Artificial proteins and their use

Abstract

The present invention relates to a synthetic peptide comprising, from the N-terminus to the C-terminus, the following elements: a) a first cell-penetrating peptide or a functional fragment or derivative thereof, or a bioactive variant, and b) a second peptide having agonist activity for the OR1 and OR2 receptors or a functional fragment or derivative thereof, or a bioactive variant.

Description

Technical Field

[0001] The present invention relates to a synthetic peptide comprising, from the N-terminus to the C-terminus, a first cell-penetrating peptide or a functional fragment or derivative thereof, or a bioactive variant, and a second peptide having orexin receptor-1 (OR1) and orexin receptor-2 (OR2) agonist activity or a functional fragment or derivative thereof, or a bioactive variant. The present invention also relates to the therapeutic use of said synthetic peptide.

Background Art

[0002] Orexin A and B (OXA and OXB), also known as hypocretin 1 and 2 (de Lecea et al., 1998), are peptides produced by a population of hypothalamic neurons that have maximal activity during active wakefulness and minimal activity during rapid eye movement (REM) sleep (Mileykovskiy et al., 2005). Orexin binds to two G-protein coupled receptors, a receptor called OR1 that is selective for OXA, and a receptor called OR2 that is non-selective for OXA and OXB (Sakurai et al., 1998). OR1 and OR2 are widely expressed in the central nervous system (CNS) in accordance with the extensive projection of orexinergic neurons (Peyron et al., 1998) (Leonard and Kukkonen, 2014). The orexinergic CNS system regulates multiple physiological functions, including wake and sleep behavior, energy homeostasis, and the autonomic control of the cardiovascular system (Bastianini and Silvani, 2018; Grimaldi et al., 2014; Sakurai, 2007). OR1 and OR2 are also expressed outside the CNS, in sites including adipose tissue, the male genital system (Leonard and Kukkonen, 2014), the heart (Perez et al., 2015), and the bone marrow (McAlpine et al., 2019). Orexin receptors outside the CNS may be involved in the pathophysiology of heart failure (Perez et al., 2015) and atherosclerosis (McAlpine et al., 2019), and may bind orexin that leaks into the systemic circulation after its release into the CNS by hypothalamic neurons that produce orexin (McAlpine et al., 2019). The OXA peptide is relatively protected from peptidase inactivation through two disulfide bonds with an N-terminal pyroglutamic acid residue and a C-terminal amidation (Sakurai et al., 1998). In contrast, OXB is a linear peptide with a free N-terminus (Sakurai et al., 1998) and is rapidly metabolized in the blood (Kastin and Akerstrom, 1999) and also in the cerebrospinal fluid (Yoshida et al., 2003). The permeability of the blood-brain barrier (BBB) to OXA is still debated.OXA has been reported to rapidly penetrate into the mouse brain by simple diffusion (Kastin and Akerstrom, 1999), and the results were consistent with other experiments conducted in rats (Kodama and Kimura, 2002; Van de Bittner et al., 2018) and functional data obtained in dogs (John et al., 2000). However, another study reported only a very small (<1%) penetration of OXA into the brain in mice and rats (Bingham et al., 2001), and this conclusion was also supported by data obtained in dogs (Fujiki et al., 2003).

[0003] Narcolepsy type 1 (NT1) is a severe and rare (prevalence 14 / 100,000 subjects (Scheer et al., 2019)) neurological disorder (Peyron et al., 2000) associated with almost complete loss of function of orexinergic neurons, probably due to autoimmune damage (Mahoney et al., 2019). NT1 is characterized by a wide range of signs and symptoms including excessive daytime sleepiness, fragmented sleep with increased muscle tone, increased tendency and decreased latency of REM sleep with episodes of sleep-onset REM sleep (SOREM), cataplexy (loss of muscle tone during wakefulness, often triggered by positive emotions), tendency to obesity, and non-dipper blood pressure profile (Grimaldi et al., 2014; Mahoney et al., 2019). Clinical states similar to human NT1 occur in dogs as a result of mutations in the gene encoding OR2 or as a sporadic form due to orexin deficiency (Mignot, 2014). The clinical picture of NT1 is recapitulated in both orexinergic neuron gene-disrupted OX-ATX3 transgenic mice (Hara et al., 2001) and orexin knockout (OX-KO) mice (Chemelli et al., 1999). Data from double knockout (KO) mice of OR1 and OR2 have shown that the lack of orexin binding to OR1 and OR2 is required for the mouse phenotype that recapitulates the full clinical picture of human NT1, in which OR2 plays a major role (Hasegawa et al., 2014; Willie et al., 2003). Consistent with these data, orexin gene therapy improves the pathological phenotypes of OX-ATX3 mice (Mieda et al., 2004) and OX-KO (Liu et al., 2008).

[0004] Despite the existing knowledge about the physiology of the orexinergic system, currently available therapies for NT1 do not rely on orexin replacement and all have significant limitations in terms of efficacy and side effects (S. W. Black et al., 2017). The preclinical data on the efficacy of orexin replacement therapy are heterogeneous. In dogs with the familial form of NT1, the therapeutic efficacy of intravenous OXA has been reported (John et al., 2000), but not confirmed (Fujiki et al., 2003). Intravenous or intraspinal administration of OXA was also not effective in single dogs with the sporadic form of NT1 (Schatzberg et al., 2004). On the other hand, intracerebroventricular (ICV) (Mieda et al., 2004) or intraspinal (Kaushik et al., 2018) administration of OXA has been shown to be effective in reducing cataplexy in OX-KO mice, where ICV administration of OXA also increased arousal (Mieda et al., 2004). In a pilot study in NT1 patients, intranasal administration of OXA has been reported to result in brain penetration of OXA comparable to that obtained intravenously in rats (Van de Bittner et al., 2018) and a decrease in REM sleep time and the incidence of SOREM (Baier et al., 2011). Overall, these studies indicate that the efficacy of systemically administered OXA is at best very limited, with insufficient permeability of the BBB indicated as a limiting factor (S. W. Black et al., 2017).

