WO2021126990A1 - Oxytocin derivatives with improved properties - Google Patents

Abstract

The present invention provides novel oxytocin derivative compounds that contain a fatty acid moiety that is conjugated to a modified oxytocin scaffold. The oxytocin derivative compounds of the invention are potent oxytocin agonists with substantially improved stability. Also provided in the invention are therapeutic methods of using the compounds in the treatment of various diseases, e.g., metabolic disorders such as obesity.

Description

OXYTOCIN DERIVATIVES WITH IMPROVED PROPERTIES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 62/948,560 (filed December 16, 2019). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

[0002] Oxytocin (OT) is a 9-residue cyclic peptide endogenously synthesized in the hypothalamus. The peptide interacts with the oxytocin receptor (OTR), a member of Class I G-protein-coupled receptor family (GPCR), and elicits both peripheral and central actions. OT is best known for its role in lactation and parturition, as well as its role in social behaviors in humans as “the Love Hormone”, promoting trust and bonding. In addition, OT has been shown to prevent skeletal muscle ageing, improve myocardium recovery after ischemic injury, and important roles in preventing chronic pain, osteoporosis, and diabetes. OT has also been studied extensively as a potential therapy for obesity. Mice lacking either OT or OTR developed late onset obesity, and administration of OT into rodents, monkeys, and humans prompted a reduction in food intake and body weight. Currently, there are multiple phase II clinical trials ongoing, trying to assess the effect of intranasal OT in obese adults and children (NCT03043053, NCT02849743, NCT03119610). However, exploration of the potential therapeutic benefits of OT has been hampered by its short half-life in plasma (2-3 min), which makes the peptide impractical for diseases like obesity that require chronic treatments. [0003] There is an unmet need in the art for potent and long-acting OT analog or derivative compounds that are suitable for various therapeutic applications. The instant invention addresses this and other needs. SUMMARY OF THE INVENTION

[0004] In one aspect, the invention provides oxytocin (OT) derivative compounds that have improved properties relative to wildtype OT or carbetocin. In some embodiments, the oxytocin derivative compound has a half-life that is substantially longer (e.g., 2-, 5-, or 10-fold longer) than that of oxytocin or carbetocin. In some embodiments, the OT derivative compounds of the invention have a structure shown below.

In this structure, R designates a fatty acid (FA)-based moiety. In some embodiments, the FA moiety is FA1, FA2 or FA3 as shown below. In some embodiments, the employed FA moiety is FA4, FA8, FA10, FA11, FA12, or FA13 exemplified herein. In still some other embodiments, the FA chain used for generating the OT derivative compounds can be selected from FA5, FA6, FA7, FA9, FA14, FA15, and FA16 described herein.

[0005] In a specific embodiment, the OT derivative compound is OT-12, which contains moiety FA3 attached to the OT scaffold shown above. In some other embodiments, the OT derivative compound of the invention can be OT-13, OT-14, OT- 15, OT-16, OT-17 or OT-18 exemplified herein. In still some other embodiments, the OT derivative compound of the invention can be OT-19, OT-20, OT-21, OT-22, OT- 23, OT-24 or OT-25 described herein. [0006] In some related aspects, the invention provides pharmaceutical compositions and therapeutic kits that contain one or more of the OT derivative compounds described herein. In another aspect, the invention provides therapeutic methods of using the OT derivative compounds in the treatment of various diseases or disorders. In some embodiments, the compounds are employed in the treatment of metabolic diseases such as obesity. In some embodiments, the employed compound is OT-12 exemplified herein. In some other embodiments, the employed compound can be any one selected from OT-13, OT-14, OT-15, OT-16, OT-17 and OT-18 as exemplified herein.

[0007] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0008] Figure 1. Chemical structures of OT and its synthetic analog carbetocin (CT).

[0009] Figure 2. Structures of bromoacetamide and the FA moieties for improved serum albumin binding and half-life extension.

[0010] Figure 3. Screening the conjugation site of the acetamide (ACM) and the FA1 moiety against the in vitro activation of OTR in the presence of 10% serum. CHO- K1 b-arrestin cells treated with the peptides at varying concentrations for 90 min, and the luminescent signals were acquired. Assays were performed in triplicate and the dose-response curves were fitted to log-agonist vs response-variable slope in Prism to generate the ECso values. Compounds with agonistic activity < 10% were considered inactive.

[0011] Figure 4. Screening the in vitro activity of the FA conjugated, /V-methylated peptides OT-7, 8 and 9 on OTR. CHO-K1 b-arrestin cells treated with the peptides at varying concentrations for 90 min, and the luminescent signals were acquired. Assays were performed in triplicate and the dose-response curves were fitted to log-agonist vs response-variable slope in Prism to generate the EC 50 values.

[0012] Figure 5. Screening the in vitro activity of the FA conjugated peptides OT- 10, OT-11, and OT-12 on OTR. CHO-K1 b-arrestin cells treated with the peptides at varying concentrations for 90 min, and the luminescent signals were acquired. Assays were performed in triplicate and the dose-response curves were fitted to log-agonist vs response-variable slope in Prism to generate the EC50 values.

[0013] Figure 6. PK profiles of peptide OT-12 after a single s.c. administration (1 mg kg ¹) in mice. (A) mean plasma concentration; (B) mean plasma and brain concentration.

[0014] Figure 7. Effects of a single s.c. administration of peptide OT-12 (0.1 mg kg ¹) on: (A) food intake; (B) body weight, compared to carbetocin (0.5 mg kg ¹) and vehicle control in regular chow fed mice (10-week old C57BL/6 male, n = 6 per group). Cumulative food consumption was measured at the beginning of dark phase and at subsequent time points at 1, 3, 6, 24 and 48 hours. Body weight was determined daily up to 2 days post administration. [0015] Figure 8. Efficacy of peptide OT-12 in DIO mouse model. Effects on: (A) food intake; (B) body weight, compared to carbetocin and vehicle control in DIO mice (21-week old mice, n = 6 per group) treated daily with s.c. administered OT-12 (0.05 and 0.1 mg kg ¹), carbetocin (0.1 mg kg ¹), and vehicle (saline). * Significantly different from vehicle control (P < 0.05).

DETAILED DESCRIPTION

[0016] The present invention is derived in part from studies undertaken by the inventors to develop a series of OT derivatives which incorporate different MEG-fatty acid moieties and demonstrate strong in vitro agonistic activity toward OTR. The OT derivatives developed by the inventors (e.g., OT-12) are potent, long acting OTR full agonist, with potent in vivo efficacy in food intake and body weight reduction in diet induced obesity model, demonstrating its advantage and potential therapeutic impact for chronic diseases like obesity. Due to the injection site depot and tight binding to the serum albumin in the circulation, these OT derivatives also demonstrated much improved PK properties, with a long terminal half-life of 24 h in mice. Such a long half-life in rodents can translate into a human half-life of more than 159 h, long enough for a once-weekly or once-biweekly administration for human chronic diseases. As exemplified herein, the long-acting agonists of the invention such as OT-12 exhibit potent anorexigenic and body weight reducing effects in a diet-induced obesity mouse model, superior to the known stabilized, long acting OT analog carbetocin. Further, the s.c. administration of OT-12 resulted in a sustained level of plasma concentrations for greater than 24 h, indicative of extended half-life and long lasting anorexigenic effects. [0017] The invention accordingly provides OT derivative compounds with improved biological and/or pharmaceutical properties as demonstrated herein. The invention also provides therapeutic methods of using these compounds in the treatment of a number of diseases and disorders, e.g., obesity. The following disclosure provides more a detailed guidance for making and using the OT derivative compounds of the invention.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0019] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[0020] The term “derivative” or “variant” is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., the cyclic or linearized OT peptide) but which has been modified in a targeted and controlled manner, by modifying a specific substituent (e.g., an amino acid residue) of the reference molecule. Such modification includes attachment of a separate agent or moiety to the reference molecule, and/or replacing the substituent with an alternate substituent. Compared to the reference molecule, a derivative or variant would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of candidate OT derivative compounds or variants of the invention to identify derivatives having improved traits (such as higher serum stability or binding affinity for a target molecule) can be performed in accordance with the present disclosure and/or methods well known in pharmaceutical chemistry.

[0021] Administration "in conjunction with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. [0022] The term “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents with cells. Contacting can occur in vitro, e.g., combining an agent with a cell or combining two cells in a test tube or other container. Contacting can also occur in vivo, e.g., by targeted delivery of an agent to a cell inside the body of a subject.

[0023] The term “subject” refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.

[0024] The term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., a metabolic disease), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereol) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0025] The OT derivative or analog compounds of the invention are comprised of a modified oxytocin scaffold and a fatty acid moiety that is chemically conjugated to the modified OT scaffold. While maintaining comparable or similar OTR agonist activities, these compounds have improved pharmaceutical properties relative to that of wildtype oxytocin, e.g., longer-acting profile. In some embodiments, the oxytocin derivative compounds have a half-life that is substantially longer (e.g., 2-, 5-, or 10-fold longer) than that of oxytocin.

