Compositions for intermittent administration of calmodulin inhibitors

Abstract

Provided herein are compositions and dosing regimens of a calmodulin inhibitor and a cytochrome p450 inhibitor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 359,350 filed on July 8, 2022 and U.S. Provisional Application No. 63 / 456,017 filed on March 31, 2023, each of which is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure also provides a method of administering a combination of a calcineurin inhibitor (e.g., cyclosporine) and a cytochrome p450 enzyme inhibitor (e.g., ritonavir) in intermittent dosing (e.g., once a week) to reduce, prevent the onset of, or slow the development of an autoimmune, alloimmune, inflammatory, and / or mitochondrial disorder. In some embodiments, the methods and compositions described herein can be used to reduce, slow, or prevent the onset of an autoimmune, alloimmune, and inflammatory disorder associated with atopy, perianal fistula, canine pemphigus foliaceus, sebaceous adenitis, granulomatous meningoencephalitis, inflammatory bowel disease, or stomatitis. Background Art

[0004] Calcineurin inhibitors are used as treatments for a variety of alloimmune, autoimmune and inflammatory disorders in both humans and non-human animals. They have also shown promise as treatments for disorders associated with mitochondrial dysfunction through their effects on mitochondrial flux [Fournier et al.]. However, their use is limited by the frequency of side effects associated with them and the frequency with which patients need to take their doses [Azzi et al.]. Summary of the invention

[0005] Calcineurin inhibitors are commonly used as treatments for disorders related to the immune system, including, for example, cyclosporine for psoriasis [Ellis et al.] or tacrolimus for preventing liver transplant rejection [Haddad et al.]. However, these treatments are limited by the side effects associated with calcineurin inhibitors and the frequency with which patients need to take their doses. In cats, for example, cyclosporine is associated with nausea and must be taken at least once daily [Roberts et al.], while tacrolimus is associated with anemia and must also be taken twice daily [Kyles et al.]. Therefore, there is a need for new calcineurin inhibitor formulations that can alleviate these undesirable conditions.

[0006] Calcineurin inhibitors are metabolized via cytochrome p450 enzymes. Without being bound by theory, the present disclosure encompasses the insight that cytochrome p450 inhibitors can result in a longer half-life of the calcineurin inhibitor in the body and a slower rate of decline in the levels of the calcineurin inhibitor in the bloodstream when administered simultaneously with the calcineurin inhibitor [Dresser et al].

[0007] In one aspect, the present disclosure provides a method for alleviating autoimmunity, alloimmunity, inflammatory disorders and mitochondrial disorders, the method comprising administering a calcineurin inhibitor or a pharmaceutically acceptable salt thereof and a cytochrome p450 inhibitor or a pharmaceutically acceptable salt thereof to a subject or a biological sample. In some embodiments, the autoimmunity, alloimmunity or inflammatory disorder is associated with elevated levels of lymphokines or interleukins. In some embodiments, the mitochondrial disorder is associated with a mitochondrial permeability transition pore (MPTP).

[0008] In another aspect, the present disclosure provides a method for preventing side effects associated with the metabolism of a calcineurin inhibitor, the method comprising contacting calcineurin with a calcineurin inhibitor or a pharmaceutically acceptable salt thereof; and contacting cytochrome p450 with a cytochrome p450 inhibitor or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the calcineurin inhibitor is selected from the group consisting of cyclosporin, tacrolimus, and pimecrolimus, and analogs or derivatives thereof. In some embodiments, the calcineurin inhibitor is cyclosporin.

[0010] In some embodiments, the calcineurin inhibitor is administered orally at a dose of about 0.2 mg / kg body weight / week.

[0011] In some embodiments, the cytochrome p450 inhibitor is amiodarone, chloroquine, cimetidine, clomipramine, diphenhydramine, fluoxetine, fluphenazine, haloperidol, paroxetine, perphenazine, propafenone, propoxyphene, quinacrine, quinidine, sertraline, terbinafine, thioridazine, amiodarone, amprenavir, clarithromycin, danazol, delavirdine, diltiazem, efavirenz, erythromycin, ethinyl estradiol, fluconazole, fluvoxamine, grapefruit juice, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, quinine, ritonavir, saquinavir, synercid, troleandomycin, verapamil or zafirlukast. In some embodiments, the cytochrome p450 inhibitor is ritonavir.

[0012] In some embodiments, the cytochrome p450 inhibitor is administered at a dose of about 1 mg / kg / week.

[0013] In certain aspects, the present disclosure provides a method of treating an autoimmune, alloimmune, inflammatory, or mitochondrial disorder, the method comprising identifying a subject experiencing one or more of those disorders; administering to the subject once weekly a calcineurin inhibitor, or a pharmaceutically acceptable salt thereof; and a cytochrome p450 inhibitor, or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION

[0014] Calcineurin inhibitors

[0015] Calcineurin inhibitors are widely used in autoimmune, alloimmune and inflammatory disorders. For example, cyclosporine is used to prevent organ transplant rejection, an alloimmune disorder, and is used to treat chronic idiopathic urticaria, an inflammatory disorder. Without being bound by theory, it is understood that the mechanism by which calcineurin inhibitors are able to treat these disorders is by binding to the cytosolic protein cyclophin in lymphocytes, thereby inhibiting calcineurin in the calcineurin-phosphatase pathway. This reduces the activity of T cells, an important type of white blood cell. This can also have a largely down-regulatory effect on the immune system [Reynolds et al.].