[0005] The development of OR2-selective non-peptidergic agonists that can pass through the BBB has made great progress. Compound 30 (Nagahara et al., 2015), renamed YNT-185 (Irukayama-Tomobe et al., 2017), although its efficacy is limited, has been shown to increase wake time and decrease SOREM in OX-KO mice after intraperitoneal administration. Compound TAK-925 has been shown to increase the time spent awake after subcutaneous (SC) injection in control wild-type (WT) mice (Yukitake et al., 2019) and has undergone a phase 1 trial in NT1 patients using intravenous infusion (Tanaka et al., 2020). Compound TAK-988 is orally bioavailable, can increase the time spent awake and decrease cataplexy in OX-ATX3 mice (Kimura, Ishikawa, and Suzuki, 2020), and has been shown to increase the time spent awake in non-human primates (Kimura, Ishikawa, Hara et al., 2020). Although all of these selective OR2 receptor agonists have the potential to contribute to NT1 therapy, insights into the physiology of the orexinergic system and the pathophysiology of NT1 indicate that it is necessary to re-integrate the binding of orexin to both OR1 and OR2 receptors to fully elucidate the NT1 clinical picture. In particular, the severity of cataplexy is moderate in OR2-KO mice, where orexin binding to OR1 is preserved, but the severity of cataplexy is high in OX-KO mice, where orexin and its binding to both OR1 and OR2 are completely absent (Willie et al., 2003). In OR1 and OR2 double KO mice, OR2 expression in the dorsal raphe via viral vectors prevented cataplexy and potentially activated serotonergic neurons of this structure that physiologically express both OR1 and OR2, but still could not prevent excessive REM sleep during the active time (dark period) (Hasegawa et al., 2014).Recent data have shown a tendency for atherosclerosis in OX-KO mice due to a lack of activation of OR1 receptors expressed by hematopoietic precursors in the bone marrow (McAlpine et al., 2019). There is no previously obtained evidence regarding the development of agonists of OR1 and OR2 that can pass through the BBB after systemic administration and are effective in the treatment of NT1.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, it is necessary to provide new molecules that are effective in the treatment of NT1.

Means for Solving the Problems

[0008] The inventors have now found an agonist of OR1 and OR2, herein defined as OX-DRAGON (TAT-OXA(4-33)), which is effective for cataplexy, a characteristic symptom of NT1, by systemic SC administration.

[0009] OX-DRAGON is an artificial peptide consisting of a truncated sequence (4-33) of the natural sequence of OXA (the sequence is preferably characterized by the sequence of SEQ ID NO: 1) fused at the N-terminus with the cell-penetrating TAT peptide sequence of type 1 human immunodeficiency virus (HIV) (preferably characterized by the sequence of SEQ ID NO: 7). Like the natural OXA protein, OX-DRAGON has a C-terminal amidated residue and two disulfide bridges (14-20 and 15-22).

[0010] OXA is preferably characterized by a sequence identified by NCBI accession number 1R02_A (protein).

[0011] TAT is preferably characterized by a sequence contained in a sequence identified by NCBI accession numbers BAA12992.1 (protein) and D86068.1 (nucleotide sequence).

[0012] The structure of OX-DRAGON is adapted to be able to cross the BBB after systemic administration and to bind to and activate both orexin receptors. From in vitro and in vivo experiments, it has been shown that OX-DRAGON crosses the cell membrane, acts as an agonist for OR1 and OR2, and exhibits a significant anti-cataplectic effect after SC administration in OX-KO mice, a mouse model of NT1 that has been verified.

[0013] OX-DRAGON also acts on CNS neurons expressing orexin receptors and peripheral extracerebral cells expressing such receptors and is thus designed to have the potential to fully elucidate the clinical picture of NT1 (John et al., 2000; Mieda et al., 2004) (Figure 1). The TAT peptide crosses the BBB (Bolhassani et al., 2017; Schwarze et al., 1999; Trazzi et al., 2018) and has already been successfully employed for the brain delivery of brain-derived neurotrophic factor (BDNF), a peptide whose receptor is expressed in the cell membrane in the same way as OR1 and OR2 (Verheij et al., 2016; Wu et al., 2015). Based on the data available in the literature, OXA may have a limited ability to cross the BBB (John et al., 2000; Kastin and Akerstrom, 1999; Kodama and Kimura, 2002; Van de Bittner et al., 2018). Such ability may cooperate with the effect of TAT, which further increases the ability of OX-DRAGON to cross the BBB (Kimura, Ishikawa, Hara et al., 2020; Kimura, Ishikawa and Suzuki, 2020; Tanaka et al., 2020; Yukitake et al., 2019). Side effects regarding the immunogenicity or toxicity of the TAT peptide have not been reported so far (Bolhassani et al., 2017). The structure of OXA is completely conserved between humans (Homo sapiens) and mice (Mus musculus) (Sakurai et al., 1998), which supports its non-clinical testing in mice. Also, OXA is highly resistant to peptidases (Kastin and Akerstrom, 1999; Sakurai et al., 1998; Yoshida et al., 2003). OX-DRAGON contains the C-terminal amidated sequence of OXA, which is important for binding to OR1 and OR2. The truncated 4-33 sequence of the OXA peptide contained in OX-DRAGON does not have the N-terminal pyroglutamic acid residue that exists in native OXA but is technically difficult to bind to the TAT peptide sequence.However, there is evidence that the 4-33 truncate sequence of OXA produces half-maximal responses (EC50) equal to approximately 1 / 7 of those of the native OXA protein and maintains significant agonist capabilities for OR1 and OR2 at a concentration and with the same receptor selectivity profile (EC50 OR1 / OR2 = 1.6) (Lang et al., 2004). In vitro experiments with cells of neuronal cell lines expressing human OR1 or OR2 have shown that OX-DRAGON has a receptor selectivity profile that favors OR2 more (EC50 OR1 / OR2 = 6.5) and has similar capabilities to OXA for OR1 and OR2. Experiments in the mouse model of NT1 have also shown that OX-DRAGON is effective after SC administration. The SC administration route is safe, tolerable, and has been successfully used in millions of people, including pediatric-aged individuals, for long-term insulin therapy for diabetes.

[0014] Accordingly, an object of the present invention is a synthetic peptide comprising, from the N-terminus to the C-terminus, the following elements: a) a first cell-penetrating peptide or a functional fragment or derivative thereof, or a bioactive variant, and b) a second peptide having agonist activity for OR1 and OR2 or a functional fragment or derivative thereof, or a bioactive variant Preferably, the second peptide comprises or consists of a fragment of the protein orexin A (OXA), and preferably the fragment is

[0015] i. the sequence PDCCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 1), or ii. - the sequence CCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 2) or - the sequence RQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 3) or - the sequence TCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 4) or - the sequence SCRLYELLHGAGNHAAGILTL (SEQ ID NO: 5) or - - The array RLYELLHGAGNHAAGILTL (sequence number 6) comprising or consisting of a fragment of sequence number 1, or iii. an array comprising or consisting of an array having at least 70% percent identity with an array comprising or consisting of sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5 or sequence number 6 comprising or consisting of, preferably the second peptide is amidated at the C-terminus, preferably the second peptide preferably exhibits disulfide bridges at positions 3-9 and 4-11 of sequence number 1 or positions 1-7 and 2-9 of sequence number 2.

