The Emerging Therapeutic Potential of Kisspeptin and Neurokinin B

PCOS is a heterogeneous condition affecting 2% to 13% ( 88 ) of women of reproductive age and is currently diagnosed by the Rotterdam criteria ( 170 ). PCOS is associated with adverse endocrine, reproductive, metabolic (insulin resistance, dyslipidemia), and psychological features ( 170 ). Despite its high prevalence and substantial clinical burden, current treatment strategies for PCOS are suboptimal as they rely on treatment of symptoms rather than the underlying pathophysiological process. The lack of mechanism-based treatments is attributable to the complex and unclear etiology of PCOS, and hence defining the causative factors driving PCOS pathogenesis has been of interest. A cardinal feature of PCOS is androgen excess driven by increased GnRH and LH) pulsatility ( 313 . As hypothalamic ARC KP-neurokinin B-dynorphin (KNDy) neurons regulate GnRH pulse generation and express androgen receptors, KNDy neurons have been implicated in mediating the androgenic effects of PCOS ( Fig. 5 ). Indeed, NKB and KP gene expression are increased in some PCOS-like animal models, thus suggesting that overactivity of KNDy neurons is responsible for the increased GnRH pulsatility observed in PCOS ( 35 , 173 ). Additionally, patients with PCOS with inactivating variants in the NKB gene ( TAC3 ) or NKB receptor (TACR3) have low baseline LH secretion and low LH pulse frequency ( 8 ). However, women with functionally null TAC3 can still conceive and mice lacking NKB (gene or receptor) can generate LH pulses, thus indicating that GnRH impairment is reduced rather than abolished ( 8 , 314 ). This diminished action that NKB inhibition has on GnRH pulsatility is of therapeutic benefit as it enables GnRH pulsatile secretion to be normalized rather than terminated. Thus, there has been great interest in the use of NKB signaling blockade as a therapeutic agent in targeting the central pathophysiology of LH hypersecretion and hyperandrogenism in PCOS. Considering NK3Rs have a high binding affinity for NKB and are highly expressed in humans, antagonists of NK3R have been the preferential developmental agents for PCOS treatment ( 8 ). In peripubertal DHT-induced PCOS mice, NK3R antagonism (MLE4901) improved several metabolic parameters (eg, adiposity, adipocyte hypertrophy, glucose tolerance) but failed to ameliorate reproductive phenotypes (eg, ovarian acyclicity) ( 315 ). NK3R antagonist treatment reduced adipocyte area without affecting food intake, energy expenditure, or locomotor activity, but altered metabolic status by using carbohydrate as the predominant fuel source ( 315 ). In parallel, NK3R antagonism also reduced circulating leptin levels ( 315 ). Although NK3R blockade did not alter fasting glucose levels, NK3R antagonism reduced the effects of DHT-induced hyperglycemia ( 315 ). The lack of a reproductive phenotype may be due to KNDy neurons not being hyperactive in this model of PCOS (chronic DHT), as other models of androgenization (eg, prenatal) do recapitulate KNDy neuronal overactivity. Alternatively, the dose of the NK3R antagonist may have been inadequate and was unable to overcome the elevated androgens observed in this chronic DHT model. In a randomized, multicenter clinical trial, women with PCOS received the NK3R antagonist MLE4901 (also known as AZD4901) at doses of either 20 mg/day, 40 mg/day, or 80 mg/day, or placebo for 28 days ( 131 ) (see Table 2C ). Women receiving 80 mg/day of MLE4901 demonstrated a 52% baseline-adjusted reduction in the AUC of LH, a 79% reduction in basal LH secretion, and an LH pulse decrease of 3.6 pulses/8 hours, compared to placebo ( 131 ). Similarly, total testosterone and free testosterone levels were reduced by 29% and 19%, respectively ( 131 ). These effects were marked following 7 days of treatment and continued to be effective until the end of treatment (28 days) in women who did not ovulate during the study ( 131 ). A more recent study using a similar dose of MLE4901 (40 mg orally twice a day for 7 days) demonstrated a reduction in LH secretion (from 6.5 to 4.0 IU/L), LH pulse frequency (from 0.8 to 0.5 pulses/hour) and FSH levels (2.5 to 2 IU/L) compared to placebo in women with PCOS ( 98 ). Another NK3R antagonist, fezolinetant (60 mg daily or 180 mg daily for 12 weeks), reduced the LH:FSH ratio and suppressed hyperandrogenism in women with PCOS ( 132 ). While both doses reduced LH and FSH throughout the study, only fezolinetant 180 mg daily reduced testosterone levels at all time points, thus indicating a dose-dependent response ( 132 ). Overall, fezolinetant 180 mg/day reduced testosterone by 33%, LH by −10.17 IU/L, and FSH by −1.46 IU/L, while fezolinetant 60 mg/day reduced testosterone by 17% nmol/L, LH by −8.21 IU/L, and FSH by −0.92 IU/L ( 132 ). No changes were