Patent Description:
The field of the present invention relates to lipophilic muramyl dipeptide enantiomer compositions and methods for reducing inflammation, promoting growth and enhancing feed conversion in animals including humans.

Muramyl dipeptide (MDP) is the minimal structure that is preserved in all bacterial peptidoglycans (PGN). It consists of N-acetyl muramic acid (ether of N-acetylglucosamine and D-lactic acid) linked with peptide bonds to L-alanine and D-γ-glutamate or D-isoglutamine (MacDonald <NUM>).

PGN has long been known to promote an inflanunatory response. The MDP subcomponent of PGN was found to be the minimal chemical structure required to elicit inflammation. MDP is also required for the adjuvant activity of Freund's complete adjuvant, an emulsion of a mycobacterial extract (MacDonald <NUM>). As an adjuvant, MDP promotes a strong immune reaction that is used to boost the effectiveness of vaccines when injected together with vaccine antigens.

While stimulating extraintestinal inflammation, MDP has anti-inflammatory effects in the intestinal tract, and protects mice from experimentally induced colitis (Watanabe <NUM>; Watanabe <NUM>).

The intestinal anti-inflammatory properties of MDP provide opportunities for therapeutic applications (Strober <NUM>). However, MDP is hydrophilic and rapidly removed via kidney excretion from circulation, thus requiring high-dosed and repeated administration in order to mediate nonspecific resistance to infection or adjuvant activity (Fogler <NUM>).

These unfavorable pharmacokinetics and serious side effects prompted many chemical modifications of MDP to correct for these shortcomings. Most successful among those were lipid modifications of MDP that increased both potency and half-life of MDP (Parant <NUM>; Matsumoto <NUM>; Fogler <NUM>).

Covalent lipid MDP conjugates thus have demonstrated several advantages including improved oral bioavailability, enhanced tumor targeting and therapeutic potency, reduced toxicity, and enhanced drug loading into delivery carriers such as liposomes (Fidler <NUM>; Irby <NUM>).

Surprisingly, the complete muramyl dipeptide molecule is not required for biological activity with lipophilic conjugated MDP. Even the L-alanine-D-isoglutamine dipeptide MDP moiety without N-acetyl muramic acid retains immunomodulating activity of MDP, when covalently conjugated to lipophilic moieties. For instance, Gobec (<NUM>) successfully replaces N-acetyl muramyl with acyl moieties in acyl-glycine-L-alanine-D-glutamate MDP analogs. Penney (<NUM>) removes the muramyl moiety altogether in octadecyl L-alanine-D-isoglutamine that still retains strong immunomodulating activity.

Furthermore, both Penney (<NUM>) and Gobec (<NUM>) demonstrate that D-isoglutamine can be replaced in the lipophilic desmuramyl dipeptides with D-glutamine or D-glutamate without loss of function.

Enhanced growth in animals is measured either by growth in mass per unit of time or by growth in mass per unit of nutrition; the latter is sometimes referred to as feed conversion. Promotion of growth by either measure is economically useful in the production of animal protein for consumption by humans and other animals because it reduces the amount of time or feed required to obtain equal gains in body mass.

Antibiotics fed at subinhibitory doses have been used for a long time as growth promoters to enhance growth in agricultural production animals. They presumably work by releasing components from intestinal bacteria (postbiotics), including MDP, which suppress inflammation within the intestinal tract. This mechanism most likely has evolved to protect animals from damaging responses to the trillions of gut-dwelling bacteria.

Due to the widespread induction of antibiotic resistance in bacteria by use of antibiotics as growth promoters, replacement of antibiotics as growth promoters is highly desirable. Nalle and Kaltenboeck teach that low-dose oral administration of potent lipophilic MDP analogs improves growth rates and feed conversion in animals (Nalle <NUM>), presumably due to reduction of asymptomatic intestinal inflammation, and thus of the whole-body systemic inflammatory status.

Production of the N-acetyl muramic acid MDP intermediate by multi-step chemical synthesis is difficult, rendering lipophilic MDP analogs too expensive for use as growth promoters in livestock. This makes immunomodulating lipophilic desmuramyl dipeptides prime candidates for non-antibiotic growth promotion in animals.

Sidwell (<NUM>) and Penney (<NUM>) show that octadecyl D-alanine-L-glutamine, a stereochemical mirror image molecule (enantiomer) of the lipophilic octadecyl L-alanine-D-glutamine desmuramyl dipeptide, is an even stronger immunomodulator than octadecyl L-alanine-D-glutamine.

