TW202444344A - Therapeutic formulations for kidney disease - Google Patents

Abstract

The present invention provides a therapeutic formulation adapted to deliver a daily dose of repagermanium or propagermanium, optionally in combination with an AT1R blocker, and use of such therapeutic formulations for the treatment of kidney diseases.

Description

Therapeutic formulations for kidney disease

The present invention relates to therapeutic formulations containing repagermanium or propagermanium and methods of use thereof, and therapeutic formulations containing AT ₁ R blocking agents and methods of use thereof. Repagermanium or propagermanium can be delivered together with an AT ₁ R blocking agent in a therapeutic combination regimen.

Therapies based on inhibitors of the chemokine receptor 2 (CCR2) pathway or _AT1R blockers are known for diseases such as kidney diseases and respiratory diseases, optionally in combination therapy.

Repagd or propagd can be used as CCR2 pathway inhibitors. Repagd and propagd were previously described in the literature as polymeric compounds. The drug substance 3-[(2-carboxyethyl-oxogermanium alkyl)oxy-oxogermanium alkyl] propionic acid exhibits polymorphism and can exist in different solid forms. However, the inventors did not observe any pharmacological effects of Repagd during the manufacture or testing of the drug substance.

Polymorphs of 3-[(2-carboxyethyl-1,2-dioxygermanium)oxy-1,2-dioxygermanium]propionic acid include rapagermanium, propagermanium, proxigermanium, Ge-132, germanium sesquioxide, 2-carboxyethylgermanium sesquioxane, SK-818, and bis(2-carboxyethylgermanium) sesquioxide.

The two most characterized solid forms, rapa-germanium and propa-germanium, have a common core structure (Mizuno et al., 2015). The two APIs have different International Non-patent Names (INNs), the INN for rapa-germanium is INN #6259 and the INN for propa-germanium is INN #6260. They consist of different stacks of monomer molecules of 3-[(2-carboxyethyl-oxo-germanium)oxy-oxo-germanium]propionic acid, with different structural units coordinated by H bonds. X-ray diffraction has shown that rapa-germanium has an infinite sheet structure, which is significantly different from the polymeric ladder structure of propa-germanium, which is composed of eight-membered rings connected in series by Ge-O bonds. In solid form, propagate germanium and rapagate germanium are different; however, in solution, when the structural units are dissolved, they are identical.

The angiotensin type 1 receptor ( _AT1R , AT1R, angiotensin II receptor type ₁ ) is a G protein-coupled receptor. Despite their structural differences, _AT1R blockers have traditionally been considered therapeutically interchangeable.

However, for various diseases, the dosing regimen of rapamyr or propamyr and _AT1R blocking agents remains to be determined. It is necessary to establish a suitable dosing regimen for rapamyr or propamyr and _AT1R blocking agents; or at least provide a new dosing regimen to supplement the previously known dosing regimen. The present invention aims to provide an improved or alternative dosing regimen for rapamyr or propamyr and _AT1R blocking agents. rapamyr or propamyr can be delivered together with _AT1R blocking agents in a therapeutic combination regimen.

The preceding discussion of the background art is intended only to facilitate understanding of the present invention. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge at the priority date of the present application.

The present invention provides a therapeutic formulation suitable for delivering a daily dose of 200 mg to 280 mg of rapamyr or propamyr.

The present invention further provides a therapeutic formulation suitable for delivering a daily dose of a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a therapeutic formulation suitable for delivering a daily dose of: (a) a therapeutically effective amount of rapamycin or propamycin; and (b) a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

Preferably, the daily dose of propagate or repagold is 200 mg to 280 mg.

Preferably, the _AT1R blocking agent is selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan.

Preferably, the daily dose of the AT ₁ R blocker is 2 mg to 800 mg.

The present invention further provides a dosing regimen comprising administering 200 mg to 280 mg of rapamyr or propamyr daily.

The present invention further provides a dosing regimen comprising administering a daily dose of a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

The present invention further provides a dosing regimen comprising administering a daily dose of: (a) a therapeutically effective amount of rapamycin or propamycin; and (b) a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: (a) a therapeutic formulation suitable for delivering 200 mg to 280 mg of rapamyr or propamyr per day.

The present invention further provides a method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: (a) a therapeutically effective amount of an AT ₁ R inhibitor, wherein the affinity of the AT 1 R inhibitor to the CCR2-AT ₁ R complex is equal to or greater than the affinity of the AT ₁ R inhibitor to AT ₁ R alone.

The present invention further provides a method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: (a) a therapeutically effective amount of rapamyr or propamyr daily; and (b) a therapeutically effective amount of an _AT1R blocker, wherein the affinity of the AT1R blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides the use of 200 mg to 280 mg of rapamyr or propamyr in a daily dose for preparing a formulation for treating, improving or preventing kidney disease in an individual.

The present invention further provides the use of 200 mg to 280 mg of rapamyr or propamyr in a daily dose for treating, improving or preventing kidney disease in an individual.

The present invention further provides the use of a therapeutically effective amount of an AT ₁ R inhibitor for preparing a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the inhibitor has an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R inhibitor for AT ₁ R alone.

The present invention further provides the use of a therapeutically effective amount of an AT ₁ R inhibitor for treating, ameliorating or preventing a kidney disease in an individual, wherein the inhibitor has an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R inhibitor for AT ₁ R alone.

The present invention further provides the use of the following substances for preparing a formulation for treating, ameliorating or preventing kidney disease in an individual: (a) a therapeutically effective amount of rapamyr or propamyr and (b) a therapeutically effective amount of an _AT1R blocker, wherein the affinity of the AT1R blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for AT1R alone.

The present invention further provides the use of the following substances for treating, ameliorating or preventing kidney diseases in an individual: (a) a therapeutically effective amount of rapamyr or propamyr, and (b) a therapeutically effective amount of an _AT1R blocker, wherein the affinity of the AT1R blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a therapeutically effective amount of rapamycin or propamycin for use in a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the rapamycin is administered to the individual simultaneously or sequentially with a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a therapeutically effective amount of an _AT1R blocker for use in a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone, wherein the at least one _AT1R blocker is administered to the individual simultaneously or sequentially with rapamycin or propamycin.

The present invention further provides a kit for treating or preventing kidney disease in an individual, the kit comprising: (a) 200 mg to 280 mg daily dose of rapamyr or propamyr; and (b) instructions for use.

The present invention further provides a kit for treating or preventing kidney disease in an individual, the kit comprising: (a) a therapeutically effective amount of an AT ₁ R blocker, the affinity of the blocker for CCR2-AT ₁ R complex being equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone; and (b) instructions for use.

The present invention further provides a kit for treating or preventing kidney disease in an individual, the kit comprising: (a) a therapeutically effective amount of rapamycin or propamycin; (b) a therapeutically effective amount of an _AT1R blocker, the affinity of the blocker for the CCR2- _AT1R complex being equal to or greater than the affinity of the _AT1R blocker for _AT1R alone; and (c) instructions for use.

Preferably, the rapamycin or propamycin is provided in a daily dose of 200 mg to 280 mg of rapamycin or propamycin.

Preferably, the _AT1R blocking agent is selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan.

Preferably, the subject to be treated is a human mammal.

Preferably, the kidney disease is a kidney disease associated with proteinuria.

Preferably, the kidney disease is selected from the list comprising focal segmental glomerulosclerosis (FSGS; including idiopathic (primary) FSGS, secondary FSGS, hereditary FSGS and FSGS of any other cause), renal fibrosis, immunoglobulin A nephropathy (IgAN), Alport's syndrome, chronic kidney disease (including chronic kidney disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic)) and renal failure diseases (including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, primary nephrotic proteinuria and vascular hypertension).

Preferably, the _AT1R blocker is not EXP3174.

Invention Detailed Description

Although CCR2 inhibitors and/or _AT1R blockers have been used in a variety of therapeutic settings, much remains unknown regarding dosing regimens that provide effective therapeutic outcomes.

