Inhibiting Kiss1 Neurons With Kappa Opioid Receptor Agonists to Treat Polycystic Ovary Syndrome and Vasomotor Symptoms

Abstract

Context: Recent evidence suggests that vasomotor symptoms (VMS) or hot flashes in the postmenopausal reproductive state and polycystic ovary syndrome (PCOS) in the premenopausal reproductive state emanate from the hyperactivity of Kiss1 neurons in the hypothalamic infundibular/arcuate nucleus (KNDy neurons).

Objective: We demonstrate in 2 murine models simulating menopause and PCOS that a peripherally restricted kappa receptor agonist (PRKA) inhibits hyperactive KNDy neurons (accessible from outside the blood-brain barrier) and impedes their downstream effects.

Design: Case/control.

Setting: Academic medical center.

Participants: Mice.

Interventions: Administration of peripherally restricted kappa receptor agonists and frequent blood sampling to determine hormone release and body temperature.

Main outcome measures: LH pulse parameters and body temperature.

Results: First, chronic administration of a PRKA to bilaterally ovariectomized mice with experimentally induced hyperactivity of KNDy neurons reduces the animals' elevated body temperature, mean plasma LH level, and mean peak LH per pulse. Second, chronic administration of a PRKA to a murine model of PCOS, having elevated plasma testosterone levels and irregular ovarian cycles, suppresses circulating levels of LH and testosterone and restores normal ovarian cyclicity.

Conclusion: The inhibition of kisspeptin neuronal activity by activation of kappa receptors shows promise as a novel therapeutic approach to treat both VMS and PCOS in humans.

Keywords: KOR; PCOS; hot flashes; hypothalamus; menopause.

Figure 1.

KOR agonists inhibit LH secretion without central effects. The synthetic KOR agonist (±) U50 488 (A) and the peripherally restricted KOR agonist [D-Ala²]-Dynorphin A (1-13)-NH₂ (AlaDyn) (B) significantly decrease LH release. Pretreatment with the KOR antagonist nor-BNI blocked the action of AlaDyn (C). The inhibitory effect on LH was replicated by the peripherally restricted KOR agonist (PRKA) difelikefalin (D). On a tail suspension test, neither (±) U50 488 nor difelikefalin induced any discernable central effects as assessed by the tail suspension test at the doses tested (E). Values are presented as median (middle line) ± max/min (box and whiskers). One-way ANOVA followed by Dunnett’s post hoc test: (A) n = 6, F (4, 24) 0.4844; (B) n = 6, F (3, 20) 2.249; (C) n = 6, F (2, 15) 0.4753; (D) n = 3, F (3, 8) 1.611. For (E) n = 8, 2-way ANOVA followed by Dunnett’s test, F (2, 21) 0.028. Statistical values are compared with time 0 (A, B, and D), the baseline LH (C) or the control group (E): *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.001.

Figure 2.

PRKAs increase the response of GnRH neurons to kisspeptin. AlaDyn (A) and difelikefalin (B) amplified the response of GnRH neurons and gonadotropes to mouse kisspeptin-10. Values expressed as mean ± SEM. Groups with different letters are significantly different. Two-way ANOVA with repeated measures and Fisher’s post hoc test. In (A) n = 4-5/group, F (2, 10) = 9.502 P = 0.0049; for (B) n = 4/group, F (2, 9) = 12.85, P = 0.0023.

Figure 3.

Effect of 24 hours of difelikefalin treatment on LH pulsatility and body temperature in OVX mice. OVX mice were implanted with osmotic minipumps delivering target 10 nM, 30 nM, or 100 nM steady-state plasma concentrations of difelikefalin for a week (n = 5/group). (A) Depicts 2 representative samples from each group. (B) Baseline LH release during 180 minutes, F (3, 15) = 10.73, P = 0.0005. (C) Peak of LH pulses observed during 180 minutes, F (3, 16) = 2.109, P = 0.1393. (D) Number of LH pulses in 180 minutes, F (3, 16) = 2.492, P = 0.097. One-way ANOVA followed by Fisher’s post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 4.

Effects of 1 week treatment with difelikefalin on LH pulsatility and body temperature in OVX mice. OVX mice were implanted with osmotic minipumps delivering target 10 nM, 30 nM, or 100 nM steady-state plasma concentrations of difelikefalin for a week (n = 5/group). (A) Depicts 2 representative samples of LH and body temperature (Tb) from each treatment group (n = 5/group). (B) Baseline LH release during 180 minutes, F (3, 15) = 2.061, P = 0.1486. (C) Peak of LH pulses observed during 180 minutes, F (3, 16) = 1.992, P = 0.1558. (D) Number of LH pulses in 180 minutes, F (3, 16) =0.8015, P = 0.5111. One-way ANOVA followed by Fisher’s post hoc test. *P < 0.05.