[0026] In various embodiments, the modified oxytocin scaffold can have one or more of the following changes: removal of the N-terminal Cys residue and replacement of the disulfide bond with athioether bridge, substitution of Leu at position 7 with Cys, and substitution of Pro at position 6 with Gly. In some embodiments, the modified oxytocin scaffold contains all of these alterations. These modifications are intended to provide attachment site for conjugation of a fatty acid moiety and to improve selectivity for OT receptor while not substantially impacting agonistic activity and potency. In some embodiments, the oxytocin derivative compounds of the invention are long- acting, e.g., with a plasma half-life that is at least 50%, 75%, 1 fold, 2 fold, 5 fold, 10 fold or more longer than the half-life of oxytocin. For example, the compounds can have a half-life that is more than 10 fold longer than oxytocin.

[0027] In some preferred embodiments, the modified OT scaffold in the derivative compounds of the invention has a structure (I) shown below:

In this modified OT scaffold structure, the arrow indicates the engineered Cys residue where the fatty acid moiety is to be attached.

[0028] Various fatty acid moieties can be employed in the construction of the OT derivative compounds of the invention. In some embodiments, the employed fatty acid moieties exhibit high binding affinities to serum albumin. Via Cys thioether linkage, such fatty acid moieties are capable of significantly increasing the in vivo half-lives of various conjugated peptides. Some specific fatty acid molecules that can be used are known in the art. See, e.g., Yang et ak, Proc. Natl. Acad. Sci. USA 2016, 113 (15), 4140-4145; Yang et al., J. Med. Chem. 2018, 61 (7), 3218-3223; and Lau et ak, J. Med. Chem. 2015, 58 (18), 7370-7380.

[0029] In some embodiments, the fatty acid moiety conjugated to the modified OT scaffold contains a structure of FA1, FA2 or FA3 shown below as Br-FAl, Br-FA2 or Br-FA3:

Attachment of these specific moieties to modified OT scaffold of structure (I) above result respectively in OT derivative compounds OT-10, OT-11 and OT-12 as exemplified herein. As described herein, these specifically exemplified compounds have OT agonist activities that are comparable to that of oxytocin while displaying substantially improved pharmacokinetic properties, including serum stability.

[0030] In some other embodiments, the fatty acid moiety conjugated to the modified OT scaffold contains a structure of as shown in any one of FA4, FA8, FA10, FA11, FA12, and FA13 as described herein. In still some other embodiments, the fatty acid moiety conjugated to the modified OT scaffold contains a structure of as shown in any one of FA5, FA6, FA7, FA9, FA14, FA15 and FA16 as described herein.

[0031] Generation of the modified OT scaffold, preparation of the fatty acid moieties, and conjugation of the FA moieties to the OT scaffold can all be readily performed in accordance with the specific protocols disclosed herein and/or methods well known in the art. For example, the modified OT compound scaffold, compound of structure (I), can be generated based on the experimental procedure described in the Examples herein. Attachment of a multiethylene glycol (MEG) linker to a fatty acid moiety and functionalization with bromoacetamide can be readily performed in accordance with the protocols described herein for synthesizing Br-FAl, Br-FA2, Br- FA3, Br-FA4, Br-FA8, Br-FAl 0, Br-FAl 1, Br-FA12, and Br-FAl 3. Detailed experimental protocols for conjugation of a functionalized FA moiety to an OT scaffold are also described herein for the synthesis of OT derivative compounds OT4-OT12. [0032] In addition to the synthesis of the OT derivative compounds, agonist activity of the compounds can be assessed with any suitable assay systems known in the art or specifically exemplified herein. For example, OTR agonist activities of the compounds can be examined via the in vitro b-arrestin oxytocin assay, in vitro cAMP assay for V2 agonist effect, and/or in vitro calcium flux assay for Via and Vib agonist effect described herein. Similarly, pharmacokinetic properties (e.g., half-life) and in vivo activities of the OT derivative compounds can be assessed via animal studies as described herein. These include using lab animals (e.g., mice) administered with the compounds to study pharmacokinetics. For example, plasma half-life of a candidate OT derivative compound can be readily determined in mice administered with the compound using the assays exemplified in Example 2 herein. As described in Example 3 herein, in vivo biological studies exemplified herein can allow one to ascertain the effect of a candidate compound on food intake and body weight. Thus, once candidate OT derivatives are synthesized based on the present disclosure, the in vitro and vivo studies described herein can enable identification of compounds with satisfactory OT agonist activities and improved pharmacokinetic properties (e.g., plasma stability). [0033] Due to their potent oxytocin agonist activity and substantially improved serum stability, the OT derivative compounds can be employed in various clinical or therapeutic applications. The invention accordingly provides methods of using these compounds in various prophylactic or therapeutic treatment of human or non-human subjects. These include, e.g., treatment of metabolic diseases, Prader-Willi syndrome (PWS) and autism. Typically, a subject afflicted with a disease or disorder described herein (e.g., obesity) or at risk of developing the symptoms of the disorder is administered with an OT derivative compound of the invention (e.g., OT-12) that is provided in a pharmaceutic composition. Generally, the treatment should enable a subject to obtain a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing the disease or sign or symptom thereof. It can also be therapeutic in terms of a partial or complete cure for the disorder and/or adverse effect that is attributable to the disorders.

[0034] In some embodiments, the OT derivative compounds of the invention can be used in place of oxytocin for treating metabolic diseases, e.g., obesity, diabetes, non alcoholic fatty liver disease (NDFLD) and nonalcoholic steatohepatitis (NASH). Oxytocin enhances glucose uptake and lipid utilization in adipose tissue and skeletal muscle. Animal studies revealed that deficiencies m oxytocin signaling and oxytocin receptor expression lead to obesity despite normal food intake, motor activity and increased leptin levels. In addition, plasma oxytocin concentration is notably lower in obese individuals with diabetes, which may suggest an involvement of the oxytocin system in the pathogenesis of cardiometabolic disease. More recently, small scale clinical trials demonstrated that intranasal administration of oxytocin was associated with significant weight loss as well as improvements in insulin sensitivity and pancreatic b-ce!l responsivity in human subjects. Due to their improved properties over OT, the OT derivative compounds of the invention (e.g., OT-12) are useful in improving peripheral insulin sensitivity, pancreatic function and lipid homeostasis.

They provide alternative and better therapeutic agents in obesity and diabetes management.

[0035] In some embodiments, die OT derivative compounds of the invention can be used in place of oxytocin for treating PWS. Oxytocin signaling plays a role in neurode ve! opment in early infancy, contributing to long-term social and cognitive abilities. Studies have shown a deficiency of oxytocin-producing neurons in the brains of individuals with PWS, and there is also stron evidence in animal models to support the possible therapeutic benefit of oxytocin for PWS. Thus, the OT derivative compounds of the invention may be readily employed in treating subjects afflicted with PWS.

[0036] In some other embodiments, the OT derivative compounds of the invention can be used in place of oxytocin for treating autism. Oxytocin w as once understood solely as a neuropeptide with a central role in social bonding, reproduction, parturition, lactation and appetite regulation. Recently, a series of studies raised the possibility that some ASD symptoms may be relieved by administration of oxytocin. Studies with typically developed (TD) individuals have shown that oxytocin modulates their neural responses to social stimuli, enhances social perception, and promotes social interactions with modulation of functional connectivity. Previous preliminary behavioral studies in individuals with ASD in small sample sizes (N < 20) have supported oxytocin’s potential as a therapy for some of the core symptoms of ASD. Restricted and repetiti ve behaviors were significantly reduced by oxytocin infusion. Social interactions and social recognition were significantly enhanced by intranasal administration of oxytocin to individuals with ASD when the social interactions mostly consisted of nonverbal information, such as facial expressions and eye gazes. Thus, the OT derivative compounds of the invention can be useful as alternative means for treating or ameliorating symptoms associated with autism.

[0037] In various embodiments, the OT derivative compounds of the invention (e.g., OT-12) can be administered to a subject in conjunction with one or more other known therapeutic agents used for treating the specific disease or disorder afflicted by the subject. For example, for treating obesity, the OT derivative compounds can be used in combination with orlistat (Xenical, Alii), lorcaserin (Belviq), phentermine- topiramate (Qsymia), naltrexone-bupropion (Contrave), and liraglutide (Saxenda). Other suitable therapy for treating obesity can also be used together with the OT derivative compound for treating obesity, e.g., behavior therapy. Similarly, for treating type 2 diabetes, various known diabetes medications (e.g., Metformin, sulfonylureas, meglitinides, thiazolidinediones and DPP-4 inhibitors) and/or insulin therapy can also be used in combination. In these combination therapies, the different treatments can be administered to the subjects either simultaneously (concurrently) or consecutively m any order.

