Lipophilic enantiomers of desacetylglucosamine muramyl dipeptide with anti-inflammatory and growth-promoting activities

Abstract

Compositions and methods for reducing inflammation in vertebrates and promoting growth and feed conversion in animals by administering an effective amount of a composition comprising a mirror image molecule (enantiomer) of a lipophilic muramyl dipeptide (MDP) derivative (i.e., desmuramyl dipeptide) that does not contain the N-acetylglucosamine portion of muramyl dipeptide (MDP). The field of the invention relates to lipophilic muramyl dipeptide enantiomer compositions and methods for reducing inflammation, promoting growth, and enhancing feed conversion in animals, including humans.

Description

[Technical Field]

[0001] Cross-reference to priority application This application claims the benefit of U.S. Provisional Patent Application No. 63 / 000,364, filed March 26, 2020, entitled "Lipophilic Enantiomers of Desacetylglucosamine Muramyl Dipeptide with Anti-Inflammatory and Growth Promoting Activity," the entire contents of which are incorporated herein by reference.

[0002] Field FIELD OF THE INVENTION The field of the invention relates to lipophilic muramyl dipeptide enantiomer compositions and methods for reducing inflammation, promoting growth, and enhancing feed conversion in animals, including humans. [Background technology]

[0003] background Muramyl dipeptide (MDP) is the smallest structure conserved in all bacterial peptidoglycans (PGNs). It consists of N-acetylmuramic acid (an ether of N-acetylglucosamine and D-lactic acid) linked by a peptide bond to L-alanine and D-γ-glutamate or D-isoglutamine (MacDonald 2005).

[0004] PGN has long been known to promote inflammatory responses. The MDP moiety of PGN was found to be the minimal chemical structure required to induce inflammation. MDP is also required for the adjuvant activity of Freund's complete adjuvant (an emulsion of mycobacterial extract) (MacDonald 2005). As an adjuvant, MDP promotes a strong immune response that is used to boost vaccine efficacy when injected along with vaccine antigens.

[0005] While stimulating extraintestinal inflammation, MDP has anti-inflammatory effects in the intestinal tract and protects mice from experimentally induced colitis (Watanabe 2008; Watanabe 2014).

[0006] The intestinal anti-inflammatory properties of MDP offer opportunities for therapeutic applications (Strober 2013). However, MDP is hydrophilic and rapidly cleared from the circulation via renal excretion, thus requiring repeated administration of high doses to mediate nonspecific resistance to infection or adjuvant activity (Fogler 1985).

[0007] These unfavorable pharmacokinetics and severe side effects have prompted numerous chemical modifications of MDP to correct these shortcomings, the most successful of which has been lipid modifications of MDP, which increase both its potency and half-life (Parant 1980; Matsumoto 1983; Fogler 1985).

[0008] Covalent lipid-MDP conjugates have therefore shown several advantages, including improved oral bioavailability, enhanced tumor targeting and therapeutic efficacy, reduced toxicity, and enhanced drug loading into delivery carriers such as liposomes (Fidler 1987; Irby 2017).

[0009] Surprisingly, the intact muramyl dipeptide molecule is not required for the biological activity associated with lipophilically conjugated MDP. Even the L-alanine-D-isoglutamine dipeptide MDP moiety without N-acetylmuramic acid retains the immunomodulatory activity of MDP when covalently conjugated to a lipophilic moiety. For example, Gobec (2016) successfully replaced N-acetylmuramyl with the acyl moiety in the acyl-glycine-L-alanine-D-glutamate MDP analog. Penney (1999) completely removed the muramyl moiety in octadecyl L-alanine-D-isoglutamine, which still retains strong immunomodulatory activity.

[0010] Furthermore, both Penney (1999) and Gobec (2016) have shown that D-isoglutamine can be replaced by D-glutamine or D-glutamate in lipophilic desmuramyl dipeptides without loss of function.

[0011] Enhanced growth in animals is measured either by mass growth per unit of time or by mass growth per unit of nutrition; the latter is sometimes referred to as feed conversion. Promoting growth by either means is economically useful in the production of animal protein for consumption by humans and other animals because it reduces the time or amount of feed required to obtain an equivalent gain in body weight.

[0012] Antibiotics, given at subinhibitory doses, have long been used as growth promoters to enhance growth in agricultural production animals. They likely function by releasing components (including MDPs) derived from gut bacteria (postbiotics) that suppress inflammation in the intestinal tract. This mechanism has most likely evolved to protect animals from damaging responses to the trillions of inhabiting gut bacteria.

[0013] Due to the widespread induction of bacterial antibiotic resistance through the use of antibiotics as growth promoters, alternatives to antibiotics as growth promoters are highly desirable. Nalle and Kaltenboeck teach that low-dose oral administration of a potent lipophilic MDP analog improves growth rate and feed conversion in animals, likely due to a reduction in subclinical intestinal inflammation and therefore the overall systemic inflammatory state (Nalle 2017).

