Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus

Definitions

• Metabolic disorders generally refer to a broad array of disorders characterized by defects that interfere with the body's metabolism, the chemical processes by which a body transforms proteins, carbohydrates and fats into energy. Metabolic disorders may include disorders resulting from altered glucose metabolism. Examples of metabolic disorders include obesity, metabolic syndrome, impaired glucose tolerance, and dyslipidemias.
• a metabolic disorder can also result from a diseased or dysfunctional organ. Diabetes is an example of a metabolic disorder resulting from a diseased and/or dysfunctional organ, the pancreas.
• Obesity which is defined in general terms as an excess of body fat relative to lean body mass, is a serious contributor to increased morbidity and mortality. Obesity, which is most commonly caused by excessive food intake coupled with limited energy expenditure and/or lack of physical exercise, often accompanies various glucose metabolism disorders. Obesity increases the likelihood of an individual developing various diseases, such as diabetes mellitus, hypertension, atherosclerosis, coronary artery disease, gout, rheumatism and arthritis.
• Obesity is often associated with psychological and medical morbidities, the latter of which includes increased joint problems, vascular diseases such as coronary artery disease, hypertension, stroke, and peripheral vascular disease. Obesity also causes metabolic abnormalities such as insulin resistance and Type II diabetes (non-insulin-dependent diabetes mellitus (NIDDM)), hyperlipidemia, and endothelial dysfunction.
• NIDDM non-insulin-dependent diabetes mellitus
• SUMMARY Methods of treating a metabolic disorder in a subject are provided. Aspects of the method include administering an effective amount of an N-lactoyl-amino acid to the subject. Also provided are pharmaceutical formulations including an amount of an N-lactoyl-amino acid effective to treat a metabolic disorder.
• FIG.1 A-D shows Lac-Phe is robustly induced in blood plasma after a single bout of mouse treadmill running.
• A Schematic of the speed and incline protocol for treadmill running in mice.
• B T-stat values of all blood plasma peaks detected by untargeted metabolomics in post- run versus sedentary mice.
• FIG.2 A-I shows Lac-Phe suppresses food intake and obesity and improves glucose homeostasis.
• A-B Cumulative food intake (A) and ambulatory activity (B) of 22-week male DIO mice following injection of either vehicle (blue) or Lac-Phe (red, 50 mg/kg, intraperitoneal [IP]).
• C-D Cumulative food intake (C) and change in body weight (D) of 22-week male DIO mice treated daily with vehicle (blue) or Lac-Phe (red, 50 mg/kg/day, intraperitoneal [IP]).
• E Glucose tolerance test (1 g/kg glucose) of vehicle- or Lac-Phe treated mice.
• GTT was performed one day after the last Lac-Phe dose on day 10 following a 6 h fast.
• F-G Tissue weights (F) and representative images of adipose tissues (G) from mice after 10 days of vehicle or Lac-Phe treatment.
• H Average daily food intake (left) and change in body weight (right) of 15-week male DIO mice after 5-day treatment with vehicle (black), Lac-Phe (red, 50 mg/kg/day, IP) or vehicle-treated pair-fed mice (blue).
• FIG.3 A-H shows CNDP2- and lactate-dependent biosynthesis and secretion of Lac- Phe from macrophages in vitro.
• A Lac-Phe levels in conditioned media and cell lysate of RAW264.7 cells.
• B Anti-CNDP2 or anti-beta tubulin Western blotting of cell lysates from WT or CNDP2-KO RAW264.7 cells.
• C Lac-Phe levels in conditioned media and cell lysate of WT and CNDP2-KO RAW264.7 cells.
• D Anti-CNDP2 or anti-beta tubulin Western blotting of cell lysates from primary peritoneal macrophages isolated from WT or CNDP2-KO mice.
• E Lac- Phe levels in conditioned media and lysate of WT and CNDP2 KO primary peritoneal macrophages.
• C-F Cumulative daily food intake (C), body weight (D), tissue weights (E), and representative images of adipose tissues (F) of WT (blue) and CNDP2-KO (red) mice under an obesigenic diet/exercise training regimen in which mice were fed high fat diet (60% kcal from fat) and exercised by treadmill running 5 days/week (see Methods). Tissue weights and images were taken on day 41.
• N 6/group.
• FIG.5 A-J shows Lac-Phe acts directly on AgRP+ and POMC+ neurons in culture.
• A Representative action potential firing traces of AgRP+ neurons after treatment with vehicle (left) or Lac-Phe (50 ⁇ M, right).
• B,C Effects of the indicated concentration of Lac-Phe on action potential firing frequency (B) and hyperpolarization (C) of AgRP+ neurons.
• (D) Representative electrophysiology recording of AgRP+ neurons after treatment with Lac-Phe (50 ⁇ M) in the presence of an inhibitor cocktail (tetrodotoxin: 1 ⁇ M, bicuculline: 50 ⁇ M, DNQX: 20 ⁇ M, and D- AP5: 50 ⁇ M).
• E Response ratio of AgRP+ neurons after treatment with various concentrations of Lac-Phe alone or in the presence of an inhibitor cocktail
• G,H Effects of the indicated concentrations of Lac-Phe on action potential firing frequency (G) and hyperpolarization (H) of POMC+ neurons.
• FIG.6 A-F shows robust and sustained elevation of Lac-Phe following human exercise.
• B False discovery rate of exercise-regulated metabolites (dark blue), lipids (grey), or proteins (light blue) from blood plasma in response to acute treadmill running.
• Lac-Phe A previously unassigned metabolite with a chemical formula matching that of Lac-Phe is shown in red.
• C Time course of Lac-Phe and lactate in blood in humans following exercised or kept sedentary.
• E Time course of Lac-Phe levels pre and post- exercise following the sprint (red), resistance (blue), and endurance (light blue) trial.
• F Correlation of plasma Lac-Phe and lactate levels immediately pre- and post-exercise across the three exercise modalities. Data are shown as mean ⁇ SEM, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
• FIG.8 A-C shows additional metabolic parameters of obese mice acutely treated with Lac-Phe.
• FIG.9 A-C shows characterization of Lac-Phe production in vitro.
• A Lac-Phe levels in conditioned media from a panel of cell lines after overnight incubation.
• B Percent abundance of the indicated metabolite in cell lysates or conditioned media of RAW264.7 cells after overnight incubation.
• C Diagram of the CNDP2-catalyzed Lac-Phe biosynthesis reaction.
• FIG.10 A-D shows the effect of Lac-Phe on serotonin and SF1 neurons in brain slices.
• A,B Representative action potential firing trace of Lac-Phe (50 ⁇ M) treatment of 5-HT+ neurons (A) and the response ratio (B).
• C,D Representative action potential firing trace of Lac- Phe (50 ⁇ M) treatment of SF1+ neurons (C) and the response ratio (D).
• N 14-15 neurons/group.
• FIG.11 A-D shows additional characterization of plasma Lac-Phe levels in humans.
• FIG.12 A-E shows additional characterization of the mouse exercise plasma metabolome.
• A Fold change of metabolites detected in our untargeted metabolomics analysis that are known to be increased with physical activity. Basal and exercised conditions are indicated in blue and red respectively.
• C Absolution quantification of Lac-Phe in mouse plasma under sedentary (blue) and exercised (red) conditions.
• D Total ion count of the 20 N-lactoyl amino acid conjugates in the plasma of mice under basal (blue) and exercised (red) conditions.
• FIG.13 A-J shows Lac-Phe acts directly on AgRP+ and POMC+ neurons in culture and contains similar information to FIG.5 (above).
• A Representative action potential firing traces of AgRP+ neurons after treatment with vehicle (left) or Lac-Phe (50 ⁇ M, middle) or Lac-Phe (50 ⁇ M) in the presence of an inhibitor cocktail (tetrodotoxin: 1 ⁇ M, bicuculline: 50 ⁇ M, DNQX: 20 ⁇ M, and D-AP5: 50 ⁇ M, right).
• C Quantification of the firing frequency of AgRP+ neurons before and after Lac-Phe treatment.
• FIG.14 A-E shows human exercise induces sustained elevations of Lac-Phe and negative correlations with BMI and fat mass.
• B False discovery rate of exercise-regulated metabolites (dark blue), lipids (grey), or proteins (light blue) from blood plasma in response to acute treadmill running. A previously unassigned metabolite with a chemical formula matching that of Lac-Phe is shown in red.
• (C) Time course of Lac-Phe (red), lactate (blue), and phenylalanine (light blue) in the plasma of subjects pre and post-exercise.
• (D) Associations of circulating Lac-Phe levels 30 minutes post-exercise with demographics and physiological markers.
• (E) Associations of circulating Lac-Phe levels 30 minutes post-exercise with BMI (left) and estimated fat mass (right).
• FIG.15 shows Lac-Phe is also present in humans and changes in circulating levels with exercise and food intake.
• FIG.16 shows peak exercised-induced Lac-Phe is negatively associated with body mass index and fat mass. Associations of circulating Lac-Phe levels 30 minutes post-exercise with BMI (left), estimated fat mass (middle), and percent fat mass (right).
• FIG.17 shows administration of Lac-Phe to diet-induced obese mice suppresses appetite and body weight and improves glucose homeostasis.
• FIG.19 A-C shows Lac-Phe is a metabolite secreted from macrophages in a lactate and CNDP2-dependent manner.