[0038] Various other therapeutic agents can be employed in combination with the OT derivative compounds of the invention in the treatment of metabolic diseases or other disorders described herein. These additional agents include, e.g., other known diabetes drugs, insulin and analogs, DPP4 inhibitors, SGLT2 inhibitors, GLP1R, GIPR and GCGR single and dual and triple agonists, hypoglycemic drugs and biguanidine drugs, insulin secretogogues and sulfonyl urea drugs, TZD drugs, FGF21 and analogs, leptin or leptin analogs, amylin and amylin analogs, anti-inflammatory drugs, cyclosporine A or FK506, 5-ASA, statins or any combination thereof.

[0039] The OT derivative compounds described herein can be administered directly to subjects in need of treatment. However, these therapeutic compounds are preferably administered to the subjects in pharmaceutical compositions.

Pharmaceutical compositions of the invention can be prepared and administered to a subject by any methods well known in the art of pharmacy. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics , Hardman et al., eds., McGraw- Hill Professional (10^th ed., 2001); Remington : The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999).

[0040] Pharmaceutical compositions of the invention contain a therapeutically effective amount of an OT derivative compound of the invention (e.g., OT-12), which is formulated with at least one pharmaceutically acceptable carrier. In addition, the pharmaceutical compositions of the invention may also be formulated to include other medically useful drugs or biological agents. The pharmaceutically acceptable carrier is any carrier known or established in the art. Exemplary pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free saline solution. Other forms of pharmaceutically acceptable carriers that can be utilized for the present invention include binders, disintegrants, surfactants, absorption accelerators, moisture retention agents, absorbers, lubricants, fillers, extenders, moisture imparting agents, preservatives, stabilizers, emulsifiers, solubilizing agents, salts which control osmotic pressure, diluting agents such as buffers and excipients usually used depending on the use form of the formulation. These are optionally selected and used depending on the unit dosage of the resulting formulation.

[0041] A therapeutically effective amount of the therapeutic compounds varies depending upon the disorder that a subject is afflicted with, the severity and course of the disorder, whether the treatment is for preventive or therapeutic purposes, any therapy the subject has previously undergone, the subject's clinical history and response to the therapeutic compound, and other known factors of the subject such as age, weight, etc. Thus, the therapeutically effective amount or dose must be determined empirically in each case. This empirical determination can be made by routine experimentation. A typical therapeutic dose of the OT derivative compound is about 5- 100 mg per dose, e.g., 10 mg per dose. For any given condition or disease, one can prepare a suitable composition which contains an OT derivative compound in accordance with the present disclosure and knowledge well known in the art , e.g., Springhouse, Physician's Drug Handbook, Lippincott Williams & Wilkins (12^th edition, 2007). Depending on the specific disorder and relevant conditions of the subject to be treated, single or multiple administrations of the pharmaceutical composition of the invention can be carried out with the dose levels and pattern being selected by the treating practitioner. [0042] The invention also provides kits useful in therapeutic applications of the compositions and methods disclosed herein. Typically, the kits of the invention contain one or more OT derivative compounds described herein. The kits can further comprise a suitable set of instructions relating to the use of the compounds for the various therapeutic or prophylactic applications described herein. The pharmaceutical composition of the invention can be present in the kits in any convenient and appropriate packaging. The instructions in the kits generally contain information as to dosage, dosing schedule, and route of administration for the intended therapeutic goal. The containers of kits may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The kits may further include a device suitable for administering the pharmaceutical composition according to a specific route of administration.

EXAMPLES

[0043] The following examples are offered to illustrate, but not to limit the present invention.

Example 1. Peptide design and in vitro activity

[0044] Aiming to increase the stability and in vivo half-life of OT, we designed a strategy based on the mono-conjugation with multiethylene glycol (MEG) linker and fatty acid (FA) moieties via a Cys thioether linkage. While fatty acids modulate the peptide’s half-life through their intrinsic affinity toward serum proteins, the MEG spacer keeps the alkyl chain away from OT, preventing potential interference in its binding toward OTR.

[0045] Three types of fatty acid-based linkers were therefore utilized in our designs, functionalized with a-bromo-acetamide to aid the conjugation of FA with a strategically positioned thiol in the peptide scaffold (Figure 2). FA1 has a short multi ethylene glycol (MEG) spacer linked to myristic acid (Cl 4 chain), and FA2 has a longer MEG spacer conjugated with an octadecanedioic diacid (Cl 8 chain). On the other hand, FA3 has two discrete MEG spacers combined with a lysine and an octadecanedioic diacid (Cl 8 chain). The presence of the terminal carboxylic acid (FA2/FA3) and the internal carboxylic acid (FA3) provides optimum serum protein binding and long in vivo half-life. [0046] We first attempted positional scanning to identify an amino acid position that can be substituted with a Cys residue, for conjugating the FAs moieties. The N- terminal Cys was removed and the disulfide bond was replaced by a thioether bridge, similar to carbetocin (Figure 1). Scheme 1 summarizes the synthesis of peptide OT-1 as an example of the synthetic route used to prepare peptides OT-1 to OT-12. Synthesis of the OT analogs was successfully completed over multiple steps, starting from commercially-supplied linear peptides synthesized by Fmoc-SPPS: (i) solution-phase, head-to-side chain macrolactamization between the /V-terminus and the L-linked butyrate at the side chain of Cys(5) using PYBOP/DIPEA, (ii) selective removal of the L'-acetamidomethyl (Acm) protecting group using AgOTf, and (iii) peptide conjugation using FA in a mixture of 30 mM NaHCOs/CFECN (v/v; 3:1).

Scheme 1. Synthesis of peptide OT-1. Reagents and conditions: i) PyBOP, DIPEA, DMF, 3 h; ii) AgOTf, anisole, TFA, 0 °C, 90 min then 1 M CFECOOH, DTT, r.t., 4 h; iii) Bromoacetamide, CFECN, 30 mM NaHC03, r.t., 4 h, 58% yield.

[0047] To pinpoint the optimal position for conjugation, a Cys residue was placed at position 7 (replacing Leu), 2 (replacing lie), or 3 (replacing Gin) of the OT-based peptide scaffold and conjugated to an acetamide (ACM) moiety to yield OT analogs OT-1, OT-2, and OT-3, respectively (Figure 3). To test the agonistic activity toward human OTR, we developed a 384-well b-arrestin assay based on PathHunter^RGPCR platform (DiscoverX Corporation). Briefly, CHO-K1 b-arrestin cells overexpressed with hOTR cells were treated with OT, carbetocin and our synthetic analogs in dose response in the presence of 10% fetal bovine serum (FBS). Upon OTR activation by the agonists, b-arrestin is recruited to OTR which forced the complementation of the two enzyme fragments and activation of the b galactosidase activity. The increase in enzyme activity can be measured using chemiluminescent PathHunter Detection Reagents, thus reporting the OTR activation. Out of the three synthesized peptides, OT- 1 exhibited full agonist activity with nanomolar potency (ECso = 4.80 ± 2.35 nM), comparable to OT (ECso = 3.22 ± 0.25 nM). Replacement of Gln(3) with Cys-ACM resulted in OT-3 as a partial agonist (47% of maximum OTR activation) and 4-5 fold loss of potency (EC 50 = 14.42 ± 4.23 nM), while replacing Ile(2) with Cys-ACM resulted in an inactive OT-2. To see whether this observation is true for longer linker and fatty acids, we conjugated the same three Cys mutants with Br-FAl and obtained OT-4, 5 and 6 (Figure 3, table). Indeed, conjugation of the FA1 moiety onto Cys(7) of the peptide scaffold resulted in OT-4, a full agonist with EC50 of 5.28 ± 1.76 nM (Figure 3). And similar to previous findings, conjugation of FA1 onto Cys mutants inside the cyclic ring resulted in peptides OT-5 and OT-6, with significant reduction of the potency and efficacy. Therefore, Leu(7) to Cys is tolerated for conjugation without perturbation to the receptor binding. Our findings are also in agreement with previous reports of modifying Leu(7) for fluorescent or metabolically stable OTR agonists.

[0048] We synthesized Gly(8) W-methylated OT analog and conjugated with FA1, FA2, and FA3 via Cys(7), resulting in OT-7, OT-8, and OT-9, respectively. In our N^a- methylated peptides, Pro-6 was also replaced with Gly, as this modification was shown to be well tolerated in the oxytocin backbone with improved selectivity toward OTR ³¹- ⁴². OT-7, OT-8, and OT-9 were tested in the aforementioned OTR- b-arrestin assay, and we were surprised to see a significant reduction of agonistic activity and potency for these molecules, relative to OT (Figure 4). As enhanced serum binding by the fatty acid moieties on the peptides may affect receptor activation, we tested these peptides in the absence of serum to reflect the true affinity toward OTR. As shown in Figure 4, the in vitro potency of the W-methylated peptides was significantly improved in the absence of serum, more prominent for FA2 and FA3 (with > 20-fold improvement), indicating tighter binding of FA2/3 to the serum albumin than FA1. Nevertheless, these modifications resulted in partial agonists toward OTR, regardless of the fatty acid identities, with more than 5-fold loss of potency compared to OT, even in the absence of serum albumin.