While counterintuitive, Zhou (<NUM>) shows that enantiomeric mirror image D-peptides of natural receptor-binding L-peptides bind their cognate receptor as strongly, or even more strongly, than the natural peptides. Additionally, such D-peptide enantiomers are biologically highly active because they are much more stable than their L-counterparts, being resistant to degradation due to the absence of naturally degrading enzymes.

The binding of enantiomeric peptides to the cognate receptor, as well as the increased stability of such naturally not occurring peptides, explains the strong biological effect of octadecyl D-alanine-L-glutamine desmuramyl dipeptide (BCH-<NUM>). This dipeptide also avoids the complicated synthesis of the anisotropic cyclic carbohydrate moiety of N-acetyl muramic acid and thus is a prime cost effective MDP analog candidate for use as growth promoter in animals.

As a lipophilic desmuramyl MDP enantiomer, octadecyl D-alanine-L-glutamine desmuramyl dipeptide has two shortcomings: i) it lacks the lactic acid moiety of N-acetyl muramic acid that links the N-acetylglucosamine moiety of muramic acid to the dipeptide (Jeanloz <NUM>), thus may forfeit some binding strength to its cognate receptor; ii) it contains the high-melting octadecyl aliphatic lipid that is suboptimal for cell membrane insertion (cellular targeting), intra-membrane transport, and intracellular release (Spector <NUM>; van Meer <NUM>), and thus disfavors the intracellular esterase cleavage of the ester bond (Hatfield <NUM>) between dipeptide and lipid that intracellularly releases the active dipeptide component of octadecyl D-alanine-L-glutamine.

Accordingly, there is a need for compounds that maximize the immunomodulatory activity of lipophilic desmuramyl MDP enantiomers.

Described herein is an oligopeptide analog of MDP (also referred to herein as an oligopeptide or a compound) comprising an L-lactate-D-alanine-L-glutamine moiety. In some cases, the analog does not contain the N-acetylglucosamine moiety of MDP. The oligopeptides described herein can have the following formula:
<CHM>
or a pharmaceutically acceptable acid or salt thereof, wherein R<NUM> is substituted or unsubstituted alkyl or substituted or unsubstituted aryl; R<NUM>, R<NUM>, R<NUM>, and R<NUM> are each independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl; and X is O or NR<NUM>, wherein R<NUM> is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. Optionally, X is O. Optionally, R<NUM> is C<NUM>-C<NUM> linear alkyl or an amino acid. In some cases, the oligopeptide has one of the following structures:
<CHM>.

In some cases, the oligopeptide has one of the following structures:
<CHM>
wherein R<NUM> is substituted or unsubstituted alkyl or substituted or unsubstituted aryl; and Y is O or NR<NUM>, wherein R<NUM> is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. Optionally, the oligopeptide is L-lactate-D-alanine-L-glutamine-hexadecyl ester.

Also described herein are compositions comprising a compound as described herein. Optionally, the composition is a pharmaceutical composition comprising at least one oligopeptide described herein and a pharmaceutically acceptable carrier. In some cases, the composition comprises at least one oligopeptide as described herein and animal feed. The at least one oligopeptide can be present in the composition in an amount of from about <NUM>/kg to <NUM>/kg. Optionally, the composition further comprises an additive used in an animal diet (e.g., an enzyme, a probiotic. a prebiotic, an antioxidant, an antibiotic growth promoter, a coloring agent, or a combination thereof).

Further described herein are methods for reducing intestinal inflammation in a human, comprising administering a pharmaceutical composition as described herein to a human having intestinal inflammation, wherein the administration reduces the intestinal inflammation. The methods can further comprise selecting a human having a disease or condition associated with intestinal inflammation (e.g., inflaminatory bowel disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, or a bacterial infection). Methods for promoting growth in animal are also provided herein, wherein the methods comprise administering the compounds or compositions as described herein, wherein the administration enhances the growth of the animal. Also provided herein are methods for enhancing feed conversion in an animal, wherein the methods comprise administering the compounds or compositions as described herein, wherein the administration enhances the feed conversion in the animal.

The details of one or more embodiments are forth in the drawings and the description below.

Provided herein are compositions containing a lipophilic enantiomer of desacetylglucosamine muramyl dipeptide (MDP) (e.g., L-lactate-D-alanine-L-glutamine-hexadecyl ester). This compound improves i) binding strength to the cognate intracellular receptor(s) by enlarging the dipeptide via the additional mirror <NUM>-lactate moiety; and ii) maximizes intracellular targeting and active dipeptide release via the increased membrane fluidity provided by the lower-melting hexadecyl (palmityl) aliphatic lipid.