observed in E2 and progesterone levels, endometrial thickness, follicle development, or menstrual cycle irregularity over the 12-week study ( 132 ). The lack of ovulation may have been due to the increased suppressive effects of fezolinetant on NK3R signaling. To avoid this, a different dose or shorter duration of therapy of fezolinetant may be more successful in restoring ovulation. Overall, manipulation of neuroendocrine signaling with NK3R antagonism may provide novel therapeutic approaches to treat specific phenotypic features of PCOS. Uterine fibroids and endometriosis are common disorders of the reproductive system affecting up to 80% and 15% of women of reproductive age, respectively ( 316 , 317 ). Uterine fibroids are benign, smooth muscle tumors of the uterus, whereas endometriosis is the presence of endometrial glands or stroma-like lesions outside the uterine cavity ( 316 , 317 ). Both conditions cause severe symptoms including abnormal uterine bleeding, chronic pelvic pain, and infertility ( 316 , 317 ). Women with early-age menarche and short menstrual cycle length are at high risk of developing these conditions, which suggests that continuous exposure of the endometrium and myometrium to estrogen is a key pathological driver of the disease ( 316 , 317 ). Thus, suppressing E2 levels through downregulation of the HPG axis using GnRH modulators (agonists and antagonists) is a clinically validated therapeutic approach for the treatment of these disorders ( 318 , 319 ). However, the approved duration of GnRH therapy is restricted due to its castrating effects and consequent menopausal-like symptoms, including bone loss and vasomotor hot flashes ( 320 , 321 ). An ideal therapy would be one that offers a more refined modulation of the HPG axis and lowers estrogenic drive to endometriosis and fibroid cell growth without causing the adverse events that are associated with current treatments. Indeed, lowering E2 levels to a range between 110 and 184 pmol/L has been recommended to be effective in reducing the symptoms of uterine fibroids and endometriosis ( 322 , 323 ). One such novel therapeutic approach is to use NKB receptor antagonists to reduce LH while preserving FSH secretion (see Fig. 5 ). In OVX ewes, NK3R antagonism (MRK-08) decreased LH pulse frequency while maintaining FSH concentrations ( 324 ). Likewise, in castrated nonhuman primates (Macaca fascicularis) , repeated daily dosing of the NK3R antagonist (ESN364) decreased plasma LH levels, inhibited the LH surge, but did not change FSH concentrations ( 325 ). NK3R blockade also lowered E2 levels in a dose-dependent manner, although nadir levels of E2 were maintained well above menopausal levels ( 325 ). Several NK3R antagonists have also shown similar patterns of gonadotropin secretion (reduced LH with preserved FSH) in healthy women (see Table 2 ). For instance, AZD4901 (also known as MLE4901, formerly AZD2624) reduced E2 levels, endometrial thickness and folliculogenesis ( 326 ) during the follicular phase. In the early mid-follicular phase, AZD4901 resulted in reduced basal LH levels and a delayed LH-surge (by 7 days), without altering LH pulse frequency ( 327 ). Another NKB antagonist, fezolinetant (ESN364), led to a dose-dependent (doses 40-120 mg once daily for 21 days) reduction in LH but not FSH, and reduced endometrial thickness. The dual NK1,3R antagonist elinzanetant (40, 80, and 120 mg once daily) administered orally over a full menstrual cycle safely reduced serum LH in a dose-dependent manner, although in a nonsignificant trend ( 21 ). Progesterone levels consistent with ovulation were reduced, especially during the luteal phase of the cycle ( 21 ). Moreover, the highest dose of 120 mg of elinzanetant once a day lowered E2 to a level ideal for treating uterine fibroids and endometriosis, and lengthened menstrual cycles from 27 to 34 days ( 21 ). Thus, NKB antagonism is a promising treatment option and studies are now required to evaluate their use in women with uterine disorders. Menopause is the complete cessation of menstruation due to ovarian insufficiency and occurs between ages 45 and 55 years ( 328 ). Hot flashes and sweats, collectively known as VMS, are the most debilitating symptom described by more than 80% of women during the menopausal transition ( 20 ). On average, symptoms last for 7 years, but they can persist, with 1 in 10 women experiencing symptoms for up to 12 years ( 328 ). Although hormone replacement therapy or menopausal hormone therapy is an effective treatment for VMS, it is contraindicated in women at high risk of breast and endometrial cancer as well as thromboembolic disease ( 20 ). Therefore, alternative treatments that can safely and effectively alleviate VMS are desired. The median preoptic nucleus (MnPO) of the hypothalamus is the control center for