[0016] Calcineurin inhibitors have also been used to treat disorders associated with mitochondrial dysfunction. For example, cyclosporine has been used to treat muscular dystrophy (a disorder associated with mitochondrial dysfunction) [Hicks et al.]. Without being bound by theory, it is understood that the mechanism by which calcineurin inhibitors treat mitochondrial disorders is by inhibiting MPTP, thereby increasing mitochondrial viability [Halestrap et al.].

[0017] Unfortunately, high levels of calcineurin inhibitors may also lead to side effects. For example, it has been found that blood concentrations of cyclosporine greater than 250 ng / mL can lead to long-term side effects in severe ulcerative colitis, including hypertension and nephrotoxicity [Pham et al.]. However, this may be difficult to maintain because blood concentrations of cyclosporine spike within the first 2 hours after dosing and then decline rapidly within 4 hours after dosing [Gomez et al.]. Similar results have been found with other calcineurin inhibitors.

[0018] It has been previously discovered that if a calcineurin inhibitor (e.g., cyclosporine) is given some time after a cytochrome p450 inhibitor (e.g., itraconazole), the dosing schedule for cyclosporine can be extended to up to 4 hours. However, it was thought that the only way to extend the dosing schedule for cyclosporine to a once-daily regimen was by co-administering the calcineurin inhibitor and cytochrome p450 inhibitor with cola [Wimberley et al].

[0019] Furthermore, it was previously thought that the only way to improve the pharmacokinetic linearity of calcineurin inhibitors was by changing the formulation of the calcineurin inhibitor, such as the cyclosporine microemulsion formulation known as Neoral [Mueller et al].

[0020] The present disclosure encompasses the insight that cytochrome p450 inhibitors, if administered simultaneously or nearly simultaneously with a calcineurin inhibitor, can allow for a longer sustained concentration of calcineurin in the bloodstream and a less dramatic drop in concentration within the first 4 hours of administration. Calcineurin inhibitors are metabolized by cytochrome p450, and the inhibition of cytochrome p450 in combination with an active drug results in a longer drug half-life.

[0021] The present disclosure also encompasses the insight that administering a cytochrome p450 inhibitor simultaneously or nearly simultaneously in combination with a calcineurin inhibitor would allow for dosing frequencies of, for example, once a week, twice a week, etc., thereby improving patient compliance.

[0022] The presently claimed methods and compositions alleviate a variety of inflammatory, alloimmune, autoimmune and / or mitochondrial disorders.

[0023] The present disclosure encompasses the insight that the combination of inhibition of calcineurin and inhibition of cytochrome p450 can reduce inflammation, alloimmunity and autoimmunity associated with high lymphocyte levels in some embodiments. The present disclosure also encompasses the insight that the combination of inhibition of calcineurin and inhibition of cytochrome p450 can reduce mitochondrial dysfunction.

[0024] Composition

[0025] In certain aspects, the methods described herein include the manufacture and use of pharmaceutical compositions and medicaments comprising a compound identified as an active ingredient by the methods described herein. Also included are the pharmaceutical compositions themselves.

[0026] In one or more embodiments, the calcineurin inhibitor is selected from the group consisting of cyclosporin, tacrolimus, and pimecrolimus, and analogs or derivatives thereof. In some embodiments, the calcineurin inhibitor is cyclosporin.

[0027] In one or more embodiments, the cytochrome p450 inhibitor is amiodarone, chloroquine, cimetidine, clomipramine, diphenhydramine, fluoxetine, fluphenazine, haloperidol, paroxetine, perphenazine, propafenone, propoxyphene, quinacrine, quinidine, ritonavir, sertraline, terbinafine, thioridazine, amiodarone, amprenavir, clarithromycin, danazol, delavirdine, diltiazem, efavirenz, erythromycin, ethinyl estradiol, fluconazole, fluvoxamine, grapefruit juice, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, quinine, ritonavir, saquinavir, sinostatin, troleandomycin, verapamil or zafirlukast. In some embodiments, the cytochrome p450 inhibitor is ritonavir.

[0028] In some embodiments, the compositions disclosed herein include other compounds, drugs and / or agents for treating alloimmunity, autoimmunity and inflammatory diseases. For example, in some cases, the compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five or less than ten) compounds.

[0029] In some cases, the compositions disclosed herein are formulated for use as pharmaceutical compositions or for use in pharmaceutical compositions. Such compositions are formulated or adapted to be administered to a subject via any route (e.g., any route approved by the Food and Drug Administration (FDA)). Exemplary methods are described in FDA's CDER Data Standards Manual Version 004 (which can be obtained at fda.give / cder / dsm / DRG / drg00301.htm). Pharmaceutical compositions described herein can be formulated for oral, parenteral or transdermal delivery. The compounds disclosed herein can also be combined with other pharmaceutical agents.

