Used to reduce CB 1 Peptide compounds with side effects of receptor agonists

Abstract

The present invention relates to a compound of formula (I): AA 1 ‑AA 2 ‑D‑Ile‑AA 3 ‑D‑Met‑D‑Tyr‑D‑Ala‑D‑Tyr‑D‑Val‑D‑Ala‑Gly‑D‑Ile‑D‑Leu‑D‑Lys‑D‑Arg‑D‑Trp‑NH2 or a pharmaceutically acceptable salt thereof, a method for obtaining the same and therapeutic indications thereof.

Description

[0001] The present invention relates to the field of pharmacological treatment with CB1 receptor agonists. Background Art

[0002] Nearly one in five Europeans suffer from chronic pain, which affects their work, social and family life and causes nearly 500 million workdays lost each year, costing the European economy at least €34 billion. The analgesic market is expected to grow to €100 billion by 2019, highlighting the need for new complementary medicines that can effectively relieve pain in chronic patients without undesirable side effects.

[0003] Activation of cannabinoid CB1 receptors (CB1R) by large CB1 receptor agonists or partial agonists such as Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient in cannabis sativa, produces pharmacological responses of great interest for therapeutic purposes, including treatment and / or prevention of pain, such as pain associated with severe migraine, osteoarthritis, endometriosis, rheumatoid arthritis, fibromyalgia, back pain, knee pain, neuropathic pain of peripheral or central origin, orexigenic treatment, as an antiemetic during chemotherapy for cancer treatment, for the treatment of glaucoma, for the treatment of spasticity in patients with multiple sclerosis, and for the treatment of movement disorders, including dyskinesia, Huntington's disease and Parkinson's disease, among others (see Kandasamy R et al., Eur J Pharmacol. 2018 Jan 5;818:271-277; Baron EP et al., Headache Pain. 2018 May 24;19(1):37; Russo J et al., Headache Pain. 2018 May 24;19(2):43-47). EB et al., Cannabis Cannabinoid Res. 2016 Jul 1;1(1):154-165; Miller S et al., Invest Ophthalmol Vis Sci. 2018 Dec 3;59(15):5904-5911; Escudero-Lara A, et al., IASP (International Association for the Study of Pain) 7th World Congress on Pain. Boston, USA. 2018 Sep 12-16; Abstract ID: 471100; La Porta C et al., Pain. 2015 Oct;156(10):2001-12; Maldonado R. et al., Pain. 2016 Feb;157 Suppl 1:S23-32; Engeli S., Handb Exp Pharmacol.2012;(209):357-81;Al-Ghezi ZZ et al.Front Immunol.2019 Aug 21;10:1921;Allen D et al.,Clin J Oncol Nurs.2019 Feb 1;23(1):23-26;Buhmann C et al.,J Neural Transm (Vienna).2019 Jul;126(7):913-924;Valdeolivas S et al.,Int J Mol Sci.2017 Mar 23;18(4); Donovan KA et al., J Adolesc Young Adult Oncol. 2019 Aug 22 [Epub ahead of print]).

[0004] However, this CB1R activation is associated with a variety of negative effects: such as impaired memory and altered other mental responses such as anxiety, as well as disrupted social interactions - significant consequences in cannabis users that constitute a significant drawback to the use of cannabinoids as therapeutic agents.

[0005] For this reason, there is great medical interest in harnessing the beneficial effects of THC and other CB1 receptor agonists.

[0006] In the absence of 5-HT 2A Receptor (5-HT 2A Behavioral studies in mice revealed a significant role for 5-HT in both the beneficial analgesic effects of THC and its deleterious amnesic properties. 2A Specific effects of THC, such as memory deficits, changes in anxiety, and social interaction, have been reported to be in the presence of 5-HT 2A R, but its acute hypolocomotor, hypothermic, anxiogenic, and analgesic effects are not ( X.et al; PLoS Biol. 2015 Jul 9;13(7):e1002194).

[0007] CB1R and 5-HT have been shown 2A R form heteromers that are expressed and functionally active in specific brain regions implicated in memory impairment, and disruption of heteromer formation is a valuable strategy leading to the selective elimination of memory impairment and other undesirable side effects caused by exposure to CB1R agonists such as THC ( X.et al; PLoS Biol. 2015 Jul 9;13(7):e1002194).

[0008] It has also been disclosed that a synthetic peptide containing the sequences of transmembrane helices 5 and 6 of the CB1 receptor fused to a cell-penetrating peptide is able to disrupt the binding of the receptor to 5-HT in vivo. 2A Heterogeneity of R.

[0009] The inventors have now discovered new synthetic peptides that are not only able to interfere with the activity of cannabinoid receptor CB1R and serotonin receptor 5HT more effectively than previously known peptides, but also 2A R-formed heteromers also have higher hydrolytic stability and can effectively penetrate the blood-brain barrier. Summary of the invention

[0010] In a first aspect, the present invention provides a compound of formula (I):

[0011] AA 1 -AA 2 -D-Ile-D-Tyr-AA 3 -D-Tyr-D-Ala-D-Tyr-D-Val-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2

[0012] (I)

[0013] AA 1 represents an amino acid selected from the group consisting of D-tryptophan (D-Trp), 1-naphthylalanine (D-1-Nal) and 2-naphthylalanine (D-2-Nal);

[0014] AA 2 represents an amino acid selected from the group consisting of D-leucine (D-Leu), D-norleucine (D-Nle) and D-phenylalanine (D-Phe); and

[0015] AA 3 represents an amino acid selected from D-methionine (D-Met) and D-norleucine (D-Nle); or a pharmaceutically acceptable salt thereof, wherein:

[0016] In a specific embodiment, AA 1 Denotes D-tryptophan (D-Trp).