body temperature regulation and downstream thermoregulatory pathways ( 19 ). This thermoregulatory center is dysregulated during menopause and results in the activation of inappropriate heat dissipation responses including VMS ( 19 ). As ARC KNDy neurons project onto both NK3R expressing neurons in the MnPO and GnRH neurons in the median eminence, they have been implicated in the pathogenesis of menopausal VMS (see Fig. 5 ) ( 19 ). E2 deficiency increases LH pulsatility and hot flashes, and this close temporal relationship between temperature and reproduction is mediated by KNDy neuronal activity ( 19 ). Indeed, while ovariectomy (E2-deficient state) increased ARC KNDy gene expression and neuronal hypertrophy, E2 supplementation reversed it ( 24 , 329 , 330 ), suggesting that E2 withdrawal leads to increased KNDy expression in rodents. Furthermore, tract tracing studies revealed that KNDy neurons project to the MnPO (the thermoregulatory center) and GnRH axons in the median eminence of the hypothalamus ( 331 ). The MnPO, which is altered by E2 and temperature, also expresses NK3R mRNA and protein ( 332 ), thus indicating KNDy neurons influence heat dissipation responses through projections to NK3R-expressing neurons in the MnPO. Notably, direct activation of NK3R in the MnPO by an NKB agonist (senktide) reduced core body temperature and activated heat dissipation effectors (tail skin vasodilatation) ( 333 ). Likewise, NKB agonist administration increased tail skin vasodilatation in OVX mice, however, this effect was lost following E2 replacement, suggesting that E2 lowers the sensitivity of the thermoregulatory center to NKB/NK3R signaling ( 334 ). Furthermore, selective toxin ablation of ARC KNDy neurons reduced both cutaneous vasodilatation and LH secretion in female mice ( 335 ), thus supporting the role of KNDy neurons in mediating temperature and reproduction regulation. Additionally, while E2 replacement restored body temperature regulation in OVX rats with intact KNDy neurons, this was not observed in KNDy-ablated OVX rats ( 335 ). These studies strongly support NKB and NK3R signaling as important mediators of postmenopausal flushing, and therefore this pathway could be targeted for future therapies. KNDy neurons in the infundibular nucleus of the hypothalamus are hypertrophied and overexpressed during E2-deficient states such as menopause ( 336 ). Furthermore, genome-wide association studies revealed that menopausal women with VMS had single-nucleotide variations in the TACR3 locus, the gene that encodes NK3R ( 337 ). Additionally, NKB has been shown to induce hot flashes in healthy women to a similar degree as those experienced by women in menopause ( 130 ). These data indicate that antagonism of NKB/NK3R signaling could provide a novel, nonhormone-based approach for the management of menopausal hot flashes. The NK3R antagonist, MLE4901 (oral pavinetant), was the first drug to demonstrate a reduction in the number (by 45%) and severity of weekly hot flashes experienced by menopausal women ( 133 ) (see Table 2D ). Another NK3R antagonist, fezolinetant (ESN364, oral 90 mg twice daily for 12 weeks), reduced VMS scores (fezolinetant: −26.5 vs placebo: −12.2) and improved VMS severity and quality-of-life measures ( 134 ). Furthermore, all doses of fezolinetant (30 mg once daily to 90 mg twice daily), except the lowest one, reduced moderate/severe VMS (>2 per day) by 4 and 12 weeks ( 135 ). A more recent phase 3 trial involving fezolinetant 30 mg or 45 mg once daily reduced the severity of VMS at week 4 (−0.15 to −0.19) and week 12 (−0.24 to −0.2). Furthermore, the improvements in VMS frequency and severity were sustained over 52 weeks ( 137 ). The dual NK1R/NK3R antagonist NT-814 (elinzanetant, dose 150 mg once daily for 2 weeks) also reduced hot flashes (−84%) vs placebo (37%) in menopausal women ( 136 ). While NK1R antagonism alone is ineffectual in attenuating VMS, its antiemetic and anxiolytic effects may benefit the poor sleep quality that women experience during menopause ( 338 ). Indeed, nocturnal awakening due to night sweats in menopause was reduced following NT-814 (−81%) compared to placebo (32%) ( 136 ). NK3R antagonists display distinct side-effect profiles. For instance, MLE4901 was discontinued following its association with transient increases in liver enzymes. Although ESN364 and NT-814 have been associated with headaches, gastrointestinal disturbance, and fatigue, no clinically significant effects on liver enzymes have been reported. Furthermore, E2 levels ( 134 ) and endometrial thickness or hyperplasia ( 135 ) remain unaffected, indicating that NK3R action is independent of effects on ovarian hormones ( 134 ). These data demonstrate that NK3R antagonists provide a safe and efficacious treatment option for managing menopausal women with VMS.

