Palmitoylated analogue of prolactin-releasing peptide for intranasal administration

Abstract

Palm

Description

Technical field Lipidized analogues of prolactin-releasing peptide are anorexigenic agents which, after peripheral, subcutaneous or intraperitoneal administration, reduce food intake and blood glucose levels and are therefore potential anti-obesity compounds. The present invention relates to intranasal administration of a specific analogue which was found to surprisingly allowing to achieve the anorexigenic and anti-obesity effects using a significantly lower dose of the active ingredient. Background art Obesity is a growing problem in developing and developed countries for which there is as yet no effective therapy. The only drug available over-the-counter is Orlistat, an intestinal lipase inhibitor that reduces fat absorption from the small intestine. However, it has side effects such as diarrhea. Sibutramine, a serotonin and noradrenaline reuptake inhibitor that induces satiety, has been withdrawn from the market and has been found to increase the risk of heart disease. Recently, two analogues of glucagon-releasing peptide 1 (glucagon-like peptide 1, GLP-1), liraglutide (Saxenda) and semaglutide (Wegovy), have entered clinical practice; however, the effects of these agents are insufficient and the development of other potential anti-obesity agents is highly desirable. In the last decade of the 20th century, a number of substances have been discovered that fundamentally affect the regulation of energy metabolism, and among them the neuropeptide PrRP (prolactin-releasing peptide). Two forms of PrRP are found naturally, one containing 31 amino acids (PrRP31) and the other 20 amino acids (PrRP20) [Hinuma S, Habata Y, et al. Nature, 393(6682), 272-276 (1998)]. The discovery of PrRP in the hypothalamic nuclei of the PVN and DMN (paraventricular and dorsomedial nuclei), which are important for maintaining energy balance, has led to the consideration of PrRP as a factor regulating food intake [Lawrence C, Celsi F, et al. Nat Neurosci, 3(7), 645-646 (2000)]. The anorexigenic effect of PrRP31 became apparent after its injection into the third cerebral ventricle, the anterior wall and fundus of which are formed by the hypothalamus (called intracerebroventricular administration, ICV). In rats, there was a significant short-term reduction in food intake, which further caused weight loss [Lawrence C, Liu Y, et al. Am J Physiol Regul Integr Comp Physiol, 286(1), R101-107 (2004)]. Genetically modified animals (knock-out, KO animals with the deletion of gene encoding PrRP or its receptor GPR10) have also been used to investigate the function of PrRP in the body [Bjursell M, Lenneras M, et al. Biochem Biophys Res Commun, 363(3), 633-638 (2007)]. Mice with the deletion of gene for PrRP suffer from hyperphagia and late onset obesity. There is no reduction in energy expenditure in these individuals as their body temperature and oxygen consumption are comparable to controls [Takayanagi Y, Matsumoto H, et al. J Clin Invest, 118(12), 4014-4024 (2008)]. In our previous studies, fatty acid binding at the N-terminus of PrRP31 was found to increase the affinity and functionality of both the natural peptide and its analogues of both forms by an order of magnitude (EP 2872124). The effect was observed in both in vitro and in vivo experiments. A statistically very significant reduction in food intake in fasted mice after administration of lipidized PrRP31 analogues was also newly achieved after peripheral (subcutaneous, SC or intraperitoneal, IP) administration [Mrazikova L, Neprasova B, et al. Front Pharmacol, 12, 779962 (2021), Maletinska L, Nagelova V, et al. Int J Obes (Lond), 39(6), 986-993 (2015), Prazienkova V, Holubova M, et al. PLoS One, 12(8), e0183449-e0183449 (2017)]. The most potent analogue of the natural peptide PrRP31 was palmitoylated at position 11 via the gamma-glutamic acid linker (palmn-PrRP31) (EP 3094643). This analogue has demonstrated anti-obesity and anti-diabetic effects after chronic peripheral administration in several mouse and rat models of high-fat diet-induced obesity [Maletinska L, Nagelova V, et al. Int J Obes (Lond), 39(6), 986-993 (2015), Cermakova M, Pelantova H, et al. J Proteome Res, 18(4), 1735-1750 (2019), Holubova M, Zemenova J, et al. J Endocrinol, 229(2), 85-96 (2016)]. Disclosure of the Invention One aspect of the invention is a palmitoylated analogue of prolactin-releasing peptide of the formula 1: SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH2 I X2(palm)                                  (1) (SEQ ID NO. 1) wherein palm is palmitic acid and X2 is gamma-glutamic acid, for use as a drug for intranasal administration. An aspect of the invention provides the palmitoylated analog of prolactin-releasing peptide of formula 1, for use as an anti-obesity drug or a drug for the treatment of hyperglycemia and / or diabetes, wherein the peptide of formula 1 is administered intranasally. An aspect of the invention provides a pharmaceutical composition for intranasal administration to humans, comprising as an active component the pharmaceutically active amount of palmitoylated prolactin-releasing peptide analogue of formula 1, and further comprising at least one pharmaceutically acceptable excipient destined for intranasal formulations, such as a solvent, a carrier, a mucoadhesive and / or a permeation enhancer. The palmitoylated peptide of formula 1 (also abbreviated as “palm1 ’-PrRP31”) showed a highly significant (P < 0.001) reduction in food intake in fasted mice after peripheral (SC, subcutaneous) administration at a dose of 5 mg / kg, and surprisingly shows the same reduction in food intake when administered intranasally at an order of magnitude