[0014] The generation of N-acetylmuramic acid MDP intermediates by multistep chemical synthesis is difficult, making lipophilic MDP analogs too expensive for use as growth promoters in livestock, making the immunomodulatory lipophilic desmuramyl dipeptide a promising candidate for non-antibiotic growth promotion in animals.

[0015] Sidwell (1995) and Penney (1999) showed that octadecyl D-alanine-L-glutamine, the stereochemical enantiomer of the lipophilic octadecyl L-alanine-D-glutamine desmuramyl dipeptide, is an even more potent immunomodulator than octadecyl L-alanine-D-glutamine.

[0016] Counterintuitively, Zhou (2002) showed that mirror-image D-peptides of the enantiomers of natural receptor-binding L-peptides bind their cognate receptors as well as, or even more strongly than, the natural peptides. Furthermore, such D-peptide enantiomers are highly biologically active because they are much more stable than their L-counterparts and resistant to degradation due to the absence of naturally occurring degrading enzymes.

[0017] The binding of enantiomeric peptides to their cognate receptors and the increased stability of such unnatural peptides explain the potent biological effects of octadecyl D-alanine-L-glutamine desmuramyl dipeptide (BCH-527). This dipeptide also avoids the complex synthesis of the anisotropic cyclic carbohydrate moiety of N-acetylmuramic acid and is therefore a promising, cost-effective MDP analog candidate for use as a growth promoter in animals.

[0018] As a lipophilic desmuramyl MDP enantiomer, octadecyl D-alanine-L-glutamine desmuramyl dipeptide has two drawbacks: i) it lacks the N-acetylmuramic acid lactic acid moiety that connects the N-acetylglucosamine moiety of muramic acid to the dipeptide (Jeanloz 1970), thus potentially losing some of its binding strength to its cognate receptor; ii) it contains a high-melting octadecyl aliphatic lipid that is suboptimal for cell membrane insertion (cellular targeting), intramembrane transport, and intracellular release (Spector 1985; van Meer 2008), thus hindering intracellular esterase cleavage of the ester bond between the dipeptide and lipid, which releases the active dipeptide component of octadecyl D-alanine-L-glutamine intracellularly (Hatfield 2016).

[0019] Thus, there is a need for compounds that maximize the immunomodulatory activity of the lipophilic desmuramyl MDP enantiomers. Summary of the Invention [Means for solving the problem]

[0020] Abstract Described herein are oligopeptide analogs (also referred to herein as oligopeptides or compounds) of MDP that contain an L-lactate-D-alanine-L-glutamine moiety. In some cases, the analogs do not contain the N-acetylglucosamine moiety of MDP. The oligopeptides described herein have the following formula: [ka] or a pharmaceutically acceptable acid or salt thereof, wherein R 1 is a substituted or unsubstituted alkyl or a substituted or unsubstituted aryl; R 2 , R 3 , R 4 , and R 5are each independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; X is O or NR 6 where R 6 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. Optionally, X is O. Optionally, R 1 is C1-C 18 A linear alkyl or amino acid. In some cases, the oligopeptide has the following structure: [ka] The ion implantation device has one of the following:

[0021] In some cases, the oligopeptide has the following structure: [ka] where R 7 is substituted or unsubstituted alkyl or substituted or unsubstituted aryl; Y is O or NR 8 where R 8 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. Optionally, the oligopeptide is L-lactate-D-alanine-L-glutamine-hexadecyl ester.

[0022] Also described herein are compositions comprising the compounds described herein. Optionally, the compositions are pharmaceutical compositions comprising at least one oligopeptide described herein and a pharmaceutically acceptable carrier. In some cases, the compositions comprise at least one oligopeptide described herein and animal feed. The at least one oligopeptide may be present in the composition in an amount of about 0.01 mg / kg to 5 mg / kg. Optionally, the compositions further comprise an additive used in animal diets (e.g., enzymes, probiotics, prebiotics, antioxidants, antibiotic growth promoters, colorants, or combinations thereof).

[0023] Further described herein is a method for reducing intestinal inflammation in a human, comprising administering a pharmaceutical composition as described herein to a human with intestinal inflammation, wherein the administration reduces intestinal inflammation. The method may further comprise selecting a human with a disease or condition associated with intestinal inflammation (e.g., inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, or a bacterial infection). Also provided herein is a method for promoting growth in an animal, comprising administering a compound or composition as described herein, wherein the administration enhances the growth of the animal. Also provided herein is a method for enhancing feed conversion in an animal, comprising administering a compound or composition as described herein, wherein the administration enhances feed conversion in the animal.

