Combination compositions and methods to treat polycystic kidney disease

Abstract

Aspects of the invention are drawn to compositions and methods for treating polycystic kidney disease (PKD) by administering difelikefailin and, optionally, tolvaptan. Difelikefalin not only potentiates the effect of tolvaptan but also allows a reduced daily dose of tolvaptan to achieve the same protection of renal function and kidney size, but with less polyuria and polydipsia. Thus, the combination of half dose tolvaptan and difelikefalin is superior to tolvaptan alone in maintaining GFR and TKV and causes less side effects.

Description

Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 COMBINATION COMPOSITIONS AND METHODS TO TREAT POLYCYSTIC KIDNEY DISEASE

[0001] This application claims priority from U.S. Provisional Application No. 63 / 543,749 filed on October 12, 2023, the entire contents of which is hereby incorporated by reference.

[0002] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights. GOVERNMENT INTERESTS

[0004] This invention was made with government support under W81XWH-22-1-0046 awarded by the Medical Research and Development Command. The government has certain rights in the invention. FIELD OF THE INVENTION

[0005] This invention is directed to compositions and methods of treating polycystic kidney disease by administering difelikefalin and, optionally, tolvaptan. BACKGROUND

[0006] Polycystic kidney disease (PKD) is a pathology characterized with variable size cysts growing in the kidneys, which replace normal nephron tissue, structure, and function. Water and electrolyte imbalance, including fluid overload and hyponatremia, can occur in patients with PKD. Progression of the disease leads to chronic renal failure and need for dialysis and kidney transplantation.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 SUMMARY OF THE INVENTION

[0007] Aspects of the invention are drawn to a method of treating a subject afflicted with polycystic kidney disease (PKD). In embodiments, PKD comprises autosomal dominant polycystic kidney disease (ADPKD).

[0008] In embodiments, the method comprises administering to the subject a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof.

[0009] Optionally, the method further comprises administering to the subject a therapeutically effective amount of tolvaptan. In embodiments, the therapeutically effective amount of tolvaptan is reduced as compared to a standard daily dose of tolvaptan. For example, the reduced daily dose of tolvaptan comprises ½ of the standard daily dose.

[0010] In embodiments, one or more adverse effects of tolvaptan are reduced. For example, the adverse effect comprises polyuria, polydipsia, or both.

[0011] In embodiments treating comprises decreasing circulating blood levels of vasopressin, maintaining glomerular filtration rate, reducing kidney fibrosis, decreasing blood urea levels, decreasing cysts number, decreasing cysts size, decreasing serum creatinine levels, or a combination thereof.

[0012] Aspects of the invention are further drawn towards the use of a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof to treat a subject afflicted with polycystic kidney disease. Optionally, the method further comprises administering to the subject a therapeutically effective amount of tolvaptan.

[0013] Aspects of the invention are further drawn towards the use of a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament to treat a subject afflicted with polycystic kidney disease. Optionally, the method further comprises administering to the subject a therapeutically effective amount of tolvaptan.

[0014] Aspects of the invention are also drawn towards a method of decreasing an adverse effect of treatment with tolvaptan. For example, the adverse effect comprises polyuria, polydipsia, or both.

[0015] In embodiments, the method comprises administering to a subject a reduced daily dose of tolvaptan relative to a standard daily dose and an effective amount of difelekfeline. For example, the reduced daily dose of tolvaptan comprises ½ of the standard daily dose. BRIEF DESCRIPTION OF FIGURESDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0016] FIGURE 1 shows the effect of daily treatment of ADPKD mice for a period of three- months with vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), and difelikefalin plus ½ tolvaptan (D + ½T) on change in glomerular filtration rate (GFR). At 6 months of age, the results show that chronic treatment of ADPKD mice with D + T, D + ½ T, N, or T prevented the decline of glomerular filtration rate in vehicle treated mice during the disease progression. *, P < 0.05, compared with V. MediBeacon transdermal GFR technology.

[0017] FIGURE 2 shows the effect of daily treatment of ADPKD mice (same mice for which data is shown in Figure 1) for a period of three months with vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), and difelikefalin plus ½ tolvaptan (D+ ½ T) on change in total kidney volume (TKV) and percent (%) kidney weight / body weight (%KW / BW). At 6 months of age, the results show that chronic treatment of ADPKD mice with D + T or D + ½ T decreased total kidney volume in the early phase of ADPKD. Total kidney volume = length x width x thickness x π / 6.

[0018] FIGURE 3 shows changes in 24-hr urine osmolality (panel A), 24-hr urine volume (panel B), 24-hr water intake (panel C), 24-hr free water clearance (panel D), 24-hr urinary sodium excretion (panel E), and 24-hr urinary potassium excretion (panel F) in ADKPD mice treated for 3-months with the vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), or difelikefalin plus ½ tolvaptan (D + ½ T). In ADPKD mice, treatment with D + ½ T decreased urine osmolality but did not cause the dramatic and sustained increase in urine volume and water intake that was produced by T alone. *, P < 0.05, compared with V;Т, P< 0.05, compared with T.