The present invention provides therapeutic formulations comprising: (i) rapagal or propagal suitable for delivery at 200 mg to 280 mg rapagal or propagal daily; (ii) an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone; or (iii) rapagal or propagal suitable for delivery at 200 mg to 280 mg rapagal or propagal daily and an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. Repa Germanium or Propa Germanium preparations

The inventors surprisingly discovered that the dosage of propagate or repagd required for therapeutic efficacy falls within a narrow dosage and treatment regimen.

RapaGermanium and Propagermanium block pathways involved in MCP-1-induced migration, monocyte activation, and tropism. RapaGermanium and Propagermanium further block the function of cellular structures involved in tropism (such as microfilaments, microtubules, and intermediate filaments) and surface proteins (such as glycosylphosphatidic acid inositol (GPI) anchored proteins, and more specifically CD55, CD59, and CD16).

The INN for rapamycin is INN #6259 and propamycin is INN #6260. Propagold has been used as an active agent against chronic hepatitis and has been shown to specifically inhibit the MCP-1-induced tropism of monocytes in vitro through a mechanism that appears to require glycosylphosphatidic acid inositol (GPI) anchoring proteins such as CD55, CD59, and CD16 (Yokochi, S. (2001) Journal of Interferon and Cytokine Research 21:389-398). Other names for propargyl and/or ripargyl include 3-[(2-carboxyethyl-oxogermanium alkyl)oxy-oxogermanium alkyl] propionic acid, proguanine, Ge-132, bis(2-carboxyethylgermanium)sesquioxide (CEGS), 2-carboxyethylgermaniumsesquioxane, SK-818, organic germanium, germanium sesquioxide, 3,3´-(1,3-dioxo-1,3-digermaniumoxanediyl)bispropionic acid, 3-oxygermanium alkylpropionic acid polymer and poly-trans-(2-carboxyethyl)germaniumsesquioxane. In the present invention, the term "ripargyllium" is defined to include "propargyl".

The inventors have found that a dose of, for example, 80 mg twice daily causes the blood concentration of rapamycin or propamycin to drop too low before the next dose to provide a therapeutic effect. Furthermore, contrary to expectations, a dose of, for example, 160 mg twice daily results in a reduced therapeutic effect. Thus, the therapeutic effect of rapamycin or propamycin is maximized at moderate doses. This general trend can be seen in Figure 1.

Thus, the present invention provides a therapeutic formulation suitable for delivering a daily dose of 200 mg to 280 mg of propagation germanium or rhaponticillin.

The present invention further provides a dosing regimen comprising administering 200 mg to 280 mg of rapamyr or propamyr daily.

Repag-germanium or propagate-germanium may suitably be delivered at 220 mg to 260 mg daily, more preferably 240 mg daily.

Preferably, the formulation comprising rapamycin or propamycin is delivered twice daily (BID). Thus, the formulation can be used in a dosing regimen preferably comprising rapamycin or propamycin in a dose of 100 mg to 140 mg twice daily. Although dosing regimens of three or more times daily are contemplated, individuals receiving treatment are unlikely to accept and adhere to such regimens. If dosing is three or more times daily, the total daily dose should still be 200 mg to 280 mg per day, more preferably 240 mg per day.

If rapamycin or propamycin is administered to children under 12 years of age, a proportionally reduced dose may be administered to achieve the same exposure and pharmacokinetic results as the adult dose. Preferably, the proportionally reduced dose is determined based on body weight. The adult dose of 200 mg to 280 mg rapamycin per day is based on an average 70 kg adult. For children under 12 years of age, the dose can be calculated based on their 70 kg body weight.

The therapeutic formulations of the present invention comprising rapaggerm or propaggerm have been developed based on pharmacokinetic data, in particular, based on the half-life of rapaggerm and propaggerm in vivo and the _Cmax of rapaggerm and propaggerm.

Based on the decreased tropism of cells expressing CCR2, this specific dose and exposure of rapamycin or propamycin is necessary to achieve peak therapeutic efficacy. Angiotensin type 1 receptor blocker activity

The present inventors surprisingly discovered that, although _AT1R blockers are traditionally considered to be interchangeable, there are some exceptions based on the interaction of _AT1R blockers with the CCR2- _AT1R complex.

Angiotensin type 1 receptor (AT ₁ R, AT1R, angiotensin II receptor type 1) is a G protein-coupled receptor.

The phrase "angiotensin type 1 receptor inhibitor" (also known as angiotensin receptor blocker or ARB) is understood to mean an agent or compound that can inhibit or partially inhibit the activation of _AT1R . This includes antagonists, inverse agonists and negative allosteric modulators of _AT1R .

The term "AT ₁ R blocker" includes a pharmaceutically acceptable salt of an AT ₁ R blocker. Alternatively, the AT ₁ R blocker may be an antibody blocker of AT ₁ R.

For example, the _AT1R inhibitor can be selected from the group comprising irbesartan (e.g. Avapro®), eprosartan (e.g. Teveten®), losartan (e.g. Cozaar®), valsartan (e.g. Diovan®), telmisartan (e.g. Micardis®), candesartan (e.g. Atacand®), olmesartan (e.g. Benicar®), azilsartan (e.g. Edarbi®), sparsentan (e.g. Filspari), fimasartan (e.g. Kanarb®) and ZD-7115. For example, the angiotensin receptor inhibitor can be irbesartan or valsartan.

Taylor et al. (2011) (Journal of the American Society of Hypertension 13(9):677-686) reviewed the angiotensin II AT ₁ receptor blockers (also called AT ₁ R blockers or ARBs) and noted that "[t]here are no major differences in efficacy or other clinical characteristics among older drugs in this class." Of note, the differences in characteristics appear to be related to interactions with other pathways rather than core angiotensin II AT ₁ receptor blockade. According to Taylor, although "there are some pharmacological differences among ARBs, and some of these drugs have specific effects independent of vasopressin II receptor blockade, there is no strong evidence that these drugs translate into unique protection against target organ damage due to hypertension." Therefore, with respect to their effects on _AT1R blockade, ARBs are functionally interchangeable despite their structural differences.

As Taylor noted in his first paragraph, ARBs were first commercialized in the 1990s, and eight specific ARBs are listed that are marketed in the United States. Despite the extended use of this class of drugs, physicians appear to view these drugs as a group of drugs that are largely interchangeable. While there are some specific subtleties in the exact pharmacology of some of the drugs in the group, these do not appear to be related to their angiotensin II AT ₁ receptor activity. Furthermore, all pharmacological products behave slightly differently in some patients, but some subgroup variability does not preclude their general use in treatment.

_AT1R blockers preferably inhibit or partially inhibit _AT1R . Preferably, _AT1R blockers interact directly with _AT1R and do not include therapeutic agents that act upstream of _AT1R to prevent the production of functional ligands but do not interact with _AT1R itself, such as angiotensin converting enzyme inhibitor (ACEi) therapeutic agents.

The term "AT ₁ R blocker" includes a pharmaceutically acceptable salt of an AT ₁ R blocker. Alternatively, the AT ₁ R blocker may be an antibody blocker of AT ₁ R.

As an example, the angiotensin receptor inhibitor may be irbesartan. Irbesartan is an angiotensin type 1 receptor antagonist, also known as 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazolyl-5-yl)phenyl]phenyl}methyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The angiotensin receptor inhibitor may be sparsentan. Sparsentan, also known as 2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(4,5-dimethyl-1,2-oxazol-3-yl)benzenesulfonamide, is a combined selective antagonist of the angiotensin II type 1 receptor ( _AT1R ) and the endothelin A receptor.

The CCR2-AT1R complex has been previously identified as a target for the treatment of kidney disease and other diseases. The inventors have previously demonstrated that ARBs consistently and highly inhibit phosphoinositide signaling of the _AT1R -CCR2 heteromer. It has also been demonstrated that the CCR2- _AT1R complex leads to activation of the Gαi protein and recruitment of β-arrestin.