KP was first discovered in 1996 as a tumor-suppressor and initially termed “ metastin ” due to its antimetastatic action in malignant melanoma cell lines ( 22 ). It later acquired the name “kisspeptin” in homage to its discovery in Hershey (Pennsylvania, USA), which is the hometown of the famous chocolate “Hershey's Kisses” ( 22 ). The gene for KP in humans is called “ KISS1 ” with the suppressor sequence denoted by “SS.” While KISS1 is used to indicate the gene in humans, Kiss1 is used for nonhuman KP genes ( 23 ). In 2003, KP's obligatory role in regulating hypothalamic GnRH neuronal function was first described in 2 landmark reports by de Roux et al and Seminara et al ( 1 , 2 ). In humans, KP is predominantly expressed in 2 distinct hypothalamic nuclei: the infundibular nucleus ( 24 , 25 ) (analogous to the ARC in rodents ( 26 )) and the rostral preoptic area (POA) ( 24 , 25 ) (analogous to the POA, including the anteroventral periventricular area, AVPV, and periventricular nucleus (PeN) in rodents ( 26 )). KP is also expressed within the limbic system (in the amygdala, caudate nucleus, cingulate gyrus, globus pallidus, hippocampus, medial and superior frontal gyrus, nucleus accumbens, parahippocampal gyrus, putamen, striatum, substantia nigra, and thalamus) ( 16 , 17 ) and has been recognized to play a role in mood and sexual behaviors. Beyond the brain, KISS1 messenger RNA (mRNA) is also highly expressed in the placenta (particularly by syncytiotrophoblasts ( 27 , 28 )), gonads ( 16 , 29 ), adipose tissue ( 16 ), pancreas ( 16 , 29 ), liver ( 29 ), small intestine ( 29 ), and bone (particularly osteoblasts) ( 30 ) (see Fig. 2 ). The KISS1 gene is mapped to the long arm of chromosome 1 (1q32-q41) and comprises 4 exons of which only 2 are translated ( 31 ). The resultant 145 amino acid prepropeptide is then posttranslationally cleaved into biologically active KP peptides of different amino acid lengths indicated by their suffix, for example, KP-54, -14, -13, and -10 ( 17 , 29 , 31 ). All native KP peptides share a common C-terminal decapeptide sequence, equivalent to KP-10, which includes a terminal RF-amide sequence (Arg-Phe-NH2) ( 17 ). This C-terminal amide sequence is important for the binding and activation of the KP receptor. In particular, amidation of the C-terminal is essential for receptor activation, with higher binding affinities observed with KP-10 (Ki = 0.042 nM) and KP-54 (Ki = 0.34 nM) than a C-terminally unamidated form (Ki = 640 nM) ( 29 ). KP-10 has a shorter terminal half-life than KP-54 (t 1/2 3 vs 28 minutes) ( 7 , 13 , 32 ). Other RF-amide family members such as neuropeptide FF, prolactin (PRL)-releasing peptide, and neuropeptide Y (NPY) do not activate the KP receptor ( 33 ). The KP receptor (encoded by KISS1R ) was described in 1999 ( 23 ), and was previously known as hOT7T175 ( 29 ), AXOR12 ( 16 ), or GPR54 ( 22 ). The KP receptor is a 398-amino acid peptide encoded by a gene on chromosome 19 (19p13.3) with 5 coding exons interrupted by 4 introns ( 16 ). The KP receptor is part of the rhodopsin-like family of G protein–coupled receptors (GPCRs), which is the largest group of GPCRs, and binds its ligand in the binding site within the transmembrane domain ( 16 ). KP has a single high-affinity binding site at the human KP receptor (dissociation constant, Kd, 1.9 ± 0.4 nM using 500 nM of 125I-KP10) ( 17 ) and induces a biphasic response in downstream signaling, with an acute response (lasting ∼5 minutes) and a prolonged phase (lasting >30 minutes) ( 34 ). While KISS1R is expressed in similar areas of the body as KISS1 , it is also expressed at low levels in tissues including the stomach, thymus, spleen, lung, gonads, heart, kidney, adrenal gland, bone, and fetal liver ( 16 , 29 , 35 ) (see Fig. 2 ). During the basal state (in the absence of KP), the KP receptor couples to Gα q/11 at the cell surface, which triggers KP-independent signaling and downstream activation of phospholipase C (PLC), the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG), and intracellular calcium mobilization ( 36 ). In the presence of KP, the KP receptor displays increased Gα q/11 signaling through recruitment of GPCR serine/threonine kinases (GRK2) and β-arrestin from the cytosol to the plasma membrane ( 36 ). GRK2 phosphorylates the KP receptor (at the intracellular loop and carboxyl terminus) and subsequently facilitates the binding of β-arrestin while preventing further coupling to G proteins ( 37 , 38 ). β-Arrestin subsequently induces receptor desensitization by uncoupling the KP receptor from Gα q/11 and simultaneously triggers receptor sequestration by trafficking the desensitized KP receptor to the cell surface clathrin-coated pit ( 36 ). The sequestered KP receptor (linked to β-arrestin) undergoes β-arrestin–dependent signaling, resulting in receptor internalization and the formation of clathrin-coated vesicles ( 36 ). Following this, the KP receptor dissociates from β-arrestin and is either resensitized and recycled back to the cell surface (ready to signal) or targeted for degradation ( 36 ) ( Fig. 3 ). Prolonged KP receptor signaling is also dependent on the continuous influx of calcium into the cell as well as maintaining a dynamic pool of receptors at the cell surface including both recycled and nonrecycled receptors ( 36 ). While the KP receptor mainly signals