antagonists may include naloxone and naltrexone. Opioid agonist-antagonists may include buprenorphine.

[0193] As used herein, the term “delayed puberty” refers to a condition where a subject has an extension of dormancy of the reproductive axis. For most children, this is a transient condition, and they will spontaneously enter puberty.

[0194] As used herein, the term “reference level of luteinizing hormone” refers to a level of luteinizing hormone (LH) against which another level of LH is compared, e.g., to make a diagnostic and / or therapeutic determination for a subject having a disorder. In some embodiments, the reference level of LH may be a baseline level of LH that is measured in the subject prior to administration of a stimulating dose or a provocative dose of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof. In some embodiments, the reference level of LH may be an average level of LH in patients with the disorder who have not been administered the stimulating dose or provocative dose. In some embodiments, the reference level of LH may be an average level of LH in patients that do not have the disorder. In some embodiments, the reference level of LH is a normal or a low normal level of LH. In some embodiments, the reference level of LH may be between about 0.5 I U / L to about 110 IU / L. The absolute quantification of LH may be measured against an international standard.

[0195] As used herein, the term “stimulating dose” refers to a dose (e.g., a single bolus) of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof described herein that is sufficient to elicit an increase in luteinizing hormone in a healthy subject. In some embodiments, the stimulating dose is a dose of a kisspeptin or a pharmaceutically acceptable salt thereof. In some embodiments, the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg. In some embodiments, the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg.

[0196] As used herein, the term “provocative dose” refers to a dose (e.g., a single bolus) of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof that is sufficient to elicit an increase in luteinizing hormone in a subject with a disorder that is likely to respond to a therapy including a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof. In some embodiments, the provocative dose is a dose of a kisspeptin-10 or a pharmaceutically acceptable salt thereof. In some embodiments, the provocative dose is administered intravenously at a dose of between about 1 pg / kg to about 1 pg / kg. In some embodiments, the provocative dose is administered subcutaneously at a dose of between about 10 pg / kg to about 10 pg / kg.

[0197] As used herein, the term “greater than” refers to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, or greater, in a level (e.g., a level of LH) detected by the methods described herein, as compared to a reference level.

[0198] A “response” of a patient or a patient’s “responsiveness” to treatment or therapy, for example a therapy including a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof, refers to the clinical or therapeutic benefit imparted to a patient. For example, a patient having a disorder who is responsive to a therapy including a kisspeptin analog or aPATENT

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[0201] pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof may show observable and / or measurable reduction in or absence of one or more symptoms.

[0202] As used herein, a “surfactant” refers to a surface-active agent, preferably a nonionic surfactant. Examples of nonionic surfactants include polysorbates (e.g., polysorbate 20 (PS20) or polysorbate 80 (PS80)), polyethers (e.g., polypropylene glycol (PG)), poloxamers, polyoxyethelene alkyl ethers, alkyl phenyl polyoxyethylene ethers, orsorbitan esters.

[0203] As used herein, the term “tonicity agent” refers to a pharmaceutically acceptable excipient that is used to control the osmolarity of a pharmaceutical composition. Non-limiting examples of a tonicity agent include sugars, amino acids, or salts. In some embodiments, the tonicity agent is a sugar. In some embodiments, the sugar is mannitol, sucrose, glucose, glycerol, sorbitol, lactose, dextrose, or trehalose. In some embodiments, the sugar is mannitol. In some embodiments, the tonicity agent is polyethylene glycol, propylene glycol, or a poloxamer.

[0204] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein.

[0205] BRIEF DESCRIPTION OF THE DRAWINGS

[0206] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0207] FIG. 1 shows the effect of different doses of kisspeptin analog administration on LH levels in WT male mice (n=5 / group). SEQ ID NO: 1 was subcutaneously injected at a dose of either 60 pmol or 12 pmol / 100 µL H2O. Blood samples were collected 15 minutes prior to injection, and then at times 0 (right before the injection) 15, 30, 60, 90, 180, 360, and 600 minutes after injection.

[0208] FIG. 2 shows the effect of different doses of kisspeptin or kisspeptin analog administration on LH levels in WT male mice (n=5 / group). SEQ ID NOs: 1 or 3 were subcutaneously injected at a dose of 1 pmol / 100 µL H2O. Blood samples were collected at times 0, 15, 30, 180, 360, and 600 minutes after injection.

[0209] FIG. 3 shows the effect of different doses of kisspeptin or kisspeptin analog administration on LH levels in WT male mice (n=5 / group). SEQ ID NOs: 1 or 3 were subcutaneously injected at a dose of 6 pmol / 100 µL H2O. Blood samples were collected at times 0, 15, 30, 180, 360, 600, and 720 minutes after injection.

[0210] FIG. 4 shows the effect of kisspeptin analog administration on LH levels in WT male mice (n=5 / group). SEQ ID NOs: 1 or 3 were subcutaneously injected at a dose of 6 nmol in 100 µL H2O. Blood samples were collected 15 minutes prior to injection, and at times 0 (right before the injection), 15, 30, 60, 90, 180, and 360 minutes after injection.PATENT

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[0213] FIGS. 5A-5B show the effect of kisspeptin or kisspeptin analog administration on LH levels in WT male mice (n=9 / group). SEQ ID NOs: 1-3 were intraperitoneally injected at a dose of 6 nmol / 100 pL saline (FIG. 5A) or 0.6 nmol / 100 pL saline (FIG. 5B). Blood samples were collected 30 and 15 minutes prior to the injection, then, at times 0 (right before the injection), 15, 30, 60, and 90 minutes after injection.

[0214] FIGS. 6A-6B show the effect of kisspeptin analog administration in a mouse model of polycystic ovary syndrome (PCOS). FIG. 6A shows a decrease in basal LH levels in the prenatal androgenization (PNA) + SEQ ID NO: 1 (12 pmol / 100pL saline, subcutaneous administration) group compared to PNA control. FIG. 6B shows an increased number of primary follicles in the ovaries of the PNA + SEQ ID NO: 1 (12 pmol / 100pL saline, subcutaneous administration) group compared to PNA control.

[0215] FIG. 7 A shows a chromatogram of 2 ng / mL SEQ ID NO: 1 and SEQ ID NO: 2 analysis by liquid chromatography mass spectrometer (LC-MS). FIG. 7B shows a chromatogram of 50 ng / mL SEQ ID NO: 1 and SEQ ID NO: 2 analysis by LC-MS. Experiments were performed as described in Example 8.

[0216] FIGS. 8A-8B show LH levels and change in LH levels, respectively, after administration of various solvents to adult (4 to 5 month-old) C57BL / 6 male mice. Experiments were performed as described in Example 10.

[0217] FIGS. 9A-9C show testis weight or % testes / body weight (BW) after implantation of non-GnRH primed adult (4 month old) Kissi KO male with iPRECIO pumps previously filled with SEQ ID NO: 3 peptide at 0.26 mg / kg (8 pL / hour) in sterile water. Experiments were performed as described in Example 11. Mice 5651 and 5620 were treated with SEQ ID NO: 3 and mouse 5654 was given the control.

[0218] FIGS. 10A-10D show LH levels after implantation of non-GnRH primed adult (4 month old) Kissi KO male with iPRECIO pumps previously filled with SEQ ID NO: 3 peptide at 0.26 mg / kg (8 pL / hour) in sterile water. Experiments were performed as described in Example 11. Mice 5651 and 5620 were treated with SEQ ID NO: 3 and mouse 5654 was given the control.

[0219] FIGS. 11A-11B show LH levels after administration of gonad intact adult (3 month-old) C57BL / 6 female mice (n=7-10 / group) at metestrus / diestrus phases with SEQ ID NO: 1 or SEQ ID NO: 2.

[0220] Experiments were performed as described in Example 12.

[0221] FIGS. 12A-12B show LH levels after administration of adult (6-8 month-old) C57BL / 6 male mice (n=9 / group) with SEQ ID NO: 1 or SEQ ID NO: 2. Experiments were performed as described in Example 13.

[0222] FIGS. 13A-13C show body weights of adult (3 month-old) C57BL / 6 female mice (n=10 / group) ovariectomized (OVX) followed by subcutaneous (sc) implantation of Alzet pump (0.11 pL / hour release) filled with SEQ ID NO: 1 at the dose of 28 pg / ml in PT30 solution. Experiments were performed as described in Example 14.

[0223] FIG. 14 shows LH levels of adult (3 month-old) C57BL / 6 female mice (n=10 / group) ovariectomized (OVX) followed by sc implantation of Alzet pump (0.11 pL / hour release) filled with SEQ ID NO: 1 at the dose of 28 pg / ml in PT30 solution. Experiments were performed as described in Example 14.

[0224] FIGS. 15A-15B show number of peaks and amplitude LH peaks 7 days post-Alzet of Adult (3 month-old) C57BL / 6 female mice (n=10 / group) ovariectomized (OVX) followed by sc implantation of Alzet pump (0.11 pL / hour release) filled with SEQ ID NO: 1 at the dose of 28 pg / ml in PT30 solution.

[0225] Experiments were performed as described in Example 14.PATENT

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[0228] FIG. 16 shows LH levels after subcutaneous administration of adult (4-5-month-old, with an average of weight of 21.3 g) Kiss1 KO female (at constant diestrus) mice (n=13) with SEQ ID NO: 2 at the doses of 0.817 pg / kg (high: corresponding to 16.0 pmoles in 100 pl of PT30 solution), 0.163 pg / kg (medium; corresponding to 3.2 pmoles in 100 pl of PT30 solution), and 0.033 pg / kg (low; corresponding to 0.64 pmoles in 100 pl of PT30 solution) in PT30 solution. Experiments were performed as described in Example 16.

[0229] FIG. 17 shows LH levels after administration of SEQ ID NO: 2 to non-GnRH primed adult Kissi KO male (3-5-month-old, with an average of weight of 22.1 g) mice (n=5 / group, PT30 solution -vehicle-vs. SEQ ID NO: 2) subcutaneously implanted with iPRECIO pumps, previously filled with SEQ ID NO: 2 in PT30 solution at the dose of 0.817 pg / kg / pulse (0.3 pl / pulse over 2 min, with one pulse every 90 min). Experiments were performed as described in Example 17.

[0230] FIG. 18 shows LH levels after subcutaneous administration of gonadal intact adult (3-4-month-old, with an average weight of 17.44 g before start of 24-hr fasting) C57BL / 6 female mice with SEQ ID NO: 2 at the doses of 0.817 pg / kg (high; corresponding to 16.0 pmoles in 100 pl of PT30 solution), 0.163 pg / kg (medium; corresponding to 3.2 pmoles in 100 pl of PT30 solution) and 0.033 pg / kg (low; corresponding to 0.64 pmoles in 100 pl of PT30 solution) in PT30 solution. Experiments were performed as described in Example 18.

[0231] FIGS. 19A-19I show in vitro characterization, pharmacokinetics, and acute in vivo activity of KISS1R agonists SEQ ID NO: 1 and SEQ ID NO: 2. (FIG. 19A) p-arrestin recruitment assay in human KISSIR-expressing cells. SEQ ID NO: 1 and SEQ ID NO: 2 demonstrate subnanomolar potency (EC50= 0.9 nM and 1.1 nM, respectively) and supramaximal efficacy compared to kisspeptin-10 (KP-10; SEQ ID NO: 3; EC50= 39.3 nM). Data represent mean ± SEM from n=3 independent experiments performed in duplicate. Curves fitted using four-parameter nonlinear regression. (FIG. 19B) Calcium flux assay measuring Gq-protein-dependent activation in mouse Kiss1r-expressing cells. SEQ ID NO: 1 and SEQ ID NO: 2 show enhanced potency (EC50= 2.0 nM and 2.2 nM, respectively) compared to SEQ ID NO: 3 (EC50= 30.6 nM). Data represent mean ± SEM from n=3 independent experiments. Dose-response curves analyzed by four-parameter nonlinear regression. (FIG. 19C) Target engagement in GT1-7 mouse hypothalamic neurons measured by ERK1 / 2 phosphorylation relative to p-catenin. SEQ ID NO: 1 and SEQ ID NO: 2 at 0.1 pM achieve comparable or greater signaling than SEQ ID NO: 3 at 1 pM. Dotted line indicates serum-starved baseline. Data represent mean ± SEM from n=3 independent experiments. (FIG.

[0232] 19D) Plasma pharmacokinetics of SEQ ID NO: 1 (100 pg / kg) following subcutaneous (SC) and intravenous (IV) administration in female C57BL / 6 mice. Plasma concentrations were quantified by LC-MS / MS with an LLOQ of 10 pg / mL and acceptance criteria of ±20% accuracy and <20% CV. Data represent mean ± SEM, n=2-3 mice per timepoint. (FIG. 19E) Plasma pharmacokinetics of SEQ ID NO: 2 (200 pg / kg) following SC and IV administration. Bioanalytical parameters as in (D). Data represent mean ± SEM, n=2-3 mice per timepoint. (FIG. 19F) Experimental timeline for acute LH secretion studies showing SC compound administration and serial tail blood sampling. (FIG. 19G) Acute LH response in intact male mice following equimolar SC injection (6 nmol) of SEQ ID NO: 3, SEQ ID NO: 1, or SEQ ID NO: 2. Both agonists induce significantly greater LH responses than SEQ ID NO: 3. Data represent mean ± SEM, n=8-10 mice per group. Statistical analysis by two-way ANOVA with Tukey’s post hoc test.PATENT

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[0235] Different letters indicate statistically significant differences (P < 0.05). (FIG. 19H) Dose-response LH secretion following SEQ ID NO: 1 in female mice during diestrus. Doses: High (0.767 pg / kg), Medium (0.153 pg / kg), Low (0.031 pg / kg). Data represent mean ± SEM, n=8-10 mice per group. Statistical analysis by two-way ANOVA with Sidak’s post hoc test. Different letters indicate statistically significant differences (P < 0.05). (FIG. 191) Dose-response LH secretion following SEQ ID NO: 2 in intact male mice. Doses: High (0.817 pg / kg), Medium (0.163 pg / kg), Low (0.033 pg / kg). Data represent mean ± SEM, n=8-10 mice per group. Statistical analysis by two-way ANOVA with Sidak's post hoc test. Different letters indicate statistically significant differences (P < 0.05).

[0236] FIGS. 20A-20E show continuous SEQ ID NO: 1 delivery suppresses elevated LH pulsatility in ovariectomized mice. (FIG. 20A) Experimental timeline. Adult female C57BL / 6 mice were ovariectomized (OVX) on Day -7. Osmotic minipumps (Alzet Model 1004) delivering continuous SEQ ID NO: 1 (28 pg / mL) or vehicle (PT30 solution) were implanted subcutaneously on Day 0. Tail blood sampling was performed on Days 7 and 14 post-pump implantation. (FIG. 20B) Mean LH levels across the experimental timeline. SEQ ID NO: 1 treatment (blue) significantly suppresses post-OVX LH elevation compared to vehicle controls (pink). Data represent individual values with mean ± SEM, n=10 mice per group. ****p < 0.0001 by two-way ANOVA with Sidak's post hoc test. (FIG. 20C) Representative individual LH pulsatility profiles from vehicle-treated (top) and SEQ ID NO: 1 -treated (bottom) mice during 2-hour serial blood sampling at 10-minute intervals on Day 7 post-pump implantation. Asterisks (*) denote detected LH pulses identified using PULSAR Otago analysis. (FIG. 20D) Quantification of LH pulse frequency. SEQ ID NO: 1 treatment significantly reduces the number of LH pulses per 120-minute sampling period. Data represent individual values with mean ± SEM, n=10 mice per group. *P < 0.05 by Mann-Whitney U test. (FIG. 20E) Quantification of LH pulse amplitude. SEQ ID NO: 1 treatment significantly reduces pulse amplitude. Data represent individual values with mean ± SEM, n=10 mice per group. ***P < 0.001 by Mann-Whitney U test.

[0237] FIGS. 21A-21E show characterization of the prenatal androgen (PNA) mouse model. (FIG. 21 A) Timeline for PNA model generation. Pregnant dams received SC injections of dihydrotestosterone (DHT; 250 pg in 100 pL sesame oil) or vehicle on gestational days 16-18. Offspring were weaned at postnatal day 21, monitored for puberty markers, and estrous cyclicity was assessed in adulthood before experimental treatment. (FIG. 21B) Puberty onset markers. Left: Day of vaginal opening (VO). Right: Day of first estrus. PNA mice show significantly advanced puberty. Data represent individual values with mean ± SEM: Control n=10, PNA n=8. ***P < 0.001 by unpaired t-test. (FIG. 21C) Representative estrous cycle profiles over 14 days in Control (top) and PNA (bottom) mice, showing disrupted cyclicity with prolonged diestrus / metestrus (M / D) phases in PNA animals. (FIG. 21 D) Quantification of estrous cycle phases over 14 days. PNA mice spend significantly more time in diestrus and less time in proestrus and estrus compared to controls. Data represent individual values with mean ± SEM: Control n=11, PNA n=15. ****p < 0.0001 by two-way ANOVA with Sidak’s post hoc test. (FIG. 21 E) Mean LH levels in adult Control and PNA mice showing elevated basal LH in the PNA model. Data represent individual values with mean ± SEM: Control n=11, PNA n=15.

[0238] FIGS. 22A-22D show SEQ ID NO: 1 normalizes LH levels and improves ovarian morphology in a prenatal androgen-exposed PCOS model. (FIG. 22A) Experimental timeline. Prenatal androgen (PNA)PATENT

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[0241] mice and control littermates received twice-daily (BID; 9 AM and 6 PM) subcutaneous injections of SEQ ID NO: 1 (12 pmol in 100 pL) or vehicle for 30 days beginning in adulthood. Tail blood was collected on Days 1, 10, and 30 (pre-treatment and 3-hour post-treatment). Ovarian histology was performed at study termination. (FIG. 22B) Mean LH levels at Day 30 (pre-treatment). PNA mice treated with vehicle show elevated LH compared to controls, while SEQ ID NO: 1 treatment normalizes LH to control levels. Data represent individual values with mean ± SEM: Control n=8, PNA+veh n=6, PNA+SEQ ID NO: 1 n=6. *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey's post hoc test. (FIG. 22C) Representative ovarian histology. Control ovaries display multiple corpora lutea (CL) indicating recent ovulation. PNA+vehicle ovaries show absence of CL. PNA+SEQ ID NO: 1 ovaries lack CL but show improved follicular organization, more primary follicles (arrowheads). (FIG. 22D) Quantification of ovarian parameters. Left: Number of primary follicles. Center: Number of corpora lutea. Right: Ovary weight normalized to body weight. Data represent individual values with mean ± SEM: Control n=8, PNA+veh n=6, PNA+SEQ ID NO: 1 n=6. *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey's post hoc test.

[0242] FIGS. 23A-23G show detailed characterization of SEQ ID NO: 1 treatment effects in the PNA model. (FIGS. 23A-23C) LH levels at Day 1, Day 10, and Day 30 showing pre-treatment (left) and 3-hour post-treatment (right) values. Initial SEQ ID NO: 1 -induced LH elevation diminishes overtime in PNA mice while maintaining normalized basal levels. Data represent individual values with mean ± SEM: Control n=8, PNA+veh n=6, PNA+SEQ ID NO: 1 n=6. **P < 0.01 by one-way ANOVA with Tukey's post hoc test.

[0243] (FIG. 23D) Estrous cycle profiles during the 30-day treatment period. Top: Control + vehicle. Middle: PNA + SEQ ID NO: 1. Bottom: PNA + vehicle. Yellow shading indicates pre-treatment period; cyan indicates SEQ ID NO: 1 treatment period. Cyclicity remains disrupted in both PNA groups regardless of treatment.

[0244] (FIG. 23E) Number of estrous phases. (FIG. 23F) Hormonal and metabolic parameters at study termination. From left to right: Testosterone, AMH (anti-Mullerian hormone), FSH. No significant differences among groups. Data represent individual values with mean ± SEM: Control n=8, PNA+veh n=6, PNA+SEQ ID NO: 1 n=6. (FIG. 23G) Body weight at study termination. PNA+vehicle mice show increased body weight compared to Control and PNA+SEQ ID NO: 1 groups. Data represent individual values with mean ± SEM: Control n=8, PNA+veh n=6, PNA+SEQ ID NO: 1 n=6. *P < 0.05 by one-way ANOVA with Tukey's post hoc test.

[0245] FIGS. 24A-24D show SEQ ID NO: 2 stimulates LH secretion in models of hypothalamic amenorrhea and kisspeptin deficiency. (FIG. 24A) Experimental timeline for fasting study. Wild-type C57BL / 6 female mice were fasted for 24 hours prior to SC administration of SEQ ID NO: 2, with serial tail blood sampling continuing through 600 minutes (34 hours total fasting). (FIG. 24B) Dose-dependent LH response to SEQ ID NO: 2 in 24-hour fasted wild-type female mice. SEQ ID NO: 2 doses: High (0.817 pg / kg), Medium (0.163 pg / kg), Low (0.033 pg / kg). Data represent mean ± SEM, n=10 mice per group. Statistical analysis by two-way ANOVA with Sidak's post hoc test. Different letters indicate statistically significant differences (P < 0.05). (FIG. 24C) Schematic representation of Kissi knockout (KO) female mouse phenotype showing anovulation, closed vagina, pubertal failure, low gonadotropins, ovarian and uterine atrophy, low estrogen, and infertility. (FIG. 24D) Dose-dependent LH response to acute SEQ ID NO: 2 administration in non-GnRH-primed Kissi KO female mice. SEQ ID NO: 2 doses as in (B). DataPATENT

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[0248] represent mean ± SEM, n=4 mice per group. Statistical analysis by two-way ANOVA with Sidak’s post hoc test. Different letters indicate statistically significant differences (P < 0.05).

[0249] FIGS. 25A-25C show pulsatile SEQ ID NO: 2 delivery restores reproductive function in Kissi -deficient male mice. (FIG. 25A) Experimental timeline. Non-GnRH-primed adult Kissi KO male mice received programmable iPRECIO pumps (SMP-310R) implanted subcutaneously on Day -3. Pumps were activated on Day 1 to deliver pulsatile SEQ ID NO: 2 (0.817 pg / kg / pulse; 0.3 pL / pulse over 2 minutes, every 90 minutes) or vehicle for 7 days. Blood and tissues were collected at study termination.

[0250] (FIG. 25B) Serum LH (left) and testosterone (right) levels at Day 7. Pulsatile SEQ ID NO: 2 significantly elevates both LH and testosterone compared to vehicle-treated Kissi KO males. Data represent individual values with mean ± SEM, n=5 mice per group. **P < 0.01 by Mann-Whitney U test. (FIG. 25C) Representative testis images and quantification. Left: Gross appearance of testes from vehicle-treated (small, underdeveloped) and SEQ ID NO: 2-treated (enlarged) Kissi KO males. Right: Testis weight normalized to body weight. Data represent individual values with mean ± SEM, n=5 mice per group. **P < 0.01 by Mann-Whitney U test.

[0251] FIGS. 26A-26F show pulsatile kisspeptin-10 administration induces dose-dependent LH pulsatility in individuals with idiopathic hypogonadotropic hypogonadism. (FIG. 26A) Clinical study design. Following outpatient GnRH priming (25 ng / kg SC every 2 hours for 6 days), participants received escalating IV boluses of kisspeptin-10 (0.313, 0.939, 3.13, and 13.19 pg / kg) administered every 2 hours at the Clinical Research Center. Blood was sampled every 10 minutes for 44-52 hours to capture LH pulse dynamics. A final GnRH bolus assessed pituitary responsiveness. (FIG. 26B) Representative LH profiles from two IHH participants showing responses to escalating kisspeptin doses. Left: Male participant with FGFR1 p. Arg507LeufsTer38 variant. Right: Female participant with SOX10 p. Asp167Val variant. Colored traces indicate different kisspeptin doses. a=0.313 pg / kg, b=0.939 pg / kg, c=3.13 pg / kg, and d=13.19 pg / kg. Dots indicate individual LH measurements. (FIG. 26C) Mean LH amplitude across kisspeptin doses. Significant dose-dependent increase in LH amplitude. Data represent box plots with median, interquartile range, and min / max, n=10 participants, ns = not significant, *P < 0.05, ****p < 0.0001 by Friedman test with Dunn's post hoc comparison to lowest dose. (FIG. 26D) Mean number of LH pulses (defined by Santen and Bardin criteria) detected per 4 kisspeptin boluses at each dose level. Data represent box plots, n=10 participants. P = 0.01 by one-way ANOVA. (FIG. 26E) Change in LH amplitude between first and last bolus at each kisspeptin dose, indicating progressive sensitization. Data represent individual values with mean, n=10 participants. P = 0.02 by one-way ANOVA. (FIG. 26F) Mean LH levels pre- and post-kisspeptin administration demonstrating sustained elevation following pulsatile kisspeptin exposure. Data represent individual paired values with connecting lines, n=8 participants. ****p < 0.0001 by Wilcoxon matched-pairs signed rank test.

[0252] FIG. 27 shows body weight in the OVX study. Body weight changes across the experimental timeline in vehicle-treated (left) and SEQ ID NO: 1 -treated (right) OVX mice. Data represent mean ± SEM, n=10 mice per group. ****p < 0.0001 by two-way ANOVA with Sidak’s post hoc test comparing to intact baseline.PATENT

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[0255] DETAILED DESCRIPTION OF THE INVENTION

[0256] The invention provides methods for treatment of disorders (e.g., reproductive disorders, hypoactive sexual arousal disorder, respiratory disease, metabolic disorders, bone disorders, VMS, liver disorders, neurodegenerative disorders, or sequelae of neurotropic viruses), involving administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described herein. In some embodiments, the methods involve administering to the subject a therapeutically effective amount of a kisspeptin (e.g., kisspeptin-10) or a pharmaceutically acceptable salt thereof. Also provided herein are methods of diagnosing congenital HH and methods of determining whether a subject will respond to a therapy including a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceuticaily acceptable salt thereof, or a pharmaceutical composition described herein. In some embodiments, the diagnostic methods involve administering to the subject a kisspeptin or a pharmaceutically acceptable salt thereof.