However, it is not clear whether ARBs retain their potency to inhibit Gαi protein activation or β-arrestin recruitment when acting on the CCR2- _AT1R complex compared to the _AT1R alone. This is important because inhibition of the _AT1R by ARBs is required for clinical efficacy. In Gαi protein dissociation assays, the potency of ARBs against the _AT1R alone is generally found to correlate well with the potency of ARBs against the co-stimulated CCR2- _AT1R complex.

However, it was surprisingly found that compound EXP3174 was less potent than _AT1R alone against the CCR2- _AT1R complex as expected. Therefore, in the presence of _AT1R -CCR2 complex, _AT1R blocker EXP3174 does not function as effectively as other _AT1R blockers. This suggests that the affinity of ARBs for CCR2- _AT1R complex is not significant. ARBs with lower potency against CCR2- _AT1R complex compared to _AT1R alone may have reduced clinical effectiveness.

The _AT1R blocker EXP3174 is a metabolite of losartan that has been widely used in research and is reported to be more potent than losartan in blocking angiotensin II-induced cellular responses (Sachinidis et al. (1993) EXP3174, a metabolite of losartan (MK 954, DuP 753) is more potent than losartan in blocking the angiotensin II-induced responses in vascular smooth muscle cells J Hypertens 11(2):155-62). EXP3174 was used as an experimental _AT1R blocker in combination with a CCR2 pathway inhibitor in WO2012094703.

The present invention therefore provides a formulation comprising a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

For purposes of the present invention, an _AT1R blocker having the same or greater affinity for the CCR2- _AT1R complex is considered to be an AT1R blocker affinity where the affinity for the CCR2- _AT1R complex is not lost more than 3.5-fold compared to the affinity for _AT1R .

The present invention further provides a dosing regimen comprising administering a daily dose of a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

Preferably, the AT ₁ R blocking agent is selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan. Preferably, the AT ₁ R blocking agent is not EXP3174.

If the affinity of an AT ₁ R blocker for the CCR2-AT ₁ R complex is lower than for AT ₁ R alone, the results of a dose determination experiment may not be translated into a clinical setting. The therapeutic dose of an AT ₁ R blocker with a lower affinity for the CCR2-AT ₁ R complex than for AT ₁ R alone will be higher than the dose expected and determined in an experimental setting using only AT ₁ R alone because in an individual the AT ₁ R is provided as a CCR2-AT ₁ R complex. Therefore, more of an AT ₁ R blocker with a lower affinity for the CCR2-AT ₁ R complex than for AT ₁ R alone will be required to achieve the same therapeutic effect in vivo.

Preferably, the _AT1R blocker is provided at 2 mg to 800 mg per day, provided in one or more doses. Even more preferably, the _AT1R blocker is provided at 5 mg to 320 mg per day. For example, the _AT1R blocker is irbesartan, and is administered at a dose of 75, 150 or 300 mg per day, provided in one or more doses. Alternatively, the _AT1R blocker is valsartan, and is administered at a dose of 80 mg, 160 mg or 320 mg per day, provided in one or more doses.

As mentioned above, as described by Taylor et al. (2011), _AT1R blockers are functionally interchangeable, although the dosage of each active substance is different. The reader skilled in the art will use their knowledge of the dosage range of each _AT1R blocker to establish the appropriate dosage for use in the present invention. Table 1 : Recommended daily dosage of _AT1R blockers ARB Daily dose Irbesartan 50-300mg Valsartan 30-320 mg Losartan 10-100 mg Candesartan 2-32 mg Telmisartan 10-80 mg Olmesartan 5-40 mg Eprosartan 300-800mg Azisartan 10-80 mg Fimasartan 30-120 mg Combination formulations

Without being bound by any theory, it is proposed that CCR2 pathway inhibition as well as _AT1R signaling is preferred for clinical effectiveness. The combination of a CCR2 pathway inhibitor with an ARB whose affinity for the CCR2- _AT1R complex is equal to or greater than that for _AT1R alone is therefore preferred.

Examples of such combinations include combinations of rapamycin or propamycin with ARBs, namely candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan and valsartan, and the dual ARB endothelin receptor antagonist spassentan. Such combinations do not include combinations of rapamycin or propamycin with EXP3174.

The present invention further provides a formulation suitable for delivering: a) a therapeutically effective amount of rapamycin or propamycin; and b) a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

The present invention further provides a formulation suitable for delivering: a) 200 mg to 280 mg daily dose of rapamycin or propamycin; and b) a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a dosing regimen comprising administering a daily dose of: a) 200 mg to 280 mg of rapamycin or propamycin; b) a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

The present invention further provides a dosing regimen comprising administering a daily dose of: c) 200 mg to 280 mg of rapamycin or propamycin; d) a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone.

The _AT1R blocker having an affinity for the CCR2- _AT1R complex equal to or greater than the affinity of the _AT1R blocker for _AT1R alone may be selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan. Preferably, the AT1R blocker is not EXP3174.

For purposes of the present invention, equal or greater affinity of an _AT1R blocker for the CCR2- _AT1R complex is considered an AT1R blocker affinity where the affinity for the CCR2- _AT1R complex is not lost more than 3.5-fold compared to the affinity for AT1R.

The therapeutically effective amount of the above _AT1R inhibitor is preferably 2 mg to 800 mg per day.

The pharmaceutical formulations described above may include excipients, solvents, carriers and other pharmaceutically acceptable ingredients as appropriate.

As used herein in the context of the pharmaceutical formulation of the present invention, the term "ingredient" means an AT ₁ R blocker or rapamyridine.

As used herein, the term "inhibit" means a decrease below the detectable limit compared to a reference. The phrase includes blocking, delaying or hindering an action to prevent an adverse result.

As used herein, the term "partial inhibition" means any decrease within detectable limits compared to a reference. The phrase includes blocking, delaying or hindering an effect to prevent an adverse outcome.

Rapagen and the _AT1R inhibitor may be the same active agent, such as a bispecific antibody.

Both _AT1R blockers and rapamyridine can be pharmaceutically acceptable salts of the corresponding active agents. Pharmaceutically and veterinarily acceptable salts include salts that retain the biological effectiveness and properties of the compounds disclosed herein and are not biologically or otherwise undesirable. In many cases, the compounds disclosed herein are capable of forming acid salts and/or base salts due to the presence of amine and/or carboxyl groups or groups similar thereto. Acceptable base addition salts can be prepared from inorganic bases and organic bases. By way of example only, salts derived from inorganic bases include sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as, by way of example only, alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trialkenylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(substituted The invention also includes amines of the type described herein and the like, wherein at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines wherein two or three of the substituents together with the amine nitrogen form a heterocyclic or heteroaryl group.

Pharmaceutically and veterinarily acceptable acid addition salts can be prepared from inorganic and organic acids. By way of example, inorganic acids that can be used include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. By way of example, organic acids that can be used include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, apple acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

Pharmaceutically or veterinarily acceptable salts of the compounds useful in the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of such compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or in a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences. 17th edition, Mack Publishing Company, Easton, Pa. (1985), page 1418, the disclosure of which is incorporated herein by reference. Examples of such acceptable salts are iodide, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetyloxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, γ-Hydroxybutyrate, β-Hydroxybutyrate, Butyne-1,4-diacid salt, Hexyne-1,4-diacid salt, Hexyne-1,6-diacid salt, Caproate, Caprylate, Chloride, Cinnamate, Citrate, Decanoate, Formate, Fumarate, Glycolate, Heptanoate, Hippurate, Lactate, Appleate, Maleate, Hydroxymaleate , malonate, mandelate, methanesulfonate, niacinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, hydrogen sulfate, pyrophosphate, Sulfates, sulfites, bisulfites, sulfonates, benzenesulfonates, p-bromobenzenesulfonates, chlorobenzenesulfonates, propanesulfonates, ethanesulfonates, 2-hydroxyethanesulfonates, methanesulfonates (merhanesulfonate), naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartaric acid salts and the like. Disease or illness

Generally, a range of diseases associated with interleukins can be treated by the methods of the present invention. This includes diseases associated with increased or decreased interleukin production and/or increased or decreased cellular responsiveness to interleukins. Interleukin-related diseases should also be understood as diseases or disorders in which interleukin receptors exhibit abnormal characteristics and are targets for specific pathogens or pharmacological interventions.