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

Described herein are oligopeptide analogs of desacetylglucosamine muramyl dipeptide (MDP). The analogs can include an L-lactate-D-alanine-L-glutamine moiety bonded to an organic lipid molecule, and any pharmaceutically acceptable acid or salt thereof. In some cases, the analog does not contain the N-acetylglucosamine moiety of MDP.

In some cases, the compounds described herein includes Formula I:
<CHM>
, wherein:.

In Formula I, R<NUM> is substituted or unsubstituted alkyl or substituted or unsubstituted aryl. Optionally, R<NUM> is a C<NUM>-C<NUM> linear alkyl. Optionally, R<NUM> is an amino acid, such as a lysine group (D-lysine or L-lysine).

Also in Formula I, R<NUM>, R<NUM>, R<NUM>, and R<NUM> are each independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.

Additionally in Formula I, X is O or NR<NUM>, wherein R<NUM> is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

Optionally, the compounds of Formula I can include compounds according to Structure I-A:
<CHM>.

In Structure I-A, R<NUM> is defined as above for Formula I.

Optionally, the compounds of Formula I can include compounds according to Structure I-B:
<CHM>.

In Structure I-B, R<NUM> is defined as above for Formula I.

Optionally, the compounds of Formula I can include compounds according to Structure I-C or Structure I-D:
<CHM>.

In Structure I-C and Structure I-D, R<NUM> is substituted or unsubstituted alkyl or substituted or unsubstituted aryl. Also in Structure I-C and Structure I-D, Y is O or NR<NUM>, wherein R<NUM> is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

The alkyl can be a straight-chain alkyl or a branched-chain alkyl. In some cases, the straight-chain alkyl can be a C<NUM>-C<NUM> alkyl (e.g., a C<NUM>-C<NUM> alkyl or a C<NUM> - C<NUM> alkyl). Examples of suitable alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl. heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl. pentadecyl, hexadecyl. heptadecyl, or octadecyl. In some cases, the oligopeptide is 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 as hexadecyl (<NUM>)-<NUM>-amino-<NUM>-[[(2R)-<NUM>-[[(<NUM>)-<NUM>-hydroxypropanoyl]amino]propanoyl]amino]-<NUM>-oxo-pentanoate.

Optionally, the aryl group includes a phenyl group. Optionally, the aryl group can include additional fused rings, for example, naphthalene, anthracene, and pyrene. The aryl and heteroaryl groups can be attached at any position on the ring, unless otherwise noted.

The alkyl and aryl groups used herein can be substituted or unsubstituted. As used herein, the term substituted includes the addition of a functional group to a position attached to the main chain of the alkyl or aryl group, e.g., the replacement of a hydrogen by one of these molecules. Examples of substitution groups 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 the alkyl or aryl group has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear hexadecyl (-(CH<NUM>)<NUM>-CH<NUM>).

The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

Variations on Formula I and the compounds described herein include the addition. subtraction, or movement of the various constituents as described for each compound. when one or more chiral centers are present in a molecule, all possible chiral variants are included. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in <NPL>.

Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of ordinary skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., <NUM>H-NMR or <NUM>C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

Exemplary methods for synthesizing compounds as described herein are provided in Example <NUM> below, depicting the synthesis of LDPP by way of example.

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

In some cases, the composition includes a compound of Formula I as described herein, such as, for example, LDPP, and animal feed. Any suitable animal feed can be used, including animal feed that includes one or more of maize, 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 from about <NUM>/kg to <NUM>/kg (e.g., <NUM>/kg to <NUM>/kg, <NUM>/kg to <NUM>/kg, <NUM>/kg to <NUM>/kg, or <NUM>/kg to <NUM>/kg). In some examples, the compound of Formula I, such as LDPP, can be included in a composition including animal feed in an amount of <NUM>/kg, <NUM>/kg. <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, <NUM>/kg, or <NUM>/kg. The animal feed composition can further include additives used in animal diets, including enzymes, probiotics, prebiotics, antioxidants, antibiotic growth promoters, and coloring agents.

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, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Optionally, the compositions described herein can administered orally, topically, intranasally, intravenously, subcutaneously. intradermally, transdermally, intramucosally, intramuscularly, by inhalation spray, rectally, nasally, sublingually, buccally, vaginally or via an implanted reservoir.