[0049] Replacing the /V“-MeGly with Gly and conjugated to FA1, FA2 and FA3 yielded OT-10, OT-11, and OT-12 (Figure 5). To our satisfaction, these new OT analogs displayed potent in vitro activity and full agonism toward OTR. In particular, peptide OT-12, conjugated to FA3, exhibited the best in vitro potency (EC 50 = 2.58 ± 0.41 nM). In addition, we observed a 75-fold shift of the potency in the presence of 10% FBS (EC50 = 192.6 ± 70.9 nM), signifying an enhanced interaction with serum albumin, improved stability, and potentially long in vivo half-life. Furthermore, the presence of two extra carboxylate moieties on the FA3 rendered OT-12 more soluble in aqueous conditions compared to OT-10 and OT-11, facilitating downstream formulation and in vivo testing.

[0050] Oxytocin is known to also bind and activate vasopressin receptor subtypes, which may cause unwanted side effects such as anti diuresis and local vasoconstriction at the site of application. We therefore tested the capability of OT-12 with respect to activating human V i_a and V i_b receptor subtypes using a calcium assay, and V2 receptor using cAMP assay. [Arg8] -Vasopressin (AVP) was used as an internal control for the vasopressin ViaR and V2R assays, while vasopressin was used as an internal control for the VibR assay. As opposed to endogenous oxytocin which can activate the vasopressin receptor subtypes at nanomolar concentrations (EC50 of 10, 240, and 7.3 nM for hVia, hVib, and I1V2 receptors, respectively), OT-12 was inactive at the human ViaR and VibR (Table 1). Additionally, considerable reduction in potency at the human V2R was also observed. Overall, OT-12 shows superior in vitro pharmacological profile compared to oxytocin and was therefore chosen as a lead candidate for the in vivo experiments.

Table 1. In vitro activity of OT-12 in vasopressin receptors.

Measured ECso

Assay Source component (M)

Receptor human recombinant

Via (CHO cells) Intracellular [Ca²⁺] >1000 human recombinant

Vlb (RBL cells) Intracellular [Ca²⁺] >1000 v₂ human recombinant

(CHO cells) cAMP_ 1.8 x 10⁶

Example 2. Pharmacokinetic study in mice

[0051] To determine the in vivo half-life of our lead candidate, OT-12, which has potent and full agonistic activity toward OTR, we injected C57BL/6 male mice with a subcutaneous (s.c.) dose of peptide (1 mg kg ¹) and measured its plasma concentration at specific time points. Peptide concentrations in plasma were determined using an LC- MS based bioanalytical method (Figure 6A). Consistent to our expectation, introduction of the best fatty acid moiety FA3 resulted in a plasma half-life of 24.2 h for OT-12, which is a significant increase in comparison to OT and carbetocin (2-3 min and 40 min, respectively). We also observed delayed onset (Tmax = 7 h) and high maximal plasma concentration (Cmax = 5281 nM), confirming its increased stability and injection site depot effect. With 1 mg kg ¹ dosing, at our last time point, there is still more than 850 nM present in the circulation, much higher than our in vitro potency for OT-12, suggesting a requirement of much lower dose for our in vivo efficacy studies.

[0052] To further characterize the PK properties of OT-12, we also measured its brain exposure after a single s.c. administration in mice. The brain to plasma ratio of the peptide was 0.005 after 7 hours, and then fell to zero after 24 h (Figure 6B).

Example 3. In vivo efficacy of peptide OT-12

[0053] To investigate whether the long acting OT-12 results in significant anorexigenic effects, we measured acute food consumption in C57BL/6 male mice (10 weeks old, n = 6 per group) fed with standard chow diet. Single s.c. dose of OT-12 was administered at 0.1 mg kg ¹ and efficacy on food intake and body weight was compared to mice treated with carbetocin at 0.5 mg kg ¹) or vehicle control. Carbetocin treatment showed a reduction in food intake at early time points which diminished by 24 h. In contrast, OT-12 treatment showed a delayed and long-lasting effect on food intake, which is consistent with its PK profile of delayed Tmax and long T1/2 (Figure 7A). Given the prolonged suppressive effect on acute food consumption, a single dose of OT-12 resulted in a long-lasting reduction of body weight for two days compared to vehicle control (Figure 7B). In contrast, carbetocin treatment resulted in an increase of body weight compared to vehicle control, possibly due to stress response and feedback compensation which leads to temporary overeat and weight gain. See, e.g., Onaka et al., J. Neuroendocrinol. 201931(3) :e 12700.

[0054] Previously, the anorexigenic effect of oxytocin has been shown to be stronger in obese rodents and obese individuals compared to normal-weight individuals. This could be due to the higher-affinity binding state of OTR in the presence of cholesterol, which is found in higher concentrations in subjects on high-fat diet. Based on the suppressive effects on food intake and body weight gain observed in the acute model (Figure 7), we investigated the anorexigenic effect of OT-12 in the diet-induced obesity (DIO) mouse model (21 weeks old C57BL/6 mice on high fat diet for 15 weeks, n = 6 per group). OT-12 was administered subcutaneously, once daily for 9 days at 0.05 and 0.1 mg kg ¹, compared to carbetocin at 0.1 mg kg ¹ and vehicle, and food intake and body weight were recorded daily (Figure 8). Dose related reduction of food intake and body weight was observed for the OT-12 treatment, while carbetocin only showed a modest trend in this DIO study. Even the lower dose of OT-12 (0.05 mg kg ¹) showed better efficacy than carbetocin, which illustrates the advantage and potential therapeutic impact of long acting OTR agonist in chronic diseases like obesity.

Example 4. Additional OT derivative peptides

[0055] To identify other oxytocin derivatives with improved properties, a number of additional compounds were designed and synthesized. These compounds contain the same OT analog scaffold as seen in OT-12 but with different fatty acid chains. Structures of the additional FA chains are shown below. Table 2 lists several examples of the new OT derivative compounds and their structural properties along with OT-12.

Table 2. Structure components and activities of OT derivatives

[0056] The FA chains used for generating these new OT derivative compounds are:

[0057] Structural properties of these compounds were examined via LC-MS as described herein. A summary of the data is noted below.

[0058] Peptide OT-13: White powder (2.23 mg, 39% yield from the conjugation reaction); Rt = 12.30 min (Q-TOF); HR-MS = in z 1177.604 ([M+H]+), calculated: 1176.604 [0059] Peptide OT-14: White powder (4.27 mg, 72% yield from the conjugation reaction); Rt = 10.31 min (Q-TOF); HR-MS = in z 1207.575 ([M+H]+), calculated: 1206.578

[0060] Peptide OT-15: White powder (3.15 mg, 38.4% yield from the conjugation reaction); Rt = 10.64 min (Q-TOF); HR-MS = in z 1668.841 ([M+H]+), calculated: 1667.815

[0061] Peptide OT-16: White powder (3.81 mg, 47% yield from the conjugation reaction); Rt = 9.89 min (Q-TOF); HR-MS = in z 1640.777 ([M+H]+), calculated: 1639.784

[0062] Peptide OT-17: White powder (3.13 mg, 38% yield from the conjugation reaction); Rt = 13.32 min (Q-TOF); HR-MS = in z 1666.899 ([M+H]+), calculated: 1665.872

[0063] Peptide OT-18: White powder (2.64 mg, 33% yield from the conjugation reaction); Rt = 12.58 min (Q-TOF); HR-MS = in z 1638.856 ([M+H]+), calculated: 1637.841

[0064] The various FA-containing conjugates for making the OT derivative compounds are synthesized as described below.

[0065] Synthesis of Br-FA4: Tetradecyl amine (0.5 g, 2.3 mmol) and bromoacetic anhydride (0.61 g, 2.3 mmol) were dissolved in 10 mL of DCM at 0 °C. DIEA (0.17 mL, 1 mmol) was then added and the reaction mixture was then stirred for 2 h. The solvent was then removed, and the product was extracted with EtOAc (3 x 15 mL). The organic layer was successively washed with sat. NaHCCh, cooled HC1 (1 M) and brine, dried over Na2SC>4, filtered, and concentrated. Purification by flash column chromatography on silica gel provided 0.62 g of Bt-FA4 as a white solid in 81% product yield. MS (ES+) m/z 334.2 [M+H]+, calculated: 334.2 [M+H]+

[0066] Synthesis of Br-FA8: 14-Aminotetradecanoic acid (0.5 g, 2.0 mmol) and bromoacetic anhydride (0.52 g, 2.0 mmol) were dissolved in 10 mL of DCM at 0 °C. DIEA (0.17 mL, 1 mmol) was then added and the reaction mixture was then stirred for 2 h. The solvent was then removed, and the product was extracted with EtOAc (3 x 15 mL). The organic layer was successively washed with sat. NaHC03, cooled HC1 (1 M) and brine, dried over Na2S04, filtered, and concentrated. Purification by flash column chromatography on silica gel provided 0.54 g of Bt-FA8 as a white solid in 74% product yield. MS (ES+) m/z 364.1 [M+H]+, calculated: 364.1 [M+H]+

[0067] Synthesis of Br-FAIO: A solution of Fmoc-Lys(ivDde)-OH (60 mg, 100 pmol) and DIPEA (70 pL, 400 pmol) in CH2CI2/DMF (v/v; 2:1, 3 mL) was added to pre-swollen (CH2CI2, 3 mL, 20 min) 2-chlorotrityl chloride resin (100 mg, 80 pmol) and the mixture was shaken for 30 min at room temperature, filtered, and washed with DMF (4 x 3 mL) and 10 CHrChld x 3 mL). The resin was then treated with CH3OH/ CH2CI2/DIPEA (v/v/v; 2:17:1, 5 mL) for 30 min to cap the unreacted trityl chloride sites, dried under vacuum, and stored in a desiccator.