[0030] In some aspects, the present disclosure provides a kit comprising one or more compositions comprising cyclosporine and / or ritonavir and / or itraconazole (in separate compositions or in a single composition). The kit may also include instructions for physicians and / or patients, syringes, needles, boxes, bottles, vials, etc.

[0031] In some cases, the methods described herein include administering an effective amount of one or more compositions as described above, comprising a calcineurin inhibitor and a cytochrome p450 inhibitor (as part of a single composition, or as separate compositions). As used herein, the terms "effective amount" and "effective treatment" refer to an amount or concentration of one or more drugs that effectively induces a desired effect or physiological result within its administration range over a period of time (including acute or chronic administration and periodic or continuous administration).

[0032] In some cases, the composition comprises a calcineurin inhibitor (e.g., cyclosporin), a cytochrome p450 inhibitor (e.g., ritonavir), and a pharmaceutically acceptable carrier, adjuvant, and / or vehicle. In some cases, the composition described herein further comprises one or more additional therapeutic agents in an amount effective to achieve modulation of a disease or disease symptom.

[0033] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compounds of the present disclosure and does not destroy the pharmacological activity of the compounds of the present disclosure when administered in a dose sufficient to deliver a therapeutic amount of the compounds and is non-toxic. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with drug administration.

[0034] The composition is typically formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0035] The composition can be in the form of a solution or powder for inhalation and / or nasal administration. Such compositions can be prepared using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil including synthetic monoglycerides or diglycerides can be used. Fatty acids (such as oleic acid and its glyceride derivatives) can be used for the preparation of injections, and natural pharmaceutically acceptable oils, such as olive oil or castor oil (especially in their polyoxyethylene forms) can also be used for the preparation of injections. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tweens or Span and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

[0036] The composition can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions and / or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier and / or suspending agent. If necessary, certain sweeteners and / or flavoring agents and / or coloring agents can be added.

[0037] Alternatively or additionally, the pharmaceutical composition may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0038] In some embodiments, the present disclosure provides methods for administering a composition comprising a calcineurin inhibitor (e.g., cyclosporine) and a composition comprising a cytochrome p450 inhibitor (e.g., ritonavir), each of which comprises a pharmaceutical composition disclosed herein (hereinafter denoted as 'X') in the following methods: Substance X for use as a medicament for treating one or more diseases or conditions disclosed herein (e.g., inflammation, referred to as 'Y' in the following examples). Use of substance X in the manufacture of a medicament for treating Y; and substance X for treating Y.

[0039] In some cases, the therapeutic compositions disclosed herein may be formulated for sale in, imported into, and / or exported from the United States.

[0040] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0041] dose

[0042] In some embodiments, the method of treating an autoimmune, alloimmune, or inflammatory disorder comprises administering a calcineurin inhibitor and a cytochrome p450 inhibitor.

[0043] In some embodiments, the calcineurin inhibitor and the cytochrome p450 inhibitor are administered contemporaneously.

[0044] In some embodiments, the calcineurin inhibitor and the cytochrome p450 inhibitor are administered sequentially.

[0045] In some aspects of the disclosure, the calcineurin inhibitor and the cytochrome p450 inhibitor are administered separately (ie, in separate dosage forms).

[0046] In some embodiments, the calcineurin inhibitor is administered in an amount of about 0.1 mg / kg body weight / week, about 0.5 mg / kg body weight / week, about 1 mg / kg body weight / week, about 2 mg / kg body weight / week, or about 4 mg / kg body weight / week. In some embodiments, the cytochrome p450 inhibitor is administered in an amount of 1 mg / kg / week, 2 mg / kg / week, 5 mg / kg / week, or 10 mg / kg / week. In some embodiments, the cytochrome p450 inhibitor is administered in an amount of 5 mg / week, 10 mg / week, or 20 mg / week. The compound can be administered separately or together, including as part of a treatment regimen.

[0047] In some aspects of the present disclosure, the calcineurin inhibitor is cyclosporin and the cytochrome p450 inhibitor is ritonavir. In some aspects, cyclosporin and ritonavir are administered separately (i.e., separate dosage forms). In some embodiments, cyclosporin is administered in an amount of about 0.1 mg / kg body weight / week, about 0.5 mg / kg body weight / week, about 1 mg / kg body weight / week, about 2 mg / kg body weight / week, or about 4 mg / kg body weight / week. In some embodiments, ritonavir is administered in an amount of 1 mg / kg / week, 2 mg / kg / week, 5 mg / kg / week, or 10 mg / kg / week. In some embodiments, ritonavir is administered in an amount of 5 mg / week, 10 mg / week, or 20 mg / week. Compounds as described herein can be administered separately or together, including as part of a treatment regimen.