[0019] FIGURE 4 shows the urine osmolality measured in 24-hr urine samples collected from ADPKD mice that received different doses of nalfurafine (Panel A) or difelikafelin (Panel B): vehicle (V); nalfurafine (240, 560, 700, 800, or 1000 µg / kg, i.p.); difelikefalin (10, 16, 32, 56, or 100 µg / kg, i.p.). The arrows indicate the dose of each kappa opioid selected for use in further studies.

[0020] FIGURE 5 shows the experimental protocol (panel A) and changes in urine osmolality (panel B) and urine volume (panel C) measured in ADPKD mice (3 months old) following a single dose administration of difelikefalin (D), tolvaptan (T), half dose of tolvaptan (½ T), difelikefalin plus tolvaptan (D+T), or difelikefalin plus ½ tolvaptan (D + ½ T). Urine was collected during baseline (-4-0 hrs), 4 hrs post drug injection (0-4 hrs), and at 24 hours after dosing (5-24 hrs). Note that data are not shown for treatment of mice with nalfurafine plus tolvaptan since this combination treatment caused all mice to fall asleep.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0021] FIGURE 6 shows the design of the 3-month experimental protocol that was used for the Early Stages of ADPKD studies in which data is presented in Figures 1-3. Three-month old PKD mice were randomly divided into groups and were treated daily (dose titrated to decrease urine osmolality) for 3-months with difelikefalin, tolvaptan, combination of difelikefalin and tolvaptan (D + T), difelikefalin and ½ dose of tolvaptan (D + ½ T) or vehicle.

[0022] FIGURE 7 shows the design of the 3-month experimental protocol that was used for the Late Stages of ADPKD studies in which data is presented in Figures 8-10.

[0023] FIGURE 8 shows the effect of 3-month treatment of ADPKD mice with vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), and difelikefalin plus ½ tolvaptan (D+½T) on change in glomerular filtration rate (GFR). At 9 months of age chronic treatment of ADPKD mice with D + T, D + ½ T, N, or T prevented decline of glomerular filtration during the disease progression. *, P < 0.05, compared with V at respective time point. MediBeacon transdermal GFR technology.

[0024] FIGURE 9 shows effect of 3-month treatment of ADPKD mice (same mice for which data is shown in Figure 8) with vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), and difelikefalin plus ½ tolvaptan (D + ½ T) on change in total kidney volume (TKV) and percent (%) kidney weight / body weight (%KW / BW). At 9 months of age there were no significant changes in total kidney volume or percent kidney weight / body weight between any of the treatment groups versus V in the late phase of ADPKD. Total kidney volume = length x width x thickness x π / 6.

[0025] FIGURE 10 shows changes in 24-hr urine osmolality (panel A), 24-hr urine volume (panel B), 24-hr water intake (panel C), 24-hr free water clearance (panel D), 24-hr urinary sodium excretion (panel E), and 24-hr urinary potassium excretion (panel F) in ADKPD mice treated for 3-months (6 to 9 months of age) with the vehicle (V), tolvaptan (T), nalfurafine (N), difelikefalin (D), difelikefalin plus tolvaptan (D+T), or difelikefalin plus ½ tolvaptan (D + ½ T). At 9 months, treatment of late stage ADPDKmice with D + ½ T decreased urine osmolality but did not cause the increase in urine volume and water intake that was produced by T alone. *, P < 0.05, compared with V. Note that in panel C, the values measured monthly for the 24-hr water intake in the D + T group were essentially the same as in the T group (i.e, the orange line / values for D + T are hidden by the blue lines for T). DETAILED DESCRIPTION OF THE INVENTION

[0026] Polycystic kidney disease (PKD) is a pathology characterized with variable size cysts growing in the kidneys, which replace normal nephron tissue, structure, and function.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 Water and electrolyte imbalance, including fluid overload and hyponatremia, can occur in patients with PKD. Progression of the disease leads to chronic renal failure and need for dialysis and kidney transplantation.

[0027] Aspects of the invention are drawn to compositions and methods for treating polycystic kidney disease (PKD) by administering difelikefailin and, optionally, tolvaptan. Difelikefalin not only potentiates the effect of tolvaptan but also allows a reduced daily dose of tolvaptan to achieve the same protection of renal function and kidney size, but with less polyuria and polydipsia. Thus, the combination of half dose tolvaptan and difelikefalin is superior to tolvaptan alone in maintaining GFR and TKV and causes less side effects.

[0028] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0029] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0030] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0031] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0032] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0033] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0034] Aspects of the invention are drawn to the use of difelikefalin to treat a subject afflicted with polycystic kidney disease (PKD).

[0035] Difelikefalin is a kappa opiod receptor agonist (KOR) that produces a water diuresis (aquaresis) by inhibiting the central release of vasopressin (AVP). Plasma AVP is elevated and participates in mediating autosomal dominant polycystic kidney disease (ADPKD).