• A Lac-Phe levels in conditioned media from a panel of cell lines after overnight incubation.
• B Lac-Phe levels in conditioned media and cell lysate of RAW264.7 cells.
• C- F Body weight, (C) cumulative food intake (D), images (E), and tissue weights (F) of WT (blue) and CNDP2-KO (red) mice under an obesigenic diet/exercise training regimen in which mice were fed high fat diet (60% kcal from fat) and exercised by treadmill running 5 days/week (see Methods).
• N 8-9/group. Data are shown as mean ⁇ SEM, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
• FIG.21 shows CNDP2-KO mice have abolished Lac-Phe levels in blood.
• aspects of the method include administering an effective amount of an N-lactoyl-amino acid to the subject.
• pharmaceutical formulations including an amount of an N-lactoyl-amino acid effective to treat a metabolic disorder.
• METHODS methods of treating a metabolic disorder in a subject are provided.
• the methods may include administering an effective amount of an N-lactoyl-amino acid to the subject.
• the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., obesity.
• treating includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder.
• Treatment is generally “effective” if one or more symptoms or clinical markers are reduced.
• treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
• Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
• treatment also includes providing relief from the symptoms or side-effects of the disease.
• a metabolic disorder may be “treated” if at least one symptom of the metabolic disorder is expected to be or is alleviated, terminated, slowed, or prevented.
• a metabolic disorder may be also “treated” if recurrence or progression of the metabolic disorder is reduced, slowed, delayed, or prevented. Any suitable N-lactoyl-amino acid or combination of N-lactoyl-amino acids may be administered in the subject methods.
• N-lactoyl-amino acids or “lac-amino acids” refer to compounds, e.g., metabolites, that are formed from lactate and amino acids, e.g., by the action of protease cytosolic nonspecific dipeptidase 2 (CNDP2).
• the N-lactoyl-amino acid may be one that is produced by the body or one that is synthetically produced.
• the amino acid in the N-lactoyl-amino acid(s) that are administered may be any suitable amino acid including, e.g., phenylalanine, isoleucine, valine, glycine, methionine, glutamic acid, tryptophan, alanine, asparagine, glutamine, histidine, serine, proline, threonine, cysteine, lysine, arginine, tyrosine, aspartic acid, leucine, etc.
• the N-lactoyl- amino acid(s) administered in the subject methods are those that are present (e.g., detectable) in the body during or after physical activity.
• the N-lactoyl-amino acid(s) administered in the subject methods are those where the levels of the N-lactoyl-amino acid(s) are elevated or decreased in the body, e.g., blood plasma, during or after physical activity.
• the subject methods include administering an analog of any of the N-lactoyl- amino acids described herein.
• the N-lactoyl-amino acid is N-lactoyl- phenylalanine (e.g., Lac-Phe).
• the N-lactoyl-amino acid is N-lactoyl-leucine (e.g., Lac-Leu).
• the N-lactoyl-amino acid is N-lactoyl-isoleucine (e.g., Lac- Ile). In some instances, the N-lactoyl-amino acid is N-lactoyl-valine (e.g., Lac-Val). In some instances, a plurality of N-lactoyl-amino acids are administered including, e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more N-lactoyl-amino acids.
• the N-lactoyl-amino acid is not N-lactoyl-methionine (e.g., Lac-Met). Any suitable amount of the N-lactoyl-amino acid may be administered. In some instances, an amount effective to treat a metabolic disease or associated condition, e.g., an effective amount, is administered. In some instances, an amount of each N-lactoyl-amino acid in a plurality of N-lactoyl-amino acids effective to treat a metabolic disease or associated condition is administered. For example, where a plurality of N-lactoyl-amino acids are administered, the effective amount for each of the N-lactoyl-amino acids may be administered.
• the effective amount includes an amount of the N-lactoyl-amino acid that when administered produces a plasma concentration of the N-lactoyl-amino acid comparable (e.g., equivalent) to that observed in the subject during or after physical activity.
• the effective amount may be equivalent to the amount of the N-lactoyl-amino acid present in the body, e.g., blood plasma, of the subject during or after physical activity.
• the effective amount includes an amount of the N-lactoyl-amino acid that when administered produces a plasma concentration of the N-lactoyl-amino acid comparable (e.g., equivalent) to that observed in the subject during or after a recovery period after physical activity (e.g., a period of time after physical activity that ranges from 1 minute to 5 hours including, e.g., from 1 minute to 4 hours, from 1 minute to 3 hours, from 1 minute to 2 hours, from 1 minute to 1 hour, from 1 minute to 30 minutes, or from 1 minute to 15 minutes).
• the effective amount may be equivalent to the amount present in the body, e.g., plasma, during or after a recovery period after physical activity.
• Physical activity may include any amount of physical activity over a period of time. In some instances, physical activity includes an amount of physical activity performed over a period of time ranging from 1 minute to 2 hours including, e.g., from 1 minute to 1 hour, or from 1 minute to 30 minutes. Physical activity may include, e.g., any activity that raises the heart rate above a resting heart rate, physical movement, exercise, any activity performed to maintain or achieve physical fitness, etc.
• the effective amount includes a single unit dose of the N-lactoyl-amino acid. In some instances, the effective amount includes one or more unit doses of the N-lactoyl-amino acid including, e.g., two or more doses, three or more doses, four or more doses, etc.
• a single dose is administered. In some instances, multiple doses, e.g., two or more, three or more, etc., are administered.
• the effective amount may range from 1 mg/kg to 500 mg/kg including, e.g., from 1 mg/kg to 400 mg/kg, from 1 mg/kg to 300 mg/kg, from 1 mg/kg to 200 mg/kg, from 1 mg/kg to 100 mg/kg.
• the methods include administering an amount of the N-lactoyl-amino acid(s) effective to induce a physical activity associated outcome in a subject (e.g., effective to cause the subject to experience a physical activity associated outcome).
• physical activity associated outcome is meant an outcome, change, or effect (e.g., biological, physical, and/or chemical) equivalent to that induced in the subject by physical activity (e.g., equivalent in magnitude and longevity).
• Physical activity associated outcomes of interest include, but are not limited to, weight loss, prevention or treatment of metabolic disorders and associated conditions, improved glucose homeostasis, improved thinking or cognition, mood improvement, reduction in the severity of mood disorders (e.g., anxiety and depression), prevention of or slowing the progress of neurodegenerative diseases (e.g., dementia), improvement in sleep, lower risk of cancer, among others.
• the methods may include administering an amount of the N-lactoyl-amino acid effective to induce a reduction in body weight in the subject equivalent to what would be induced by physical activity.
• the methods may include administering an amount of the N-lactoyl-amino acid effective to induce an improvement in glucose homeostasis in the subject equivalent to what would be induced by physical activity.
• the methods may include administering an amount of the N-lactoyl-amino acid effective to induce a mood improvement in the subject equivalent to what would be induced by physical activity.
• the methods may include administering an amount of the N-lactoyl-amino acid effective to induce a reduction in the severity of a mood disorder in the subject equivalent to what would be induced by physical activity. In yet another example, the methods may include administering an amount of the N-lactoyl-amino acid effective to induce a reduction in the severity of a neurodegenerative disorder (e.g., prevention of the development of the disorder or slowing of the progression of the disorder) in the subject equivalent to what would be induced by physical activity. In some embodiments, the methods include administering an amount of the N-lactoyl-amino acid effective to treat a nervous system disorder and/or associated conditions.
• a neurodegenerative disorder e.g., prevention of the development of the disorder or slowing of the progression of the disorder
• the nervous system disorder may include, e.g., mood or psychiatric disorders (e.g., anxiety, depression, bipolar disorder, seasonal affective disorder, etc.) or a neurodegenerative disease (e.g., dementia, Alzheimer’s disease, Parkinson’s disease, etc.).
• the effective amount may be any of the amounts described herein.
• the N-lactoyl-amino acid(s) may be administered according to a dosing schedule.
• the effective amount is administered once to the subject.
• the effective amount is administered once a day to the subject.
• the effective amount is administered multiple times a day to the subject.
• the effective amount is administered once a day over a period of time ranging from 1 day to 60 days, e.g., from 1 day to 10 days, from 1 day to 7 days, from 1 day to 5 days, or from 1 day to 3 days. In some instances, the effective amount is administered from 1 time to 5 times per day including, e.g., 1 time to 3 times per day, 2 times to 5 times per day, or 3 times to 5 times per day, over a period of time ranging from 1 day to 14 days, e.g., from 1 day to 10 days, from 1 day to 7 days, from 1 day to 5 days, or from 1 day to 3 days.
• the methods include administering the N-lactoyl-amino acid, e.g., N-lactoyl-phenylalanine, or combination of N-lactoyl-amino acids in combination with one or more therapies for treating a metabolic disorder and/or associated condition, e.g., obesity.
• the methods include administering the N-lactoyl-amino acid in combination with an active agent (or a combination of one or more active agents) for treating the metabolic disorder.
• the methods include administering the N-lactoyl-amino acid in combination with an active agent for treating obesity.
• Active agents of interest include, but are not limited to, orlistat, lorcaserin, phentermine-topiramate, naltrexone-bupropion, liraglutide, phentermine, benzphetamine, diethylpropion, phendimetrazine, among others.