[0048] In some embodiments, cyclosporin is administered in an amount of about 1.0-2.5 mg / kg. In some embodiments, cyclosporin is administered in an amount of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / kg. In some embodiments, cyclosporin is administered in an amount of about 5-15 mg. In some embodiments, cyclosporin is administered in an amount of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 29 mg. In some embodiments, ritonavir is administered in an amount of 5 mg, 10 mg, or 20 mg. The compound can be administered separately or together, including as part of a treatment regimen. In some embodiments, a single dose of cyclosporine and ritonavir is administered, followed by administration of a different dose after a period of time (eg, one, two, three, four, five, six, or one week).

[0049] In some embodiments, the subject receives a specific dose in a few days, weeks, months or years. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor according to an intermittent dosing regimen (for example, a frequency less than once a day, such as once every other day, twice a week, once a week, twice a month, once a month, twice a year, once a year, etc.). In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor every other day. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor twice a week. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor once a week. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor once a week. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor once a week. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor twice a month. In some embodiments, the subject receives a calcineurin inhibitor and a cytochrome inhibitor once a month. In some embodiments, the subject receives the calcineurin inhibitor and the cytochrome inhibitor every other month. In some embodiments, the subject receives the calcineurin inhibitor and the cytochrome inhibitor twice a year. In some embodiments, the subject receives the calcineurin inhibitor and the cytochrome inhibitor once a year.

[0050] In some embodiments, the calcineurin inhibitor is cyclosporin and the cytochrome p450 inhibitor is ritonavir. In some embodiments, cyclosporin and ritonavir or itraconazole are administered separately or together as a single daily dose on a daily basis, a weekly basis, or some other basis. In some embodiments, the subject receives a specific dose over a period of weeks, months, or years. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc.

[0051] Treatment

[0052] The methods described herein include methods for treating disorders associated with elevated lymphocyte levels (e.g., organ transplant rejection) or mitochondrial dysfunction (e.g., muscular dystrophy). Typically, the methods include administering a therapeutically effective amount of a calcineurin inhibitor (e.g., cyclosporin) in combination with a cytochrome p450 inhibitor as described herein (e.g., itraconazole) to a subject (e.g., a mammalian subject, such as a human subject) who is in need of such treatment or has been determined to be in need of such treatment.

[0053] In some cases, method can include selecting a human subject who has or has had an illness or disease. In some cases, suitable subjects include, for example, a subject who has or has had an illness or disease but has made the disease or its aspects disappear, presents disease symptoms and reduces (for example, relative to other subjects (for example, most subjects) suffering from the same illness or disease), and / or survives an extended period of time under the illness or disease (for example, relative to other subjects (for example, most subjects) suffering from the same illness or disease), for example, in an asymptomatic state (for example, relative to other subjects (for example, most subjects) suffering from the same illness or disease).

[0054] The methods disclosed herein can be applied to a wide range of species, such as humans, non-human primates (eg, monkeys), horses, cows, pigs, sheep, deer, elk, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

[0055] The terms "treat", "treating", or "treatment" as applied to isolated cells include subjecting cells to any kind of process or condition or performing any kind of manipulation or procedure on cells. The term "treating" as applied to a subject means providing medical or surgical attention, care, or management to an individual who is typically ill or injured or at increased risk of illness relative to the average member of a population and who is in need of such attention, care, or management.

[0056] In some embodiments, the term "treating" or "treatment" refers to administering an effective amount of a composition, such as a composition comprising a calcineurin inhibitor and a composition comprising a cytochrome p450 inhibitor, to a subject so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, such as a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (partial or complete), whether detectable or undetectable. Treatment may refer to prolonged survival compared to expected survival when not receiving treatment. Therefore, those skilled in the art recognize that treatment may improve the disease state, but may not be a complete cure of the disease. In some embodiments, treatment may be a preventive treatment, in which a composition as disclosed herein is administered to a subject at risk of developing inflammation as disclosed herein. In some embodiments, treatment is "effective" if the progression of the disease is reduced or stopped.

[0057] The term "subject" as used herein refers to any animal. In some cases, the subject is a mammal. In some cases, as used herein, the term "subject" refers to a person (e.g., a man, a woman, or a child). In some embodiments, the "subject" is a non-human animal, such as a non-human primate (e.g., a monkey), a horse, a cow, a pig, a sheep, a deer, an elk, a goat, a dog, a cat, a rabbit, a guinea pig, a hamster, a rat, and a mouse.

[0058] In some cases, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample to obtain an indication that the subject is suitable for selection. In some cases, the subject can be, for example, confirmed or identified by a health care professional as having or suffering from an illness or disease. In some cases, the positive immune response to an illness or disease can be shown from patient records, family history, and / or an indication of a positive immune response detected. In some cases, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate, and a second party can test the sample. In some cases, the subject can be selected and / or recommended by a practitioner (e.g., a general practitioner). In some cases, subject selection can include obtaining a sample from a selected subject and storing the sample and / or using methods disclosed herein. The sample can include, for example, a cell or a cell population.

[0059] In some cases, the method of treatment may include a single administration, multiple administrations, and repeated administrations as needed to prevent or treat a disease or disorder suffered by a subject. In some cases, the method of treatment may include assessing the level of disease in a subject before, during, and / or after treatment. In some cases, treatment may continue until a decrease in the level of disease in a subject is detected.