via Gα q/11 , it can also activate the extracellular signal-regulated kinase 1/2 (ERK1/2) β-arrestin–dependent pathway that also contributes to GnRH secretion ( 39 ). Additionally, the KP receptor can form homodimers, heterodimers, or even oligomers with modified actions ( 40 ). For instance, the KP receptor heterodimerizes with the G protein estrogen receptor, which reduces its expression at the cell surface and decreases KP receptor signaling ( 40 ). KP receptor induces differential responses in downstream signaling. KP has a high-affinity binding site for the human KP receptor and induces a biphasic response in downstream signaling, with an acute (lasting ∼5 minutes) and prolonged response (lasting >30 minutes). KISS1R (coupled to Gαq/11) triggers the activation of PLC and subsequent recruitment of secondary intracellular messengers, IP3 and DAG, which in turn mediate intracellular calcium release. DAG additionally activates PKC and induces downstream phosphorylation of ERK 1 and 2. Kisspeptin binding results in the recruitment of β-arrestin and GPCR serine/threonine kinases (GRK2), which leads to desensitization and internalization of the kisspeptin receptor (through uncoupling of Gαq/11). β-Arrestin traffics the desensitized KISS1R to the clathrin-coated pit resulting in sequestration, which results in β-arrestin–dependent signaling. Internalized KISS1R eventually dissociates from β-arrestin and the majority of kisspeptin receptors become resensitized and traffic back to the cell surface, thus maintaining a continuous pool of receptors at the cell surface which are ready to signal while a lesser population of KISS1R are targeted for degradation. DAG; diacylglycerol; ERK, extracellular signal-related kinase; GRK2, GPCR serine/threonine kinases; IP3, inositol triphosphate; KISS1R, kisspeptin receptor gene; KP, kisspeptin; PKC; protein kinase C; PLC, phospholipase C. Figure created with BioRender.com . The KP receptor is vulnerable to tachyphylaxis, whereby the receptor response is reduced following repeated doses or continuous high doses of KP administration ( 37 ). For instance, in agonadal juvenile and adult male monkeys, a 98-hour intravenous (IV) infusion of KP-10 induced a maximal LH response at 3 hours; however, a rapid decline then followed by 12 hours ( 41 , 42 ). Moreover, an additional bolus of GnRH but not KP-10 resulted in an LH rise, thus indicating that tachyphylaxis is occurring at the level of the KP receptor ( 41 , 42 ). Likewise, in women with hypothalamic amenorrhea (HA), twice-daily administration of KP-54 resulted in a reduced LH response within a few days ( 43 ). Interestingly, KP's responsiveness was maintained with a twice-weekly dosing interval suggesting that chronic stimulation with KP is possible using an appropriate dosing protocol ( 43 ). Furthermore, although tachyphylaxis occurs after persistent high-dose exogenous KP, this may not be the case with physiological endogenous KP. Indeed, optogenetic activation of KP neurons in the rostral periventricular area of the third ventricle (RP3V) of female mice can persistently stimulate GnRH neuronal firing ( 44 ). NKB was first discovered as a central regulator of reproduction in 2009, whereby loss-of-function variants in either NKB or its receptor (NK3R) were identified in 4 of 9 multiplex families affected by hypogonadotropic hypogonadism using genome-wide single-nucleotide polymorphism (now called single-nucleotide variation) analysis ( 8 ). In humans, NKB is predominantly expressed in the infundibular nucleus, anterior hypothalamic area septal region, diagonal band of Broca, bed nucleus of the stria terminalis, amygdala, and neocortex ( 45 ). The gene encoding NKB ( TAC3 in higher primates and Tac2 in rodents) is located on chromosome 12 and is divided into 7 exons, 5 of which are translated to form the preprotachykinin B peptide ( 46-48 ). Following proteolytic cleavage, this precursor peptide leads to, first, proneurokinin B, and then NKB (initially contained in exon 5) ( 46 ). NKB belongs to the tachykinin family of peptides, which is characterized by a common C-terminal amino-acid sequence (Phe-X-Gly-Leu-Met-NH2) and includes substance P, neurokinin A, and NKB, as well as neuropeptide K, neuropeptide γ, and hemokinin-1 ( 46 , 48 ). Three tachykinin receptors have been identified, NK1R, NK2R, and NK3R, with the latter having a longer amino acid sequence ( 46 ). The genes encoding the 3 tachykinin receptors are all divided into 5 exons with identical distribution of intronic sequences ( 46 ). NKB is an agonist for all 3 receptors; however, it exhibits strong preferential binding for NK3R (encoded by TACR3 ) ( 49 , 50 ). Following NKB binding, NK3Rs are activated and result in increased intracellular Ca 2+ (through inositol phospholipid hydrolysis) and increased intracellular cyclic adenosine monophosphate levels (through adenylate cyclase activation), and are then internalized ( 51 ). Like NKB, NK3R is also expressed within the central nervous system and spinal cord, although it has also been reported in the uterus, mesenteric vein, gut neurons, and placenta ( 45 ). NK3Rs also display species differences and exert differing actions. For instance, while NK3R antagonists have similar potency on NK3Rs in the gerbil, guinea pig, dog, and human, they have lower activity on NK3R in the rat and mouse ( 52 ).