[0024] The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0025] [Figure 1]FIG. 1 is a schematic diagram showing the synthesis of L-lactate-D-alanine-L-glutamine palmityl ester (lactate-dipeptide-palmityl ester, LDPP). [Figure 2] Figure 2 contains bar graphs showing the evaluation of the growth-promoting effect of L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) in broiler chickens. (A) Experimental diets were fed starting on day 21 and ending on day 44 (constant time for 24 days). (B) Weight gain per chicken over time. Data shown are mean ± 95% confidence interval (95% CI). (C) Total feed consumed per chicken over time, mean ± 95% CI. (D) Feed conversion ratio, determined by dividing total feed consumed by total weight gain for all chickens over time for each group. Error bars represent the 25th to 75th percentiles of calculated feed conversion for individual pens. (E) Modeling of the time required for feed to achieve the same weight gain as untreated controls (1,653 g constant weight gain). (F) Weight gain per chicken over constant weight gain, mean ± 95% CI. (G) Total feed consumed per chicken for a given weight gain, mean ± 95% CI. (H) Feed conversion ratio per given weight gain for each treatment group. Morbidity and mortality rates were not significantly different between groups. Relevant differences between treatment groups are indicated by dashed brackets and corresponding p-values. [Figure 3]Figure 3 contains bar graphs showing the evaluation of the growth-promoting effect of L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) in nursery pigs. (A) Experimental diets were fed starting on day 0 and ending on day 42 (constant time for 42 days). (B) Weight gain per pig over time. Data shown are mean ± 95% confidence interval (95% CI). (C) Total feed consumed per pig over time, mean ± 95% CI. (D) Feed conversion ratio, determined by dividing total feed consumed by total weight gain of all pigs over time for each group. Error bars represent the 25th to 75th percentiles of calculated feed conversion for individual pens. (E) Modeling of the time to feed required to achieve the same weight gain as untreated controls (22.076 kg constant weight gain). (F) Weight gain per pig over constant weight gain, mean ± 95% CI. (G) Total feed consumed per pig for a given weight gain, mean ± 95% CI. (H) Feed conversion ratio per given weight gain for each treatment group. Morbidity and mortality rates were not significantly different between groups. Significant differences between treatment groups are indicated by bold p-values. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description Provided herein are compositions containing lipophilic enantiomers of desacetylglucosamine muramyl dipeptide (MDP) (e.g., L-lactate-D-alanine-L-glutamine-hexadecyl ester). This compound i) improves binding strength to cognate intracellular receptors by bulking up the dipeptide via an additional mirror-image l-lactate moiety; and ii) maximizes intracellular targeting and active dipeptide release via increased membrane fluidity provided by the low-melting point hexadecyl(palmityl) aliphatic lipid.

[0027] When administered to humans or animals, compositions containing lipophilic enantiomers of MDP reduce inflammation, promote growth, and improve feed conversion. Thus, methods for reducing inflammation in humans and animals, and methods for promoting growth and enhancing feed conversion in animals are provided. According to the methods, lipophilic enantiomers of MDP are combined with a pharmaceutically acceptable acid or addition salt thereof, a pharmaceutical carrier, or animal feed. This is then administered to the animal or human in an amount sufficient to achieve the desired reduction in inflammation, promotion of growth, or improvement in feed conversion.

[0028] I. Compound Described herein are oligopeptide analogs of desacetylglucosamine muramyl dipeptide (MDP). The analogs may contain an L-lactate-D-alanine-L-glutamine moiety bound to an organic lipid molecule, and any pharmaceutically acceptable acid or salt thereof. In some cases, the analogs do not contain the N-acetylglucosamine moiety of MDP.

[0029] In some cases, the compounds described herein have the formula I: [ka] Contains, where:

[0030] In Formula I, R 1 is substituted or unsubstituted alkyl or substituted or unsubstituted aryl. Optionally, R 1 is C1-C 18 It is a linear alkyl. If necessary, R 1 is an amino acid, for example, a lysine group (D-lysine or L-lysine).

[0031] Also in formula I, R 2 , R 3 , R 4 , and R 5are each independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl.

[0032] Further, in Formula I, X is O or NR 6 where R 6 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

[0033] Optionally, the compound of Formula I has the structure IA: [ka] The compound may include a compound according to

[0034] In structure IA, R 1 is defined as above for Formula I.

[0035] Optionally, the compound of Formula I has the structure IB: [ka] The compound may include a compound according to

[0036] In structure IB, R 1 is defined as above for Formula I.

[0037] Optionally, the compound of Formula I has Structure IC or Structure ID: [ka] The compound may include a compound according to

[0038] In Structure IC and Structure ID, R 7 is a substituted or unsubstituted alkyl or a substituted or unsubstituted aryl. Also, in Structures IC and ID, Y is O or NR 8 where R8 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

[0039] The alkyl may be a straight chain alkyl or a branched chain alkyl. In some cases, the straight chain alkyl is a C-C 18 Alkyl (e.g., C2-C 17 Alkyl or C3-C 16 The oligopeptide may be L-lactate-D-alanine-L-glutamine-hexadecyl ester, also referred to herein as L-lactate-D-alanine-L-glutamine palmityl ester (lactate-dipeptide-palmityl ester, LDPP) or hexadecyl(2S)-5-amino-2-[[(2R)-2-[[(2S)-2-hydroxypropanoyl]amino]propanoyl]amino]-5-oxo-pentanoate.

[0040] Optionally, the aryl group comprises a phenyl group. Optionally, the aryl group can comprise additional fused rings, such as naphthalene, anthracene, and pyrene. The aryl and heteroaryl groups can be attached at any position on the ring, unless otherwise noted.