[0036] As used can refer to a geneticdisorder characterized by the growth of numerous cysts in the kidney. PKD cysts can slowly reduce much of the mass of kidneys reducing kidney function and leading to kidney failure. PKD can be classified as two major inherited forms of PKD which are autosomal dominant PKD and autosomal recessive PKD, while the non-inherited PKD can be referred to as acquired cystic kidney disease. Biomarkers can be determined and / or measured by non-invasive imaging, such as reduction of renal cysts by non-invasive imaging, or genetic testing, such as genetic testing for PKD1 and PKD2 genes.

[0037] In embodiments, the PKD is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by a genetic fault that disrupts the normal development of some of the cells in the kidney and causes cysts to grow. Faults in 1 of 2 different genes are known to cause ADPKD. The affected genes are: PKD1, which accounts for around 78% of cases. PKD2, which accounts for around 15% of cases.

[0038] As used herein, the term “treating” or “to treat” or “treatment” can refer to clinical intervention in an attempt to alter the natural course of the subject being treated. For example, “treating a disease” can comprise curing a disease, preventing a disease, reducing theDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 incidence of a disease, ameliorating symptoms of a disease, or slow down the progression of the disease.

[0039] In the case of PKD, “treating” polycystic kidney disease can be indicated by decreasing circulating blood levels of vasopressin, maintaining glomerular filtration rate, reducing kidney fibrosis, decreasing blood urea levels, decreasing cysts number, decreasing cysts size, decreasing serum creatinine levels, or a combination thereof.

[0040] In embodiments, treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., prior to an identifiable disease, disorder, and / or condition), and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0041] As used herein, "subject" can refer to a mammal, preferably human. The patient may or may not have received previous treatment for the instant or other indications, such as tolvaptan, angiotensin converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers (ARBs), statins, acetaminophen, opoids, tramadol, antibiotics. The terms “subject”, “individual”, and “patient” can be used interchangeably.

[0042] As used herein, the phrase “administering” (or “administer” or “administration”) can refer to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified care giver. Non-limiting examples of routes of administration include oral or parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, intranasal, transdermal (topical), transmucosal, and rectal administration.

[0043] Embodiments as described herein can be administered to a subject in the form of a pharmaceutical compositions suitable for the intended route of administration. For example, such pharmaceutical compositions can be in a form adapted to oral, subcutaneous, parenteral (intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocularly, or pulmonary administration. For example, such pharmaceutical compositions can be in a form adapted for administration by a peripheral route or is suitable for oral administration or suitable for parenteral administration. For example, such pharmaceutical composition can be in a form suited for local or systemic injection. Other routes of administration are subcutaneous, intraperitoneal and intravenous. Such pharmaceutical compositions can be prepared in a manner well-known to the person skilled in the art, e.g., as described in “Remington's Pharmaceutical Sciences”, Alfonso R. Gennaro (Ed.), MarkDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 Publishing Company, Easton, Pa., U.S.A., and in “Drugs and the Pharmaceutical Sciences” series, Marcel Dekker.

[0044] Pharmaceutical compositions as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0045] In embodiments, pharmaceutical compositions as described herein can be administered to a subject chronically. “Chronic administration” can refer to administration of the pharmaceutical composition in a continuous manner, such as to maintain the therapeutic effect (activity) over a prolonged period of time.

[0046] Embodiments as described herein can be administered to a subject in the form of a pharmaceutical compositions comprising a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for human pharmaceutical or therapeutic use. Pharmaceutically acceptable carriers can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. The term carrier may also encompass any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. Pharmaceutical compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the compound or active agent based on the weight of the total composition including the carrier.

[0047] The pharmaceutical carrier or diluent employed can be a conventional solid or liquid carrier. When a solid carrier is used for oral administration, the preparation can be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose.

[0048] When a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquidDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 suspension or solution. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

[0049] The carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0050] The pharmaceutical compositions can be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The pharmaceutical composition can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, or calcium chloride.

[0051] In embodiments, the pharmaceutical composition can be administered to the subject in a therapeutically effective amount. In embodiments, a therapeutically effective amount of difelekfalin can comprise a dose of about 1ug / kg to about 500 ug / kg. In some embodiments, a therapeutically effective amount of difelekfalin can comprise a dose of about 5 ug / kg to about 200 ug / kg. In some embodiments, a therapeutically effective amount of difelekfalin can comprise a dose of about 10ug / kg to about 112ug / kg.