The disease associated with interleukin is preferably an inflammatory disease, or a disease resulting from or associated with inflammation. The inflammatory disease, or a disease resulting from or associated with inflammation may be a kidney disease.

The inflammatory disease or the disease resulting from or associated with inflammation to be treated, improved or prevented is preferably a kidney disease. More preferably, the kidney disease is a kidney disease associated with proteinuria. Kidney disease may be selected from the list comprising: focal segmental glomerulosclerosis (FSGS; including idiopathic (primary) FSGS, secondary FSGS, hereditary FSGS and FSGS of any other cause), renal fibrosis, immunoglobulin A nephropathy (IgAN), Alport's syndrome, chronic kidney disease (including chronic kidney disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic)) and renal failure disorders (including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, primary nephrotic proteinuria and renovascular hypertension).

The term "prevention" includes administering the formulations of the invention to individuals who have symptoms indicating that they are at risk for developing the above-mentioned kidney diseases, but who may not be diagnosed with the kidney disease at the time of treatment. For example, an individual may have elevated blood creatine or urine protein, but has not yet been diagnosed with the kidney disease.

The term "amelioration" includes administration of the formulations of the invention to an individual who already has a renal disease and is at risk of developing a post-morbid condition. Measurement of Inhibition

Effects of (i) rapamycin or propamycin, (ii) _AT1R blockers, or (iii) a combination of rapamycin or propamycin and _AT1R blockers on the CCR2 pathway and/or _AT1R blockers Inhibition or partial inhibition of CCR2 can be measured using in vitro methods described herein and include, but are not limited to, biochemical or cellular assays for assessing in vitro tropism migration of neutrophils expressing CCR2 and other cells known in the art, as well as measuring phosphoinositide production, extracellular regulated kinase (ERK) phosphorylation, cAMP production, label-free techniques (such as using impedance, photorefractive index, or charge redistribution), G protein coupling using proximity reporter systems or other methods, beta-arrestin recruitment or mediated signaling, transcription factor-based reporter systems, microscopic visualization using fluorescent labels, using antibodies to assess receptor cellular localization (such as enzyme-linked immunosorbent assay), using bioluminescence resonance energy transfer to assess cellular localization and trafficking, and fluorescence activated cell sorting.

Inhibition or partial inhibition of the CCR2 pathway and/or _AT1R by (i) rapamycin or propamycin, (ii) an _AT1R blocker, or (iii) a combination of rapamycin or propamycin and an _AT1R blocker can be measured using in vivo methods, including but not limited to measuring the levels of cells and interleukins in lung exudate, measuring lung function (including physical lung function capacity) using spirometry-based tests, or measuring lung function output using blood gas or other biochemical measures, or improvement in functional benefit, including clinical benefit measured by quantitative methods such as a walk test or qualitative methods such as patient-reported outcome assessments. Inhibition or partial inhibition can be indicated by qualitative improvements in lung structure, as measured by one or more of the above endpoints.

In a preferred embodiment, the overall efficacy of the pharmaceutical formulation is greater than the efficacy of either component when either is administered without the other component. Thus, the combination formulation can be administered _in a single dose, including a subtherapeutic dose, or less frequently than either component could be administered as a single compound.

Preferably, the total efficacy of the pharmaceutical formulation is greater than the sum of the efficacy of the _AT1R blocker and rapamyr or propamyr when either component is administered without any other component. More preferably, a synergistic effect of efficacy is observed when the _AT1R blocker and rapamyr or propamyr are administered simultaneously or sequentially.

Alternatively, the total efficacy of the pharmaceutical formulation is equal to the sum of the efficacy of the _AT1R blocker and rapamycin or propamegerium when either component is administered without any other component. As another preferred embodiment of this alternative, when the _AT1R blocker and rapamycin or propamegerium are administered simultaneously or sequentially, an additive effect of efficacy is observed.

In another alternative, the total efficacy of the pharmaceutical formulation is less than the sum of the efficacy of the _AT1R blocker and rapamyr or propamyr when either component is administered without any other component. In yet another embodiment, although the combination efficacy is less than the sum of the efficacy of the _AT1R blocker and rapamyr or propamyr when each component is administered without any other component, the treatment provides greater efficacy than single treatments of either _AT1R blocker or rapamyr or propamyr administered alone.

Preferably, the two components are administered simultaneously (e.g., as two tablets taken together, or as a single tablet with each component formulated together) or sequentially (e.g., one tablet taken after the other). The doses of each component can be taken together (simultaneously), or sequentially and within seconds, minutes, days, weeks or months of each other.

One component of the combination of the invention may already be administered to an individual, for example as a standard of care treatment. In this case, the second component of the combination of the invention is administered as the second component in a treatment regimen to provide a therapeutic combination of the invention.

The present invention further provides a method for treating, improving or preventing kidney disease, which comprises the step of administering to an individual: a) a therapeutic formulation suitable for delivering 200 mg to 280 mg of rapamyr or propamyr daily.

The present invention further provides a method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: i) a therapeutically effective amount of an AT ₁ R inhibitor, wherein the affinity of the AT 1 R inhibitor to the CCR2-AT ₁ R complex is equal to or greater than the affinity of the AT ₁ R inhibitor to AT ₁ R alone.

The present invention further provides a method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: i) a therapeutically effective amount of rapamyr or propamyr daily; and ii) a therapeutically effective amount of an _AT1R blocker, wherein the affinity of the AT1R blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a method for treating, ameliorating or preventing kidney disease, comprising the step of administering to a subject: i) a therapeutic formulation suitable for delivering 200 mg to 280 mg of rapamycin or propamycin per day; and ii) a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The subject to be treated is preferably a mammal, including a human mammal.

Repagold and the _AT1R blocker may be administered in the same dosage form or in separate dosage forms.

Rapaxil and/or _AT1R blocker can be an antibody inhibitor or blocker of the corresponding receptor. Rapaxil and _AT1R blocker can be the same active agent, such as a bispecific antibody. Rapaxil and/or _AT1R blocker can be a pharmaceutically acceptable salt of Rapaxil and/or _AT1R blocker.

Repagold and the _AT1R blocker may be administered simultaneously or sequentially.

One component of the combination of the invention may already be administered to an individual, for example as a standard of care treatment. In this case, the second component of the combination of the invention is administered as the second component in a therapy to provide the therapeutic combination of the invention.

Although not intended to be limited to any particular mode of action, in a preferred embodiment, when CCR2 is associated with the angiotensin receptor _AT1R , rapamycin has greater affinity and/or potency and/or efficacy when interacting with CCR2 or modulating its downstream pathways. For example, CCR2 and the angiotensin receptor _AT1R can be associated with a CCR2/ _AT1R complex.

Preferably, the combination of the present invention provides a lower dose of the _AT1R blocker or rapamyr than when either is used alone. For example, one or both of the _AT1R blocker or rapamyr may be provided at a subtherapeutic dose. This would advantageously reduce the negative safety profile of _AT1R or rapamyr while providing the same therapeutic benefit.

In another preferred embodiment, when Repag-Germanium and an _AT1R blocker are administered to a subject simultaneously or sequentially, the affinity, potency and/or efficacy of the combination is greater than the affinity, potency and/or efficacy achieved when Repag-Germanium is not administered in combination with an _AT1R blocker (whether simultaneously or sequentially). In another preferred embodiment, when Repag-Germanium and an _AT1R blocker are administered to a subject in combination (whether simultaneously or sequentially), a synergistic effect (e.g., as measured by affinity, potency and/or efficacy) is achieved.