[0017] In a specific embodiment, AA 2 Denotes D-leucine (D-Leu).

[0018] In a specific embodiment, AA 3 Denotes D-tyrosine (D-Tyr).

[0019] In a specific embodiment, the compound is selected from:

[0020] D-Trp-D-Leu-D-Ile-D-Tyr-D-Met-D-Tyr-D-Ala-D-Tyr-D-Val-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp or a pharmaceutically acceptable salt thereof.

[0021] In a second aspect, the present invention relates to a pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. Pharmacy in the context of the present invention relates to human and veterinary medicine.

[0022] In a third aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament in combination with a CB1 receptor agonist.

[0023] In a specific embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a CB1 receptor agonist for use in treating or preventing diseases in animals including humans that are susceptible to improvement by agonism of CB1 receptors.

[0024] In a specific embodiment, the disease susceptible to improvement by agonism of CB1 receptors is selected from pain associated with severe migraine, osteoarthritis, endometriosis, rheumatoid arthritis, fibromyalgia, back pain, knee pain, neuropathic pain of peripheral or central origin, appetite therapy, as an antiemetic during chemotherapy, for the treatment of glaucoma, treatment of spasticity in patients with multiple sclerosis, and treatment of movement disorders, including dyskinesia and Parkinson's disease, etc.

[0025] In a specific embodiment, the treatment and / or prevention is capable of reducing memory impairment and / or anxiety changes caused by a CB1 receptor agonist.

[0026] In a fourth aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in reducing the side effects of a CB1 receptor agonist in a patient treated with a CB1 receptor agonist, such as impaired memory or other psychoactive responses, such as changes in anxiety and disturbances in social interactions. The patient is considered to be a mammal, in particular a human suffering from a disease that is susceptible to improvement by administration of a CB1 receptor agonist, in particular pain, pain associated with severe migraine, osteoarthritis, endometriosis, rheumatoid arthritis, fibromyalgia, back pain, knee pain, neuropathic pain of peripheral or central origin, anorectic treatment, as an antiemetic during chemotherapy, for the treatment of glaucoma, for the treatment of spasticity in patients with multiple sclerosis, and for the treatment of movement disorders, including dyskinesia and Parkinson's disease, among others.

[0027] In particular embodiments of the third, fourth, fifth and sixth aspects of the invention, the CB1 receptor agonist is a full agonist or a partial agonist.

[0028] In more specific embodiments of the third, fourth, fifth and sixth aspects of the present invention, the CB1 receptor agonist is selected from endocannabinoids produced by the mammalian body, such as 2-arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE, noladin ether), anandamide (N-arachidonoylethanolamine or AEA), N-arachidonoyl dopamine, palmitoylethanolamide (PEA); phytocannabinoids, such as 11-hydroxy-Δ8-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, cannabinol, Δ8-tetrahydrocannabinol, Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC), as well as synthetic cannabinoids such as AM-2201 (1-(5-fluoropentyl)-3-(1-naphthoyl)indole), AM-678 (1-pentyl-3-(1-naphthoyl)indole), arachidonic acid-2'-chloroacetamide (ACEA), CP55,940 ([(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol]), HU-210, JWH-018 (1-pentyl-3-(1-naphthoyl)indole), JWH-073, linoleoylethanolamide, levofloxacin (CP50,556-1), nabilone, Nabiximols and WIN 55,212-2, especially the plant-derived cannabinoid THC.

[0029] In other words, the present invention relates to a method for treating or preventing diseases susceptible to agonism of CB1 receptors in animals including humans by administering a pharmaceutical or veterinary composition comprising a compound of formula (I). Methods for treating the above-mentioned specific diseases and disorders are specific embodiments of the present invention.

[0030] In a fifth aspect, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a CB1 receptor agonist for the preparation of a medicament for treating or preventing a disease in animals including humans that is susceptible to improvement by CB1 receptor agonism.

[0031] In a specific embodiment, the treatment and / or prevention is capable of reducing memory impairment and / or anxiety changes caused by a CB1 receptor agonist.

[0032] In a sixth aspect, the present invention relates to a method for treating or preventing a disease susceptible to CB1 receptor agonism in animals including humans by administering to the animal a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a CB1 receptor agonist.

[0033] In a specific embodiment, the treatment and / or prevention is capable of reducing memory impairment and / or anxiety changes caused by a CB1 receptor agonist.

[0034] According to the fifth aspect of the present invention, the compound of formula (I) can be prepared by a method comprising the following steps:

[0035] a) providing amino acids (D-Ala, D-Arg, Gly, D-Ile, D-Leu, D-Lys, D-Met, D-1-Nal, D-2-Nal, D-Nle, D-Phe, D-Trp, D-Tyr, D-Val) forming part of the peptide,

[0036] b) protecting the amino acids D-Lys and D-Trp with Boc,

[0037] c) protecting the amino acid D-Arg with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl,

[0038] d) protecting the amino acid D-Thr with a tert-butyl group,

[0039] e) in the presence of excess 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate In the presence of (HBTU) and excess DIEA, the amino acid and the protected amino acid are assembled in Fmoc-Rink-amide resin with DMF as solvent, resulting in coupling with excess Fmoc-amino acid,

[0040] f) cleaving the protected side groups of the peptide and the peptide from the resin, preferably using CF3COOH / H2O / 3,6-dioxa-1,8-octanedithiol (DODT) / triisopropylsilane.

[0041] As used herein, "a compound of formula (I) in combination with a CB1 receptor agonist" refers to a fixed combination of a compound of formula (I) and a CB1 receptor agonist in one unit dosage form, or a kit comprising a first composition comprising a compound of formula (I) and a second composition comprising a CB1 receptor agonist for combined administration, wherein the compound of formula (I) and the CB1 receptor agonist can be administered independently at the same time or separately at time intervals that particularly allow for a reduction in side effects of the CB1 receptor agonist, or any combination thereof.