[0257] I. Peptides

[0258] Provided herein are kisspeptin or kisspeptin analogs or pharmaceutically acceptable salts thereof that may be used in any of the methods described herein. The kisspeptin analogs described herein have improved potency and stability compared to the native kisspeptin. The kisspeptin analogs or pharmaceutically acceptable salts thereof may contain both natural and unnatural amino acids.

[0259] Commonly accepted abbreviations that are used in the art as well as herein to represent unnatural amino acids include the following:

[0260] D-Phe(2,4-DiCI): 2,4-dichloro-D-phenylalanine

[0261] Arg(Me): N-w-methyl-arginine

[0262] HoPro: homoproline, S-homoproline, S-Pip, S-high Proline, or (S)-piperidine-2-formic acid A6c: 1-amino-cyclohexyl formic acid

[0263] azaGly: azaglycine

[0264] Ac: acetyl

[0265] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes the sequence of Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (SEQ ID NO: 1). In other embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof is Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (SEQ ID NO: 1). The structure of SEQ ID NO: 1 is shown below.PATENT

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[0268] OH

[0269]

[0270] (SEQ ID NO: 1)

[0271] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof includes the sequence of Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (SEQ ID NO: 2). In other embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof is Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (SEQ ID NO: 2). The structure of SEQ ID NO: 2 is shown below.

[0272]

[0273] In some embodiments, the kisspeptin or pharmaceutically acceptable salt thereof is the native kisspeptin-10 (KP-10), which has the sequence of Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe (SEQ ID NO: 3). The structure of SEQ ID NO: 3 is shown below.

[0274]

[0275] NO: 3).

[0276] In some embodiments, the kisspeptin analog is a kisspeptin analog disclosed in WO2020247936A2 or W02019007383A1 (wherein the kisspeptin analogs described in each are hereby specifically incorporated by reference).

[0277] In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof may sensitize KISS1R to activation. In some embodiments, the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof may desensitize KISS1R to activation.PATENT

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[0280] Those skilled in the art will appreciate that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, atropisomers, tautomers) or isotopic (e.g., in which one or more atoms has been substituted with a different isotope of the atom, such as hydrogen substituted for deuterium) forms. Unless otherwise indicated or clear from context, a sequence can be understood to represent any such isomeric or isotopic form, individually or in combination.

[0281] Accordingly, the compositions and methods of the disclosure include (where possible) individual diastereomers, enantiomers, epimers, tautomers, and atropisomers of the compounds disclosed herein, and mixtures of diastereomers and / or enantiomers thereof including racemic mixtures.

[0282] II. Formulations

[0283] The invention provides pharmaceutical compositions that include kisspeptin analogs (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof with improved recovery.

[0284] Advantageously, the pharmaceutical compositions described herein have minimized loss of kisspeptin analogs (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof due to adsorption to containers, transfer apparatuses, and drug delivery pumps. In some embodiments, over 90% recovery is achieved from the aforementioned devices.

[0285] The pharmaceutical compositions may have any suitable concentration of the kisspeptin analog (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof. For example, the concentration of the kisspeptin analog or a pharmaceutically acceptable salt thereof can be about 0.01 ng / mL to about 100 mg / mL (e.g., about 0.01 ng / mL to about 0.5 ng / mL, about 0.5 ng / mL to about 1 ng / mL, about 1 ng / mL to about 50 ng / mL, about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 pg / mL to about 50 pg / mL, about 50 pg / mL to about 100 pg / mL, about 100 pg / mL to about 500 pg / mL, about 500 pg / mL to about 1 mg / mL, about 1 mg / mL to about 50 mg / mL, or about 50 mg / mL to about 100 mg / mL).

[0286] In some embodiments, the pharmaceutical compositions may have a pH of about 3.5 to about 8.0 (e.g., about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, or about 7.5 to about 8.0). In some embodiments, the pharmaceutical compositions may have a pH of about 4.0 to about 6.0. In some embodiments, the pharmaceutical compositions may have a pH of about 5.0.

[0287] In some embodiments, the pharmaceutical compositions may include a buffer. Any suitable buffer may be used. In some embodiments, the buffer is an acetate buffer (e.g., a sodium acetate buffer), a phosphate buffer, a succinate buffer, a histidine buffer, a citrate buffer (e.g., a sodium citrate buffer), a Tris buffer (e.g., a Tris-HCI buffer), a carbonate buffer, or a combination thereof. In some embodiments, the buffer is an acetate buffer, such as a sodium acetate buffer.

[0288] Any suitable concentration of the buffer can be used. For example, in some embodiments, the buffer may be present at a concentration of about 0.1 mM to about 100 mM (e.g., about 0.1 mM to about 0.5 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 15 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 30 mM to about 35 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 50 mM to about 55 mM, about 55 mM to about 60 mM, about 60 mM to about 65 mM, about 65 mMPATENT

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[0291] to about 70 mM, about 70 mM to about 75 mM, about 75 mM to about 80 mM, about 80 mM to about 85 mM, about 85 mM to about 90 mM, about 90 mM to about 95 mM, or about 95 mM to about 100 mM). In some embodiments, the buffer may be present at a concentration of 1 mM to about 10 mM. In some embodiments, the buffer may be present at a concentration of about 5 mM.

[0292] In some embodiments, the pharmaceutical composition further comprises a tonicity agent, in some embodiments, the tonicity agent is a sugar, an amino acid, or a salt. In some embodiments, the tonicity agent is a sugar. In some embodiments, the sugar is mannitol, sucrose, glucose, glycerol, sorbitol, lactose, dextrose, or trehalose. In some embodiments, the sugar is mannitol, in some embodiments, the tonicity agent is polyethylene glycol, propylene glycol, or a poloxamer.

[0293] Any suitable concentration of the tonicity agent can be used. For example, in some embodiments, the tonicity agent may be present at a concentration of about 1 mg / mL to about 100 mg / mL (e.g., about 1 mg / mL to about 5 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 15 mg / mL, about 15 mg / mL to about 20 mg / mL, about 20 mg / mL to about 25 mg / mL, about 25 mg / mL to about 30 mg / mL, about 30 mg / mL to about 35 mg / mL, about 35 mg / mL to about 40 mg / mL, about 40 mg / mL to about 45 mg / mL, about 45 mg / mL to about 50 mg / mL, about 50 mg / mL to about 55 mg / mL, about 55 mg / mL to about 60 mg / mL, about 60 mg / mL to about 65 mg / mL, about 65 mg / mL to about 70 mg / mL, about 70 mg / mL to about 75 mg / mL, about 75 mg / mL to about 80 mg / mL, about 80 mg / mL to about 85 mg / mL, about 85 mg / mL to about 90 mg / mL, about 90 mg / mL to about 95 mg / mL, or about 95 mg / mL to about 100 mg / mL). In some embodiments, the tonicity agent may be present at a concentration of about 10 mg / mL to about 70 mg / mL. In some embodiments, the tonicity agent may be present at a concentration of about 40 mg / mL.

[0294] In some embodiments, the tonicity agent may be present at a concentration of about 200 mOsm / kg to about 500 mOsm / kg (e.g., about 200 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 400 mOsm / kg, or about 400 mOsm / kg to about 500 mOsm / kg). In some embodiments, the tonicity agent may be present at a concentration of about 200 mOsm / kg to about 600 mOsm / kg (e.g., about 200 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 400 mOsm / kg, about 400 mOsm / kg to about 500 mOsm / kg, or about 500 mOsm / kg to about 600 mOsm / kg). For example, in some embodiments, the tonicity agent may be mannitol, which may be present at a concentration of about 200 mOsm / kg to about 300 mOsm / kg (e.g., about 220 mOsm / kg).

[0295] In some embodiments, the pharmaceutical composition further comprises one or more surfactants. In some embodiments, one or more surfactants is a nonionic surfactant. Examples of suitable nonionic surfactants include polysorbates (e.g., polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), or polysorbate 80 (PS80)), polyethers (e.g., polypropylene glycol (PG), polyoxyethylene 4 lauryl ether (Brij 30), polyoxyethylene acetyl ether (Brij 58), or polyoxyl 40 stearate), poloxamers, polyoxyethelene alkyl ethers, alkyl phenyl polyoxyethylene ethers, orsorbitan esters (e.g., sorbitan trioleate, or sorbitan monooleate). In some embodiments, the nonionic surfactant is a polysorbate (e.g., PS20 or PS80). In some embodiments, the polysorbate is PS80. In some embodiments, the nonionic surfactant is a polyether (e.g., PG). In some embodiments, the polyether is PG.PATENT

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[0298] Any suitable concentration of the surfactant can be used. For example, in some embodiments, each of the one or more surfactants may be present at a concentration of about 0.01% to about 20% (e.g., about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, or about 15% to about 20%). In some embodiments, each of the one or more surfactants may be present at a concentration of about 0.01% to about 1% (e.g., about 0.1%). In some embodiments, each of the one or more surfactants may be present at a concentration of about 1% to about 10% (e.g., about 5%).

[0299] In some embodiments, the pharmaceutical composition further comprises a stabilizer. In some embodiments, the stabilizer is a sugar, such as mannitol, sucrose, trehalose, or sorbitol. In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is a polyol, such as a polyoxyethylene (e.g., PEG300 or PEG400), propylene glycol (PG), a poloxamer (e.g., P407 or P188), an amino acid, a covalent cation (e.g., Mg2+), a small molecule (e.g., EDTA or nicotinamide), a surfactant (e.g., PS80, PS20, or a glucoside such as hexyl-, octyl-, decyl- or dodecyl glucoside), or a cyclodextrin (e.g., a beta-cyclodextrin, such as hydroxypropyl beta- cyclodextrin or sulfobutyl ether beta-cyclodextrin).

[0300] Any suitable concentration of the stabilizer can be used. For example, in some embodiments, the stabilizer may be present at a concentration of about 1 mg / mL to about 100 mg / mL (e.g., about 1 mg / mL to about 5 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 15 mg / mL, about 15 mg / mL to about 20 mg / mL, about 20 mg / mL to about 25 mg / mL, about 25 mg / mL to about 30 mg / mL, about 30 mg / mL to about 35 mg / mL, about 35 mg / mL to about 40 mg / mL, about 40 mg / mL to about 45 mg / mL, about 45 mg / mL to about 50 mg / mL, about 50 mg / mL to about 55 mg / mL, about 55 mg / mL to about 60 mg / mL, about 60 mg / mL to about 65 mg / mL, about 65 mg / mL to about 70 mg / mL, about 70 mg / mL to about 75 mg / mL, about 75 mg / mL to about 80 mg / mL, about 80 mg / mL to about 85 mg / mL, about 85 mg / mL to about 90 mg / mL, about 90 mg / mL to about 95 mg / mL, or about 95 mg / mL to about 100 mg / mL). In some embodiments, the stabilizer (e.g., mannitol) may be present at a concentration of about 40 mg / mL.

[0301] In some embodiments, the pharmaceutical composition may include about 1 ng / mL to about 100 mg / mL kisspeptin analog (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, about 0.1 mM to about 100 mM buffer (e.g., about 5 mM buffer), about 1 mg / mL to about 100 mg / mL tonicity agent (e.g., about 40 mg / mL tonicity agent), about 0.01 % to about 1.0% polysorbate surfactant (e.g., about 0.1% polysorbate surfactant), and about 1% to about 10% polyether (e.g., about 5% PG), and has a pH of about 3.5 to about 8.0 (e.g., about 5.0).

[0302] In some embodiments, the pharmaceutical composition may include about 0.01 ng / mL to about 100 mg / mL kisspeptin analog (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, about 0.1 mM to about 100 mM sodium acetate buffer (e.g., about 5 mM sodium acetate buffer), about 1 mg / mL to about 100 mg / mL mannitol (e.g., about 40 mg / mL mannitol), about 0.01 % to about 1.0% PS80 (e.g., about 0.1% PS80), and about 1% to about 10% PG (e.g., about 5% PG), and has a pH of about 4.0 to about 6.0 (e.g., about 5.0).PATENT

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[0305] III. Combination Therapies

[0306] 1. GnRH Agonists / Antagonists

[0307] In some embodiments, any of the methods described herein may further comprise administering to the subject a gonadotropin-releasing hormone receptor (GnRHR) agonist or antagonist. GnRHR agonists bind to and activate GnRHR signaling. Examples of GnRHR agonists include leuprolide (LUPRON®), gonadorelin, goserelin (ZOLADEX®), nafarelin, histrelin (SUPPRELIN®, VANTAS®), triptoreli n (TRELSTAR®), and buserelin. GnRHR antagonists bind to GnRHR but do not activate GnRHR signaling. Examples of GnRHR antagonists include abarelix (PLENAXIS®), cetrorelix (CETROTIDE®), degarelix (FIRMAGON®), elagolix (ORLISSA®), ganirelix (ORGALUTRAN®), and relugolix (RELUMINA®, ORGOVYK®).

[0308] 2. Neurokinin Agonists / Antagonists

[0309] In some embodiments, any of the methods described herein may further comprise administering to the subject a neurokinin receptor agonist or antagonist. Neurokinin receptor agonists bind to and activate neurokinin receptor signaling. Neurokinin receptor antagonists bind to the neurokinin receptor but do not activate neurokinin receptor signaling. Examples of neurokinin receptor antagonists include fezolinetant (VEOZAH®), aprepitant, rolapitant, casopitant, fosaprepitant, netupitant, and maropitant.

[0310] 3. Opioid Agonists / Antagonists

[0311] In some embodiments, any of the methods described herein may further comprise administering to the subject an opioid agonist, antagonist, or agonist-antagonist. Opioid agonists bind to and activate opioid receptor signaling. Examples of opioid agonists include morphine, methadone, and alfentanil. Opioid antagonists bind to the opioid receptor but do not activate opioid receptor signaling. Examples of opioid antagonists include naloxone and naltrexone. Opioid agonist-antagonists have agonistic activity at some opioid receptors and antagonistic activity at other opioid receptors. Examples of opioid agonistantagonists include buprenorphine.

[0312] IV. Methods of Treatment

[0313] 1. Reproductive Disorders

[0314] In some embodiments, the invention provides methods of treating a subject having a reproductive disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. A reproductive disorder is a condition or disease involving the reproductive axis, which refers to the hypothalamus, pituitary gland, and gonadal glands (the hypothalamic-pituitary-ovarian axis in females and the hypothalamic-pituitary-testicular axis in males).

[0315] a. Suppressed Reproductive Axis

[0316] In some embodiments, the reproductive disorder is associated with a suppressed reproductive axis, which is a condition where the subject has lowered levels of a reproductive health biomarker. ThePATENT

[0317] Attorney Docket No.: 51796-005WO3

[0318] BWH Ref.: 2024-010

[0319] kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a reproductive disorder with pulsatile administration, which sensitizes the KISS1R receptor to activation, increasing levels of the reproductive health biomarker, and activating the reproductive axis, in some embodiments, the reproductive health biomarker is a downstream marker of reproductive health, i.e., a marker of overall health, such as bone density or basal body temperature. In some embodiments, the subject may be a female, and the reproductive health biomarker may be LH, FSH, inhibin, estradiol, AMH, a marker of egg quality, or a marker of embryo quality. In some embodiments, the reproductive health biomarker is phenotypic, such as external signs of puberty onset (Tanner scale), sexual desire, follicle size, uterine lining (e.g., endometrial lining), cervical mucus, or vaginal moisture. Administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein to a female subject may result in follicle growth, improve the manner in which eggs grow, improve the number of eggs that grow, improve the quality of the eggs that grow, and / or improve the ability of eggs to ovulate. Alternatively, the subject may be a male, and the reproductive health biomarker may be LH, FSH, inhibin, or testosterone, in some embodiments, the reproductive health biomarker is phenotypic, such as external signs of puberty onset (Tanner scale), sexual desire, semen analysis, or testicular size. Administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein to a male subject may result in spermatogenesis.

[0320] In some embodiments, the reproductive disorder may be hypogonadotropic hypogonadism (HH). HH is a condition where the subject produces little or no sex hormones due to a lack of signaling from the pituitary gland or hypothalamus. In some embodiments, the HH may be congenital HH, in which the subject was on a trajectory since birth to never go through puberty. Congenital HH may be congenital idiopathic HH (normosmic). Congenital HH may be associated with an inability to smell, which is referred to as congenital Kallmann syndrome (anosmic / hyposmic). In some embodiments, the HH may be acquired HH, which refers to a postnatal onset of disorders that damage or alter the function of gonadotropin-releasing hormone (GnRH) neurons and / or pituitary gonadotroph cell. In some embodiments, the acquired HH is hypothalamic amenorrhea. In some embodiments, the acquired HH is hyperprolactinemia. In some embodiments, the acquired HH may be associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse. In some embodiments, the reproductive disorder may be abnormal pregnancy, infertility, or a reproductive disorder treated with an IVF protocol.

[0321] b. Hyperactive Reproductive Axis

[0322] In some embodiments, the reproductive disorder is associated with a hyperactive reproductive axis, which is a condition where the subject has elevated levels of a reproductive health biomarker. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a reproductive disorder with continuous administration, which desensitizes the KISS1RPATENT

[0323] Attorney Docket No.: 51796-005WO3

[0324] BWH Ref.: 2024-010

[0325] receptor to activation, decreasing levels of the reproductive health biomarker, and suppressing the reproductive axis. The subject may be a female, and the female reproductive health biomarker may be LH, FSH, inhibin, estradiol, AMH, external signs of puberty onset (Tanner scale), sexual desire, follicle size, bone density, uterine lining (e.g., endometrial lining), cervical mucus, vaginal moisture, basal body temperature, a marker of egg quality, or a marker of embryo quality. Administration of the polypeptide to a female subject may decrease follicle growth. Alternatively, the subject may be a male, and the male reproductive health biomarker may be LH, FSH, inhibin, testosterone, external signs of puberty onset (Tanner scale), sexual desire, semen analysis, bone density, or testicular size. Administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein to a male subject may modify spermatogenesis or the potential for spermatogenesis.

[0326] In some embodiments, the reproductive disorder may be precocious puberty, sex hormone- dependent cancer, gender dysphoria, polycystic ovary syndrome (PCOS), endometriosis, uterine fibroid, or abnormal pregnancy.

[0327] In some embodiments when treating PCOS, administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein decreases levels of LH without significantly decreasing levels of FSH. PCOS is typically characterized by disproportionately high levels of LH compared to FSH and it is considered that the elevated LH participates in driving excess male hormone levels in the blood. Thus, PCOS is often referred to as a state of “LH predominance” compared to FSH. As women move through reproductive life and their FSH levels begin to rise, some PCOS symptoms improve (e.g., improved menstrual cycling). It is thought that, without being bound by theory, a more normal balance of LH and FSH may be responsible for this improved cycling in older but still reproductive aged women.

[0328] 2. Hypoactive Sexual Arousal Disorder

[0329] In some embodiments, the invention provides methods of treating a subject having a hypoactive sexual desire disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. Hypoactive sexual arousal disorder is a condition in which desire for sexual activity is persistently or recurrently diminished or absent, causing marked distress or interpersonal difficulties. Sexual arousal disorder can be caused by psychological / emotional factors, relationship factors, illness, medications / drugs (e.g., analgesics, such as opioids) and hormone changes. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a hypoactive sexual desire disorder with pulsatile administration, which sensitizes the KISS1 receptor to activation.

[0330] 3. Respiratory Disease

[0331] In some embodiments, the invention provides methods of treating a subject having a respiratory disease, the method including administering to the subject a therapeutically effective amount of aPATENT

[0332] Attorney Docket No.: 51796-005WO3

[0333] BWH Ref.: 2024-010

[0334] kisspeptin analog ora pharmaceutically acceptable salt thereof, kisspeptin-10 o a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. Respiratory diseases may include any diseases of the airway and lungs that affect human respiration. For example, a respiratory disease may be asthma. The kisspeptin analog or a pharmaceutically acceptabie sait thereof, kisspeptin-10 ora pharmaceutically acceptable salt thereof, ora pharmaceutical composition described herein may be administered to the subject having a respiratory disease with pulsatile administration, which sensitizes the KISS1R receptor to activation.

[0335] 4. Metabolic Disorder

[0336] In some embodiments, the invention provides for methods of treating a subject having a metabolic disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a metabolic disorder with pulsatile administration, which sensitizes the KISS1R receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a metabolic disorder with continuous administration, which desensitizes the KISS1R receptor to activation. In some embodiments, the metabolic disorder is obesity. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the metabolic disorder is metabolic dysfunction-associated steatotic liver disease (MASLD), which is a condition in which fat (e.g., lipids) builds up in the liver due to causes other than excessive alcohol consumption, in some embodiments, the MASLD may progress to metabolic dysfunction-associated steatohepatitis (MASH).

[0337] 5. Liver Disorder

[0338] In some embodiments, the invention provides for methods of treating a subject having a liver disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a liver disorder with pulsatile administration, which sensitizes the KISS1R receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a liver disorder with continuous administration, which desensitizes the KISS1R receptor to activation. An exemplary liver disorder is metabolic dysfunction-associated steatotic liver disease (MASLD), which is a condition in which fat (e.g., lipids) builds up in the liver due to causes other than excessive alcohol consumption. In some embodiments, the MASLD may progress to metabolic dysfunction-associated steatohepatitis (MASH).PATENT

[0339] Attorney Docket No.: 51796-005WO3

[0340] BWH Ref.: 2024-010

[0341] 6. Bone disorder

[0342] In some embodiments, the invention provides for methods of treating a subject having a bone disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein described herein. In some embodiments, the bone disorder may be osteoporosis. Osteoporosis is a bone disease in which bone mineral density decreases, which may lead to fractures. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described herein may be administered to the subject having a bone disorder with pulsatile administration, which sensitizes the KISS1 receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a bone disorder with continuous administration, which desensitizes the KISS1 receptor to activation.

[0343] 7. Vasomotor Symptoms (VMS)

[0344] In some embodiments, the invention provides for methods of treating a subject having vasomotor symptoms (VMS), the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. VMS refers to hot flashes, night sweats, palpitation of the heart rhythm, joint pain, and muscular weakness. In a particular embodiment, the subject is female and the vasomotor symptoms are hot flashes and night sweats. The hot flashes or night sweats may be associated with menopause or hormonal dependent cancers such as breast cancer (tamoxifen) in females or prostate cancer (androgen deprivation therapy) in males. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having VMS with pulsatile administration, which sensitizes the KISS1 receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having VMS with continuous administration, which desensitizes the KISS1R receptor to activation.

[0345] 8. Neurodegenerative disorder

[0346] in some embodiments, the invention provides for methods of treating a subject having a neurodegenerative disorder, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a neurodegenerative disorder with pulsatile administration, which sensitizes the KISS1 RPATENT

[0347] Attorney Docket No.: 51796-005WO3

[0348] BWH Ref.: 2024-010

[0349] receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a neurodegenerative disorder with continuous administration, which desensitizes the KISS1R receptor to activation, in some embodiments, the neurodegenerative disorder may be Alzheimer’s disease.

[0350] 9. Sequelae of neurotropic viruses

[0351] In some embodiments, the invention provides for methods of treating a subject having a sequela of a neurotropic virus, the method including administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0352] Neurotropic viruses are viruses that affect the nervous system. An exemplary neurotropic virus is SARS CoV-2. Neurotropic viruses may cause sequelae that affect the central nervous system, which may include brain fog.

[0353] The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a sequela of a neurotropic virus with pulsatile administration, which sensitizes the KISS1 R receptor to activation. Alternatively, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered to the subject having a sequela of a neurotropic virus with continuous administration, which desensitizes the KISS1R receptor to activation.

[0354] 10. Methods of treating a subject suspected of having congenital HH

[0355] In some embodiments, the invention proves for methods of treating a subject suspected of having congenital HH, comprising: (a) providing a reference level of LH; (b) administering to the subject a stimulating dose of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, wherein: (I) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg; or (ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg; (c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose; and (d) when the level of LH in the subject after administration of the stimulating dose is not different from the reference level of LH, administering to the subject a pulsatile therapy regimen of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof effective to activate the reproductive axis, thereby treating congenital HH.

[0356] In some embodiments, the invention proves for methods of treating a subject having a disorder who is likely to respond to a therapy comprising a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, the method comprising: (a) providing aPATENT

[0357] Attorney Docket No.: 51796-005WO3

[0358] BWH Ref.: 2024-010

[0359] reference level of LH; (b) administering to the subject a provocative dose of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; (c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose; and (d) when the level of LH in the subject after administration of the provocative dose is greater than the reference level of LH, administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, thereby treating the disorder.

[0360] V. Dosing and Administration

[0361] 1. Pulsatile Administration

[0362] The administration of the therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be by pulsatile, wherein a single bolus of drug is released followed by a period of little / no release (lag phase). This cycle occurs in a defined frequency. Pulsatile administration may be used in the treatment of disorders where agonistic stimulation of KISS1R activation would be beneficial (e.g., a reproductive disorder (e.g., HH (e.g., congenital HH (e.g., congenital idiopathic HH (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic)) or acquired HH (e.g., hypothalamic amenorrhea, hyperprolactinemia, or acquired HH associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse)), abnormal pregnancy, infertility, or a reproductive disorder treated with an IVF protocol), hypoactive sexual arousal disorder, VMS, a respiratory disease (e.g., asthma), a metabolic disorder or liver disorder (e.g., obesity, diabetes, MASLD, or MASH), a bone disorder (e.g., osteoporosis), a neurodegenerative disorder (e.g., Alzheimer’s disease), or a sequela of a neurotropic virus). During pulsatile administration, the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein sensitizes the KISS1 receptor to activation. Biomarkers may be monitored throughout treatment.

[0363] In some embodiments, the therapeutically effective amount may be between about 0.05 pg / kg to about 90 ng / kg (e.g., between about 0.05 pg / kg to about 0.1 pg / kg, e.g., between about 0.1 pg / kg to about 0.5 pg / kg, e.g., between about 0.5 pg / kg to about 1 pg / kg, e.g., between about 1 pg / kg to about 5 pg / kg, e.g., between about 5 pg / kg to about 10 pg / kg, e.g., between about 10 pg / kg to about 50 pg / kg, e.g., between about 50 pg / kg to about 100 pg / kg, e.g., between about 100 pg / kg to about 500 pg / kg, e.g., between about 500 pg / kg to about 1 ng / kg, e.g., between about 1 ng / kg to about 5 ng / kg, e.g., between about 5 ng / kg to about 10 ng / kg, e.g., between about 10 ng / kg to about 50 ng / kg, e.g., between about 50 ng / kg to about 90 ng / kg). In some embodiments, the therapeutically effective amount is between about 10 pg / kg to about 1 ng / kg.

