Prediction and treatment of rapidly progressive kidney disease

Abstract

The present disclosure relates to a method for predicting whether a patient is at risk of developing rapidly progressive kidney disease (FPRD) and treating the patient to avoid such risk, comprising determining the concentration of one or more endocannabinoids, including arachidonoylethanolamide (AE), in a whole blood, serum or plasma sample from the patient, and correlating the concentration of POEA in the sample with the likelihood that the patient will develop FPRD.

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to methods for predicting whether a patient is at risk for developing rapidly progressive kidney disease (FPRD) and for treating the patient to avoid such risk. [Background technology]

[0002] (background) Chronic kidney disease (CKD) is highly prevalent, but identifying patients at high risk for rapid CKD progression or rapidly progressive kidney disease (FPRD) in the short term before they reach end-stage renal failure remains a challenge. Whether factors associated with rapid progression vary with diabetic status is also not fully understood. Summary of the Invention [Means for solving the problem]

[0003] Thus, there is a need in the art to identify when a patient will develop rapidly progressive kidney disease so that early therapeutic intervention can be performed.

[0004] (Abstract) In one embodiment, the present disclosure relates to a method for predicting whether a patient is at risk of developing fast progressive kidney disease (FPRD), comprising determining the concentration of one or more endocannabinoids selected from arachidonoylethanolamide (AE), D-erythro sphingosine (DeSph), dihomo-γ-linolenoylethanolamide (DhγLE), docosatetraenoylethanolamide (DE), linoleoylethanolamide (LE), palmitoylethanolamide (PE) and palmitoleoylethanolamine (POEA) in a whole blood, serum or plasma sample from the patient, and correlating the concentration of POEA in the sample with the likelihood that the patient will develop FPRD. The patient may suffer from diabetes or pre-diabetes, and the diabetes may be severe insulin resistant diabetes (SIRD).

[0005] In various embodiments of the present disclosure, the correlating step includes determining that the patient will develop FPRD if the level of POEA in the sample is greater than about 0.1 mg, or the correlating step includes determining that the patient will develop FPRD if the level of POEA in the sample is greater than about the 90th percentile of FPRD in a population of diabetic patients.Furthermore, the correlating step may include determining that the patient will develop FPRD if the level of POEA in the sample is greater than about the 90th percentile of FPRD in a population of patients suffering from pre-diabetes.Furthermore, the correlating step may include predicting that the patient will develop FPRD within 1 year to 5 years if the level of POEA in the sample is greater than about 0.1 mg.

[0006] Another embodiment of the present disclosure includes a method for identifying a diabetic patient who will develop FPRD, comprising determining a concentration of palmitoleoylethanolamide (POEA) in a whole blood, serum, or plasma sample from the patient, and determining that the patient will develop FPRD if the concentration of POEA in the sample is above about the 90th percentile of POEA concentrations in a population of diabetic patients.

[0007] Additionally, embodiments of the present disclosure include a method of treating a patient at increased risk for FPRD, the method comprising: (a) determining the concentration of palmitoylethanolamide (POEA) in a sample of whole blood, serum or plasma from the patient; (b) determining that the patient has an increased risk of developing FPRD if the concentration of POEA in the sample is above about the 90th percentile of POEA concentrations in a population of diabetic patients; and (c) treating the patient with an antagonist of the cannabinoid 1 (CB1) receptor. Includes.

[0008] Yet another aspect of the present disclosure includes a method of treating a patient with SIRD to avoid progression to FPRD, the method comprising: (a) determining the concentration of palmitoylethanolamide (POEA) in a sample of whole blood, serum or plasma from the patient; (b) determining a predicted onset of FPRD based on the concentration of POEA in the sample; and (c) treating the patient with an antagonist of the cannabinoid 1 (CB1) receptor. Includes.

[0009] In various embodiments of the disclosure, treatment with an agonist of the CB1 receptor includes treatment with an antibody that binds to the CB1 receptor or an antigen-binding fragment that binds to the CB1 receptor.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of the embodiments of the present disclosure may be read in conjunction with the following drawings. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows an overview of the cohort, in which 526 age- and sex-matched patients were stratified into severe insulin-resistant diabetes (SIRD; HOMA-IR>5) and non-SIRD (HOMA-IR<5) groups for assessment of endocannabinoid levels.

[0012] [Diagram 2] FIG. 2 shows the chemical structures of seven of the thirteen endocannabinoids that were measured as described herein.

[0013] [Figure 3-1] Figures 3A-C show the distribution of endocannabinoids in the entire cohort, with the y-axis showing the number of patients and the x-axis showing the endocannabinoid levels in relative units. The putative endocannabinoid 3 was later identified as palmitoleoylethanolamide (POEA). [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0014] [Figure 4] FIG. 4 shows that certain endocannabinoids are significantly elevated (p≦0.05) in patients with severe insulin-resistant diabetes (SIRD) versus age-, sex- and BMI-matched subjects without SIRD ("non-SIRD").

[0015] [Diagram 5]Figure 5 shows the association between elevated specific endocannabinoids in SIRD patients and HOMA-IR, blood glucose, age, BMI and baseline glomerular filtration rate (GFR). The presence of a circle at the intersection between a SIRD factor and a specific endocannabinoid indicates a significant association, with larger circles indicating increased significance. Light and dark indicate the magnitude of the effect (darker shading indicates a larger effect). Although not apparent in the grayscale figure, color indicates the direction of the correlation, with red being a positive correlation and blue being a negative correlation.

[0016] [Figure 6] FIG. 6 shows that GFR declines moderately faster over time in patients with SIRD compared to patients without SIRD (left panel), and that GFR declines significantly faster in patients with FPRD compared to patients without FPRD (right panel).

[0017] [Figure 7] FIG. 7 shows that while most endocannabinoids tested (see FIGS. 3A-C) showed some level of association with FPRD, only palmitoleoylethanolamide (POEA) was predictive of FPRD.

[0018] [Figure 8] Figure 8 shows the association between FPRD and other aspects of kidney disease, including glomerular filtration rate (GFR), Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), proteinuria, and combinations thereof. Both p-value and hazard ratio calculations show that FPRD is independent of these factors.