[0068] Deprotection of the Fmoc group was accomplished by treating the resin (50 mg, 40 pmol) with 20% v/v piperidine/DMF (5 mL) for 15 min twice with consecutive DMF washes after each addition. The resin was then treated with 14-(tert-butoxy)-14- oxotetradecanoic acid (63 mg, 200 pmol) using HATU (76 mg, 200 pmol), and DIPEA (35 pL, 200 pmol) in DMF (5 mL) for 2 h or repeated until a negative ninhydrin test was observed. After washing with DMF, the resin was treated with 2% hydrazine in DMF (5 mL, 2 x 5 min). Positive ninhydrin test was observed. The resin was then washed as described above. The resin was then treated with Fmoc-NH-dPEG2- COOH (200 pmol), HATU (76 mg, 200 pmol), and DIPEA (35 pL, 200 pmol) in DMF (5 mL) for 2 h or repeated until a negative ninhydrin test was observed. The resin was then washed as described above. The Fmoc group was then removed as described above and the resin treated again with a fresh mixture of activated Fmoc-NH-dPEG2-COOH for an additional 2 h. Bromoacetic anhydride (55 mg, 200 pmol) and DIPEA (35 pL, 200 pmol) in CH2CI2 (2 mL) were then added to the resin and the mixture was stirred for 30 min. After washing with CH2CI2, the product was cleaved from the resin using TF A//Pr3 S i H/H2O/ C H2CI 2 (v/v; 5:10:10:75, 5 mL) for 1 h. The resin was removed by filtration, washed with TFA (2 x 3 mL) and the combined filtrate was concentrated under reduced pressure, dissolved in CH3CN/H2O (v/v; 1:1) and lyophilized. The crude peptide was then purified using preparative HPLC column (Phenomenex, Prep Cl 8, 300A, 50 x 250 mm) and the desired compound Br-FAIO was obtained as a white solid (23 mg, 34% yield). MS (ES+) m/z 825.4 [M+H]+, calculated: 825.4 [M+H]+

[0069] Synthesis of Br-FAll: Br-FAll was synthesized using Fmoc-SPPS using a similar procedure to Br-FAIO, using the fatty acid 12-(tert-butoxy)-12-oxododecanoic acid. The crude peptide was then purified using preparative HPLC column (Phenomenex, Prep Cl 8, 300A, 50 x 250 mm) and the desired compound Br-FAll was obtained as a white solid (24 mg, 37% yield). MS (ES+) m/z 797.4 [M+H]+, calculated: 797.3 [M+H]+ [0070] Synthesis of Br-FA12: Br-FA12 was synthesized using Fmoc-SPPS using a similar procedure to Br-FAIO, using the fatty acid palmitic acid. The crude peptide was then purified using preparative HPLC column (Phenomenex, Prep C18, 300A, 50 x 250 mm) and the desired compound Br-FA12 was obtained as a white solid (29.7 mg, 45% yield). MS (ES+) m/z 823.5 [M+H]+, calculated: 823.4 [M+H]+ [0071] Synthesis of Br-FA13: Br-FA12 was synthesized using Fmoc-SPPS using a similar procedure to Br-FAIO, using the fatty acid myristic acid. The crude peptide was then purified using preparative HPLC column (Phenomenex, Prep Cl 8, 300A, 50 x 250 mm) and the desired compound Br-FA13 was obtained as a white solid (20.4 mg, 32% yield). MS (ES+) m/z 795.4 [M+H]+, calculated: 795.4 [M+H]+ [0072] Other than OT derivative compounds exemplified above (e.g., OC-10 through OT-18), additional OT derivatives suitable for the invention can be readily designed and synthesized with alternative FA chains. Some of these designed compounds are shown in Table 3. Table 3. Designed OT derivative compounds

[0073] Structures of the respective FA chains for generating these designed OT derivative compounds are shown below in the context of Br-FAs.

Example 5. Some protocols employed in the exemplified studies [0074] Peptide Synthesis: Peptide OT-12 is used here as an example of the synthetic procedure used for the synthesis of the carbetocin analogs. Refer to Scheme 5 in Example 5 for the synthetic route used for the synthesis of oxytocin agonist OT-12. To a solution of the commercially-obtained peptide (100 mg, 98.8 pmol, 1.0 eq; final peptide concentration 12 mM) in DMF was added PyBOP (61.7 mg, 118.6 pmol, 1.2 eq) and DIPEA (34.4 pL, 197.7 pmol, 2.0 eq). The reaction mixture was agitated 3 h at room temperature, then diluted with H2O and lyophilized. Semi-preparative HPLC afforded the cyclized product S6 as white fluffy solid (70 mg, 71% yield). The purified intermediate (70 mg, 70.4 pmol, 1.0 eq) was then dissolved in cold TFA (14.1 mL, 200 mL per mmol peptide) containing anisole (281.6 pL, 4 mL per mmol peptide). AgOTf (361.8 mg, 1.41 mmol, 20 eq) was then added and the mixture was agitated for 1.5 h at 4°C. Cold Et20 was then added to precipitate the peptide silver salt. The solution was isolated by centrifugation and re-suspended in 1 M aqueous acetic acid (12 mL). DTT (434.4 mg, 2.82 mmol, 40 eq) was then added and the mixture was agitated at room temperature for 4 h. The peptide was again isolated using cold ether and purified using semi-preparative HPLC to afford the product as white fluffy solid (45.5 mg, 70% yield). To a solution of the cysteine free peptide (45.5 mg, 49.2 pmol, 1.0 eq; final concentration of 2 mM) in CH3CN/3O mM NH4HCO3 (v/v; 1:3, 24.6 mL) was added FA3 (65.1 mg, 73.8 pmol, 1.5 eq) and TCEP (28.2 mg, 98.4 pmol, 2 eq). The reaction mixture was agitated at room temperature for 4h. Semi preparative HPLC afforded peptide OT-12 as a white fluffy solid (43.8 mg, 51.6 % yield, 98% pure).

[0075] in vitro b-arrestin oxytocin assay: b-Arrestin oxytocin assay was performed as per manufacturer’s instruction (DiscoverX Corporation). The PathHunter® CHO-K1 human OTR b-Arrestin Cells are cultured in cell culture medium offered by DiscoverX. Cells were harvested and plated in 384-well assay plates with a density of 5000 cells per well. After incubating for 12 h at 37 °C, 5% CO2, culture medium was exchanged into AssayComplete Cell Plating Reagent containing 10% or 0% FBS. Oxytocin and its analogs were dissolved in OPTIMEN medium with serial dilutions and then added to the assay plate for 90 min at 37 °C. The assay plates were assayed using working detection solution and continued incubating for 1 h at room temperature in the dark. Luminance signals were read using EnVision Multilabel Plate Reader System (PerkinElmer, USA). Data analysis was performed with GraphPad Prism.

[0076] in vitro calcium flux assay for Vi_a and Vi_b agonist effect: Calcium No Wash^plus Assay was performed by Eurofin Pharma Discovery Services. Briefly, CHO cells stably expressing human recombinant V i_a and RBL cells stably expressing human recombinant VIB were treated with OT-12 in dose response at RT to measure intracellular calcium level. luM of AVP and 0. luM of Vasopressin were used as control for V i_a and V IB agonist effect. The EC50 values were determined by non-linear regression analysis of the concentration-response curve generated with mean replicate values using Hill equation curve fitting.

[0077] in vitro cAMP assay for V2 agonist effect: cAMP assay was performed by Eurofin Pharma Discovery Services. Briefly, CHO cells stably express human recombinant V2 were treated with OT-12 for 30 min at RT then cAMP level was measured using HTRF method. 1 nM of AVP was used as control. The EC50 values were determined by non-linear regression analysis of the concentration-response curve generated with mean replicate values using Hill equation curve fitting.