[0364] The therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical compositionPATENT

[0365] Attorney Docket No.: 51796-005WO3

[0366] BWH Ref.: 2024-010

[0367] described herein may also be higher. In some embodiments, the therapeutically effective amount is between about 90 ng / kg to about 5 pg / kg. In some embodiments, the therapeutically effective amount is between about 90 ng / kg to about 0.5 pg / kg. In some embodiments, the therapeutically effective amount is between about 0.5 pg / kg to about 1 pg / kg. In some embodiments, the therapeutically effective amount is between about 1 pg / kg to about 5 pg / kg.

[0368] In some embodiments, the therapeutically effective amount is about 5 pg / kg or greater. In some embodiments, the therapeutically effective amount is between about 5 pg / kg to about 30 pg / kg. In some embodiments, the therapeutically effective amount is between about 5 pg / kg to about 10 pg / kg. In some embodiments, the therapeutically effective amount is between about 10 pg / kg to about 20 pg / kg. In some embodiments, the therapeutically effective amount is between about 20 pg / kg to about 30 pg / kg.

[0369] The administration frequency of the boluses of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be determined based on normative data regarding GnRH induced LH pulses. The pulsatile administration may be at a frequency of between about 20 minutes to about 4 days (e.g., between about 20 minutes to about 40 minutes, e.g., between about 40 minutes to about 1 hour, e.g., between about 1 hour to about 2 hours, e.g., between about 2 hours to about 4 hours, e.g., between about 4 hours to about 8 hours, e.g., between about 8 hours to about 16 hours, e.g., between about 16 hours to about 1 day, e.g., between about 1 day to about 2 days, e.g., between about 2 days to about 3 days, e.g., between about 3 days to about 4 days). In some embodiments, the pulsatile administration is at a frequency of between about 45 minutes to about 2 hours.

[0370] The pulsatile administration may be over a period of at least about 1 day (e.g., at least about 2 days, e.g., at least about 3 days, e.g., at least about 4 days, e.g., at least about 5 days, e.g., at least about 6 days, e.g., at least about 1 week, e.g., at least about 2 weeks, e.g., at least about 1 month, e.g., at least about 1.5 months, e.g., at least about 2 months, e.g., at least about 6 months, e.g., at least about 1 year, e.g., at least about 2 years).

[0371] In some embodiments, a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered chronically, e.g., for a period of days, weeks, months, or years. The treatment can be continued until, and optionally stopped or reduced when, a desired outcome, e.g., fertility, or development of secondary sexual characteristics, has been achieved. In some embodiments, the treatment continues for months and years until desired effects are achieved. For example, for ovulation induction in women, a course could be anywhere from 1 month to 6 months. As another example, for spermatogenesis induction in men, a course could be anywhere from 3 to 24 months. As another example, for pubertal induction and / or maintenance of reproductive endocrine function, treatment could be for years. Sensitivity to kisspeptin increases with repetitive exposure; thus in some embodiments, the present methods can include repeated administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein to increase sensitivity to the point where endogenous kisspeptin secretion produces meaningful responses, to induce reversal of the kisspeptin resistance.PATENT

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[0373] BWH Ref.: 2024-010

[0374] For example, the boluses may be released every 45 minutes for 2 weeks or the boluses may be released every 2 hours for 2 days. In another example, the boluses may be released every 3 hours for 1 month. In yet another example, the boluses may be released every 5 hours for 6 months. In a further example, the boluses may be released every 3 days for 2 years. In yet another example, a single bolus administration every 2 days for 1 week.

[0375] In some embodiments, at least 1 bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein is released (e.g., at least 2 boluses, e.g., at least 4 boluses, e.g., at least 8 boluses, e.g., at least 16 boluses, e.g., at least 25 boluses, e.g., at least 50 boluses, e.g., at least 100 boluses, e.g., at least 250 boluses, e.g., at least 500 boluses, e.g., at least 1000 boluses, e.g., at least 5000 boluses, e.g., at least 10,000 boluses, at least 25,000 boluses).

[0376] In some embodiments, the invention involves administration of a single bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, which is useful for treating any disorder described herein. In some embodiments, the invention involves administration of a single bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, which is useful as an ovulatory trigger during IVF. In some embodiments, the invention involves administration of a single bolus of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein for enhancing libido.

[0377] The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered subcutaneously, intravenously, or intramuscularly by programmable body pump to treat the disorder, e.g., a reproductive disorder, VMS, a metabolic disorder, a bone disorder, or a liver disorder. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered intranasally to treat the disorder, e.g., hypoactive sexual arousal disorder. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered by inhalation to treat the disorder, e.g., a respiratory disease. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered intrathecally to treat the disorder, e.g., a neurogenerative disorder or a sequela of a neurotropic virus.

[0378] 2. Continuous Administration

[0379] The administration of the therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be continuous, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein is consistently administered over an extended period.PATENT

[0380] Attorney Docket No.: 51796-005WO3

[0381] BWH Ref.: 2024-010

[0382] Continuous administration may be used in the treatment of disorders where antagonistic attenuation of KISS1 activation would be beneficial (e.g., a reproductive disorder (e.g., precocious puberty, sex hormone-dependent cancer, gender dysphoria, PCOS, endometriosis, uterine fibroid, or abnormal pregnancy), a metabolic disorder (e.g., diabetes, obesity, MASLD, or MASH), a liver disorder (e.g., MASLD or MASH), a bone disorder (e.g., osteoporosis), VMS, a neurodegenerative disorder (e.g., Alzheimer’s disease), or a sequela of a neurotropic virus). KISS1 R desensitizes and internalizes when the ligand is continuously bound to the receptor. Thus, continuous administration can effectively induce the inhibition (partial or total) of the gonadotropic axis.

[0383] In some embodiments, the therapeutically effective amount may be between about 0.05 pg / kg / h to about 250 ng / kg / h (e.g., between about 0.05 pg / kg / h to about 0.1 pg / kg / h, e.g., between about 0.1 pg / kg / h to about 0.5 pg / kg / h, e.g., between about 0.5 pg / kg / h to about 1 pg / kg / h, e.g., between about 1 pg / kg / h to about 5 pg / kg / h, e.g., between about 5 pg / kg / h to about 10 pg / kg / h, e.g., between about 10 pg / kg / h to about 50 pg / kg / h, e.g., between about 50 pg / kg / h to about 100 pg / kg / h, e.g., between about 100 pg / kg / h to about 500 pg / kg / h, e.g., between about 500 pg / kg / h to about 1 ng / kg / h, e.g., between about 1 ng / kg / h to about 5 ng / kg / h, e.g., between about 5 ng / kg / h to about 10 ng / kg / h, e.g., between about 10 ng / kg / h to about 50 ng / kg / h, e.g., between about 50 ng / kg / h to about 100 ng / kg / h, e.g., between about 100 ng / kg / h to about 250 ng / kg / h). In some embodiments, the therapeutically effective amount is between about 10 pg / kg / h to about 1 ng / kg / h.

[0384] The continuous administration may be over a period of at least about 2 hours (e.g., at least about 4 hours, e.g., at least about 8 hours, e.g., at least about 16 hours, e.g., at least about 24 hours, e.g., at least about 48 hours, e.g., at least about 72 hours, e.g., at least about 1 week, e.g., at least about 2 weeks, e.g., at least about 1 month, e.g., at least about 2 months, e.g., at least about 6 months, e.g., at least about 1 year, e.g., at least about 2 years). For example, the continuous administration may be over a period of about 16 hours. In yet another example, the continuous administration may be over a period of about 24 hours.

[0385] In some embodiments, a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered chronically, e.g., for a period of days, weeks, months, or years. The treatment can be continued until, and optionally stopped or reduced when, a desired outcome, e.g., fertility, or development of secondary sexual characteristics, has been achieved. In some embodiments, the treatment continues for months and years until desired effects are achieved. For example, for ovulation induction in women, a course could be anywhere from 1 month to 6 months. As another example, for spermatogenesis induction in men, a course could be anywhere from 3 to 24 months. As another example, for pubertal induction and / or maintenance of reproductive endocrine function, treatment could be for years. Sensitivity to kisspeptin increases with repetitive exposure; thus in some embodiments, the present methods can include repeated administration of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein to increase sensitivity to the point where endogenous kisspeptin secretion produces meaningful responses, to induce reversal of the kisspeptin resistance.PATENT

[0386] Attorney Docket No.: 51796-005WO3

[0387] BWH Ref.: 2024-010

[0388] The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered subcutaneously, intravenously, or intramuscularly by programmable body pump to treat the disorder, e.g., a reproductive disorder, VMS, a metabolic disorder, a liver disorder, or a bone disorder. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein may be administered intrathecally to treat the disorder, e.g., a neurogenerative disorder or a sequela of a neurotropic virus.

[0389] In some embodiments, pulsatile administration is so frequent as to mimic continuous administration. For example, when the frequency of pulsatile administration is less than about 20 minutes (e.g., about 2 minutes, about 4 minutes, about 6 minutes, about 8 minutes, about 10 minutes, about 12 minutes, about 14 minutes, about 16 minutes, about 18 minutes), KISS1 desensitizes.

[0390] VI. Diagnostics

[0391] 1. Methods of Diagnosing Congenital HH

[0392] Also provided herein are methods for identifying a subject as having, being at risk for, or having an attenuated form of, congenital HH (e.g., congenital Kallmann syndrome (anosmic / hyposmic)). These methods are useful for distinguishing between a normal prepubertal child (e.g., a child who has constitutional delayed puberty but will eventually undergo puberty on their own without intervention) from a child with congenital HH. Children with delayed puberty who later progress through puberty are often indistinguishable at first presentation from those who have congenital HH, and years of waiting may be needed before a given child’s outcome is known. As a result, patients, families, and providers can be faced with a conundrum when deciding between two common management approaches for delayed puberty: treatment with sex steroids or watchful waiting without intervention.

[0393] Being a provocative test, the diagnostic test provides a method to measure a child’s future potential for GnRH secretion. The kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein can elicit LH responses in children who will eventually progress through puberty at a time when they appear prepubertal on physical examination and daytime laboratory evaluation (e.g., a test to assess an individual’s capacity for GnRH secretion).

[0394] The methods comprise: (a) providing a reference level of LH; (b) administering a stimulating dose of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, wherein (i) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg (e.g., between about 0.05 pg / kg to about 0.1 pg / kg, e.g., between about 0.1 pg / kg to about 0.5 pg / kg, e.g., between about 0.5 pg / kg to about 1 pg / kg, e.g., between about 1 pg / kg to about 5 pg / kg, e.g., between about 5 pg / kg to about 10 pg / kg, e.g., between about 10 pg / kg to about 50 pg / kg, e.g., between about 50 pg / kg to about 100 pg / kg, e.g., between about 100 pg / kg to about 500 pg / kg, e.g., between about 500 pg / kg to about 1 ng / kg, e.g., between about 1 ng / kg to about 5 ng / kg, e.g., between about 5 ng / kg to about 10 ng / kg, e.g., between about 10 ng / kg to about 50 ng / kg, e.g., between about 50 ng / kg to about 90 ng / kg); or (ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5PATENT

[0395] Attorney Docket No.: 51796-005WO3

[0396] BWH Ref.: 2024-010

[0397] pg / kg to about 900 ng / kg (e.g., between about 0.5 pg / kg to about 1 pg / kg, e.g., between about 1 pg / kg to about 5 pg / kg, e.g., between about 5 pg / kg to about 10 pg / kg, e.g., between about 10 pg / kg to about 50 pg / kg, e.g., between about 50 pg / kg to about 100 pg / kg, e.g., between about 100 pg / kg to about 500 pg / kg, e.g., between about 500 pg / kg to about 1 ng / kg, e.g., between about 1 ng / kg to about 5 ng / kg, e.g., between about 5 ng / kg to about 10 ng / kg, e.g., between about 10 ng / kg to about 50 ng / kg, e.g., between about 50 ng / kg to about 100 ng / kg, e.g., between about 100 ng / kg to about 500 ng / kg, e.g., between about 500 ng / kg to about 900 ng / kg); (c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose; (d) comparing the reference level of LH to the level of LH in the subject after administration of the stimulating dose; and (e) identifying a subject with delayed puberty who has a level of LH after administration of the stimulating dose that is below or not different from the reference level of LH (e.g., less than 3.5, 3, 2.5, or 2 times the reference level) as having congenital HH.

[0398] In some embodiments, a stimulating dose of a kisspeptin or a pharmaceutically acceptable salt thereof described herein is administered to the subject.

[0399] In some embodiments, the reference level of LH may be a baseline level of LH that is measured in the subject prior to administration of the stimulating dose. In some embodiments, the reference level of LH may be an average level of LH in patients with congenital HH who have not been administered the stimulating dose. In some embodiments, the reference level of LH may be an average level of LH in patients that do not have congenital HH. In some embodiments, the reference level of LH is a normal or a low normal level of LH. In some embodiments, the reference level of LH may be between about 0.5 IU / L to about 110 IU / L. The absolute quantification of LH may be measured against an international standard.

[0400] In some embodiments, the method further comprises treating the subject with a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, wherein the treating is as described herein.

[0401] 2. Methods of Determining Patient Response to Therapy

[0402] Also provided herein are methods for determining whether a patient having a disorder is likely to respond to a therapy including a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0403] The methods comprise: (a) providing a reference level of LH; (b) administering to the subject a provocative dose of the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein is administered intravenously or subcutaneously; (c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose; (d) comparing the reference level of LH to the level of LH in the subject after administration of the provocative dose; and (e) identifying a subject who has a level of LH after administration of the provocative dose thatPATENT

[0404] Attorney Docket No.: 51796-005WO3

[0405] BWH Ref.: 2024-010

[0406] is greater than the reference level of LH (an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, or greater, compared to the reference level) as likely to respond to the therapy.

[0407] In some embodiments, a provocative dose of a kisspeptin or a pharmaceutically acceptable salt thereof described herein is administered to the subject.

[0408] In some embodiments, the reference level of LH may be a baseline level of LH that is measured in the subject prior to administration of the provocative dose. In some embodiments, the reference level of LH may be an average level of LH in patients with the disorder who have not been administered the provocative dose. In some embodiments, the reference level of LH may be an average level of LH in patients that do not have the disorder, in some embodiments, the reference level of LH is a normal or a low normal level of LH. In some embodiments, the reference level of LH may be between about 0.5 IU / L to about 110 IU / L. The absolute quantification of LH may be measured against an international standard.

[0409] In some embodiments, the provocative dose is administered intravenously at a dose of between about 1 pg / kg to about 1 pg / kg (e.g., between about 1 pg / kg to about 5 pg / kg, e.g., between about 5 pg / kg to about 10 pg / kg, e.g., between about 10 pg / kg to about 50 pg / kg, e.g., between about 50 pg / kg to about 100 pg / kg, e.g., between about 100 pg / kg to about 500 pg / kg, e.g., between about 500 pg / kg to about 1 ng / kg, e.g., between about 1 ng / kg to about 5 ng / kg, e.g., between about 5 ng / kg to about 10 ng / kg, e.g., between about 10 ng / kg to about 50 ng / kg, e.g., between about 50 ng / kg to about 100 ng / kg, e.g., between about 100 ng / kg to about 500 ng / kg, e.g., between about 500 ng / kg to about 1 pg / kg). In some embodiments, the provocative dose is administered subcutaneously at a dose of about 0.313 pg / kg.

[0410] In some embodiments, the provocative dose is administered subcutaneously at a dose of between about 10 pg / kg to about 10 pg / kg (e.g., between about 10 pg / kg to about 50 pg / kg, e.g., between about 50 pg / kg to about 100 pg / kg, e.g., between about 100 pg / kg to about 500 pg / kg, e.g., between about 500 pg / kg to about 1 ng / kg, e.g., between about 1 ng / kg to about 5 ng / kg, e.g., between about 5 ng / kg to about 10 ng / kg, e.g., between about 10 ng / kg to about 50 ng / kg, e.g., between about 50 ng / kg to about 100 ng / kg, e.g., between about 100 ng / kg to about 500 ng / kg, e.g., between about 500 ng / kg to about 1 pg / kg, e.g., between about 1 pg / kg to about 5 pg / kg, e.g., between about 5 pg / kg to about 10 pg / kg). In some embodiments, the provocative dose is administered intravenously at a dose of about 3.13 pg / kg.

[0411] In some embodiments, the provocative dose is administered intramuscularly. The disorder may be a reproductive disorder, hypoactive sexual arousal disorder, respiratory disease, metabolic disorder, liver disorder, bone disorder, VMS, neurodegenerative disorder, or a sequela of a neurotropic virus. The reproductive disorder may be a reproductive disorder (e.g., HH (e.g., congenital HH (e.g., congenital idiopathic HH (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic)) or acquired HH (e.g., acquired HH associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, hyperprolactinemia, analgesic or psychiatric medications, or substance abuse)).

[0412] In some embodiments, the method further comprises treating the subject with a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof, kisspeptin-10 or aPATENT

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[0415] pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, wherein the treating is as described herein.

[0416] EXAMPLES

[0417] The following examples are provided as a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and is not intended to limit the scope of what the inventors regard as their invention.

[0418] Table of Contents

[0419] Example 1 Mouse models and sample collection for Examples 2-7

[0420] Example 2 Testing doses of SEQ ID NO: 1

[0421] Example 3 Testing doses of SEQ ID NO: 1

[0422] Example 4 Testing doses of SEQ ID NO: 1

[0423] Example 5 Testing doses of SEQ ID NO: 1

[0424] Example 6 LH levels following kisspeptin or kisspeptin analog administration

[0425] Example 7 Twice Daily (BID) treatment of SEQ ID NO: 1 in PNA mice (model of PCOS) Example 8 Formulation development for SEQ ID NOs: 1 and 2

[0426] Example 9 Study design, formulation, dosing, and study duration for Examples 10-14 Example 10 Testing SEQ ID NO: 3 solvents

[0427] Example 11 Testing IPRECIO pumps

[0428] Example 12 Dose response in WT female mice

[0429] Example 13 Dose response in WT male mice

[0430] Example 14 Ovariectomized (OVX) mice study

[0431] Example 15 Materials and methods for Examples 16-18

[0432] Example 16 Dose response in Kissi KO female mice (IHH model)

[0433] Example 17 IHH males iPRECIO SEQ ID NO: 2

[0434] Example 18 Hypothalamic amenorrhea dose response SEQ ID NO: 2 WT female mice Example 19 Kisspeptin analog testing

[0435]

[0436] Example 1. Mouse models and sample collection

[0437] This Example provides materials and methods used in Examples 2-7.

[0438] Animals

[0439] Mice were housed at the Harvard Institutes of Medicine (HIM) animal facilty. All mice were maintained in constant conditions of light, 12 hr light / dark cycle, at standard temperature (22-24°C) and given ad libitum access to standard laboratory mice feed (5L0D; LabDiet) and water, unless mentioned otherwise. Kissi KO mice were generated by crossing heterocygote B6(129S4)-Kiss1tm1.1(cre / EGFP)Rpa / J mice, commonly named as KissCre: GFP (v2), obtained from The Jackson Laboratory.PATENT

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[0442] A mouse model of PCOS based on the prenatally androgenization (PNA) model was generated as previously described in McCarthy et al., J Clin Endocrinol Metab 107: e328-e347 (2022). Briefly, C57BL / 6 wild-type (WT) female mice were paired with adult WT males. Female mice were daily screening for vaginal plugs. The first day of gestation was considered to be the day after the plug was found.

[0443] Pregnant females were treated on days 16 through 18 of gestation with either DHT (250 pg / 100 pL in sesame oil, subcutaneously (sc)) to generate PNA mice or vehicle (sesame oil, Sigma) to generate the control group.

[0444] Sample collection

[0445] For mouse genotyping, ear samples were taken and subsequently analyzed by qPCR through the Transnetyx automated genotyping system. Reproductive tissues (i.e., ovaries and uterus) were weighed and dissected out, immediately upon the decapitation of the mice. Trunk blood was collected after decapitation to obtain serum for further hormonal assessments. Serum testosterone, follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH) were measured at the University of Virginia Ligand Assay Core with the Testosterone Mouse & Rat I BL ELISA (reportable average range 10-1600 ng / dl; sensitivity of 10 ng / dl), the Ultra-Sensitive MR FSH ELISA (reportable average range 0.016 - 8.0 ng / ml) and the AMH Mouse and Rat (ANSH) ELISA assays (reportable average range 3.3 - 210.0 ng / mL), respectively. Ovaries were included in 4% paraformaldehyde (PFA) to further be sectioned and stained with hematoxylin and eosin at the Harvard Histopathology core. Type of follicles, corpus lutea and cysts were counted in every ovary.

[0446] For hormone LH assays, blood samples were obtained from the mouse-tail at different timepoints along the experiment and / or after intraperitoneal (ip) or subcutaneous (sc) injections. Briefly, 4 pL of whole blood from mouse tail were diluted in 116 pL of phosphate buffered saline (PBS 1X, pH 7.4) with 0.05 % Tween 20, snap-frozen on dry ice and stored at -80°C until assay using a super-sensitive LH ELISA (Steyn et al., Endocrinology 154: 4939-4945 (2013)). Samples were assayed duplicated. For intra-and inter-assays variability, reference LH (0.25 ng / ml) was included in replicates, repeated within each assay and across separate assays, respectively.

[0447] For LH pulsatile secretion assessment, mice were previously habituated to be daily handled to later performing the tail-tip bleeding every in 8-min interval during 3 hours.

[0448] Assessment of pulsatile LH secretion

[0449] Basal and mean LH secretion as well as the number of pulses were measured. LH pulses were defined as those displaying an increase of 125% over basal LH values. Basal LH levels were determined as the average of the 6 lowest LH measurements from each mouse, based on previously validated protocols (McQuillan et ai., Endocrinology 160: 1480-1491 (2019)).

[0450] Statistical Analysis

[0451] All analyses were performed with GraphPad Prism Software. Data are expressed as the mean ± standard error of the mean (SEM) for each group. Unpaired Mann Whitney test and / or 1- or 2-wayPATENT

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[0454] ANOVA test followed by Tukey and / or Fisher’s post hoc test were used to assess variation among experimental groups, * p < 0.05; ** p < 0.01; p < 0.001; p < 0.0001.

[0455] Peptide Synthesis and Purification

[0456] All peptides were synthesized according to Lu etai. (1981) J. Org. Chem, 46: 3433. SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized and purified according to methods described in U. S. Patent No. US11807660B2 (see, e.g, Embodiments 2, 6, and 7 of US11807660B2), incorporated herein by reference. SEQ ID NO: 3 was synthesized using solid phase peptide synthesis (SPSS).

[0457] Example 2. Testing doses of SEQ ID NO: 1

[0458] Adult (6 month-old) WT male mice (n=5 / group) were subcutaneously injected with SEQ ID NO: 1 at 60 pmol or 12 pmol / 100 µL H2O (4 groups in total). Blood samples were collected 15 minutes prior to injection, and then at times 0 (right before the injection) 15, 30, 60, 90, 180, 360 and 600 minutes after sc injection. LH content was assessed as described in Example 1.

[0459] Results are shown in FIG. 1. Single doses of the kisspeptin analogs resulted in persistent activation of Kissi r demonstrated by LH responses lasting for several hours.

[0460] Example 3. Testing doses of SEQ ID NO: 1

[0461] Adult (7 month-old) WT male mice (n=5 / group) were subcutaneously injected with SEQ ID NO: 1 or SEQ ID NO: 3 at 1 pmol / 100 µL H2O (4 groups in total). Blood samples were collected at times 0, 15, 30, 180, 360 and 600 minutes after sc injection. LH content was assessed as described in Example 1.

[0462] Results are shown in FIG. 2. Single doses of the kisspeptin analogs resulted in persistent activation of Kissi r and durable LH responses.

[0463] Example 4. Testing doses of SEQ ID NO: 1

[0464] Adult (8 month-old) WT male mice (n=5 / group) were sc injected with SEQ ID NO: 1 or SEQ ID NO: 3 at 6 pmol / 100 µL H2O. Blood samples were collected at times 0, 15, 30, 180, 360, 600 and 720 minutes after sc injection. LH content were assessed as as described in Example 1.

[0465] Results are shown in FIG. 3. Single doses of these molecules resulted in persistent activation of Kissi rand durable LH responses.

[0466] Example 5. Testing doses of SEQ ID NO: 1

[0467] Adult (6 month-old) WT male mice (n=5 / group) were subcutaneously injected with SEQ ID NO: 1 or SEQ ID NO: 3 at 6 nmol in 100 µL H2O. Blood samples were collected 15 minutes prior to injection, and at times 0 (right before the injection) 15, 30, 60, 90, 180 and 360 minutes after sc injection. LH content was assessed as described in Example 1.

[0468] Results are shown in FIG. 4. Single doses of the kisspeptin analogs resulted in persistent activation of Kissi r and durable LH responses.PATENT

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[0470] BWH Ref.: 2024-010

[0471] Example 6. LH levels following kisspeptin or kisspeptin analog administration

[0472] Adult (6 month-old) WT male mice (n=9 / group) were intraperitoneally injected with SEQ ID NO: 3 (native kisspeptin-10), SEQ ID NO: 1, or SEQ ID NO: 2 at the dose of 6 nmol / 100 pL saline (FIG. 5A) or 0.6 nmol / 100 pL saline (FIG. 5B). Blood samples were obtained from the mouse-tail 30 and 15 minutes prior to the injection, then, at times 0 (right before the injection), 15, 30, 60 and 90 minutes after intraperitoneal kisspeptin or kisspeptin analog injection. LH content was assessed as described in Example 1.

[0473] Results are shown in FIGS. 5A-5B. Single doses of the kisspeptin analogs resulted in durable LH responses. At the doses used, SEQ ID NO: 1 and SEQ ID NO: 2 resulted in persistent activation of Kissi r.