[0016] Preferably, the first peptide comprises or consists of the TAT peptide of the HIV-1 virus, preferably the TAT peptide comprises or consists of an array having at least 70% percent identity with an array comprising or consisting of the sequence YGRKKRRQRRR (sequence number 7), preferably the first peptide comprises or consists of sequence number 7, or the first peptide is the following sequence: LLIILRRRIRKQAHAHSK (sequence number 9), RRLSYSRRRF (sequence number 10), YARKAARQARA (sequence number 11), GLAFLGFLGAAGSTMGAWSQPKKKRKV (sequence number 12), KETWWETWWTEWSQPKKRKV (sequence number 13), MVRRFLVTLRIRRACGPPRVRV (sequence number 14), MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (sequence number 15), KLALKLALKALKAALKLA (sequence number 16), LSTAADMQGVVTDGMASGLDKDYLKPDD (sequence number 17), DPKGDPKGVTVTVTVTVTGKGDPKPD (sequence number 18), PFVYLI (SEQ ID NO: 19), MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 20), RKKRRRESRKKRRRES (SEQ ID NO: 21) or KCFQWQRNMRKVRGPPVSCIKR (SEQ ID NO: 22) comprises or consists of a sequence including one of the above, or a sequence having at least 70% percent identity.

[0017] Preferably, the peptide of the present invention comprises or consists of SEQ ID NO: 8 (YGRKKRRQRRRPDCCRQKTCSCRLYELLHGAGNHAAGILTL) or a functional fragment, equivalent, variant, mutant, derivative, or functional recombinant or synthetic analog thereof, and the sequence preferably has disulfide bridges at positions 14 - 20 and 15 - 22 of SEQ ID NO: 8, and the C-terminus is amidated.

[0018] Preferably, the peptide of the present invention comprises or consists of SEQ ID NO: 8 (YGRKKRRQRRRPDCCRQKTCSCRLYELLHGAGNHAAGILTL) and the sequence has disulfide bridges at positions 14 - 20 and 15 - 22 of SEQ ID NO: 8, and the C-terminus is amidated.

[0019] Preferably, the peptide of the present invention has an anti-cataplectic effect and / or the following functions: - Promote wakefulness or counteract drowsiness - Reduce body weight and glucose intolerance in the state of obesity - Improve myocardial function in heart failure - Prevent atherosclerosis - Have an anti-inflammatory effect - Have an analgesic effect - Have a chemotherapeutic effect on colon cancer and neuroblastoma and has at least one of the above functions.

[0020] A further object of the present invention is a pharmaceutical composition comprising a peptide disclosed herein, and at least one pharmaceutically acceptable vehicle, preferably for subcutaneous administration.

[0021] Another object of the present invention is an isolated nucleic acid encoding a peptide disclosed herein, or a recombinant expression vector containing said nucleic acid.

[0022] A further object of the present invention is a host cell comprising and / or expressing a peptide disclosed herein, or a nucleic acid or vector disclosed herein.

[0023] A further object of the present invention is for use as an analgesic for the medical management of, for example, pain, post-stroke pain and chemotherapy-induced pain, etc., in drug-resistant pain conditions, chronic pain, in patients with or without NT1, for the treatment and / or prevention of type 1 narcolepsy, type 2 narcolepsy, idiopathic hypersomnia, obesity, or related cardiometabolic comorbidities including atherosclerosis, heart failure, for example inflammation in septic shock, neuroinflammation, or intestinal barrier-level inflammation in, for example, ulcerative colitis, tumors and metastases, for example colon cancer and neuroblastoma, a peptide or pharmaceutical composition or nucleic acid, or vector, or cell disclosed herein.

[0024] Preferably, the peptide defined herein is for subcutaneous administration.

[0025] A further object of the present invention is a method for producing a peptide of the present invention, the method comprising the steps of transforming a host cell with an expression vector encoding the peptide, culturing the host cell under conditions that allow expression of the fusion protein, and optionally recovering and purifying the fusion protein.

[0026] The present invention also includes an array disclosed herein, a functional fragment or derivative thereof, and an array having at least 70% percent identity.

[0027] The present invention will be disclosed by way of non-limiting examples with reference to the following drawings.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0029] In the context of the present invention, synthetic peptide also means artificial peptide, fusion peptide, or conjugate.

[0030] In the context of the present invention, a preferred cell-penetrating peptide is preferably TAT characterized by the sequence YGRKKRRQRRR (SEQ ID NO: 7), for example (Elmquist et al., 2001; Rousselle et al., 2001; Stalmans et al., 2015): pVEC: LLIILRRRIRKQAHAHSK (SEQ ID NO: 9) Any cell-penetrating peptide known to those skilled in the art, such as SynB3:RRLSYSRRRF (SEQ ID NO: 10), can be used.

[0031] Other cell-penetrating peptides that can be used are

[0032] [Table 1]

[0033] as follows.

[0034] In the context of the present invention, preferred OR1 and OR2 receptor-binding sequences are PDCCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 1), which has a C-terminal amidated residue and two disulfide bridges (3-9 and 4-11), corresponding to the OXA protein truncation sequence 4-33 (Lang et al., 2004).

[0035] Other OR1 and OR2 receptor-binding sequences that can be used are the reduced sequence of SEQ ID NO: 1 or fragments thereof, with or without disulfide bridges, or the following truncated OXA sequences (Lang et al., 2004), all of which are intended to have a C-terminal amidated residue, and the above preferred sequence (SEQ ID NO: 1): OXA(6-33) CCRQKTCSCRLYELLHGAGNHAAGILTL SEQ ID NO: 2 OXA(8-33) RQKTCSCRLYELLHGAGNHAAGILTL SEQ ID NO: 3 OXA(11-33) TCSCRLYELLHGAGNHAAGILTL SEQ ID NO: 4 OXA(13-33) SCRLYELLHGAGNHAAGILTL SEQ ID NO: 5 OXA(15-33) RLYELLHGAGNHAAGILTL SEQ ID NO: 6 Optionally reduced (i.e., without disulfide bridges) portions or fragments thereof.

[0036] The present invention also encompasses modifications to an intermediate sequence between the sequence of a cell-penetrating peptide, perhaps with a side chain, and the sequence of an orexin-agonistic agonist.

[0037] The intermediate sequence can include, for example, peptide tags and / or functional sequences, such as endosomal escape or protease resistance sequences (Eldridge et al., 2009; Li et al., 2020; Lotze et al., 2016; Varkouhi et al., 2011; Wadia et al., 2004).

[0038] The term "functional derivative" is used herein to mean a chemical derivative of a disclosed peptide that has the same physiological function as the corresponding unmodified counterpart or has the same function in vitro in a functional assay (e.g., in one of the assays disclosed herein or in one of the examples disclosed herein).

[0039] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as subcutaneous, intradermal, intravenous, transdermal (topical), transmucosal, and rectal, inhalation, intranasal administration.

[0040] The present invention also relates to a polynucleotide encoding a peptide as defined herein, a vector comprising the above polynucleotide, and a genetically engineered host cell expressing a peptide as defined above. Preferably, the polynucleotide is selected from the group consisting of RNA or DNA, and preferably the polynucleotide is DNA.

[0041] Preferably, the vector is an expression vector selected from the group consisting of plasmids, virus particles, and phages.

[0042] Preferably, the host cell is selected from the group consisting of bacterial cells, fungal cells, insect cells, animal cells, and plant cells, and preferably the host cell is an animal cell.