[0060] As used herein, the term "administer / administering / administration" refers to implantation, absorption, ingestion, injection or inhalation of the drug of the present invention, regardless of its form. In some cases, one or more compounds disclosed herein can be topically (e.g., nasally) and / or orally administered to a subject. For example, the methods herein include administering an effective amount of a compound or a compound composition to achieve a desired or stated effect. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including: the activity of the specific compound used, age, body weight, general health status, sex, diet, time of administration, excretion rate, drug combination, severity and course of disease, illness or symptom, patient's treatment of the disease, illness or symptom, and the judgment of the treating physician.

[0061] After administration, the subject can be evaluated to detect, assess or determine its disease level. In some cases, treatment can continue until a change (e.g., a decrease) in the subject's disease level is detected.

[0062] After improvement of the patient's condition (e.g., a change (e.g., a decrease) in the subject's disease level), a maintenance dose of a compound, composition, or combination of the present disclosure may be administered, if necessary. Subsequently, the dose or frequency of administration, or both, may be reduced, depending on the symptoms, to a level at which the improved condition is maintained. However, upon any recurrence of disease symptoms, the patient may require intermittent treatment on a long-term basis.

[0063] definition

[0064] Neurodegeneration refers to any disease that results in the progressive death of nerve cells.

[0065] Mitochondrial disorders refer to any disease caused by mitochondrial dysfunction.

[0066] Inhibitor: As used herein, the term "inhibitor" refers to an entity, condition, or event whose presence, level, or extent is associated with a reduced level or activity of a target. In some embodiments, an inhibitor may act directly (in which case it exerts its effect directly on its target, such as by binding to the target); in some embodiments, an inhibitor may act indirectly (in which case it exerts its effect by interacting with and / or otherwise altering a modulator of the target, such that the level and / or activity of the target is reduced). In some embodiments, an inhibitor is an inhibitor whose presence or level is associated with a reduced level or activity of a target relative to a particular reference level or activity (e.g., observed under appropriate reference conditions such as the presence of a known inhibitor or the absence of the inhibitor in question).

[0067] As used herein, inhibitor refers to an inhibitor, and inhibition refers to the activity of an inhibitor.

[0068] Regulation: Regulation refers to changing, increasing or decreasing the activity of an organelle or cell.

[0069] Antagonist: One skilled in the art will appreciate that the term "antagonist," as used herein, can be used to refer to an agent, condition, or event whose presence, level, degree, type, or form is associated with a decrease in the level or activity of another agent (i.e., an inhibited agent or target). In general, an antagonist can be or include an agent of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity that exhibits the relevant inhibitory activity. In some embodiments, an antagonist can be direct (in which case it exerts its effect directly on its target); in some embodiments, an antagonist can be indirect (in which case it exerts its effect by means other than binding to its target; for example, by interacting with a modulator of the target such that the level or activity of the target is altered).

[0070] Agonist: Those skilled in the art will appreciate that the term "agonist" may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form is associated with an increase in the level or activity of another agent (i.e., an agonized or target agent). In general, an agonist may be or include an agent of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity that exhibits the relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its effect directly on its target); in some embodiments, an agonist may be indirect (in which case it exerts its effect by means other than binding to its target; for example, by interacting with a modulator of the target such that the level or activity of the target is altered).

[0071] Administration: As used herein, the term "administering" generally refers to applying or delivering to a subject or system. One of ordinary skill in the art reading this disclosure will understand that, for example, a variety of routes can be used to administer a composition; for example, some compositions can be administered by one or more routes (such as ocular, oral, parenteral, topical, etc.). In some specific embodiments, administration can be bronchial (e.g., by bronchial instillation), buccal, dermal (which can be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In addition, in some embodiments, the present disclosure describes the administration of behavioral therapy, for example, via interaction with a counselor (e.g., a therapist) and / or with a device or computing system as described herein. In some embodiments, administration may involve intermittent administration, administration or interaction (e.g., multiple doses separated in time, e.g., in some embodiments, the frequency is less than once a day, such as once every other day, twice a week, once a week, twice a month, once a month, twice a year, once a year, etc.). In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are administered to subjects according to an intermittent dosing regimen. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are administered to subjects at a frequency less than once a day. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are each administered to subjects once a week. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are each administered to subjects twice a week. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are each administered to subjects once a month. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are each administered to subjects twice a month. In some embodiments, calcineurin inhibitors and cytochrome p450 inhibitors are each administered to subjects twice a year. In some embodiments, the calcineurin inhibitor and the cytochrome p450 inhibitor are each administered to the subject once a year.

[0072] Cyclosporine is a chemical compound with the following structure

[0073]

[0074] Ritonavir is a chemical compound with the following structure:

[0075]

[0076] About: When used in this article when referring to a value, the term "about" refers to a value similar to the value mentioned in the context. Generally, those skilled in the art who are familiar with the context will understand the relevant degree of variation covered by "about" in this context. For example, in some embodiments, the term "about" can cover a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0077] Pharmaceutically acceptable salt: The term "a pharmaceutically acceptable salt" or "pharmaceutically acceptable salts" refers to salts formed from acidic and basic groups of a pharmaceutically active compound. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0079] Other features and advantages of the disclosure will be apparent from the following detailed description and drawings, and from the claims.