KP and upstream NKB govern the reproductive endocrine axis through their critical role in regulating GnRH neuronal activity and stimulating GnRH pulsatile secretion. Their fundamental role in reproductive hormone secretion has opened several avenues for their use in diagnosing and treating several pubertal, reproductive, metabolic, bone, and behavioral disorders. For instance, KP induces lower LH increases in patients with CHH than in those with CDGP or in healthy controls. Additionally, higher circulating KP levels are observed in CPP, thus highlighting KP's utility in diagnosing puberty-related disorders. KP levels rise linearly with advancing pregnancy, and therefore it could be developed as a promising marker for predicting pregnancy complications. In particular, the reduced KP levels associated with miscarriage and IUGR could enable its use in risk-stratifying women presenting with possible complications during pregnancy. MAFLD/NASH is associated with upregulated hepatic-KP signaling and increased circulating KP concentrations; therefore, KP measurements could potentially be used to discriminate patients with MAFLD/NASH from healthy controls. Thus, assessing gonadotropin responses to KP or measuring circulating KP levels directly could aid in the diagnosis of common disorders. However, further studies to validate KP's diagnostic accuracy are necessary. KP-based therapies have been extensively explored over the past decade. In hypogonadal disorders such as HA, hyperprolactinemia, and diabetes-induced hypogonadism, KP induces gonadotropin increases that could restore reproductive function. KP and KP receptor agonists also mirror the physiological ovulatory mid-cycle LH surge and thus could be used therapeutically to induce oocyte maturation during IVF protocols in women seeking fertility. Further studies evaluating KP's safety and efficacy in comparison to current agents, especially in women at high risk of OHSS, are warranted. The intricate connections between KP neurons and hypothalamic neurons involved in appetite regulation have implicated a potential role for KP in obesity-related disorders. Although absence of KP has been associated with increased BW, KP's effects on appetite in animals and humans remain unclear. KP receptor agonism has also been shown to alleviate hepatic steatosis and fibrosis and thus could play an important role in suppressing the progression of hepatic lipogenesis in patients with MAFLD. With regard to bone metabolism, KP enhances osteoblastogenesis and inhibits osteoclast activity in vitro, and therefore could be used as a complementary treatment for osteoporosis. KP also has potential as a therapy for men and women with psychosexual dysfunction, as it has been shown to enhance sexual brain processing and associated physiological and behavioral measures of sexual function in patients with distressing low sexual desire. NKB antagonism, in particular potent NK3 receptor antagonists, have emerged as an advantageous therapeutic tool for treating PCOS, uterine fibroids, and endometriosis through their unique ability to partially suppress (and not abolish) the reproductive endocrine axis. Additionally, the critical interaction between NKB and the hypothalamic thermoregulatory center has resulted in the development of NKB antagonists as efficacious nonhormonal treatment options for women with menopausal VMS. Since the pivotal discoveries of KP and NKB's role in reproduction in 2003 and 2009, respectively, there has been an abundance of basic science and translational studies demonstrating their function in the pathophysiology of several disorders including reproduction, metabolism, bone, and behavior. The wealth of evidence accumulated over the past 2 decades, alongside the development of potent KP and NKB antagonist-based therapies, has provided the opportunity for these peptide hormones to be investigated as promising diagnostic and management tools in the coming years.

In healthy adult men, acute administration of KP-54 induced dose-dependent increases in circulating LH and, to a lesser degree, FSH ( 13 ) ( Table 1A ). In particular, KP-54 (IV infusion 0.24 nmol/kg/h over 90 minutes) increased mean LH levels 2.6-fold higher than placebo ( 13 ). Similarly, an IV bolus of KP-10 (0.77 nmol/kg) potently evoked LH secretion from 4.1 to 12.4 ± IU/L and a continuous IV infusion (3.07 nmol/kg/h) of KP-10 led to persistent LH secretion over 22.5 hours ( 76 ). The shorter isoform, KP-10, has a briefer half-life and duration of gonadotropin release, with LH levels rising within 30 to 40 minutes after an IV bolus administration (0.3 to 1.0 nmol/kg) ( 77 ). In a direct equimolar comparison between KP-54 and KP-10 (hypothalamic stimulation) against GnRH (pituitary stimulation), LH and FSH responses were greater following GnRH, then KP-54, and then KP-10 ( 32 ). Although GnRH is more potent than KP, KP is hypothesized to induce the release of GnRH from a limited endogenous pool ( 126 ), which could be preferable when stimulating reproductive hormone secretion in a clinical context where there is an unwanted risk of overstimulation. Clinical trials involving kisspeptin LH (5.2 ± 0.8 to 14.1 ± 1.7 IU/L) LH pulse frequency (0.7 ± 0.1 to 1.0 ± 0.2 pulses/h) higher mean postglucose load insulin secretion 4.1 µU/mL higher disposition index (IVGTT-DI) 2768 ± 