• the therapies that may be used in combination with the N-lactoyl-amino acid to treat the metabolic disorder include any one of or a combination of any of the following therapies: physical activity, a dietary plan (e.g., a low fat diet, low calorie diet, intermittent fasting, etc.), use of a weight loss device, and surgical intervention (e.g., bariatric surgery).
• Weight loss devices may include, e.g., an electrical stimulation system (e.g., a device that blocks nerve activity between the stomach and brain), a gastric balloon system (e.g., one or more balloons placed in the stomach), and/or a gastric emptying system (e.g., a pump and tube to drain food from the stomach after a meal).
• the one or more therapies include a small molecule agent.
• Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
• Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing the screening protocols.
• the one or more therapies include a protein or a fragment thereof or a protein complex.
• the one or more therapies include an antibody binding agent or derivative thereof.
• antibody binding agent includes polyclonal or monoclonal antibodies or fragments that are sufficient to bind to an analyte of interest.
• the antibody fragments can be, for example, monomeric Fab fragments, monomeric Fab' fragments, or dimeric F(ab)'2 fragments.
• antibody binding agent molecules produced by antibody engineering, such as single-chain antibody molecules (scFv) or humanized or chimeric antibodies produced from monoclonal antibodies by replacement of the constant regions of the heavy and light chains to produce chimeric antibodies or replacement of both the constant regions and the framework portions of the variable regions to produce humanized antibodies.
• the one or more therapies include an enzyme or enzyme complex.
• the one or more therapies includes a phosphorylating enzyme, e.g., a kinase.
• the one or more therapies includes a complex including a guide RNA and a CRISPR effector protein, e.g., Cas9, used for targeted cleavage of a nucleic acid.
• the one or more therapies includes a nucleic acid.
• the nucleic acids may include DNA or RNA molecules.
• the nucleic acids modulate, e.g., inhibit or reduce, the activity of a gene or protein, e.g., by reducing or downregulating the expression of the gene.
• the nucleic acid may be a single stranded or double-stranded and may include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
• the one or more therapies includes intracellular gene silencing molecules by way of RNA splicing and molecules that provide an antisense oligonucleotide effect or an RNA interference (RNAi) effect useful for inhibiting gene function.
• RNAi RNA interference
• gene silencing molecules such as, e.g., antisense RNA, short temporary RNA (stRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA), snRNA, snoRNA, and other RNAi-like small RNA constructs, may be used to target a protein-coding as well as non-protein-coding genes.
• the nucleic acids include aptamers (e.g., spiegelmers).
• the nucleic acids include antisense compounds.
• the nucleic acids include molecules which may be utilized in RNA interference (RNAi) such as double stranded RNA including small interfering RNA (siRNA), locked nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, etc.
• RNAi RNA interference
• small interfering RNA small interfering RNA
• LNA locked nucleic acid
• PNA peptide nucleic acid
• the N-lactoyl-amino acid(s) may be administered by any suitable means.
• administering includes in vivo administration as well as direct administration to tissues ex vivo.
• administration is, for example, oral, buccal, parenteral (e.g., intravenous, intraarterial, subcutaneous), intraperitoneal (i.e., into the body cavity), topically, e.g., by inhalation or aeration (i.e., through the mouth or nose), or rectally systemic (i.e., affecting the entire body).
• parenteral e.g., intravenous, intraarterial, subcutaneous
• intraperitoneal i.e., into the body cavity
• topically e.g., by inhalation or aeration (i.e., through the mouth or nose), or rectally systemic (i.e., affecting the entire body).
• topically may include injection, insertion, implantation, topical application, or parenteral application.
• the N-lactoyl-amino acid is administered in a pharmaceutical formulation or as a pharmaceutically acceptable composition in which one or more N-lactoyl- amino acids may be mixed with one or more carriers, thickeners, diluents, buffers, preservatives, surface active agents, excipients and the like.
• Pharmaceutical compositions may also include one or more additional active ingredients such as antimicrobial agents, anti- inflammatory agents, anesthetics, and the like in addition to the one or more N-lactoyl-amino acids.
• the N-lactoyl-amino acid composition includes, e.g., a derivative or analog of an N-lactoyl-amino acid.
• “Derivatives” include pharmaceutically acceptable salts and chemically modified agents. “Analogs” include a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog may be a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
• the pharmaceutical compositions may be administered by any route commonly used to administer pharmaceutical compositions.
• administration may be done topically (including opthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injection.
• the pharmaceutical composition including the N- lactoyl-amino acid may be stored at any suitable temperature. In some cases, the N-lactoyl- amino acid composition is stored at temperatures ranging from 1o C to 30 oC, from 2o C to 27 oC, or from 5oC to 25 oC.
• the N-lactoyl-amino acid composition may be stored in any suitable container, as described in detail below.
• the metabolic disorder treated by the subject methods may vary.
• metabolic disorder refers to any disorder associated with or aggravated by impaired or altered glucose regulation or glycemic control, such as, for example, insulin resistance.
• a “metabolic disorder” refers to any disorder associated with or aggravated by impaired or altered glucose regulation or glycemic control, such as, for example, insulin resistance.
• Such disorders include, but are not limited to, diabetes, hyperglycemia, obesity, etc.
• Metabolic disorders and conditions associated with metabolic disorders that can be treated according to the methods described herein include but are not limited to overweight, obesity, hyperphagia, diabetes (inclusive of type 1 diabetes and type 2 diabetes), type 2 diabetes, impaired glucose tolerance, insulin resistance, hyperinsulinemia, dyslipidemia, hypertension, metabolic syndrome.
• the disorder treated by the subject methods may also be obesity and metabolic syndrome associated disorders, such as but not limited to, meningioma, adenocarcinoma, multiple myeloma, kidney cancer, endometrium cancer, ovarian cancer, colorectal cancer, pancreatic cancer, stomach cancer, gallbladder cancer, liver cancer, breast cancer, thyroid cancer, and any other obesity associated cancers.
• obesity and metabolic syndrome associated disorders such as but not limited to, meningioma, adenocarcinoma, multiple myeloma, kidney cancer, endometrium cancer, ovarian cancer, colorectal cancer, pancreatic cancer, stomach cancer, gallbladder cancer, liver cancer, breast cancer, thyroid cancer, and any other obesity associated cancers.
• osteoarthritis Including osteoarthritis, stroke, gallbladder disease, chronic kidney disease, and coronary artery disease.
• mental disorders such as clinical depression and anxiety, bipolar disorder, panic disorder, and agoraphobia.
• the term “obesity” refers to a condition characterized by an excess of body fat.
• the operational definition of obesity may be based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg/m 2 ).
• BMI Body Mass Index
• Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m 2 , or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m 2 .
• An “obese subject” is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m 2 or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m 2 .
• a “subject at risk of obesity” is an otherwise healthy subject with a BMI of 25 kg/m 2 to less than 30 kg/m 2 or a subject with at least one co-morbidity with a BMI of 25 kg/m 2 to less than 27 kg/m 2 .
• the increased risks associated with obesity may occur at a lower BMI in people of Asian descent.
• “obesity” refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m 2 .
• an “obese subject” in these countries refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m 2 .
• a “subject at risk of obesity” is a person with a BMI of greater than 23 kg/m 2 to less than 25 kg/m 2 .
• the metabolic disorder is an obesity-related metabolic disorder.
• the term “obesity-related disorders” encompasses disorders that are associated with, caused by, or result from obesity.
• obesity-related disorders include overeating and bulimia, diabetes, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, breast, prostate, endometrial and colon cancer, heart disease, cardiovascular disorders, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack.
• Other examples include pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass.
• obesity-related disorders include metabolic syndrome, also known as syndrome X, insulin resistance syndrome, type II diabetes, impaired fasting glucose, impaired glucose tolerance, inflammation, such as systemic inflammation of the vasculature, atherosclerosis, hypercholesterolemia, hyperuricaemia, as well as secondary outcomes of obesity such as left ventricular hypertrophy.
• Obesity-related metabolic disorders may further include, e.g., hypertension, osteoarthritis, Type II diabetes mellitus, increased blood pressure, stroke, and heart disease.
• Obesity-related disorders also include the liver abnormalities associated with obesity such as steatosis or non-alcoholic fatty liver disease (NAFLD) a rising cause of cirrhosis associated to obesity and metabolic syndrome.
• NAFLD non-alcoholic fatty liver disease
• NAFLD can present as simple steatosis or evolve towards inflammation and steatohepatitis (NASH), with a 20% risk of cirrhosis after 20 years.
• “Dyslipidemia” is a major risk factor for coronary heart disease (CHD).
• High density lipoprotein (HDL) cholesterol with either normal or elevated levels of low density (LDL) cholesterol is a significant risk factor for developing atherosclerosis and associated coronary artery disease in humans.
• Dyslipidemia is often associated with obesity. Additional obesity related disorders are described in, e.g., U.S. Patent No.8394969, the disclosure of which is incorporated herein by reference in its entirety.
• the metabolic disorder is diabetes.
• Diabetes refers to a group of metabolic diseases characterized by high blood sugar (glucose) levels which result from defects in insulin secretion or action, or both. Diabetes is classified according to the types of disease into insulin dependent diabetes (IDDM; type I diabetes) and non-insulin dependent diabetes (NIDDM; type II diabetes). “Type 2 diabetes” refers to one of the two major types of diabetes, the type in which the beta cells of the pancreas produce insulin, at least in the early stages of the disease, but the body is unable to use it effectively because the cells of the body are resistant to the action of insulin. In later stages of the disease the beta cells may stop producing insulin. Type 2 diabetes is also known as insulin-resistant diabetes, non-insulin dependent diabetes and adult-onset diabetes.