[0052] In embodiments, a therapeutically effective amount of difelekfalin can comprise a dose of about 0.005 mg / kg to about 1000 mg / kg. In some embodiments, a therapeutically effective amount can comprise a dose of about 0.005 mg / kg to about 10 mg / kg. In some embodiments, a therapeutically effective amount can comprise a dose of about 0.25 mg / kg to about 2 mg / kg. In some embodiments, the therapeutically effective amount is at least about 0.001 mg / kg at least about 0.0025 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.1 mg / kg body weight, at least about 0.25 mg / kg body weight, at least about 0.5 mg / kg body weight, at least about 0.75 mg / kg body weight, at least about 1 mg / kg body weight, at least about 2 mg / kg body weight, at least about 3 mg / kg body weight, at least about 4 mg / kg body weight, at least about 5 mg / kg body weight, at least about 6 mg / kg body weight, at least about 7 mg / kg body weight, at least about 8 mg / kg body weight, at least about 9 mg / kg body weight, at least about 10 mg / kg body weight, at least about 15 mg / kg body weight, at least about 20 mg / kg body weight, at least about 25 mg / kg body weight, at least about 30 mg / kg body weight, at least about 40 mg / kg body weight, at least about 50 mg / kg body weight, at least about 75 mg / kg body weight, at least about 100 mg / kg body weight, at least about 200 mg / kg body weight, at least about 250 mg / kg body weight, at least about 300 mg / kg bodyDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 weight, at least about 3500 mg / kg body weight, at least about 400 mg / kg body weight, at least about 450 mg / kg body weight, at least about 500 mg / kg body weight, at least about 550 mg / kg body weight, at least about 600 mg / kg body weight, at least about 650 mg / kg body weight, at least about 700 mg / kg body weight, at least about 750 mg / kg body weight, at least about 800 mg / kg body weight, at least about 900 mg / kg body weight, or at least about 1000 mg / kg body weight.

[0053] The skilled artisan will recognize that the dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the biological effect desired; and rate of excretion.

[0054] Aspects of the invention further comprise administering to the subject a therapeutic combination comprising difelikefalin and tolvaptan.

[0055] Tolvaptan is a V2 AVP receptor antagonist which acts exclusively in the renal collecting ducts to block the water-retaining effects of AVP and is use clinically to treat ADPKD.

[0056] Adverse of tolvaptan. The term “adverse effects” can refer to an unwanted, unpleasant, harmful, or potentially harmful drug effect. Adverse effects can be mild, moderate, or severe. Examples of adverse effects associated with tolvaptan includes polyuria (e.g., frequent and abnormally large amount of urination), polydipsia (e.g., excessive thirst or urge to drink fluids constantly), osmotic demyelination syndrome (e.g., reduction in blood sodium levels too quickly which can cause serious nerve damage), xerostomia (e.g., dry mouth), dehydration, hypovolemia, constipation, and liver damage. Regarding the latter, the FDA has issued a black box warning indicating that tolvaptan may cause liver damage, sometimes serious enough to require liver transplantation or to cause death.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0057] Referring to Figure 3, for example, combination treatment of ADPKD mice with difelekfalin and a reduced dose tolvaptan decreased urine osmolality but did not cause the dramatic and sustained (e.g., 3 months) increase in urine volume (polyuria) and water intake (polydipsia) that was produced by the standard daily dose of tolvaptan alone. This is of merit since the combination treatment of ADPKD mice for 3 months with difelekafalin and reduced daily dose tolvaptan was equally effective as full dose tolvaptan alone in preventing a decline in glomerular filtration rate (Figure 1).

[0058] Accordingly, aspects of the invention are drawn to methods of treating a subject afflicted with polycystic kidney disease while also reducing one or more adverse effects (e.g., polyuria, polydipsia, liver damage) associated with a standard daily dose of tolvaptan. In embodiments, the adverse effect can be reduced by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, by at least about 99%, or by at least about 100%.

[0059] Initiating tolvaptan treatment in humans should be considered for patients having demonstrated rapid progression or who are likely to have rapid progression. Tolvaptan can be prescribed to adult ADPKD patients aged <50 years with chronic kidney disease (CKD) stages 1-3a (eGFR>45 mL / min / 1.73 m2) who have demonstrated or who are likely to have rapidly progressing disease. In embodiments, CKD stage may be interpreted in conjunction with age. In addition to careful assessment of which patients may benefit most from tolvaptan, other considerations should be considered before administering ths drug to a patient for management of ADPKD, which include the following: elevated liver enzymes and / or signs or symptoms of liver injury prior to initiation of treatment that meet the requirements for permanent discontinuation of tolvaptan, volume depletion, hypernatraemia, inability to perceive or repond to thirst. To date, there are no established short-term markers of the efficacy of tolvaptan treatment in patients with ADPKD. Therefore, the dose of tolvaptan prescribed by physicans is that which has been used in the TEMPO clinical trial (ClinicalTrials.gov number, NCT00428948) which demonstrated treatment efficacy in ADPKD patients, i.e. starting tolvaptan at a standard total daily dose of 60 mg (given 45 mg in the morning and 15 mg in the afternoon), and uptitrated when tolerated to a standard daily dose of 90 mg / day (60 / 30 mg) or a standard daily dose of 120 mg / day (90 / 30 mg), respectively.