Although not intended to be limited to any particular mode of action, in a preferred embodiment, when the angiotensin receptor AT ₁ R is associated with CCR2, the AT ₁ R blocker has greater affinity and/or potency and/or efficacy when interacting with the angiotensin receptor AT ₁ R. For example, CCR2 and the angiotensin receptor AT ₁ R can be associated as a CCR2/AT ₁ R complex. In another preferred embodiment, when the AT ₁ R blocker is administered to an individual simultaneously or sequentially with rapagra, the affinity, potency and/or efficacy of the combination is greater than the affinity, potency and/or efficacy achieved when the AT ₁ R blocker is not administered in combination with rapagra (whether simultaneously or sequentially). In yet another preferred embodiment, when the _AT1R blocker is administered in combination with rapamycin (either simultaneously or sequentially) to a subject, a synergistic effect (eg, as measured by affinity, potency, and/or efficacy) is achieved.

For purposes of the present invention, an _AT1R blocker having the same or greater affinity for the CCR2- _AT1R complex is considered to be an AT1R blocker affinity where the affinity for the CCR2- _AT1R complex is not lost more than 3.5- fold compared to the affinity for _AT1R .

The dosage forms provided by the present invention may further include vials, cartridges, containers, tablets or capsules, which contain the pharmaceutical formulation of the present invention and dosage instructions for administering the dosage form to an individual to treat, ameliorate or prevent a disorder or disease.

The amount of each active ingredient that can be combined with the carrier material to produce a single dose will vary depending on the host to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to 1 g of each active compound and an appropriate and convenient amount of carrier material, which may comprise about 5% to 95% of the total formulation. Dosage unit forms typically contain about 0.5 mg to 500 mg of active ingredient.

Preferably, the _AT1R blocker is provided at 2 mg to 800 mg per day, provided in one or more doses. Even more preferably, the _AT1R blocker is provided at 75 mg to 300 mg per day. For example, the _AT1R blocker is irbesartan, and is administered at 75, 150 or 300 mg per day, provided in one or more doses.

Preferably, rapamerium is provided in a dose of 200 mg to 280 mg rapamerium or propamerium per day, provided in one or more doses. Even more preferably, rapamerium or propamerium is provided in a dose of 220 mg to 260 mg per day, more preferably 240 mg per day, provided in one or more doses.

The dosage of each active agent may be provided in a single dosage form or in two separate dosage forms and may contain 2 mg to 800 mg of an AT ₁ R blocker and 200 mg to 280 mg of rapamyr or propamyr. The dosage of both active substances may be provided in a single dosage form or in two separate dosage forms and may contain (i) 2 mg to 800 mg daily of an AT ₁ R blocker and (ii) 200 mg to 280 mg daily of rapamyr or propamyr. The AT ₁ R blocker may be irbesartan and the dosage form may contain about 300 mg daily of irbesartan. Alternatively, the _AT1R blocker is valsartan and is administered at a daily dose of 80 mg, 160 mg or 320 mg, provided in one or more doses.

However, it should be understood that the specific dose for any particular individual will depend on a variety of factors, including the activity of the specific compound employed, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disorder or condition being treated.

In various aspects, the formulations of the present invention can be administered by injection, or prepared for oral, pulmonary, nasal administration or any other form of administration. Preferably, the formulations are administered, for example, intravenously, subcutaneously, intramuscularly, intraorbitally, intraocularly, intraventricularly, intracranially, intracapsularly, intraspinally, intracisternally, intraperitoneally, intracheekally, rectally, vaginally, intranasally or by aerosol administration.

In one aspect, the mode of administration is at least suitable for the form in which the formulation is prepared. The mode of administration that produces the most effective response can be determined empirically, and the modes of administration described below are given as examples only and are not intended to limit the delivery method of the formulation of the present invention in any way. All formulations provided are commonly used in the pharmaceutical industry and are well known to practitioners with appropriate qualifications. Oral dosage form

Contemplated for use herein are oral solid dosage forms, which are generally described in Martin, Remington's Pharmaceutical Sciences, 18th edition (1990 Mack Publishing Co. Easton PA 18042), Chapter 89, which is incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, buccal tablets or lozenges, cachets, or pellets. In addition, liposomes or protein-like encapsulation can be used to formulate the formulations of the present invention (e.g., protein-like microspheres reported in U.S. Patent No. 4,925,673). Liposome encapsulation can be used and liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). Marshall in Modern Pharmaceutics, Chapter 10, Banker and Rhodes, eds., (1979) gives a description of possible solid dosage forms for therapeutic agents, which is incorporated herein by reference. Typically, the formulation will include the compound described as part of the present invention (or a chemically modified form thereof) and inert ingredients that allow protection from the gastric environment and release of the biologically active material in the intestine.

The oral dosage form of the present invention may be a liquid, suspension or other appropriate dosage form, which is delivered directly into the feeding tube of a patient who is in a coma and/or dependent on a ventilator and cannot swallow tablets, etc.

For the rapamycin or _AT1R blockers of the present invention, the release site can be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Those skilled in the art have available formulations that will not dissolve in the stomach but will release the material in the duodenum or other locations in the intestine. In one aspect, the release will avoid the deleterious effects of the gastric environment by protecting the formulation or by releasing the compound outside the gastric environment, such as in the intestine.

Coatings or coating mixtures may also be applied to tablets that are not intended to protect against the stomach. This includes, but is not limited to, sugar coatings, or coatings that make the tablet easier to swallow. Exemplary capsules consist of a hard shell (such as gelatin) for delivery of dry therapeutics, i.e., powders; for liquid forms, soft gelatin shells may be used. In some aspects, the outer shell material of the cachet is thick starch or other edible paper. For pills, buccal tablets, molded tablets, or tablet triturates, moist massing techniques are also contemplated, but are not limited to.

The present invention further provides an oral sustained-release pharmaceutical formulation comprising a therapeutically effective pharmaceutical formulation according to the present invention and a release-delaying agent.

In one aspect of the present invention, the release delaying agent is a water-soluble, water-swellable and/or water-insoluble polymer. Specifically, the water-soluble polymer is selected from the group comprising: ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, enteric coating; and semipermeable membrane. In another aspect of the present invention, the release delaying agent is a non-polymer release delaying agent. More specifically, the non-polymer release delaying agent is hydrogenated castor oil. According to the known procedures known in this art, the formulation of the present invention can be ground or granulated and compressed into tablets or encapsulated into capsules.

To ensure complete gastric resistance, a coating that is impermeable to at least pH 5.0 is used. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and Shellac. These coatings can be used as mixed films.

As used herein, the term "sustained release" means that the amount of the therapeutic compound after oral ingestion is gradually but continuously or continuously released over a relatively extended period of time. The release may start from the stomach after the pharmaceutical formulation passes through the stomach until the pharmaceutical formulation reaches the intestine and continue after the pharmaceutical formulation reaches the intestine. The phrase "sustained release" also means delayed release, in which the release of the therapeutic compound does not start immediately when the pharmaceutical formulation reaches the stomach, but is delayed for a period of time, for example, until the pharmaceutical formulation reaches the intestine. After reaching the intestine, the increase in pH may then trigger the release of the therapeutic compound from the pharmaceutical formulation.

As used herein, the term "release delaying agent" means a substance that reduces the rate at which a therapeutic compound is released from a pharmaceutical formulation when taken orally. Release delaying agents can be polymeric or non-polymeric. Release delaying agents can be used according to any of several sustained release systems, including, for example, diffusion systems, dissolution systems, and/or osmotic systems.

In some embodiments, the therapeutic agent is included in the formulation as fine particles in the form of granules or pellets of about 1 mm in diameter. In some embodiments, the formulation of the material for capsule administration is a powder, a slightly compressed plug or even a tablet. In one embodiment, the therapeutic agent can be prepared by compression.

Colorants and flavorings may be included as an option. For example, the compound may be formulated (such as but not limited to by liposome or microsphere encapsulation) and then further included in an edible product such as a refrigerated beverage containing colorants and flavorings.