• Type I diabetes refers to a condition that results from an autoimmune-mediated destruction of pancreatic ⁇ cells with consequent loss of insulin production, which results in hyperglycemia. Type I diabetics require insulin replacement therapy to ensure survival.
• diabetes disorders may refer to complications due to diabetes. For example, complications such as retinopathy, nephropathy and neuropathy develop with angiopathy as a prime factor in diabetic individuals. Treatment may result in various outcomes.
• treatment of obesity and obesity-related disorders refers to the administration of the N-lactoyl-amino acid or combinations of N-lactoyl-amino acids as described herein to reduce or maintain the body weight of an obese subject.
• One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the compounds or combinations as described herein. Another outcome of treatment may be preventing regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy and preventing weight gain from cessation of smoking. Another outcome of treatment may be decreasing the occurrence of and/or the severity of obesity-related diseases. Yet another outcome of treatment may be decreasing the risk of developing diabetes in an overweight or obese subject.
• the treatment may result in a reduction in food or calorie intake by the subject, including a reduction in total food intake, or a reduction of intake of specific components of the diet such as carbohydrates or fats; and/or the inhibition of nutrient absorption; and/or the inhibition of the reduction of metabolic rate.
• the treatment may result in weight reduction in patients in need thereof.
• the treatment may also result in an alteration of metabolic rate, such as an increase in metabolic rate, rather than or in addition to an inhibition of the reduction of metabolic rate; and/or in minimization of the metabolic resistance that normally results from weight loss.
• the methods prevent the development of obesity or obesity-related disorders in a subject.
• Prevention of obesity and obesity-related disorders refers to the administration of the N-lactoyl amino acid or combinations of N-lactoyl-amino acids to reduce or maintain the body weight of a subject at risk of obesity.
• One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the compounds or combinations of the present invention.
• Another outcome of prevention may be preventing regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and/or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity.
• the methods reduce food intake of the subject, e.g., during and/or after treatment.
• food intake is meant the amount of food consumed by the subject. In some instances, food intake is measured in kcal over a period of time, e.g., kcal/day.
• the food intake is cumulative food intake over a period of time ranging from 1 day to 14 days, e.g., from 1 day to 10 days, from 1 day to 7 days, from 1 day to 5 days, or from 1 day to 3 days. In some instances, the food intake is average daily food intake, e.g., over a period of time ranging from 1 day to 14 days, e.g., from 1 day to 10 days, from 1 day to 7 days, from 1 day to 5 days, or from 1 day to 3 days. In some instances, the methods reduce food intake by the subject compared to (e.g., relative to) a control. In some instances, the methods reduce food intake by the subject compared to the food intake of the subject before treatment.
• cumulative food intake is reduced by 10% to 90% including, e.g., by 10% to 80%, by 10% to 70%, by 10% to 60%, by 10% to 50%, by 10% to 40%, by 10% to 30%, or by 10% to 20%.
• average daily food intake is reduced by 10% to 90% including, e.g., by 10% to 80%, by 10% to 70%, by 10% to 60%, by 10% to 50%, by 10% to 40%, by 10% to 30%, or by 10% to 20%.
• the methods reduce the body weight of the subject, e.g., during and/or after treatment. In some instances, the methods reduce the body weight of the subject compared to (e.g., relative to) a control.
• the methods reduce the body weight of the subject compared to the body weight of the subject before treatment. In some instances, the methods reduce the average body weight of the subject over a period of time ranging, e.g., from 1 day to 14 days, e.g., from 1 day to 10 days, from 1 day to 7 days, from 1 day to 5 days, or from 1 day to 3 days. In some instances, the methods reduce the average body weight of the subject compared to a control. In some instances, the methods reduce the average body weight of the subject compared to the average body weight of the subject before treatment. In some instances, body weight is reduced by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• average body weight is reduced by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods improve glucose regulation in a subject, e.g., during and/or after treatment.
• the methods may improve the body’s ability to regulate glucose.
• the term “glucose regulation” or “regulation of glucose metabolism” as used herein refer to processes by which a cell, tissue, organ, organ system, or whole organism maintains glucose homeostasis by altering, e.g., increasing or decreasing, specific processes of glucose metabolism.
• Glucose metabolism or glucose metabolic processes encompass processes involving glucose synthesis, processing, transport, uptake, utilization, or storage, and includes gluconeogenesis and glycolysis.
• Specific aspects of glucose metabolism and regulation include expression of glucose transporters or enzymes which facilitate movement of glucose across a cell membrane and retention or secretion of glucose by a cell; alteration in expression and/or activity of enzymes involved in glucose utilization or formation, including, e.g., glycolytic and gluconeogenic enzymes; and alteration of glucose distribution within body or culture fluids, including, e.g., interstitial (i.e. extracellular) and intracellular fluids, blood, urine, and the like.
• the methods improve glucose homeostasis in the subject, e.g., during and/or after treatment.
• the term “glucose homeostasis” refers to maintenance of normal glucose levels, e.g., normal blood glucose levels, in an organism.
• the methods improve glucose homeostasis in the subject compared to (e.g., relative to) a control.
• the methods improve glucose homeostasis in the subject compared to the glucose homeostasis in the subject before treatment.
• the methods improve glucose clearance, e.g., from circulation, in the subject compared to a control.
• the methods improve glucose clearance in the subject compared to the glucose clearance in the subject before treatment.
• Improved glucose clearance may include increased glucose clearance. Increased glucose clearance may reduce blood glucose levels. In some instances, glucose clearance is improved by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%. In some embodiments, the methods reduce adipose tissue mass (e.g., the amount of adipose tissue or fat) in the subject.
• adipose tissue refers to fat including, e.g., the connective tissue that stores fat. Adipose tissue contains multiple regenerative cell types, including, e.g., adipose derived stem cells (ASCs) and endothelial progenitor and precursor cells.
• ASCs adipose derived stem cells
• Types of adipose tissue of interest include, but are not limited to, white adipose tissue and brown adipose tissue.
• the methods reduce adipose tissue mass compared to a control.
• the methods reduce adipose tissue mass in a subject compared to the adipose tissue mass in the subject before treatment.
• the methods reduce adipose tissue by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods reduce adipose tissue by 10% to 50% including, e.g., by 10% to 40%, by 10% to 30%, by 10% to 20%, by 20% to 50%, by 30% to 50%, or by 40% to 50%.
• the methods reduce the amount of white fat compared to a control.
• the methods reduce the amount of white fat in a subject compared to the amount of white fat in the subject before treatment.
• the methods reduce white fat by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods reduce white fat by 10% to 50% including, e.g., by 10% to 40%, by 10% to 30%, by 10% to 20%, by 20% to 50%, by 30% to 50%, or by 40% to 50%. In some instances, the methods reduce the amount of brown fat compared to a control. In some instances, the methods reduce the amount of brown fat in a subject compared to the amount of brown fat in the subject before treatment. In some instances, the methods reduce brown fat by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods reduce brown fat by 10% to 50% including, e.g., by 10% to 40%, by 10% to 30%, by 10% to 20%, by 20% to 50%, by 30% to 50%, or by 40% to 50%. In some instances, the methods reduce the amount of smaller epididymal fat compared to a control. In some instances, the methods reduce the amount of smaller epididymal fat in a subject compared to the amount of smaller epididymal fat in the subject before treatment. In some instances, the methods reduce smaller epididymal fat by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods reduce smaller epididymal fat by 10% to 50% including, e.g., by 10% to 40%, by 10% to 30%, or by 10% to 20%. In some instances, the methods reduce the amount of subcutaneous inguinal fat compared to a control. In some instances, the methods reduce the amount of subcutaneous inguinal fat in a subject compared to the amount of subcutaneous inguinal fat in the subject before treatment. In some instances, the methods reduce subcutaneous inguinal fat by 1% to 50% including, e.g., by 1% to 40%, by 1% to 30%, by 1% to 20%, or by 1% to 10%.
• the methods reduce subcutaneous inguinal fat by 10% to 50% including, e.g., by 10% to 40%, by 10% to 30%, by 10% to 20%, by 20% to 50%, by 30% to 50%, or by 40% to 50%.
• the “control,” as used herein in its conventional sense may be any suitable control.
• the control includes a subject, e.g., a subject with a metabolic disorder, to whom an effective amount of an N-lactoyl-amino acid has not been administered.
• the control may be a subject that has the same metabolic disorder(s) and/or associated conditions as the treated subject.
• the control includes a subject to whom an effective amount of an N-lacotyl-amino acid is not administered, where the subject has a metabolic disorder or a combination of metabolic disorders and/or associated conditions that match those of the subject to whom the effective amount is administered.
• the control subject has characteristics (e.g., age, sex, height, weight, race, diet, etc.) that are shared by the treated subject.
• the outcomes for a subject as described herein are measured relative to the subject before treatment.
• Embodiments of the methods can be practiced on any suitable subject.
• a subject of the present invention may be a “mammal” or “mammalian”, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans.
• the methods may be applied to human subjects of both genders and at any stage of development (i.e., neonates, infant, juvenile, adolescent, adult), where in certain embodiments the human subject is a juvenile, adolescent or adult.