[0060] Accordingly, the standard daily dose of Tolvaptan can be about 60 mg, between about 60 mg and about 90 mg, about 90 mg, between about 90 mg – about 120 mg, or about 120 mg. The skilled artisan will recognize that the term “standard daily dose” can refer to anyDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 required daily dose of a drug given to a subject for treating or preventing the disease to be treated or prevented.

[0061] As described herein, embodiments comprise methods of decreasing an adverse effect associated with the standard daily dose of tolvaptan by administering a reduced daily dose of tolvaptan together with difelekfalin. As used herein, the term “reduced daily dose” can refer to a daily dose of a drug given to a subject that is lower than the standard daily dose described herein. In embodiments, the reduced daily dose of tovaptan comprises less than about ¼ of the standard daily dose, about ¼ of the standard daily dose, less than about 1 / 3 of the standard daily dose, about 1 / 3 of the standard daily dose, less than about 1 / 2 of the standard daily dose, about ½ of the standard daily dose, less than about 2 / 3 of the standard daily dose, about 2 / 3 of the standard daily dose, less than abou 3 / 4 of the standard daily dose, about ¾ of the standard daily dose, less than the standard daily dose or about the standard daily dose. For example, if the standard daily dose is about 60 mg of tolvaptan, then a reduced daily dose of about ½ is about 30 mg of tolvaptan. For example, if the standard daily dose is about 120 mg of tolvaptan, then a reduced daily dose of about ½ is about 60 mg of tolvaptan.

[0062] In embodiments, the therapeutic effectiveness of the reduced daily dose of tolvaptan (in combination witih difelekfaline) is the same or about the same of as the therapeutic effectiveness of the standard daily dose of tolvaptan. As used herein, the term “therapeutically effective amount” can refer to that amount of the therapeutic agent sufficient to realize a biological effect, such as treating a disease or disorder such as polycystic kidney disease. The skilled artisan will recognize that the therapeutically effective amount can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the biological effect desired; and rate of excretion.

[0063] When applied to an individual active ingredient administered alone, a therapeutically effective dose can refer to that ingredient alone. When applied to a combination, a therapeutically effective dose can refer to either each of the active ingredients separately, or the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In embodiments, the compounds of the therapeutic combination (e.g., difelikefalin and tolvaptan) may be administered in combination or separately, and if separately, simultaneously or sequentially over a period of time as determined by a qualified care giver.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0064] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0065] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 EXAMPLES

[0066] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1

[0067] New Therapy for Early-Stage Polycystic Kidney Disease: Combination of Difelikefalin and Tolvaptan

[0068] We have reported that difelikefalin, which is an agonist of kappa opioid receptors, produces a centrally mediated water diuresis presumably by inhibiting the central release of vasopressin (AVP). Since plasma AVP is elevated and participates in mediating polycystic kidney disease (PKD), we validated that difelikefalin is superior to the V2 AVP antagonist, tolvaptan, in slowing progression of early stage PKD with less adverse effects.

[0069] Three-month old PKD mice were randomly divided into 4 groups and were treated daily (dose titrated to decrease urine osmolality) for 3-months with either difelikefalin, tolvaptan, combination of difelikefalin and tolvaptan (D+T), or vehicle. Glomeruli filtration rate (GFR; beginning, midterm, endpoint) and total kidney volume (TKV, calculated as length, width, and depth of kidneys after mice were sacrificed) were measured in each group. Metabolic studies were also performed to measure 24-hour urine output, urine osmolality, and water intake.

[0070] The results shown in Figure 1 demonstrated that in contrast to vehicle treatment, difelikefalin, tolvaptan, and D+T each prevented a significant decline in GFR during the 3- month PKD disease progression. However, at the termination of study only D+T markedly reduced TKV compared with vehicle treated PKD mice (D+T versus V, 188 ± 12 vs 228 ±10 mm3, resp.).

[0071] In other studies, it was shown that the same age PKD mice treated for 3-months with difelikefalin combined with a half dose of tolvaptan (D+1 / 2T) was as efficacious in preventing a decline in GFR over the 3 month treatment (Figure 1). Suppressingly, the combination treatment of difelikefalin and ½ dose tolvaptan decreased TKV to the same level as that produced by D+T (Figure 2). As shown in Figure 3, compared to levels in vehicle treated PKD mice, water intake and urine output were significantly elevated in PKD miceDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 treated with tolvaptan and D+T. Importantly, PKD mice treated with D+1 / 2T had significantly reduced urine output and water intake than PKD mice treated with tolvaptan alone or D+T. Together, these findings suggest that difelikefalin not only potentiates the effect of tolvaptan but also allows a reduced daily dose of tolvaptan to achieve the same protection of renal function and kidney size, but with less polyuria and polydipsia. Thus, the combination of half dose tolvaptan and difelikefalin is superior to tolvaptan alone in maintaining GFR and TKV and causes less side effects. EXAMPLE 2

[0072] The combination of Tolvaptan and Difelikefalin in treating polycystic kidney disease