[0042] As used herein, the expression "CB1 receptor agonist" refers to compounds that are capable of activating the CB1 cannabinoid receptor, including ligands, endogenous cannabinoids produced by the mammalian body, such as 2-arachidonoylglycerol (2-AG), 2-arachidonoylglycerol ether (2-AGE, noladin ether), anandamide (N-arachidonoylethanolamine or AEA), N-arachidonoyl dopamine, palmitoylethanolamide (PEA); phytocannabinoids , such as 11-hydroxy-Δ8-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, cannabinol, Δ8-tetrahydrocannabinol, Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC), and synthetic cannabinoids such as AM-2201 (1-(5-fluoropentyl)-3-(1-naphthoyl)indole), AM-678 (1-pentyl-3-(1-naphthoyl)indole), arachidonic acid-2'-chloroacetamide (ACEA), CP 55,940 ([(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol]), HU-210, JWH-018 (1-pentyl-3-(1-naphthoyl)indole), JWH-073, linoleoylethanolamide, levofloxacin (CP 50,556-1), nabilone, Nabiximols and WIN55,212-2.

[0043] In a preferred embodiment, the CB1 cannabinoid receptor agonist is Δ9-tetrahydrocannabinol (THC). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A transwell system for performing an in vitro BBB translocation assay is described.

[0045] Figure 2 The results of evaluating peptide internalization by confocal microscopy as described in Example 7 are depicted. DETAILED DESCRIPTION

[0046] As used herein, the expression "pharmaceutically acceptable" as used herein relates to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects (e.g., humans) without significant toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within sound medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts and include, for example, preservatives, agglutinating agents, wetting agents, emollients, and antioxidants.

[0047] As used herein, the expression "pharmaceutically acceptable salt" refers to any salt that can (directly or indirectly) provide a compound described herein when administered to a patient. For example, a pharmaceutically acceptable salt of a compound provided herein is synthesized by a parent compound containing an alkaline or acidic part by conventional chemical methods. In general, such salts are prepared, for example, by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred. Examples of acid addition salts include inorganic acid addition salts, such as, for example, hydrochlorides, hydrobromides, hydroiodides, sulfates, nitrates, phosphates, and organic acid addition salts, such as, for example, acetates, trifluoroacetates, maleates, fumarates, citrates, oxalates, succinates, tartrates, malates, mandelates, mesylates and p-toluenesulfonates. Examples of base addition salts include inorganic salts such as, for example, sodium salts, potassium salts, calcium salts and ammonium salts, and organic base salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkyleneethanolamine, triethanolamine and basic amino acid salts.

[0048] Throughout the specification and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprise" covers the situation of "consisting of..."

[0049] Peptide synthesis

[0050] The synthesis of the peptides according to the present invention and the peptides of Reference Examples can be performed by a conventional peptide synthesis method.

[0051] The methods described in the following paragraphs have actually been used to prepare the peptides described in the examples and can be used to prepare any peptide according to the invention.

[0052] The peptides were assembled on 50 μmol (0.106 g; 0.47 mmol / g) of Fmoc-Rink-amide ChemMatrix resin in a Prelude instrument (Protein Technologies, Tucson, AZ) running an optimized Fmoc synthesis protocol. Side chain protections were Boc (for D-Lys, Trp, and D-Trp), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (for D-Arg), and tert-butyl (for Glu, Ser, Thr, Tyr, and D-Tyr). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorosulfate was used. In the case of HBTU (5-fold excess) and DIEA (10-fold excess), double coupling was systematically performed with a 5-fold excess of Fmoc amino acids in DMF as solvent for 5+5 min.

[0053] The deprotection and cleavage mixture was CF3COOH / H2O / 3,6-dioxa-1,8-octanedithiol (DODT) / triisopropylsilane (94:2.5:2.5:1 v / v, 90 min). The synthesized product was isolated by precipitation with cold ether, centrifuged at 4°C for 3×10 min, extracted in H2O and lyophilized.

[0054] Peptide analysis and purification

[0055] The following protocol has been used to purify and analyze the peptides described in this application.

[0056] The lyophilized crude peptide was reconstructed in a direct manner (HO, acidified with HOAc). In a LC-20AD system (Shimadzu, Kyoto, Japan), on a Luna C18 column (4.6×50mm, 3μm, Phenomenex, Torrance, CA, USA), the crude peptide was analyzed by HPLC. A linear gradient of solvent B (0.036% v / v TFA in CHCN) to solvent A (0.045% v / v TFA in H2O) was eluted over 15min. In a Shimadzu LC-8A instrument, on a Luna C18 (10x 250mm, 10μm, Phenomene) column, a linear gradient of solvent B (0.1% v / v TFA in CHCN) to solvent A (0.1% v / v TFA in H2O) was used, with a flow rate of 7mL / min, over 30min, with UV detection at 220nm, the peptide was purified by preparative HPLC. Fractions with satisfactory purity (>95%) obtained by analytical HPLC were pooled, lyophilized and reanalyzed for identity by HPLC-MS on C18 (4.6×150 mm column, 3.5 μm, Phenomenex) in a Shimadzu LC-MS2010EV instrument, eluting by a linear gradient from solvent B (0.08% v / v HCOOH in CH3CN) to solvent A (0.1% v / v TFA in H2O) over 15 min at a flow rate of 1 mL / min.