• a pharmaceutical formulation may include an N-lactoyl- amino acid (or a combination of N-lactoyl-amino acids) or a pharmaceutically acceptable salt thereof, and one or more of pharmaceutically acceptable carriers or excipients.
• a pharmaceutical formulation includes an amount of an N-lactoyl-amino acid effective to treat a metabolic disease; and an excipient.
• the N- lactoyl-amino acid may be any suitable N-lactoyl-amino acid or combination of N-lactoyl amino acids as described herein.
• the N-lactoyl-amino acid is N- lactoyl-phenylalanine.
• the amount of N-lactoyl-amino acid may be any suitable amount according to any of the embodiments described herein.
• the pharmaceutical formulations may be administered in combination with any of the therapies (e.g., therapies for treating a metabolic disorder) as described herein.
• the pharmaceutical formulation may be formulated for administration by any suitable means.
• the composition is formulated for administration orally, intradermally, intramuscularly, parenterally, intravenously, intra- arterially, intracranially, subcutaneously, intraorbitally, intraventricularly, intraspinally, intraperitoneally, or intranasally.
• the pharmaceutical formulations or compositions can be formulated into various dosage forms, including tablets, powders, fine granules, granules, dry syrups, capsules, liquid compositions, etc.
• the pharmaceutical formulation is a capsule or tablet.
• the pharmaceutic formulation is a parenteral formulation.
• the pharmaceutical formulation is an intraperitoneal formulation. Additives and diluents normally utilized in the pharmaceutical arts can optionally be added to the pharmaceutical formulation.
• thickening granulating, dispersing, flavoring, sweetening, coloring, and stabilizing agents, including pH stabilizers, other excipients, anti-oxidants (e.g., tocopherol, BHA, BHT, TBHQ, tocopherol acetate, ascorbyl palmitate, ascorbic acid propyl gallate, and the like), preservatives (e.g., parabens), and the like.
• preservatives include, but are not limited to, benzylalcohol, ethylalcohol, benzalkonium chloride, phenol, chlorobutanol, and the like.
• Some useful antioxidants provide oxygen or peroxide inhibiting agents for the formulation and include, but are not limited to, butylated hydroxytoluene, butylhydroxyanisole, propyl gallate, ascorbic acid palmitate, ⁇ - tocopherol, and the like.
• Thickening agents such as lecithin, hydroxypropylcellulose, aluminum stearate, and the like, may improve the texture of the formulation.
• a container for holding the N-lactoyl-amino acid formulation or N-lactoyl-amino acid pharmaceutical composition may be configured to hold any suitable volume of the N-lactoyl-amino acid formulation or composition.
• the size of the container may depend on the volume of N-lactoyl-amino acid composition to be held in the container.
• the container may be configured to hold an amount of N-lactoyl-amino acid composition ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg
• the container is configured to hold an amount of N- lactoyl-amino acid composition ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In certain instances, the container is configured to hold a volume (e.g., a volume of a liquid N-lactoyl-amino acid composition) ranging from 0.1 ml to 200 ml.
• a volume e.g., a volume of a liquid N-lactoyl-amino acid composition
• the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml.
• a volume e.g., a volume of a liquid
• the container is configured to hold a volume (e.g., a volume of a liquid N-lactoyl-amino acid composition) ranging from 0.1 ml to 200 ml.
• the shape of the container may also vary.
• the container may be configured in a shape that is compatible with the assay and/or the method or other devices used to perform the assay.
• the container may be configured in a shape of typical laboratory equipment used to perform the assay or in a shape that is compatible with other devices used to perform the assay.
• the container is a liquid container.
• the liquid container is a vial or a test tube. In certain cases, the liquid container is a vial.
• the liquid container is a test tube. In some instances, the container is a blister pack. As described above, embodiments of the container can be compatible with the N- lactoyl-amino acid composition.
• suitable materials for the containers include, but are not limited to, glass and plastic.
• the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX TM ), fused quartz glass, fused silica glass, and the like.
• suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.
• the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container.
• the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container.
• the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container.
• the seal is made of a resilient material to provide a barrier (e.g., a water-tight and/or air-tight seal) for retaining a sample in the container.
• Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container.
• Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof.
• the seal is a septum pierceable by a needle, syringe, or cannula.
• the seal may also provide convenient access to a sample in the container, as well as a protective barrier that overlies the opening of the container.
• the seal is a removable seal, such as a threaded or snap-on cap or other suitable sealing element that can be applied to the opening of the container.
• a threaded cap can be screwed over the opening before or after a sample has been added to the container.
• the subject methods and formulations find use in applications, e.g., clinical applications, involving metabolic disorders and one or more conditions associated with metabolic disorders. In some embodiments, the methods and formulations find use in applications where it is desirable to treat a metabolic disorder and one or more conditions associated with metabolic disorders including, e.g., obesity, an obesity related disorder, diabetes, etc.
• the methods and formulations find use in applications where it is desirable to prevent the development or occurrence of a metabolic disorder and one or more conditions associated with metabolic disorders. In certain embodiments, the methods and formulations find use in applications where weight loss for a subject is desirable. In certain embodiments, the methods and formulations find use in applications where improving glucose homeostasis in a subject is desirable. In certain embodiments, the methods and formulations find use in applications where it is desirable to induce a physical activity associated outcome in a subject. In certain embodiments, the methods and formulations find use in applications where it is desirable to treat a nervous system disorder. The methods and formulations may also find use in combination with other therapies and treatments for any of the disorders and associated conditions described herein.
• Example 1 A lactate-derived exercise-inducible metabolite that suppresses food intake and obesity Materials and Methods Cell line cultures. All cell lines were obtained from ATCC and grown at 37°C with 5% CO 2 . RAW 264.7, HEK293T, HEK293A, C2C12, F442A, 3T3-L1, Caco2, and SW48 were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (pen/strep). AML-12 cells were grown in DMEM with 10% FBS and pen/strep supplemented with Insulin-Transferrin-Selenium.
• DMEM Dulbecco’s modified Eagle’s medium
• FBS fetal bovine serum
• pen/strep penicillin/streptomycin
• T84 cells were grown in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) with 5% FBS and pen/strep.
• Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 DMEM/F-12
• FBS pen/strep.
• General animal information Animal experiments were performed according to procedure approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). Mice were maintained in 12-hr light-dark cycles at 22 °C and ⁇ 50% relative humidity and fed a standard irradiated rodent chow diet. Where indicated, high-fat diet (D12492, Research Diets 60% kcal from fat) was used.
• C57BL/6J stock no.000664
• C57BL/6J DIO mice stock no. 380050
• mice were purchased from Charles River Laboratory.
• Whole body CNDP2 knockout mice (catalog number, C57BL/6NCrl-Cndp2em1(IMPC)Mbp/Mmucd, RRID: MMRRC_043492-UCD) were obtained from the Mutant Mouse Regional Resource Center, a NCRR-NIH funded strain repository.
• compounds including Lac-Phe, lactate, and phenylalanine, compounds were dissolved in 18:1:1 (by volume) of saline/Kolliphor EL (Sigma Aldrich)/DMSO.
• mice were administered to mice daily via intraperitoneal injections at 5 ⁇ l/g body weight at the indicated doses.
• mice were mock injected with the vehicle for 3-5 days until body weights have stabilized.
• mice were fasted for 6 hours then injected with glucose at 10 ⁇ l/g body weight.
• a dose of 1 g/kg was used for the GTT of vehicle and Lac-Phe treated obese mice.
• Chemicals. L-Phenylalnine (AAA-1323814) was purchased from Fisher Scientific, sodium L-lactate (L7022) was purchased from Sigma. The synthesis of non-commercially available Lac-Phe is described below. Synthesis of Lac-Phe (N-lactoyl phenylalanine).
• mice were exercised 5 days/week, Monday through Friday while on high fat diet (60% kcal from fat) and were allowed to rest the remaining 2 days/week. Treadmill running was performed at a constant 5° incline and began at a speed of 6 m/min. Speed was increased by 2 m/min every 5 minutes until a maximum speed of 30 m/min. Mice were stopped upon reaching exhaustion as described above and run times were normalized between the two groups of mice. Preparation of plasma samples for LC-MS analysis. Plasma was collected from mice via a submandibular bleed into lithium heparin tubes (BD, 365985) and immediately transferred onto ice.
• BD lithium heparin tubes
• the blood was centrifuged at 4 °C at 5000 rpm for 5 min and the top layer of plasma was aliquoted and frozen at -80 °C.
• 150 ul of a 2:1 mixture of acetonitrile/methanol was added to 50 ⁇ l of plasma.
• the mixture was centrifuged at 4 °C for 10 min at 15,000 rpm and the supernatant was transferred to a LC-MS vial.
• Untargeted metabolomics measurements were performed on an Agilent 6520 Quadrupole Time-of-Flight (Q-TOF) LC/MS.
• Mass spectrometry analysis was performed using electrospray ionization (ESI) in negative mode.
• ESI electrospray ionization
• the dual ESI source parameters were set as follows, the gas temperature was set at 250 °C with a drying gas flow of 12 l/min and the nebulizer pressure at 20 psi.
• the capillary voltage was set to 3500 V and the fragmentor voltage set to 100 V.
• Separation of polar metabolites was conducted on a Luna 5 ⁇ m NH2100 ⁇ LC column (Phenomenex 00B-4378-E0) with normal phase chromatography.