484 units Abbreviations: AUC, area under the curve; AUCROC, area under receiver operating characteristic curve; CDGP, constitutional delay of growth and puberty; CHH, congenital hypogonadotropic hypogonadism; CPP, central precocious puberty; E2, estradiol; EP, ectopic pregnancy; F, female; FSH, follicle-stimulating hormone; GA, gestational age; GABA, γ-aminobutyric acid; GDM, gestational diabetes mellitus; fMRI, functional magnetic resonance imaging; GnRH, gonadotropin-releasing hormone; HA, hypothalamic amenorrhea; HCG, human chorionic gonadotropin, IUGR, intrauterine growth restriction; IV, intravenous; IVF, in vitro fertilization; IVGTT-DI, intravenous glucose tolerance test—disposition index; KP, kisspeptin; LH, luteinizing hormone; M, male; NAFL, nonalcoholic fatty liver; MAFLD, metabolic fatty liver disease; NASH, nonalcoholic steatohepatitis; OHSS, ovarian hyperstimulation syndrome; PE, preeclampsia; PRL, prolactin; RCT, randomized controlled trial; SC, subcutaneous, SGA, small for gestational age. The pulsatile secretion of GnRH is critical for reproductive function. Indeed, KP-10 (IV infusion 3.07 nmol/kg/hour over 22.5 hours) increased LH pulse frequency from 0.7 to 1.0 pulses per 1 hour in men ( 76 ). KP has also been shown to reset the “GnRH pulse generator” in healthy men but not women ( 75 ). KP-10 (IV bolus 0.24 nmol/kg) resulted in sustained GnRH neuronal activation lasting approximately 17 minutes and immediately induced an LH pulse (irrespective of the timing of the preceding endogenous pulse) and increased the LH pulse amplitude by 2.4-fold ( 75 ). Furthermore, the following native pulse was delayed by an interval approximating the usual interpulse interval, indicating that KP-10 had reset the schedule of pulses ( 75 ). In healthy premenopausal women, acute administration of KP-54 (subcutaneous; SC bolus 0.4 nmol/kg) increased circulating LH during all phases of the menstrual cycle, with the highest LH levels being observed during the preovulatory (20.64 ± 2.91 IU/L) compared to the follicular (0.12 ± 0.17 IU/L) or luteal (2.17 ± 0.79 IU/L) phases of the cycle ( 14 ) (Table 1B ). Similarly, while KP-10 (IV bolus 10 nmol/kg) increased gonadotropins during the preovulatory phase (mean area under the curve; AUC: LH = 30.3 ± 7.7 IU/L, FSH = 6.9 ± 0.9 IU/L), it was least sensitive during the follicular phase ( 77 ). However, KP-54 (SC bolus 0.30-0.60 nmol/kg) can still increase LH pulsatility (by 2.33 pulses per 4 hours) during the follicular phase in premenopausal women ( 80 ). The effects of chronic KP administration have also been evaluated in healthy women. For instance, twice-daily KP54 (SC bolus 6.4 nmol/kg) injections for 1 week increased maximal change in LH from baseline on day 7 (8.6 ± 3.4 IU/L), day 11 (8.3 ± 2.4 IU/L), and day 14 (12.7 ± 8.1 IU/L) of the menstrual cycle ( 81 ). Furthermore, an infusion of KP54 (SC 0.3-1.0 nmol/kg/hour over 8 hours) induced a mean LH increase (>8 IU/L) during the early-follicular phase ( 82 ). KP receptor analogues have been shown to stimulate longer LH responses and are similarly cost-effective to manufacture ( 83 ). For example, MVT-602 (formerly known as TAK-448) generated similar LH amplitude responses as KP-54 during the follicular phase, but the peak LH level was later at approximately 21 hours compared to KP-54 (∼5-hours), resulting in a 4-fold increase in the AUC of LH secretion ( 83 ). Although NKB administration increased LH concentration in male juvenile monkeys ( 127 ), no significant changes in circulating LH, FSH, or testosterone concentrations were observed in healthy men during either a 90-minute (doses 0.04-5.12 nmol/kg/hour), a 4-hour (doses 2.56 and 5.12 nmol/kg/hour), or 8-hour (dose 5.12 nmol/kg/hour) IV infusion of NKB ( 128 ) ( Table 2A ). Similarly, no significant differences in either LH pulsatility or mean LH, FSH, or E2 levels have been observed in healthy premenopausal women ( 128 ). Interestingly, NKB induced vasoactive effects in healthy men (IV infusion 10.24 nmol/kg/hour) ( 128 ) and in 80% of premenopausal healthy women (IV infusion 5.12 nmol/kg/hour) ( 130 ) (see Table 2B ). These data highlighted the potential of NKB-signaling blockade for the management of vasomotor symptoms (VMS) in postmenopausal women and/or following cancer therapy (eg, breast or prostate cancer). Thereafter, several safe and efficacious NKB receptor (mainly NK3R) antagonists have been investigated for this indication, which are discussed in later sections of this review. Recent in vitro data have also suggested that the NKB receptor, NK1R, may have a role in promoting breast ( 128 ) and non–small cell lung cancer ( 138 ), hence it is possible that antagonists against NK1R could have a therapeutic role in addition to the relief of VMS. Clinical trials involving neurokinin B and NKB antagonism LH AUC by 52.0% (95% CI, 29.6%-67.3%) LH pulses by 3.55 LH pulses/8 h (95% CI, 2.0-5.1) Total testosterone by 28.7% (95% CI, 13.9%-40.9%) LH (4.0 ± 0.4 vs 6.5 ± 0.8 IU/L) LH pulse frequency (0.5 ± 0.1 vs 0.8 ± 0.1 pulses/h) FSH secretion (2.0 ± 0.3 vs 2.5 ± 0.4 IU/L) Abbreviations: AUC, area under the curve; E2, estradiol; FSH, follicle-stimulating hormone; IV, intravenous; KP, kisspeptin; LH, luteinizing hormone; NK3R, neurokinin 3 receptor; NKB, neurokinin B; PCOS, polycystic ovary syndrome; VMS, vasomotor symptoms.