lower dose (0.01 and 0.1 mg / kg). Moreover, the peptide of formula 1 at 0.3 mg / kg intranasally (IN) after repeated administration to obese Wistar Kyoto (WKY) rats on a high-fat diet (HFD) or obese C57BL / 6N mice on HFD reduced weight and decreased blood glucose (as an indicator of prediabetes or Type 2 diabetes) to the same extent as after IP (intraperitoneal) administration in a dose 5mg / kg. To date, no lipidized neuropeptide analogues have been published that exhibit such pronounced anti-obesity and glucose-lowering effects after intranasal administration. The results obtained by the experiments shown herein below in the Examples chapter can be summarized as follows: - In a feeding test in mice, palm"-PrR.P3 l significantly reduced food intake after both SC and IN administration, with the dose for IN administration being 1-2 orders of magnitude lower than for SC administration to achieve the same effect. This is the first ever description of a modified neuropeptide that reduces food intake after intranasal administration. - In a test of chronic administration of palmn-PrRP31 to obese rats, the IP administered dose of 5 mg / kg once daily was as effective as the IN dose of 0.3 mg / kg once daily and caused a highly significant weight reduction when administered for 30 days. - In the test of chronic administration of palmn-PrRP31 to obese rats, a significant decrease in blood glycated hemoglobin was then measured after 30 days of IN administration as an indicator of glucose reduction and improvement in type 2 diabetes. - In a test of chronic administration of palmn-PrRP31 to obese mice, the SC administered dose of 5 mg / kg twice daily as well as the IN dose of 0.3 mg / kg twice daily caused a highly significant weight reduction when administered for 14 days. - Intranasal administration has the advantage of allowing a wider pharmaceutical use than injectable administration, its fundamental advantage is non-invasiveness. Brief description of drawings Figure 1 shows the time dependence of food intake after SC and IN administration of palmitoylated PrRP31 analogue (palmn-PrRP31) to fasted C57BL / 6 mice. The x-axis plots time in minutes, the y-axis plots cumulative food intake in grams (n = 6). Significance is ***P < 0.001 vs. saline (one-way ANOVAfollowed by Dunnett's post-hoc test). • - saline, A - palm11-PrRP31 at 0.01 mg / kg IN, o - palmn-PrRP31 at 0.1 mg / kg IN, ■ - palmn-PrRP31 at 5 mg / kg SC. Figure 2 shows the change in weight of obese WKY rats on high-fat diet after chronic (30 days) administration of palmn-PrRP31 at a dose of 5 mg / kg IP once daily or IN at a dose of 0.3 mg / kg once daily plus saline after IP or IN administration. Time in days is plotted on the x-axis and change in weight of rats compared to weight at the beginning of the experiment (n = 5-8) on the y-axis. Significance is ***P<0.001 vs. saline (one-way ANOVAfollowed by Dunnett's post-hoc test). • - saline, □ - palmll-PrR.P3 l at 0.3 mg / kg IN, ■ - palmn-PrRP31 at 5 mg / kg IP. Figure 3 shows the glycated hemoglobin (HbAlc) level as an indicator of long-term blood glucose reduction in obese WKY rats after 30 days of treatment with palmll-PrR.P3 l at a dose of 5 mg / kg IP once daily or IN at a dose of 0.3 mg / kg once daily, as well as saline after IP or IN administration (n = 5-8). The y-axis shows HbAlc values as a percentage of total hemoglobin. □ - saline, ■ - palmH-PrRP3 l at 0.3 mg / kg IN, ■ - palmll-PrR.P3 l at 5 mg / kg IP. Significance is *P < 0.05, ***P < 0.001 vs. saline (one-way ANOVA followed by Dunnett's post-hoc test). Figure 4 shows the change in weight of obese C57BL / 6J mice on high-fat diet after chronic (14 days) administration of palm"-PrR.P3 l at a dose of 5 mg / kg SC twice daily or IN at a dose of 0.3 mg / kg twice daily plus saline after IP or IN administration. Time in days is plotted on the x-axis and change in weight of mice compared to weight at the beginning of the experiment (n = 6) on the y-axis. Significance is ***P<0.001 vs. saline (one-way ANOVA followed by Dunnett's post-hoc test). • - saline SC, o - saline IN, □ - palmn-PrRP31 at 0.3 mg / kg IN, ■ -palmn-PrRP31 at 5 mg / kg SC. Examples Abbreviations used: ANOVA - analysis of variance IN - intranasal IP - intraperitoneal SC - subcutaneous PrRP - prolactin-releasing peptide Methods used in tests with PrRP analogue: The peptide with the sequence SRTHRHSMEIK(N-y-E(N-palmitoyl)) TPDINPAWYASRGIRPVGRF-NH2 (SEQ ID NO. 1, palmn-PrRP31) was synthesized by the solid-phase synthesis method according to the procedure of Prazienkova et al. 2017 using the Fmoc strategy on an ABI 433A synthesizer (Applied Biosystems, Foster City, CA, USA). Lipidization with an adjacent fatty acid was performed prior to cleavage of the peptide from the resin as described [Maletinska L, Nagel ova V, et al. Int J Obes (Lond), 39(6), 986-993 (2015)]. In most studies on small laboratory animals (i.e. rats, mice), drug solutions dissolved usually in saline are used and pipetted dropwise (3-10 pl) into the animal's nasal cavity. The animal is maintained in the supine position for the duration of the application. The advantage of this method is that it is non-invasive and the animal does not need to be anesthetized during the procedure. However, the drug solution injected through the nasal cavity must first cross the respiratory epithelium and the vomeronasal organ (VNO), where some loss may occur [Veronesi MC, Kubek DJ, et al. Methods Mol Biol, 789, 303-312 (2011)]. Animals without anesthesia may also be prone to sneezing, which could further reduce the accuracy of the method. However, we did not observe any of these complications in our experiments. Example 1: Food intake test after SC or IN administration of palmu-PrRP31 Male mice of strain C57BL / 6 (Charles River, Germany) were kept in the accredited animal facility of the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic (CAS), Prague in the facility of the Academy of Sciences in Krc at a temperature of 22 ± 2°C and had free access to food and tap water. The rhythm of light / dark was 12 / 12 h (light onset 6:00). The animals were treated according to the Act on the Protection of Animals against Cruelty (Czech Act No. 246 / 1992 Coll.). Males were fed a standard Ssniff R / M-H diet (Ssniff Spezialdiaten GmbH, Soest, Germany). 