The compounds described herein or derivatives thereof can be provided in a pharmaceutical composition. In some cases, the compositions are pharmaceutical compositions that include a compound of Formula I and a pharmaceutically acceptable carrier. Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

The compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., <NPL>). Examples of physiologically acceptable carriers include buffers, such as phosphate buffers. citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about <NUM> residues) polypeptides; proteins, such as serum albumin. gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone: amino acids such as glycine. glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc. ; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF: Florham Park, NJ).

Compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

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

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 is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose. alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate. potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

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

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds 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 compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral or intravenous administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol. ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, <NUM>,<NUM>-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol. polyethyleneglycols. and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

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

Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide. bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

As described above, the one or more compounds described herein can be provided with a nebulizer, which is an instrument that generates very fine liquid particles of substantially uniform size in a gas. The liquid containing the one or more compounds described herein can be dispersed as droplets about <NUM> or less in diameter in the form of a mist. The small droplets can be carried by a current of air or oxygen through an outlet tube of the nebulizer. The resulting mist can penetrate into the respiratory tract of the patient.

Additional inhalants useful for delivery of the compounds described herein include intra-oral sprays, mists, metered dose inhalers, and dry powder generators (See <NPL>). For example, a powder composition containing the one or more compounds as described herein, with or without a lubricant, carrier, or propellant, can be administered to a patient. The delivery of the one or more compounds in powder form can be carried out with a conventional device for administering a powder pharmaceutical composition by inhalation.

Compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.

Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, and inhalants. The compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.

As noted above, the compositions can include one or more of the compounds described herein or pharmaceutically acceptable salts thereof. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the 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 a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the 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, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammoniun, quaternary ammonium, and amine cations including, but not limited to anunonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See <NPL>).

Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder. The effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary administrations for an animal or human at a dose that delivers the active compound to the subject in an amount between about <NUM> x (BW/<NUM>)<NUM>/<NUM> µg and <NUM>,<NUM> x BW/<NUM>)<NUM>/<NUM> µg per day, wherein BW is the body weight of the subject in grams. This amount may be administered in a single dose or in the form of individual divided doses, such as from <NUM> to <NUM> times per day.

Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound employed. the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject. the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells, tissues and/or organs of a subject.

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 a pharmaceutically acceptable salt thereof. The expression "effective amount," when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example, an amount that results in enhanced growth or feed conversion.

Methods for promoting growth in an animal are provided herein, along with methods for enhancing feed conversion in an animal. The methods comprise administering a compound or composition as described herein to the animal. The administration can enhance the growth of the animal and/or enhance the feed conversion of the animal as compared to a control (an animal not administered a compound or composition as described herein).

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

The methods described herein are useful for treating the diseases and conditions described herein in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary application.

Also provided herein are kits for promoting growth in an animal are provided herein, along with methods for enhancing feed conversion in an animal. A kit can include any of the compounds or compositions described herein. For example, a kit can include a compound of Formula I. A kit can further include one or more additional agents, such as animal feed and/or animal feed supplements. A kit can include an oral formulation of any of the compounds or compositions described herein. A kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions, and/or a carrier.

Also provided herein are kits for treating or preventing a disease or condition associated with intestinal inflammation in a subject. A kit can include any of the compounds or compositions described herein. For example, a kit can include a compound of Formula I. A kit can further include one or more additional agents, such as anti-inflammatory agents. A kit can include an oral formulation of any of the compounds or compositions described herein. A kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions, and/or a carrier.

As used herein the terms treatment. treat, or treating refer to a method of reducing one or more symptoms of a disease or condition. Thus in the disclosed method, treatment can refer to a <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% 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 <NUM>% reduction in one or more symptoms or signs of the disease in a subject as compared to a control. As used herein, control refers to the untreated condition. Thus the reduction can be a <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or any percent reduction in between <NUM>% and <NUM>% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.

As used herein, references to decreasing, reducing, or inhibiting include a change of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.), farmed birds (chickens, turkeys, pigeons, geese, etc.), and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein. Non-human subjects are also referred to as animals in this disclosure.

The following examples will serve to farther illustrate the present invention without, at the same time, however, constituting any limitation thereof.

L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) was synthesized according to the method detailed below, and depicted in <FIG>.