[0041] As used herein, alkyl and aryl groups may be substituted or unsubstituted. As used herein, the term substituted includes the addition of a functional group to the position attached to the backbone of the alkyl or aryl group, e.g., replacing a hydrogen with one of these moieties. Examples of substituents include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. Conversely, as used herein, the term unsubstituted indicates that the alkyl or aryl group has a full complement of hydrogens, i.e., its level of saturation is balanced with no substitution (e.g., linear hexadecyl (-(CH2) 15 -CH3))

[0042] II. Methods of Making Compounds The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using a variety of synthetic methods. At least some of these methods are known in the field of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0043] Variations to the compounds of Formula I and described herein include the addition, subtraction, or movement of various components as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Furthermore, compound synthesis may involve the protection or deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in Wuts, Greene's Protective Groups in Organic Synthesis, 5th Edition, Wiley & Sons, 2014 (which is incorporated herein by reference in its entirety).

[0044] The reactions to produce the compounds described herein can be carried out in a solvent, which can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reactions are carried out. The reactions can be carried out in one solvent or a mixture of more than one solvent. The formation of products or intermediates can be monitored according to any suitable method known in the art. For example, the formation of products can be monitored by spectrophotometric means (e.g., nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography (TLC)).

[0045] An exemplary method for synthesizing compounds as described herein is provided in Example 1, which shows by way of example the synthesis of LDPP.

[0046] III. Preparations Also described herein are compositions comprising a compound of Formula I as described herein (e.g., at least one oligopeptide analog of MDP) and a carrier. Optionally, the composition comprises L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) and a carrier.

[0047] In some cases, the composition includes a compound of Formula I (such as LDPP) as described herein and animal feed. Any suitable animal feed can be used, including animal feed containing one or more of corn, sorghum, wheat, barley, oats, soybean meal, fish meal, and / or whey. Optionally, the compound of Formula I can be included in the composition in an amount of about 0.01 mg / kg to 5 mg / kg (e.g., 0.05 mg / kg to 4.5 mg / kg, 0.1 mg / kg to 4 mg / kg, 0.15 mg / kg to 3.5 mg / kg, or 0.2 mg / kg to 3 mg / kg). In some examples, a compound of formula I (e.g., LDPP) may be included in a composition comprising an animal feed in an amount of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, or 5.0 mg / kg. The animal feed composition may further comprise additives used in animal diets, including enzymes, probiotics, prebiotics, antioxidants, antimicrobial growth promoters, and colorants.

[0048] The compositions described herein may be suitable for oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or implanted reservoir administration. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Where appropriate, the compositions described herein may be administered orally, topically, intranasally, intravenously, subcutaneously, intradermally, transdermally, intramucosally, intramuscularly, by inhalation spray, rectally, nasally, sublingually, buccally, vaginally, or via an implanted reservoir.

[0049] The compounds described herein or their derivatives can be provided in pharmaceutical compositions. In some cases, the composition is a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable carrier. Depending on the intended mode of administration, the pharmaceutical composition can be in a solid, semi-solid, or liquid dosage form (e.g., tablet, suppository, pill, capsule, powder, liquid, or suspension), preferably in a unit dosage form suitable for single administration of a precise dosage. The composition comprises a therapeutically effective amount of the compounds described herein or their derivatives in combination with a pharmaceutically acceptable carrier, and may further comprise other medicinal agents, drugs, carriers, or diluents. Pharmaceutically acceptable means a substance that is not biologically or otherwise undesirable and can be administered to an individual in conjunction with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is included.

[0050] The composition may comprise one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term carrier includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other substance known in the art for use in pharmaceutical formulations. The choice of carrier for use in the composition depends on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these substances is described, for example, in Remington: The Science and Practice of Pharmacy, edited by Adeboye Adejare, 23rd Edition, Academic Press (2021). Examples of physiologically acceptable carriers include buffers such as phosphate buffer, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and / or non-ionic surfactants (e.g., TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS® (BASF; Florham Park, NJ)).

[0051] The composition comprising the compound described herein or its derivative suitable for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into sterile injectable solution or dispersion.Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oil (e.g., olive oil) and injectable organic ester (e.g., ethyl oleate).Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0052] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispensing agents. Prevention of microbial action can be enhanced by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, can also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0053] Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are mixed with at least one inert conventional excipient (or carrier) (e.g., sodium citrate or disodium phosphate), or with (a) a filler or extender (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), (b) a binder (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia), (c) a humectant (e.g., glycerol), (d) a disintegrant (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate), (e) a solution retarder (e.g., glycerol), (f) a disintegrant (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate), (g) a dissolution retarder (e.g., glycerol), (h) a disintegrant (e.g., glycerol), (i) a disintegrant (e.g., glycerol), (j ... The pharmaceutical composition is mixed with (i) a lubricant (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof), (ii) a binder (e.g., PEG-10 ...

[0054] Solid compositions of a similar type may also be employed in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as fillers.