[0474] Example 7. Twice daily (BID) treatment of SEQ ID NO: 1 in PNA mice (mode! of PCOS)

[0475] After weaning, post-natal day (PND)-21 control (n=10) and PNA (n=8) mice were confirmed for phenotypic markers of puberty onset on a daily basis. Pubertal development indices that were monitored included BW, age of vaginal opening (VO), considered an external marker of puberty in females and age of the first estrus, a marker of first ovulation in female mice. During adulthood, PND-90 control (n=8) and PNA (n=12) mice were monitored daily to assess threir estrous cyclicity for 2 weeks. Tail blood samples were collected during 3 hr to determine LH pulsatile secretion as detailed above. Before initiating the treament, PNA and control mice were monitored daily during 2 more weeks to assess their estrous cycles again. After that period, PNA mice were divided into 2 groups; one group (PNA + SEQ ID NO: 1; n=6) was given BID (mornings at 9.00 am and evenings at 5.00 pm) sc injections of SEQ ID NO: 1 at a dose of 12 pmol in 100 pL saline; and the second PNA group (PNA + vehicle, n=6) was BID treated with vehicle (100 pL saline). Control (n=8) mice were also BID treated with vehicle (veh). Blood samples were collected before and 15 minutes after the first bolus of the experiment (day 1) and before and after 15 minutes of the first bolus on days 3, 10, 20 and 30. after one month of treatment, tail blood sampling was performed on those mice to assess pulsatile LH secretion. Body weight measurements were monitored before the treatment and on days 10, 20 and 30 of the treatment. At the end of the experiment, control and PNA mice were euthanized. Trunk blood was collected and gonads were weighed and collected for further analysis as described in Example 1.

[0476] Results in FIG. 6A show reduced basal LH levels after BID peripheral administration of the long acting SEQ ID NO: 1 (12 pmol / 100pL saline sc) compared to PNA control. Results in FIG. 6B indicate the potential to treat the features of PCOS by attenuating Kissi r signaling.

[0477] Example 8. Formulation development for SEQ ID NOs: 1 and 2

[0478] Objective

[0479] SEQ ID NO: 1 and SEQ ID NO: 2 (the active pharmaceutical ingredients or the APIs) are small peptides containing chemically modified unnatural amino acids with a molecular weight of approximately in the range of 1200-1350 Daltons. This Example describes the development of a formulation for these APIs to prevent or minimize the loss of API due to adsorption to the containers, transfer apparatus, and drug delivery pump. The target is to achieve >90% recovery from the above mentioned devices. ThisPATENT

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[0481] BWH Ref.: 2024-010

[0482] Example also describes the improvement of the current analytical method, liquid chromatography mass spectrometry (LC-MS), to increase the sensitivity and limit of quantitation (LOQ).

[0483] Summary

[0484] Method verification: The Mass Spectrometry (MS) signal (signal to noise ratio) improved considerably with a higher ratio of acetonitrile (ACN) in sample dilution solution for analysis. As a result, 50% ACN in water was selected as the dilution solution before injection. With the 50% ACN in water as sample dilution solution, the limit of quantitation (LOQ) for SEQ ID NO: 1 and SEQ ID NO: 2 was 2 ng / mL.

[0485] Adsorption study: A new formulation was developed for SEQ ID NO: 1 and SEQ ID NO: 2 resulting in better recovery of API in plastic containers compared with glass viais. Furthermore, SEQ ID NO: 1 and SEQ ID NO: 2 demonstrated improved recovery in Type I Plus vials (2 mL medium borosilicate glass tubing coated film injection bottie) compared to Toplyo (2 mL medium borosilicate glass tubing coated film injection bottle) and Standline (2 mL medium borosilicate glass injection bottle) vials.

[0486] Formulation Screening:

[0487] Polysorbate 20 (PS20) or polysorbate 80 (PS80) and propylene glycol (PG) as excipients in the formulation were effective in preventing or considerably minimizing the adsorption of SEQ ID NO: 1 and SEQ ID NO: 2 to containers made of glass or plastic.

[0488] For SEQ ID NO: 1, 5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.05% PS80 + 5% PG was best performed in lead formulation (PT18), which showed no or minimum adsorption (recovery >90%) at 10 ng / mL at 40°C over a 2 week duration.

[0489] For SEQ ID NO: 2, 5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.05% PS80 + 5% PG was best performed in lead formulation (PT18) which showed no or minimum adsorption (recovery >90%) at 10 ng / mL at 40°C over a 2 week duration.

[0490] In-use study:

[0491] Plastic vial (Container): The recovery of 5 ng / mL SEQ ID NO: 1 and 20 ng / mL SEQ ID NO: 2 with lead formulation (PT30: 5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.1% PS80 + 5% PG) in pump was above 90% at 25°C over 24 hours (1 day).

[0492] Syringes (transfer device): The recovery of 5 ng / mL SEQ ID NO: 1 and 20 ng / mL SEQ ID NO: 2 with lead formulation (PT30: 5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.1% PS80 + 5%PG) in syringes was above 90% at 25°C over 24 hours (1 day).

[0493] Pump (drug delivery device): The recovery of SEQ ID NO: 1 (10 ng / mL) and SEQ ID NO: 2 (20 ng / mL) with lead formulation (PT30: 5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.1% PS80 + 5% PG) in pump was above 90% at 37°C over 28 days.

[0494] The lead formulation and drug product profile were as follows:

[0495] Table 1. SEQ ID NO: 1 formulation

[0496] Component Concentration Description SEQ ID NO: 1 10 ng / mL Active ingredient Mannitol 40% Excipient Polysorbate 80 (PS80) 0.1% Excipient

[0497]

[0498] PATENT

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[0501] Component Concentration Description Propylene glycol (PG) 5% Excipient

[0502] Sodium acetate tri hydrate 0.046% Buffer agent

[0503] Acetic Acid 0.010% Buffer agent

[0504] Pure water Quantum satis (q.s.) Solvent

[0505]

[0506] Table 2. SEQ ID NO: 2 formulation

[0507] Component Concentration Description SEQ ID NO: 2 20 ng / mL Active ingredient Mannitol 40% Excipient Polysorbate 80 (PS80) 0.1% Excipient

[0508] Propylene glycol (PG) 5% Excipient

[0509] Sodium acetate trihydrate 0.046% Buffer agent

[0510] Acetic Acid 0.010% Buffer agent

[0511] Pure water Quantum satis (q.s.) Solvent

[0512]

[0513] Materials and Methods

[0514] SEQ ID NO: 1 and SEQ ID NO: 2 (white powder) were stored at 4°C and protected from light. Reagents and excipients are provided in Table 3.

[0515] Table 3. Reagents and excipients.

[0516] Name Grade Vendor Lot No. Mannitol EP, BP, USP, JP Merck MP21016596 Polysorbate 80 (PS80) USP, EP, ChP Croda 0002173081 Polysorbate 20 (PS20) USP, JP, EP, ChP Croda 0002125303 Propylene glycol (PG) USP, EP, ChP Hedinger 232010 Sodium acetate tri hydrate Injectable Kirsch 2022094713 Acetic Acid Injectable Merck K54818200 L-Histidine EP, JP, USP Merck K52170752014 L-histidine monohydrochloride monohydrate EP / BP. JP Merck K55899454 Sodium hydroxide (NaOH) EP / BP, JP, NF Merck MB1962520307 Hydrochloric acid (HCI) EP, BP, JP, NF, ChP Merck Z0917965 Acetonitrile (ACN) >99.9% CNW D802094

[0517]

[0518] Analytical methods are provided in Tables 4 and 5.

[0519] Table 4. UPLC method-1

[0520] Instrument: Waters TQ-S of LC^

[0521]

[0522] PATENT

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[0524] BWH Ref.: 2024-010

[0525] ACQUITY UPLC® Peptide CSHTM C18, 2.1 x150mm, 1.7pm Column

[0526] (PN: 186006938)

[0527] Wavelength 220 nm

[0528] Column Temp. 35°C

[0529] Flow Rate 0.3 mL / min

[0530] Injection Volume 0.5 pL or 1pL

[0531] Mobile Phase A 0.1% TFA in H2O

[0532] Mobile Phase B 0.1% TFA in ACN

[0533] Diluent 50% ACN

[0534] HPLC

[0535] Time (min) A (%) B (%) 0.00 70 30

[0536] 1.00 70 30

[0537] 7.00 40 60 Gradient program

[0538] 7.10 5 95

[0539] 9.00 5 95

[0540] 9.10 70 30

[0541] 11.00 70 30

[0542] Run Time 11.0 min

[0543] Acquisition

[0544] Time 0 to 11.0 min Source ESI Polarity Positive MS-Tune Capillary (kV) 3.0 Source Temp 150 °C Mode

[0545] Desolvation Temp. 350 °C Desolvation Gas Flow (L / hr) 650 Cone Gas Flow(L / hr) 50 API Parent (M / Z) Daughter (M / Z) Cone (V) Collision (V) MS-MRM SEQ ID NO: 1 619.53 98.21 4 34 SEQ ID NO: 2 638.44 84.30 4 38

[0546]

[0547] Table 5. UPLC method-2

[0548] Instrument: Waters TQ-S of LC-MS (PDS-PF-LCMS-02)

[0549] ACQUITY™ PREMIER BEH C18, Column

[0550] 2.1 x50mm, 1.7pm (PN: 186009452) Wavelength 220 nm

[0551] HPLC

[0552] Column Temp. 35°C

[0553] Flow Rate 0.3 mL / min

[0554] Injection Volume 1 pL

[0555]

[0556] PATENT

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[0558] BWH Ref.: 2024-010

[0559] Mobile Phase A 0.1% TFA in H2O

[0560] Mobile Phase B 0.1% TFA in ACN

[0561] Diluent 50% ACN

[0562] Time (min) A {%) B {%) 0.00 70 30 0.30 70 30 Gradient 2.70 40 60 program 3.00 5 95

[0563] 3.50 5 95 3.80 70 30 4.50 70 30 Run Time 4.5 min

[0564] Acquisition

[0565] Time 0 to 4.5 min Source ESI Polarity Positive Capillary (kV) 3.0

[0566] Source Temp 150°C

[0567] MS-Tune

[0568] Desolvation

[0569] 350°C

[0570] Mode Temp.

[0571] Desolvation

[0572] 650

[0573] Gas Flow (L / hr)

[0574] Cone Gas Flow

[0575] 50

[0576] (L / hr)

[0577] API Parent (M / Z) Daughter (M / Z) Cone (V) Collision (V) MS-MRM SEQ ID NO: 1 619.53 98.21 4 34

[0578] SEQ ID NO: 2 638.44 84.30 4 38

[0579]

[0580] Preliminary method verification

[0581] Diluent screening

[0582] To improve the MS signal (sensitivity) of SEQ ID NO: 1 and SEQ ID NO: 2 at low concentration (ng / mL), different ratios of ACN in sample diluent was screened. SEQ ID NO: 1 and SEQ ID NO: 2 in 2.5%, 5%, 10%, 20%, 30% and 50% of ACN were tested at 100 ng / mL by LC-MS.

[0583] Results: The MS signal notably improved with a higher percent (%) of ACN (Table 6).

[0584] Conclusion: 50% ACN was selected as dilution for LC-MS injection.PATENT

[0585] Attorney Docket No.: 51796-005WO3

[0586] BWH Ref.: 2024-010

[0587] Table 6. Peak area of 100 ng / mL samples with different diluents Sample Peak Area

[0588] Diluent

[0589] concentration SEQ ID NO: 1 SEQ ID NO: 2

[0590] 2.5% AON NS NS

[0591] 5%ACN NS NS

[0592] 10%ACN 28.993 0.485

[0593] 100 ng / mL

[0594] 20%ACN 1624.968 422.225

[0595] 30%ACN 1960.255 1017.277

[0596] 50%ACN 2001.168 1134.585

[0597]

[0598] Note: NS = no signal was detected.

[0599] Preliminary method verification

[0600] A system suitability test, linearity and recovery test was performed on SEQ ID NO: 1 and SEQ ID NO: 2 solutions within the range of 2 ng / mL to 160 pg / mL.

[0601] Results: For SEQ ID NO: 1 and SEQ ID NO: 2, the LOQ was 2 ng / mL. System suitability (Table 7), linearity (Table 8) and recovery (Table 9) for all compounds were acceptable (FIG. 7A and FIG. 7B).

[0602] Conclusion: Based on the linearity (dynamic) range, for SEQ ID NO: 1 and SEQ ID NO: 2, concentration analysis can be performed reliably within the concentration range of 2 ng / mL to 160 pg / mL.

[0603] Table 7. RSD% results of SEQ ID NO: 1 and SEQ ID NO: 2

[0604] Peak Area

[0605] Item Cone. No.

[0606] SEQ ID NO: 1 SEQ ID NO: 2 LOQ_1 2 ng / mL 01 33.853 16.225

[0607] 02 24.453 15.606

[0608] 03 22.965 10.005

[0609] 04 30.018 18.164

[0610] 05 33.038 20.265 Average 28.8654 16.053 RSD% 17.1% 23.9%

[0611] S / N 26 13

[0612] LOQ_2 50 ng / mL 01 805.108 474.837

[0613] 02 780.944 442.425

[0614] 03 794.32 398.024

[0615] 04 744.888 534.146

[0616] 05 794.027 472.812 Average 783.8574 464.4488 RSD% 3.0% 10.7%

[0617] S / N 280 82

[0618]

[0619] Note: NS = no signal was detectedPATENT

[0620] Attorney Docket No.: 51796-005WO3

[0621] BWH Ref.: 2024-010

[0622] Table 8. Linearity resuits of SEQ ID NO: 1 and SEQ ID NO: 2

[0623] Peak Area

[0624] Item Cone.

[0625] SEQ ID NO: 1 SEQ ID NO: 2 2 ng / mL 28.865 16.053 Linearity-Low 5 ng / mL 92.884 49.949 concentration 10 ng / mL 168.121 92.207

[0626] 20 ng / mL 358.25 210.064 50 ng / mL 827.425 525.951 Slope 16.542 10.654 Intercept 7.270 -6.538 Correl 0.999 0.999 Linearity-Middle 0.1 pg / mL 1818.563 1098.506 concentration 0.2 pg / mL 3657.676 2199.017 0.5pg / mL 8889.642 5580.887 1 pg / mL 19872.549 12106.065 1.5 pg / mL 32872.781 20000.58 2 pg / mL 44698.91 27684.439 Slope 22721.260 14000.415 Intercept -1435.426 -922.118 Correl 0.998 0.998 Linearity-High 20 pg / mL 583083.688 370362.719 concentration 50 pg / mL 1461667.375 933345.375

[0627] 100 pg / mL 2792807.550 1792411.550 160 pg / mL 4033430.750 2629785.25 Slope 24613.578 16114.782 Intercept 187127.133 102006.728 Correl 0.997 0.998

[0628]

[0629] Table 9. Recovery results of SEQ ID NO: 1 and SEQ ID NO: 2

[0630] Sample Recovery (to liner-100 pg / ml) Item Cone.

[0631] SEQ ID NO: 1 SEQ ID NO: 2 Recovery- 1 pg / mL 1 96.09% 101.11% Middle 2 96.51% 100.80% concentration 3 102.27% 105.25% Average 98.29% 102.39% Recovery-High 100 1 88.80% 93.42% concentration pg / mL 2 91.79% 96.41%

[0632] 3 92.96% 98.76%

[0633]

[0634] PATENT

[0635] Attorney Docket No.: 51796-005WO3

[0636] BWH Ref.: 2024-010

[0637] Sample Recovery (to liner-100 pg / ml)

[0638] Item Cone.

[0639] SEQ ID NO: 1 SEQ ID NO: 2 Average 91.19% 96.19%

[0640]

[0641] Adsorption study

[0642] An adsorption study was conducted to select suitable consumables and containers for sample preparation and appropriate packaging materials.

[0643] For the absorption study during sample preparation, 1 mg / mL stock solution of SEQ ID NO: 1 and 1 mg / mL stock solution of SEQ ID NO: 2 were diluted with 50% AON to 2 ng / mL, 50 ng / mL, 1 pg / mL and 100 pg / mL solution. Each of the four solutions (100 pL each) were stored in a tube, vial, and pipette tip at 25°C. Recovery (as % of initial pre-load solution) was tested after 1 day.

[0644] For the absorption study during package and storage, 1 mg / mL stock solution of SEQ ID NO: 1 and 1 mg / mL stock solution of SEQ ID NO: 2 were diluted with pure water to 2 ng / mL, 50 ng / mL, 1 pg / mL and 100 pg / mL solution. The four solutions (0.15 mL for 100 pg / mL and 1.0 mL for rest of the concentrations) were stored in three types of vials (TopLyo, Type I plus and Standard line, see Table 10) at 25°C. Sample solutions from the vials were mixed with AON (1:1, v:v) before LC-MS injection.

[0645] Recovery (as % of pre-vial solution) was tested over 24 hours (after 1 day).

[0646] Table 10. Information of package material

[0647] Material Information Manufacturer / Cat No HPLC inner tube Plastic HPLC inner tube-250 pL Agilent / 5182-0549 Glass HPLC inner tube-250 pL Agiient / 5183-2085 Plastic 4 mL Polypropylene Centrifuge tube Zongyi / 45000218 Glass 2 mL medium borosilicate glass Agilent / 5191 -5611 TopLyo 2 mL medium borosilicate glass SCHOTT / 1579611 Vials

[0648] Type I plus tubing coated film injection bottle SCHOTT / 1678760

[0649] 2 mL medium borosilicate glass

[0650] Standard Line SCHOTT / 1519190 injection bottle

[0651] Pipette tips Plastic-200 pL Cellpro / 800201 Polypropylene

[0652] Plastic-1 mL KIRGEN / KG1313

[0653]

[0654] Results:

[0655] For HPLC inner tube, SEQ ID NO: 1 and SEQ ID NO: 2 at 2 ng / mL showed better recovery in plastic than glass. SEQ ID NO: 1 and SEQ ID NO: 2 at 50 ng / mL to 100 pg / mL showed good recovery (>98%) in both plastic and glass tube (Table 11).

[0656] For vials, SEQ ID NO: 1 at 2 ng / mL showed better recovery from plastic vials than glass vials. SEQ ID NO: 2 at 2 ng / mL showed similar recovery in plastic tube (76%) and glass tube (69%). All peptides at 50 ng / mL to 100 pg / mL showed good recovery in both plastic tube and glass tube (recovery% >95%).PATENT

[0657] Attorney Docket No.: 51796-005WO3

[0658] BWH Ref.: 2024-010

[0659] For pipette tips, SEQ ID NO: 1 and SEQ ID NO: 2 at 2 ng / mL was not detected. SEQ ID NO: 1 and SEQ ID NO: 2 at 50 ng / mL showed similar recovery in both plastic tube and glass tube (recovery% >99%). The peptides at 1 pg / mL and 100 pg / mL showed good recovery in 0.2 mL pipette tip and 1 mL pipette tip (Recovery%>90%).

[0660] For package, SEQ ID NO: 1 and SEQ ID NO: 2 showed the best recovery in Type I plus glass vial than TypoLyo vial or Standline vial.

[0661] Conclusion:

[0662] Based on the recovery data (Table 11 and Table 12) plastic tube, plastic vial and 1 mL plastic pipette tips were selected during sample preparation, and Type I plus glass vial was selected as primary package.

[0663] Table 11. Adsorption of SEQ ID NO: 1 and SEQ ID NO: 2 in materials at 25°C over one day duration

[0664] Recovery (%) at 25°C - 1 day Concentration Material

[0665] SEQ ID NO: 1 SEQ ID NO: 2 HPLC inner Plastic 81.6% 57.8%

[0666] tube Glass 58.7% 23.3%

[0667] 2 ng / mL Vial Plastic 90.3% 76.1%

[0668] Glass 57.5% 69.2% Pipette tips Plastic-200 pL NS NS

[0669] Plastic-1 mL NS NS

[0670] HPLC inner Plastic 109.3% 102.0%

[0671] tube Glass 105.5% 103.8%

[0672] 50 ng / mL Vial Plastic 101.2% 108.0%

[0673] Glass 100.6% 95.1% Pipette tips Plastic-200 pL 110.6% 99.3%

[0674] Plastic-1 mL 107.4% 115.6% HPLC inner Plastic 112.9% 110.2%

[0675] tube Glass 106.5% 106.0%

[0676] 1 pg / mL Vial Plastic 112.2% 110.6%

[0677] Glass 107.4% 105.5% Pipette tips Plastic-200 pL 114.3% 110.4%

[0678] Plastic-1 mL 118.8% 116.9% HPLC inner Plastic 109.8% 108.2%

[0679] tube Glass 107.2% 104.7%

[0680] 100 pg / mL

[0681] Vial Plastic 110.0% 107.4%

[0682] Glass 109.3% 106.0% Pipette tips Plastic-200 pL 111.0% 108.7%

[0683]

[0684] PATENT

[0685] Attorney Docket No.: 51796-005WO3

[0686] BWH Ref.: 2024-010

[0687] Recovery (%) at 25°C - 1 day Concentration Material

[0688] SEQ ID NO: 1 SEQ ID NO: 2 Plastic-1 mL 117.8% 117.7%

[0689]

[0690] Note: NS = no signal was detected.

[0691] Table 12. Adsorption of SEQ ID NO: 1 and SEQ ID NO: 2 in package at 25°C over one day duration Recovery (%) at 25°C - 1 day Concentration Material

[0692] SEQ ID NO: 1 SEQ ID NO: 2 Standard Line NS NS

[0693] 2 ng / mL

[0694] TopLyo NS NS

[0695] Type I plus NS NS

[0696] Standard Line 3.22% NS

[0697] 50 ng / mL

[0698] TopLyo 18.45% NS

[0699] Vial Type I plus 35.81% 19.89%

[0700] Standard Line 63.40% 38.54%

[0701] 1 pg / mL

[0702] TopLyo 90.81% 67.50%

[0703] Type I plus 88.94% 72.93% Standard Line 95.83% 89.95%

[0704] 100 pg / mL

[0705] TopLyo 96.74% 94.29%

[0706] Type I plus 99.48% 97.46%

[0707]

[0708] Note: NS = no signal was detected.

[0709] Formulation stability study

[0710] Formulation pre-stability test

[0711] 1 mg / mL stock solution of SEQ ID NO: 1 and 1 mg / mL stock solution of SEQ ID NO: 2 were diluted with formulation solutions to 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL sample solutions. Sample solutions (1.0 ml_) were stored at 60°C in Type I plus 2R vial. Recovery (as % of pre vial solution) was tested at one day (over 24 hours).

[0712] Results:

[0713] PS80 and PG were able to effectively prevent or considerably minimize the adsorption of SEQ ID NO: 1 (Table 13) and SEQ ID NO: 2 (Table 14) at 60°C over one day (24 hours). There was no considerable difference in recovery between pH 4.5 and pH 5.0 of SEQ ID NO: 1 and SEQ ID NO: 2 under 60°C after one day. 5mM sodium-acetate was better than 5mM Histidine at recovery of SEQ ID NO: 1 and SEQ ID NO: 2 under 60°C after one day.

[0714] Conclusion:

[0715] The combination of PS80 and PG was selected for the following formal formulation stability study.PATENT

[0716] Attorney Docket No.: 51796-005WO3

[0717] BWH Ref.: 2024-010

[0718] Table 13. Stability of SEQ ID NO: 1 in PT01 to PT10 at 60°C over one day duration Formulation Composition Recovery % of SEQ ID NO: 1 No. PT 10 20 50 100 200 pH Buffer Excipients

[0719] No. ng / mL ng / mL ng / mL ng / mL ng / mL pH

[0720] 01 PT01 5.2% NS 2.8% 3.0% 4.8%

[0721] 4.5

[0722] 5mM 40 mg / mL mannitol

[0723] pH

[0724] 02 PT02 sodium2.9% 5.0% 1.3% 2.7% 0.0%

[0725] 5.0

[0726] acetate

[0727] pH 40 mg / mL mannitol

[0728] 03 PT03 75.7% 59.1% 103.7% 98.8% 98.2%

[0729] 5.0 +0.05% PS80

[0730] pH 5mM

[0731] 04 PT04 78.0% 59.2% 88.3% 75.4% 84.8%

[0732] 5.0 Histidine

[0733] pH

[0734] 05 PT05 80.0% 42.0% 107.3% 95.9% 102.1%

[0735] 4.5 40 mg / mL mannitol

[0736] pH +0.02% PS80

[0737] 06 PT06 71.7% 91.4% 98.8% 78.3% 92.8%

[0738] 5.0

[0739] pH

[0740] 07 PT07 5mM 72.6% 76.3% 102.2% 91.6% 100.1%

[0741] 5.5

[0742] sodium- pH 40 mg / mL mannitol

[0743] 08 PT08 acetate 83.0% 104.9% 110.4% 99.0% 103.9%

[0744] 5.0 +0.02%PS20+5%PG

[0745] pH 40 mg / mL mannitol

[0746] 09 PT09 92.9% 112.4% 112.6% 112.4% 111.0%

[0747] 5.0 +0.02%PS80+5%PG

[0748] pH 40 mg / mL

[0749] 10 PT10 14.4% 17.8% 30.6% 29.0% 51.5%

[0750] 5.0 mannitol+5%PG

[0751] 5mM

[0752] pH 40 mg / mL

[0753] 11 PT11 sodium24.9% 40.4% 46.3% 55.8% 51.2%

[0754] 5.0 mannitol+10%PG

[0755] acetate

[0756]

[0757] Note: NS = no signal was detected.