[0073] We have reported that difelikefalin, an agonist of kappa opioid receptors (KOR), produces a centrally mediated water diuresis presumably by inhibiting the central release of vasopressin (AVP) (Pharmacol Res. 2022 Nov; 185:106470. Doi 10.1016 / j.phrs.2022. Epub 2022 Oct 3. PMID: 36202183). This is of clinical significance since plasma AVP is elevated and plays a key role in mediating autosomal dominant polycystic kidney disease (ADPKD) (Muller et al., Nephrol Dial Transplant 2022, 37:825-839; PMID: 35134221). Currently, tolvaptan, a V2 AVP receptor antagonist which acts exclusively in the renal collecting ducts to block the water-retaining effects of AVP is used clinically to treat ADPKD. However, by inhibiting the central secretion / release of AVP into the blood, which would then attenuate / prevent actions of AVP throughout the body (e.g., within the brain, blood, systemic and renal blood vessels, and other sites in the kidneys other than only the collecting ducts), we hypothesized that difelikefalin would be superior to the V2 AVP receptor antagonist, tolvaptan, in slowing progression of early stage ADPKD and have fewer adverse effects.

[0074] Methods: For these studies, 3-month-old ADPKD mice were divided into 4 groups and animals were treated daily (dose titrated to decrease urine osmolality) for 3-months with either 1) difelikefalin, 2) tolvaptan, 3) combination of difelikefalin and tolvaptan (D+T), or 4) vehicle. Initial daily doses of drugs were as follows: difelikefalin (56 ug / kg, i.p.); tolvaptan (0.4 mg / mouse oral); combination difelikefalin and tolvaptan (56 ug / kg, i.p. and 0.4 mg / mouse oral, resp.) and vehicle. Glomeruli filtration rate (GFR; beginning, midterm, endpoint) and total kidney volume (TKV, calculated as length, width, and depth of kidneys after mice were sacrificed) were measured in each group. Metabolic studies were also performed to measure 24-hour water intake, urine output and osmolality, and urinary sodium / potassium excretion.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1

[0075] The results demonstrated that in contrast to vehicle treatment, difelikefalin, tolvaptan, and D+T each prevented a significant decline in GFR during the 3-month ADPKD disease progression (Figure 1). However and unexpected (Figure 2), at the termination of study only the combination of D+T markedly reduced TKV compared with vehicle treated ADPKD mice (188 ± 12 vs 228 ±10 mm3, resp.). As shown in Figure 3b, while tolvaptan alone caused a marked and sustained increase in urine output in ADPKD mice for the duration of the 3- month study, treatment with D+T increased urine output at 1 month and thereafter was lower in magnitude than that produced by tolvaptan. These findings are of importance since currently, risk prediction in ADPKD relies on TKV and is complemented by genetic information and clinical complications (Muller et al., Nephrol Dial Transplant 2022, 37:825-839). Thus, while difelikefalin and tolvapatan administered alone were effective in preventing a decline in renal function (GFR), our data clearly indicate that combination treatment of mice with D+T was superior in protecting renal function and in decreasing TKV, the latter being a prime indicator in ADPKD risk prediction. Finally, compared to vehicle levels, water intake remained significantly elevated throughout the 3-month study in ADPKD mice treated with tolvaptan and D+T (Figure 3c).

[0076] The findings above are of high clinical importance in regards to new approaches to treat ADPKD; however, there remain concerns regarding the therapeutic use of tolvaptan in patients, particularly when administered chronically. Commonly reported adverse effects of tolvaptan (SAMSCA®, Jynarque®) include production and renal excretion of an abnormally large amount of urine (polyuria), exsessive thirst and desire to drink (polydipsia), abnormal urination during the day (pollakiuria), dry mouth and dehydration, hypovolemia, and constipation. Along these lines, in our findings described above, ADPKD mice treated with the combination tolvaptan and D+T showed a marked increase in water intake that was sustained over the 3-month study. Further, as noted in the black box warning on the medication label, “Tolvaptan can cause serious and potentially fatal liver injury. Acute liver failure requiring liver transplantation has been reported.” The frequency of clinically apparent liver injury during therapy was one reason for the delay of formal approval of long term tolvaptan therapy for ADPKD; Consequently, guidelines state that tolvaptan should not be used to treat ADPKD outside of the FDA-approved risk evaluation and mitigation strategy (REMS) for ADPKD patients (https: / / www.drugs.com / mtm / jynarque.html).

[0077] Due to the potential of lifestyle modifying (polyuria and polydipsia) and serious (liver injury) adverse effects of tolvaptan, without wishing to be bound by theory, drug therapy with a lower dose of tolvaptan in combination with a kappa opioid receptor agonist providesDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 superior renal protection in ADPKD mice due to the dual renal (V2-receptor antagonism) and central (inhibition of AVP secretion) actions of these compounds, respectively, with less adverse effects. To validate this, we completed studies in groups of ADPKD mice (3-months of age) to establish an effective dose of the kappa opioid agonists, nalfurafine and difelikefalin to be used in further studies. As shown in Figure 4, based on the ability to suppress 24-hour urine osmolality, an i.p. dose of 560 µg / kg nalfurafine and 56 µg / kg difelikefalin were selected for subsequent investigations.