16 h before the application of palmn-PrRP31, the mice were fasted with free access to water. Application of saline and palmn-PrRP31 after dissolution in saline was performed by SC at 5 mg / kg (volume 0.15 ml / mouse) or IN using a fine-tip pipette at 0.01 and 0.1 mg / kg (5 pl / mouse). Twenty minutes after peptide injection, mice were given pre-weighed food. The food was then weighed every 30 minutes for 7 hours. The experiment was performed at least twice for each dose of palm"-PrRP3 l and one group of mice consisted of at least 6 mice. The results are expressed as a percentage of food intake compared to the saline-injected control group. Graph-Pad Prism Software (San Diego, CA, USA), one-way ANOVA followed by Dunnett's post-hoc test was used for statistical evaluation. Differences in food intake between individuals injected with saline (control) and those injected with palm11-PrRP31 were considered statistically significant at P < 0.05. The results are summarized in Figure 1. Palm1 '-PrRP31 reduced food intake in fasted mice very significantly and over a long period of time, to the same extent after SC administration at 5 mg / kg as after IN administration at 0.01 and 0.1 mg / kg, i.e. 50 and 500 times lower, respectively. Example 2: Test for changes in weight and glycated hemoglobin after chronic administration of palm11-PrRP31 IP or IN to obese WKY rats Male WKY rats (Charles River, Germany) were kept in the accredited animal facility of the Institute of Physiology of the CAS, v.v.i., Prague, in the facility of the Academy of Sciences in Krc at a temperature of 22 ± 2°C and had free access to food and tap water. The light / dark rhythm was 12 / 12 hours (light onset 4:00). The animals were treated according to the Act on the Protection of Animals against Cruelty (Act No. 246 / 1992 Coll.). Males were fed a high-fat diet D12492 (Research Diets, USA) containing 60% fat, 20% protein and 20% carbohydrate. From 8 weeks of age, they were fed this diet for 4 months. They were then divided into groups of 5 animals (IP administration of saline or palmll-PrR.P3 l in saline at 5 mg / kg) or 8 animals (IN administration of saline or palm"-PrRP3l in saline at 0.3 mg / kg). The compound was administered IP or IN once daily 1 hour before lights out for 30 days. Throughout the period, the weight of the rats was monitored 3 times a week and at the end, glycated hemoglobin was measured in blood drawn from the tail. Glycated hemoglobin (HbAlc) was determined using the Tina-quant HbAlc Gen. 3 kit (Roche, Mannheim, Germany). Graph-Pad Prism Software (San Diego, CA, USA), one-way ANOVA followed by Dunnetf s post-hoc test was used for statistical evaluation. Differences in food intake between individuals injected with saline and those injected with palm"-PrR.P3 l were considered statistically significant at P < 0.05. The results are summarized in Figures 2 and 3. Palm1 '-PrRP31 reduced the weight of rats after chronic administration to the same extent after both IP and IN administration, but IN administration was performed at a dose of 0.3 mg / kg, 16 times lower than the dose for IP administration. Furthermore, significantly reduced glycated hemoglobin was measured at the end of the experiment after both IN and IP administration. Example 3: Test for changes in weight after chronic administration of palm11-PrRP31 SC or IN to obese C57BL / 6 mice Male C57BL / 6 mice (Charles River, Germany) were kept in the accredited animal facility of the Institute of Organic Chemistry and Biochemistry of the CAS, Prague, in the facility of the Academy of Sciences in Krc at a temperature of 22 ± 2°C and had free access to food and tap water. The light / dark rhythm was 12 / 12 hours (light onset 4:00). The animals were treated according to the Act on the Protection of Animals against Cruelty (Act No. 246 / 1992 Coll.). Males were fed at-house-made high-fat diet containing 60% fat (lard), 20% protein and 20% carbohydrate. From 8 weeks of age, they were fed this diet for 4 months. They were then divided into groups of 6 animals (SC administration of saline or palm"-PrR.P3 l in saline at 5 mg / kg and IN administration of saline or palm1 '-PrRP31 in saline at 0.3 mg / kg). The compound was administered SC or IN twice daily 1 hour before lights out for 14 days. Throughout the period, the weight of the mice was monitored 3 times a week. Graph-Pad Prism Software (San Diego, CA, USA), one-way ANOVA followed by Dunnett's post-hoc test was used for statistical evaluation. Differences in food intake between individuals injected with saline and those injected with palm"-PrRP3 l were considered statistically significant at P < 0.05. 5 The result is summarized in Figure 4. Palm"-PrR.P3 l after chronic administration into obese mice reduced body weight of mice, the SC administered dose of 5 mg / kg twice daily as well as the IN dose of 0.3 mg / kg twice daily caused a highly significant weight reduction when administered for 14 days. 10 Industrial applicability Palm"-PrR.P3 l is an anti-obesity agent which is especially suitable for the extremely convenient intranasal administration by the patient himself. Intranasal administration increases patient compliance. 15