• Mobile phases were as follows: Buffer A, 95:5 water/acetonitrile with 0.2% ammonium hydroxide and 10 mM ammonium acetate.
• Buffer B acetonitrile.
• the LC gradient started at 100% B with a flow rate of 0.2 ml/min from 0-2 min. The gradient was then increased linearly to 50% A/50% B at a flow rate of 0.7 ml/min from 2-20 minutes. From 20-25 minutes the gradient was maintained at 50% A/50% B at a flow rate of 0.7 ml/min.
• Targeted measurements of Lac-AAs Targeted measurements of performed on an Agilent 6470 Triple Quadrupole (QQQ) LC/MS. Mass spectrometry analysis was performed using electrospray ionization (ESI) in negative mode.
• ESI electrospray ionization
• the AJS ESI source parameters were set as follows, the gas temperature was set at 250 °C with a gas flow of 12 l/min and the nebulizer pressure at 25 psi.
• the sheath gas temperature was set to 300 °C with the sheath gas flow set at 12 l/min.
• the capillary voltage was set to 3500 V. Separation of polar metabolites was performed as described above in the untargeted metabolomics section. Multiple reaction monitoring (MRM) was performed for the indicated metabolites with the listed dwell times, fragmentor voltage, collision energies, cell accelerator voltages, and polarities. Differentiation of cell lines. C2C12 cells were plated in 12-wells and grown until 80-90% confluence.
• CNDP2-KO RAW 264.7 cells After induction, cells were maintained in 5 ⁇ g/ml insulin and 1 ⁇ M rosiglitazone for the remaining 4-6 days.
• Generation of CNDP2-KO RAW 264.7 cells The plentiCRISPRv2 system developed by the Zhang lab was used to generate the CNDP2-KO RAW 264.7 cell line.
• the sgRNA used was 5’-CAGTGAAATGAGATCCGTCA-3’(SEQ ID NO:01).
• oligonucleotides for the sgRNA and reverse complement sequences were synthesized and cloned into the plentiCRISPRv2 vector (Forward oligo, 5’- CACCGCAGTGAAATGAGATCCGTCA-3’ (SEQ ID NO:02); reverse oligo, 5’- AAACTGACGGATCTCATTTCACTG C-3’ (SEQ ID NO:03)).
• Lentivirus particles were generated in the HEK293T cell line using polyfect for the co-transfection of the cloned plentiCRISPRv2 plasmid with the viral packing psPAX2 plasmid, and viral envelope pMD2.G plasmid.
• a parental plentiCRISPRv2 plasmid was used as a control. Lentiviral supernatants were harvested after 24 hours and filtered through a 0.45 uM filter. The supernatant was then mixed in a 1 to 1 ratio with polybrene to a final concentration of 8 ug/ml polybrene. This mixture was added to RAW 264.7 cells at 40-50% confluence in 6-well plates. Transduced cells were transferred to a 10 cm plate followed by selection with 5 ug/ml of puromycin for 3-6 days. Primary peritoneal macrophage isolation.
• mice were injected with 2 ml of 3% brewer thioglycollate medium (Fisher, B11716) and the macrophage elicitation allowed to proceed for 3 days.
• thioglycollate medium Fisher, B11716
• mice were euthanized with CO 2 and the abdominal skin peeled back to expose the peritoneal wall.10 ml of ice-cold calcium and magnesium free DPBS was injected into the peritoneal cavity, taking care not to puncture the organs. The mice were gently massaged and the fluid aspirated from the peritoneum using the same syringe and needle. The fluid was dispensed into a 50 ml falcon tube on ice.
• Cells were then centrifuged at 4 °C for 10 minutes at 2,000 xg and the supernatant removed to obtain the cell pellet.100 ⁇ l of a 2:1:1 mixture of acetonitrile/methanol/water mixture was used to lyse the cells and precipitate large proteins. The mixture was centrifuged at 4 °C for 10 minutes at 15,000 rpm and the supernatant was transferred to a LC-MS vial. Western blot analysis. Cells were collected and lysed by sonication in RIPA buffer containing 1:100 HALT protease inhibitor. Cell lysates were centrifuged at 4 °C for 10 minutes at 13,000 rpm to remove residual cell debris.
• Protein concentrations of the supernatant were normalized using the Pierce BCA protein assay kit and combined with 4 x NuPAGE LDS Sample Buffer with 10 mM DTT. Samples were then boiled for 10 minutes at 95 °C. Prepared samples were run on a NuPAGE 4-12% Bis-Tris gel then transferred to nitrocellulose membranes. Blots were blocked for 30 minutes at room temperature in Odyssey blocking buffer. Primary antibodies (rabbit anti-CNDP2 and rabbit anti-Beta-Tubulin) were added to Odyssey blocking buffer at a ratio of 1:1000. Blots were incubated in the indicated primary antibodies overnight while shaking at 4 °C.
• blots were washed 3 times with PBS-T, 10 minutes each before staining with the secondary antibody for 1 hour at room temperature.
• the secondary antibody used was a goat anti-rabbit antibody diluted in blocking buffer to a ratio of 1:10,000.
• the blot was washed 3 times with PBS-T before being imaged with the Odyssey CLx Imaging System.
• Generation of CNDP2 KO animals CNDP2 KO and wild type animals were generated via heterozygous breeding crosses. Genotyping was performed as follows: Tail clippings were obtained from littermates and boiled for 30 minutes at 95 °C in 100 ⁇ l of 50 mM NaOH to extract genomic DNA.
• PCR reactions were performed by using primers for either the CNDP2 WT allele (Forward: 5’- CAGATGGCTCGGAGATACCAC-3’ (SEQ ID NO:04), Reverse: 5’- TTCCCGCTCCACCAAGGTGAAG-3’ (SEQ ID NO:05)) or CNDP2 KO allele (Forward: 5’- GCTCTGTAAGGGAAAGAGATGACCC-3’ (SEQ ID NO:06), Reverse: 5’- AATAGGACATACCCAGTTCTGTGAGG-3’ (SEQ ID NO:07)).
• the Promega GoTaq master mix was used for the PCR reaction.
• Each 25 ⁇ l reaction consisted of 12.5 ⁇ l of the promega master mix (M7122), 2.5 ⁇ l of a 10 ⁇ M mixture of forward and reverse primers, 2 ⁇ l of genomic DNA, and 8 ⁇ l of ultrapure water.
• the thermocycling program on BioRad C1000 Touch Thermo Cycler began with an initial 30 seconds at 95°C, followed by cycles of 30 seconds at 98°C, 30 seconds at 58 °C, and 45 seconds at 72 °C, followed by 5 minutes at 72 °C and finally held at 4 °C.
• PCR reactions for WT primers consisted of 30 cycles while PCR reactions for KO primers consisted of 48 cycles.
• AgRP-IRES- Cre/Rosa26-LSL-tdTOMATO mice for recordings from AgRP neurons in the arcuate nucleus of hypothalamus (ARH); POMC-CreER/Rosa26-LSL-tdTOMATO mice (with 200 mg/kg tamoxifen induction at 8-12 weeks) were used for recordings from POMC neurons in the ARH; SF1- Cre/Rosa26-LSL-tdTOMATO mice were used for recordings from SF1 neurons in the ventromedial hypothalamus (VMH); TPH2-CreER/Rosa26-LSL-tdTOMATO mice (with 200 mg/kg tamoxifen induction at 8-12 weeks) were used for recording from 5-HT neurons in the dorsal raphe nucleus (DRN).
• ARH arcuate nucleus of hypothalamus
• POMC-CreER/Rosa26-LSL-tdTOMATO mice with 200 mg/kg t
• mice Males and females, 8-16 weeks old were anesthetized with isoflurane and transcardially perfused with a modified ice-cold sucrose-based cutting solution (pH 7.4; containing 10 mM NaCl, 25 mM NaHCO 3 , 195 mM sucrose, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO 4 , 2 mM sodium pyruvate, 0.5 mM CaCl 2 , and 7 mM MgCl 2 , bubbled continuously with 95% O 2 and 5% CO 2 ). The mice were then decapitated, and the entire brain was removed and immediately submerged in the cutting solution.
• a modified ice-cold sucrose-based cutting solution pH 7.4; containing 10 mM NaCl, 25 mM NaHCO 3 , 195 mM sucrose, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO 4 , 2
• the slices were recovered for ⁇ 30 minutes at 32 °C and then maintained at room temperature for another 1 hour in oxygenated (95% O 2 and 5% CO 2 ) artificial cerebrospinal fluid (ACSF, pH 7.4; containing 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 1.2 mM MgCl 2 , 11.1 mM glucose, and 21.4 mM NaHCO 3 ) before recording.
• Slices were transferred to the recording chamber at 32 °C and perfused continuously with oxygenated ACSF at a flow rate of 1.8–2.0 ml/min. Slices were allowed to equilibrate for at least 5 min before recording.
• tdTOMATO-labeled neurons in the ARH, VMH or DRN were visualized using epifluorescence and infrared–differential interference contrast (IR-DIC) imaging on an upright microscope (Eclipse FN-1, Nikon) equipped with a moveable stage (MP-285, Sutter Instrument).
• Patch pipettes with resistances of 3–5 M ⁇ were filled with intracellular solution (pH 7.3) containing 128 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl 2 , 0.05 mM GTP (sodium salt), and 0.05 mM ATP (magnesium salt).