[0078] PK Study in mice: peptide OT-12 (1 mg kg ¹) in 10 mM phosphate buffered saline (pH 8.1) was administered to male C57BL/6 mice by the s.c. route. 70 pL blood was collected from retro orbital or saphenous vein at each time point and the samples were transferred into microcentrifuge tubes containing 2 pL of K2EDTA (0.5 M) as anti-coagulant and placed on wet ice until processed for plasma. Blood samples were processed for plasma by centrifugation at approximately 4 °C, 3000 g 15 min within half an hour of collection. Plasma samples were then stored in polypropylene tubes, quickly frozen over dry ice and kept at -70 ± 10 °C. The concentrations of peptides in plasma at each time point were determined using a bioanalytical method by LC-MS. Peptide concentrations in plasma were obtained and plotted against time points to obtain in vivo half-life of each peptide, using WinNonLin Phoenix software (Pharsight Corp, St. Louis, MO). Due to volume/sampling limitations in mice, sparse sampling was used. Therefore, a single PK profile was obtained by combining concentrations from various animals and PK parameter estimates were averaged. Perfused brain samples were also harvested and frozen down for bioanalysis at 7 h and 24 h using standard procedures.

[0079] Effects on acute food intake and body weight after a single administration in normal mice: Male C57BL/6 mice (Jackson Lab, Bar Harbor, ME; age 10 weeks) were housed 2 per cage and maintained in a reverse light cycle room with ad libitum access to water and normal chow. Food was removed 2 h prior to the start of dark phase and animals were treated subcutaneously with either vehicle (DPBS, pH 8.5; GibCo; cat# 14190-144) or with the peptides 15 minutes before the start of food monitoring. Cumulative food consumption was measured at the beginning of dark phase (T = 0) and at subsequent time points at 1, 3, 6, 24 and 48 hours. Body weight was determined daily up to 2 days post-oxytocin administration.

[0080] Effects on food intake and body weight after sub-chronic treatment in diet-induced obese (DIO) mice: DIO mice (Jackson Lab, Bar Harbor, ME; age 21 weeks) housed 2 per cage in regular 12 h light/dark cycle with ad libitum access to water and HFD (D12492, Research Diets, New Brunswick, NJ) were subcutaneously treated daily with either vehicle (DPBS, pH 8.5, GibCo; cat# 14190-144), or peptide OT-12 at two doses (0.05 mg kg ¹ and 0.1 mg kg ¹ ), or carbetocin (Sigma- Aldrich, St. Louis, MO) at 0.1 mg kg ¹. Food intake and body weight were measured for up to 9 days. Serum lipids profile were determined after 13 days of treatment.

[0081] Animal care and use of statistical analysis. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of California Institute for Biomedical Research (Calibr) and strictly followed the NIH guidelines for humane treatment of animals. The results are expressed as the mean ± SE using GraphPad Prism 6, and the data were compared using the unpaired Student’s t test. Where appropriate, data were compared using repeated measures or one-way analysis of variance, followed by the Student-Newman- Keuls post hoc test. Groups of data were considered to be significantly different if P < 0.01.

Example 6. General methods and detailed experimental protocols [0082] General Methods:

[0083] Materials: Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification. Peptides were purchased from Cellmano Biotech Limited, Innopep or Shanghai Dechi Biosciences Co. Trifluoroacetic acid (TFA), A A -di isopropyl ethyl amine (DIPEA), piperidine, hydrazine, triisopropylsilane (/PnSiH). dithiothreitol (DTT), silver trifluoromethanesulfonate (AgOTl), chloroform-d6, dimethylsulfoxide (DMSO), hydrochloric acid (HC1), myristic acid, (Benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PYBOP), anisole, and tris(2- carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO) octadecanedioic acid mono-tert-butyl ester was purchased from Astatech (Bristol, PA). Bromoacetic anhydride, A A-di methyl form amide (DMF), formic acid, and diethyl ether was purchased from Thermo Scientific (Fair Lawn, NJ). Fmoc-Lys(ivDde)-OH was purchased from Combi-Blocks (San Diego, CA). 2- chlorotrityl chloride resin was purchased from NovaBiochem (San Diego, CA). 2-(7- Aza- 1 A-benzotriazole- 1 -yl)- 1.1.3.3- tetramethyluronium hexafluorophosphate (HATU) was purchased from Chem-Impex International (Wood Dale, IL). Acetonitrile (CfbCN), ethyl acetate (EtOAc), dichloromethane (CH2CI2), and methanol (CH3OH) were purchased from Fisher Scientific (Fair Lawn, NJ). All MEG building blocks were acquired from Quanta BioDesign Ltd. (Plain City, OH).

[0084] All reactions involving air or moisture sensitive reagents, or intermediates were performed under an inert atmosphere of nitrogen or argon. All solvents used in the purifications were of HPLC/UHPLC grade. Reactions were monitored by liquid chromatography -mass spectrometry (LC-MS) or by thin-layer chromatography (TLC) on Merck 50 x 100 mm silica gel 60 aluminum sheets using an aqueous solution of KMnCU.

[0085] Chromatography conditions: Flash chromatography purifications were performed on silica gel (40 pm, RediSepRf from Teledyne Isco) prepacked columns on a CombiFlash Rf from Teledyne Isco. The purified final compounds were eluted as single and symmetrical peaks, thereby confirming a purity of >95%.

[0086] Semi-preparative chromatography was performed on a Shimadzu with a Phenomenex Luna column (Cie, 100 A pore size, 10 pm particle size, 250 x 10.0 mm, flow: 4 mL min ¹) or on a Agilent 1200 with a Phenomenex Luna column (Cie, 100 A pore size, 5 pm particle size, 150 x 21.2 mm, flow: 20 mL min ¹).

[0087] Nuclear magnetic resonance spectra were recorded on a Bruker 400 system using DMSO-d6, CDCh or CD3OD as a solvent. Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal standard. Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multi pi et, dd = doublets of doublet, br = broad. Coupling constants (J values) are given in hertz (Hz). Low resolution mass spectra were recorded on a Waters Acquity UPLC with a Phemomenex Luna Omega Cie column (Cie, 100 A pore size, 1.6 pm particle size, 50 x 2.1 mm, flow: 0.4 mL min ¹). The solvent system used was A (0.1% formic acid in H2O) and B (0.1% formic acid in CH3CN). The gradient used for peptide analysis is described in the table below.

Time %A %B

(minutes)

0 90 10

1 10 90

1.6 10 90 1.7 90 10

2 90 10

[0088] High resolution mass HR-MS were recorded on an Agilent 6520 accurate- mass quadrupole-time-of-light (QTOF) instrument equipped with reverse phase liquid chromatography and an electrospray ionization (ESI) source. The column used was Aeris Widepore column (XB-Cis, 3.6 pm particle size, 150 x 2.1 mm, flow: 0.5 mL min ¹). The solvent system used was A (0.1% formic acid in H2O) and B (0.1% formic acid in CH3CN). The gradient used for peptide analysis is described in the table below.

Time %A %B

(minutes)

0 95 5 2 95 5

12 40 60

13 20 80

14 80 20

15 20 80

16 80 20 17 5 95 20 95 5

[0089] Synthesis of FA moieties

[0090] Synthesis ofFAl:

Scheme 2. Synthesis of FA1. Reagents and conditions: i) myristic acid, B0C-NH-PEG2- COOH, HATU, DIPEA, DMF, r.t, 90 min; ii) TFA, r.t, 30 min; iii) bromoacetic anhydride, CH2CI2, 4 °C, 30 min then r.t., 90 min.

[0091] Compound SI: Myristic acid (184 mg, 0.81 mmol, 1.0 eq) was dissolved in DMF (4 mL). To this solution, HATU (338 mg, 0.89 mmol, 1.1 eq) and DIPEA (154 pL. 0.89 mmol, 1.1 eq) were added followed by the addition of B0C-NH-PEG2-COOH (200 mg, 0.81 mmol, 1.0 eq). The reaction mixture was then stirred for 1.5 h, the solvent was removed, and the product was then dissolved in EtOAc. The organic layer was successively washed with sat. NaHCCb, 1 M HC1 and brine, dried over Na2SC>4, filtered, and concentrated. Purification by flash column chromatography on silica gel provided the desired compound SI as a white solid (254 mg, 0.55 mmol, 69%).

¾ NMR (400 MHz, CDCb) d 3.66 - 3.54 (m, 8H), 3.49 (q, J= 5.2 Hz, 2H), 3.35 (d, J = 6.1 Hz, 2H), 2.20 (t, J= 7.7 Hz, 2H), 1.63-1.58 (m, 2H), 1.47 (s, 8H), 1.33-1.24 (m, 21H), 0.90 (t, J = 6.9 Hz, 3H).