In one aspect, the volume of the therapeutic agent can be diluted or increased with an inert material. Such diluents can include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts can also be used as fillers as appropriate, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

In other embodiments, a disintegrant is included in the therapeutic formulation to form a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch-based disintegrant Explotab. Also contemplated are sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acidic carboxymethylcellulose, natural sponge, and bentonite. Another form of disintegrant is an insoluble cation exchange resin. Powdered gels may also be used as disintegrants and binders as appropriate, and these include but are not limited to powdered gels such as agar, karaya gel or astragalus gel. Alginic acid and its sodium salt may also be used as a disintegrant.

Binders are contemplated to bind the therapeutic compound together to form a hard tablet and include, but are not limited to, materials from natural products such as gum arabic, gum tragacanth, starch, and gelatin. Other binders include, but are not limited to, methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) are contemplated for use in alcohol solutions to granulate the therapeutic agent.

Anti-friction agents may be optionally included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may optionally be used as a layer between the therapeutic and the mold wall, and these may include, but are not limited to: stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Exemplary soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, and Carbowax 4000 and 6000.

Glidants may be added as appropriate, which can improve the flow characteristics of the compound during formulation and help rearrangement during compression. Glidants may include, but are not limited to, starch, talc, fumed silica, and hydrated aluminosilicates.

To help the therapeutic agent dissolve into the aqueous environment, a surfactant may be added as a wetting agent in certain embodiments. The surfactant may include, for example, but not limited to, anionic cleaners such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic cleaners may be used as appropriate and may include, but not limited to, benzalkonium chloride or benzethhomium chloride. A list of potential nonionic detergents that can be included in the formulation as surfactants is lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. When used, these surfactants can be present in the formulation of the compound alone or as a mixture in different ratios.

Additives that may enhance the absorption of the compound may be desirable. Such additives include the fatty acids oleic acid, linoleic acid, and linolenic acid.

Controlled release formulations may be desirable. In some aspects, the compound may be incorporated into an inert matrix that allows release by diffusion or leaching mechanisms (i.e., resins). In some aspects, slowly degrading matrices may also be incorporated into the formulation. Another controlled release form of this therapeutic agent is by a method based on the OROS™ therapeutic system (Alza Corp), where the drug is enclosed in a semipermeable membrane that allows water to enter and push the drug out through a small opening due to osmosis. Some enteric coatings also have a delayed release effect.

In other aspects, a mixture of materials may be used to provide an optimal film coating. Film coating may be performed, for example but not limited to, in a pan coater or in a fluidized bed or by compression coating. Dosing Schedule

It should be understood that in certain aspects, the formulations of the present invention may be administered as a bolus dosing schedule, or preferably, in a multiple dosing schedule. A multiple dosing schedule is a regimen in which the primary delivery course may be 1 to 10 divided doses, followed by other doses at subsequent time intervals as needed to maintain or intensify the treatment, as appropriate. The dosing regimen will also be determined, at least in part, by the needs of the individual and the judgment of the practitioner.

Therefore, the present invention provides tablets, capsules, liquids, suspensions or other oral dosage forms comprising the pharmaceutical formulations of the present invention.

The _AT1R blocker and repa Germanium or propagate Germanium may be delivered in the same formulation, or may be delivered in separate formulations.

The _AT1R blocker and repaq or propaguergium may be in the same dosage form, or may be in separate dosage forms.

A subject to whom an _AT1R blocker and rapamycin are administered may have already received an active agent and the other therapeutic components of the invention may be administered according to the invention.

The present invention provides the use of 200 mg to 280 mg of a daily dose of rapamyr or propamyr for preparing a formulation for treating, improving or preventing a kidney disease in an individual.

The present invention provides a use of 200 mg to 280 mg of rapamyr or propamyr in a daily dose for treating, improving or preventing kidney disease in an individual.

The present invention provides the use of a therapeutically effective amount of an AT ₁ R inhibitor for preparing a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the inhibitor has an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R inhibitor for AT ₁ R alone.

The present invention provides a use of a therapeutically effective amount of an AT ₁ R inhibitor for treating, improving or preventing a kidney disease in an individual, wherein the inhibitor has an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R inhibitor for AT ₁ R alone.

The present invention also provides the use of (a) a therapeutically effective amount of repagd or propaguervate and (b) a therapeutically effective amount of an _AT1R blocker for manufacturing a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention also provides the use of (a) a therapeutically effective amount of rapamycin or propamycin and (b) a therapeutically effective amount of an _AT1R blocker for treating, ameliorating or preventing kidney disease in an individual, wherein the affinity of the blocker for CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone.

The present invention further provides a therapeutically effective amount of rapamyr or propamyr for use in a formulation for treating, ameliorating or preventing kidney disease, wherein the at least one _AT1R blocker and rapamyr or propamyr are administered to the subject simultaneously or sequentially.

The present invention further provides a therapeutically effective amount of rapagent or propagent for use in a formulation for treating, ameliorating or preventing kidney disease, wherein the rapagent and a therapeutically effective amount of an _AT1R blocker having an affinity for CCR2- _AT1R complex equal to or greater than the affinity of the _AT1R blocker for _AT1R alone are administered to the subject simultaneously or sequentially.

The therapeutically effective amount of the above-mentioned rapamyr or propamyr is preferably 200 mg to 280 mg of rapamyr or propamyr per day.

The AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone may be selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan. Preferably, the AT ₁ R blocker is not EXP3174.

The therapeutically effective amount of the above _AT1R inhibitor is preferably 2 mg to 800 mg per day.

Repag-germium or propaguermium and the _AT1R blocker may be administered in the same dosage form or in separate dosage forms.

Repag Germanium or propaguertin and/or _AT1R blocker can be an antibody inhibitor or blocker of the corresponding receptor. Repag Germanium or propaguertin and _AT1R blocker can be the same active agent, such as a bispecific antibody. Repag Germanium or propaguertin and/or _AT1R blocker can be a pharmaceutically acceptable salt of Repag Germanium, propaguertin and/or _AT1R blocker.

Repag-Gr or Propag-Gr and the _AT1R blocker can be administered simultaneously or sequentially .

The present invention provides a kit for treating or preventing kidney disease, the kit comprising: a) 200 mg to 280 mg daily dose of rapamycin or propamycin; and b) instructions for use.

The present invention further provides a kit for treating or preventing kidney disease, the kit comprising: a) a therapeutically effective amount of an AT ₁ R blocker, the affinity of the blocker for the CCR2-AT ₁ R complex being equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone; and b) instructions for use.

The present invention provides a kit for treating or preventing kidney disease, the kit comprising: a) a therapeutically effective amount of rapamycin or propamycin; b) a therapeutically effective amount of an _AT1R blocker, the affinity of the blocker for the CCR2- _AT1R complex being equal to or greater than the affinity of the _AT1R blocker for _AT1R alone; and c) instructions for use.

The therapeutically effective amount of the above-mentioned rapamyr or propamyr is preferably 200 mg to 280 mg of rapamyr or propamyr per day.

The AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone may be selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and sparsentan. Preferably, the AT ₁ R blocker is not EXP3174.

The contents of the kit may be freeze-dried, and the kit may additionally contain a solvent suitable for resolubilizing the freeze-dried components. The individual components of the kit will be packaged in separate containers, and associated with such containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

When the components of the kit are provided in one or more liquid solutions, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. For in vivo use, the expression construct may be formulated into a pharmaceutically acceptable injectable formulation. In this case, the container member itself may be an inhaler, syringe, pipette, eye dropper or other similar device from which the formulation may be administered to an animal's diseased area, such as the lungs, injected into the animal's body, or even applied to the kit and mixed with the other components of the kit.

The components of the kit may also be provided in dry or freeze-dried form. When the reagents or components are provided in dry form, they are generally reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container member. Regardless of the number or type of containers, the kit of the present invention may also contain or package an instrument useful for assisting in the injection/administration or placement of the final compound formulation into the animal. Such an instrument may be an inhaler, a syringe, a pipette, a tweezer, a measuring spoon, an eye dropper, or any such medically approved delivery tool. General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all steps, features, formulations and compounds mentioned or indicated in the specification, individually or collectively, and any and all combinations or any two or more steps or features.