[0055] Solid dosage forms (e.g., tablets, dragees, capsules, pills, and granules) can be prepared with coatings and shells (e.g., enteric coatings and others known in the art). They can contain opacifying agents and can be of such composition that they release the active compound(s) in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound can also be in microencapsulated form (if appropriate) with one or more of the above-mentioned excipients.

[0056] The liquid dosage form for oral or intravenous administration of the compounds described herein or their derivatives includes pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.In addition to active compound, liquid dosage form can contain inert diluent (for example, water or other solvent), solubilizer and emulsifier commonly used in this field (for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan), or the mixture of these substances.

[0057] Besides such inert diluents, the composition can also include additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0058] Suspensions may contain, in addition to the active compound, further agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0059] As described above, one or more compounds described herein can be provided with a nebulizer, which is a device that generates very fine liquid particles of substantially uniform size in a gas. A liquid containing one or more compounds described herein can be dispersed as droplets of about 5 mm or smaller in diameter in the form of a mist. The small droplets can be carried by a stream of air or oxygen through the outlet tube of the nebulizer. The resulting mist can penetrate the patient's respiratory tract.

[0060] Additional inhalation agents useful for delivery of the compounds described herein include intra-oral sprays, mists, metered-dose inhalers, and dry powder generators (see Gonda, J. Pharm. Sci. 89:940-945, 2000, which is incorporated herein by reference in its entirety, at least for the inhalation delivery methods taught therein). For example, a powder composition containing one or more compounds as described herein, with or without a lubricant, carrier, or propellant, can be administered to a patient. Delivery of the one or more compounds in powder form can be carried out using conventional devices for administering powder pharmaceutical compositions by inhalation.

[0061] The composition of the compounds described herein or their derivatives for rectal administration is, if necessary, a suppository, which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or a suppository wax (which is solid at ordinary temperatures but liquid at body temperature, and therefore melts in the rectal or vaginal cavity and releases the active ingredient).

[0062] The dosage form for topical administration of the compound described herein or its derivatives includes ointment, powder, spray and inhalant.Under sterile conditions, the compound described herein or its derivatives are mixed with physiologically acceptable carrier and any preservative, buffer or propellant that may be required.Ophthalmic preparations, ointments, powders and solutions are also contemplated as being within the scope of the above-mentioned compositions.

[0063] As noted above, the composition may contain one or more of the compounds described herein or their pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds described herein or their derivatives, where possible, zwitterionic forms of the compounds described herein, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., and effective for their intended use, commensurate with a reasonable benefit / risk ratio. The term "salt" refers to relatively non-toxic inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compound in its free base form with an appropriate organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, methanesulfonate, and laurylsulfonate. These may include cations based on alkali metals and alkaline earth metals (e.g., sodium, lithium, potassium, calcium, magnesium, etc.), as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See S.M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least with respect to the compositions taught herein).

[0064] Administration of the compounds and compositions described herein or their pharmaceutically acceptable salts can be carried out for a period of time effective to treat the disorder, using a therapeutically effective amount of the compounds and compositions described herein or their pharmaceutically acceptable salts as described herein. The effective amount of the compounds and compositions described herein or their pharmaceutically acceptable salts as described herein can be determined by one skilled in the art and is about 0.01 x (BW / 20) 3 / 4 μg~10,000×BW / 20) 3 / 4 Exemplary administration of the active compound to an animal or human includes administration in a dose that delivers the active compound to the subject in an amount between 100 μg / day and 100 μg / day (where BW is the subject's body weight in grams). This amount can be administered in a single dose or in the form of individual divided doses (e.g., 1-4 times per day).

[0065] Those skilled in the art will understand that the specific dose level and dosing frequency for any particular subject may vary and will depend on a variety of factors, including the activity of the specific compound used, the metabolic stability and length of action of that compound, the species, age, weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0066] The exact dosage to be used in the formulation also depends on the route of administration and the severity of disease or disorder, and should be determined by the judgment of the physician and the circumstances of each individual subject.Effective dosage can be extrapolated from the dose-response curve obtained from in vitro or animal model test systems.In addition, depending on the route of administration, those skilled in the art know how to determine the dosage that produces the plasma concentration for the desired response level in cells, tissues and / or organs of the subject.

[0067] IV.How to use Provided herein are methods that include administering to a subject an effective amount of one or more of the compounds or pharmaceutical compositions described herein, or pharmaceutically acceptable salts thereof. The phrase "effective amount," when used to describe the amount of a compound in a method, refers to the amount of compound that achieves the desired pharmacological or other effect, such as the amount that results in enhanced growth or feed conversion.

[0068] A method for promoting growth in animals is provided herein, along with a method for enhancing feed conversion in animals.The method comprises administering a compound or composition as described herein to animals.The administration can enhance the growth of the animal and / or enhance the feed conversion of the animal compared to a control (an animal that is not administered a compound or composition as described herein).