[0043] The peptides of the present invention are in the form of linear and multimeric synthetic or recombinant peptides in any chemical, physical, and / or biological form that maintains their function.

[0044] The peptides of the present invention can be synthesized and used in branched form as multiple antigenic peptides (MAPs), as disclosed, for example, in U.S. Patent No. 5,229,490.

[0045] All amino acids in the peptide can have the same stereochemistry. For example, the peptide can consist of only L-amino acids or only D-amino acids. Alternatively, the peptide can contain a combination of L and D amino acids. Partially or fully retroinverse peptides are also included in the present invention (Rai, 2019).

[0046] The peptides of the present invention can be in dimeric or multimeric form. Examples of spacers included in the dimer or multimer herein include ester bonds (-CO-O-, -O-CO-), ether bonds (-O-), amide bonds (NHCO, CONH), sugar chain-based linkers, polyethylene glycol linkers, peptide linkers, etc. Examples of peptide linkers include linkers containing at least one of the 20 natural amino acids that make up proteins. The number of amino acids in the linker peptide can be, for example, but not limited to, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4. Examples of peptide linkers include arginine dimer, arginine trimer, arginine tetramer, lysine dimer, lysine trimer, lysine tetramer, glycine dimer, glycine trimer, glycine tetramer, glycine pentamer, glycine hexamer, alanine-alanine-tyrosine-leucine (AAY), isoleucine-leucine-alanine (ILA), arginine-valine-lysine-arginine (RVKR), etc. The spacer can be divalent or polyvalent.

[0047] When the peptides of the present invention are multimeric, branched polyvalent linkers (e.g., dendrimers), metal complexes, etc. can be used for crosslinking.

[0048] Peptides as defined above, or derivatives or variants of the peptides of the present invention are also included in the present invention. Suitably, "derivatives" or "variants" include those in which the amino acids occurring in the sequence are structural analogs of natural - origin amino acids. The amino acids used in the sequence can also be derivatized or modified, for example labeled, provided that the function of the peptide is not significantly and detrimentally affected. Derivatives and variants as disclosed above can be prepared during peptide synthesis or by post - production modification, or, if the peptide is in recombinant form, using known techniques of site - directed mutagenesis, random mutagenesis or enzymatic cleavage and / or nucleic acid ligation. In the context of the present invention, variants also include variants having antagonist activity against OR1 or OR2 or both OR1 and OR2.

[0049] Functional "fragments" of the present invention can be obtained by truncation, for example by removal of one or more amino acids from the N - terminus and / or by removal of one or more amino acids within the sequence. Such fragments can be derived from the sequences disclosed herein or from functionally equivalent peptides disclosed herein.

[0050] Suitably, functional variants or derivatives of the present invention have an amino acid sequence having more than 70% homology or identity, for example 75% or 80%, preferably more than 85%, for example more than 90% or 95% homology or identity with the sequences disclosed herein.

[0051] The polynucleotides or peptides disclosed herein can also be defined with respect to a more specific range of identity and / or similarity to those exemplified herein. Sequence identity can typically be higher than 70%, more preferably higher than 80%, even more preferably higher than 90%, and can be higher than 95%. The identity and / or similarity of the sequences can be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of the sequences exemplified herein. Unless otherwise specified, when used herein, the percentage of sequence identity and / or similarity between two sequences can be determined using the algorithm derived from Karlin and Altschul (Karlin and Altschul, 1990), modified as (Karlin and Altschul, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (Altschul et al., 1990). A BLAST search can be performed with the NBLAST program, score = 100, word length = 12 to obtain sequences having the desired percentage of sequence identity. For comparison purposes, Gapped BLAST can be used to obtain a gapped alignment / alignment of the sequences with gaps, as disclosed in (Altschul et al., 1997). When using BLAST and Gapped BLAST, the default parameters of each program (NBLAST and XBLAST) can be used. See the NCBI / NIH website.

[0052] The peptides of the present invention can be chemically modified, for example post-translationally modified, as defined herein. For example, the peptides of the present invention can be glycosylated or can contain modified amino acid residues. The peptides of the present invention can be various forms of polypeptide derivatives, including conjugates with starch and polypeptides.

[0053] Chemically modified peptides also include those having one or more residues chemically derivatized from the reaction of functional side chains. Such derivatized side chains include those obtained to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzyloxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, and formyl groups. Free carboxylic acid groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.

[0054] Cyclic peptides, i.e., peptides of the present invention linked by covalent bonds to form a ring, are also included as chemically modified peptides.

[0055] Those containing one or more amino acid derivatives of natural origin of the 20 standard amino acids are also included as chemically modified peptides. For example, 4-hydroxyproline can be substituted for proline, or homoserine can be substituted for serine.

[0056] The peptides of the present invention may have a marking label. Suitable labels include radioisotopes, fluorescent labels, enzyme labels, or other protein labels such as biotin.

[0057] All formulas provided herein are also intended to represent both the unlabeled and isotopically labeled forms of the peptide. Isotopically labeled peptides have a structure represented by the formulas provided herein, except that one or more atoms are replaced with atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the peptides of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine. Pharmaceutically acceptable solvated compounds according to the present invention include those in which the crystallization solvent is isotopically substituted, such as D2O, d6-acetone, d6-DMSO.

[0058] For use according to the present invention, the peptides disclosed above can be prepared by conventional synthetic modes, including genetic or chemical means.

[0059] Synthetic techniques such as solid-phase Merrifield-type synthesis may be preferred for reasons of purity, antigen specificity, the complete absence of unwanted by-products, and ease of production. Techniques suitable for solid-phase peptide synthesis are well known to those skilled in the art (see, for example, Fields and Noble, 1990; Merrifield, 1969). Chemical synthesis may be carried out by methods well known in the art, involving a periodic set of selective deprotection reactions of the functional groups of the terminal amino acids and coupling of selectively protected amino acid residues, followed by complete deprotection of all final functional groups. The synthesis may be carried out in solution or on a solid support using a suitable solid phase known in the art.

[0060] In an alternative embodiment, the peptides of the invention may be produced or administered in the form of polynucleotides that can encode and express them. Such polynucleotides can be synthesized according to methods well known in the art, such as those disclosed in (Green et al., 2012). Such polynucleotides can be used in vitro or in vivo in the production of the peptides of the invention. Such polynucleotides can then be administered or used in the treatment of NT1 or another disease or condition disclosed herein.

[0061] The present invention also includes an expression vector containing such a polynucleotide sequence. Such expression vectors are typically constructed in the field of molecular biology and may, for example, be necessary to enable the expression of the peptides of the invention and may involve the use of plasmid DNA and appropriate primers, promoters, enhancers, and other elements, such as a polyadenylation signal, positioned in the correct orientation. However, other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, the inventors refer to (Green et al., 2012).