[0758] Table 14. Stability of SEQ ID NO: 2 in PT01 to PT10 at 60°C over one day duration Formulation Composition Recovery % of SEQ ID NO: 2 No. PT 10 20 50 100 200 pH Buffer Excipients

[0759] No. ng / mL ng / mL ng / mL ng / mL ng / mL pH NS NS 0.6% NS 0.2% 01 PT01

[0760] 4.5

[0761] 5mM 40 mg / mL mannitol

[0762] pH NS NS NS 0.2% NS 02 PT02 sodium5.0

[0763] acetate

[0764] pH 40 mg / mL mannitol 76.5% 46.8% 69.9% 75.2% 72.9% 03 PT03

[0765] 5.0 +0.05% PS80

[0766]

[0767] PATENT

[0768] Attorney Docket No.: 51796-005WO3

[0769] BWH Ref.: 2024-010

[0770] Formulation Composition Recovery % of SEQ ID NO: 2 No. PT 10 20 50 100 200 pH Buffer Excipients

[0771] No. ng / mL ng / mL ng / mL ng / mL ng / mL pH 5mM 62.3% 45.9% 56.6% 54.4% 63.6% 04 PT04

[0772] 5.0 Histidine

[0773] pH 53.6% 22.9% 68.1% 67.9% 73.5% 05 PT05

[0774] 4.5 40 mg / mL mannitol

[0775] pH +0.02% PS80 39.8% 73.5% 52.7% 58.5% 67.1% 06 PT06

[0776] 5.0

[0777] pH 69.1% 44.1% 69.2% 72.3% 71.5% 07 PT07 5mM

[0778] 5.5

[0779] sodium- pH 40 mg / mL mannitol 56.7% 61.7% 81.1% 77.9% 84.0% 08 PT08 acetate

[0780] 5.0 +0.02%PS20+5%PG

[0781] pH 40 mg / mL mannitol 70.9% 67.0% 75.1% 78.9% 86.5% 09 PT09

[0782] 5.0 +0.02%PS80+5%PG

[0783] pH 40 mg / mL 4.4% 1.1% 8.3% 11.9% 18.1% 10 PT10

[0784] 5.0 mannitol+5%PG

[0785] 5mM 3.9% 6.1% 11.6% 17.3% 20.6% pH 40 mg / mL

[0786] 11 PT11 sodium5.0 mannitol+10%PG

[0787] acetate

[0788]

[0789] Note: NS = no signal was detected.

[0790] Formulation stability testing

[0791] 1 mg / mL stock solution of SEQ ID NO: 1 and 1 mg / mL stock solution of SEQ ID NO: 2 were diluted with formulation solution to 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL and 1 pg / mL sample solutions. Recovery % was tested at initial, 1 week, and 2 weeks.

[0792] Results:

[0793] SEQ ID NO: 1 (Table 15) at -20°C over 2 weeks showed good recovery (>95%) with PT09 and PT12 to PT20. At 40°C over 2 weeks, 10 ng / mL SEQ ID NO: 1 showed good recovery (>80%) with PT09 and PT12 to PT20, except for formulation containing Histidine buffer (recovery of PT14: 76%). 50 ng / mL and 1 pg / mL SEQ ID NO: 1 showed good recovery (>95%) with PT09 and PT12 to PT20 at 40°C over 2 weeks. There was no considerable difference among pH 4.5, pH 5.0 and pH 5.5 with SEQ ID NO: 1.

[0794] SEQ ID NO: 2 (Table 16) at -20°C over 2 weeks showed good recovery (>80%) with PT09 and PT15 to PT20. At 40°C over 2 weeks, 10 ng / mL SEQ ID NO: 2 showed good recovery (>90%) with PT12, PT13, PT15 and PT18. SEQ ID NO: 2 had compatibility issues with histidine (PT14), and showed better recovery results at pH 5.0 than pH 4.5 or pH 5.5.

[0795] Conclusion:

[0796] PT18 (5 mM acetate buffer, pH 5.0, 40 mg / mL mannitol + 0.05% PS80+5% PG) was recommended as the lead formulation for SEQ ID NO: 1 and SEQ ID NO: 2.Table 15. Stability of SEQ ID NO: 1 in different formulations

[0797] Recovery% of SEQ ID NO: 1

[0798] Formulation Composition

[0799] Initial -20°C 1W 40°C 1W 40°C 2W No 1 1 PT 10 50 1 10 50 1 10 50 10 50

[0800] pH buffer Excipients pg / mL pg / mL No. ng / mL ng / mL pg / mL ng / mL ng / mL pg / mL ng / mL ng / mL ng / mL ng / mL

[0801] PT 103.2 102.3 104.2 118.0 100.2 103.1 108.3 01 5.0 97.6% 108.3% 118.5% 107.6% 101.7%

[0802] 09 40 mg / mL % % % % % % %

[0803] 5mM

[0804] PT mannitol 105.5 113.4 103.5 109.6 02 4.5 sodium96.5% 99.5% 98.2% 110.9% 108.7% 98.1% 96.5% 114.9%

[0805] 12 +0.02%PS80+ % % % % acetate

[0806] PT 5% PG 101.3 102.0 110.8 100.4 108.1 101.9 03 03 98.6% 102.9% 103.3% 106.8% 94.3% 100.4%

[0807] 13 % % % % % %

[0808] 40 mg / mL

[0809] PT 5mM mannitol 100.6 103.3 100.0 04 5.0 86.9% 108.5% 95.4% 103.4% 110.8% 69.0% 94.1% 76.3% 99.2%

[0810] 14 Histidine +0.02%PS80+ % % %

[0811] 5% PG

[0812] PT 40 mg / mL 106.0 106.6 111.2 117.2 113.4 05 4.5 87.7% 101.2% 99.6% 113.0% 110.5% 88.5% 110.0%

[0813] 15 mannitol % % % % % PT +0.02%PS80+ 105.8 102.4 101.7 114.0 110.9 06 5.0 5mM 89.9% 100.8% 99.5% 110.4% 104.4% 81.6% 104.3%

[0814] 16 10% PG % % % % % sodium- PT 40 mg / mL 104.9 109.6 113.5 106.5 112.3 07 4.5 acetate 91.2% 105.3% TBD 111.3% 111.2% 89.0% 116.1%

[0815] 17 mannitol % % % % % PT +0.05%PS80+ 104.5 103.0 118.2 101.1 113.6 113.6 08 5.0 101.3% 104.3% 108.1% 109.2% 89.0% 113.3%

[0816] 18 5% PG % % % % % %

[0817]

[0818] Recovery% of SEQ ID NO: 1

[0819] Formulation Composition

[0820] Initial -20°C 1W 40°C 1W 40°C 2W No 1 1 PT 10 50 1 10 50 1 10 50 10 50

[0821] pH buffer Excipients pg / mL pg / mL No. ng / mL ng / mL pg / mL ng / mL ng / mL pg / mL ng / mL ng / mL ng / mL ng / mL

[0822] PT 40 mg / mL 107.1 111.3 107.1 115.8 09 4.5 87.1% 101.7% 109.3% 112.4% 97.5% 98.2% 99.8% 101.6%

[0823] 19 mannitol % % % % PT +0.05%PS20+ 104.2 114.8 104.9 100.6 110.6 118.4 114.0 10 5.0 106.0% 108.8% 108.8% 114.3% 114.5%

[0824] 20 5% PG % % % % % % %

[0825]

[0826] Table 16. Stability of SEQ ID NO: 2 in different formulations

[0827] % Recovery of SEQ ID NO: 2

[0828] Formulation Composition

[0829] initial -20°C 1W 40°C 1W 40°C 2W

[0830] No 1 1 PT 10 50 1 10 50 1 10 50 10 50

[0831] pH buffer Excipients pg / mL pg / mL No. ng / mL ng / mL pg / mL ng / mL ng / mL pg / mL ng / mL ng / mL ng / mL ng / mL

[0832] PT

[0833] 01 5.0 40 mg / mL 83.6% 85.4% 97.9% 102.4% 103.5% 98.8% 102.5% 98.4% 97.2% TBD 102.6% 94.2% 09

[0834] 5mM mannitol

[0835] PT

[0836] 02 4.5 sodium+0.02% 98.0% 82.9% 97.1% 86.6% 102.7% 101.3% 109.0% 99.6% 99.1% 100.3% 107.7% 95.3% 12

[0837] acetate PS80+5%

[0838] PT

[0839] 03 03 PG 90.2% 79.7% 91.6% 76.4% 84.4% 97.9% 96.3% 71.6% 54.7% 107.8% 71.1% 35.1% 13

[0840] PT 5mM 40 mg / mL

[0841] 04 5.0 47.1% 86.8% 93.4% 56.1% 100.9% 101.2% 8.9% 69.9% 95.3% 2.7% 86.7% 75.6% 14 Histidine mannitol

[0842]

[0843] PATENT

[0844] Atorney Docket No.: 51796-005WO3

[0845] BWH Ref.: 2024-010

[0846] % Rf SEQ ID NO 2ecovery o:

[0847] Fli Ciittormuaonomposon

[0848] Iiiltna

[0849] N 1o

[0850] 50 10 50 10 1 10 1 50 10

[0851] / L Eii bffHt pgmxcpensuer p

[0852] / L / L / L / L / L / L / L / L / L ngm ngm ngm ngm pgm ngm pgm ngm ngm

[0853] 002% +.

[0854] PS805%+

[0855] PG

[0856] 1065 1105 40 / L mgm..

[0857] 1097% 930% 927% 992% 1029% 1023% 837% 985% 894% 902% 45 05...........

[0858] % %ilt manno

[0859] 002% +.

[0860] 1006 1129..

[0861] PS8010% 967% 842% 916% 909% 989% 1037% 1208% 975% 830% 938% 50 06+...........

[0862] % %

[0863] PG

[0864] 1071 1032 PT..

[0865] 1170% 843% 930% 924% 1036% 1042% 1204% 1006% 915% 592% 45 07...........

[0866] % % 5M 17m

[0867] di soum1077 1003 1035...

[0868] tt 982% 1117% 1144% 1037% 1075% 1262% 1006% 954% 774% 50 08 aceae..........

[0869] § % % %

[0870] o

[0871] © 1080 1060..

[0872] s 776% 764% 850% 999% 1069% 994% 1038% 986% 917% 872% 45 09...........

[0873] % %

[0874] | / iu6u ■ 1043 1093.. j / iuBrl - 1002% 829% 977% 1072% 1048% 970% 857% 739% 50......... OS % % MZ OoOfr Mt OoOfr

[0875] r~ _ '-p -J _ t: o o sP-- m ° E £

[0876] i s a £ CT C £ + 0 k W o «>Q" § d 8 °- o xt E “ + Q_ §E +£

[0877] H in H co H o> H o t S' Q. v- CL CL v- CL CL Od o V”

[0878]

[0879] PATENT Attorney Docket No.: 51796-003001

[0880] BWH Ref.: 2024-010 In-use study

[0881] Plastic vial compatibility study

[0882] A stock solution of SEQ ID NO: 1 and SEQ ID NO: 2 at 1 mg / mL were diluted with formulation solutions of PT30 to 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL concentrations. Sample solutions were stored at 25°C in plastic vial. Recovery % was tested at 1 hour and 24 hours.

[0883] Results: The recovery of SEQ ID NO: 1 and SEQ ID NO: 2 with PT30 formulation was more than 90% at 25°C over 1 day (Table 17).

[0884] Conclusion: Plastic vial is recommended for sample preparation.

[0885] Table 17. Recovery% of SEQ ID NO: 1 and SEQ ID NO: 2 with lead formulation in plastic vial Formulation Composition Recovery (%)

[0886] Filling API at 25° C No.

[0887] Volume

[0888] PT concentration

[0889] API pH buffer Vehicle 1h 24h No

[0890] 40 mg / mL 5 ng / mL 100.26 104.31 SEQ ID 5mM

[0891] 1 PT30 pH 5.0 mannitol

[0892] NO: 1 sodium+0.1% PS80+5%

[0893] acetate 1.0 mL 10 ng / mL 103.12 99.18

[0894] PG

[0895] (Plastic

[0896] 40 mg / mL vials) 20 ng / mL 99.81 107.08 SEQ ID 5mM

[0897] 2 PT30 pH 5.0 mannitol

[0898] NO: 2 sodium+0.1% PS80+5%

[0899] acetate 50 ng / mL 112.33 106.55

[0900] PG

[0901]

[0902] Syringe compatibility study

[0903] A stock solution of SEQ ID NO: 1 and SEQ ID NO: 2 at 1 mg / mL were diluted with vehicle (formulation) solution of PT30 to 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL solution. Sample solutions were stored at 25°C in both BD syringes and Kangcheng syringes. Recovery % was tested at initial (TO), and over 30 min, 1 hour, 4 hours and 24 hours duration.

[0904] Results: The recovery of SEQ ID NO: 1 and SEQ ID NO: 2 with PT30 formulation was above 90%, compared to presyringe solution, in both of BD (Cat No. 300841) and Kangcheng syringe (Cat No.

[0905] 20163140236) over 1 day duration (Table 18).

[0906] Conclusion: The selected formulation has no considerable loss of API due to adsorption to either brand of syringes. Recommend using 10 ng / mL SEQ ID NO: 1 and 20 ng / mL SEQ ID NO: 2 for the pump compatibility test.Table 18. Recovery % of SEQ ID NO: 1 and SEQ ID NO: 2 with lead formulation in syringes

[0907] Formulation Composition Recovery (%)

[0908] Filling API

[0909] PT

[0910] No. Volume Condition concentration

[0911] API pH buffer Vehicle Syringe type

[0912] No TO 30min 1h 4h 1day BD 89.04 105.66 101.18 107.97 101.92 40 mg / mL

[0913] pH 5 ng / mL Kangcheng 110.14 106.43 105.73 101.30 108.96 1 PT30 mannitol

[0914] SEQ ID 5.0

[0915] +0.1% PS80+ BD 102.75 111.37 111.18 107.85 99.22 NO: 1 5mM 5% PG 10 ng / mL Kangcheng 105.89 92.76 99.89 97.36 111.05 sodium- 1 mL 25°C

[0916] acetate BD 104.77 95.17 105.88 96.68 96.68

[0917] 40 mg / mL

[0918] SEQ ID pH 20 ng / mL Kangcheng 108.09 93.58 110.75 100.48 102.94 2 PT30 mannitol

[0919] NO: 2 5.0

[0920] +0.1% PS80+ BD 94.63 100.74 103.00 102.14 105.84 5% PG 50 ng / mL Kangcheng 103.47 98.19 97.66 107.16 115.68

[0921]

[0922] PATENT

[0923] Attorney Docket No.: 51796-003001

[0924] BWH Ref.: 2024-010

[0925] Delivery pump compatibility study

[0926] A stock solution of SEQ ID NO: 1 or SEQ ID NO: 2 at 1 mg / mL was diluted with formulation solution of PT30 to prepare sample solution containing 10 ng / mL SEQ ID NO: 1 and 20 ng / mL SEQ ID NO: 2. Each sample solution was injected into pumps by BD syringe and stored at 37°C. Recovery % was tested in a plastic vial at initial timepoint under 25°C and in an Alzet osmotic delivery pump at a rate of 0.11 pL / hr (Model 1004) at initial (TO), 1 day, 7 day, 14 day and 28 day duration at 37°C.

[0927] Results: The recovery of 10 ng / mL SEQ ID NO: 1 and 20 ng / mL SEQ ID NO: 2 with PT30 formulation was above 90%, compared to initial timepoint, in delivery pump over 28 day duration (Table 19).

[0928] Conclusion: There was no considerable adsorption of APIs to the delivery pump for SEQ ID NO: 1 and SEQ ID NO: 2 in PT30 formulation.Table 19. Recovery% of SEQ ID NO: 1 and SEQ ID NO: 2 with lead formulation in delivery pump

[0929] Formulation Composition Cone. Recovery(%) at 37°C No. Filling volume Condition

[0930] PT No API pH buffer Vehicle ng / ml TO in vial TO in pump 1d 7d 14d 28d 33d 1 SEQ ID 10 119.36 96.81 112.19 108.28 115.50 119.29 N / A NO: 1 5mM sodiumPT30 pH 5.0 40 mg / mL mannitol 0.1 mL 37°C

[0931] 2 SEQ ID acetate

[0932] +0.1%PS80+5%PG 20 105.02 99.36 100.69 105.70 110.45 113.03 N / A NO: 2

[0933]

[0934] PATENT

[0935] Attorney Docket No.: 51796-005WO3

[0936] BWH Ref.: 2024-010-02

[0937] Example 9. Study design, formulation, dosing, and study duration

[0938] This Example provides materials and methods used in Examples 10-14.

[0939] Peptide Synthesis and Purification

[0940] All peptides were synthesized according to Lu et al. (1981) J. Org. Chem, 46: 3433. SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized and purified according to methods described in U. S. Patent No. US11807660B2 (see, e.g, Embodiments 2, 6, and 7 of US11807660B2), incorporated herein by reference. SEQ ID NO: 3 was synthesized using solid phase peptide synthesis (SPSS).

[0941] Ethics Statement. All procedures were approved by the Harvard Medical Area Standing Committee on the Use of Animals in Research and Teaching in the Harvard Medical School Center for Animal Resources and Comparative Medicine.

[0942] Animals. Mice were housed at the Harvard Institutes of Medicine (HIM) animal facility. All mice were maintained in constant conditions of light, 12 hr light / dark cycle, at standard temperature (22-24°C) and given ad libitum access to standard laboratory mice (5L0D; LabDiet) and water, unless mentioned otherwise. Kiss1 KO mice were generated by crossing heterozygote B6(129S4)-Kiss1tm1.1(cre / EGFP)Rpa / J mice, commonly named as KissCre:GFP(v2), obtained from The Jackson Laboratory.

[0943] Sample collection. For mouse genotyping, ear samples were taken and subsequently analyzed by qPCR through the Transnetyx automated genotyping system. For LH pulsatile secretion assessment, mice were previously habituated to be daily handled to later performing the tail-tip bleeding every 10-min interval during 2 hours. Blood samples were obtained from the mouse-tail at different timepoints along the experiment and / or after subcutaneous (sc) injections. Briefly, 4 pL of whole blood from mouse tail were diluted in 116 pL of phosphate buffered saline (PBS 1X, pH 7.4) with 0.05 % Tween 20, snap-frozen on dry ice and stored at -80°C until assay using a super-sensitive LH ELISA (Steyn et al. Endocrinology. 2013 Dec;154(12):4939-45). Samples were assayed duplicated. For intra- and inter-assays variability we included reference LH (0.25 ng / ml) in replicates, repeated within each assay and across separate assays, respectively. Values of LH were normalized using the reference LH (0.25 ng / ml) across assays performed on the same day. Any data point below the lowest measurable concentration on the standard curve (the limit of detection) was included as being at the lowest detectable value for that assay.

[0944] Assessment of pulsatile LH secretion. Mean LH secretion, the number of pulses and the amplitude of LH pulses were measured. LH pulses were determined by using the reformulated PULSAR Otago platform (pulsar.otago.ac.nz / pulsar / ) created to analyze pulsatile LH secretion (Porteous et al. Endocrinology. 2021 Nov 1; 162(11 ):bqab165). The parameter settings for ovariectomized mice were the following: smoothing 0.7, peak split 2.5, level of detection 0.04, amplitude distance 3, and assay variability 0, 2.5, and 3.3. The G values were G (1) 3.5, G (2) 2.6, G (3) 1.9, G (4) 1.5 and G (5) 1.2, based on previous reference article (Porteous et al. Endocrinology. 2021 Nov 1; 162(11 ):bqab165).

[0945] ELISA PK studies. Adult C57BL / 6 male mice were subcutaneously (sc) and / or intraperitoneally (ip) injected with SEQ ID NO: 2 and serum was collected at different timepoints (i.e., 1-, 5- and 15 min; 1-,PATENT

[0946] Attorney Docket No.: 51796-005WO3

[0947] BWH Ref.: 2024-010-02

[0948] 2-, 3-, 5-, 8- and 24 hours after sc and / or ip administration) in an external facility. Serum samples were diluted (1:20) in phosphate buffered saline (PBS 1X, pH 7.4) with 0.05 % Tween 20 to assess the LH content as previously described.

[0949] In Example 10 (testing SEQ ID NO: 3 solvents), adult (4 to 5 month-old) C57BL / 6 male mice were subcutaneously injected with SEQ ID NO: 3 for 1 day at the dose of 0.26 mg / kg in either water (n=9), saline (n=9) and / or Na-acetate 40 mg / mL mannitol (n=8). Blood samples were collected prior to the injection (baseline), and at times 15, 30, 60 and 90 minutes after sc SEQ ID NO: 3 peptide injection. LH content were assessed as previously detailed in Sample Collection section.

[0950] In Example 11 (testing iPRECIO pumps), non-GnRH primed adult (4 month old) Kiss1 KO malemice (n=3 (n=1 control iPRECIO no activated-, n=2 SEQ ID NO: 3 treated)) were subcutaneously implanted with iPRECIO pumps (SMP-310R), previously filled with SEQ ID NO: 3 at 0.26 mg / kg (8 pL / hour) in sterile water. Mice 5651 and 5620 were treated with SEQ ID NO: 3 and mouse 5654 was given the control. The duration of the study was 5 days, with pulses every 1 hour (740pL total at 8 pL / pulse), and dosing PD measured on day 5. LH pulsatile secretion assessment was performed for 2 hours before and after 3.6 days of iPRECIO pump implantation, as previously described in Sample Collection section. Once euthanized, testicles were dissected out and weighed. Body weight was monitored throughout the experiment.

[0951] In Example 12 (Dose response in WT female mice), gonad intact adult (3 month-old) C57BL / 6 female mice (n=7-10 / group) at metestrus / diestrus phases were sc treated with either SEQ ID NO: 1 or SEQ ID NO: 2 at a high, medium and / or low dose. The doses used for SEQ ID NO: 1 were: 0.767 pg / kg (high), 0.153 pg / kg (medium) and 0.031 pg / kg (low), in 100 pL of PT30 solution. For SEQ ID NO: 2, the doses were: 0.817 pg / kg (high), 0.163 pg / kg (medium) and 0.033 pg / kg (low), in 100 pLof PT30 solution. Blood samples were collected before the sc administration (baseline) and at 15, 30, 60, 90, 180, 360 and 600 minutes after sc peptide administration. LH content was assessed as previously detailed in Sample Collection section.

[0952] In Example 13 (Dose response in WT male mice), adult (6-8 month-old) C57BL / 6 male mice (n=9 / group) were sc treated with either SEQ ID NO: 1 or SEQ ID NO: 2 at a high, medium and low doses. The doses used for SEQ ID NO: 1 were: 0.639 pg / kg (high), 0.128 pg / kg (medium) and 0.026 pg / kg (low), in 100 pL of PT30 solution. For SEQ ID NO: 2, the doses were: 0.681 pg / kg (high), 0.136 pg / kg (medium) and 0.027 pg / kg (low), in 100 pL of PT30 solution. Blood samples were collected before the sc administration (baseline) and at 15, 30, 60, 90, 180, 360 and 600 minutes after sc peptide administration. LH content was assessed as previously detailed in Sample Collection section.

[0953] In Example 14 (OVX study), adult (3 month-old) C57BL / 6 female mice (n=10 / group) were ovariectomized (OVX) followed by sc implantation of Alzet model 1004 pump (continuous infusion, 100 pL at 0.11 pL / hour release) filled with the SEQ ID NO: 1 peptide at the dose of 28 pg / ml in PT30 solution. Alzet pump implantation were performed for 4 weeks, starting 7 days upon OVX. Blood samples were collected before and 1 week post OVX, and 1- and 2 days after Alzet pumps were implanted to further measurements of the LH content. For LH pulsatile assessment, blood samples were collected for 2 hoursPATENT

[0954] Attorney Docket No.: 51796-005WO3

[0955] BWH Ref.: 2024-010-02

[0956] with a 10 min-interval between sampling on day 7 and 14 after Alzet model 1004 pumps were implanted. Control pumps were filled with PT30 solution. Body weight was monitored throughout the experiment.

[0957] Statistical Analysis. All analyses were performed with GraphPad Prism Software. Data are expressed as the mean ± standard error of the mean (SEM) for each group. Unpaired Mann Whitney test and / or 1- or 2-way ANOVA test followed by Tukey, Dunnett and / or Fisher's post hoc test were used to assess variation among experimental groups. Significance level was set at P < 0.05.

[0958] Example 10. Testing SEQ ID NO: 3 solvents

[0959] Experiments were performed according to the materials and methods described in Example 9. Results are shown in FIGS. 8A-8B. SEQ ID NO: 3 induced a greater LH response when formulated with water compared to saline or a Mannitol solution.

[0960] Example 11. Testing iPRECIO pumps

[0961] Experiments were performed according to the materials and methods described in Example 9. Results are shown in FIGS. 9A-9C and 10A-10D. SEQ ID NO: 3 induced elevations of LH and increase in testes mass in Kiss1 KO IHH model animals that received up to 4 days of pulsatile doses.

[0962] Example 12. Dose response in WT female mice

[0963] Experiments were performed according to the materials and methods described in Example 9. Results are shown in FIGS. 11A-11B.

[0964] Dose response studies in adult WT female mice revealed a powerful, dose-related, stimulatory effect on luteinizing hormone (LH) secretion after the subcutaneous (sc) administration of SEQ ID NO: 1 and SEQ ID NO: 2.

[0965] Example 13. Dose response in WT male mice

[0966] Experiments were performed according to the materials and methods described in Example 9. Results are shown in FIGS. 12A-12B.

[0967] Dose response studies in adult WT male mice revealed a powerful, dose-related, stimulatory effect on luteinizing hormone (LH) secretion after the subcutaneous (sc) administration of SEQ ID NO: 1 and SEQ ID NO: 2. The LH response from SC dosing of SEQ ID NO: 1 was more pronounced in male animals than in female animals.

[0968] Example 14. Ovariectomized (OVX) mice study

[0969] Due to the long-acting effect of SEQ ID NO: 1, its ability to attenuate signaling was studied in a model of hyperstimulated hypothalamic-pituitary-gonadal (HPG) axis, such as one week ovariectomized (OVX) mice, through continuous delivery using Alzet minipumps.

[0970] Experiments were performed according to the materials and methods described in Example 9. Results are shown in FIGS. 13A-13C, 14, and 15A-15B.PATENT

[0971] Attorney Docket No.: 51796-005WO3

[0972] BWH Ref.: 2024-010-02

[0973] One day of treatment was sufficient to significantly attenuate mean LH content. After 7 days of treatment, LH attenuation was evident in mean LH, amplitude of LH pulses and a trend to decrease the number of pulses per hour in comparison to control OVX mice was also observed. Taken together, the pre-clinical data discussed above identify these KISS1 R agonists as potential therapeutic tools to activate the reproductive axis in conditions of insufficient GnRH release, i.e. HH and HA, or to decrease axis activity in conditions of excessive activation, with potential applications in PCOS. These agonists represent an approach to treat reproductive conditions that significantly affect women's health with high unmet medical needs.