[0057] abbreviation:

[0058] The following abbreviations have been used in this application:

[0059] Ala: L-Alanine

[0060] D-Ala: D-Alanine

[0061] D-Arg: D-Arginine

[0062] Glu: L-glutamic acid

[0063] Gly: Glycine

[0064] Ile: L-isoleucine

[0065] D-Ile: D-isoleucine

[0066] Leu: L-Leucine

[0067] D-Leu: D-Leucine

[0068] D-Lys: D-lysine

[0069] Met: L-methionine

[0070] D-Met: D-Methionine

[0071] D-1-Nal: 1-naphthylalanine ((2R)-2-amino-3-naphth-1-ylpropionic acid)

[0072] D-2-Nal: 2-naphthylalanine ((2R)-2-amino-3-naphth-2-ylpropionic acid)

[0073] D-Nle: D-norleucine

[0074] Phe: L-phenylalanine

[0075] D-Phe: D-phenylalanine

[0076] Ser: L-serine

[0077] Thr:L-Threonine

[0078] Trp: L-Tryptophan

[0079] D-Trp: D-Tryptophan

[0080] Tyr: L-Tyrosine

[0081] D-Tyr: D-Tyrosine

[0082] Val: L-Valine

[0083] D-Val: D-Valine

[0084] CF: 5(6)-carboxyfluorescein residue of the following formula:

[0085] -Lys(CF)-NH2: a residue of the formula:

[0086]

[0087] -D-Lys(CF)-NH2: a residue of the formula:

[0088]

[0089] Fmoc-Lys(Mtt)-OH

[0090]

[0091] Example

[0092] Reference Example 1:

[0093] A peptide of the formula Glu-Thr-Tyr-Leu-Met-Phe-Trp-Ile-Gly-Val-Thr-Ser-Val-Leu-Leu-Leu-Phe-Ile-Val-Tyr-Ala-Tyr-Met-Tyr-Ile-Leu-Trp-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH2 (SEQ ID NO 1-NH2) was prepared using the general synthetic method described above.

[0094] This peptide corresponds to the transmembrane region 5 (TM5) of the cannabinoid receptor 1 coupled to the cell penetrating peptide sequence Tat (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) (SEQ ID NO 2).

[0095] HPLC:t R 8.10 min (40-90% linear gradient from B to A over 15 min). ESI-MS; m / z 1576.45 [M+3H] +3 ,1182.65[M+4H] +4 ,946.30[M+5H] +5 ,788.70[M+6H] +6 ,676.20[M+7H] +7 ,591.75[M+8H] +8 (Calculated MW: 4727.76).

[0096] Reference Example 2:

[0097] A peptide of the formula Val-Tyr-Ala-Tyr-Met-Tyr-Ile-Leu-Trp-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2 was prepared using the general synthetic method described above.

[0098] This peptide is derived from a subsequence of amino acids 19 to 27 of TM5 coupled to the cell penetrating peptide sequence D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2.

[0099] HPLC:t R 5.24 min (30-60% linear gradient from B to A over 15 min). ESI-MS; m / z: 1023.60 [M+2H] +2 ,682.75[M+3H] +3 ,511.20[M+4H] +4 ,411.90[M+5H] +5 ,340.90[M+6H] +6 ,292.00[M+7H] +7 (Calculated MW: 2045.52).

[0100] Reference Example 3:

[0101] A peptide of the formula D-Trp-D-Leu-D-Ile-D-Tyr-D-Met-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2 was prepared using the general synthetic method described above.

[0102] This peptide is derived from the anti-enantiomer form (D-Trp-D-Leu-D-Ile-D-Tyr-D-Met) of the subsequence of amino acids 23 to 27 of TM5 coupled to the cell penetrating peptide sequence D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2. By the anti-enantiomer form of the peptide sequence it is understood that all amino acids of the new peptide sequence are replaced by their D-enantiomers and the order of amino acids in the sequence is reversed.

[0103] HPLC:t R 6.33 min (20-60% linear gradient from B to A over 15 min). ESI-MS; m / z: 1549.70 [M+1H] +1 ,775.45[M+2H] +2 ,517.35[M+3H] +3 ,389.95[M+4H] +4 (Calculated MW: 1548.96).

[0104] Embodiment 1:

[0105] A peptide of the formula D-Trp-D-Leu-D-Ile-D-Tyr-D-Met-D-Tyr-D-Ala-D-Tyr-D-Val-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2 was prepared using the general synthetic method described above.

[0106] This peptide is derived from the anti-enantiomer form of the subsequence of amino acids 19 to 27 of TM5 coupled to the cell penetrating peptide D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2 (D-Trp-D-Leu-D-Ile-D-Tyr-D-Met-D-Tyr-D-Ala-D-Tyr-D-Val). It can be understood from the anti-enantiomer form of the peptide sequence that all amino acids of the new peptide sequence are replaced by their D-enantiomers and the order of amino acids in the sequence is reversed.

[0107] HPLC:t R 7.10 min (30-60% linear gradient from B to A over 15 min). ESI-MS; m / z: 1023.90 [M+2H] +2 ,682.70[M+3H] +3 ,512.85[M+4H] +4 ,409.90[M+5H] +5 ,340.95[M+6H] +6 ,293.00[M+7H] +7 (Calculated MW: 2045.52).

[0108] Embodiment 2:

[0109] The peptide of Example 1 has been labeled by attaching a -Lys(CF)-NH2 residue to its carboxyl terminus.

[0110] The carboxyl terminus of the peptide was labeled (incorporation of 5(6)-carboxyfluorescein) by adding an additional labeled lysine to the peptide sequence. For this purpose, Fmoc-Lys(Mtt)-OH was used.

[0111] In said compound, the side chain of lysine is protected by a methyltrityl (Mtt) protecting group which can be selectively deprotected in the resin in the presence of 1% TFA (all other side chain protecting groups are stable under these conditions).

[0112] After condensation of the compound with the carboxyl terminal amino acid of the peptide to be labeled, the methyl triphenyl group is removed and the ε-NH2 group of the "excess" lysine side chain reacts with the -COOH group of 5(6)-carboxyfluorescein to obtain the labeled peptide. Finally, the carboxyl terminal group is amidated.