Figure 5.

Effect of difelikefalin on body temperature and body weight. Body temperature (Tb) was recorded every 10 minutes for 180 minutes at 2 time points: 24 hours and 1 week after initiation of treatment (A). Target 100 nM plasma concentration of difelikefalin significantly decreased Tb at both time points, F (3, 24) = 6.076, P = 0.0032, 2-way ANOVA followed by Fisher’s post hoc test. (B) Difelikefalin reduces the rate of increase in body weight observed following OVX, F (3, 16) = 3.064, P = 0.0582, 1-way ANOVA followed by Fisher’s post hoc test. *P < 0.05, ***P < 0.001.

Figure 6.

PNA replicates a model of PCOS in adult female mice. DHT treatment prenatally (PNA) delays puberty onset in females as observed by vaginal opening (VO) (A, B) and first estrous (C). Because of the older age, body weight (BW) was significantly elevated at the time of VO in PNA mice (D), although the BW of age-matched littermates was not different at any age (E). In 2 representative examples of estrous cycles in controls (treated with oil prenatally) and PNA mice are depicted showing disruption of the estrous cycle with prolonged phases in diestrus (F). Values are presented as median (middle line) ± max/min (box and whiskers). Control group n = 6; PNA n = 14. Student t test. In (B) ***P = 0.001, in (C) *P = 0.0194; in (D) ****P < 0.0001.

Figure 7.

Difelikefalin restores estrous cyclicity, decreases serum levels testosterone (T) levels, and induces ovulation in PNA mice. One month of chronic exposure to difelikefalin 100 nM targeted plasma concentration restored estrous cyclicity in PNA mice (A), as observed by the number of estrous cycles in 28 days (F (2, 10) = 4.990, P = 0.0314, n = 4-5, 1-way ANOVA followed by Dunnett’s post hoc test) (B) and time spent in estrus vs diestrus (F (2, 20) = 50.69, P < 0.0001, n = 4-5, 2-way ANOVA followed by Tukey’s post hoc test) (C). In (D), representative samples of ovarian histology from each group are depicted. The number of corporal lutea (CL) was quantified in the middle section of each ovary (E). (F) T levels at the end of the treatment (28 days) (F (2, 12) = 7.738, P = 0.0069, n = 5/group, 1-Way ANOVA followed by Fisher’s post hoc test). *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 8.

Effect of 1 month of difelikefalin treatment on Kiss1, Tac2, Tacr3, and Pdyn expression in the MBH and POA. PNA significantly increased Kiss1 expression in the MBH, which was restored by difelikefalin (100 nM targeted plasma concentration) treatment for a month (A) F (2, 12) = 9.531, P = 0.0033. The expression of Tac2 (B), Pdyn (C), and Tacr3 (D) were not significantly altered. In the POA, difelikefalin treatment significantly increased the expression of Kiss1 (E) (F (2, 12) = 5.4, P = 0.0205) but not Tacr3 (F). One-way ANOVA followed by Fisher’s post hoc test, n = 5/group. *P < 0.05, **P < 0.01.

Figure 9.

Effect of 1 week of difelikefalin treatment on LH pulsatility in PNA mice. (A) Depicts 2 representative samples of LH pulses from each group after 1 week of difelikefalin treatment (100 nM targeted plasma concentration). (B) Baseline LH levels during 240 min (F (2, 41) = 21.86, P < 0.0001). (C) Mean peak LH release of pulses during 240 minutes, (F (2, 41) = 19.94, P < 0.0001). (D) Number of LH pulses in 240 min, F (2, 16) = 0.6283, P = 0.5461. One-way ANOVA followed by Fisher’s test. *P < 0.05, ****P < 0.0001. Control n = 3; PNA + vehicle n = 3; PNA + difelikefalin; n = 5. (These mice had intact ovaries, and estrus was the only cycle phase that had a sufficient number of animals to compare between treatment groups [ie, 3 or more mice at the same cycle phase].).

Figure 10.

Effect of 1 month of difelikefalin treatment on LH pulsatility in PNA mice. (A) Depicts 2 representative samples of LH pulses from each group after 1 month of difelikefalin treatment (100 nM targeted plasma concentration). (B) Baseline LH levels during 240 minutes (F (2, 37) = 3.397, P = 0.0442). Mean peak LH release of pulses during 240 minutes, (F (2, 37) = 23.76, P < 0.0001). (D) Number of LH pulses in 240 minutes, F (2, 15) = 1.077, P = 0.3656. (C) One-way ANOVA followed by Fisher’s test. *P < 0.05, ****P < 0.0001. Control n = 3; PNA + vehicle n = 4; PNA + difelikefalin n = 3. (These mice had intact ovaries, and diestrus was the only cycle phase that had a sufficient number of animals to compare between treatment groups [ie, 3 or more mice at the same cycle phase].).