[0069] The compounds and compositions described herein or their pharmaceutically acceptable salts are useful for treating and / or preventing diseases or conditions associated with intestinal inflammation.Therefore, a method for reducing intestinal inflammation in a human is provided herein, the method comprising administering a composition as described herein (e.g., a pharmaceutical composition as described herein) to a human with intestinal inflammation, wherein the administration reduces intestinal inflammation.Optionally, the human has or is at risk of developing inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, or bacterial infection (e.g., Clostridium difficile infection).The method can further comprise selecting a human with a disease or condition associated with intestinal inflammation (e.g., inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, or bacterial infection).

[0070] The methods described herein are useful for treating the diseases and conditions described herein in humans (including, but not limited to, pediatric and geriatric populations) and in animals (e.g., veterinary applications).

[0071] V. Kit Also provided herein is a kit for promoting growth in animals, along with a method for enhancing feed conversion in animals.The kit may include any of the compounds or compositions described herein.For example, the kit may include a compound of Formula I.The kit may further include one or more additional active ingredients (e.g., animal feed and / or animal feed supplements).The kit may include an oral formulation of any of the compounds or compositions described herein.The kit may further include instructions for using the kit (e.g., instructions for treating a subject), a container, a means for administering the compound or composition, and / or a carrier.

[0072] Also provided herein is a kit for treating or preventing a disease or condition associated with intestinal inflammation in a subject.The kit may include any of the compounds or compositions described herein.For example, the kit may include a compound of Formula I.The kit may further include one or more additional drugs (e.g., anti-inflammatory agents).The kit may include an oral formulation of any of the compounds or compositions described herein.The kit may further include instructions for using the kit (e.g., instructions for treating a subject), a container, a means for administering the compound or composition, and / or a carrier.

[0073] As used herein, the terms treatment, treating, or treating refer to a method of reducing one or more symptoms of a disease or condition. Thus, in the methods of the present disclosure, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs of the disease in a subject compared to a control. As used herein, a control refers to an untreated condition. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or symptoms of the disease or condition.

[0074] As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action (e.g., administration of a composition or therapeutic agent) that inhibits or delays the onset or severity of one or more symptoms of the disease or disorder, taken before or at about the same time as a subject begins to exhibit one or more symptoms of the disease or disorder.

[0075] As used herein, references to decrease, reduce, or inhibit include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater change compared to a control level. Such terms can, but do not necessarily, include complete elimination.

[0076] As used throughout, subject refers to an individual. Preferably, the subject is a mammal (e.g., a primate, and more preferably, a human). Non-human primates are also subjects. The term subject includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), poultry (chickens, turkeys, pigeons, geese, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses and medical preparations are contemplated herein. Non-human subjects are also referred to as animals in this disclosure.

[0077] Throughout this application, various publications are referenced, the disclosures of which in their entireties are incorporated herein by reference.

[0078] The following examples serve to further illustrate the present invention, but at the same time do not constitute any limitation thereof. On the contrary, it should be clearly understood that, after reading and understanding the description herein, it may be necessary to resort to various embodiments, modifications and equivalents thereof that may be suggested to those skilled in the art without departing from the spirit of the present invention. [Example]

[0079] Example 1: Synthesis of L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) was synthesized according to the method detailed below and shown in FIG.

[0080] The hexadecyl ester of tert-butyloxycarbonyl (BOC) L-glutamine was prepared in step a by esterification of BOC-glutamine (1) with 1-hexadecanol (2) in the presence of dicyclohexylcarbodiimide in tetrahydrofuran to give hexadecyl BOC-L-glutamine (3). The BOC protecting group was removed in step b by treating intermediate (3) dissolved in methylene chloride with hydrogen chloride gas to give hexadecyl L-glutamine hydrochloride (4). Intermediate (4) was dissolved in N,N-dimethylformamide, and N,N-diisopropylethylamine was added, followed by BOC D-alanine (5) and the coupling agent, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), to give the BOC-protected hexadecyl dipeptide 6 in step c. Intermediate 6 was purified by column chromatography, and the BOC group was removed in step d by treating the product with hydrogen chloride gas to give hexadecyl dipeptide 7. To intermediate 7 dissolved in N,N-dimethylformamide in the presence of N,N-diisopropylethylamine and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hexafluorophosphate benzotriazole tetramethyluronium, HBTU), lithium L-lactate 8 was added, and the final product, L-lactate-D-alanine-L-glutamine hexadecyl ester 9, was obtained in step e. The white L-lactate-D-alanine-L-glutamine palmityl ester had a MW of 513.72 g / mol. 1 It was greater than 98% pure as determined by 1 H-nuclear magnetic resonance analysis.

[0081] Example 2: Effect of LDPP on growth rate and feed conversion in chickens The objective of this study was to evaluate whether dietary supplementation with 0.2 mg / kg dietary L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) would increase growth rate and / or improve feed conversion in broiler chickens, i.e., promote growth by causing broiler chickens to grow faster and / or require less feed for the same amount of weight gain. A secondary objective was to compare the effects of LDPP with those of bacitracin, an industry-standard growth-promoting antibiotic.