[0113] This slightly modified peptide has been used to evaluate in vitro BBB translocation and internalization of the peptide.

[0114] Test Method

[0115] A. Trypsin digestion

[0116] The stability of the peptides to the action of trypsin has been tested by the following method.

[0117] 1000 μg / 1000 μL trypsin solution (Thermo Fisher Scientific) was prepared by suspending the lyophilized enzyme in 50 mM ammonium bicarbonate buffer (pH 8.2). Peptides (1 μg / μL) dissolved in the same buffer were incubated with trypsin (peptide: trypsin ratio = 100: 1) at 37°C for 24 h. Samples were collected at different time points and digestion was terminated by adding 20% ​​(v / v) of 0.5% formic acid in H2O. Samples were stored at -20°C until analyzed by reverse phase HPLC-MS. The experiment was performed in duplicate and the data were fitted with GraphPad Prism.

[0118] B.bEnd.3 cell culture

[0119] bEnd.3 brain endothelioma cells ( CRL-2299TM) at 75cm 2 In T-flasks, cells were grown in monolayer in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotic solution (GibcoTM, USA). Cells were cultured at 37°C in a humid atmosphere of 95% air and 5% CO2 (MCO-18AIC (UV), Sanyo, Japan), and culture medium was changed every other day. When converging with trypsin-EDTA (GibcoTM, USA), cells were harvested from cell culture T-flasks and used for different determinations (cell proliferation, BBB translocation in vitro and bimolecular fluorescence complementation in vitro).

[0120] C. Cell viability assay

[0121] Study of in vitro cytotoxicity of the compounds to be evaluated - The same cell line as in the in vitro BBB model, i.e. bEnd.3 cells, was used to evaluate possible toxicity. The bEnd.3 cell line was exposed to increasing concentrations of the compounds for 24 h.

[0122] Has been tested according to the manufacturer's instructions as described below. Cell viability assay to test the cytotoxicity of peptides on bEnd.3 cells.

[0123] The assay is based on the ability of living cells to reduce resazurin to resorufin, a highly fluorescent metabolite. Non-living cells cannot produce resorufin. Therefore, using this method, it is possible to distinguish between metabolizing and non-metabolizing cells and indirectly determine the cytotoxicity of different compounds. bEnd.3 cells were seeded at 10,000 cells / 100 μL / well in a 96-well plate (Corning, USA) and incubated for 24 h. After removing the culture medium, 100 μL of peptide previously diluted in DMEM medium (range 0.01-50 μM) was added to the wells. After 24 h of incubation, 20 μL of The reagents were added to each well and incubated for 3 h at 37°C in a humidified atmosphere of 5% CO2. exc =560nm and λ em =590nm fluorescence intensity was measured. The culture medium and the culture medium containing 1% Triton X-100 were used as positive control (100% cell viability) and negative control (0% cell viability), respectively. The cell viability (%) was determined using the following equation:

[0124]

[0125] Among them, F p is the fluorescence intensity of peptide-treated cells, F NC is the fluorescence intensity of the negative control, and F PC is the fluorescence intensity of the positive control.

[0126] Experiments were performed with independently grown cell cultures on different days.

[0127] D. In vitro BBB translocation assay

[0128] bEnd.3 cells were grown in culture T-flasks until confluent. TM, USA) were carefully harvested and seeded at 5,000 cells / well into 1% fibronectin-coated tissue culture inserts (transparent polyester (PET) membrane with 1.0 μm pores) of 24-well plates (BD Falcon, USA). The medium was changed every two days throughout 10 days. After 10 days, the cells were washed twice with 1x PBS and once with phenol red-free DMEM medium (Gibco / Thermo Fisher, USA). Then, the peptide diluted to a final concentration of 1 μM in phenol red-free DMEM was added to the apical side of the in vitro BBB model. The experiments were performed with independently grown cell cultures on different days.

[0129] Translocation of 5(6)-carboxyfluorescein labeled peptides was determined by fluorescence emission.

[0130] The in vitro BBB model consists of a transwell system with an insert in which bEnd.3 cells are grown, separating two compartments (see Figure 1 ). The insert, or apical side, corresponds to the blood side of the blood-brain barrier, while the basolateral side (bottom of the chamber) corresponds to the brain side of the barrier.

[0131] To study the translocation ability of the peptide, 1 μM of the 5(6)-carboxyfluorescein-labeled peptide to be evaluated was added to the apical side of the above-mentioned in vitro BBB model. The apical volume and the basolateral volume were collected after 24 h of cultivation, and the fluorescence in those samples was measured in a plate reader, respectively. The internalization percentage was obtained by the difference between the total fluorescence and the sum of the apical and basolateral fluorescence.

[0132] After 24 h of incubation, samples from the apical and basolateral sides were collected and analyzed. Fluorescence was measured using an Infinite F200 TECAN plate reader. The percentage of translocation (%) was calculated using the following equation:

[0133]

[0134] F i is the collected fluorescence intensity, F 细胞 is the fluorescence intensity collected from cells incubated without peptide, F 肽 is the fluorescence intensity of the total peptide initially added to the apical side of the well.

[0135] Endothelial barrier integrity was assessed by permeability of fluorescently labeled dextran (FD) with a molecular weight of 4 kDa (FD4). The clearance of the fluorescent probe from the apical side in the presence or absence of the labeled peptide provided information about the BBB integrity.

