Intranasal kisspeptin administration rapidly stimulates gonadotropin release in humans

Abstract

Background: Kisspeptin administration by intravenous or subcutaneous routes activates hypothalamic gonadotropin-releasing hormone (GnRH) neurons and is being developed to treat reproductive disorders. However, these invasive routes markedly limit patient acceptability and clinical use. Recent rodent data has identified a large GnRH population within the olfactory system communicating directly with hypothalamic GnRH neurons. Intranasal kisspeptin administration may be able to capitalise on this novel pathway and thus offer a potential non-invasive approach to stimulate reproductive hormones. Herein, we examine intranasal kisspeptin using human, pharmaceutical, and rodent studies.

Methods: Reproductive hormone profiles were measured after intranasal kisspeptin administration in healthy volunteers and patients with reproductive disorders as part of a randomised, double-blinded, crossover, placebo-controlled clinical study. Pharmaceutical testing evaluated the chemical stability and nasal kisspeptin delivery, and rodent studies provided mechanistic insight.

Findings: Intranasal kisspeptin-54 rapidly stimulates gonadotropin release in healthy men and women, and in patients with a common reproductive disorder (hypothalamic amenorrhoea), without any side effects or adverse events encountered. Specifically, intranasal kisspeptin (at 12.8 nmol/kg) induced clinically-significant mean maximal increases above baseline in serum luteinising hormone in all study groups: 4.4 ± 0.6 IU/L (mean difference = 3.1 IU/L [95% CI, 1.2-4.9], P = 0.002 vs. placebo) in healthy men; 1.4 ± 0.3 IU/L (mean difference = 1.0 IU/L [95% CI, 0.4-1.7], P = 0.004 vs. placebo) in healthy women; 4.4 ± 0.2 IU/L (mean difference = 4.3 IU/L [95% CI, 2.7-6.0], P < 0.001 vs. placebo) in patients with hypothalamic amenorrhoea. Kisspeptin-54 was delivered effectively via nasal spray and was stable for up to 60 days at 4 °C. Mirroring the human effects, intranasal kisspeptin-54 in adult C57BL/6J male mice stimulates luteinising hormone release. Further mechanistic insights reveal the accumulation of fluorescently-tagged kisspeptin in the olfactory epithelium, as well as the presence of kisspeptin receptors in olfactory bulb GnRH neurons, implicating the involvement of these extra-hypothalamic GnRH neurons in the pathway mediating intranasal kisspeptin's effects on reproductive hormones.

Interpretation: We demonstrate the clinical potential for intranasal kisspeptin delivery as the first non-invasive method to robustly and safely stimulate gonadotropins with kisspeptin and potentially transform the management of reproductive disorders.

Funding: National Institute for Health and Care Research (NIHR)/NIHR Imperial Biomedical Research Centre/Medical Research Council (MRC).

Keywords: Fertility; Kisspeptin; Neuroendocrinology; Neuropeptides; Reproduction.

Fig. 1

Intranasal administration of kisspeptin stimulates reproductive hormone secretion in healthy men. (a) Protocol schematic: Participants completed five study visits each, receiving the following five interventions via the intranasal route: 3.2, 6.4, 12.8, and 25.6 nmol/kg of kisspeptin-54, and 0.9% saline (placebo). After self-administration of kisspeptin-54 or placebo at timepoint 0 min, serum levels of LH, FSH, and testosterone were measured every 15 min for 4 h (b, e and h): Mean (±SEM) change from baseline in serum LH (IU/L) (b), serum FSH (IU/L) (e), and serum testosterone (nmol/L) (h) in healthy men receiving intranasal administration of kisspeptin-54 or placebo. Groups were compared by two-way ANOVA with post-hoc Bonferroni multiple comparison test (asterisk denotes statistical significance for individual kisspeptin doses vs. placebo administration). (c, f and i): Mean (±SEM) area under the curve (AUC) of the change in serum LH (h.IU/L) (c), serum FSH (h.IU/L) (f), and serum testosterone (h.nmol/L) (i) after intranasal administration of kisspeptin-54 or placebo in healthy men. Groups were compared by one-way ANOVA with post-hoc Bonferroni multiple comparison test (asterisk denotes statistical significance for individual kisspeptin doses vs. placebo administration). (d, g and j) Mean (±SEM) maximum increase from baseline in serum LH (IU/L) (d), serum FSH (IU/L) (g), and serum testosterone (nmol/L) (j) after intranasal administration of kisspeptin-54 or placebo in healthy men. Groups were compared by one-way ANOVA with post-hoc Bonferroni multiple comparison test (asterisk denotes statistical significance for individual kisspeptin doses vs. placebo administration). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 12.