[0082] Experimental design Newly hatched female Ross 708 broiler chickens were housed in single flocks in floor pens with used bedding for 21 days. All chickens were fed a standard, crushed, untreated Aviagen 708 starter and grower feed containing 80% recommended crude protein and no anticoccidial supplements during this time. After 3 weeks, the chickens were grouped into 38 replicate floor pens of 25 chickens with new bedding. Chickens were fed a standard, crushed finisher Aviagen 708 finisher feed containing 100% protein and 0.0125% amprolium anticoccidial for 24 days, from day 21 to termination on day 44. Feed and water were available ad libitum throughout the study. Thirteen pens were assigned to each of the untreated controls (diet without supplements) and LDPP treatments (0.2 mg LDPP / kg diet), and 12 pens were assigned to the bacitracin treatment (50 mg bacitracin / kg diet). The experimental unit was the pen rather than the individual chicken. Chicken weights per pen were determined on days 21 and 44. Finisher feed intake was recorded, and residual feed was determined on day 44, at which time the experiment was terminated.

[0083] statistical analysis For constant time analyses of the full 24-day duration of the feeding experiment, the overall (true) feed conversion for each treatment group was determined by dividing the total feed consumed by the total weight gain of all pens in each experimental group.

[0084] For constant weight gain analysis, the time on feed for the bacitracin- and LDPP-treated groups was modeled to match the weight gain of the untreated control group. Based on the close matching of body weight and weight gain for standard female Ross 708 broilers, weight gain per chicken on the final day of the experiment, day 44, was calculated as the pen weight gain from days 21 to 44 / percentage of surviving chickens per pen, 0.04781. The mean weight gain for each treatment group was adjusted to the control mean by repeatedly subtracting the same percentage of the calculated day 44 weight gain for all pens in the treatment until a single fractional day was found that resulted in a weight gain that matched the control group. Similarly, feed consumption per chicken on day 44 was calculated as the pen feed intake from days 21 to 44 / percentage of surviving chickens per pen, 0.05867. The average feed intake per treatment group was then calculated by subtracting the fractional daily feed uptake found earlier from each pen.

[0085] Weight gain and feed consumption data were analyzed by one-way ANOVA with Tukey's Honest True Difference correction for multiple comparisons. Group differences in feed conversion ratio were assessed from pen FCR data by the nonparametric Mann Whitney U test.

[0086] Results and Conclusions LDPP (supplemented at 0.2 mg / kg feed) significantly improved the growth rate of broiler chickens by increasing the weight gain of LDPP-treated chickens by 4.4%, to 1,725 ​​g, compared to 1,653 g for untreated control chickens. This resulted in a highly significant, almost 1-day faster growth for LDPP-treated chickens compared to controls (23.13 days vs. 24 days). The feed conversion ratio of LDPP-treated versus untreated chickens improved from 1.818 to 1.805 at a constant time, with a stronger improvement of 1.7%, to 1.787, at constant weight gain. The growth-promoting effect of LDPP was significantly stronger than that of bacitracin (an industry-standard growth-promoting antibiotic), with a significantly higher feed conversion ratio of 1.827 at constant weight gain. The results are shown in Figure 2.

[0087] Example 3: Effect of LDPP on growth rate and feed conversion in pigs The objective of this experiment was to evaluate whether dietary supplementation with 0.2 mg / kg feed of L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) would increase growth rate and / or improve feed conversion in newly weaned piglets.

[0088] Experimental design Male pigs (castrated (barrow)) were used in this study. The pigs were weaned at approximately 3 months of age and transferred to a nursery where they were randomly assigned to 24 pens in the nursery with 4 pigs per pen. One of two dietary treatments, an untreated control (diet without supplements) or LDPP treatment (0.2 mg LDPP / kg diet), was assigned to each pen, resulting in 12 pens with 4 pigs each used to evaluate the effect of each diet. A premix of the treatment compound was added to the mixed diet at 0.1% and pelleted. The phase 1 diet was fed at 6 pounds per pig from day 0 to approximately day 8 after weaning. On day 8 after weaning, the pigs were switched to a phase 2 diet of 12 pounds per pig, which was terminated on approximately day 18. Once the phase 2 diet was consumed, the pigs were switched to a phase 3 diet and maintained until the end of the study on day 42. No antibiotics were added to any diet. Diets were formulated to meet or exceed all nutritional requirements based on 2012 NRC specifications. Pigs had free access to food and water. Pigs were weighed individually on days 0 and 42 of the study. Feed intake per pen was monitored during the weigh-in period. Individual pig weights were obtained, with the pen being the experimental unit. The study was terminated on day 42, and untreated pigs were retained in the food chain, while treated pigs were euthanized.

[0089] statistical analysis Pen data were converted to individual pig data by dividing by the number of pigs. For the three pens where pigs were euthanized, time-fractional pig counts were used. Weight gain and calculated feed consumption data were analyzed by pairwise T-test. Overall (true) feed conversion for each treatment was determined by dividing the total feed consumed by the total weight gain of all pens for each treatment. Treatment differences in feed conversion were statistically evaluated by the nonparametric Mann Whitney U-test of pen feed conversion data.