[0062] Thus, the peptide can be provided by transporting such a vector into cells and enabling transcription from the vector. Suitably, the polynucleotide for use in the present invention, or in a vector in the present invention, can be operably linked to a control sequence capable of providing expression of the coding sequence by a host cell, i.e., the vector can be an expression vector. The term "operably linked" refers to juxtaposition wherein the disclosed components are in a relationship that enables them to function in their intended manner. A regulatory sequence such as a promoter "operably linked" to a coding sequence is arranged so that expression of the coding sequence is obtained under conditions compatible with the regulatory sequence.

[0063] The vector can be, for example, a plasmid, virus or phage vector having an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a promoter regulator. The vector may contain one or more selectable marker genes, such as the ampicillin resistance gene in the case of a bacterial plasmid, or a resistance gene for a fungal vector. The vector can be used, for example, in vitro for the production of DNA or RNA, or can be used to transfect or transform a host cell, such as a mammalian cell. The vector can also be adapted for use in vivo, for example to enable in vivo expression of a polypeptide.

[0064] The present invention also includes cells modified to express the peptide of the present invention. Such cells preferably include taxonomically higher eukaryotic cell lines such as mammalian cells or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacterial cells. Specific examples of cells that can be modified by inserting a vector encoding the peptide of the present invention include mammalian HEK293T, CHO, HeLa and COS cells. Expression can also be obtained in transformed oocytes. Suitable peptides can be expressed in cells of transgenic non-human animals, especially mice.

[0065] The present invention also relates to antibodies (monoclonal or polyclonal) and antigen-binding fragments thereof (e.g., F(ab)2, Fab, and Fv fragments, i.e., fragments of the "variable" region of an antibody that contain the antigen-binding site) that bind to a peptide as defined above, i.e., bind to an epitope present on the peptide, and thus bind selectively and specifically to such a peptide and can be used in the method of the present invention.

[0066] The peptides of the present invention may be employed alone as a monotherapy or in combination with other therapeutic agents for the prevention and / or treatment of the diseases described above or below.

[0067] Compositions comprising one or more peptides or polypeptides disclosed herein are also encompassed by the present invention. Such compositions typically include a pharmaceutically acceptable vehicle. As used in the present invention, the term "pharmaceutically acceptable vehicle" includes, but is not limited to, saline, solvents, dispersion media, coating agents, antibacterial and antifungal agents, absorption-delaying agents, and isotonic agents, among others, that are compatible with pharmaceutical administration. Additional compounds may also be incorporated into the compositions.

[0068] Compositions can be prepared by methods known in the art of pharmacy. Generally, the compositions can be formulated to be compatible with their intended route of administration. The formulations can be solid or liquid. Administration can be systemic or local. In some embodiments, local administration can be advantageous for the management of targeted site-specific diseases. Local therapy can directly provide clinically effective high concentrations at the treatment site and is less likely to cause systemic side effects.

[0069] Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular) and topical (e.g., on the skin, inhalation, transmucosal, and intranasal) administration. Dosage forms suitable for topical administration can include nasal sprays, metered-dose inhalers, dry powder inhalers, or nebulizers. Solutions or suspensions can contain the following components: a sterile diluent such as water for administration, physiological saline, volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; electrolytes such as sodium ions, chloride ions, potassium ions, calcium ions, and magnesium ions, and agents for isotonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The composition can be enclosed, for example, in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0070] The composition can include a sterile aqueous solution or dispersion, and a sterile powder for the immediate preparation of a sterile solution or dispersion. For intravenous administration, suitable carriers include human albumin, physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, NJ), or phosphate buffered saline. Typically, the composition should be sterile and, when suitable for injection, should be fluid to allow easy injectability. The composition should be stable under the conditions of manufacture and storage and should be preserved to prevent contamination with microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium, including, for example, albumin, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, etc., and sodium chloride. Sustained absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin. Sterile solutions can be prepared by incorporating the active compound (e.g., a peptide or polynucleotide disclosed herein) in the required amounts into a suitable solvent with the components or combinations of components listed above, and then, if necessary, subjecting the mixture to sterile filtration. Generally, dispersions are prepared by incorporating the active compound, which includes a dispersion medium such as those listed above and other components, into a sterile vehicle. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation that can be used include vacuum drying and freeze drying, which produce powders of the active ingredient and any other desired components from a previously filtered and sterilized solution.

[0071] For enteral administration, the composition can be administered, for example, by nasogastric tube, enema, colonoscopy, or orally. Oral compositions may contain an inert diluent or an edible vehicle. For the purpose of oral therapeutic administration, the active compound may be incorporated into an excipient and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid vehicle. A pharmaceutically compatible binder may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primojel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate or orange flavoring.

[0072] For administration by inhalation, the active compound can be delivered in the form of an aerosol spray, a nebulizer or an inhaler, such as a nasal spray, a metered-dose inhaler or a dry powder inhaler. Systemic administration can also be obtained by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for permeating the barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, surfactants, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be effected using a nasal spray or a nasal suppository. For transdermal administration, the active compound can be formulated in an ointment, a salt, a gel or a cream, which are generally known in the art. An example of transdermal administration is iontophoretic transport to the dermis or other relevant tissues. The active compound can also be prepared in the form of a suppository (e.g., together with conventional suppository bases such as cocoa butter and other glycerides) or a retention enema for rectal delivery. The active compound can be prepared with a carrier that protects the compound from rapid excretion from the body, such as a controlled-release formulation containing an implant. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyester and polylactic acid can be used. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. Transport reagents such as lipids, cationic lipids, phospholipids, liposomes, microencapsulation and nanoparticles can also be used.

[0073] As is common practice, the composition will usually be accompanied by instructions or printed instructions for use in the treatment of the disorder in question.

[0074] Those skilled in the art will select the form of administration and the effective dosage by choosing appropriate diluents, adjuvants and / or excipients.

[0075] When the peptide of the present invention is combined with other active ingredients, the active ingredients may be formulated separately in a preparation of a single component in one of the forms disclosed above and then provided as a combined preparation administered simultaneously or at different times, or may be formulated together in a preparation of two or more components.

[0076] The peptides as defined above can be administered to a patient, for example, at a total daily dose of 0.1 to 500 mg / kg body weight per day. The dosage unit composition may contain an amount that is a multiple so as to constitute a daily dose.

Example

[0077] 1) The synthesis of OX-DRAGON is feasible. Two batches of OX-DRAGON were synthesized by an external commercial organization (Genscript, www.genscript.com), and their purity was ≧98.0% and the net peptide content was ≧68.0% (Figure 2). The peptide is highly soluble (<=10 mg / mL) in water, DPBS, and DMSO.