Fig. 2

Intranasal administration of kisspeptin stimulates reproductive hormone secretion in healthy women. (a) Protocol schematic: Participants completed two study visits each, receiving 12.8 nmol/kg of kisspeptin-54 and placebo via the intranasal route. After self-administration of kisspeptin-54 or placebo at timepoint 0 min, serum levels of LH, FSH, oestradiol, and progesterone were measured every 15 min for 4 h. (b and e): Mean (±SEM) change from baseline in serum LH (IU/L) (b) and serum FSH (IU/L) (e) in healthy women receiving intranasal administration of kisspeptin-54 or placebo. Groups were compared by two-way ANOVA with post-hoc Bonferroni multiple comparison test (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). (c and f): Mean (±SEM) area under the curve (AUC) of the change in serum LH (h.IU/L) (c) and serum FSH (h.IU/L) (f) after intranasal administration of kisspeptin-54 or placebo. Groups were compared by paired t-tests (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). (d and g): Mean (±SEM) maximum increase from baseline in serum LH (IU/L) (d) and serum FSH (IU/L) (g) after intranasal administration of kisspeptin-54 or placebo. Groups were compared by paired t-tests (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). ∗P < 0.05, ∗∗P < 0.01. N = 12.

Fig. 3

Intranasal administration of kisspeptin stimulates reproductive hormone secretion in patients with hypothalamic amenorrhoea. (a) Protocol schematic: Participants completed two study visits each, receiving 12.8 nmol/kg of kisspeptin-54 and placebo via the intranasal route. After self-administration of kisspeptin-54 or placebo at timepoint 0 min, serum levels of LH, FSH, oestradiol, and progesterone were measured every 15 min for 4 h. (b and e): Mean (±SEM) change from baseline in serum LH (IU/L) (b) and serum FSH (IU/L) (e) in patients with HA receiving intranasal administration of kisspeptin-54 or placebo. Groups were compared by two-way ANOVA with post-hoc Bonferroni multiple comparison test (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). (c and f): Mean (±SEM) area under the curve (AUC) of the change in serum LH (h.IU/L) (c) and serum FSH (h.IU/L) (f) after intranasal administration of kisspeptin-54 or placebo. Groups were compared by paired t-tests (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). (d and g): Mean (±SEM) maximum increase from baseline in serum LH (IU/L) (d) and serum FSH (IU/L) (g) after intranasal administration of kisspeptin-54 or placebo. Groups were compared by paired t-tests (asterisk denotes statistical significance for kisspeptin 12.8 nmol/kg vs. placebo administration). (h and i): Mean (±SEM) maximum increase from baseline in serum LH (IU/L) (h) and serum FSH (IU/L) (i) after intranasal administration of kisspeptin-54 at 12.8 nmol/kg in healthy women (presented in red) and women with hypothalamic amenorrhoea (presented in blue). Groups were compared by unpaired t-tests (asterisk denotes statistical significance for gonadotropin responses in healthy women vs. women with hypothalamic amenorrhoea). ∗P < 0.05, ∗∗∗P < 0.001. N = 10 (women with HA) and N = 12 (healthy women).

Fig. 4

Pharmaceutical studies. (a) Deposition profile of kisspeptin-54 in 0.9% saline solution nasal spray. Proportion of recovered dose depositing in the nasal vestibule, turbinates, olfactory region, and nasopharynx sections of the Alberta Idealised Nasal Inlet, an idealised replica of the adult nasal cavity. Recovery was 96.5% of the emitted dose; data represent N = 3 with SEM. (b and c) Size and shape of kisspeptin-54 powder particles measured using the Morphologi 4. (b) Shape of representative kisspeptin-54 powder particles in the 20–50-micron circular equivalent diameter size range. (c) Particle size distribution of kisspeptin-54 powder particles (N = 55,421), showing 0.5–113 μm circular equivalent diameter range and over 40% of the particles in the optimal 20–50 μm range for nasal delivery.