The hexadecyl ester of tert-butyloxycarbonyl (BOC) L-glutamine was prepared in step a by esterification reaction of BOC-glutamine (<NUM>) with <NUM>-hexadecanol (<NUM>) in tetrahydrofuran, in the presence of dicyclohexyl carbodiimide. to yield hexadecyl BOC-L-glutamine (<NUM>). The BOC protecting group was removed in step b by treatment of intermediate (<NUM>) dissolved in methylene chloride with hydrogen chloride gas to yield the hydrochloride salt of hexadecyl L-glutamine (<NUM>). Intermediate (<NUM>) was dissolved in N,N-dimethylformamide, N,N-diisopropylethylamine was added followed by BOC D-alanine (<NUM>) and the coupling agent benzotriazole-<NUM>-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), to yield in step c the BOC-protected hexadecyl dipeptide <NUM>. Intermediate <NUM> was purified by column chromatography and the BOC group was removed in step d by treatment of the product with hydrogen chloride gas to yield hexadecyl dipeptide <NUM>. To intennediate (<NUM>) dissolved in N,N-dimethylformamide in the presence of N. N-diisopropylethylamine and <NUM>-(<NUM>H-benzotriazol-<NUM>-yl)-<NUM>,<NUM>,<NUM>,<NUM>-tetramethyluronium hexafluorophosphate (Hexafluorophosphate Benzotriazole Tetramethyl Uronium, HBTU), lithium L-lactate (<NUM>) was added, and step e yielded the final product L-lactate-D-alanine-L-glutamine hexadecyl ester (<NUM>). The white L-lactate-D-alanine-L-glutamine palmityl ester with a MW of <NUM>/mol was greater than <NUM>% pure, as determined by <NUM>H-Nuclear Magnetic Resonance analysis.

The objective of this experiment was to evaluate if supplementation of feed with L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) at <NUM>/kg feed increases the growth rate and/or improves the feed conversion in broiler chickens, i.e., if it promotes growth by making broiler chickens grow faster and/or require less feed for the same amount of gain in body weight. A secondary objective was to compare the effect of LDPP to that of bacitracin, an industry-standard growth promoting antibiotic.

Freshly hatched female Ross <NUM> broiler chickens were housed for as single flock for <NUM> days on a floor pen with used bedding. All chickens received crumbled untreated standard Aviagen <NUM> starter and grower feeds during this time, with <NUM>% recommended crude protein and without anti-coccidial supplement. After <NUM> weeks, the chickens were grouped into <NUM> replicate floor pens of <NUM> chickens with fresh bedding. Chickens were fed for <NUM> days from day <NUM> through termination on day <NUM> standard crumbled finisher Aviagen <NUM> finisher feed with <NUM>% protein and <NUM>% amprolium anti-coccidial inclusion. Feed and water were available ad libitum throughout the trial. Thirteen pens each were assigned to untreated controls (feed without supplement) and LDPP-treatment (<NUM> LDPP/kg feed), and <NUM> pens were assigned to bacitracin treatment (<NUM> bacitracin/kg feed). The experimental unit was the pen rather than individual chickens. Pen weights of chickens were determined on day <NUM> and <NUM>. Finisher feed uptake was be recorded. and on day <NUM> residual feed was detennined, and the experiment was terminated.

For constant time analyses of the complete <NUM>-day duration of the feeding experiment, the overall (true) feed conversion for each treatment group was determined by dividing total consumed feed by total weight gains of all pens of each treatment group.

For constant body weight gain analyses, the time on feed of the bacitracin and LDPP treated groups was modeled to match the weight gain of the untreated control group. Based on the closely matching body weights and weight gains of standard female Ross <NUM> broilers, body weight gains per chicken on the last day <NUM> of the experiment were calculated as the <NUM> fraction of pen body weight gain day <NUM>-<NUM>/surviving chickens per pen. The mean body weight gain of each treatment group was adjusted to the control mean by iteratively subtracting the same fractions of the calculated day <NUM> body weight gain of all pens of a treatment until a single fractional day was found that produced a weight gain matching with the control group. Similarly, feed consumption per chicken on day <NUM> was calculated as the <NUM> fraction of pen feed uptake day <NUM>-<NUM>/surviving chickens per pen. Mean feed uptake per treatment group was then calculated by subtracting the previously found fractional daily feed uptake from each pen.

Body weight gain and feed consumption data were analyzed by one-way ANOVA and Tukey Honest True Difference correction for multiple comparisons. Group differences in feed conversion rates were evaluated from pen FCR data by non-parametric Mann Whitney U test.