[0019] [Figure 9]Figure 9A shows the Kaplan-Meier curves of the incidence of FPRD in a population of SIRD patients. The population (n=526) was divided into two groups, one including the top 10% of patients stratified by POEA concentration (n=53) and the other including the bottom 90% of patients (n=473). Patients with highly elevated POEA were significantly more likely to develop FPRD than those with lower POEA concentrations (p=0.0021). Figure 9B shows an overview of the patients used for the Kaplan-Meier curves.

[0020] [Figure 10-1] Figures 10A-B show triple quadrupole mass spectrometry of putative endocannabinoid #3, which confirms its identity as palmitoleoylethanolamide (POEA). [Figure 10-2] Same as above.

[0021] [Figure 11] FIG. 11 shows that treatment of patients with highly elevated POEA with nimacimab significantly reduced POEA levels at a dose of 0.6 mg / kg of nimacimab, which is a peripheral antagonist of the CB1 receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] (explanation) Generally, the present disclosure relates to a method for determining, predicting, and / or prognosticating whether a patient is at risk of developing rapidly progressive kidney disease (FPRD), and also relates to treating such patients to delay, prevent, or alleviate FPRD and related symptoms. The method includes determining the concentration of one or more cannabinoids in a whole blood, serum, or plasma sample from the patient. The treatment includes administering a therapeutically effective amount of a cannabinoid 1 (CB1) receptor antagonist to the patient identified in the diagnostic method of the present disclosure.

[0023] Before describing examples of embodiments of the present disclosure in further detail, a number of terms are defined. The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and / or" unless specifically stated otherwise. The phrase "at least one" has the same meaning as the phrase "one or more." Furthermore, the use of the term "including" as well as other forms (e.g., "includes" and "included") is not limiting. Additionally, terms such as "element" or "component" include both elements or components that include one unit and elements or components that include multiple units unless specifically stated otherwise.

[0024] "Fast Progressing Renal Disease" or "FPRD" refers to a patient condition defined as a glomerular filtration rate (GFR) decline of greater than 5 units / year resulting in a GFR of less than 60.

[0025] Cannabinoid 1 (CB1) receptor is a member of the G protein-coupled receptor (GPCR) superfamily.CB1 receptor is expressed in the central nervous system (CNS), lung, liver, adipose tissue and kidney, and has been implicated in many human diseases, including obesity, diabetes, fibrosis, liver disease, cardiovascular disease, cancer, pain, multiple sclerosis spasticity, and glaucoma, among others.Cannabinoid 1 receptor may also be referred to herein as "CB1 receptor" or simply "CB1".

[0026] As used herein, the term "antibody" refers to a binding protein having at least one antigen-binding domain, and the term "antibody" includes monoclonal antibody fragments and / or variants thereof, including recombinant polypeptides, fusion proteins, and immunoconjugates. Thus, the terms "antibody", "antibody fragment", and "antibody variant" are used interchangeably herein. Examples of antibody fragments of the present disclosure include, but are not limited to, Fab fragments consisting of VL, VH, CL, and CHI domains; Fc fragments consisting of VH and CHI domains; Fv fragments consisting of VL and VH; dAb fragments consisting of VH domains; isolated CDR regions; F(ab')2 bivalent fragments containing two linked Fab fragments; and single chain molecules (scFv). The CB1 receptor-binding antibodies provided herein can be generated from any species, including, but not limited to, mouse, rat, rabbit, primate, llama, and human. These CB1 receptor-binding antibodies can be chimeric, humanized, or fully human antibodies.

[0027] The antibodies and antigen-binding fragments thereof disclosed herein are specific for the cannabinoid 1 (CB1) receptor. By "specific for", it is meant that the antibodies and fragments thereof bind to the CB1 receptor with greater affinity (i.e., lower binding affinity Kd value) than any other target. Thus, antibodies and fragments thereof selective for the CB1 receptor bind to the CB1 receptor with greater affinity (i.e., lower binding affinity Kd value) than any other cannabinoid receptor or any other GPCR or any other target. These antibodies and fragments or variants thereof may have binding affinity Kd values ​​for the CB1 receptor in the range of about 0.01 nM to about 500 nM, about 0.02 nM to about 250 nM, about 0.02 nM to about 200 nM, about 0.05 nM to about 100 nM, about 0.05 nM to about 50 nM. The antibodies and fragments thereof may have a binding affinity Kd value for the CB1 receptor of about 500nM, about 250nM, about 200nM, about 150nM, about 100nM, about 75nM, about 50nM, about 25nM, about 10nM, about 5nM, about 1nM, about 500pM, about 250pM, about 100pM, about 50pM, or about 10pM. The antibodies and fragments thereof may have a binding affinity Kd value for the CB1 receptor of about 100nM or less, about 75nM or less, about 50nM or less, about 10nM or less, about 1nM or less, about 500pM or less, or about 100pM or less.

[0028] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Biol. Chem., 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), and Chothia et al., J. Mol. Biol., 196:901-917 (1987) and MacCallum et al., J. Mol. Biol., 262:732-745 (1996), whose definitions include overlapping amino acid residues or subsets of amino acid residues when compared against each other. Kabat's definition is based on sequence variability. The IMGT unique numbering for all IG V-regions and TR V-regions of all species relies on the high conservation of the structure of the variable regions (Lefranc, Mp et al., Dev comp. Immunol., 27:55-77, 2003). The IMGT numbering starts after aligning more than 5,000 sequences and combines them taking into account the framework and CDR definitions. The Clothia definition is based on the location of the structural loop regions. The Contact definition (MacCallum et al.) is based on the analysis of complex crystal structures and antibody-antigen interactions. The amino acid residues that comprise the CDRs defined by each of the above cited references are shown for comparison. In one embodiment disclosed herein, the term "CDR" is a CDR as defined by this Kabat definition. In another embodiment disclosed herein, the CDR is a CDR as defined by IMGT.

[0029] As used herein, the term "antagonist" refers to a compound that inhibits, reduces or prevents the signaling activity of another compound at a receptor site, and more generally, to a compound that reduces or prevents the activation and / or signaling activity of a receptor.