All documents, references, patent applications or patents cited in this article are expressly incorporated into this article in their entirety by reference, which means that the reader should read them and regard them as part of this article. For the sake of brevity, the documents, references, patent applications or patents cited in this article will not be repeated in this article.

Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein are incorporated herein by reference and may be used in practicing the present invention.

The scope of the present invention is not limited to any specific embodiments described herein. Such embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the present invention as described herein.

The invention described herein may include a range of one or more values (e.g., size, displacement, and field strength, etc.). A range of values should be understood to include all values within the range, including values defining the range, and values adjacent to the range that result in the same or substantially the same result as the value of that value adjacent to the boundary defining the range. Therefore, unless otherwise indicated, the numerical parameters given in the specification and patent application range are approximate values, which may vary depending on the desired properties sought to be obtained by the present invention. Therefore, "about 80%" means "about 80%", also "80%". At a minimum, each numerical parameter should be interpreted based on the number of significant digits and ordinary rounding methods.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises,” “comprised,” “comprising,” and similar terms may have the meanings ascribed to them by U.S. patent law; for example, they may mean “includes,” “included,” “including,” and similar terms; and terms such as “consisting essentially of” and “consists essentially of” have the meanings ascribed to them by U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements that are found in the prior art or that affect the basic or novel characteristics of the invention.

Other definitions of selected terms used herein can be found in the embodiments of the present invention and are applicable throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The term "active agent" may refer to one active agent, or may cover two or more active agents.

The following examples are used to more fully describe the manner of using the above invention and to illustrate the best mode expected for implementing various aspects of the invention. It should be understood that these methods are by no means intended to limit the true scope of the invention, but are presented for illustrative purposes.

Other features of the present invention are more fully described in the following non-limiting examples. This description is included for the sole purpose of illustrating the present invention. It should not be construed as limiting the broad description of the present invention as described above. Example 1 Pharmacokinetic Modeling Methods for Rapa Germanium

Pharmacokinetic data are available from studies DMX-200-101, DMX-200-201, DMX-200-202, and DMX-200-203. Table 2 : Pharmacokinetic data Project Research 101 Research 202 Research 203 Research Phase Number DMX-200-101 Phase I DMX-200-202 Phase II DMX-200-203 Phase II Individuals with PK 13 8 45 Description/ Patient Group Open label, two-way crossover/healthy volunteers A double-blind, randomized, placebo-controlled, 2-way crossover study to evaluate the safety and efficacy of RPG in FSGS patients receiving irbesartan A Phase 2, double-blind, randomized, placebo-controlled, crossover study to evaluate the safety and efficacy of RPG in patients with DKD receiving irbesartan Route Dosage/ Formulation Oral single dose administration: - IR formulation without food1) - ER formulation without food - ER formulation with food Two 16-week treatment periods of repeated-dose oral RPG 120 mg bid IR in period 1 and placebo in period 2, or repeated-dose oral placebo in period 1 with crossover and 120 mg RPG bid IR in period 2 Two 12-week treatment periods of repeated oral doses of RPG 120 mg bid IR in period 1 and placebo in period 2, or repeated oral doses of placebo in period 1 crossed over and 120 mg RPG bid IR in period 2 Number of samples ±16 PK plasma samples per subject per treatment; 7 urine sampling intervals Before both periods of dosing, at weeks 1, 8, 15, and 16; ± 24-hour urine collection samples at weeks 8, 15, and 16 Before both periods of dosing, at weeks 1, 6, 11, and 12; ± 24-hour urine collection samples at weeks 8, 15, and 16

In all studies, blood samples were analyzed using liquid chromatography tandem mass spectrometry (LC–MS/MS). This method involves the addition of deuterated PPG (d ₂ -PPG) as an internal standard (IS) and liquid-liquid extraction using chloroform for sample preparation. Samples were analyzed on a LC-MS/MS system consisting of a Shimadzu LC20AD and Thermo Fisher Hypercarb columns using gradient elution and an API 4000 mass spectrometer. The mass spectrometer was operated in negative charge ionization (ESI) mode and was used to monitor the conversion of the former exogenous to the product ions of PPG and d ₂ -PPG, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL.

The pharmacological analysis was performed in several steps, driven by the suitability of clinical data for PK model development. 1. An initial structural population PK model was developed using data from healthy volunteers (Study 101). 2. This model was subsequently updated using sparse data from patients in Studies 202 and 203, and the role of covariates was explored. 3. Model validation was performed. 4. Finally, the developed model was used to simulate the effects of covariates on key PK parameters and potential exposures.

SAS software (version 9.4) was used to prepare the analytic data sets. Population PK analyses were performed using NONMEM (version 7.5.0) and the one-order conditional estimation with interaction (FOCE) method. PsN (version ≥5.0.0) was used to perform NONMEM runs and to run visual prediction checks (VPC), sampling importance resampling (SIR), and automated stepwise covariate modeling (SCM). R software (version ≥4.0.2) was used for exploratory analyses, creation of goodness-of-fit (GOF) plots, and summary statistics. Campsis (version 1.1.0), an R-based application software provided by Calvagone, was used for simulations.

NONMEM version 7.5.0 was used with the GNU Fortran (GCC) compiler version 9.3.0, running on an Ubuntu 20.04 LTS system with Linux kernel version 5.4.0-72-generic on an Intel Xeon E5-2640 v3 @ 8 HT 2.60GHz, using a fault-tolerant VMWare ESXi 7.0u1 platform with 8 vCPUs for the hypervisor and Intel Xeon E5-2680 v4 14HT 2.40GHz for the compute nodes. Fortran compiler optimization options were: -static -w -O3 -ffast-math. All software was used in a validated environment.

In mixed effects modeling, population parameters are classified as follows: Fixed effect parameters θ (theta) are used for structural population parameters (e.g., Ka, CL, V, F, etc.), as well as for including certain parameters that relate population parameters to physiological or demographic variables (covariates) in the analysis. Random effect parameters η (eta) are used to describe inter-individual variability (IIV) in fixed effect parameters, and can also be used to describe inter-occasion variability (IOV) if appropriately included in the model. The η parameters are assumed to be normally distributed random variables with mean zero and variance-covariance matrix Ω (omega). The square roots of the diagonal elements of this matrix provide estimates of the IIV. Off-diagonal elements (if not fixed to zero) are usually transformed into correlations. The random effect parameter ε (epsilon) is used to describe residual variability. The residual variation includes within-subject error, analytical error, potential error due to model missspecification, and any other errors not accounted for by the structural elements such as θ and η. The residual variation parameters are assumed to be normally distributed random variables with mean zero and standard deviation σ (sigma).

Different structural models were combined with different residual models and, if necessary, with different estimation methods to identify the most appropriate mixed-effects model. Parameter estimates were performed using first-order conditional estimation (FOCE) with the INTERACTION option to account for the interaction between the between-individual random effect η and the residual random effect ε. An exponential model was used to describe the between-individual variability (IIV) of the model parameters, assuming that the individual pharmacokinetic parameters were log-normally distributed: PP mm = PP TTTT × ff ηηwhere PI is a single parameter value, P TV is the typical population estimate of that parameter, and η is the individual deviation from the population estimate with mean 0 and variance ω2. Correlations between the random effect parameters were investigated by estimating some selected off-diagonal elements of the ω matrix. Inter-site variability (IOV) of the parameters F and CL/F was assessed to potentially explain variability associated with steady-state PK observations performed at completely different visits during the study. The data were modeled using the log-transformed two-sided (LTBS) approach, whereby residual errors were incorporated as additive errors on a logarithmic scale. Separate residual error terms were explored between healthy individuals and patients.