[0974] Example 15. Materials and methods

[0975] This Example provides materials and methods used in Examples 16-18.

[0976] Ethics Statement. All procedures were approved by the Brigham and Women's Hospital Institutional Animal Care and Use Committee (IACUC) in the Center for Comparative Medicine.

[0977] Animals. Mice were housed at the Harvard Institutes of Medicine (HIM) animal facility. All mice were maintained in constant conditions of light, 12 hr light / dark cycle, at standard temperature (22-24°C) and given ad libitum access to standard laboratory mice (5L0D; LabDiet) and water, unless mentioned otherwise. Kissi KO mice were generated by crossing heterozygote B6(129S4)-Kiss1tm1.1(cre / EGFP)Rpa / JmiC6Jcommonly named as Kiss1Cre:GFP(v2), obtained from The Jackson Laboratory.

[0978] Sample collection. For mouse genotyping, ear samples were taken and subsequently analyzed by qPCR through the Transnetyx automated genotyping system. For LH pulsatile secretion assessment, mice were previously habituated to be daily handled to later performing the tail-tip bleeding every 10-min interval for 2 hours. Blood samples were obtained from the mouse-tail at different timepoints along the experiment and / or after subcutaneous (sc) injections. Briefly, 4 pl of whole blood from mouse tail were diluted in 116 pl of phosphate buffered saline (PBS 1X, pH 7.4) with 0.05 % Tween 20, snap-frozen on dry ice and stored at -80°C until assay using a super-sensitive LH ELISA (Steyn et al., Endocrinology. 2013 Dec;154(12):4939-45). Samples were assayed duplicated. For intra- and inter-assays variability, reference LH (0.25 ng / ml) was included in replicates, repeated within each assay and across separate assays, respectively. Values of LH were normalized using the reference LH (0.25 ng / ml) across assays performed on the same day. Any data point below the lowest measurable concentration on the standard curve (the limit of detection) was included as being at the lowest detectable value for that assay.

[0979] In Example 16 (Dose response in Kissi KO female mice (IHH model)), adult (4-5-month-old, with an average of weight of 21.3 g) Kiss1 KO female (at constant diestrus) mice (n=13) were injected subcutaneously (sc) with SEQ ID NO: 2 at the doses of 0.817 pg / kg (high; corresponding to 16.0 pmoles in 100 pl of PT30 solution), 0.163 pg / kg (medium; corresponding to 3.2 pmoles in 100 pl of PT30 solution), and 0.033 pg / kg (low; corresponding to 0.64 pmoles in 100 pl of PT30 solution) in PT30 solution. Blood samples were collected before the sc administration (baseline) and at 15, 30, 60, 90, 180, 360 and 600 minutes after sc administration of SEQ ID NO: 2. LH content was assessed as previously detailed in Sample Collection section.PATENT

[0980] Attorney Docket No.: 51796-005WO3

[0981] BWH Ref.: 2024-010-02

[0982] In Example 17 (IHH males IPRECIO SEQ ID NO: 2), non-GnRH primed adult Kiss1 KO male (3-5-month-old, with an average of weight of 22.1 g) male mice (n=5 / group, PT30 solution -vehicle- vs. SEQ ID NO: 2) were sc implanted with iPRECIO pumps, previously filled with SEQ ID NO: 2 in PT30 solution at the dose of 0.817 pg / kg / pulse (0.3 pl / pulse over 2 min, with one pulse every 90 min). Blood samples were collected at day 1 before first SEQ ID NO: 2 release (time -20, -10 and 0) and every 10 min for 90 min after the first bolus for pulsatile LH assessment. At day 5, blood samples were collected as described for day 1, before the first bolus and up to 90 min sampling with 10 min-intervals between bleedings. At day 7, blood samples were collected at time 0 and 10 min after the first release of the day. Mice were euthanized at this time (10 min after the first bolus of day 7). Retro-orbital blood was collected at the time of the euthanasia. Brain was dissected and collected in dry ice to further analysis. Testis were dissected out, weighed and split into two groups, one pair was collected in dry ice for molecular analysis and the other one was embedded in Bouin’s solution for further histological analysis. Body weight was monitored throughout the experiment.

[0983] In Example 18 (Hypothalamic amenorrhea dose response SEQ ID NO: 2 WT female mice), gonadal intact adult (3-4-month-old, with an average weight of 17.44 g before start of 24-hr fasting) C57BL / 6 female mice (n=10 / group) were fasted for 24 hr and subsequently sc injected with SEQ ID NO: 2 at the doses of 0.817 pg / kg (high; corresponding to 16.0 pmoles in 100 pi of PT30 solution), 0.163 pg / kg (medium; corresponding to 3.2 pmoles in 100 pl of PT30 solution) and 0.033 pg / kg (low; corresponding to 0.64 pmoles in 100 pl of PT30 solution) in PT30 solution. Blood samples were collected after 24 hr of fasting (time 0) and at 15, 30, 60, 90, 180, 360 and 600 minutes of sc SEQ ID NO: 2 administration. Mice were kept in under fasting through the end of blood sampling (600 minutes from sc administration female mice, i.e. 34 hours of fasting). LH content was assessed as previously detailed in Sample Collection section. Body weight was monitored throughout the experiment.

[0984] Statistical Analysis. All analyses were performed with GraphPad Prism Software. Data are expressed as the mean ± standard error of the mean (SEM) for each group. Unpaired Mann Whitney test and / or 1- or 2-way ANOVA test followed by Tukey, Dunnett and / or Fisher’s post hoc test were used to assess variation among experimental groups. Significance level was set at P < 0.05.

[0985] Example 16. Dose response in Kissi KO female mice (IHH model)

[0986] Experiments were performed according to the materials and methods described in Example 15. Results are shown in FIG. 16.

[0987] Adult (4-5-mo nth-o Id, with an average of weight of 21.3 g) Kissi KO female (at constant diestrus) mice were subcutaneously injected with SEQ ID NO: 2 at the doses of 0.817 pg / kg (high), 0.163 pg / kg (medium) and 0.033 pg / kg (low) pmoles in 100 pl of PT30 solution. Blood samples were collected before the subcutaneous administration (baseline) and at 15, 30, 60, 90, 180, 360 and 600 minutes after subcutaneous administration of SEQ ID NO: 2. LH content was assessed as previously described.PATENT

[0988] Attorney Docket No.: 51796-005WO3

[0989] BWH Ref.: 2024-010-02

[0990] Example 17. IHH males iPRECIO SEQ ID NO: 2

[0991] Experiments were performed according to the materials and methods described in Example 15. Results are shown in FIG. 17 and in Table 20.

[0992] Non-GnRH primed adult Kiss1 KO male (3–5-month-old, with an average of weight of 22.1 g) male mice (n=5 / group, PT30 solution -vehicle- vs. SEQ ID NO: 2) were subcutaneously implanted with iPRECIO pumps, filled with SEQ ID NO: 2 in PT30 solution at the dose of 0.817 pg / kg / pulse (0.3 pl / pulse over 2 min, with one pulse every 90 min). Blood samples were collected at day 1 before first SEQ ID NO: 2 release (time -20, -10 and 0) and every 10 min for 90 min after the first bolus for pulsatile LH assessment. At day 5, blood samples were collected as described for day 1, before the first bolus and up to 90 min sampling with 10 min-intervals between bleedings. At day 7, blood samples were collected at time 0 and 10 min after the first release of the day. Mice were euthanized at this time (10 min after the first bolus of day 7). Retro-orbital blood was collected at the time of the euthanasia. Brain was dissected and collected in dry ice to further analysis. Testis were dissected out, weighed and split into two groups, one pair was collected in dry ice for molecular analysis and the other one was embedded in Bouin’s solution for further histological analysis. Body weight was monitored throughout the experiment.Table 20. Body weights (BW) and testicle weights.

[0993] BW BW at the

[0994] Number Weight iPRECIO weight - BW Testicle Testicle Mean Testis

[0995] Group before end of the Testis / Bw*100 mouse iPRECIO BWwith iPRECIO change 1 (g) 2 (g) Testis %

[0996] iPRECIO experiment

[0997] 1 23.2 24.7 3.4 21.3 -1.9 0.0036 0.0039 0.00375 0.375 0.017605634 2 24.5 25.5 3.4 22.1 -2.4 0.006 0.0056 0.0058 0.58 0.026244344 Control 3 22.3 22.7 3.4 19.3 -3 0.0035 0.0033 0.0034 0.34 0.01761658

[0998] 4 20.3 22.7 3.4 19.3 -1 0.0032 0.003 0.0031 0.31 0.016062176 5 20.7 23.5 3.4 20.1 -0.6 0.0031 0.003 0.00305 0.305 0.015174129 1 21 24.3 3.4 20.9 -0.1 0.0168 0.0136 0.0152 1.52 0.072727273 2 23.6 25.7 3.4 22.3 -1.3 0.0092 0.0101 0.00965 0.965 0.043273543 SEQ ID

[0999] 3 21.9 25.2 3.4 21.8 -0.1 0.01 0.0096 0.0098 0.98 0.044954128 NO: 2

[1000] 4 22.1 26 3.4 22.6 0.5 0.0133 0.0126 0.01295 1.295 0.057300885 5 22 24.6 3.4 21.2 -0.8 0.0071 0.0068 0.00695 0.695 0.032783019

[1001]

[1002] PATENT

[1003] Attorney Docket No.: 51796-005WO3

[1004] BWH Ref.: 2024-010-02

[1005] Example 18. Hypothalamic amenorrhea dose response SEQ ID NO: 2 WT female mice Experiments were performed according to the materials and methods described in Example 15. Results are shown in FIG. 18.

[1006] Gonadal intact adult (3-4-month-old, with an average weight of 17.44 g before start of 24-hr fasting) C57BL / 6 female mice (n=10 / group) were fasted for 24 hr and subsequently subcutaneously injected with the SEQ ID NO: 2 peptide at the doses of 0.817 pg / kg (high), 0.163 pg / kg (medium) and 0.033 pg / kg (low) pmoles in 100 pl of PT30 solution. Blood samples were collected after 24 hr of fasting (time 0) and at 15, 30, 60, 90, 180, 360 and 600 minutes of subcutaneous administration of SEQ ID NO: 2. Mice were kept in under fasting through the end of blood sampling (600 minutes from subcutaneous administration in female mice, i.e. 34 hours of fasting). LH content was assessed as previously described. Body weight was monitored throughout the experiment.

[1007] Example 19. Kisspeptin analog testing

[1008] Objective

[1009] This Example is directed towards testing kisspeptin analogs, building on several aforementioned examples.

[1010] Dysregulated gonadotropin-releasing hormone (GnRH) pulsatility underlies diverse reproductive disorders, from the elevated LH pulse frequency in polycystic ovary syndrome (PCOS) to the absent pulsatility in idiopathic hypogonadotropic hypogonadism (IHH). This Example describes the development and characterization of KISS1R agonists with distinct pharmacokinetic profiles designed to address these opposing pathophysiologies. Through screening and optimization, we identified SEQ ID NO: 1, a molecule for sustained receptor activation, and SEQ ID NO: 2, an agonist suitable for pulsatile stimulation. Both compounds demonstrated subnanomolar potency, high selectivity for KISS1R, and favorable ADME properties including enhanced plasma stability relative to the native kisspeptin-10 parent molecule. In wild-type mice, subcutaneous administration induced robust, dose-dependent LH secretion with duration profiles matching their pharmacokinetic design. Continuous SEQ ID NO: 1 infusion suppressed the elevated LH pulsatility characteristic of ovariectomized mice, while twice-daily semi-acute administration in a prenatal androgen-exposed PCOS model normalized LH levels and partially improved ovarian morphology and follicular parameters. Conversely, pulsatile SEQ ID NO: 2 delivery to Kiss1-deficient (Kiss1 KO) male mice restored gonadotropin secretion, testosterone production, and testicular development. To establish clinical proof-of-concept, we administered pulsatile kisspeptin-10 to individuals with IHH, demonstrating dose-dependent induction of LH pulsatility. These findings establish KISS1R agonism as a therapeutic strategy across the spectrum of GnRH-related reproductive disorders and provide pharmacological tools with properties suited to distinct clinical applications.

[1011] Results

[1012] Characterization of KISS1R agonists

[1013] To develop KISS1 R agonists with pharmacological properties optimized for distinct therapeutic applications, we designed and synthesized a library of 326 peptide analogs based on the C-terminalPATENT

[1014] Attorney Docket No.: 51796-005WO3

[1015] BWH Ref.: 2024-010-02

[1016] active sequence of kisspeptin. Primary screening using a fluorescence resonance energy transfer (FRET)-based KISS1 R binding assay identified 81 compounds with EC50values below 0.1 nM. From this subset, we selected two compounds (SEQ ID NO: 1 and SEQ ID NO: 2) based on their exceptional potency and favorable predicted pharmacokinetic properties. SEQ ID NO: 1 demonstrated an EC50of 0.011 nM, while SEQ ID NO: 2 had an EC50of 0.021 nM in the primary binding assay.

[1017] To confirm functional activity, we evaluated both compounds in complementary cellular assays. In a PathHunter p-arrestin recruitment assay using KISS1R-expressing U2OS cells, SEQ ID NO: 1 and SEQ ID NO: 2 displayed EC50values of 0.9 nM and 1.1 nM, respectively, representing approximately 40-fold greater potency than kisspeptin-10 (SEQ ID NO: 3; EC50= 39.3 nM; Table 21). Notably, both compounds exhibited supramaximal efficacy compared to SEQ ID NO: 3, achieving higher maximal response levels even at substantially lower concentrations (FIG. 19A). In a calcium flux assay measuring Gq-protein-dependent Kissi r activation in CHO-K1 mouse receptor-expressing cells, SEQ ID NO: 1 and SEQ ID NO: 2 similarly demonstrated potent activation with EC50values of 2.0 nM and 2.2 nM, respectively, compared to 30.6 nM for SEQ ID NO: 3 (FIG. 19B, Table 21). Target selectivity was confirmed by demonstrating no appreciable agonist activity at the closely related melanin-concentrating hormone receptor 1 (MCHR1 / Mchr1) for either compound (Table 22).

[1018] Table 21. p -arrestin and Ca++flux EC50s (nM) for human KISS1R and mouse Kissi r. Values represent EC50 derived from four-parameter nonlinear regression of concentration-response curves. Data from n=3 independent experiments performed in duplicate.

[1019] Assay SEQ ID NO: 3 SEQ ID NO: 1 SEQ ID NO: 2

[1020] KISS1R p-Arrestin 39.3 0.9 1.1

[1021] Kissi r Ca++ flux 30.6 2 2.2

[1022]

[1023] Table 22. Binding assay for MCHR1 and Mchrl. Selectivity assessment by p-arrestin assay against melanin-concentrating hormone receptors. Values represent percent activity at 3 pM compound concentration. Negative values indicate no agonist activity.

[1024] Assay SEQ ID NO: 3 SEQ ID NO: 1 SEQ ID NO: 2

[1025] MCHR1 4.8 -1.1 -8.4

[1026] Mchrl 0.5 0.3 0.1

[1027]

[1028] Intracellular target engagement was further validated by measuring ERK1 / 2 phosphorylation in Kissi r-expressing GT1-7 mouse hypothalamic neurons. Treatment with SEQ ID NO: 1 or SEQ ID NO: 2 at 0.1 pM induced ERK1 / 2 phosphorylation comparable to or greater than that achieved with SEQ ID NO: 3 at 1 pM, demonstrating 10-fold enhanced signaling potency (FIG. 19C).

[1029] ADME profiling revealed favorable drug-like properties for both compounds (Table 23). SEQ ID NO: 1 and SEQ ID NO: 2 demonstrated high aqueous solubility (67-79 mg / mL across multiple buffers)PATENT

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[1032] with greater than 90% recovery at concentrations as low as 5 ng / mL when formulated for subcutaneous administration. Importantly, both compounds exhibited significantly enhanced plasma stability compared to native kisspeptin. In human plasma, SEQ ID NO: 1 had a half-life exceeding 90 hours and SEQ ID NO: 2 showed no detectable degradation as tested. In rat plasma, SEQ ID NO: 1 and SEQ ID NO: 2 displayed half-lives of 9.4 hours and 5.3 hours, respectively, representing 471 -fold and 266-fold improvements over SEQ ID NO: 3 (T1 / 2 = 0.02 hours). Both compounds demonstrated reasonable hepatic microsomal stability (SEQ ID NO: 1 T1 / 2 > 2 hours; SEQ ID NO: 2 T1 / 2 = 11.8 hours). SEQ ID NO: 1 showed moderate plasma protein binding (81.1%), while SEQ ID NO: 2 exhibited high binding (96.7%).

[1033] Table 23. ADME profiling summary for SEQ ID NO: 3, SEQ ID NO: 1, and SEQ ID NO: 2. Solubility measured at 100 pM initial concentration in indicated buffers. Plasma stability expressed as T1 / 2 at 37°C. Liver microsome stability determined in human hepatic microsomes with NADPH cofactor. Protein binding assessed by equilibrium dialysis in human plasma. N / D = not determined; INF = infinite (no detectable degradation).

[1034] Assay SEQ ID NO: 3 SEQ ID NO: 1 SEQ ID NO: 2 Solubility (mg / mL)

[1035] H2O 101 79.4 68.1

[1036] Glucose 95.5 67.4 70.3

[1037] Saline 90.0 68.8 66.5

[1038] Plasma Stability T½(h)

[1039] Mouse N / D 1.45 1.45

[1040] Rat 0.02 9.42 5.32

[1041] Human N / D 90.3 INF

[1042] Liver Microsomes T½(min) N / D 142.9 710.9

[1043] Protein Binding (%) N / D 81.1 96.7

[1044]

[1045] Mouse pharmacokinetic studies following subcutaneous and intravenous dosing confirmed distinct profiles for the two compounds (FIGS. 19D-19E; Table 24). SEQ ID NO: 1 (100 pg / kg) achieved a Cmax of 315,000 pg / mL with a terminal half-life of 1.18 hours and clearance of 45.6 mL / hr. SEQ ID NO: 2 (200 pg / kg) demonstrated higher peak exposure (Cmax = 600,165 pg / mL) with faster clearance (97.9 mL / hr) and a shorter half-life (0.9 hours). These pharmacokinetic profiles (SEQ ID NO: 1 with slower clearance enabling sustained exposure, and SEQ ID NO: 2 with rapid clearance suitable for pulsatile dosing) aligned with our design objectives for therapeutics targeting distinct reproductive pathophysiologies.

[1046] Table 24. PK Parameters for SEQ ID NO: 1 and SEQ ID NO: 2 following subcutaneous and intravenous administration in female C57BL / 6 mice. SEQ ID NO: 1 dosed at 100 pg / kg; SEQ ID NO: 2 dosed at 200 pg / kg. Plasma concentrations quantified by LC-MS / MS using solid-phase extraction with an LLOQ of 10 pg / mL. Acceptance criteria: ±20% accuracy for calibration standards; <20% CV for precision of qualityPATENT

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[1049] control samples. Parameters calculated using non-compartmental analysis. Cmax - maximum observed concentration; AUCinf = area under the curve extrapolated to infinity; Vss - volume of distribution at steady state; CL = clearance. n=2-3 mice per timepoint.

[1050] Half-life Cmax AUCinf Vss CL

[1051] Compound (hr) (pg / mL) (hr*pg / mL) (mL) (ml / hr)

[1052] SEQ ID NO: 1 1.18 315000 46000 8.54 45.6

[1053] SEQ ID NO: 2 0.9 600165 40842 8.9 97.9

[1054]

[1055] SEQ ID NO: 1 and SEQ ID NO: 2 induce robust LH secretion in wild-type mice

[1056] Having established the in vitro potency and favorable pharmacokinetic properties of SEQ ID NO: 1 and SEQ ID NO: 2, we next evaluated their ability to stimulate the reproductive axis in vivo. Intact wild¬ type male mice received single subcutaneous injections of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 at equimolar doses (6 nmol), and LH levels were measured over 90 minutes (FIG. 19F). Both SEQ ID NO: 1 and SEQ ID NO: 2 induced significantly greater LH responses compared to SEQ ID NO: 3 (P < 0.0001; FIG. 19G). Up to 90 min, the response kinetics between SEQ ID NO: 1 and SEQ ID NO: 2 was similar, showing in both cases an upwards trend at the end of the experiment, while SEQ ID NO: 3 returned to baseline by 60 min post injection.

[1057] Dose-response studies were conducted in both female mice during diestrus and intact male mice using three dose levels: High 0.767-0.817 pg / kg, Medium 0.153-0.163 pg / kg, Low 0.031-0.033 pg / kg. Because the previous experiment showed increasing LH levels at 90 min, here we extended the sampling to 10h (600 min). Both compounds demonstrated robust, dose-dependent LH stimulation in both sexes (P < 0.0001; FIGS. 19H-19I). The response patterns were consistent with the pharmacokinetic differences observed between compounds, with SEQ ID NO: 1 producing more sustained elevations and SEQ ID NO: 2 generating more transient responses. These results confirmed that both KISS1R agonists are capable of potently stimulating the hypothalamic-pituitary-gonadal axis in vivo, with distinct temporal profiles that suggest utility for different therapeutic applications.

[1058] Continuous SEQ ID NO: 1 suppresses elevated LH in ovariectomized mice.

[1059] Although ovariectomy (OVX) does not model the ovarian and androgenic milieu of PCOS, it provides a well-established neuroendocrine context in which removal of gonadal steroid feedback produces markedly elevated GnRH / LH output and increased LH pulse frequency / amplitude. In this setting, the hypothalamic-pituitary axis operates in a high-drive regime that is useful to test the core mechanistic premise of our approach: whether sustained kissl r agonism can induce a controlled adaptive response that lowers excessive gonadotropin output. Thus, OVX mice were used here as an in vivo platform to probe “hyperdrive” regulation of LH dynamics and to evaluate whether continuous exposure shifts the system toward a lower-output state, while recognizing that translation to PCOS will additionally require consideration of ovarian steroidogenesis and the broader endocrine-metabolic context.

[1060] Based on the established principle that continuous KISS1 activation leads to receptor desensitization and inhibition of the reproductive axis we hypothesized that sustained delivery of the long-PATENT

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[1063] acting compound SEQ ID NO: 1 at the appropriate dose would attenuate the elevated gonadotropin secretion in OVX mice through partial downregulation of the KISS1 R without complete desensitization / shutdown of the reproductive axis (FIG. 20A).

[1064] Seven days post-ovariectomy, mice received subcutaneous osmotic minipumps delivering continuous SEQ ID NO: 1 or vehicle. Serial blood sampling revealed that SEQ ID NO: 1 treatment resulted in sustained suppression of mean LH levels to -25-50% of that in vehicle-treated controls at days 1, 7, and 14 of treatment (P < 0.0001; FIG. 20B). To characterize the effects on pulsatile secretion, we performed 10-minute interval blood sampling over a 2-hour period. Individual LH pulse profiles demonstrated marked attenuation of pulsatility in SEQ ID NO: 1-treated animals compared to controls (FIG. 20C). Quantitative analysis revealed that SEQ ID NO: 1 treatment significantly reduced LH pulse frequency and amplitude (P < 0.001) (FIGS. 20D-20E). Body weight (BW) was similar between both groups (FIG. 27). These findings demonstrate that continuous KISS1R agonist exposure effectively decreases the hyperactive gonadotropin secretion characteristic of OVX mice to a profile of LH release that resembles that of an ovary intact mouse, and support the potential utility of long-acting KISS1 R agonists for conditions characterized by elevated GnRH pulsatility.

[1065] SEQ ID NO: 1 normalizes reproductive parameters in a prenatal androgen PCOS model.

[1066] To evaluate therapeutic efficacy in a clinically relevant disease model, we utilized the prenatal androgen (PNA) mouse model, which recapitulates key features of PCOS including elevated LH, disrupted estrous cyclicity, and polycystic ovarian morphology (FIG. 21A). Exposure to dihydrotestosterone (DHT) during late gestation (embryonic days 16-18) resulted in female offspring with advanced puberty onset, as evidenced by earlier vaginal opening and first estrus (P < 0.001; FIG. 21 B).

[1067] Adult PNA mice displayed markedly disrupted estrous cyclicity with prolonged time in diestrus (P < 0.0001; FIG. 21C).

[1068] To test the efficacy of a clinically translational delivery paradigm of SEQ ID NO: 1, PNA mice received twice-daily (BID) subcutaneous injections of SEQ ID NO: 1 (12 pmol / 100 pL) orvehicle for 30 days (FIG. 22A). After 30 days of treatment, SEQ ID NO: 1 normalized basal LH levels to those of control mice (FIG. 22B), indicating that kisslr activation can correct the central neuroendocrine dysfunction characteristic of the PNA model. While all animals initially responded to SEQ ID NO: 1 injections with an increase in LH release (FIG. 23A), this effect decreased in PNA mice by day 10 of treatment, which remained at control LH levels without signs of hypogonadism (FIGS. 23B-23C). Estrous cyclicity, which was disrupted by PNA, remained unchanged following treatment (FIG. 23D), and corpora lutea were absent in both PNA and SEQ ID NO: 1-treated groups (FIG. 22C), confirming that ovulation was not restored. Testosterone, AMH and FSH levels were also unaffected by treatment (FIG. 23F). Despite the lack of restored cyclicity, SEQ ID NO: 1-treated mice exhibited reduced ovarian size and increased primary follicle numbers compared to untreated PNA mice (FIG. 22D), suggesting that chronic kisslr agonism relieves the ovary from sustained gonadotropin overstimulation and partially rescues ovarian morphology and early follicular dynamics. The persistence of acyclicity and anovulation despite normalized tonic LH likely reflects the inability of twice-daily dosing to reconstitute gonadotropin patterns,PATENT

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[1071] including appropriate FSH fluctuations and the preovulatory LH surge, required for follicle maturation and ovulation. Testosterone (T) levels were not elevated in PNA mice relative to controls and were unaffected by treatment, indicating that the ovarian phenotype in this model is driven primarily by gonadotropin dysregulation rather than hyperandrogenism. Finally, the absence of a T increase does not rule out a valid PNA-PCOS phenotype, since circulating androgen elevations are variable across PNA implementations, age, and assays, and neuroendocrine / cyclicity defects can occur even when serum T is not clearly elevated. Collectively, these results demonstrate that targeting the kissl r pathway can correct central neuroendocrine dysfunction and ameliorate aspects of ovarian pathology in a PCOS-like model, while also suggesting that alternative dosing regimens designed to restore physiological gonadotropin dynamics may be necessary to fully reinstate reproductive cyclicity.