[0030] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventive measures. A subject in need of treatment refers to a subject who already has a disease or disorder, as well as a subject who may develop a disease or disorder, and the purpose is to prevent, delay, or reduce the disease or disorder. The method of "treatment" disclosed herein uses administering to a subject (e.g., a subject who has been determined to be at risk of developing FPRD or a subject who is suspected of being at risk of developing FPRD) an antibody or antigen-binding fragment disclosed herein to prevent, cure, delay, reduce the severity, or alleviate one or more symptoms, or to prolong the subject's survival beyond that expected in the absence of such treatment.

[0031] As used herein, the term "subject" refers to a mammal, such as a rodent, feline, canine, primate, or human.

[0032] "Therapeutically effective amount" as used herein refers to the amount of a compound or composition required to provide therapeutic and / or prophylactic benefit to a subject. The therapeutically effective amount varies depending on the subject and disease state to be treated, the subject's weight and the subject's age, the severity of the disease state, the mode of administration, etc., and can be easily determined by those skilled in the art. The administration regimen can be adjusted to provide an optimal therapeutic response. An effective amount is also an amount in which any toxic or harmful effects (i.e., side effects) of the antibody or its antigen-binding fragment are minimized or outweighed by beneficial effects.

[0033] Now, looking at various aspects herein, the present disclosure includes both a method of diagnosing (e.g., predicting, determining, and / or prognosing) whether a patient is at risk or at risk for developing rapidly progressive kidney disease (FPRD), as well as a method of treating a patient identified by such a diagnosis. The diagnostic aspect of the present disclosure includes determining the concentration of one or more cannabinoids (which may be one or more of the following: arachidonoylethanolamide (AE), D-erythro sphingosine (DeSph), dihomo-γ-linolenoylethanolamide (DhγLE), docosatetraenoylethanolamide (DE), linoleoylethanolamide (LE), palmitoylethanolamide (PE), and palmitoleoylethanolamide (POEA)) in a biological sample from the subject. In an embodiment, the sample is a whole blood, serum, or plasma sample from the patient. The sample may also be a urine sample. Patients identified by the disclosed diagnostic methods can be treated with a therapeutically effective amount of a CB1 receptor antagonist. Many, but not all, patients with FPRD or at risk for FPRD have diabetes, including severe insulin resistant diabetes (SIRD).

[0034] In various aspects of the present disclosure, the cannabinoid measured in a patient sample (e.g., whole blood, plasma, serum, or urine) is POEA. A patient who does not currently show symptoms of FPRD may be determined to be at risk of developing FPRD based on the concentration or amount of cannabinoids in the sample. For example, the concentration of FPRD in the sample may be associated with the patient's risk of developing FPRD. In some embodiments, a quantitative determination of cannabinoids in the sample may be associated with the patient's level of risk of developing FPRD. In other embodiments, the patient is qualitatively determined to be at risk of developing FPRD when the level of cannabinoids in the sample is above a threshold concentration. Furthermore, the method of the present disclosure may predict how long before a patient will experience an onset of FPRD.

[0035] In some embodiments, the present disclosure provides a method for determining a diagnosis or prognosis or a method of treatment in an individual, comprising: i) determining the concentration of an endocannabinoid in a sample from the individual or in a series of samples from the individual; and ii) determining a diagnosis, prognosis, or method of treatment in the individual based on the concentration in the sample or the series of samples. In some embodiments of the methods of the present disclosure, step ii) comprises analysis such as comparing the concentration or series of concentrations to normal values ​​for the endocannabinoid concentration, comparing the concentration or series of concentrations to a predefined threshold level, comparing the concentration or series of concentrations to a baseline value, and determining the rate of change of concentration for the series of concentrations.

[0036] In some embodiments of the method of the present disclosure, step ii) comprises comparing the concentration of the cannabinoid in the sample with a predetermined threshold concentration, and determining a method of diagnosis, prognosis, or treatment if the sample concentration is greater than the threshold level.In some embodiments of the method of the present disclosure, the threshold concentration is determined by determining a threshold percentile concentration of the cannabinoid in a population of individuals suffering from diabetes or pre-diabetes.The threshold concentration can be, for example, the 80th, 85th, 90th, 95th, 96th, 97th, 98th, or 99th percentile of the level of endocannabinoid concentration in the population. In some embodiments of the methods of the present disclosure, the endocannabinoid is selected from arachidonoylethanolamide (AE), D-erythro sphingosine (DeSph), dihomo-γ-linolenoylethanolamide (DhγLE), docosatetraenoylethanolamide (DE), linoleoylethanolamide (LE), palmitoylethanolamide (PE) and palmitoleoylethanolamide (POEA).

[0037] In some embodiments of the disclosed method, the endocannabinoid is POEA and the threshold concentration is the 90th percentile of POEA in a population of patients suffering from diabetes or pre-diabetes. In some embodiments of the disclosed method, the concentration or range of concentrations is determined at or near the time the individual presents to a medical professional with symptoms or potential symptoms of diabetes or SIRD.

[0038] The disclosed method includes correlating the level of endocannabinoids with the risk of a patient (e.g., a patient suffering from diabetes or pre-diabetes) developing FPRD within 1 to 5 years. For example, in one embodiment of the disclosure, a patient is determined to be at risk of developing FPRD if the level of POEA in a plasma sample is greater than about 0.1 mg, about 1.5 mg, or about 2.0 mg. In some embodiments, the patient has diabetes or SIRD.

[0039] (Method of treatment)

[0040] The method of treatment according to the present disclosure includes treating an individual identified as being at risk of developing FPRD.The treatment can include administering to the at-risk individual a therapeutically effective amount of a CB1 receptor antagonist sufficient to prevent FPRD, to prolong the time to onset of FPRD, or to slow the progression of FPRD in patients already suffering from FPRD.

[0041] In one aspect, diagnosis according to the present disclosure may be performed 1 to 5 years prior to the onset of FPRD, particularly 1, 2, 3, 4, or 5 years prior to the onset of a rapid decline in the patient's GFR, which is indicative of FPRD. In some embodiments, treatment according to the present disclosure may postpone the onset of FPRD indefinitely or for up to 20, 10, 5, 4, 3, 2, or 1 year. In patients experiencing FPRD, slowing the progression of FPRD may include slowing the decline in the patient's GFR by 1, 2, 3, 4, or 5 or more units per year.