Fig. 5

Intranasal administration of kisspeptin stimulates LH release in a dose-dependent manner in adult male mice. (a) Protocol schematic: A single volume of 30 μL of either 0.9% saline (placebo) or different concentrations of kisspeptin-54 (1, 3, 12.8, 30, or 50 nM solution; equivalent to 0.001–0.05 nmol/kg) was delivered into one nostril at timepoint 0 min. LH levels were assessed using tail-tip serial blood sampling, with the first sampling point at timepoint −5 min, followed by sampling every 10 min over 70 min. (b) Mean (±SEM) plasma LH (ng/mL) in adult male mice receiving intranasal administration of placebo or kisspeptin-54 at timepoint 0 min (dotted line). Shaded grey area indicates putative presence of the peptide in circulation. Group analysis revealed the presence of a dose-dependent effect of kisspeptin-54 on LH levels with a pronounced induction of LH secretion by 12 nM (^ΦP < 0.05), 30 nM (∗∗∗∗P < 0.0001, ∗∗P < 0.01, ∗P < 0.05) and 50 nM (^####P < 0.0001, ^#P < 0.05) compared with 0.9% saline group levels (two-way ANOVA with Fisher’s LSD test). N = 5 per group except 12.8 nM where N = 6 and 30 nM where N = 7. (c) Integrated LH response to kisspeptin-54 is represented by area under the curve (A.U.C.) at different doses of kisspeptin-54. Groups were compared by one-way ANOVA with Fisher’s LSD test (∗∗P < 0.01, ∗P < 0.05). N = 5 per group except 12.8 nM where N = 6 and 30 nM where N = 7. (d) Experimental protocol schematic: Mice were injected intranasally with a fluorescently tagged kisspeptin-54 (kiss-54-D2) and sacrificed 25 min later to assess compound diffusion into the brain. After sacrifice and transcardial perfusion with 4% paraformaldehyde dissolved in PBS 0.01 M, pH 7.4, heads were removed and decalcified prior to whole-head tissue-clearing and light-sheet microscopy imaging. (e) Mean (±SEM) plasma LH (ng/mL) in adult male mice after intranasal administration of fluorescently-tagged kisspeptin-54 (kiss-54-D2) at 1 nMol. Intranasal kiss-54-D2 was administered at timepoint 0 min, with blood sampling for plasma LH at 5- and 25-min post-administration. N = 2. (f) Solvent-based tissue clearing, and light-sheet imaging revealed the distribution of kisspeptin-54-D2 (red) in a 3.5 mm-thick 3D sagittal projection of an optically cleared mouse head. (g and h). Single-plane optical re-slices of the 3D volume facilitates precise visualisation and confirmed that kisspeptin-54-D2 remained restricted to the nasal compartment without reaching deeper brain structures. White = tissue auto-fluorescence, scale bars = 1 mm. 3v, third ventricle; crb, cerebellum; cx, cortex; hip, hippocampus; hyp, hypothalamus; me, median eminence; ob, olfactory bulb; oe, olfactory epithelium; pit, pituitary; sep, septum; th, thalamus.

Fig. 6

GnRH neurons located in the olfactory bulb of male mice express Kiss1r mRNA. (a) Fluorescent in situ hybridisation (RNAScope) for kisspeptin receptor mRNA (Kiss1r, red) and GnRH-1 mRNA (Gnrh1, white) shows Kiss1r-expressing GnRH neurons in the murine olfactory bulb of both naïve (b) and sexually experienced males (c). Scale bars = 20 μm (a); 10 μm (b, c). (a) boxed area magnified in (b). Gml, glomerular layer; onl, olfactory nerve layer. (d) Quantitative analysis showing that the entire population of GnRH neurons located in the olfactory bulb expresses Kiss1r in both naïve and sexually experienced males. N = 4.