• the neurons located on the surface of the slice were selected to record and the puff pipette was always put at a 100 ⁇ m horizontal and 100 ⁇ m vertical distance from the recorded neurons.
• the puff strength was maintained at a same level using a repeatable pressure pulse system (Picospritzer III, Parker).
• the ACSF solution containing 1 ⁇ M tetrodotoxin (TTX, Tocris), 50 ⁇ M bicuculline (Tocris), 20 ⁇ M DNQX (Tocris) and 50 ⁇ M D-AP5 (Tocris) was used to block the majority of presynaptic inputs.
• Each neuron was recorded for at least 1 minute at baseline and only the neurons with stable baseline were used to test the Lac-Phe treatment.
• the values of RM and firing frequency were averaged in baseline and in a 1 minute range containing the point with the maximal change in resting membrane potential after Lac-Phe puff in POMC, SF1 and 5-HT neurons.
• the values of RM and firing frequency were averaged in 40 seconds at each time point indicated in the figures.
• a neuron was considered depolarized or hyperpolarized if a change in membrane potential was at least 2 mV, whereas values between a 2 mV was defined as “irresponsive”. Human exercise study- single bout of acute treadmill running.
• Body composition was measured by dual-energy x- ray absorptiometry (Lunar DPX-IQ DEXA Scanner, Lunar Corporation, WI, USA). Maximal oxygen uptake (VO2 peak) was measured with an incremental ramp test on a Monark Ergomedic 893E bicycle (Monark, Sweden), to evaluate training status. The test was comprised of 5 minutes at 100 W and 5 minutes at 150 W, followed by a 25 W increase per min until exhaustion. Expired air was collected during the test, using an online gas analyzer (CareFusion, MasterScreen-CPX, Germany). Prior to each trial, subjects were asked to refrain from strenuous physical activity for a minimum of 48 hours.
• a venflon catheter (BD Venflon TM Pro Safety, Helsingborg, Sweden) was inserted in an antecubital vein for blood sampling at rest, acutely after exercise (0), and during recovery from exercise (15, 60, 120 and 180 minutes) After centrifugation, plasma was pipetted into aliquots (200 ⁇ l) and stored at -80°C until further analysis. All subjects underwent three identical experimental trials, only separated by the exercise modalities performed which were: 1) an endurance exercise trial (END), 2) a sprint exercise trial (SPT) and 3) a resistance exercise trial (RES). The trials were performed in a randomized order, and each trial was separated by at least 10 days. Endurance exercise trial (END).
• END Endurance exercise trial
• the END trial consisted of 90 minutes of continuous cycling at 55 % VO2 peak. The load was established during preliminary testing, although VO 2 measurements were conducted and evaluated during the trial, to ensure the estimated load elicited 55% VO 2 peak, and to account for a potential drift in VO 2 during exercise. In case of insufficient or excessive loading, adjustments were implemented.
• Sprint exercise trial SPT
• the SPT trial consisted of a 5-min warmup at 50 W, followed by three bouts of 30-s all-out sprint (Wingate tests) on an ergometer bike. Each Wingate test was interspersed by 4 min of active recovery on 5 W.
• Resistance exercise trial (RES). The RES trial was based on bilateral knee extension exercise.
• the trial was initiated by a warmup consisting of 3 sets of 10 repetitions with a load corresponding to 50% of the 10-RM load. Each of the warm-up sets were interspersed by 2 min of rest. Following the warm-up 6 sets of 10 repetitions were performed at a load corresponding to 10-RM with each set interspersed by 2 min of rest. Statistics. All data was expressed as mean ⁇ SEM unless otherwise specified. A student’s t-test was used for pair-wise comparisons. Unless otherwise specified, statistical significance was set as P ⁇ 0.05. Results Regular physical activity is a powerful intervention that can reduce obesity and confer protection against obesity-associated metabolic diseases (1–4). Conversely, physical inactivity increases the risk of developing obesity and type 2 diabetes (5, 6).
• cardiometabolic benefits is comparable, and in some cases even greater, than first line pharmacological therapies such as metformin (7–9).
• the mechanisms responsible for the cardiometabolic benefits of exercise are incompletely understood but are likely to extend beyond activity-associated increases in energy expenditure alone.
• One endocrine mechanism that underlies the metabolic benefits of physical activity is the exercise-inducible production of circulating signaling molecules.
• These secreted factors have been proposed to mediate tissue crosstalk and function as molecular transducers of the metabolic benefits of physical activity (10).
• candidate approaches have identified several bioactive circulating metabolites that mediate salutary remodeling of adipose and muscle tissues following exercise training (11–14). However, untargeted strategies to systematically identify novel metabolites has been more limited.
• Lac-Phe N-lactoyl- phenylalanine
• Lac-Phe is an exercise-inducible signaling metabolite that mediates the anti- obesity effects of physical activity.
• untargeted metabolomic profiling by liquid chromatography-mass spectrometry (LC-MS) of acetonitrile/methanol-extracted blood plasma was performed in mice following an acute bout of treadmill running (Fig.1 A).
• the treadmill speed and incline were increased every three minutes until the mice reached exhaustion (see Methods).
• the untargeted metabolomics pipeline does not require a priori knowledge of the chemical structures of the peaks detected, thereby enabling a broader and more unbiased sampling of the exercise-regulated changes in blood plasma.
• metabolites previously detected in targeted mass spectrometry experiments to be robustly increased by physical activity including intermediates of the TCA cycle (e.g., succinate, fumarate, malate) as well as products of nucleotide catabolism (e.g. urate), were found in the metabolomics dataset to be increased by 2- to 4-fold in exercise relative to sedentary conditions (Fig.1 B and Fig.7 A). From the 7,752 peaks detected in this metabolomics experiment, the most statistically significant change was in an unknown peak of mass-to-charge ratio (m/z) of 236.0928 and retention time ⁇ 10.5 min (Fig.1 C). This metabolite was robustly elevated by > 5-fold relative to control mice (P ⁇ 0.001).
• intermediates of the TCA cycle e.g., succinate, fumarate, malate
• products of nucleotide catabolism e.g. urate
• Lac-Phe as one of the most robustly induced plasma metabolites following acute treadmill running in mice. Lac-Phe suppresses food intake and obesity Lac-Phe is a poorly studied metabolite of unknown function.
• metabolic chambers were used to determine the effect of acute Lac-Phe administration (50 mg/kg, intraperitoneal [IP]) to diet- induced obese (DIO) mice. Importantly, these experiments were performed at a dose of Lac- Phe such that peak plasma concentrations were comparable to that observed after a single bout of exercise training (plasma levels 30 min post injection, mean ⁇ SEM, 3.8 ⁇ M ⁇ 1.9 ⁇ M).
• Fig.2 A acute Lac-Phe administration suppressed food intake by 52% compared to vehicle-treated mice over a 12 h post-administration period (mean ⁇ SEM, vehicle 6.4 ⁇ 0.6 kcal/mouse; Lac-Phe 3.0 ⁇ 1.0 kcal/mouse).
• ambulatory activity was not different between groups (Fig.2 B), demonstrating that the suppression of food intake was not simply due to reduction in overall movement.
• Acute Lac-Phe treatment did not alter oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), or respiratory exchange ratio (RER, Fig. 8 A-C).
• Lac-Phe was chronically administered to DIO mice (50 mg/kg/day, intraperitoneal [IP], once dose per day, see Methods) and changes in food intake and body weight were monitored over a 10- day period.
• chronic Lac-Phe treated animals exhibited reduced cumulative food intake compared to control mice (mean ⁇ SEM, Lac-Phe 2.1 ⁇ 0.1 g/mouse/day; vehicle 2.9 ⁇ 0.1 g/mouse/day, Fig.2 C).
• Lac-Phe is composed of two “halves,” lactate and phenylalanine. Identical experiments to those described above were performed to directly compare Lac-Phe with lactate and phenylalanine (50 mg/kg, IP daily). Lac-Phe once again suppressed food intake and body weight compared to control mice (Fig.2 I). By contrast, lactate or phenylalanine administration at equivalent doses were identical to vehicle treated mice in both food intake and body weight (Fig.2 I).
• CNDP2 a cytosolic enzyme called CNDP2 has been shown to catalyze the condensation of lactate and phenylalanine to generate Lac-Phe in vitro (Fig.9 C) (15).
• Fig.9 C a cytosolic enzyme called CNDP2
• CNDP2-KO RAW264.7 cells were generated with CRISPR/Cas9 and loss of CNDP2 protein was validated using an anti-CNDP2 antibody (Fig.3 B).
• extracellular Lac- Phe levels were reduced by > 75% in CNDP2-KO relative to control cells.
• CNDP2 as the principal biosynthetic enzyme for Lac-Phe production in cell culture.
• These data also identify muscle- derived lactate as a candidate metabolic precursor that drives Lac-Phe production during acute exercise (Fig.3 H). Genetic ablation of Lac-Phe biosynthesis confers resistance to the anti-obesity effects of exercise training
• CNDP2-KO mice were used as a model for genetic ablation of Lac-Phe.
• an anti-CNDP2 antibody was used to determine the tissue expression of CNDP2 protein in mice. As expected, peritoneal macrophages exhibited the highest CNDP2 expression. CNDP2 protein expression was also detected at lower levels in kidney and gut (Fig.4 A).