[0078] Using the doses of difelikefalin and nalfurafine established in the experiments described above, studies were performed in ADPKD mice to determine the acute changes in renal excretory function produced by these kappa opioids alone and in combination with tolvaptan. For these studies, tolvaptan was administered at either a full dose or ½ the dose of tolvaptan (½T) that was used in the studies described above. Initially, dose-response studies were performed to determine the acute (4-hour and then remaining 20 hour) effects of difelikefalin (56 µg / kg, i.p.), tolvaptan (0.4 mg / mouse, oral), ( ½ T (0.2 mg / mouse, oral), and the combination of difelikefalin (56 ug / kg, i.p.) plus T (0.4 mg / mouse, oral) (D + ½ T), and difelikefalin (56 ug / kg, i.p.) plus ½T (0.2 mg / mouse, oral) (D + ½T) in mediating changes in 4-hr urine volume and urine osmolality.

[0079] As shown in Figure 5C, difelikefalin alone did not alter urine volume at 4 hours, and even after an additional 20 hours, urine output was only marginally increased. In contrast, injection of ½T alone significantly increased urine volume at 4 hours, yet the magnitude of diuresis was not significantly elevated further by the combination of D + ½T (Figure 5C). This was somewhat unexpected since we presumed that due to dual mechanisms of action (tolvaptan blockade of AVP in the collecting ducts, and difelikefalin inhibition of AVP secretion), the combination therapy would have produced a substantially greater diuresis. Of merit, in addition to producing diuresis via suppression of AVP secretion, kappa opioid agonists including difelikefalin and nalfurafine are recognized to decrease the renal excretion of sodium and potassium. Thus, without wishing to be bound by theory, in ADPKD mice, the addition of difelikefalin to the ½ tolvaptan dose did not result in a potentiation of the diuresis due to a concurrent kappa opioid-mediated increase in the renal tubular reabsorption of sodium / potassium, and consequently water. In comparison, however, when difelekafalin was combined with the full dose of tolvaptan used in prior studies (D+T), 4-hour urine output was markedly increased, with the level being significantly higher than the diuresis produced by D + ½T. Despite differences in the magnitude of urine output between these groups, all dose treatments except for difelikefalin and nalfurafine alone maximally suppressed urineDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 osmolality to the same level at the 4-hour period and urine osmolality remained below baseline at the end of the remaining 24-hour period (Figure 5B). Without wishing to be bound by theory, the high level of urine osmolality produced by difelikefalin and nalfurafine at 24 hours was associated with these two groups low urine volume.

[0080] To determine the effects of combination treatment of nalfurafine and tolvaptan on urine output in ADPKD mice, the above dose-response studies on 4-hr urine output were repeated. However, it was observed that when nalfurafine was administered together with tolvaptan at a ½ dose (0.2 mg / mouse, oral) or full dose (0.4 mg / mouse, oral), all mice very quickly fell asleep. Of interest, cytochrome CYP3A4 is the major isoform involved in the metabolism of both nalfurafine and tolvaptan. Therefore, we did not continue to complete the urine output studies. Without wishing to be bound by theory, because tolvaptan and nalfurafine are both metabolized by the same hepatic cytochrome P450 (CYP3A4) isozyme pathway, combination therapy of these two drugs most likely resulted in exceedingly high plasma levels of nalfurafine, which, due to its opioid activity caused sedation. This is an important finding and indicates that the potential combined use of tolvaptan together with a KOR agonist be restricted to difelikefalin or other KOR agonists that are not metabolized by the cytochrome P4503A4 enzyme pathway.

[0081] Based on these acute studies, we next investigated the long-term effects of ½T tolvaptan in combination with difelikefalin on kidney function and kidney size in ADPKD mice. Using the same experimental protocol (Figure 6), the results of these studies demonstrated that the same age PKD mice treated for 3-months with difelikefalin combined with a half dose of tolvaptan (D + ½T) was as efficacious in preventing a decline in GFR (Figure 1) and suppressing an increase in TKV as D + T (Figure 2). As shown in Figure 3, compared to vehicle levels, water intake and urine output were significantly elevated in PKD mice treated with tolvaptan and D + T. Importantly, however, PKD mice treated with D + 1 / 2T had significantly reduced urine output and water intake than PKD mice treated with tolvaptan alone or D + T (Figure 3).

[0082] Together, these findings indicate that difelikefalin not only potentiates the effect of tolvaptan but also allows a reduced daily dose of tolvaptan to achieve the same protection of renal function and kidney size, but with less polyuria and polydipsia. Thus, the combination of half dose tolvaptan and difelikefalin is superior to tolvaptan alone in maintaining GFR and TKV and causes less side effects.