LDPP, supplemented at <NUM>/kg feed, significantly improves the growth rate of broiler chickens by increasing the weight gain of LDPP-treated chickens by <NUM>% to <NUM>,<NUM> as compared to the <NUM>,<NUM> weight gain of untreated control chickens. This resulted in highly significant, nearly a day more rapid growth of the LDPP-treated chickens as compared to the controls (<NUM> vs <NUM> days). The feed conversion rate of LDPP-treated versus untreated chickens is improved at constant time from <NUM> to <NUM>, and more strongly by <NUM>% to <NUM> at constant body weight gain. The growth promoting effect of LDPP is significantly stronger than that of bacitracin, an industry-standard growth promoting antibiotic, which showed a significantly higher feed conversion rate of <NUM> at constant body weight gain. The results are shown in <FIG>.

The objective of this experiment was to evaluate if supplementation of feed with L-lactate-D-alanine-L-glutamine palmityl ester (LDPP) at <NUM>/kg feed increases the growth rate and/or improves the feed conversion in freshly weaned nursery pigs.

Male pigs (barrows) were used in this study. Pigs were weaned at approximately <NUM> weeks of age, transferred to the nursery, and randomly allotted to <NUM> nursery pens with <NUM> pigs per pen. One of <NUM> dietary treatments, untreated controls (feed without supplement) or LDPP-treatment (<NUM> LDPP/kg feed), was assigned to each pen, such that <NUM> pens of <NUM> pigs were used to evaluate the effect of each diet. Premixes of the treatment compounds were added at <NUM>% to mixed diets, which were then pelleted. Phase <NUM> diet was fed at <NUM> lb/pig from day <NUM> to approximately day <NUM> post-weaning. On day <NUM> post-weaning, pigs were switched to <NUM> lb/pig Phase <NUM> diet which expired approximately on day <NUM>. Once the Phase <NUM> diet had been consumed, pigs were switched to Phase <NUM> diet and maintained until termination of the study on day <NUM>. No antibiotics were added to any diet. Diets were formulated to meet or exceed all the nutrient requirements based on the <NUM> NRC specifications. Pigs received diets and water ad libitum. Pigs were weighed individually on days <NUM> and <NUM> of the experiment. Feed intake per pen was monitored for the weigh period. Although individual pig weights were obtained, the pen was the experimental unit. On day <NUM>, the study was terminated and untreated pigs were retained in the food chain while the treatment pigs were euthanized.

Pen data were converted into individual pig data by dividing by the number of pigs. For three pens in which pigs were euthanized, a time-fractional pig number was used. Body weight gain and calculated feed consumption data were analyzed by pairwise T-test. The overall (true) feed conversion for each treatment was determined by dividing total consumed feed by total weight gains of all pens of each treatment. Treatment differences in feed conversion were statistically evaluated by non-parametric Mann Whitney U test of pen feed conversion data.

For constant body weight gain analyses, the time on feed of the LDPP treated group was modeled to match the weight gain of the untreated control group. Daily body weights and weight gains were modeled by linear interpolation between weights on days <NUM> and <NUM>. The mean body weight gain of the LDPP treatment group was adjusted to the control mean by subtracting the calculated day <NUM> and <NUM> body weight gains, and then iteratively subtracting the same fractions of the calculated day <NUM> body weight gain of all pens of the LDPP treatment until a single fractional day was found that produced a weight gain matching with the control group. From interpolated daily body weight data, daily feed consumption was first calculated as <NUM>% of body weight. The sum of these daily feed uptakes was then divided by the actual weighed feed uptake of each pen. and daily feed uptakes were multiplied by this fraction to arrive at the precise weighed feed uptake per pen. These calculated daily feed uptakes were used to calculate feed uptake by changed times on feed for the LDPP treatment group. Mean feed uptake of the LDPP treatment group was then calculated by subtracting for each pen the previously found day <NUM> and <NUM> and the fractional day <NUM> feed uptake.

Body weight gain and feed consumption data were analyzed by one-way ANOVA and Student's T-test. Differences in feed conversion rates were evaluated from pen FCR data by non-parametric Mann Whitney U test.

Claim 1:
An oligopeptide of the following formula:
<CHM>
or a pharmaceutically acceptable acid or salt thereof, wherein:
R<NUM> is substituted or unsubstituted alkyl or substituted or unsubstituted aryl;
R<NUM>, R<NUM>, R<NUM>, and R<NUM> are each independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl; and
X is O or NR<NUM>, wherein R<NUM> is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.