[0136] E. In vitro determination of disruption of CB1R and 5-HT by bimolecular fluorescence complementation 2A R heteromer capacity

[0137] Will" X, Moreno*E, Lanfumey L, CordomíA, Pastor A, de La Torre R, Gasperini P, Navarro G, Howell LA, Pardo L, Lluís C, Canela EI, McCormick*PJ, Maldonado*R, Robledo*P. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors. PLoS Biology, 13(7): e1002194 (2015)" was used with slight modifications as described herein to determine whether peptides disrupt CB1R and 5-HT2A in vitro. 2A R heteromer capacity.

[0138] Expression vector

[0139] To obtain the YFP Venus half-truncated protein (5-HT) fused to the target receptor 2A R-cYFP, CB1R-nYFP), 5-HT 2A R was subcloned in frame with pcDNA3.1-cVenus restriction sites, which contains the sequence encoding amino acid residues 156-238 of the YFP Venus protein. In addition, CB1R was subcloned in frame with pcDNA3.1-nVenus restriction sites, which contains the sequence encoding amino acid residues 1-155 of the YFP Venus protein.

[0140] Expression of the constructs was tested by confocal microscopy and receptor fusion protein functionality was tested by ERK1 / 2 phosphorylation.

[0141] Cell culture and transient transfection

[0142] Human embryonic kidney (HEK-293T) cells obtained from ATCC were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 2 mM L-glutamine, 100 μg / ml sodium pyruvate, 100 U / ml penicillin / streptomycin, MEM non-essential amino acid solution (1 / 100) and 5% (v / v) heat-inactivated fetal bovine serum (FBS) (all supplements were from Invitrogen, Paisley, Scotland, UK). Cells were maintained at 37° C. in an atmosphere of 5% CO .

[0143] Fluorescence complementation assay

[0144] Cells grown in 6-well culture dishes were incubated with proteins encoding CB1R fused to the N-terminal fragment of YFP Venus (n-YFP) and 5-HT fused to the C-terminal fragment of YFP Venus (c-YFP) by the PEI (polyethyleneimine, Sigma) method. 2A Transient transfection of cDNA of R. The cells were incubated with the corresponding cDNA, PEI and 150 mM NaCl in serum-deficient medium for 4 h. After 4 h, the medium was replaced with fresh complete medium.

[0145] After 48h, cells were treated or not treated with the TAT-peptide (4 μM) shown at 37°C for 4h. For quantitative complementary YFPVenus expression, cells were washed twice quickly and continuously in HBSS with 10mM glucose, separated, and resuspended in the same buffer. To control cell number, the Bradford assay kit (Bio-Rad, Munich, Germany) was used to determine the sample protein concentration using bovine serum albumin diluent as a standard. 20 μg of protein was distributed into 96-well microplates (black plates with transparent bottoms, Porvair, King's Lynn, UK) and recorded in the fluorescence emission at 530nm after excitation at 500nm with Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany). Protein fluorescence was determined as the fluorescence of the sample minus the fluorescence (baseline) of untransfected cells. Expression of 5-HT 2A Cells transfected with either R-cVenus and nVenus or CB1R-nVenus and cVenus showed fluorescence levels similar to those of untransfected cells.

[0146] Example 3 (Trypsin Stability of the Compound of Example 1)

[0147] HPLC analysis of the compound of Example 1, after incubation with the provided trypsin as described above in Method A, provided the results summarized in Table 1.

[0148] Table 1

[0149] Compound % at t = 0 % at t = 24h Example 1 100.00±2,05 82.96±4.04

[0150] The compound of Example 1 proved to be highly resistant to trypsin digestion, with more than 80% of the starting peptide unchanged after 24 h.

[0151] Example 4 (Cytotoxicity of the compound of Example 1)

[0152] The cytotoxicity of the compound of Example 1 has been evaluated as described in Method B above, and the results are summarized in Table 2 below.

[0153] Table 2

[0154]

[0155]

[0156] From the above results, it can be seen that EC 50 The value is higher than 50 μM. The experiment shows that the compound of Example 1 has no significant toxicity.

[0157] Example 5 (Metathesis of the Compound of Example 2)

[0158] The translocation of the compound of Example 2 (5(6)-carboxyfluorescein labeled peptide of Example 1) was evaluated as described in Method D above.

[0159] Initially, a total of 1 μM of the peptide of Example 2 (5(6)-carboxyfluorescein labeled peptide of Example 1) was added to the apical side. After 24 h of incubation, the relative amounts of the peptides on the apical side, internalized, and basolateral sides were quantified. The amounts on the apical side and basolateral sides were normalized to the initial total amount of labeled peptide added to the apex and expressed as a percentage of the collected fluorescence dose. The internalized peptide was the difference between the initial amount added and the apical and basolateral sides. The results are shown in Table 3 below:

[0160] Table 3

[0161] Compound % Top side % Internalization % Basolateral Example 2 36.00 19.98 43.39

[0162] The data obtained showed that the peptide of Example 2 (peptide of Example 1 labeled with 5(6)-carboxyfluorescein) was able to translocate the BBB with an internalization retention rate of 19.98±7.324% up to 43.39±2.703% at 24 h.

[0163] Endothelial barrier integrity was assessed as described in Method D. Clearance of the fluorescent probe (fluorescently labeled dextran (FD) with a molecular weight of 4 kDa (FD4)) from the apical side was negligible in the presence or absence of labeled peptide, indicating a lack of fenestrations in the cell barrier and paracellular leakage.

[0164] Example 6 (In vitro determination of disruption of CB1R and 5-HT by bimolecular fluorescence complementation 2A R heteromer capacity)

[0165] The compounds of Example 1 were evaluated for disruption of CB1R and 5-HT by bimolecular fluorescence complementation according to the above method E. 2A The results are summarized in Table 4.