[0042] Antagonists suitable for treatment according to the present disclosure include anti-CB1 receptor antibodies (or binding fragments thereof). Many anti-CB1 antibodies are known in the art. See, for example, WO2019211665A1, WO2014210205A1 and WO2017058771A1 (each of which is incorporated by reference in its entirety). In addition, many anti-CB1 antibodies are described in the following table and disclosure. In one example, the antibody is nimacimab (Bird Rock Bio, Inc., La Jolla, California, USA), which is disclosed in WO2017058771A1 and further described herein.

[0043] In some embodiments of the present disclosure, patients who are determined to be at risk for FPRD are administered anti-CB1 receptor antibodies weekly, every 2 weeks, every 3 weeks, monthly, every 6 weeks, every 2 months, or once every 3 months or more, if necessary, to prevent or delay the onset of FPRD. Patients suffering from FPRD may receive the antibody on a similar schedule to delay the progression of the disease (including slowing or stopping the decline in the patient's GFR).

[0044] The anti-CB1 antibodies can be delivered to the patient in a variety of dosage forms and routes of administration as further described herein.

[0045] Pharmaceutical Composition

[0046] Administration of an anti-CB1 antibody according to the present disclosure includes administration of a pharmaceutical composition comprising an anti-CB1 antibody or a fragment thereof.

[0047] Methods for preparing the antibody or fragment thereof disclosed herein and administering it to a subject are well known to or easily determined by those skilled in the art. The route of administration of the antibody or fragment thereof disclosed herein can be oral, parenteral, by inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intravenous, intraarterial, subcutaneous, and intramuscular forms of parenteral administration can be used in certain embodiments. All these forms of administration are expressly contemplated as being within the scope disclosed herein, but the form of administration is a solution for injection (particularly for intravenous or intraarterial injection or infusion). Usually, pharmaceutical compositions suitable for injection can include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin), etc. However, in other methods consistent with the teachings herein, the polypeptide may be delivered directly to the site of a harmful cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0048] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, alcoholic / aqueous emulsions or suspensions (including saline), and buffered media. In the present disclosure, pharma- ceutically acceptable carriers include, but are not limited to, 0.01M-0.1M (e.g., 0.05M) phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., Ringer dextrose-based replenishers), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases. More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile, and the composition should be fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage, and in certain embodiments, preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be, for example, a solvent or dispersion medium, including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), as well as suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain embodiments, isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride, are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as aluminum monostearate and gelatin.

[0049] The effective dosage of the antibodies or fragments thereof disclosed herein for treating SIRD or preventing a diabetic patient from progressing to SIRD will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medicaments administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Treatment dosages may be determined using routine methods known to those skilled in the art to optimize safety and efficacy.

[0050] The subject may be administered such dosages daily, every other day, weekly, or according to any other schedule determined by empirical analysis.Exemplary treatments include administration in multiple doses over an extended period of time (e.g., at least 6 months or for the patient's life).Further exemplary treatment regimes include administration once every 2 weeks or once a month or once every 3 to 6 months.

[0051] The interval between single doses can be, for example, daily, weekly, monthly or yearly. The interval can also be irregular as indicated by measuring blood levels of endocannabinoids or therapeutic antibodies in the patient. In some methods, the dosage is adjusted to achieve a particular plasma antibody concentration (e.g., 1 μg / ml to 1000 μg / ml or 25 μg / ml to 300 μg / ml). Alternatively, the antibody or fragment thereof can be administered as a sustained release formulation, which requires less frequent administration. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and non-human antibodies. In one embodiment, the antibody or fragment thereof disclosed herein can be administered in unconjugated form. In another embodiment, the antibody disclosed herein can be administered multiple times in conjugated form. In yet another embodiment, the antibodies or fragments thereof disclosed herein may be administered in an unconjugated form followed by a conjugated form, or may be administered in a conjugated form followed by an unconjugated form.

[0052] Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the antibodies or a cocktail thereof are administered to patients not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose." In this use, the exact amount also depends on the patient's health and general immunity, but generally ranges from 0.1 mg to 250 mg per dose (e.g., 0.1 mg, 1.0 mg, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, or 250 mg per dose). In another embodiment, the patient may receive about 0.1 mg / kg to about 5.0 mg / kg (e.g., about 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg, or 5 mg / kg). Particular examples include 0.6 mg / kg, 1.2 mg / kg, and 2.5 mg / kg. Some patients continue to receive treatment for the rest of their lives.

[0053] In one embodiment, a subject may be treated with a nucleic acid molecule (e.g., in a vector) encoding a polypeptide disclosed herein. A single dose for a nucleic acid encoding a polypeptide ranges from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30 μg to 300 μg of DNA per patient. Dosages for infectious viral vectors vary from 10 virions to 100 virions or more per dose.

[0054] As discussed above, the antibodies or fragments thereof disclosed herein may be administered in a pharmacologic effective amount for the in vivo treatment of mammalian disorders. In this regard, it is understood that the disclosed antibodies or fragments thereof are formulated to facilitate administration and promote the stability of the active agent. In certain embodiments, the pharmaceutical composition according to the present disclosure comprises a pharmacologic acceptable non-toxic sterile carrier (e.g., saline, non-toxic buffers, preservatives, etc.). For the purposes of this application, a pharmacologic effective amount of the antibodies disclosed herein, complexed or uncomplexed with a therapeutic agent, is considered to mean an amount sufficient to achieve effective binding to a target and achieve a benefit (e.g., to alleviate symptoms of a disease or disorder, or to detect a substance or cell). In the case of tumor cells, the polypeptide, in certain embodiments, is capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells, providing increased death of those cells. Of course, the pharmaceutical compositions disclosed herein may be administered in a single dose or multiple doses to provide a pharmacologic effective amount of the polypeptide.

[0055] In accordance with the scope of the present disclosure, the antibody disclosed herein can be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect according to the above-mentioned treatment method. The polypeptide disclosed herein can be administered to such humans or other animals in a conventional dosage form prepared by combining the antibody disclosed herein with a conventional pharmacologic acceptable carrier or diluent according to known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmacologic acceptable carrier or diluent are determined by the amount of active ingredient with which it is combined, the route of administration and other well-known variables. Those skilled in the art will further understand that a cocktail comprising one or more polypeptides according to the present disclosure may prove to be particularly effective.