[0078] 2) OX-DRAGON is an agonist of the OR1 and OR2 receptors for orexin in vitro. OX-DRAGON did not cause any effect on control Neuro-2a cells (wild type, WT) that do not express such receptors in two stable clones of mouse neuroblastoma Neuro-2a cells expressing the human cDNA of OR1 or OR2, respectively (Holmqvist et al., 2002), and the cytoplasmic calcium ion concentration ([Ca 2+) was shown to elicit the maximum in vitro response with respect to the increase. Selective expression of the gene encoding OR1 or OR2 in the corresponding cell line, as well as the absence of expression of OR1 and OR2 in WT cells, was confirmed by individual deoxyribonuclease treatment and real-time PCR from RNA extracted from back-transcribed cells (Figure 3A). Using the Fluo-4 Direct calcium assay (Invitrogen) and a Tekan Spark microplate reader, the resulting data were normalized against the maximum response to assess the response regarding intracellular [Ca 2+ . The response was evaluated three times. From the results, the responses of OR1-expressing cells to OX-DRAGON at a concentration of 8.5 μM (Figures 3B and 3C) and particularly those of OR2-expressing cells (Figures 3B and 3D) were shown to be close to the maximum ones induced by OXA at a concentration of 14.8 μM in terms of both amplitude and time dynamics. Since there was no significant effect of OX-DRAGON on WT cells (Figure 3B), it was shown that the effect of OX-DRAGON on OR1- and OR2-expressing cells depends on the respective receptor expressed.

[0079] Using commercially available receptor assay kits (n.600240 and 600250, Cayman Chemical, USA), the concentration-response relationships of OX-DRAGON and OXA were evaluated on the OR1 and OR2 receptors of HEK-293T cells (Yamanaka et al., 2020). Data were obtained from two cell batches for OXA on OR1, one batch for OX-DRAGON on OR1, three batches for OXA on OR2, and two batches for OX-DRAGON on OR2. The data were fitted to a function of the Hill equation to evaluate the ligand concentration that gives rise to the half-maximal response (EC 50 ). From the results (Figure 4), the following EC 50 values were obtained: OXA on OR1: 43.7 nM OXA on OR2: 30.0 nM OX-DRAGON at OR1: 67.7 nM OX-DRAGON at OR2: 10.5 nM

[0080] This resulted in an estimated OR1 / OR2 EC ratio of 1.45 for OXA and 6.47 for OX-DRAGON, showing a slightly higher preference for OX-DRAGON of OR2 over OR1 for OXA and a similar receptor selectivity profile for OXA and OX-DRAGON. 50

[0081] 3) OX-DRAGON shows an effect similar to or superior to OXA on arousal and sleep after ICV administration in the OX-KO mouse model of NT1. To test the ability of OX-DRAGON to show in vivo CNS effects similar to those of OXA despite its ability to cross the BBB, in a mouse model of NT1 consisting of adult female OX-KO mice (bred in-house by the Dept Biomedical and Neuromotor Sciences, University of Bologna, genetic background N>10 C57B1 / 6J (Bastianini et al., 2011)), the electroencephalogram (EEG; bilateral frontoparietal derivation electrodes (Bastianini et al., 2011) and cannulae in both lateral ventricles (C313G / SPC and C313IC / SPC, Plastics One; anteroposterior coordinate -0.6 mm, bilateral +1.2 mm, dorsoventral -2.0 mm)) of the nuchal muscle electromyogram (EMG) were implanted under general anesthesia (isoflurane) for the experiment. Biocompatible acrylic resin protection was applied to the animal's head to protect the electrodes and cannula connectors. After 1 - 2 weeks of postoperative recovery, the animals were connected to thin electrical cables and catheters via rotating electrical and fluid connectors respectively, and the catheters themselves were connected to a precision pump for ICV injection. The rotating connectors, cables and catheters were supported by a balance arm to allow the animals to move completely freely during recordings made in a 12:12 hour light:dark cycle. Arousal, non-REM sleep and REM sleep were evaluated based on EEG and EMG tracking using previously published techniques in detail (Bastianini et al., 2011). Administration of OX-DRAGON via ICV (160 μM at 5 μL / hour during the first 6 hours of the light period, ZT0 - 6) to bypass the BBB produced a consistent effect of increased wakefulness time and decreased non-REM sleep time throughout the light period (ZT0 - 12) compared to administration of the corresponding vehicle (artificial cerebrospinal fluid; Figure 5, center). Furthermore, a marked decrease in REM sleep time continued until the end of the dark period (ZT24).The effects on wakefulness and non-REM sleep were similar to those induced by OXA ICV (160 μM at 5 μL / h, ZT0 - 6; Figure 5, left), a natural double agonist of OR1 and OR2, although the increase in wakefulness time during the ZT1 - ZT6 period was slightly lower. In contrast, ICV administration of TAT (160 μM at 5 μM / h, ZT0 - 6; Figure 6, right) in OX-KO mice did not cause significant effects on wakefulness and non-REM sleep times, but led to a significant decrease in REM sleep time during the dark period (ZT13 - 24) compared to the vehicle, although the value was slightly higher than that obtained with OX-DRAGON (Figure 5, right). This suggests that the decrease in REM sleep time during the dark period shown by OX-DRAGON via ICV in OX-KO mice cannot be attributed solely to the effect of TAT.

[0082] 4) OX-DRAGON can pass through the cell membrane. From immunofluorescence measurements using an anti-OXA antibody (16 - 33 amide, Phoenix Peptide, #H-003-36) in confocal microscopy, a diffuse cytoplasmic signal of fluorescence became apparent in WT Neuro-2a cells ((Holmqvist et al., 2002); see the second point above) treated with 74 μM OX-DRAGON for 1 hour. This indicates the internalization of OX-DRAGON into the cells after 1 hour of incubation. This result was not observed in the control experiment with 74 μM OXA (Figure 6). These data showed that the specific chemical structure of OX-DRAGON confers the ability of the molecule to pass through the cell membrane. Since the ability to pass through the endothelial cell membrane is required to cross the BBB, these experiments support the ability of OX-DRAGON to reach brain tissue by crossing the BBB after systemic administration.

[0083] 5) OX-DRAGON is effective on REM sleep after systemic subcutaneous administration in the NT1 OX-KO mouse model. Except for the absence of ICV cannula implantation, the experiment was conducted as in step 3 above. Systemic SC administration of OX-DRAGON (single injection at ZT0, 160 μM in 1 mL of saline) in OX-KO mice (bred in-house at the Department of Biomedical and Movement Neuroscience, University of Bologna, (Bastianini et al., 2011)) was found to be able to significantly reduce the time spent in REM sleep during both the light and dark periods, without showing a significant effect on the time spent awake or in non-REM sleep (Figure 7, center). SC injection of equimolar OXA (Figure 7, left) or TAT (Figure 7, right) (160 μM in 1 mL of saline) did not produce a significant effect on the time spent in REM sleep, non-REM sleep, or awake. These results indicate that OX-DRAGON is systemically effective, and its effect can be attributed to the ability of OX-DRAGON to cross the BBB. These results also suggest that the effect of SC administration of OX-DRAGON cannot be simply achieved by SC administration of OXA, probably due to a decrease in the ability of OXA to cross the BBB. Finally, these results show that, unlike after ICV administration, the effect of OXA on REM sleep after SC administration cannot be attributed to the non-specific effect of the TAT peptide.