[0166] Table 4: Bimolecular fluorescence complementation of selected compounds (I) (fluorescence units)

[0167] Example Comparison Example 1 Reference Example 1 Reference Example 2 Reference Example 3 Fluorescence Unit 21353 10637 11154 13568 16754 % vs. Control 100％ 49.8 52.2 63.5 78.5

[0168] Example 7: Evaluation of peptide internalization by confocal microscopy

[0169] bEnd.3 cells were seeded on 8-well μ-slides (Ibidi, Germany) coated with ibiTreat at 50,000 cells / 200 μL for 24 h. Then, the cells were carefully washed twice with 1×PBS, washed once with culture medium, and cultivated with labeled peptides at a final concentration of 10 μM for 2 h. After 2 h, the cells were washed twice with 1×PBS again and once with culture medium, and the nucleus and membrane were stained with Hoescht 33342 (ThermoFisher, USA) and CellsMaskTM Deep Red (ThermoFisher, USA), respectively. Nuclear and membrane dyes were added to cells at a final concentration of 5 μg / mL and a working solution of 0.5X at 37°C for 10 min. Finally, the cells were washed twice with 1×PBS and imaged.

[0170] Acquisition was performed on a confocal scanning Zeiss LSM 880 microscope (Carl Zeiss, Germany) equipped with an α Plan-Apochromat X 100 oil immersion objective (1.46 numerical aperture). Diode 405-30 and HeNe633 lasers were used to excite Hoesch 33342 (Sigma-Aldrich, Spain) and CellMaskTM Deep Red plasma membrane stain (ThermoFisher, USA). 488 nm light from an argon laser was used to excite the peptides of Example 2 (peptides of Example 1 labeled with 5(6)-carboxyfluorescein-Lys). ×0.6 zoom images were recorded at 1024×1024 resolution in normal confocal mode. ZEN software was used for image acquisition. Fiji software was used for image processing. A total of at least 12 images were obtained in three independent replicates. To confirm the internalization of the peptide of Example 2 (ie, the peptide of Example 1 labeled with 5(6)-carboxyfluorescein-Lys), bEnd.3 cells were incubated with 10 μM of the peptide at 37° C. for 2 h and examined using confocal microscopy.

[0171] A punctate pattern of peptide fluorescence was observed, indicating that the peptide was present in the vesicular compartment after internalization. Figure 2 middle.

[0172] Example 8: Determination of the effect of peptides on memory in vivo (novel object recognition test)

[0173] The method described in "Puighermanal E, Marsicano G, Busquets-Garcia A, Lutz B, Maldonado R, Ozaita A. Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling. Nature Neuroscience, 12(9): 1152-8 (2009)" was used in combination with the slightly modified peptides described herein to evaluate the effects of the peptides on memory. The results are summarized in Table 5 below.

[0174] On the first day (adaptation), mice were adapted to the V-maze for 9 min to the task. On the second day (training), mice were placed back in the V-maze for 9 min, two identical objects were presented, and the time mice spent exploring each object was recorded. After 24 h, mice were placed in the maze again for 9 min (test), one of the familiar objects was replaced with a new object, and the total time spent exploring each of the two objects (new and familiar) was calculated. Object exploration is defined as nose orientation less than 2 cm from the object. The discrimination index is calculated by the difference between the time spent exploring new objects or familiar objects divided by the total time of exploring the two objects. It is believed that a higher discrimination index reflects a higher memory retention of familiar objects, because normal mice spend more time exploring new objects than familiar objects.

[0175] The following treatments were given intravenously (IV) immediately after training: the peptide to be tested in vehicle (saline + 2% DMSO) at a dose of 20 mg peptide / kg mouse body weight; or the vehicle alone, while IP vehicle (5% v / v ethanol, 5% v / v Cremophor-EL, and 90% v / v saline) or 3 mg THC / kg mouse dissolved in IP vehicle was given by intraperitoneal injection (IP) 30 min after the peptide.

[0176] Table 5

[0177]

[0178] The results summarized in the table above show that co-administration of the compound of Example 1 reversed the adverse effects of THC on memory.

[0179] Example 9: Determination of the analgesic effect of peptides in vivo (hot plate test)

[0180] The method described in "Monory K, Blaudzun H, Massa F, Kaiser N, Lemberger T, Schütz G, Wotjak CT, Lutz B*, Marsicano G*. Genetic Dissection of Behavioural and Autonomic Effects of D9-tetrahydrocannabinol in Mice. PLOS Biology, 5(10): e269, 2007" was used in combination with the slight modification described herein to evaluate the analgesic effect of the peptide. The results are summarized in Table 6 below.

[0181] Table 6

[0182]

[0183] THC-induced analgesia was measured using a hot plate analgesia instrument (Hot / Cold Plate Test, Bioseb, USA) 60 min after intraperitoneal injection (IP) of THC dissolved in IP vehicle (5% v / v ethanol, 5% v / v Cremophor-EL and 90% v / v saline) or vehicle alone (saline + 2% DMSO), and 90 min after intravenous injection of the peptide to be tested in the first injection of IV vehicle or vehicle alone. The plate was heated to 52 ± 0.5 ° C and the time (licking and jumping latency) until the mouse showed a licking and jumping response on the plate was recorded (in seconds). A cut-off time of 240 s was set to prevent tissue damage.

[0184] Co-administration of the compound of Example 1 with THC allowed a significant analgesic effect to be achieved.

[0185] References

[0186] Non-patent literature cited in the specification

[0187] 1. X.et al;PLoS Biol.2015Jul 9;13(7):e1002194

[0188] 2. Kandasamy R, Dawson CT, Craft RM, Morgan MM. Anti-migraine effect of Δ9-tetrahydrocannabinol in the female rat. Eur J Pharmacol. 2018 Jan 5; 818: 271-277. doi: 10.1016 / j.ejphar.2017.10.054. Epub 2017 Oct 28.