[0056] In one embodiment, the antibodies and binding fragments or variants thereof disclosed herein may be administered to the subject by at least one route selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intrachondral, intracavitary, intraperitoneal, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intratympanic, intrauterine, intravesical, intravitreal, bolus, subconjunctival, intravaginal, rectal, buccal, sublingual, intranasal, and transdermal.

[0057] (Methods for detecting endocannabinoids)

[0058] The presence or amount of endocannabinoids in patient samples can be achieved by methods known to those skilled in the art. The disclosure herein is not limited to any particular detection method, including liquid chromatography mass spectrometry (LCMS). The examples herein describe the detection of 23 endocannabinoids from plasma samples and healthy controls using LCMS. Once the presence or amount of endocannabinoids is detected, the value can be used to correlate endocannabinoid levels with SIRD status and prognosis of patients who will develop FPRD. In various forms of analysis, logistic regression can be used and confounding factors, including age, sex, and BMI, can be considered. In one example, the association between endocannabinoids and rapidly progressive kidney disease can be determined, and Cox linear regression was used to consider confounding factors, including age, sex, BMI, proteinuria, HOMA-IR, and GFR. Statistical analysis can be performed in R studio.

[0059] (anti-CB1 antibody)

[0060] In addition to the antibodies described herein, in some embodiments, CB1 receptor antibodies are produced by methods known to those skilled in the art, including recovering B cells from immunized mice and generating hybridoma cells that produce CB1 receptor antibodies or using phage libraries.

[0061] In some embodiments, the antibodies and fragments provided herein are chimeric or humanized antibodies. A "chimeric antibody" is an antibody that has at least a portion of the heavy chain variable region and at least a portion of the light chain variable region from one species and at least a portion of the constant region from another species. For example, in one embodiment, a chimeric antibody can contain a mouse variable region and a human constant region. A "humanized antibody" is an antibody that contains a complementarity determining region (CDR) from a non-human antibody and a framework region and a constant region from a human antibody.

[0062] For example, the CB1 antibody provided herein may comprise CDRs derived from one or more mouse antibodies and human framework and constant regions. Thus, in one embodiment, the humanized antibody provided herein binds to the same epitope on CB1 as the mouse antibody from which the antibody's CDRs are derived. Exemplary humanized antibodies are provided herein. Additional humanized CB1 antibodies, or variants thereof, comprising the heavy and light chain CDRs provided herein, can be made using any human framework sequence and are also included in the present disclosure. In one embodiment, framework sequences suitable for use in the present disclosure include framework sequences that are structurally similar to the framework sequences provided herein. In some embodiments, the human frameworks were selected based on the homology of the VH and VK genes between the parent antibody and human germline. The selected frameworks, in some embodiments, have the highest homology with the VH and VK genes of the parent antibody, and they were also predicted to support the CDR structure predicted to be presented by the parent antibody based on computer modeling or other means.

[0063] Further modifications in the framework regions may be made to improve the properties of the antibodies provided herein. Such further framework modifications may include chemical modifications; point mutations to reduce immunogenicity or remove T-cell epitopes; or back mutations to residues in the original germline sequence. In one embodiment of the present disclosure, the humanized antibodies and fragments thereof comprise a human framework and the grafted CDRs provided herein, and do not comprise further modifications in the variable regions. A humanized antibody that does not comprise a human framework back mutation is referred to herein as H0 (e.g., P1C4-H0). In another embodiment of the present disclosure, the humanized antibodies and fragments thereof comprise a human framework and the grafted CDRs provided herein, and the amino acid at position 27 and / or 28 of the heavy chain framework region 1 is back mutated. In a further embodiment, the amino acid at position 27 is back mutated from Gly (G) to Tyr (Y); and the amino acid at position 28 is back mutated from Thr (T) to Glu (E). Humanized antibodies with such mutations at positions 27 and 28 are referred to herein as "H2" or "H2(YE)" (e.g., P1C4-H2 or P1C4-H2(YE)). In another embodiment of the disclosure, the humanized antibodies and fragments thereof comprise a human framework and grafted CDRs as provided herein, in which the amino acids at positions 27 and / or 28 in heavy chain framework region 1 and positions 60 and / or 61 in heavy chain framework region 3 are backmutated. In a further embodiment, the amino acid at position 27 is backmutated from Gly (G) to Tyr (Y); the amino acid at position 28 is backmutated from Thr (T) to Glu (E); the amino acid at position 60 is backmutated from Ala (A) to Asn (N); and the amino acid at position 61 is backmutated from Gln (Q) to Gly (G). A humanized antibody having such mutations at positions 27, 28, 60, and 61 is referred to herein as "H4" or "H4(YENG)" (e.g., P1C4-H4 or P1C4-H4(YENG)).In one embodiment of the disclosure, the antibodies and antigen-binding fragments thereof contain framework modifications (e.g., back mutations) in the light chain. For example, in one embodiment, the antibodies contain mutations at positions 45 and / or 47 of the light chain framework region 2. In a further embodiment, the amino acid at position 45 is mutated from Arg (R) to Lys (K) and the amino acid at position 47 is mutated from Leu (L) to Trp (W). The disclosure also includes humanized antibodies that bind to CB1, which include framework modifications corresponding to the exemplary modifications described herein for any suitable framework sequence, as well as other framework modifications that otherwise modify the properties of the antibody. The CB1 antibodies and fragments thereof disclosed herein may be of IgG1, IgG2, IgG3, or IgG4 isotype, or any combination thereof. The term "isotype" refers to the antibody class encoded by the heavy chain constant region genes. Furthermore, the heavy chain constant region can be derived from any species, including but not limited to mouse, rat, rabbit, hamster, guinea pig, primate, llama, or human. For example, in one embodiment, the CB1 antibodies and fragments thereof of the present disclosure comprise a human IgG1 Fc constant region. In another embodiment, the CB1 antibodies and fragments thereof comprise a human IgG2 Fc constant region, a human IgG4 Fc constant region, or a hybrid IgG2 Fc constant region-IgG4 Fc constant region.