• the residual increase in Lac-Phe with exercise likely reflects additional, CNDP2-independent pathways for Lac-Phe biosynthesis.
• WT and CNDP2-KO mice were next evaluated for food intake and obesity phenotypes. Because Lac-Phe is induced by exercise training, WT and CNDP2-KO mice were subjected to high fat diet (60% kcal from fat) in combination with a chronic treadmill running regimen (see Methods). Body weights at the beginning of this experiment were not different between WT and CNDP2-KO mice (mean ⁇ SEM, WT, 29.1 ⁇ 0.8 g; CNDP2-KO, 28.2 ⁇ 0.7 g; P > 0.05).
• CNDP2-KO mice began to exhibit increased food intake compared to control mice (Fig.4 C). This increased feeding behavior persisted until the end of the experiment on day 40. Consistent with this feeding behavior, CNDP2-KO mice also exhibited increasingly divergent body weight compared to control mice, with final body weight in CNDP2-KO mice +13% higher versus WT mice (mean ⁇ SEM, WT, 32.3 ⁇ 0.9 g; CNDP2-KO, 36.4 ⁇ 0.6 g, Fig.4 D). Importantly, total run times in this experiment were equivalent between groups (mean ⁇ SEM, WT 41 ⁇ 1.2 min/day; KO 41 ⁇ 1.7 min/day).
• Fig.5 A Representative electrophysiology recordings for AgRP neurons are shown in Fig.5 A. Consistent with its appetite suppressive effects in vivo, Lac-Phe (50 ⁇ M) rapidly inhibited orexigenic AgRP neurons, as demonstrated by decreased firing frequency rate and hyperpolarization of the resting membrane potential (Fig.5 A). The inhibitory action of Lac-Phe was dose responsive, with inhibitory activity at concentrations as low as 1 ⁇ M which is similar to physiologic levels in circulation (Fig.5 B, C).
• TTX sodium channel blocker
• DNQX the AMPA glutamatergic receptor antagonist
• D-AP5 the NMDA glutamatergic receptor antagonist
• bicuculline the GABAA receptor antagonist
• Lac-Phe acts in a cell type-specific manner.
• Lac-Phe can directly act to inhibit orexigenic AgRP neurons and stimulate anorexigenic POMC neurons in brain slices.
• Robust and sustained elevations of circulating Lac-Phe following exercise in humans To determine whether circulating Lac-Phe also exhibited exercise-inducible elevation in humans, Lac-Phe levels were measured in two independent human exercise cohorts.
• Lac-Phe was in fact slightly decreased over the same time course, thereby establishing the exercise-dependent accumulation of Lac-Phe in humans (Fig.6 C). From these data, it is concluded that Lac-Phe is also one of the most robustly increased metabolites in human plasma following an acute bout of treadmill running. To determine the generality of Lac-Phe elevation following human exercise, plasma Lac- Phe levels in a second human exercise cohort consisting of individuals that had each been tested in three distinct exercise trials were measured (endurance, sprint, and resistance, see Methods, Fig.6 D).
• Lac-Phe Blood plasma was collected prior to exercise and at several time points post-exercise, and Lac-Phe was measured from acetonitrile/methanol-extracted plasma by LC-MS/MS. Lac-Phe once again exhibited robust and sustained elevations across all three exercise modalities (Fig.6 E). Sprint exercise exhibited the most dramatic accumulation of plasma Lac-Phe (peaking at 8-fold elevation at 1 h post exercise), followed by resistance training and then endurance training. Because the magnitude of Lac-Phe elevation across exercise modalities matched that of lactate (sprint > resistance > endurance, Fig.11 D), the relationship between Lac-Phe and lactate levels was examined.
• Lac-Phe is an important circulating molecule for the anti-obesity effects of physical activity: (1) elevation of Lac-Phe to levels achieved during acute exercise are sufficient to reduce food intake and obesity; (2) genetic ablation of Lac-Phe biosynthesis in mice confers resistance to the anti-obesity effects of exercise training; and (3) Lac-Phe directly regulates the excitability of neurons previously established to regulate feeding behaviors in vitro. Further supporting these findings, Lac-Phe is identified as one of the most robustly induced molecular changes in blood plasma following diverse physical activity modalities in humans.
• Strenuous exercise has been well-documented to acutely suppress food intake in mice, a behavioral phenotype that correlates with post-exercise suppression and stimulation of neuronal activity in AgRP and POMC neurons, respectively (26–28). While specific candidate molecules, including ghrelin, have been proposed to contribute to these effects, the role of additional exercise-inducible signaling molecules on these food intake pathways has remained poorly understood.
• the data designate Lac-Phe as an exercise-inducible molecule that functions upstream of these critical central pathways that regulate feeding behaviors. In humans, the effects of exercise on appetite and food intake are more complex, depending on study cohort and the type, duration, or intensity of physical activity (29–31).
• Lac-Phe As a circulating regulator of obesity and food intake, Lac-Phe exhibits both similarities and also important differences compared to other peripherally-derived appetite-regulating hormones. Lac-Phe inhibits feeding behaviors just like many other peptide hormones including GLP-1, GDF15, CCK, PYY, and leptin. Both Lac-Phe and GDF15 are produced and secreted from similar cell types and anatomical locations, including macrophages, the intestine, and the kidney (32, 33). However, there are also important differences.
• Lac-Phe is a metabolite (MW ⁇ 250 Da), whereas the other appetite hormones are much larger polypeptides (e.g., GLP-1, MW ⁇ 3000 Da) or proteins (e.g., GDF15, MW ⁇ 15 kDa).
• myocytes and muscle tissues have been a historical focus as a source for circulating molecular transducers of physical activity (37–39). That macrophages and other CNDP2+ cell types produce and secrete Lac-Phe suggests that many additional cell types can sense and respond to physical activity (13, 40). A potential common aspect of these other exercise-responsive cell types might be their ability to directly detect or otherwise import muscle-derived lactate.
• lactate functions as a metabolic precursor for Lac-Phe biosynthesis provides both a circuit as well as a biochemical logic for the robust elevation of Lac-Phe during exercise: namely, that exercise induces lactate secretion from contracting muscles, which is rapidly imported into CNDP2+ cell types and then re-exported back into the circulation in the amidated Lac-Phe form.
• Alternative metabolic derivatizations of lactate could also in principle constitute yet additional undiscovered exercise-inducible signaling molecules.
• Lac-Phe exhibits robust and sustained elevation even at time points when lactate has returned back to baseline levels suggests that biochemical derivatization of lactate functions as an organismal second messenger system in which lactate sensing is coupled with long-lasting endocrine signaling.
• Lac-Phe unlike lactate, cannot be directly re-incorporated back into pathways of primary metabolism.
• the benefits of physical activity extend beyond metabolic health alone. In the brain, exercise enhances mood, reduces anxiety and depression, and protects against dementia and other neurodegenerative diseases (41–43). Lac-Phe, by exhibiting direct action on neurons, represents an attractive candidate factor for mediating some of these additional benefits. Pharmacological targeting of the Lac-Phe pathway may therefore be useful for the treatment of obesity and these other disorders of the nervous system.
• N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids. Proc. Natl. Acad. Sci. U. S. A.112, 6601–6606 (2015). 16. C. Wu, C. Orozco, J. Boyer, M. Leglise, J. Goodale, S. Batalov, C. L. Hodge, J. Haase, J. Janes, J. W. Huss, A. I. Su, BioGPS: An extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol.10, R130 (2009). 17.
• NPY/AgRP neurons are essentials for feeding in adult mice but can be ablated in neonates. Science (80-. ).310, 683–685 (2005). 21. Y. Aponte, D. Atasoy, S. M. Sternson, AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nat. Neurosci.14, 351–355 (2011). 22. G. J. Morton, D. E. Cummings, D. G. Baskin, G. S. Barsh, M. W. Schwartz, Central nervous system control of food intake and body weight. Nature.443, 289–295 (2006). 23.
• a method of treating a metabolic disorder in a subject comprising administering an effective amount of an N-lactoyl-amino acid to the subject.
• the N-lactoyl-amino acid is N-lactoyl-phenylalanine.
• the effective amount comprises an amount of the N-lactoyl-amino acid that when administered produces a plasma concentration of the N-lactoyl-amino acid comparable to that observed after physical activity.
• the method of any of Clauses 1-23, wherein the administering comprises intraperitoneal administration.
• 25. The method of any of Clauses 1-24, wherein the method comprises administering the N-lactoyl-amino acid in combination with one or more therapies for treating the metabolic disorder.
• 26. The method of Clause 25, wherein the one or more therapies comprises an active agent for treating the metabolic disorder.
• 28. The method of any of Clauses 25-27, wherein the one or more therapies comprises a low-calorie diet. 29. The method of any of Clauses 25-28, wherein the one or more therapies comprises surgical intervention. 30.
• a pharmaceutical formulation comprising: an amount of an N-lactoyl-amino acid effective to treat a metabolic disorder; and an excipient.
• 33. The pharmaceutical formulation of any of Clauses 31-32, wherein the effective amount ranges from 1 mg/kg to 500 mg/kg.
• 34. The pharmaceutical formulation of any of Clauses 31-33, wherein the pharmaceutical formulation is a capsule or tablet. 35.
• each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
• all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.
• a range includes each individual member.
• a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
• a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

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