R_t = 2.21 min (Agilent default 10_90 min)

MS (ES⁺) in z 459.6 ([M+H]⁺)

[0092] Compound FA1: A solution of SI (250 mg, 0.54 mmol, 1.0 eq) in CH2CI2 (2 mL) was treated with TFA (2 mL) for 30 min. The mixture was then concentrated, co-evaporated with heptane, re-dissolved in CH2CI2 (10 mL), and cooled at 0°C. DIPEA (188 pL, 1.08 mmol, 2.0 eq) was added followed by bromoacetic anhydride (154.4 g, 0.59 mmol, 1.1 eq) which was already dissolved in CH2CI2 (1 mL). The reaction mixture was then stirred for 30 min at 0 °C then 90 min at room temperature, and the solvent was removed. The product was re-dissolved in EtOAc, and the organic layer was then washed with sat. NaHC03, 1 M HC1, brine, dried over Na2S04, filtered, and concentrated. Purification by flash column chromatography on silica gel provided title compound FA1 as white solid (163 mg, 0.34 mmol, 63%). Characterization data for this compound were in agreement with that reported in the literature [1]

¾ NMR (400 MHz, CDCb) d 3.89 (s, 2H), 3.58 (dtd, J= 10.0, 5.0, 0.0 Hz, 4H), 3.49 (dp, J= 15.7, 5.1 Hz, 4H), 2.17 (t, J= 8.0 Hz, 2H), 1.74 (s, 2H), 1.62 (t, J= 7.4 Hz, 2H), 0.86 (t, J = 7.0 Hz, 3H).

MS (ES⁺) m/z 480.4 [M+H]⁺, calculated: 479.5.

[0093] Synthesis of FA2:

Scheme 3. Synthesis of FA2. Reagents and conditions: i) octadecanedioic acid mono- tert-butyl ester, B0C-NH-PEG3-NH2, HATU, DIPEA, DMF, r.t, 3 h; ii) TFA, r.t, 30 min; iii) bromoacetic anhydride, DIPEA, 0 °C, 90 min then r.t., 30 min. [0094] Compound S2: Octadecanedioic acid mono-tert-butyl ester acid (200 mg,

0.54 mmol, 1.0 eq) was dissolved in DMF (5 mL). HATU (225 mg, 0.59 mmol, 1.1 eq) and DIPEA (103 pL, 0.59 mmol, 1.1 eq) were then added followed by the addition of B0C-NH-PEG3-NH2 (157.8 g, 0.54 mmol, 1.0 eq). The reaction mixture was then stirred for 3 h, and the solvent was removed. The product was dissolved in EtOAc. The organic layer was successively washed with sat. NaHCCb, 1 M HC1, brine, dried over Na2SC>4, filtered, and concentrated. Purification by flash column chromatography on silica gel provided the desired product S2 as white solid (281 mg, 0.43 mmol, 81%).

¾ NMR (400 MHz, CDCb) d 3.76-3.61 (m, 8H), 3.63-3.54 (m, 4H), 3.48 (q, J= 5.1 Hz, 2H), 3.34 (s, 2H), 2.20 (dt, J= 9.8, 7.6 Hz, 4H), 1.67- 1.55 (m, 4H), 1.49- 1.44 (m, 17H), 1.30 (s, 6H), 1.30- 1.24 (m, 19H).

MS (ES⁺) m/z 645.5 ([M+H]⁺)

[0095] Compound FA2: A solution of compound S2 (281 mg, 0.44 mmol) in

CH2CI2 (2 mL) was treated with TFA (2 mL) for 30 min. The mixture was concentrated, co-evaporated with heptane, dissolved in CH2CI2 (10 mL), and cooled at 0 °C. DIPEA (35 pL, 0.2 mmol) was added to the reaction mixture followed by bromoacetic anhydride (26 mg, 0.1 mmol) dissolved in CH2CI2 (1 mL). The reaction mixture was then stirred for 90 min at 0 °C, then 30 min at room temperature, and the solvent was removed. The product was purified by HPLC semi-preparative to provide the desired product FA2 as a white solid (27 mg, 0.04 mmol, 10%). ¾ NMR (400 MHz, CD3OD) d 3.86 (s, 2H), 3.70-3.62 (m, 9H), 3.57 (dt, J= 11.0, 5.5

Hz, 4H), 3.40 (dt, J= 16.7, 5.4 Hz, 5H), 2.29 (t, J= 7.4 Hz, 2H), 2.21 (t, J= 7.5 Hz, 2H), 1.68-1.55 (m, 4H), 1.40-1.25 (m, 27H).

MS (ES⁺) m/z 609.3 ([M+H]⁺) [0096] Synthesis of FA3

Scheme 4. Synthesis of FA3. Reagents and conditions: i) 20% piperidine, DMF, r.t, 2 x 15 min; ii) octadecanedioic acid mono-tert-butyl ester, HATU, DIPEA, DMF, r.t., 2 h; iii) 2% hydrazine, DMF, r.t., 2 x 5 min; iv) Fmoc-dPEG2-COOH, HATU, DIPEA, DMF, r.t., 90 min; v) bromoacetic anhydride, DIPEA, CH2CI2, r.t., 30 min; vi) TFA, iPrsSiH, H2O, CH2CI2, r.t., 1 h.

[0097] A solution of Fmoc-Lys(ivDde)-OH (60 mg, 100 pmol) and DIPEA (70 pL, 400 pmol) in CH2CI2/DMF (v/v; 2:1, 3 mL) was added to pre-swollen (CH2CI2, 3 mL, 20 min) 2-chlorotrityl chloride resin (100 mg, 80 pmol) and the mixture was shaken for 30 min at room temperature, filtered, and washed with DMF (4 x 3 mL) and CH2Cl2(4 x 3 mL). The resin was then treated with CH3OH/ CH2CI2/DIPEA (v/v/v;

2: 17: 1, 5 mL) for 30 min to cap the unreacted trityl chloride sites, dried under vacuum, and stored in a desiccator. [0098] Deprotection of the fluorenylmethyloxy carbonyl [Fmoc] group was accomplished by treating the resin (50 mg, 40 pmol) with 20% v/v piperidine/DMF (5 mL) for 15 min twice with consecutive DMF washes after each addition to yield peptidyl resin S3. The resin was then treated with octadecanedioic acid mono-tert-butyl ester (74 mg, 200 pmol) using HATU (76 mg, 200 pmol), and DIPEA (35 pL, 200 pmol) in DMF (5 mL) for 2 h or repeated until a negative ninhydrin test was observed. After washing with DMF, the resin was treated with 2% hydrazine in DMF (5 mL, 2 x 5 min). Positive ninhydrin test was observed. The resin was then washed as described above to yield peptidyl resin S4. The resin was then treated with Fmoc-NH-dPEG2- COOH (200 pmol), HATU (76 mg, 200 pmol), and DIPEA (35 pL, 200 pmol) in DMF (5 mL) for 2 h or repeated until a negative ninhydrin test was observed. The resin was then washed as described above. The Fmoc group was then removed as described above and the resin treated again with a fresh mixture of activated Fmoc-NH-dPEG2-COOH for an additional 2 h. Bromoacetic anhydride (55 mg, 200 pmol) and DIPEA (35 pL, 200 pmol) in CH2CI2 (2 mL) were then added to the resin and the mixture was stirred for 30 min. After washing with CH2CI2, the product was cleaved from the resin using TF A/ /Pn S i H/H2O/ C H2CI 2 (v/v; 5:10:10:75, 5 mL) for 1 h. The resin was removed by filtration, washed with TFA (2 x 3 mL) and the combined filtrate was concentrated under reduced pressure. The resulting residue was washed several times with cold diethyl ether and was finally dried to a crude product as yellow powder under nitrogen flow. The crude peptide, (50 mg) purified using preparative HPLC column

(Phenomenex, Prep Cie, 300 A, 50 x 250 mm) equilibrated with 10% CFECN (0.1% TFA). The composition of the eluent then was ramped to 35% CFECN-water (0.1%TFA) over 1 min, and a linear gradient was initiated at a rate of 0.5% per min of CFECN (0.1% TFA) into H2O (0.1% TFA) and run for 50 min. The desired compound FA3 was obtained as a white solid (15 mg, 41% yield). Characterization data for this compound were in agreement with that reported in the literature [1]

[0099]

4.37 (dd, J= 9.1, 4.9 Hz, 1H), 3.88 (s, 2H),

3.76 (q, J= 6.2 Hz, 4H), 3.63 (d, J= 2.3 Hz, 8H), 3.57 (q, J= 5.5 Hz, 4H), 3.40 (dt, J = 9.7, 5.5 Hz, 4H), 3.21 (t, J= 6.9 Hz, 2H), 2.47 (dt, J= 13.5, 6.2 Hz, 4H), 2.28 (dt, J = 12.8, 7.5 Hz, 4H), 1.93-1.83 (m, 1H), 1.77-1.68 (m, 1H), 1.66-1.52 (m, 6H), 1.51-1.40

(m, 2H), 1.39-1.26 (m, 24H).