[0080] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0081] Example

[0082] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0083] Example 1

[0084] Materials and methods

[0085] Compounds were tested in ascending dose studies as described herein.

[0086] Modified cyclosporin (Neoral) and ritonavir were obtained.

[0087] 18 cats were screened for inclusion in the escalating dose study. Inclusion criteria were age greater than 6 months and body weight of 3.5-6 kg. Exclusion criteria were any evidence of clinically significant (in the investigator's opinion) acute or chronic disease after a detailed medical and surgical history and complete physical examination.

[0088] After admission, subjects will be divided into 3 cohorts: Cohort 1, Cohort 2, and Cohort 3. Each subject will participate in only one cohort. Each cohort will contain 6 cats.

[0089] Cohort 1 will receive an oral dose of 1.2 mg cyclosporine co-administered with a single oral dose of 5 mg ritonavir.

[0090] Cohort 2 will receive an oral dose of 1.2 mg cyclosporine co-administered with a single oral dose of 10 mg ritonavir.

[0091] Cohort 3 will receive an oral dose of 1.2 mg cyclosporine co-administered with a single oral dose of 20 mg ritonavir.

[0092] Each cohort was sampled 7 days after the oral dose of cyclosporine and the oral dose of co-administered cyclosporine and ritonavir.

[0093] result

[0094] Whole blood cyclosporine concentrations will be determined for each sample. In addition, for each sample, whole blood PK parameters will be estimated using non-compartmental analysis when appropriate: Cmax, tmax, kel, t1 / 2, AUC0-last, AUC0-inf, CL / F, and Vz / F. In addition, plasma ritonavir concentrations will be measured to confirm its presence after dosing on Day 4.

[0095] Descriptive statistics will be calculated for all relevant PK parameters: n, mean, standard deviation, minimum, median, maximum, geometric mean, and coefficient of variation.

[0096] The PK parameters - Cmax, AUC0-last, AUC0-inf - were compared between Day 1 and Day 4 using the natural logarithm of the parameters uncorrected for dose, with subject as a random effect and day as a fixed effect using an analysis of variance (ANOVA) model. Confidence intervals (CI) (90%) for the geometric mean ratio (GMR) of cyclosporine on Day 4 to Day 1 were constructed for all three parameters using log-transformed data and a two-sided t-test procedure. The GMR and 90% CI will be indexed back to the original scale. The effect of co-administration of ritonavir on cyclosporine will be evaluated from the GMR and CI.

[0097] This analysis will show a statistically significant effect of prolonging the half-life of cyclosporine suitable for a once-weekly dosing regimen.

[0098] Example 2

[0099] Materials and methods

[0100] Compounds were tested in comparative dosing studies as described herein.

[0101] Modified cyclosporin (Atopica) and ritonavir were obtained.

[0102] 14 cats were screened for inclusion in the escalating dose study. 12 of these cats were selected. Inclusion criteria were age greater than 6 months and body weight of 3.5-6 kg. Exclusion criteria were evidence of any clinically significant (in the investigator's opinion) acute or chronic disease after a detailed medical and surgical history and complete physical examination, as well as any other factors, such as recent medical treatment that, in the investigator's opinion, could affect the study objectives.

[0103] After admission, subjects were acclimatized to study conditions for 7 days before the start of the study. Subjects received 10 mg of modified cyclosporine in Torpac capsules and were followed for their illness for 7 days.

[0104] Physical measurements and clinical observations were performed throughout the study. In addition, blood samples were collected once before 7 days after dosing, once after the study period, and 15 times throughout the 7-day period. The blood samples were analyzed.

[0105] After a 10-day washout period, Phase 2 began. Subjects received 10 mg cyclosporine (Atopica) in Torpac capsules and 20 mg ritonavir solution in Torpac capsules. The same procedures were followed with regard to anthropometric measurements, clinical observations, and blood samples.

[0106] result

[0107] Whole blood cyclosporine concentrations were determined for each sample. In addition, for each sample, whole blood PK parameters were estimated using non-compartmental analysis when appropriate: Cmax, tmax, kel, t1 / 2, AUC0-last, AUC0-inf, CL / F, and Vz / F. In addition, plasma ritonavir concentrations were measured at 11 time points during the first 24 hours of Phase 2.

[0108] Descriptive statistics will be calculated for all relevant PK parameters: n, mean, standard deviation, minimum, median, maximum, geometric mean, and coefficient of variation.

[0109] PK parameters—Cmax, AUC0-last, AUC0-inf—were compared between Day 1 and Day 4 using an analysis of variance (ANOVA) model with subject as a random effect and day.

[0110] The following table provides a summary of the pharmacokinetic parameters of cyclosporine following oral administration of 10 mg cyclosporine to cats with and without ritonavir (20 mg).Two cats were excluded from the study due to dosing variability.

[0111]

[0112] *Geometric mean [geometric CV %] (N), except for Tmax where median (N) [range] is reported.