[0084] 6) OX-DRAGON shows a significant anti-cataplectic effect upon systemic SC administration in the NT1 OX-KO mouse model. Systemic administration of OX-DRAGON (single injection at ZT0, 160 μM in 1 mL saline) in OX-KO mice (in-house breeding in the Department of Biomedical and Movement Neuroscience, University of Bologna; (Bastianini et al., 2011); the experiment disclosed in the fifth point above) was found to be able to significantly reduce the total duration of cataplexy-like episodes during the dark phase (Figure 8). Such episodes were identified as a direct transition from wakefulness to REM sleep (Scammell et al., 2009) and are 100% specific for cataplexy in the mouse model of NT1 during the dark phase (Fujiki et al., 2009). The coincidence between such events of sleep polygraph tracing and the sudden behavioral transition to an immobile state was confirmed based on simultaneous video tracing in a subset of mice (n = 4) during vehicle treatment and during OX-DRAGON treatment. The effect of OX-DRAGON was due to a decrease in the number of cataplexy-like episodes with no significant change in their duration (P = 0.034, Wilcoxon one-sided test with significance of the exact Monte Carlo method). This effect was not reproduced by equimolar SC injection of either other OXA or TAT (Figure 8). These results indicate that OX-DRAGON has the potential to be a new therapeutic agent for NT1 with anti-cataplectic activity.

Industrial Applicability

[0085] NT1 has a relatively high estimated association (14 / 100,000 (Scheer et al., 2019)) compared to other rare diseases and is associated with significant human, social, and economic costs (J. Black et al., 2014). None of the currently available therapies for NT1 are based on replacement of the deficient orexin in patients and all have important limitations in terms of efficacy and side effects (S. W. Black et al., 2017). OX-DRAGON could be a new therapy for NT1 that is strictly based on the etiology and pathophysiology of the disease. In particular, OX-DRAGON is an agonist of both types of orexin receptors and could thus enable complete compensation for the lack of orexin in NT1 patients. Based on this, OX-DRAGON could have a major medical, social, and economic impact on NT1 patients and their families.

[0086] Due to the multifaceted physiological effects of orexin, OX-DRAGON has surprisingly diverse potential therapeutic applications, including in addition to NT1, type 2 narcolepsy, obesity, heart failure, awakening from anesthesia, pain and inflammation, and cancer.

[0087] In particular, TAK925, an OR2 agonist, has recently been shown to be effective in promoting awakening in patients with type 2 narcolepsy characterized by the absence of cataplexy and detectable orexin levels in cerebrospinal fluid (Tanaka et al., 2020). Similarly, considering the experiments already conducted by the inventors, OX-DRAGON could also have therapeutic utility in patients suffering from type 2 narcolepsy. Given that the prevalence of narcolepsy without cataplexy (type 2) is almost four times higher than that of NT1 (Scheer et al., 2019), this further increases the social impact of OX-DRAGON and could also extend to patients with idiopathic hypersomnia.

[0088] Obesity is a serious global health problem (Swinburn et al., 2011). The orexin system affects energy balance and metabolic control. Therefore, NT1 increases the risk of obesity, especially during childhood (Grimaldi et al., 2014; Poli et al., 2013). Promotion of the OR2-mediated signaling pathway has been shown to prevent diet-induced obesity and improve leptin sensitivity and glucose tolerance in mice (Funato et al., 2009). On the other hand, recent data also suggest a distinct role of OR1 in energy metabolism, and orexin binding to OR1 is shown to be sufficient to prevent diet-induced obesity in OR2 KO mice fed a high-fat diet (Kakizaki et al., 2019). OX-DRAGON is a dual agonist of OR1 and OR2 and may therefore be useful for the treatment of obesity and related cardiometabolic comorbidities not only in patients with NT1 and especially children, but also in the general population.

[0089] There is evidence that the human heart expresses OR2 and that these are related to the pathophysiology of heart failure (Perez et al., 2015). In particular, heart-level OR2 expression was significantly higher in human hearts with dilated or ischemic cardiomyopathy than in control hearts. This may represent a compensatory mechanism: mouse KO of OR2 shows signs of ventricular dilation disorder of the heart, but injection of OXA, a non-selective agonist of OR1 and OR2, reduces cardiac dysfunction in a mouse model of heart failure (Perez et al., 2015). From these results, it is suggested that OX-DRAGON, which is designed to be a cell membrane-permeable OR1 and OR2 agonist such as OXA that increases transcapillary transport, may also be used for the treatment of heart failure.

[0090] There is strong preclinical evidence that OXA facilitates awakening from anesthesia with propofol (Zhang et al., 2012) and isoflurane (Yang et al., 2019) by a mechanism involving OR1-mediated modulation of GABA-A receptors (Sachidanandan et al., 2017) and dorsal raphe serotonergic neurons (Yang et al., 2019). OR1 agonists have the potential to provide a tool for anesthetics to actively promote awakening from postoperative anesthesia (Zhou et al., 2018), and OX-DRAGON may be useful in this regard. These considerations may also be relevant to patients with NT1, as the available evidence is insufficient to still conclude whether such patients exhibit an increased risk before and after surgery (Hershner et al., 2019).

[0091] The first results identifying the analgesic properties of OXA (Bingham et al., 2001) were subsequently confirmed and extended, highlighting the role of OR1 at the spinal cord level (Yamamoto et al., 2002) and in the periaqueductal gray of the midbrain (Ho et al., 2011). Persistent pain and stress activate the orexinergic pathway that inhibits pain, suggesting the involvement of orexin as an endogenous pain modulator (Watanabe et al., 2005). Preclinical evidence suggests that OR1 agonists may also be effective against drug-resistant pain states such as pain after stroke (Matsuura et al., 2020) and pain induced by chemotherapy (Toyama et al., 2017). Therefore, OX-DRAGON may also be useful in the field of analgesia. Furthermore, this may be relevant to patients with NT1, in whom a high prevalence of chronic pain has been reported (Cremaschi et al., 2019; Dauvilliers et al., 2011).

[0092] OR1 agonists such as OX-DRAGON may also be useful for treating severe inflammatory conditions. Systemic administration of OXA in a mouse model of advanced ischemic shock acts on the CNS to regulate inflammation and increase survival when the BBB is vulnerable (Ogawa et al., 2016). Thus, early administration of OX-DRAGON, which can cross an intact BBB, may help act early to prevent complete ischemic shock. Also, OXA may exhibit an anti-inflammatory effect by binding to peripheral OR1 receptors. Recent data indicate that OXA can act on OR1 at the intestinal level to prevent lipopolysaccharide-induced neuroinflammation at the intestinal barrier level (Tunis et al., 2019) and can reduce inflammation in ulcerative colitis (Messal et al., 2018). Additionally, OXA acts on pre-neutrophils in the bone marrow via OR1 to attenuate bone marrow hematopoiesis and suppress the increase in circulating inflammatory monocytes and neutrophils at night, limiting the severity and extent of atherosclerosis (McAlpine et al., 2019).