[1072] Pulsatile SEQ ID NO: 2 restores reproductive function in Kissi -deficient mice.

[1073] To model the opposite pathophysiology (absent GnRH signaling as seen in IHH), we first confirmed that the short-acting compound SEQ ID NO: 2 could stimulate LH release in conditions of negative energy balance (24h fasting) that suppress endogenous kisspeptin signaling (FIG. 24A), leading to a model of hypothalamic amenorrhea (HA). Twenty-four-hour fasted wild-type female mice responded to SEQ ID NO: 2 with dose-dependent LH elevation (FIG. 24B), demonstrating compound activity under conditions of metabolic suppression of the reproductive axis.

[1074] Once the stimulatory action of SEQ ID NO: 2 on LH release was confirmed in a model of HA, we utilized Kissi knockout (Kissi KO) mice, which lack endogenous kisspeptin and consequently fail to undergo puberty, have low gonadotropin and sex steroid levels, and exhibit small gonads with impaired gametogenesis (FIG. 24C), serving as a model of congenital IHH. Acute administration of SEQ ID NO: 2 to Kissi KO female mice (never primed with GnRH) induced robust, dose-dependent LH secretion (FIG.

[1075] 24D), confirming that the downstream signaling machinery remains intact and responsive to exogenous kissl r agonism in these animals.

[1076] To test whether pulsatile kissl r agonist delivery could restore reproductive function, mimicking the physiologic pulsatile pattern of kisspeptin release from KNDy neurons, we implanted Kissl KO male mice with programmable iPRECIO pumps delivering SEQ ID NO: 2 (0.817 pg / kg / pulse) or vehicle in a pulsatile pattern over 7 days (FIG. 25A). Serial blood sampling revealed that pulsatile SEQ ID NO: 2 treatment significantly elevated LH levels compared to vehicle controls (P < 0.01; FIG. 25B).

[1077] Testosterone levels, which are suppressed in Kissl KO males, were also significantly increased by pulsatile SEQ ID NO: 2 treatment (P < 0.01; FIG. 25B), indicating restoration of testicular steroidogenic function. Examination of testes at study completion revealed that vehicle-treated Kissl KO mice exhibited the characteristic small, underdeveloped testes of kisspeptin-deficient animals. In contrast, pulsatile SEQ ID NO: 2 treatment resulted in significantly increased testicular weight relative to body weight (P < 0.01, FIG. 25C), reflecting enhanced testicular development. These findings demonstrate that pulsatile delivery of a short-acting kissl r agonist can restore the hypothalamic-pituitary-gonadal axis in a model of complete kisspeptin deficiency, providing proof-of-concept for therapeutic replacement strategies in conditions characterized by low or absent GnRH signaling.PATENT

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[1080] Pulsatile kisspeptin induces dose-dependent LH pulsatility in humans with IHH.

[1081] To establish clinical proof-of-concept that pulsatile kisspeptin administration can restore gonadotropin pulsatility in humans with deficient GnRH signaling, we conducted a study in ten individuals with idiopathic hypogonadotropic hypogonadism (IHH; 5 men, 5 women). Eighty percent of participants had Kallmann syndrome (IHH with anosmia), suggesting neurodevelopmental etiology, and fifty percent harbored deleterious variants in known IHH genes. Following a priming period with pulsatile GnRH (25 ng / kg SC every 2 hours) to ensure pituitary responsiveness, participants received escalating doses of kisspeptin-10 (SEQ ID NO: 3; 0.313, 0.939, 3.13, and 13.19 pg / kg) administered as intravenous boluses every 2 hours, with frequent blood sampling (every 10 minutes for 40 minutes post-bolus) to capture LH pulse dynamics (FIG. 26A).

[1082] Individuals with IHH demonstrated clear responsiveness to kisspeptin (FIG. 26B). Fifty percent of participants exhibited formal LH pulses, as defined by Santen and Bardin criteria, in response to kisspeptin boluses. Analysis of mean LH responses across all participants revealed significant dosedependent increases in LH amplitude (P = 0.036 to P < 0.0001; FIG. 26C). The number of LH pulses detected also increased significantly with higher kisspeptin doses (P = 0.01; FIG. 26D). The 13.19 pg / kg dose generated a detectable LH response in 90% of participants, while 3.13 pg / kg produced responses in 40% of participants.

[1083] Importantly, the magnitude of LH response to kisspeptin increased with cumulative exposure. Comparison of LH responses between the first and last bolus at each dose revealed progressive enhancement of responsiveness overtime at higher doses (P = 0.02; FIG. 26E), suggesting possible sensitization or priming effects. Furthermore, two hours after the final kisspeptin bolus, participants demonstrated significantly elevated baseline LH levels compared to pre-treatment values (P < 0.0001;

[1084] FIG. 26F), indicating sustained activation of endogenous reproductive function. One participant demonstrated new spontaneous pulse-like activity following kisspeptin exposure, suggesting restoration of endogenous GnRH pulse generator function.

[1085] Multivariate regression modeling identified significant predictors of LH response to kisspeptin. Kisspeptin dose (p = 0.034, P < 0.001), FSH pool (p = 0.413, P < 0.001), GnRH-induced LH amplitude (p = 0.015, P < 0.001), female sex (p = -0.14, P = 0.019), baseline LH (p = -0.394, P = 0.014), and sex steroid use (p = 0.406, P = 0.003) were all significant predictors, with the overall model explaining 51% of variance in LH response (r2= 0.51, P < 0.0001). These clinical findings demonstrate that individuals with IHH retain the capacity to respond to kisspeptin and establish that pulsatile KISS1R agonist administration can stimulate gonadotropin pulsatility in humans with deficient GnRH signaling, providing essential clinical validation for the therapeutic approach tested in our preclinical models.

[1086] DISCUSSION

[1087] We describe SEQ ID NO: 1 and SEQ ID NO: 2 as KISS1 R agonists that modulate the kisspeptin-GnRH axis across two ends of the GnRH pulsatility disorder spectrum. A primary screen of 326 kisspeptin analogs identified these leads based on high potency and favorable stability / ADM E properties relative toPATENT

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[1090] native peptides. Although both agonists robustly activate KISS1R in vitro, their in vivo pharmacokinetics were deliberately separated to enable distinct dosing paradigms. In this context, SEQ ID NO: 1 exhibits slower clearance and higher systemic exposure compatible with sustained receptor engagement, whereas SEQ ID NO: 2 exhibits faster clearance suited for programmable pulsatile delivery.

[1091] Mechanistically, our strategy takes advantage of the dose-dependent nature of homologous GPCR desensitization. When receptor occupancy is high and sustained, trafficking and downregulation processes can dominate, attenuating downstream signaling; when occupancy is intermittent, receptors can recover between doses, preserving responsiveness. This approach is distinct from partial agonism (limited intrinsic efficacy) and from biased agonism, in which ligands preferentially engage specific signaling pathways and trafficking outcomes. In the framework of our compounds, the therapeutic goal is not maximal receptor activation, but rather calibrated exposure that produces a predictable shift in system output (LH pulsatility) while maintaining an adequate baseline reproductive state.

[1092] Our OVX model results illustrate the therapeutic potential of calibrated, partial KISS1R downregulation. Continuous SEQ ID NO: 1 infusion reduced both LH pulse frequency and pulse amplitude while preserving detectable pulsatility, producing a partial normalization of the post-OVX hypersecretory state rather than complete suppression. This is clinically relevant because full suppression of gonadotropin secretion, as achieved by pituitary desensitization with GnRH agonists, drives profound hypogonadism and is not aligned with the therapeutic objective in PCOS (Chwalisz 2023: Conn and Crowley 1994). By contrast, our data support a dosing window in which pathological hyperpulsatility can be attenuated while maintaining residual pulsatility and baseline gonadal function.

[1093] Importantly, the same principle translated to a disease-relevant PCOS model. PNA mice exhibited ovarian features consistent with an anovulatory PCOS-like phenotype, including reduced corpora lutea and disrupted estrous cycling. Treatment with SEQ ID NO: 1 produced evidence of partial ovarian improvement (e.g., changes in follicular composition and reduced ovarian weight) without restoration of ovulation or cyclicity.

[1094] For hypogonadotropic states characterized by inadequate GnRH drive, our data support pulsatile KISS1 R agonist delivery as a replacement strategy that is biologically congruent with endogenous KNDy-driven episodic signaling. In Kissi KO mice, acute SEQ ID NO: 2 elicited robust LH secretion and programmable pulsatile delivery over seven days induced increases in LH and testosterone with concomitant testicular growth, demonstrating that intermittent dosing can preserve responsiveness while restoring downstream gonadal function. Our findings are complemented by our clinical proof-of-concept study in individuals with IHH, in which intravenous SEQ ID NO: 3 boluses produced dose-dependent increases in LH pulse amplitude and pulse detection, with evidence of progressive sensitization at higher doses and sustained elevation of baseline LH after dosing. Together, these data support the concept that a subset of IHH patients retain functional GnRH neurons and pituitary-gonadal machinery that can be engaged by KISS1R stimulation.

[1095] In conclusion, SEQ ID NO: 1 and SEQ ID NO: 2 represent pharmacologically optimized KISS1R agonists that translate insights from KNDy neuron biology into indication-matched therapeutics. By engineering exposure profiles to modulate receptor occupancy, this approach enables two clinicallyPATENT

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[1098] distinct dosing paradigms: a) calibrated partial downregulation to attenuate pathological hyperactive LH pulsatility in PCOS-like states, and b) pulsatile stimulation to restore episodic drive in hypogonadotropic conditions. This platform provides a coherent path to develop KISS1R agonists as precision neuroendocrine therapies, with dosing patterns tailored to disease mechanism and with measurable, physiology-aligned pharmacodynamic endpoints.

[1099] MATERIALS AND METHODS

[1100] Animals

[1101] Mice were housed at the Harvard Institutes of Medicine (HIM) animal facility under constant conditions with a 12-hour light / dark cycle at standard temperature (22-24°C) and provided ad libitum access to standard laboratory chow (5L0D; LabDiet) and water unless otherwise specified. C57BL / 6J mice were purchased from The Jackson Laboratory. Kissi knockout mice were generated by crossing heterozygous B6(129S4)-K / ss / tm1'(cre / EGFP)R a / jmjce( / < / ssfCreGFPv2), obtained from The Jackson Laboratory. Genotyping was performed using ear tissue samples analyzed by quantitative PCR through the Transnetyx automated genotyping system.

[1102] Compound Synthesis and Preparation

[1103] SEQ ID NO: 1 and SEQ ID NO: 2 were designed and synthesized as kisspeptin analog peptides based on the C-terminal active sequence of kisspeptin. A library of 326 peptide analogs was designed and synthesized for primary screening. Dihydrotestosterone (5a-androstan-17p-ol-3-one) was purchased from Steraloids Inc. (#A2570-000). For in vivo studies, SEQ ID NO: 1 and SEQ ID NO: 2 were formulated in PT30 solution (5 mM sodium acetate / acetic acid, 40 mg / mL mannitol, 0.1% polysorbate 80, 5% propylene glycol, pH 5.0 ± 0.05). Kisspeptin 112-121, the 10-amino-acid isoform of kisspeptin (corresponding to amino acids 112 to 121 of the preprohormone; SEQ ID NO: 3), and GnRH were synthesized using good manufacturing practices by NeoMPS (PolyPeptide Laboratories, San Diego, CA). NeoMPS provided kisspeptin 112-121 under contract to the National Institute of Child Health and Human Development. Resuspended aliquots underwent additional tests for sterility, pyrogenicity, purity, and concentration.

[1104] Primary Screening Assay

[1105] Primary screening utilized NFAT-bla CHO-K1 cells expressing KISS1R (#K1720, ThermoFisher) in a fluorescence resonance energy transfer (FRET)-based binding assay. Cells were cultured in DMEM containing 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acids, 25 mM HEPES, 100 U / mL penicillin, and 100 pg / mL streptomycin at pH 7.3. Cells were seeded at 10,000 cells / well in 384-well plates. Peptides were prepared as 50 mM working solutions in DMSO and serially diluted 1:4 to generate 10-point concentration-response curves. After 4-hour incubation at 37°C with 5% CO2, LiveBLAZER™-FRET B / G Loading Kit (#K1095, ThermoFisher) with CCF-4AM substrate was added. Fluorescence was measured using a PerkinElmer detector (Aex=409 nm, Aemi=460 nm, Aem2=530 nm). EC50 values were determined by four-parameter nonlinear regression using GraphPad Prism.PATENT

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[1108] P-Arrestin Recruitment Assay

[1109] PathHunter p-arrestin assays (DiscoverX) were performed using KISS1R-expressing U2OS cells. Cells were seeded in 20 pL total volume in white-walled 384-well microplates and incubated at 37°C prior to compound addition. Compounds were diluted to generate 5X working solutions in assay buffer, and 5 pL was added to cells. Following 90-180 minute incubation, PathHunter Detection reagent cocktail (12.5-15 pL, 50% v / v) was added for 1 hour at room temperature. Chemiluminescent signal was detected using a PerkinElmer Envision™ instrument. Vehicle concentration was 1%. SEQ ID NO: 1 and SEQ ID NO: 2 were tested at semi-log concentrations from 0.000002-10 pM. ECso values and concentration-response curves were analyzed using CBIS data analysis suite (Chemlnnovation).

[1110] Calcium Flux Assay

[1111] Gq-protein-dependent Kissi r activation was assessed by intracellular calcium mobilization. Cells were loaded with 1X Dye Loading Buffer (1X Dye, 1X Additive A, 2.5 mM freshly prepared probenecid in HBSS / 20 mM HEPES) for 30-60 minutes at 37°C. Following equilibration at room temperature in the dark for 30 minutes, compounds (4X concentration in HBSS / 20 mM HEPES) were added using a FLIPR Tetra system (MDS). Calcium mobilization was monitored for 2 minutes with compound addition at 5 seconds. SEQ ID NO: 1 and SEQ ID NO: 2 were tested at concentrations from 0.00001-1 pM. Percentage activity was calculated as: % Activity = 100% * (mean RLUtest - mean RLUvehioie) / (mean RLUMAX - mean RLUvehide).

[1112] Target Selectivity Assessment

[1113] Selectivity against melanin-concentrating hormone receptors (MCHR1 and Mchrl) was evaluated using PathHunter p-arrestin assays at 3 pM compound concentration.

[1114] Intracellular Target Engagement

[1115] Target engagement was assessed by ERK1 / 2 phosphorylation in GT1-7 mouse hypothalamic neuronal cells as described (Ayoub et al. 2014). Cells were plated at 100,000 cells / well in 12-well plates and serum-starved for 6 hours the following day. Compounds were added at 0.1 pM and 1 pM concentrations, with FBS as positive control. Cells were lysed in Cell Signaling Technologies buffer (#9803) containing protease and phosphatase inhibitors. Lysates were analyzed on 4-12% gels, and Western blots were probed with phosphorylated ERK1 / 2 (Rabbit pAb 1:1000, Cell Signaling Technologies #9101) and p-catenin (Rabbit pAb 1:2000, Cell Signaling Technologies #9562) antibodies. Images were acquired using the LICOR Odyssey platform, and target activation was quantified as the ratio of pERK1 / 2 to p-catenin normalized to serum-starved controls.

[1116] ADME Profiling

[1117] All ADME measurements utilized liquid chromatography-tandem mass spectrometry (LC-MS / MS) on a Sciex API 6500 system with positive electrospray ionization. Internal standards were imipramine orPATENT

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[1120] osalmid. Mobile phases were H2O with 0.025% formic acid and 1 mM ammonium acetate (Phase A) and methanol with 0.025% formic acid and 1 mM ammonium acetate (Phase B). Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (1.7 pm, 2.1 mm x 50 mm).

[1121] Solubility

[1122] Peptides (100 pM, 1% DMSO final) were incubated in H2O, 0.9% NaCI, or 5% glucose at room temperature with 1000 rpm shaking for 1 hour. Following centrifugation at 12,000 rpm for 10 minutes, supernatants were analyzed by LC-MS / MS. For subcutaneous formulation development, additional testing was performed in buffers containing 5 mM sodium acetate or L-histidine, mannitol, polysorbate-20 / 80, and propylene glycol at pH 3-5.5 using UPLC-MS-MRM methods.

[1123] Plasma Stability

[1124] Peptides (2 pM) were incubated in pre-warmed human, rat, or mouse plasma at 37°C. Samples were collected at 0, 5, 15, 30, 45, and 60 minutes and quenched with acetonitrile containing 1% formic acid and internal standard. Following mixing (600 rpm, 10 minutes) and centrifugation (5594g, 15 minutes), supernatants were analyzed by LC-MS / MS. Half-lives were calculated from the slope of ^[concentration] versus time.

[1125] Hepatic Microsome Stability

[1126] Peptides (1.5 pM) were incubated with human liver microsomes (0.75 mg / mL) in 0.1 M potassium phosphate buffer with 6 mM NADPH at 37°C. Samples were collected at 0, 5, 15, 30, and 45 minutes and processed as above. Intrinsic clearance was calculated using a scaling factor of 1254.2 (microsomal protein / g liver x liver weight / kg body weight).

[1127] Plasma Protein Binding

[1128] Equilibrium dialysis was performed with peptides (1 pM) in human plasma against blank dialysis buffer at 37°C with 60 rpm shaking for 5 hours. Samples from donor and receiver chambers were matrix- matched and analyzed by LC-MS / MS. Fraction bound was calculated as: % bound = 100 x ([Donorsh] - [Receivers!-]) / [Donorsh].

[1129] Pharmacokinetic Studies

[1130] Female C57BL / 6 mice received single subcutaneous (SC) or intravenous (IV) doses of SEQ ID NO: 1 (100 pg / kg) or SEQ ID NO: 2 (200 pg / kg) at 2.5 mL / kg dose volume. Vehicles were 10% DMSO in saline (SEQ ID NO: 1) or sterile water (SEQ ID NO: 2). Blood was collected at 1, 5, 15 minutes and 1, 2, 3, 5, 8, and 24 hours post-dosing and processed to plasma using K2EDTA tubes (n=2-3 animals per time point).PATENT

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[1133] Bioanalytical Methods

[1134] Plasma concentrations were quantified by LC-MS / MS using a Sciex 6500 mass spectrometer coupled to an Eksigent microLC system. Stock solutions of SEQ ID NO: 1, SEQ ID NO: 2, and internal standards were prepared in 20% acetonitrile with 0.1% formic acid. SEQ ID NO: 2 served as internal standard (IS) for SEQ ID NO: 1 analysis (2000 pg / mL final concentration); YA-3 served as IS for SEQ ID NO: 2 analysis (500 pg / mL final concentration).

[1135] Sample preparation: 50 pL plasma was spiked with 5 pL calibration working solution and 5 pL IS, diluted with 50 pL water, and applied to Oasis WCX pElution SPE plates (Waters #186002499) preconditioned with methanol and water. Wells were washed with 5% ammonium hydroxide (pH ~10) and 20% acetonitrile. Analytes were eluted with 70% acetonitrile containing 1% trifluoroacetic acid, dried under nitrogen at 50°C, and reconstituted in 60 pL of 20% acetonitrile with 0.1% formic acid.

[1136] Chromatographic separation was performed on an ACE 3 C-18 column (300 A, 1 * 50 mm) at 60°C. Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Gradient elution: 0-5 min 5— >45% B, 5-6.5 min 45— >65% B, 6.5-7.5 min 65— >95% B, 7.5-8.5 min held at 95% B, 5 8.5-8.6 min 95— >5% B, held at 5% B to 10 min. Flow rate was 75 pL / min with 50 pL injection volume.

[1137] Mass spectrometer parameters: positive ion mode MRM with 5 msec dwell time; gas 1 and 2, 40 psi; ion spray voltage, 5000 V; source temperature, 300°C; curtain gas, 40 psi. MRM transitions: SEQ ID NO: 1: 619.3— >98.1 and 619.3^331.0; SEQ ID NO: 2: 639.3^510.1, 639.3^371.2, and 639.3^907.5.

[1138] Acceptance Criteria

[1139] Calibration curves were fitted using least-squares linear regression with 1 / x2weighting.

[1140] Calibration standards ranged from 10 to 5000 pg / mL with a lower limit of quantification (LLOQ) of 10 pg / mL. Quality control (QC) samples were prepared at 10 pg / mL (LLOQ), 20, 200, and 2000 pg / mL. Acceptance criteria: calibration standard accuracy ±20% of nominal concentrations; QC sample precision 5 <20% coefficient of variation (CV); QC sample accuracy ±20% of nominal. Data were processed using MultiQuant software (Sciex).

[1141] Pharmacokinetic parameters were calculated by non-compartmental analysis. Peak areas for multiple MRM transitions were summed for quantification.

[1142] 0 Blood Sample Collection and Hormone Assays

[1143] For LH pulsatility assessment, mice were habituated to daily handling prior to serial tail-tip blood sampling at 10-minute intervals for 2-3 hours. Whole blood (4 pL) was diluted in 116 pL PBS (pH 7.4) with 0.05% Tween 20, snap-frozen on dry ice, and stored at -80°C. LH was measured in duplicate using a super-sensitive ELISA (Steyn et al. 2013). Intra- and inter-assay variability was monitored using reference 5 LH (0.25 ng / mL). Values below the limit of detection were assigned the lowest detectable value. Retro- orbital or trunk blood collected at euthanasia was used to measure testosterone, anti-Mullerian hormone (AMH), and follicle-stimulating hormone (FSH) at the University of Virginia Ligand Core.PATENT

[1144] Attorney Docket No.: 51796-005WO3

[1145] BWH Ref.: 2024-010-02

[1146] LH Pulsatility Analysis

[1147] In intact female mice, LH pulses were defined as >125% increase over basal LH values, with basal LH calculated as the mean of the 6 lowest measurements (McQuillan et al. 2019). In ovariectomized mice, LH pulses and amplitude were quantified using the PULSAR Otago platform (https: / / pulsar.otago.ac.nz / pulsar / ; (Porteous et al. 2021)) with parameters: smoothing 0.7, peak split 2.5, detection limit 0.04, amplitude distance 3, assay variability 0, 2.5, and 3.3; G values: G(1) 3.5, G(2) 2.6, G(3) 1.9, G(4) 1.5, G(5) 1.2.

[1148] Estrous Cycle Assessment

[1149] Daily vaginal smears were collected using a 20-pL pipette with 8 pL of 0.9% NaCI, transferred to microscope slides, air-dried, and stained with toluidine blue. Cycle phases were identified under 10X brightfield microscopy based on cell morphology: metestrus (mixed cornified, leukocytes, nucleated epithelial), diestrus (predominantly leukocytes), proestrus (small, round nucleated epithelial), and estrus (anucleated cornified epithelial).

[1150] Acute Dose-Response Studies

[1151] Adult gonadally intact male (3-7 months) and diestrus female (3-4 months) C57BL / 6 mice (n=8-10 / group) received SC administration of YA compounds at high, medium, and low doses. SEQ ID NO: 2: 0.817 pg / kg (high), 0.163 pg / kg (medium), 0.033 pg / kg (low). SEQ ID NO: 1: 0.767 pg / kg (high), 0.153 pg / kg (medium), 0.031 pg / kg (low). Compounds were administered in 100 pL PT30 solution. Blood samples were collected at baseline and 15, 30, 60, 90, 180, 360, and 600 minutes post-administration.

[1152] Ovariectomy and Continuous infusion Study

[1153] Adult (3-month-old) C57BL / 6 female mice (n=10 / group) were bilaterally ovariectomized (OVX). Seven days post-OVX, mice received SC implantation of Alzet osmotic minipumps (Model 1004; 0.11 pL / hour release rate) containing SEQ ID NO: 1 (28 pg / mL in PT30 solution) or vehicle. Blood samples were collected before OVX, 7 days post-OVX, and 7 and 14 days post-pump implantation. Pulsatile LH secretion was assessed on day 7 post-implantation via 10-minute interval sampling over 2 hours. Body weight was monitored throughout.

[1154] Prenatal Androgen (PNA) Model Generation

[1155] Pregnant female mice received SC injections of dihydrotestosterone (DHT; 250 pg in 100 pL sesame oil) or vehicle (sesame oil) on gestational days 16, 17, and 18. PNA (n=8) and control (n=10) female pups were weaned at postnatal day 21 and monitored daily for puberty markers including body weight, vaginal opening, and first estrus. At postnatal day 90, estrous cyclicity was monitored for 2 weeks in control (n=11) and PNA (n=15) mice.PATENT

[1156] Attorney Docket No.: 51796-005WO3

[1157] BWH Ref.: 2024-010-02

[1158] SEQ ID NO: 1 Treatment in PNA Mice

[1159] PNA mice were randomized to receive twice-daily (BID; 9 AM and 6 PM) SC injections of SEQ ID NO: 1 (12 pmoi in 100 pL sterile water; n=6) or vehicle (100 pL sterile water; n=6) for 30 days. Control mice (n=8) received vehicle BID. Blood was collected before and 3 hours after the first injection on days 1, 10, and 30. Following 30 days of treatment, pulsatile LH secretion was assessed over 3 hours. At study termination, trunk blood and gonads were collected for hormone analysis and histological examination, respectively.

[1160] Fasting Study in Wild-Type Female Mice

[1161] Adult (3-4-month-old) C57BL / 6 female mice (n=10 / group) were fasted for 24 hours prior to SC injection of SEQ ID NO: 2 at high (0.817 pg / kg), medium (0.163 pg / kg), or low (0.033 pg / kg) doses in 100 pL PT30 solution. Blood samples were collected after fasting (time 0) and at 15, 30, 60, 90, 180, 360, and 600 minutes post-injection. Fasting was maintained throughout blood sampling (34 hours total). Body weight was monitored throughout the experiment.