[0113] References

[0114] 1. Fournier, N., G. Ducet, and A. Crevat. "Action of cyclosporine onmitochondrial calcium fluxes." Journal of bioenergetics and biomembranes 19.3 (1987): 297-303.

[0115] 2.Jamil R.Azzi, Mohamed H.Sayegh, Samir G.Mallat, CalcineurinInhibitors: 40 Years Later, Can't Live Without..., The Journal of ImmunologyDecember 15, 2013, 191(12)5785-5791; DOI: 10.4049 / jimmunol.1390055

[0116] 3.Jeffrey Schiff,Edward Cole and Marcelo Cantarovich,TherapeuticMonitoring of Calcineurin Inhibitors for the Nephrologist,CJASN March 2007,2(2)374-384;DOI:https: / / doi.org / 10.2215 / CJN.03791106

[0117] 4.van Hooff,Johannes P.;Christiaans,Maarten H.L.;van Duijnhoven,EllyM.Tacrolimus and Posttransplant Diabetes Mellitus in Renal Transplantation,Transplantation:June 15th,2005-Volume 79-Issue 11-p 1465-1469 doi:10.1097 / 01.TP.0000157870.21957.E5

[0118] 5.Ellis CN,Gorsulowsky DC,Hamilton TA,et al.Cyclosporine ImprovesPsoriasis in a Double-blind Study.JAMA.1986;256(22):3110-3116.doi:10.1001 / jama.1986.03380220076026

[0119] 6.Haddad E,McAlister V,Renouf E,Malthaner R,Kjaer MS,GluudLL.Cyclosporin versus tacrolimus for liver transplanted patients.CochraneDatabase of Systematic Reviews 2006,Issue 4.Art.No.:CD005161.DOI:10.1002 / 14651858.CD005161.pub2.Accessed 20 May 2021.

[0120] 7.Niwa,Toshiro,et al.″Effect of cyclosporine and tacrolimus oncytochrome p450 activities in human liver microsomes.″Yakugaku Zasshi 127.1,2007:209-216.

[0121] 8.Dresser,G.K.,Spence,J.D.&Bailey,D.G.Pharmacokinetic-PharmacodynamicConsequences and Clinical Relevance of Cytochrome P4503A4 Inhibition.ClinPharmacokinet 38,41-57,2000.https: / / doi.org / 10.2165 / 00003088-200038010-00003

[0122] 9.Reynolds,N.J.and Al-Daraji,W.I.,Calcineurin inhibitors andsirolimus:mechanisms of action and applications in dermatology.Clinical andExperimental Dermatology,(2002),27:555-561. https: / / doi.org / 10.1046 / j.1365- 2230.2002.01148.x

[0123] 10.Hicks,Debbie,et al.″Cyclosporine A treatment for Ullrichcongenital muscular dystrophy:a cellular study of mitochondrial dysfunctionand its rescue.″Brain 132.1(2009):147-155.

[0124] 11.Halestrap,A P et al.“Cyclosporin A binding to mitochondrialcyclophilin inhibits the permeability transition pore and protects heartsfrom ischaemia / reperfusion injury.”Molecular and cellular biochemistryvol.174,1-2(1997):167-72.

[0125] 12.Pham CQ,Efros CB,Berardi RR.Cyclosporine for Severe UlcerativeColitis.Annals of Pharmacotherapy.2006;40(1):96-101.doi:10.1345 / aph.1G374

[0126] 13.Gomez,D.Y.,Wacher,V.J.,Tomlanovich,S.J.,Hebert,M.F.and Benet,L.Z.,The effects of ketoconazole on the intestinal metabolism and bioavailabilityof cyclosporine Clinical Pharmacology&Therapeutics,1995,58:15-19. https: / / doi.org / 10.1016 / 0009-9236(95)90067-5

[0127] 14.Wimberley,S.,Haug Iii,M.,Shermock,K.,Qu,A.,Maurer,J.,Mehta,A.,Schilz,R.and Gordon,S.(2001),Enhanced cyclosporine-itraconazole interactionwith cola in lung transplant recipients.Clin Transplantation,15:116-122. doi.org / 10.1034 / j.1399-0012.2001.150206.x

[0128] 15.Mueller,E.A.,Kovarik,J.M.,van Bree,J.B.et al.Improved DoseLinearity of Cyclosporine Pharmacokinetics from a MicroemulsionFormulation.Pharm Res 11,301-304(1994). doi.org / 10.1023 / A:1018923912135 .

[0129] 16.Roberts,et al.,Safety,tolerability,and pharmacokinetics of 6-monthdaily dosing of an oral formulation of cyclosporine(ATOPICA for )in cats,J.Veterinary Pharmacology and Therapeutics,37(2):161-168(2013).

[0130] 17.Kyles,et al.,Pharmacokinetics oftacrolimus after multidose oraladministration and efficacy in the prevention of allograft rejection in catswith renal transplants,Am.J.of Veterinary Research,64(7):926-934(2003).