[00100] MS (ES⁺) m/z 881.4 [M+H]⁺, calculated: 880.9 [M+H]⁺

[00101] Peptide synthesis:

Scheme 5. Synthesis of the conjugated carbetocin analog OT-12. Reagents and conditions: i) PyBOP, DIPEA, DMF, r.t, 3 h; ii) AgOTf, anisole, TFA, 0 °C, 90 min then 1 M aq CFECOOH, DTT, r.t., 4 h; iii) FA3, CFECN, 30 mM NaH HC0₃, r.t., 4h. [00102] Peptide OT-12: Peptide OT-12 is used here as an example of the synthetic procedure used for the synthesis of the carbetocin analogs. Refer to Scheme 5 for the synthetic route used for the synthesis of oxytocin agonist 9.

[00103] To a solution of the commercially-obtained peptide S5 (100 mg, 98.8 pmol,

1.0 eq; final peptide concentration 12 mM) in DMF was added PyBoP (61.7 mg, 118.6 pmol, 1.2 eq) and DIPEA (34.4 pL, 197.7 pmol, 2.0 eq). The reaction mixture was agitated 3 h at room temperature, then diluted with H2O and lyophilized. Semi preparative HPLC afforded the cyclized product S6 as white fluffy solid (70 mg, 71% yield); R_t = 7.50 min (Q-TOF); m/z (HR-MS) 995.4142 ([M+H]⁺).

[00104] The purified intermediate S6 (70 mg, 70.4 pmol, 1.0 eq) was dissolved in cold TFA (14.1 mL, 200 mL per mmol peptide) containing anisole (281.6 pL, 4 mL per mmol peptide). AgOTf (361.8 mg, 1.41 mmol, 20 eq) was then added and the mixture was agitated for 1.5 h at 4°C. Cold Et20 was then added to precipitate the peptide silver salt. The solution was isolated by centrifugation and re-suspended in 1 M aqueous acetic acid (12 mL). DTT (434.4 mg, 2.82 mmol, 40 eq) was then added and the mixture was agitated at room temperature for 4 h. The peptide was again isolated using cold ether and purified using semi-preparative HPLC to afford the product S7 as white fluffy solid (45.5 mg, 70% yield); Rt = 7.60 min (Q-TOF); m/z (HR-MS) 924.3800 ([M+H]⁺).

[00105] To a solution of the cysteine free peptide S7 (45.5 mg, 49.2 pmol, 1.0 eq; final concentration of 2 mM) in CH3CN/3O mM NH4HCO3 (v/v; 1:3, 24.6 mL) was added FA3 (65.1 mg, 73.8 pmol, 1.5 eq) and TCEP (28.2 mg, 98.4 pmol, 2 eq). The reaction mixture was agitated at room temperature for 4h. Semi preparative HPLC afforded peptide OT-12 as a white fluffy solid (43.8 mg, 51.6 % yield, 98% pure); Rt = 13.2 min (Q-TOF); m/z (HR-MS) 1725.8930 [M+H]⁺, calculated: 1724.8810 [M+H]⁺ . x 10⁷

+ TIC Scan

[00106] Analysis of the other purified analogs

[00107] Peptide OT-1: White powder (1.77 mg, 1.73 pmol, 58%); Rt = 6.85 min (Q-TOF); HR-MS = m/z 1021.42627 ([M+H]⁺), calculated: 1021.41908 ([M+H]⁺)

x10 ⁸ + TIC Scan

[00109] Peptide OT-3: White powder (1.58 mg, 1.57 pmol, 56%);

Rt = 8.32 min (Q-TOF) ; HR-MS = m/z 1006.44985 ([M+H]⁺), calculated: 1006.44456 (| IVI+HI ') cΐo ⁸ + TIC Scan

1006.44985

522.70210

il^~

1529.13665

[00110] Peptide OT-4: White powder (1.89 mg, 1.39 pmol, 60%); Rt = 12.30 min (Q-TOF); HR-MS = m/z 1362.71873 ([M+H]⁺), calculated: 1362.71207 ([M+H]⁺) x10 8 + TIC Scan 4 3 2 1

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Counts vs. Acquisition Time (min)

681.86186

1362 71873

2044.56867 2726.41162 [00111] Peptide OT-5: White powder (1.16 mg, 0.85 pmol, 46%); Rt = 12.75 min

(Q-TOF); HR-MS = m/z 1362.71829 ([M+H]⁺), calculated: 1362.71207 ([M+H]⁺)

[00112] Peptide OT-6: White powder (0.35 mg, 0.26 pmol, 21%); Rt = 13.22 min (Q-TOF); HR-MS = m/z 1347.74364 ([M+H]⁺), calculated: 1347.73756 ([M+H]⁺) x10 8 + TIC Scan 3

2

1

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Counts vs. Acquisition Time (min)

674.37421

2041.07394 2696.47310

[00113] Peptide OT-7: White powder (0.59 mg, 0.44 pmol, 20%); Rt = 12.71 min (Q-TOF); HR-MS = m/z 1358.6848 ([M+Na]⁺), calculated: 1336.6964 ([M+H]⁺) X10 2 + TIC Scan

2 4 6 8 10 12 14 16 18 20

Counts (%) vs. Acquisition Time (min)

[00114] Peptide OT-8: White fluffy solid (0.73 mg, 0.50 mihoΐ, 25% yield); Rt = 12.24 min (Q-TOF); HR-MS = m/z 1488.74 ([M+Na]⁺), calculated: 1466.7594 ([M+H]⁺) xio 2 + TIC Scan

2 4 6 8 10 12 14 16 18 20

Counts (%) vs. Acquisition Time (min)

[00115] Peptide OT-9: White powder (0.75 mg, 0.43 pmol, 22%); Rt = 11.91 min (Q-TOF); HR-MS = m/z 869.9524 ([M+2H]²⁺), calculated: 869.4483 ([M+2H]²⁺)

^{X10 2} + TIC Scan

2 4 6 8 10 12 14 16 18 20

Counts (%) vs. Acquisition Time (min) s

[00116] Peptide OT-10: White powder (1.61 mg, 1.22 pmol, 37%); Rt = 11.95 min

(Q-TOF); HR-MS = m/z 1344.6666 ([M+Na]⁺), calculated: 1323.6648 ([M+H]⁺) xio ⁸ + TIC Scan

Counts vs. Acquisition Time (min)

[00117] Peptide OT-11: White powder (1.43 mg, 0.98 pmol, 38%); Rt = 11.9 min

(Q-TOF); HR-MS = m/z 1474.7364 ([M+H]⁺), calculated: 1453.7278 ([M+H]⁺) xio 8 + TIC Scan

Counts vs. Acquisition Time (min)

[00118] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof.

[00119] It is further noted that all publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. An oxytocin derivative compound, comprising a modified oxytocin scaffold and a chemically conjugated fatty acid moiety.

2. The compound of claim 1, which is substantially longer-acting than oxytocin or carbetocin.

3. An oxytocin derivative compound, comprising a structure:

wherein R is a fatty acid (FA) moiety.

4. The compound of claim 3, wherein the FA moiety is FA1, FA2 or

FA3,

5. The compound of claim 3, wherein the FA moiety is FA3,

6. The compound of claim 3, wherein the FA moiety is FA4, FA8, FA10, FA11, FA12, or FA13 shown below,

7. The compound of claim 3, wherein the FA moiety is FA5, FA6, FA7, FA9, FA14, FA15, or FA16 described herein.

8. A pharmaceutical composition comprising the compound of claim

3.

9. A method for treating a metabolic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound

wherein R is a fatty acid (FA) moiety.

10. The method of claim 9, wherein the metabolic disorder is obesity, diabetes, NDFLD or NASH.

11. The method of claim 9, wherein the FA moiety is FA1, FA2 or FA3 shown below,

12. The method of claim 9, wherein the FA moiety is FA3,

13. The method of claim 9, wherein the FA moiety is selected from the group consisting of FA4-FA16 described herein.

14. The method of claim 9, further comprising administering to the subject an additional therapeutic agent.

15. The method of claim 13, wherein additional therapeutic agent is selected from the group consisting of a diabetes drug, insulin or an insulin analog, a DPP4 inhibitor, a SGLT2 inhibitor, an agonist of GLP1R, GIPR or GCGR, a hypoglycemic drug, a biguanidine drug, an insulin secretogogue, a sulfonyl urea drug, a TZD drug, FGF21 or an FGF21 analog, leptin or a leptin analog, amylin or an amylin analog, an anti-inflammatory drug, cyclosporine A, FK506, 5-ASA, a statin, and any combination thereof.

16. A method for improving peripheral insulin sensitivity, pancreatic function or lipid homeostasis in a subject, comprising administering to the subject a therapeutically effective amount of a compound

wherein R is a fatty acid (FA) moiety.

17. The method of claim 16, wherein the FA moiety is FA1, FA2 or FA3 shown below ^r,

18. The method of claim 16, wherein the FA moiety is FA3,

19. The method of claim 16, wherein the FA moiety is selected from the group consisting of FA4-FA16 described herein.