[0083] Additional studies were performed to investigate whether combination therapy of difelikefalin and tolvaptan would have beneficial effects in the treatment of late stage ADPKD.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 For these investigations, experiments were conducted in 6-month-old ADPKD mice that were treated for 3 months with all groups of drugs described above and then sacrificed at 9 months of age (experimental protocol shown in Figure 7). As shown in Figure 8, treatments of T, N, D, D+T, and D+1 / 2 T prevented the GFR from declining as which occurred in the V treated mice aged from 6 months to 9 months old, which agrees with findings observed in mice with early stage PKD. It is interesting that nalfurafine alone increased GFR at the midterm of study compared to that in mice treated with vehicle. Different from findings in the early stage PKD study, the combinations of D + T or D + ½T did not significantly decrease TKV or change the ratio of kidney weight to body weight, compared to mice treated with vehicle (Figure 9). As shown in Figure 10, treatments of T, N, D, D+T, or D+ ½T each lowered urine osmolality and maintained it at a lower level than observed in mice treated with vehicle. Twenty-four-hour urine volume and water intake were markedly increased over the course of the study in mice treated with T and D+T (Figure 10b and 10c). Note that in panel 10c, the values measured monthly for the 24-hr water intake in the D + T group were essentially the same as in the T group (i.e, the orange line / values for D + T are hidden by the blue lines for T). While treatment of ADPKD mice with D + ½T also increased urine volume and water intake compared to vehicle treated animals, the magnitudes of these changes were not pronounced until months 8 and 9 and importantly, except for urine output at 8 months were substantially less than that produced by T alone and D + ½T (Figure 10b and 10c). Tolvaptan monotherapy improved free water clearance, but other treatments did not cause a marked change in this parameter in these older age ADPKD mice (Figure 10d). In contrast to T, D + T and D + ½T increased urinary sodium excretion (Figure 10e).

[0084]

[0085] Together, these findings demonstrate that in comparison to vehicle treated animals, the combined administration of difelikefalin and a full or half dose of tolvaptan was highly efficacious in preventing a marked decline in GFR in mice with late stage ADPKD, yet did not significantly reduce TKV. Further, in comparison to a full dose of tolvaptan, the combined administration of difelikefalin and a reduced daily dose of tolvaptan (D + ½T) produced less adverse effects associated with excessive urination and drinking over the course of the 3-month study.

[0086] Embodiments of the invention comprise: 1. Treatment for PKD. The combination of difelikafelin and tolvaptan can achieve a greater efficacy but with a reduced daily dose amount and frequency ofDate of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 tolvaptan than giving tolvaptan alone. This combination also results in less polyuria and polydipsia. 2. Treatment for hyponatremia. The combination of difelikafelin and tolvaptan can act as water diuretics to increase water excretion from the kidney and restore the plasma osmolality of these patients. 3. Nalfurafine is approved in Japan for treatment of pruritus. Our data have demonstrated that tolvaptan can increase the sedation effect of the regular dose of nalfurafine. Therefore, a black box warning should be given when chronic kidney disease patients are prescribed both drugs. The existing technology uses tolvaptan alone to treat PKD. Embodiments described herein comprise the combination use of difelikefelin and tolvaptan to achieve greater efficacy and cause fewer side effects. ***** EQUIVALENTS

[0087] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 WE CLAIM:

1. A method of treating a subject afflicted with polycystic kidney disease (PKD), the method comprising administering to the subject a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the method further comprises administering to the subject a therapeutically effective amount of tolvaptan.

3. The method of claim 2, wherein the therapeutically effective amount of tolvaptan is reduced as compared to a standard daily dose of tolvaptan.

4. The method of claim 3, wherein the reduced daily dose of tolvaptan comprises ½ of the standard daily dose.

5. The method of claim 1, wherein PKD comprises autosomal dominant polycystic kidney disease (ADPKD).

6. The method of claim 1, wherein treating comprises decreasing circulating blood levels of vasopressin, maintaining glomerular filtration rate, reducing kidney fibrosis, decreasing blood urea levels, decreasing cysts number, decreasing cysts size, decreasing serum creatinine levels, or a combination thereof.

7. The method of claim 4, wherein one or more adverse effects of tolvaptan are reduced.

8. The method of claim 7, wherein the adverse effect comprises polyuria, polydipsia, or both.

9. Use of a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof to treat a subject afflicted with polycystic kidney disease.

10. Use of a therapeutically effective amount of difelikefalin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament to treat a subject afflicted with polycystic kidney disease.

11. A method of decreasing an adverse effect of treatment with tolvaptan, the method comprising administering to a subject a reduced daily dose of tolvaptan relative to a standard daily dose and an effective amount of difelekfeline.Date of Filing: October 15, 2024 Ref. No.: 2932719-000239-WO1 12. The method of claim 9, wherein the reduced daily dose of tolvaptan comprises ½ of the standard daily dose.

13. The method of claim 9, wherein the adverse effect comprises polyuria, polydipsia, or both.

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