[0064] (Effector functions and Fc modifications)

[0065] In some embodiments, the present disclosure provides a CB1 antibody comprising a variant Fc region. The Fc region of an antibody is the portion of the antibody that binds to Fcγ receptors (FcγR) and the complement molecule C1q. The Fc region plays a role in mediating antibody effector functions. "Effector function" as used herein with respect to antibody Fc refers to antibody functions such as, for example, C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; opsonization; transcytosis; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require that the Fc region is combined with a binding domain (e.g., an antibody variable domain), and such effector functions can be evaluated using various assays known in the art for evaluating such antibody effector functions. A variant Fc region is an Fc region that includes modifications that alter effector functions. In some embodiments, the CB1 antibody provided herein comprises an Fc region modification that reduces, impairs, or eliminates one or more effector functions. For example, in one embodiment, the antibodies and fragments thereof disclosed herein bind to CB1, and they exhibit reduced, impaired, or absent C1q binding and / or CDC and / or ADCC. The Fc modification can be an amino acid insertion, deletion, or substitution, or can be a chemical modification. For example, the Fc region modification can be performed to increase or decrease complement binding; to increase or decrease antibody-dependent cellular cytotoxicity; or to modify glycosylation. Any of the Fc modifications known in the art can be applied to the exemplary CB1 antibody disclosed herein to change effector function. Any of the Fc modifications known in the art can be applied to the CB1 receptor antibody provided herein to change effector function, antibody half-life, or other antibody properties.

[0066] In one embodiment, the CB1 antibody exhibits reduced effector function. In a further embodiment, the CB1 antibody comprises an IgG4 Fc region with a mutation at position 228. In a further embodiment, the amino acid at position 228 is mutated from serine (S) to proline (P) (i.e., S228P). In another embodiment, the CB1 antibody exhibits reduced effector function and the CB1 antibody comprises an IgG2 Fc region with a mutation at position 330 and / or 331. In a further embodiment, the amino acid at position 330 is mutated from alanine (A) to serine (S) and / or the amino acid at position 331 is mutated from proline (P) to serine (S). In a further embodiment, the CB1 antibody comprises an IgG2 Fc domain with both the A330S and P331S mutations. In another embodiment, the CB1 antibody comprises an IgG2 / IgG4 hybrid Fc region. For example, in one embodiment, the CB1 antibody comprises a CH1 and hinge region derived from IgG2 and a CH2 and CH3 region derived from IgG4.

[0067] In one embodiment, the disclosure provides an isolated nucleic acid encoding any one of the antibodies and antigen-binding fragments or variants thereof disclosed herein. In some embodiments, a vector comprising the isolated nucleic acid is provided. In some embodiments, a host cell transformed with the vector is provided. In some embodiments, the host cell is a prokaryotic cell. In further embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is a eukaryotic cell. In further embodiments, the eukaryotic cell is selected from the group consisting of a protist cell, an animal cell, a plant cell, and a fungal cell. In some embodiments, the host cell is a mammalian cell (including but not limited to 293, COS, NS0, and CHO); or a fungal cell (e.g., Saccharomyces cerevisiae); or an insect cell (e.g., Sf9). One embodiment of the disclosure provides a method of producing the antibodies and fragments or variants described herein, the method comprising culturing any one of the host cells herein in a culture medium under conditions sufficient to produce the binding protein.

[0068] In some embodiments, the anti-CB1 receptor antibody provided herein comprises the sequence provided herein or a conservative variant thereof. "Conservative variant" as used herein includes conservative amino acid substitution, amino acid insertion, or amino acid deletion. Those skilled in the art recognize that a conservative amino acid substitution is the replacement of an amino acid with another amino acid that has similar structural or chemical properties (such as similar side chain); conservative amino acid substitution, amino acid insertion, or amino acid deletion results in a sequence that retains the biological activity of the reference sequence.

[0069] Amino acid and nucleic acid sequences of anti-CB1 antibodies useful for treatment according to the present disclosure can be found in Tables 1-9, which are provided herein after the Examples. Experimental data relating to the discovery, testing and validation of these antibodies are available in WO2017058771A1. Additional anti-CB1 antibodies are known and may be useful in the methods of the present disclosure.

[0070] Nimacimab is an anti-CB1 antibody comprising a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO: 352, the CDR2 amino acid sequence of SEQ ID NO: 353, and the CDR3 amino acid sequence of SEQ ID NO: 354, and a light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO: 355, the CDR2 amino acid sequence of SEQ ID NO: 356, and the CDR3 amino acid sequence of SEQ ID NO: 357.

[0071] Additionally, nimacimab comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 340, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 337. The heavy chain amino acid sequence of the antibody is SEQ ID NO: 437, and the light chain amino acid sequence of the antibody is SEQ ID NO: 338.

[0072] In other embodiments, the anti-CB1 antibody is an antibody similar to nimacimab, but containing at least one mutation identified in Table 7. Additionally, other antibodies useful in the methods of the disclosure include the following combinations of heavy and light chain variable regions: (Combination 1) a. a heavy chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 352, a CDR2 amino acid sequence of SEQ ID NO: 353, and a CDR3 amino acid sequence of SEQ ID NO: 354, and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:694, and the CDR3 amino acid sequence of SEQ ID NO:836. (Combination 2) a. a heavy chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 352, a CDR2 amino acid sequence of SEQ ID NO: 353, and a CDR3 amino acid sequence of SEQ ID NO: 354, and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:835, and the CDR3 amino acid sequence of SEQ ID NO:836. (Combination 3) a. a heavy chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 352, a CDR2 amino acid sequence of SEQ ID NO: 353, and a CDR3 amino acid sequence of SEQ ID NO: 354, and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:694, and the CDR3 amino acid sequence of SEQ ID NO:357. (Combination 4) a. a heavy chain variable region comprising a CDR1 amino acid sequence of SEQ ID NO: 352, a CDR2 amino acid sequence of SEQ ID NO: 353, and a CDR3 amino acid sequence of SEQ ID NO: 354, and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:834, and the CDR3 amino acid sequence of SEQ ID NO:779.