Claims

1. A palmitoylated analogue of a prolactin-releasing peptide of formula 1 SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NBLX2(palm)                            (1) (SEQ ID NO. 1)wherein palm is palmitic acid residue and X2 is gamma-glutamic acid residue, for use as a medicament, wherein the palmitoylated analog of the prolactin-releasing peptide of formula 1 is administered intranasally.

2. The palmitoylated analogue of a prolactin-releasing peptide of formula 1 for use according to claim 1, wherein the palmitoylated analogue of a prolactin-releasing peptide of formula 1 is for use in the treatment of obesity, hyperglycemia, and / or diabetes, and wherein the palmitoylated analog of the prolactin-releasing peptide of formula 1 is administered intranasally.

3. A pharmaceutical composition for human or veterinary use for intranasal administration, comprising as an active ingredient a pharmaceutically active amount of a palmitoylated prolactin-releasing peptide analogue of formula 1SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH2X2(palm)(1) (SEQ ID NO. 1)wherein palm is palmitic acid residue and X2 is gamma-glutamic acid residue, and further comprising at least one pharmaceutically acceptable excipient destined for intranasal formulations.

4. The pharmaceutical composition according to claim 3, wherein the at least onepharmaceutically acceptable excipient is a mucoadhesive and / or a permeation enhancer.

5. A method of treatment of obesity, hyperglycemia and / or diabetes in a human or animal subject, comprising the steps of intranasally administering (a therapeutically effective amount of) a palmitoylated analogue of a prolactin-releasing peptide of formula 1SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH25                 X2(palm)                             (1) (SEQ ID NO. 1)wherein palm is palmitic acid residue and X2 is gamma-glutamic acid residue, to the subject in need of such treatment.10

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