KP neuronal bodies are located in 2 discrete hypothalamic nuclei in rodents: the ARC, and the RP3V, which includes the anteroventral periventricular (AVPV) and periventricular (PeN) nuclei ( 26 ). The analogous regions in humans are the infundibular nucleus and the rostral POA, respectively ( 24 , 25 ). Both ARC and RP3V KP neuronal populations innervate GnRH neurons and are responsible for regulating GnRH pulsatility and the mid-cycle LH surge, respectively ( 53-57 ) (see Fig. 1 ). The number and distribution of KP neurons differs between sexes. For instance, while female mice require high hypothalamic Kiss1 expression levels to preserve fertility, male mice need only 5% of Kiss1 expression ( 58 ). In rodents and sheep, the proportion of KP neurons in both the AVPV ( 59 ) and ARC ( 26 , 60 ) is greater in females than males. Consistent with this, the number of KP immune-positive cell bodies found in the infundibulum of human brain autopsies is 7-fold higher in women compared to men ( 24 , 25 ). KP neurons in the ARC nucleus coexpress NKB and Dyn and are hence known as Kisspeptin-Neurokinin-Dynorphin (KNDy) neurons ( 61 ). KNDy neurons are regulated in an autocrine/paracrine manner, with NKB stimulating (via NKB receptor—mainly TAC3R) ( 61 ) and Dyn inhibiting (via kappa opioid receptor) ( 25 ) neuronal activity. This synchronized episodic action results in KP release, which in turn activates distal dendrons of GnRH neurons and leads to the secretion of GnRH pulses ( 62 ). Considering KP receptors are highly expressed within GnRH neurons and absent in KNDy neurons, KP's action predominantly occurs via GnRH neurons ( 62 ). ARC-KP neurons are key regulators of GnRH pulsatile secretion and are referred to as the “GnRH pulse generator” ( 9 , 60 ). Indeed, optogenetic activation of the channel rhodopsin expressing ARC KP-neurons in Kiss1-Cre mice induced pulsatile LH secretion, whereas inhibition suppressed it ( 63 , 64 ). Likewise, knockout of greater than 90% of ARC Kiss1 neurons resulted in marked suppression of LH pulses in ovariectomized (OVX) female rats ( 65 ). However, a recent report has challenged the KNDy hypothesis suggesting that synchronization within the ARC is dependent on a “glutamate 2-transition” mechanism in male mice ( 66 ). In this model, the first transition is dependent on glutamate but gated by Dyn tone to initiate neuron synchronization, and the second transition is dependent on NKB, which potentiates that synchronization ( 66 ). ARC-KP neurons are tightly regulated by intricate feedback mechanisms in response to several modulators, including sex steroids such as estradiol (E2). In the presence of low circulating E2 levels, a negative feedback effect is exerted on ARC-KP neurons. Indeed, a recent RNA sequencing study in mice identified 1583 estrogen-responsive genes in the ARC with the majority of the genes being suppressed in response to a low E2 environment ( 67 ). While negative feedback is present continuously in males, in females it occurs during most of the follicular and luteal phases of the menstrual cycle ( 68 ). Negative feedback in response to E2 is mediated by the “nonclassic pathway,” whereby the interaction between E2 and its receptor (ERα) results in the recruitment of estrogen response element (ERE)-independent transcriptions factors ( 69 , 70 ). E2-ERα signaling leads to Kiss1 promoter histone deacetylation, which inhibits chromatin loop formation between the Kiss1 promoter and the Kiss1 gene enhancer, resulting in reduced ARC-specific Kiss1 gene expression. KP neurons in the RP3V, which includes the AVPV and PeN, innervate the soma and proximal dendrites of GnRH neurons to stimulate GnRH secretion ( 67 ). This KP neuronal network is mainly regulated by positive feedback from higher levels of E2. In the presence of high E2, RP3V-KP neurons in rodents (rostral POA neurons in humans) continuously produce GnRH leading to an LH surge ( 71 , 72 ), which occurs during the proestrus phase in rodents and during the late follicular phase (mid-cycle) in women ( 73 ). Of note, 222 genes within RP3V-KP neurons are upregulated in response to high E2 levels, demonstrating their importance to facilitating positive feedback ( 67 ). The mechanism responsible for positive feedback predominantly involves E2-ERα signaling and recruitment of cofactors to ERE in the “classic pathway” ( 69 , 70 ). In contrast to the ARC, Kiss1 promoters within the AVPV undergo histone acetylation and subsequent increased AVPV-specific Kiss1 gene expression. The role of these neurons remains uncertain in male mammals that have lower KP expression than female mammals in RP3V-KP neurons ( 74 ).