[0090] For constant weight gain analysis, the time the LDPP-treated group was fed was modeled to match the weight gain of the untreated control group. Daily body weight and weight gain were modeled by linear interpolation between the weight on day 0 and the weight on day 42. The mean weight gain of the LDPP-treated group was adjusted to the control mean by subtracting its calculated weight gains on days 42 and 41, and then repeatedly subtracting the same percentage of the calculated weight gain on day 40 for all LDPP-treated pens until a single fractional day was found that resulted in a weight gain that matched the control group. From the interpolated daily weight data, daily feed consumption was first calculated as 5% of body weight. The sum of these daily feed intakes was then divided by the actual weighed feed intake of each pen, and the daily feed intake was multiplied by this percentage to arrive at the accurately weighed feed intake per pen. These calculated daily feed intakes were used to calculate the feed intake due to the varying times that the LDPP-treated groups were fed. The mean feed intake of the LDPP treatment group was then calculated by subtracting the feed intake fractions on days 42 and 41, and day 40 found previously for each pen.

[0091] Weight gain and feed consumption data were analyzed by one-way ANOVA and Student's T-test. Differences in feed conversion ratio were assessed from pen FCR data by the non-parametric Mann Whitney U-test.

[0092] Results and Conclusions LDPP (supplemented at 0.2 mg / kg feed) significantly improved piglet growth rate by increasing the weight gain of LDPP-treated pigs by 7.9%, to 23.826 kg, compared to 22.076 kg for untreated piglets. This resulted in a highly significant 2.1-day faster growth for LDPP-treated pigs compared to controls (39.921 days vs. 42 days). Feed conversion ratio for LDPP-treated vs. untreated pigs improved by 3.8%, from 1.479 to 1.423, at constant time, with an even stronger improvement of 5.5%, to 1.396, at constant weight gain. The results are shown in Figure 3.

[0093] All references cited herein are incorporated by reference in their entirety. [ka] [ka] [ka]

[0094] Although one or more illustrative embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the inventive concept as defined by the following claims.

Claims

1. The formula: 【Chemistry 9】 or a pharma- ceutically acceptable acid or salt thereof, wherein: R １ is substituted or unsubstituted alkyl or substituted or unsubstituted aryl; R ２ , R ３ , R ４ , and R ５ are each independently selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; X is O or NR ６ where R ６ is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; An oligopeptide or a pharma- ceutically acceptable acid or salt thereof.

2. 2. The oligopeptide of claim 1, wherein X is O, or a pharma- ceutically acceptable acid or salt thereof.

3. R １ is C １ -C １８ 3. The oligopeptide of claim 1 or 2, which is a linear alkyl or amino acid, or a pharma- ceutically acceptable acid or salt thereof.

4. The oligopeptide has the following structure: 【Chemistry 10】 The oligopeptide according to any one of claims 1 to 3, or a pharma- ceutically acceptable acid or salt thereof, having the formula:

5. The oligopeptide has the following structure: 【Chemistry 11】 where R ７ is substituted or unsubstituted alkyl or substituted or unsubstituted aryl; Y is O or NR ８ where R ８ is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; 4. The oligopeptide according to any one of claims 1 to 3, or a pharma- ceutically acceptable acid or salt thereof.

6. The oligopeptide according to any one of claims 1 to 3, wherein the oligopeptide is L-lactate-D-alanine-L-glutamine-hexadecyl ester, or a pharma- ceutically acceptable acid or salt thereof.

7. A pharmaceutical composition comprising at least one oligopeptide according to any one of claims 1 to 6, or a pharma- ceutically acceptable acid or salt thereof, and a pharma- ceutically acceptable carrier.

8. A composition comprising at least one oligopeptide according to any one of claims 1 to 6 or a pharma- ceutically acceptable acid or salt thereof, and an animal feed.

9. The composition of claim 8, wherein the at least one oligopeptide or a pharma- ceutically acceptable acid or salt thereof is present in the composition in an amount of about 0.01 mg / kg to 5 mg / kg.

10. 10. The composition of claim 8 or 9, further comprising an additive used in animal diets.

11. 11. The composition of claim 10, wherein the additive comprises an enzyme, a probiotic, a prebiotic, an antioxidant, an antimicrobial growth promoter, a colorant, or a combination thereof.

12. 10. A pharmaceutical composition for use in reducing intestinal inflammation in a human, said pharmaceutical composition comprising at least one oligopeptide according to any one of claims 1 to 6 or a pharma- ceutical acceptable acid or salt thereof, and a pharma- ceutical acceptable carrier.

13. The pharmaceutical composition of claim 12, for use as described in claim 12, wherein the disease or condition associated with intestinal inflammation comprises inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, or bacterial infection.

14. A composition comprising the oligopeptide according to any one of claims 1 to 6, or a pharma- ceutically acceptable acid or salt thereof, for use in promoting growth in an animal or enhancing feed conversion in an animal.

15. A composition according to any of claims 8 to 11 for use in promoting growth in an animal or in enhancing feed conversion in an animal.

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