[0093] There is evidence that the activation of OR1 results in a pro-apoptotic effect in colon cancer and neuroblastoma cell lines (Rouet-Benzineb et al., 2004). These results were subsequently confirmed both in vitro and in vivo after xenotransplantation in nude mice, in human colon cancer cell lines and liver metastases (Voisin et al., 2011). OXA also promotes strong apoptosis in cells resistant to 5-fluorouracil, the most commonly used chemotherapy in colon cancer, and reverses tumor development when administered 7 days after inoculation in a mouse model (Rouet-Benzineb et al., 2004). OXA can promote tumor apoptosis in vivo by directly activating caspase-3. From these results, it is suggested that OX-DRAGON, which has the agonist activity of OR1 and can permeate the cell membrane and increase transcapillary transport, can be used as a therapy for colon cancer (Rouet-Benzineb et al., 2004) and neuroblastoma. Recent data on human colon cancer cell lines show that OXA induces autophagy (Wen et al., 2016) and that cell migration is decreased by the activation of OR1 and cholecystokinin A receptor, which form receptor heterodimers with them (Bai et al., 2017). Orexin-dependent apoptosis can be mediated by motifs present in both OR1 and OR2 through the involvement of the phosphotyrosine phosphatase SHP2 and the induction of mitochondrial apoptosis (Mogavero et al., 2021). (References) TIFF2025521725000002.tif250166TIFF2025521725000003.tif252166TIFF2025521725000004.tif252166TIFF2025521725000005.tif251166TIFF2025521725000006.tif252166TIFF2025521725000007.tif252166TIFF2025521725000008.tif252166TIFF2025521725000009.tif252166TIFF2025521725000010.tif252166TIFF2025521725000011.tif252166TIFF2025521725000012.tif252166TIFF2025521725000013.tif91166

Claims

1. From the N-terminus to the C-terminus, the elements are: a) a first cell-penetrating peptide or a functional fragment or derivative thereof, or a bioactive variant, and b) a second peptide having orexin receptor 1 (OR1) and orexin receptor 2 (OR2) agonist activity or a functional fragment or derivative thereof, or a bioactive variant A synthetic peptide comprising.

2. The second peptide comprises or consists of a fragment of the orexin A (OXA) protein, preferably the fragment is i. the sequence PDCCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 1), or ii. - the sequence CCRQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 2) or - the sequence RQKTCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 3) or - the sequence TCSCRLYELLHGAGNHAAGILTL (SEQ ID NO: 4) or - the sequence SCRLYELLHGAGNHAAGILTL (SEQ ID NO: 5) or - the sequence RLYELLHGAGNHAAGILTL (SEQ ID NO: 6) A fragment of SEQ ID NO: 1, comprising or consisting of, or iii. a sequence having at least 70% percent identity with a sequence comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 Comprising or consisting of, Preferably, the second peptide is amidated at the C-terminus, Preferably, the second peptide preferably exhibits disulfide bridges at positions 3-9 and 4-11 of SEQ ID NO: 1 or positions 1-7 and 2-9 of SEQ ID NO: 2, The peptide according to claim 1.

3. The first peptide comprises or consists of the human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (TAT) peptide, preferably the TAT peptide comprises a sequence having at least 70% percent identity with a sequence comprising or consisting of the sequence YGRKKRRQRRR (SEQ ID NO: 7), or consists of, preferably the first peptide comprises or consists of SEQ ID NO: 7, or the first peptide has the following sequences: LLIILRRRIRKQAHAHSK (SEQ ID NO: 9), RRLSYSRRRF (SEQ ID NO: 10), YARKAARQARA (SEQ ID NO: 11), GLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 12), KETWWETWWTEWSQPKKRKV (SEQ ID NO: 13), MVRRFLVTLRIRRACGPPRVRV (SEQ ID NO: 14), MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (SEQ ID NO: 15), KLALKLALKALKAALKLA (SEQ ID NO: 16), LSTAADMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 17), DPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID NO: 18), PFVYLI (SEQ ID NO: 19), MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 20), RKKRRRESRKKRRRES (SEQ ID NO: 21) or KCFQWQRNMRKVRGPPVSCIKR (SEQ ID NO: 22) The peptide according to claim 1 or 2, comprising or consisting of one of the above, or a sequence having at least 70% percent identity.

4. SEQ ID NO: 8 (YGRKKRRQRRRPDCCRQKTCSCRLYELLHGAGNHAAGILTL) or a functional fragment or equivalent, variant, mutant, derivative or functional recombinant or synthetic analog thereof, wherein the sequence preferably exhibits disulfide bridges at positions 14-20 and 15-22 of SEQ ID NO: 8 and the C-terminus is amidated, the synthetic peptide according to any one of claims 1 to 3.

5. SEQ ID NO: 8 (YGRKKRRQRRRPDCCRQKTCSCRLYELLHGAGNHAAGILTL) or consisting of the same, wherein the sequence exhibits disulfide bridges at positions 14-20 and 15-22 of SEQ ID NO: 8 and the C-terminus is amidated, the synthetic peptide according to any one of claims 1 to 4.

6. Having an anti-cataplectic effect and / or the following functions: - Promoting wakefulness or counteracting drowsiness - Reducing body weight and glucose intolerance in the state of obesity - Improving myocardial function in heart failure - Preventing atherosclerosis - Anti-inflammatory effect - Analgesic effect - Chemotherapeutic effect against colon cancer and neuroblastoma The peptide according to any one of claims 1 to 5, having at least one of the above.

7. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 6, preferably for subcutaneous administration, and at least one pharmaceutically acceptable vehicle.

8. An isolated nucleic acid encoding the peptide according to any one of claims 1 to 6, or a recombinant expression vector containing the isolated nucleic acid.

9. A host cell comprising and / or expressing the peptide according to any one of claims 1 to 6, or the nucleic acid according to claim 8, or the vector according to claim 8.

10. For medical use, the peptide according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the nucleic acid according to claim 8, or the vector according to claim 8, or the cell according to claim 9.

11. For use in the treatment and / or prevention of type 1 narcolepsy, type 2 narcolepsy, idiopathic hypersomnia, obesity, or related cardiometabolic comorbidities including atherosclerotic cardiovascular disease, heart failure, inflammation such as septic shock, neuroinflammation, or intestinal barrier-level inflammation such as in ulcerative colitis, tumors and metastases, such as in the treatment of colon cancer and neuroblastoma, for example, in patients with type 1 narcolepsy (NT1) if applicable, drug-resistant pain conditions such as pain, post-stroke pain, and pain induced by chemotherapy, and for the management of surgical anesthesia in the treatment of chronic pain, as an analgesic, the peptide according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 7, or the nucleic acid according to claim 8, or the vector according to claim 8, or the cell according to claim 9.

12. A peptide for use according to claim 10 or 11 for subcutaneous administration.

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