[0073] Combination 1 may comprise the light chain set forth in SEQ ID NO: 829, combination 2 may comprise the light chain set forth in SEQ ID NO: 830, combination 3 may comprise the light chain set forth in SEQ ID NO: 831, and combination 4 may comprise the light chain set forth in SEQ ID NO: 832, which may further comprise the heavy chain set forth in SEQ ID NO: 437.

[0074] Further exemplary CB1 receptor binding antibodies of the disclosure are provided herein below in Tables 1-9 and as described in WO2017058771A1. EXAMPLES

[0075] (Example) The following examples illustrate specific embodiments of the present disclosure and various uses thereof. The following examples are presented for illustrative purposes and should not be construed in any way as limiting the scope of the present disclosure.

[0076] Example 1 - Cohort Selection

[0077] A subset of 526 plasma samples was selected from a community cohort of >5000 individuals. 429 of these samples were SIRD individuals (defined as HOMA-IR>5.0). 97 control (non-SIRD) samples were also included, as described in Figure 1.

[0078] Example 2 - Sample Preparation

[0079] Plasma samples were prepared in a 96-well plate format using an automated liquid handler. Samples were protein precipitated by the addition of chilled organic solvent (methanol + 0.1% acetic acid) containing six isotopically labeled internal standards. The samples were then vortexed, centrifuged at 4°C, the supernatant removed, and dried under vacuum at 40°C. Prior to analysis, samples were resuspended in methanol:water (80:20) containing 0.1% acetic acid, heat sealed, and stored in the autosampler at 4°C awaiting impending injection.

[0080] Example 3 - Liquid Chromatography Tandem Mass Spectrometry

[0081] An Agilent Ultivo triple quadrupole mass spectrometer coupled to a 1290 infinity II liquid chromatography system was equipped with a Phenomenex Kinetex 1.7 μm C18 100A, 100×2.1 mm column and a Waters Acquity UPLC BEH Shield RP18 1.7 μm, 2.1×5 mm guard column and operated in ESI positive mode during the acquisition period. A linear gradient was used with a flow rate of 0.4 mL / min, consisting of water with 0.1% formic acid for mobile phase A and acetonitrile (ACN) 0.1% formic acid for mobile phase B.

[0082] A subset of the endocannabinoid structures measured is shown in Figure 2, and experimental results are shown in Figures 3A-C. In addition to the confirmed endocannabinoids (Figure 2, excluding palmitoleoylethanolamide (POEA)), several other unknown compounds were measured in the SIRD population ("putative endocannabinoids" 1a, 1b, 3, 7, 8, 9, 10; Figures 3A-C). The identity of putative endocannabinoid 3 was confirmed as palmitoleoylethanolamide (POEA) by liquid chromatography and triple quadrupole mass spectrometry (Figures 10A-B).

[0083] Example 4 - Statistical Analysis

[0084] For the association between endocannabinoid levels and SIRD status, logistic regression was used, accounting for confounding factors including age, sex, and BMI. For the association between endocannabinoids and rapidly progressive kidney disease, Cox linear regression was used, accounting for confounding factors including age, sex, BMI, proteinuria, HOMA-IR, and GFR. All statistical analyses were performed in R studio.

[0085] As shown in Figure 4, six circulating endocannabinoids were significantly associated with SIRD status (adjusted for age, sex, and BMI), including arachidonoylethanolamide (AE), D-erythro sphingosine (DeSph), dihomo-γ-linolenoylethanolamide (DhγLE), docosatetraenoylethanolamide (DE), linoleoylethanolamide (LE), and palmitoylethanolamide (PE). All endocannabinoids were higher in SIRD individuals than in non-SIRD controls (p<0.05). This indicated that circulating plasma endocannabinoid levels were significantly altered in individuals with SIRD.

[0086] As shown in Figure 5, of these six endocannabinoids, AE, PE, LE, DhγLE, and DE showed significant associations with BMI (p<0.05, effect size>0), while AE, LE, and DhγLE were associated with baseline GFR (p<0.05), but with more modest effect sizes (greater than 0 but less than 0.2).

[0087] Example 5 - Endocannabinoid Markers for Rapidly Progressive Renal Disease (FPRD)

[0088] Of the 429 SIRD samples (HOMA-IR>5.0) included in the study, 11.9% (n=51) of these individuals went on to develop FPRD (defined as GFR decline >5 units / year and end GFR<60; Figure 6).

[0089] One endocannabinoid (palmitoleoylethanolamide (POEA; "putative endocannabinoid 3")) was significantly associated with FPRD development, adjusting for age, sex, and BMI (p<0.01, hazard ratio 1.34; Figure 7, left panel). POEA continued to show a significant association with FPRD, independent of baseline GFR, HOMA-IR, and proteinuria (p<0.01 and hazard ratio>1.3; Figure 8). Individuals with elevated POEA at baseline had a significantly higher risk (p<0.01) of FPRD events over 6 years, as shown in the Kaplan-Meier curves in Figure 9A, indicating that POEA may be a useful biomarker for predicting FPRD events and / or identifying high-risk individuals in certain subsets of SIRD individuals who go on to develop FPRD.

[0090] Example 6 - Effect of nimacimab on POEA

[0091] Patients received nimacimab at doses of 0.6 mg / kg, 1.2 mg / kg, or 2.5 mg / kg intravenously (IV) weekly for four doses (Figure 11).

[0092] The clinical dose of nimacimab that showed one response was 0.6 mg / kg. Clinical doses of 1.2 g / kg and 2.5 g / kg were also tested, but those cohorts did not achieve high POEA levels in their subjects at baseline.

[0093] Now that the present disclosure has been described in detail with reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the disclosure as defined in the appended claims. More particularly, although certain aspects of the present disclosure have been identified herein as being particularly advantageous, it is contemplated that the present disclosure will not necessarily be limited to these particular aspects of the disclosure.