[1162] Acute SEQ ID NO: 2 in Kiss1 Knockout Female Mice

[1163] Kissi knockout female adult mice (4-5 months, n=4 / group) that had never received GnRH priming and were in constant diestrus received SC injection of SEQ ID NO: 2 at high (0.817 pg / kg), medium (0.163 pg / kg), or low (0.033 pg / kg) doses in 100 pL PT30 solution. Blood samples were collected at baseline and 15, 30, 60, 90, 180, 360, and 600 minutes post-administration.

[1164] Pulsatile SEQ ID NO: 2 Delivery in Kiss1 Knockout Male Mice

[1165] Non-GnRH-primed adult Kiss1 knockout males (3-5 months; n=5 / group) received SC implantation of programmable iPRECIO pumps (SMP-310R) on day -3. Pumps were programmed to deliver pulsatile SEQ ID NO: 2 (0.817 pg / kg / pulse; 0.3 pL / pulse over 2 minutes, one pulse every 90 minutes) or vehicle (PT30 solution) beginning on day 1 for 7 days. Tail blood was collected on day 7 prior to euthanasia for LH measurement. Retro-orbital blood was collected at euthanasia for testosterone measurement. Testes were weighed and fixed in Bouin's solution for histological analysis.

[1166] Human Clinical Study: Patients and Eligibility Criteria

[1167] Subjects were recruited from postings (e.g., clinicaltrials.gov), advertisements (Rally at Partners), and direct recruitment from the clinic practice of the Reproductive Endocrine Unit at Massachusetts General Hospital (MGH). Screening laboratory tests were performed by LabCorp, MGH clinical laboratories, and Quest Diagnostics.

[1168] Ten patients with IHH were enrolled in this study. IHH was defined as hypogonadal sex steroid levels (estradiol <20 pg / mL in women, testosterone <100 ng / dL in men) in the setting of low or inappropriately normal gonadotropin levels at age >18 years and in the absence of any identifiable medical condition that could have caused hypogonadotropic hypogonadism. All individuals with ANOS1 (formerly KAL1) mutations or prior evidence of severe midline facial defects were excluded as they werePATENT

[1169] Attorney Docket No.: 51796-005WO3

[1170] BWH Ref.: 2024-010-02

[1171] less likely to have GnRH neurons in the hypothalamus. Because prior data suggested kisspeptin responsiveness was not modulated by sex steroids, individuals were allowed to continue on current physiologic hormonal replacement therapy (testosterone therapy, oral or transdermal estradiol therapy).

[1172] Genetic Analysis

[1173] Patients who consented to genetic studies underwent whole exome sequencing of their DNA and results in known IHH genes (Supplemental Table 1) were confirmed by Sanger sequencing, as described previously. Rare sequence variants were defined as having a minor allele frequency of <0.1% in the Genome Aggregation Database. Rare sequence variants were reported if they were predicted to be damaging by at least two of three in silico prediction programs: PolyPhen-2, SIFT, and Mutation Taster.

[1174] Neuroendocrine Phenotyping in Response to Kisspeptin

[1175] This study consisted of a 52-hour visit to the Harvard Catalyst Translational Clinical Research Center at MGH. Participants were admitted to the CRC for blood sampling up to every 10 minutes to evaluate LH secretion during a 6-hour baseline, in response to 20 boluses of kisspeptin delivered at the physiologic frequency of every 2 hours, and in some individuals, a follow-up blood sampling period to assess LH secretion after exposure to kisspeptin. Since prior data suggested a possible rightward shift in kisspeptin responsiveness in individuals with IHH, each individual received 4 doses of kisspeptin: 0.313 (physiologic dose in healthy men and healthy luteal-phase women), 0.939, 3.13, and 13.19 mcg / kg. To explore if hypothalamic priming was needed to improve kisspeptin responsiveness, the 4 doses were delivered 5 times in a randomized block pattern. Following administration of kisspeptin, all patients were administered a bolus of 75 ng / kg GnRH to assess pituitary gonadotrope function.

[1176] Pituitary Priming Protocol

[1177] All patients underwent pituitary "priming" prior to undergoing the study to ensure that the pituitary gonadotropes were capable of producing an LH pulse in response to exogenous or endogenous GnRH. Patients underwent pituitary "priming" via a Crono F portable infusion pump (Canè S.p.A, Rivoli, Italy) administering GnRH 25 ng / kg subcutaneously every 2 hours for 5 to 7 days. The final dose of GnRH via the infusion pump was given 48 hours prior to CRC admission.

[1178] Human Laboratory Assays and LH Analysis

[1179] LH was measured at each time point and on an all-study quality control pool; FSH was measured on 1- or 2-hour study pools. LH and FSH were run at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core using Siemens IMMULITE 2000 (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) using previously described assays. Estradiol and testosterone were measured on 1- or 2-hour study pools by LabCorp, using electrochemiluminescence immunoassay methods (Roche Diagnostics, Indianapolis, IN).PATENT

[1180] Attorney Docket No.: 51796-005WO3

[1181] BWH Ref.: 2024-010-02

[1182] LH pulses were identified with a validated modification of the Santen and Bardin method. Pulse amplitude was calculated as the difference between time 0 of kisspeptin administration and peak of the pulse. If LH decreased after kisspeptin, the pulse amplitude was assigned a value of 0.

[1183] To evaluate changes in LH that may not meet LH pulse criteria, the directional change in LH from time 0 of kisspeptin bolus up to time 40 min (ΔLHkiss), the longest time to peak based on Santen and Bardin and previous characterization of kisspeptin-induced LH pulses, was calculated. To establish baseline variability, the largest directional change in LH from the 3, 2-hour windows of the 6-hour baseline were recorded.

[1184] Multivariate Regression Analysis

[1185] Univariate and multivariate linear regression using Stata / SE 17.0 (StataCorp LLC, College Station, Texas) was used to characterize the factors that predicted ΔLHkiss. Factors considered in this regression and evaluated using likelihood ratio test included: GnRH-LH amplitude, prior kisspeptin dose, time of kisspeptin dose, block of the kisspeptin dose, sex, baseline LH, baseline FSH, sex steroid exposure, baseline LH variability, and post-kisspeptin LH variability.

[1186] Histological Analysis

[1187] Gonads were fixed in Bouin's solution, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Ovarian follicles were classified and counted according to established criteria. Corpora lutea were identified by morphological characteristics.

[1188] Statistical Analysis

[1189] All analyses were performed using GraphPad Prism (version 10, San Diego, CA). Data are expressed as mean ± standard error of the mean (SEM). Normality and homogeneity of variance were assessed prior to selecting parametric or non-parametric tests. For unpaired two-group comparisons with non-normally distributed data, Mann-Whitney U tests were used. For experiments involving more than two groups, one-way ANOVA was performed with Tukey's or Dunnett's post hoc tests. For experiments with multiple factors or repeated measures, two-way ANOVA was performed with Sidak's post hoc test. For non-parametric repeated measures, Friedman test with Dunn's post hoc comparisons was used. Paired comparisons used Wilcoxon matched-pairs signed rank test. The significance threshold was set at P < 0.05. Specific statistical tests and P values are reported in the corresponding figure legends. EC50 values were determined by four-parameter nonlinear regression. Multivariate regression modeling was performed for clinical study data with stepwise variable selection.

[1190] OTHER EMBODIMENTS

[1191] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention andPATENT

[1192] Attorney Docket No.: 51796-005WO3

[1193] BWH Ref.: 2024-010-02

[1194] including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[1195] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[1196] Other embodiments are within the following claims.

Claims

PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02CLAIMS1. A method of treating a subject having a disorder, the method comprising administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein the administration is pulsatile.

2. The method of claim 1, wherein the therapeutically effective amount is between about 0.05 pg / kg to about 90 ng / kg.

3. The method of claim 1 or 2, wherein the therapeutically effective amount is between about 10 pg / kg to about 1 ng / kg.

4. The method of claim 1, wherein the therapeutically effective amount is between about 90 ng / kg to about 5 pg / kg.

5. The method of claim 1, wherein the therapeutically effective amount is between about 5 pg / kg to about 30 pg / kg.

6. The method of any one of claims 1-5, wherein the pulsatile administration is at a frequency of between about 20 minutes to about 4 days.

7. The method of any one of claims 1-6, wherein the pulsatile administration is at a frequency of between about 45 minutes to about 2 hours.

8. The method of any one of claims 1-7, wherein the pulsatile administration is over a period of at least about 1 day.

9. The method of any one of claims 1-8, wherein the pulsatile administration is over a period of at least about 14 days.

10. The method of any one of claims 1-9, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:(a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or(b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2).

11. The method of any one of claims 1-10, wherein the disorder is a reproductive disorder.

12. The method of claim 11, wherein the reproductive disorder is associated with a suppressed reproductive axis.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0213. The method of claim 11 or 12, wherein administration of the kisspeptin analog ora pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof activates the reproductive axis.

14. The method of any one of claims 11-13, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof enables fertility.

15. The method of any one of claims 11-14, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof increases the level of a reproductive health biomarker.

16. The method of any one of claims 11-15, wherein the subject is a female.

17. The method of claim 16, wherein the reproductive health biomarker is luteinizing hormone (LH), follicle-stimulating hormone (FSH), inhibin, estradiol, anti-mullerian hormone (AMH), external signs of puberty onset (Tanner scale), sexual desire, follicle size, bone density, uterine lining (e.g., endometrial lining), cervical mucus, vaginal moisture, basal body temperature, a marker of egg quality, or a marker of embryo quality.

18. The method of claim 16 or 17, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof results in follicle growth.

19. The method of any one of claims 16-18, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof improves the manner in which eggs grow, the number of eggs that grow, the quality of the eggs that grow, or the ability of eggs to ovulate.

20. The method of any one of claims 11-15, wherein the subject is a male.

21. The method of claim 20, wherein the reproductive health biomarker is LH, FSH, inhibin, testosterone, external signs of puberty onset (Tanner scale), sexual desire, semen analysis, bone density, or testicular size.

22. The method of claim 20 or 21, wherein administration of the kisspeptin analog ora pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof results in spermatogenesis.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0223. The method of any one of claims 11-22, wherein the reproductive disorder is hypogonadotropic hypogonadism (HH).

24. The method of claim 23, wherein the HH is congenital HH.

25. The method of claim 24, wherein the congenital HH is congenital idiopathic HH (normosmic).

26. The method of claim 24, wherein the congenital HH is congenital Kallmann syndrome (anosmic / hy posmic).

27. The method of claim 23, wherein the HH is acquired HH.

28. The method of claim 27, wherein the acquired HH is hypothalamic amenorrhea.

29. The method of claim 27, wherein the acquired HH is hyperprolactinemia.

30. The method of claim 27, wherein the acquired HH is associated with negative energy balance, malnutrition, obesity, stress, a metabolic disorder, diabetes, acute illness, hypothyroidism, excessive exercise, analgesic or psychiatric medications, or substance abuse.

31. The method of any one of claims 11-19, wherein the reproductive disorder is abnormal pregnancy.

32. The method of any one of claims 11-22, wherein the reproductive disorder is infertility or a reproductive disorder treated with an IVF protocol.

33. The method of any one of claims 1-10, wherein the disorder is hypoactive sexual arousal disorder.

34. The method of any one of claims 1-10, wherein the disorder is VMS.

35. The method of any one of claims 1-10, wherein the disorder is a respiratory disease.

36. The method of claim 35, wherein the respiratory disease is asthma.

37. The method of any one of claims 1-10, wherein the disorder is a metabolic disorder.

38. The method of claim 37, wherein the metabolic disorder is diabetes.

39. The method of claim 37, wherein the metabolic disorder is obesity.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0240. The method of claim 37, wherein the metabolic disorder is MASLD.

41. The method of claim 37, wherein the metabolic disorder is MASH.

42. The method of any one of claims 1-10, wherein the disorder is a liver disorder.

43. The method of claim 42, wherein the liver disorder is MASLD.

44. The method of claim 42, wherein the liver disorder is MASH.

45. The method of any one of claims 1-10, wherein the disorder is a bone disorder.

46. The method of claim 45, wherein the bone disorder is osteoporosis.

47. The method of any one of claims 1-10, wherein the disorder is a neurodegenerative disorder.

48. The method of claim 47, wherein the neurodegenerative disorder is Alzheimer’s disease.

49. The method of any one of claims 1-10, wherein the disorder is a sequela of a neurotropic virus.

50. The method of claim 49, wherein the neurotropic virus is SARS CoV-2.

51. A method of treating a subject having a disorder, the method comprising administering to the subject a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein the administration is continuous.

52. The method of claim 51, wherein the therapeutically effective amount is between about 0.05 pg / kg / h to about 250 ng / kg / h.

53. The method of claim 51 or 52, wherein the therapeutically effective amount is between about 10 pg / kg / h to about 1 ng / kg / h.

54. The method of any one of claims 51-53, wherein the continuous administration is over a period of at least about 2 hours.

55. The method of any one of claims 51-54, wherein the continuous administration is over a period of at least about 16 hours.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0256. The method of any one of claims 51-55, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:(a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or(b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 2).

57. The method of any one of claims 51-56, wherein the disorder is a reproductive disorder.

58. The method of claim 57, wherein the reproductive disorder is associated with a hyperactive reproductive axis.

59. The method of claim 57 or 58, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof suppresses the reproductive axis.

60. The method of any one of claims 57-59, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof decreases the level of a reproductive health biomarker.

61. The method of any one of claims 57-60, wherein the subject is a female.

62. The method of claim 61, wherein the reproductive health biomarker is LH, FSH, inhibin, estradiol, AMH, external signs of puberty onset (Tanner scale), sexual desire, follicle size, bone density, uterine lining (e.g., endometrial lining), cervical mucus, vaginal moisture, basal body temperature, a marker of egg quality, or a marker of embryo quality.

63. The method of claim 61 or 62, wherein administration of the kisspeptin analog ora pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof decreases follicle growth.

64. The method of any one of claims 57-60, wherein the subject is a male.

65. The method of claim 64, wherein the reproductive health biomarker is LH, FSH, inhibin, testosterone, external signs of puberty onset (Tanner scale), sexual desire, semen analysis, or testicular size.

66. The method of claim 64 or 65, wherein administration of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof modifies spermatogenesis or the potential for spermatogenesis.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0267. The method of any one of claims 57-66, wherein the reproductive disorder is precocious puberty, sex hormone-dependent cancer, or gender dysphoria.

68. The method of any one of claims 57-63, wherein the reproductive disorder is polycystic ovary syndrome (PCOS), endometriosis, uterine fibroid, or abnormal pregnancy.

69. The method of any one of claims 51-56, wherein the disorder is a metabolic disorder.

70. The method of claim 69, wherein the metabolic disorder is diabetes.

71. The method of claim 69, wherein the metabolic disorder is obesity.

72. The method of claim 69, wherein the metabolic disorder is MASLD.

73. The method of claim 69, wherein the metabolic disorder is MASH.

74. The method of any one of claims 51-56, wherein the disorder is a liver disorder.

75. The method of claim 74, wherein the liver disorder is MASLD.

76. The method of claim 74, wherein the liver disorder is MASH.

77. The method of any one of claims 51-56, wherein the disorder is a bone disorder.

78. The method of claim 77, wherein the bone disorder is osteoporosis.

79. The method of any one of claims 51-56, wherein the disorder is vasomotor symptoms (VMS).

80. The method of any one of claims 51-56, wherein the disorder is a neurodegenerative disorder.

81. The method of claim 80, wherein the neurodegenerative disorder is Alzheimer’s disease.

82. The method of any one of claims 51-56, wherein the disorder is a sequela of a neurotropic virus.

83. The method of claim 82, wherein the neurotropic virus is SARS CoV-2.

84. The method of any one of claims 1 -32, 34, 37-46, or 51-79, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intravenously, subcutaneously, or intramuscularly.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-0285. The method of any one of claims 1 -10 or 33, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intranasally.

86. The method of any one of claims 1-10, 35, or 36, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered by inhalation.

87. The method of any one of claims 1-10, 47-56, or 80-83, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is administered intrathecally.

88. The method of any one of claims 1 -87, further comprising administering to the subject a gonadotropinreleasing hormone receptor (GnRHR) agonist or antagonist, neurokinin receptor agonist or antagonist, or opioid agonist, antagonist, or agonist-antagonist.

89. The method of claim 88, wherein the GnRHR agonist is leuprolide, gonadorelin, goserelin, nafarelin, histrelin, triptorelin, or buserelin.

90. The method of claim 88, wherein the GnRHR antagonist is abarelix, cetrorelix, degarelix, elagolix, ganirelix, or relugolix.

91. The method of claim 88, wherein the neurokinin receptor antagonist is fezolinetant, aprepitant, rolapitant, casopitant, fosaprepitant, netupitant, or maropitant.

92. The method of claim 88, wherein the opioid agonist is morphine, methadone, or alfentanil.

93. The method of claim 88, wherein the opioid antagonist is naloxone or naltrexone.

94. The method of claim 88, wherein the opioid agonist-antagonist is buprenorphine.

95. A method of identifying a subject as having congenital HH, the method comprising:(a) providing a reference level of LH;(b) administering to the subject a stimulating dose of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, wherein:(I) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg; orPATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02(ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg;(c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose;(d) comparing the reference level of LH to the level of LH in the subject after administration of the stimulating dose; and(e) identifying a subject who has a level of LH after administration of the stimulating dose that is not different from the reference level of LH as having congenital HH.

96. The method of claim 95, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:(a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 1); or(b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2).

97. The method of claim 95 or 96, wherein the congenital HH is congenital Kallmann syndrome (anosmic / hy posmic).

98. A method of determining whether a patient having a disorder is likely to respond to a therapy comprising a therapeutically effective amount of a kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 (SEQ ID NO: 3) or a pharmaceutically acceptable salt thereof, the method comprising:(a) providing a reference level of LH;(b) administering to the subject a provocative dose of the kisspeptin analog or a pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof;(c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose;(d) comparing the reference level of LH to the level of LH in the subject after administration of the provocative dose; and(e) identifying a subject who has a level of LH after administration of the provocative dose that is greater than the reference level of LH as likely to respond to the therapy.

99. The method of claim 98, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:(a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 1); or(b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (SEQ ID NO: 2).

100. The method of claim 98 or 99, wherein the provocative dose is administered intravenously at a dose of between about 1 pg / kg to about 1 pg / kg.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02101. The method of claim 98 or 99, wherein the provocative dose is administered subcutaneously at a dose of between about 10 pg / kg to about 10 pg / kg.

102. The method of any one of claims 98-101, wherein the disorder is a reproductive disorder, hypoactive sexual arousal disorder, respiratory disease, metabolic disorder, liver disorder, bone disorder, VMS, neurodegenerative disorder, or a sequela of a neurotropic virus.

103. The method of claim 102, wherein the reproductive disorder is HH, abnormal pregnancy, infertility, precocious puberty, sex hormone-dependent cancer, gender dysphoria, PCOS, endometriosis, uterine fibroid, or a reproductive disorder treated with an IVF protocol.

104. The method of claim 103, wherein the HH is congenital HH or acquired HH.

105. The method of claim 104, wherein the congenital HH is congenital idiopathic HH (normosmic) or congenital Kallmann syndrome (anosmic / hyposmic).

106. The method of any one of claims 1-105, wherein the kisspeptin analog or pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 1.

107. The method of any one of claims 1-105, wherein the kisspeptin analog or pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 2.

108. The method of any one of claims 1-105, wherein kisspeptin-10 (SEQ ID NO: 3) is used.

109. The method of any one of claims 1-108, wherein the kisspeptin analog or pharmaceutically acceptable salt thereof or kisspeptin-10 or a pharmaceutically acceptable salt thereof is delivered to a human using a programmable pump.

110. A pharmaceutical composition comprising a kisspeptin analog ora pharmaceutically acceptable salt thereof, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of:(a) Ac-(D-Tyr)-A6c-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 1); or(b) Ac-[D-Phe(2,4-DiCI)]-HoPro-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2(SEQ ID NO: 2).

111. The pharmaceutical composition of claim 110, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 1.

112. The pharmaceutical composition of claim 110, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof comprises a sequence of SEQ ID NO: 2.PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02113. The pharmaceutical composition of any one of claims 110-112, wherein the pharmaceutical composition is an aqueous formulation having a pH of between about 3.5 to about 8.0.

114. The pharmaceutical composition of claim 113, wherein the formulation has a pH of about 4.0 to about 6.0.

115. The pharmaceutical composition of any one of claims 110-114, wherein the pharmaceutical composition comprises a buffer.

116. The pharmaceutical composition of claim 115, wherein the buffer is present at a concentration of about 1 mM to about 10 mM.

117. The pharmaceutical composition of claim 116, wherein the buffer is present at a concentration of about 5 mM.

118. The pharmaceutical composition of any one of claims 115-117, wherein the buffer is an acetate buffer.

119. The pharmaceutical composition of any one of claims 110-118, wherein the pharmaceutical composition comprises a tonicity agent.

120. The pharmaceutical composition of claim 119, wherein the tonicity agent is present at a concentration of about 1 mg / mL to about 100 mg / mL.

121. The pharmaceutical composition of claim 120, wherein the tonicity agent is present at a concentration of about 40 mg / mL.

122. The pharmaceutical composition of any one of claims 119-121, wherein the tonicity agent is mannitol.

123. The pharmaceutical composition of any one of claims 110-122, wherein the pharmaceutical composition comprises one or more surfactants.

124. The pharmaceutical composition of claim 123, wherein each of the one or more surfactants is present at a concentration of about 0.01% to about 20%.

125. The pharmaceutical composition of claim 124, wherein the one or more surfactants is polysorbate 80 (PS80).PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02126. The pharmaceutical composition of claim 125, wherein the pharmaceutical composition comprises about 0.05% to about 1% PS80.

127. The pharmaceutical composition of claim 126, wherein the pharmaceutical composition comprises about 0.1% PS80.

128. The pharmaceutical composition of any one of claims 123-127, wherein the one or more surfactants is propylene glycol (PG).

129. The pharmaceutical composition of claim 128, wherein the pharmaceutical composition comprises about 1% to about 10% PG.

130. The pharmaceutical composition of claim 129, wherein the pharmaceutical composition comprises about 5% PG.

131. The pharmaceutical composition of any one of claims 110-130, comprising about 5 mM sodiumacetate buffer, about 40 mg / mL mannitol, about 0.1% PS80, and about 5% PG, wherein the pH is about 5.0.

132. The pharmaceutical composition of any one of claims 110-131, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is present at a concentration of about 0.01 ng / mL to about 100 mg / mL.

133. A method of treating a subject having a disorder, the method comprising administering to the subject the pharmaceutical composition of any one of claims 110-132.

134. The method of claim 133, wherein the subject is a male or a female.

135. The method of claim 133 or 134, wherein the administration is pulsatile.

136. The method of claim 135, wherein the disorder is a reproductive disorder, hypoactive sexual arousal disorder, VMS, respiratory disease, metabolic disorder, liver disorder, bone disorder, neurodegenerative disorder, or a sequela of a neurotropic virus.

137. The method of claim 136, wherein the disorder is a reproductive disorder and administration of the pharmaceutical composition activates the reproductive axis, enables fertility, increases the level of a reproductive health biomarker, results in follicle growth, improves the manner in which eggs grow,PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02improves the number of eggs that grow, improves the quality of the eggs that grow, improves the ability of eggs to ovulate, or results in spermatogenesis.

138. The method of claim 133 or 134, wherein the administration is continuous.

139. The method of claim 138, wherein the disorder is a reproductive disorder, a metabolic disorder, a liver disorder, a bone disorder, a neurodegenerative disorder, abnormal pregnancy, VMS, ora sequela of a neurotropic virus.

140. The method of claim 139, wherein the disorder is a reproductive disorder and administration of the pharmaceutical composition suppresses the reproductive axis, decreases the level of a reproductive health biomarker, decreases follicle growth, or modifies spermatogenesis or the potential for spermatogenesis.

141. The method of any one of claims 133-140, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a therapeutically effective amount.

142. A method of identifying a subject as having congenital HH, the method comprising:(a) providing a reference level of LH;(b) administering to the subject the pharmaceutical composition of any one of claims 110-132, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a stimulating dose, wherein:(i) the stimulating dose is administered intravenously at a dose of between about 0.05 pg / kg to about 90 ng / kg; or(ii) the stimulating dose is administered subcutaneously at a dose of between about 0.5 pg / kg to about 900 ng / kg;(c) measuring at least one level of LH after administration of the stimulating dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the stimulating dose;(d) comparing the reference level of LH to the level of LH in the subject after administration of the stimulating dose; and(e) identifying a subject who has a level of LH after administration of the stimulating dose that is not different from the reference level of LH as having congenital HH.

143. A method of determining whether a patient having a disorder is likely to respond to a therapy comprising a therapeutically effective amount of a kisspeptin analog or a pharmaceuticaliy acceptable salt thereof, the method comprising:(a) providing a reference level of LH;PATENTAttorney Docket No.: 51796-005WO3BWH Ref.: 2024-010-02(b) administering to the subject the pharmaceutical composition of any one of claims 110-132, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a provocative dose;(c) measuring at least one level of LH after administration of the provocative dose, e.g., within about 10, 15, 20, 30, 45, or 60 minutes after administration of the provocative dose;(d) comparing the reference level of LH to the level of LH in the subject after administration of the provocative dose; and(e) identifying a subject who has a level of LH after administration of the provocative dose that is greater than the reference level of LH as likely to respond to the therapy.

144. A method of treating a subject having PCOS, the method comprising administering to the subject a therapeutically effective amount of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

145. A method of treating a subject having PCOS, the method comprising administering to the subject a therapeutically effective amount of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

146. The method of claim 144 or 145, wherein the administration is continuous.

147. A method of treating a subject having HH, the method comprising administering to the subject a therapeutically effective amount of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

148. A method of treating a subject having HH, the method comprising administering to the subject a therapeutically effective amount of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

149. The method of claim 147 or 148, wherein the administration is pulsatile.

150. A method of treating a subject having PCOS, the method comprising administering to the subject the pharmaceutical composition of any one of claims 110-132.

151. The method of claim 150, wherein the administration is continuous.

152. A method of treating a subject having HH, the method comprising administering to the subject the pharmaceutical composition of any one of claims 110-132.

153. The method of claim 152, wherein the administration is pulsatile.

154. The method of any one of claims 150-153, wherein the kisspeptin analog or a pharmaceutically acceptable salt thereof is administered at a therapeutically effective amount.

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