[0189] 3. Baron EP, Lucas P, Eades J, Hogue O. Patterns of medicinal cannabis use, strain analysis, and substitution effect among patients with migraine, headache, arthritis, and chronic pain in a medicinal cannabis cohort. HeadachePain. 2018May 24;19(1):37.doi:10.1186 / s10194-018-0862-2.

[0190] 4.Russo EB.Clinical Endocannabinoid Deficiency Reconsidered:CurrentResearch Supports the Theory in Migraine,Fibromyalgia,Irritable Bowel,andOther Treatment-Resistant Syndromes.Cannabis Cannabinoid Res.2016Jul 1;1(1):154-165.doi:10.1089 / can.2016.0009.eCollection 2016.

[0191] 5.Miller S,Daily L,Leishman E,Bradshaw H,Straiker A.Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Regulate IntraocularPressure.Invest Ophthalmol Vis Sci.2018Dec3;59(15):5904-5911.doi:10.1167 / iovs.18-24838.

[0192] 6.Escudero-Lara A,Argerich J,Cabanero D,Maldonado R.Δ9-tetrahydrocannabinol(THC)alleviates pelvic hypersensitivity in a mouse modelof endometriosis.IASP(International Association for the Study of Pain)7thWorld Congress on Pain.Boston,USA.12-16September2018,Abstract ID:471100.

[0193] 7.La Porta C,Bura SA,Llorente-Onaindia J,Pastor A,Navarrete F,García-Gutiérrez MS,De la Torre R,Manzanares J,Monfort J,Maldonado R.Role of theendocannabinoid systemin the emotional manifestations of osteoarthritispain.Pain.2015Oct;156(10):2001-12.

[0194] 8.Maldonado R, JE, D.The endocannabinoid system andneuropathic pain.Pain.2016Feb;157Suppl 1:S23-32.

[0195] 9.Engeli S.Central and peripheral cannabinoid receptors astherapeutic targets in the control of food intake and body weight.Handb ExpPharmacol.2012;(209):357-81.doi:10.1007 / 978-3-642-24716-3_17.

[0196] 10.Al-Ghezi ZZ,Miranda K,Nagarkatti M,Nagarkatti PS.Combination ofCannabinoids,Δ9-Tetrahydrocannabinol and Cannabidiol,AmelioratesExperimental Multiple Sclerosis by Suppressing Neuroinflammation ThroughRegulation of miRNA-Mediated Signaling Pathways.Front Immunol.2019Aug 21;10:1921.

[0197] 11. Allen D. Dronabinol Therapy: Central Nervous System Adverse Events in Adults With Primary Brain Tumors. Clin J Oncol Nurs. 2019 Feb 1; 23(1): 23-26.

[0198] 12. Buhmann C, Mainka T, Ebersbach G, Gandor F. Evidence for the use of cannabinoids in Parkinson's disease. J Neural Transm (Vienna). 2019Jul; 126(7):913-924.

[0199] 13. Valdeolivas S, Sagredo O, Delgado M, Pozo MA, Fernández-Ruiz J. Effects of a Sativex-Like Combination of Phytocannabinoids on Disease Progression in R6 / 2Mice, an Experimental Model of Huntington's Disease. Int J Mol Sci. 2017Mar23; 18(4).

[0200] 14. Donovan KA, Oberoi-Jassal R, Chang YD, Rajasekhara S, Haas MF, Randich AL, Portman DG. Cannabis Use in Young Adult Cancer Patients. J Adolesc Young Adult Oncol. 2019Aug 22. doi:10.1089 / jayao.2019.0039. [Epub ahead of print] Sequence Listing <110> University of Barcelona Pompeu Fabra University Autonomous University of Barcelona Joao Lobo Antunes Institute of Molecular Medicine <120> Peptide compounds for reducing the side effects of CB1 receptor agonists <130> P18316PC00 <150> EP 19382856.3 <151> 2019-10-03 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Transmembrane domain 5 (TM5) of cannabinoid receptor 1 <400> 1 Glu Thr Tyr Leu Met Phe Trp Ile Gly Val Thr Ser Val Leu Leu Leu 1 5 10 15 Phe Ile Val Tyr Ala Tyr Met Tyr Ile Leu Trp Gly Arg Lys Lys Arg 20 25 30 Arg Gln Arg Arg Arg 35 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Cell penetrating peptide sequence Tat <400> 2 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10

Claims

1. Compounds of formula (I): D-Trp-D-Leu-D-Ile-D-Tyr-D-Met-D-Tyr-D-Ala-D-Tyr-D-Val-D-Ala-Gly-D-Ile-D-Leu-D-Lys-D-Arg-D-Trp-NH2(I).

2. A pharmaceutical or veterinary composition comprising a therapeutically effective amount of a compound as defined in claim 1.

3. Use of a compound as defined in claim 1 in combination with Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC) for the preparation of a medicament for the treatment or prevention of a disease or condition susceptible to improvement by agonism of CB1 receptors in animals including humans, wherein the disease or condition susceptible to improvement by agonism of CB1 receptors is selected from the group consisting of pain associated with severe migraine, osteoarthritis, endometriosis, rheumatoid arthritis, fibromyalgia, back pain, knee pain, neuropathic pain of peripheral or central origin.

4. Use of a compound as defined in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating or preventing a disease susceptible to improvement by agonism of CB1 receptors in animals including humans in combination with Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC), wherein the disease susceptible to improvement by agonism of CB1 receptors is selected from pain associated with severe migraine, osteoarthritis, endometriosis, rheumatoid arthritis, fibromyalgia, back pain, knee pain, and neuropathic pain of peripheral or central origin.

5. The use according to claim 4, wherein the treatment or prevention is also capable of reducing memory impairment caused by Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC).

6. Use of a compound as defined in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing memory impairment caused by Δ9-tetrahydrocannabinol (dronabinol, THC or Δ9-THC).

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