Claims

1. A method of treating an alloimmune, autoimmune, inflammatory or mitochondrial disorder in a subject, the method comprising: The subject is administered a calcineurin inhibitor and a cytochrome p450 inhibitor according to an intermittent dosing schedule.

2. The method of claim 1, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered once a week.

3. The method of claim 1, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered contemporaneously.

4. The method of claim 1, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered sequentially.

5. The method of any one of claims 1-3, wherein the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus, pimecrolimus, and analogs or derivatives thereof, or pharmaceutically acceptable salts thereof.

6. The method of any one of claims 1 to 4, wherein the cytochrome p450 inhibitor is selected from the group consisting of amiodarone, chloroquine, cimetidine, clomipramine, diphenhydramine, fluoxetine, fluphenazine, haloperidol, paroxetine, perphenazine, propafenone, propoxyphene, quinacrine, quinidine, ritonavir, sertraline, terbinafine, thioridazine, amiodarone, amprenavir, clarithromycin, danazol, delavirdine, diltiazem, efavirenz, erythromycin, ethinyl estradiol, fluconazole, fluvoxamine, grapefruit juice, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, quinine, ritonavir, saquinavir, sinostatin, troleandomycin, verapamil, zafirlukast, and analogs or derivatives thereof, or pharmaceutically acceptable salts thereof.

7. The method of claim 1, wherein the calcineurin inhibitor is cyclosporine.

8. The method of claim 1, wherein the cytochrome p450 inhibitor is ritonavir.

9. The method of any one of claims 1-7, wherein the calcineurin inhibitor is administered in an amount of about 0.1 mg / kg body weight / week, about 0.2 mg / kg body weight / week, about 0.3 mg / kg body weight / week, about 0.4 mg / kg body weight / week, about 0.5 mg / kg body weight / week, about 1.0 mg / kg body weight / week, about 2.0 mg / kg body weight / week, or about 4.0 mg / kg body weight / week.

10. The method of any one of claims 1-8, wherein the cytochrome p450 inhibitor is administered in an amount of about 0.1 mg / kg body weight / week, about 0.2 mg / kg body weight / week, about 0.3 mg / kg body weight / week, about 0.4 mg / kg body weight / week, about 0.5 mg / kg body weight / week, about 1.0 mg / kg body weight / week, about 2.0 mg / kg body weight / week, or about 4.0 mg / kg body weight / week.

11. The method of any one of claims 1-8, wherein the cytochrome p450 inhibitor is administered in an amount of about 1 mg / week, about 2 mg / week, about 5 mg / week, about 10 mg / week, about 20 mg / week, or about 40 mg / week.

12. The method of any one of claims 1-11, wherein the autoimmune, alloimmune or inflammatory disorder is associated with elevated levels of lymphocytes.

13. The method of any one of claims 1-11, wherein the mitochondrial disorder is associated with the mitochondrial permeability transition pore.

14. The method of claim 12, wherein the autoimmune, alloimmune or inflammatory disorder is atopy, perianal fistula, canine pemphigus foliaceus, sebaceous adenitis, granulomatous meningoencephalitis, inflammatory bowel disease or stomatitis.

15. The method of any one of claims 1-14, wherein the calcineurin inhibitor is administered as an oral dosage form.

16. The method of any one of claims 1-15, wherein the cytochrome p450 inhibitor is administered as an oral dosage form.

17. A method of mediating a disease, condition or disorder associated with elevated lymphocyte levels in a subject, the method comprising administering to the subject a calcineurin inhibitor and a cytochrome p450 inhibitor according to an intermittent dosing regimen.

18. A method of mediating a disease, disorder or condition associated with the mitochondrial permeability transition pore in a subject, the method comprising administering to the subject a calcineurin inhibitor and a cytochrome p450 inhibitor according to an intermittent dosing regimen.

19. The method of claim 17 or 18, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered contemporaneously.

20. The method of any one of claims 17-19, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered sequentially.

21. The method of any one of claims 17-20, wherein the calcineurin inhibitor is selected from the group consisting of cyclosporine, tacrolimus and pimecrolimus and analogs or derivatives thereof, or pharmaceutically acceptable salts thereof.

22. The method of any one of claims 17-21, wherein the cytochrome p450 inhibitor is selected from the group consisting of amiodarone, chloroquine, cimetidine, clomipramine, diphenhydramine, fluoxetine, fluphenazine, haloperidol, paroxetine, perphenazine, propafenone, propoxyphene, quinacrine, quinidine, ritonavir, sertraline, terbinafine, thioridazine, amiodarone, amprenavir, clarithromycin, danazol, delavirdine, diltiazem, efavirenz, erythromycin, ethinyl estradiol, fluconazole, fluvoxamine, grapefruit juice, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, quinine, ritonavir, saquinavir, sinostatin, troleandomycin, verapamil or zafirlukast and analogs or derivatives thereof, or pharmaceutically acceptable salts thereof.

23. The method of any one of claims 17-22, wherein the calcineurin inhibitor and the cytochrome p450 inhibitor are administered once a week.

24. The method of any one of claims 1-23, wherein the subject is a non-human mammal.

25. The method of claim 24, wherein the subject is a cat or a dog.

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