[0094] The amino acid and nucleic acid sequences of the disclosure follow herein below. (array) Table 1. Nucleic acid and amino acid sequences of the heavy and light chain variable regions of exemplary CB1 receptor binding antibodies [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] Table 2. SEQ ID NOs of additional CB1 receptor binding antibodies [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 3. Exemplary humanized antibody sequences. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Table 4. Fc variant design [Table 4-1] [Table 4-2] [Table 4-3] Table 5. Permissible mutations within the PA13R3-P1C4 heavy chain CDRs [Table 5-1] [Table 5-2] Table 6. Permissible mutations within the PA13R3-P1C4 light chain CDRs [Table 6] Table 7. Sequence of permissible mutations within the PA13R3-P1C4 CDRs [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] Table 8. Sequences of light chain affinity matured P1C4 clones. [Table 8] Table 9. Summary of amino acid changes in affinity matured clones. [Table 9]

Claims

1. 1. A method for predicting whether a patient is at risk for developing rapidly progressive kidney disease (FPRD), comprising: determining the concentration of one or more endocannabinoids selected from arachidonoylethanolamide (AE), D-erythro sphingosine (DeSph), dihomo-gamma-linolenoylethanolamide (DhγLE), docosatetraenoylethanolamide (DE), linoleoylethanolamide (LE), palmitoylethanolamide (PE) and palmitoleoylethanolamine (POEA) in a sample of whole blood, serum or plasma from the patient; and Correlating the concentration of POEA in the sample with the likelihood that the patient will develop FPRD. A method comprising:

2. 10. The method of claim 1, wherein the patient is suffering from diabetes or a pre-diabetic condition.

3. 3. The method of claim 2, wherein the diabetes is severe insulin resistant diabetes (SIRD).

4. The method of any one of claims 1 to 3, wherein the step of correlating comprises determining that the patient suffers from FPRD if the level of POEA in the sample is greater than about 0.1 mg.

5. 2. The method of claim 1, wherein the correlating step comprises determining that the patient will develop FPRD if the level of POEA in the sample is above about the 90th percentile for FPRD in a population of diabetic patients.

6. 3. The method of claim 2, wherein the associating step comprises determining that the patient will develop FPRD if the level of POEA in the sample is above about the 90th percentile for FPRD in a population of patients suffering from a prediabetic condition.

7. 2. The method of any preceding claim, wherein the correlating step comprises predicting that the patient will develop FPRD within 1 year to 5 years if the level of POEA in the sample is above about 0.1 mg.

8. 1. A method for identifying a diabetic patient who will develop FPRD, comprising: determining the concentration of palmitoleoylethanolamide (POEA) in a sample of whole blood, serum or plasma from the patient; and determining that the patient will develop FPRD if the concentration of POEA in the sample is greater than about the 90th percentile of POEA concentrations in a population of diabetic patients; A method comprising:

9. 1. A method of treating a patient at increased risk for FPRD, comprising: a. determining the concentration of palmitoylethanolamide (POEA) in a sample of whole blood, serum or plasma from the patient; b. determining that the patient has an increased risk of developing FPRD if the concentration of POEA in the sample is greater than about the 90th percentile of POEA concentrations in a population of diabetic patients; and c. Treating the patient with an antagonist of the cannabinoid 1 (CB1) receptor. A method comprising:

10. 1. A method of treating a patient having SIRD to avoid progression to FPRD, comprising: a. determining the concentration of palmitoylethanolamide (POEA) in a sample of whole blood, serum or plasma from the patient; b. determining a predicted onset of FPRD based on the concentration of POEA in the sample; and c. Treating the patient with an antagonist of the cannabinoid 1 (CB1) receptor. A method comprising:

11. The antagonist is an antibody or antigen-binding fragment thereof that binds to the CB1 receptor, the antibody or antigen-binding fragment thereof comprising: a. a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:352, the CDR2 amino acid sequence of SEQ ID NO:353, and the CDR3 amino acid sequence of SEQ ID NO:354; and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:355, the CDR2 amino acid sequence of SEQ ID NO:356, and the CDR3 amino acid sequence of SEQ ID NO:

357. The method of claim 9 or claim 10, comprising:

12. the antibody or antigen-binding fragment being a. a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:340 or SEQ ID NO:341, and b. a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 337 The method of claim 9 or claim 10, comprising:

13. the antibody or antigen-binding fragment being a. a heavy chain amino acid sequence comprising SEQ ID NO:437, and b. A light chain amino acid sequence comprising SEQ ID NO:338 The method of claim 9 or claim 10, comprising:

14. The method of claim 10, wherein the antibody or antigen-binding fragment has at least one mutation identified in Table 7 or Table 6.

15. The antagonist is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a. a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:352, the CDR2 amino acid sequence of SEQ ID NO:353, and the CDR3 amino acid sequence of SEQ ID NO:354; and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:694, and the CDR3 amino acid sequence of SEQ ID NO:

836. The method of claim 9 or claim 10, comprising:

16. The antagonist is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a. a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:352, the CDR2 amino acid sequence of SEQ ID NO:353, and the CDR3 amino acid sequence of SEQ ID NO:354; and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:835, and the CDR3 amino acid sequence of SEQ ID NO:

836. The method of claim 9 or claim 10, comprising:

17. The antagonist is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a. a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:352, the CDR2 amino acid sequence of SEQ ID NO:353, and the CDR3 amino acid sequence of SEQ ID NO:354; and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:694, and the CDR3 amino acid sequence of SEQ ID NO:

357. The method of claim 9 or claim 10, comprising:

18. The antagonist is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a. a heavy chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:352, the CDR2 amino acid sequence of SEQ ID NO:353, and the CDR3 amino acid sequence of SEQ ID NO:354; and b. A light chain variable region comprising the CDR1 amino acid sequence of SEQ ID NO:833, the CDR2 amino acid sequence of SEQ ID NO:834, and the CDR3 amino acid sequence of SEQ ID NO:

779. The method of claim 9 or claim 10, comprising:

19. The method of claim 15, wherein the antibody or antigen-binding fragment thereof comprises a light chain set forth in SEQ ID NO:

829.

20. The method of claim 16, wherein the antibody or antigen-binding fragment thereof comprises a light chain set forth in SEQ ID NO:

830.

21. The method of claim 17, wherein the antibody or antigen-binding fragment thereof comprises a light chain set forth in SEQ ID NO:

831.

22. The method of claim 18, wherein the antibody or antigen-binding fragment thereof comprises a light chain set forth in SEQ ID NO:

832.

23. The method of any one of claims 19 to 22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:437.

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