Covariate model building was exploratory. Potential relationships between covariates and individual parameter estimates were initially explored graphically if the degree of η-contraction in the parameters was sufficiently low (<25%). As the model was intended for exposure simulations in children, it was decided to explain the associations between body weight and PK parameters based on the allokinetic theory already present in the base model. A fixed allokinetic exponent of 0.75 was used for clearance and compartmental flux (Q), whereas an exponent of 1.0 was used for the relationship between volume and body weight. Automated stepwise covariate modeling (SCM) using a forward addition/backward deletion procedure as implemented in PsN [20] was applied to the base model for the full analysis data set. To improve the computational time of SCM, PsN applied Taylor transformation to generate a linearized form of the model, on which the covariate function was then applied. The final model from the SCM procedure was then rerun in its original untransformed form. The following covariates were tested for association with CL and relative bioavailability (F) in the model: weight, BMI, age, race (if more than 5% of participants were non-white), sex, liver/renal function (AST, ALT, albumin, CRCL, eGFR), disease (study equivalent). In principle, the baseline covariate values were considered, however, if changes in covariates over time also needed to be considered in the covariate model, this was assessed. Correlations between covariates were assessed graphically. If two or more covariates are highly correlated, the most clinically/biologically and practically relevant covariate is tested first in the covariate analysis, and another covariate is dropped or tested only after the more relevant covariate is included in the model. For continuous covariates, linear functions or gamma functions are tested, as shown in the following equations: Where Pi is the parameter estimate for the ith patient, whose covariate (COVARi) value is centered or scaled around the typical median of the covariate (MEDCOVAR). θ is the slope (index) of the parameter-covariate relationship. Dichotomous categorical covariates are included as follows: Where θ is the coefficient of the parameter-covariate relationship. CATCOVAR takes the value of 0 or 1 for the status of the covariate value (absent/present) for a particular individual. During SCM, a forward addition/backward deletion procedure is performed. Typically, during forward selection, covariates are introduced into the model once, and only covariate model relationships that are considered significant are considered for the next step. The covariate with the highest statistical significance will be included first, and then other significant covariates from the first step will be retested. Similarly, the most important covariate at that time will be retained. This process will be repeated until there is no further improvement in the model. During backward deletion, covariates are removed one at a time. The least significant parameter will be removed from the model first, and then the process is repeated until the covariate cannot be removed without significantly worsening the model adjustment. Forward covariate selection was performed using a p-value of p<0.05 (χp=0.05, ν=1 2 =3.84) as the selection criterion. Subsequently, backward deletion was performed using a p-value of p<0.01 (χp=0.01, ν=1 2 =6.63) as the selection criterion. The clinical relevance of the covariates included in the model after the covariate search was completed and their contribution in explaining the inter-individual variability were further assessed. The clinical relevance was judged based on the change in the predicted AUC at different values of a given covariate (e.g., % change in AUC for patients with high (low) weight compared to patients with medium weight). Small changes would indicate that the covariate may have limited clinical relevance. Results

The clinical effect of reducing the protein to creatinine ratio, a biomarker of renal function, was produced by administration of a 120 mg BID dose of rapamycin, which produced a steady-state AUC of approximately 15 ug.h/mL (range 5-25) as shown in Table 3. Table 3 : Summary Statistics of Individual Steady-State Exposure (AUC _24h ) at the 240 mg Total Daily Dose for Individuals Evaluated in the Analysis Steady-state exposure in adults after a total daily dose of 240 mg 101 202 203 N (%) 13 (20.3%) 8 (12.5%) 43 (67.2%) Clearance rate (L/h) Median 40.2 18.0 20.7 Scope 24.6 - 60.1 4.6 - 36.5 5.8 - 47.6 F( Relative bioavailability ) Median 1.6 1.1 1.0 Scope 0.7 – 3.7 0.9 - 1.7 0.4 - 1.9 AUC24h (ug.h/mL) Median 9.8 16.7 11.8 Scope 5.5 – 24.9 7.8 – 45.2 3.9 – 47.7

This specific dose and exposure of rapamycin is necessary for peak renal efficacy. Example 2 ARB-CCR2 inhibitor combination that inhibits G protein activation is used to treat a disease in which the potency of the ARB against the _AT1R -CCR2 complex is not less than the expected potency of the ARB against only the _AT1R

The CCR2- _AT1R complex has previously been identified as a target for the treatment of kidney disease. Clinical effectiveness requires inhibition of both the CCR2 pathway and _AT1R signaling.

The inventors have previously demonstrated that phosphoinositide signaling of the _AT1R /CCR2 heteromer is consistently and highly inhibited by ARBs. The inventors have also demonstrated that the CCR2- _AT1R complex leads to activation of the Gαi protein and recruitment of β-arrestin.

However, it is unknown whether ARBs retain their potency in inhibiting Gαi protein activation and β-arrestin recruitment when acting on the CCR2- _AT1R complex compared to the _AT1R alone. Understanding the ability of ARBs to inhibit the _AT1R in the CCR2-AT1R complex is important because it is expected to affect clinical effectiveness.

For example, the inventors have demonstrated that AngII inhibits MCP-1-induced activation of the CCR2- _AT1R complex for Gαi1 coupling. Blocking this AngII-mediated response using an ARB is expected to eliminate this inhibition of MCP-1-induced Gαi1 signaling.

This experiment evaluates the potency of ARBs against _AT1R alone and against the co-stimulatory CCR2- _AT1R complex in a Gαi protein dissociation assay.

To determine whether ARBs retain their potency to inhibit Gαi activation, the potency of ARBs against _AT1R alone was correlated with the potency of ARBs against co-stimulated CCR2- _AT1R complexes obtained from the Gαi dissociation assay (Figure 2).

The potency of ARBs against _AT1R alone was obtained from public sources, such as the IUPHAR Pharmacology Database Guide and/or Michel et al. (2013). The potency of ARBs against the co-stimulatory CCR2- _AT1R complex was experimentally determined, as described below.

HEK293FT cells were transiently transfected with plasmids encoding Gαi1-Nluc and CCR2-YFP hemagglutinin (HA)-tagged _AT1R . 48 h after transfection, cells were incubated with the substrate for 1 h and then pre-incubated with different concentrations of ARBs (candesartan cilexetil, losartan, telmisartan, irbesartan, eprosartan, EXP3174, valsartan, olmesartan medoximil, omasartan, azilsartan kamedoxomil, or candesartan) for 30 min. Cells were then treated with 100 nM AngII and 100 nM MCP-1 or vehicle and ligand-induced BRET was measured by measuring continuous light emission at 400-475 nm and 520-540 nm. BRET signal was calculated by subtracting the ratio of 520-540 nm emission to 400-475 nm emission from a second aliquot of cells treated with ligand (ligand-induced BRET) from the same ratio for the same cells treated with vehicle.

Ligand-induced BRET versus ARB concentration was then plotted using GraphPad Prism (v9.3.1), and potency ( _pIC50 value) was obtained by fitting the sigmoidal concentration-response curve using a nonlinear regression function in GraphPad Prism (v9.3.1).

To calculate the potency of sparsentan, HEK293FT cells were transiently transfected with plasmids encoding Gαi1-Rluc8, CCR2-YFP, and hemagglutinin (HA)-tagged AT1R. 48 h after transfection, cells were incubated with substrate for 2 h, then cells were pre-incubated with sparsentan (1 µM), and filtered emission was measured for 1 s in each of the "donor wavelength window" (410-490 nm) and the "receiver wavelength window" (520-620 nm) for 30 min. Cells were then stimulated with 10 nM MCP-1 (CCL2) and various concentrations of AngII, and filtered emission was measured again for 1 h.

Ligand-induced BRET versus AngII concentration was then plotted in the absence and presence of sparsentan (1 µM) using GraphPad Prism (v9.3.1), sigmoidal concentration response curves were fit to the data using a nonlinear regression function in GraphPad Prism (v9.3.1) and sparsentan affinity estimates (pKB) were calculated as described in Furchgott ((1972) The classification of adrenoceptors (adrenergic receptors) from the perspective of receptor theory in Catecholamines, Handbook of Experimental Pharmacology (Blaschko H and Muscholl E, eds.) Vol. 33, pp. 283-335).

The potency of ARBs on the co-stimulated CCR2- _AT1R complex was then compared to the potency of ARBs on the _AT1R using linear regression in GraphPad Prism (v9.3.1). The data in Figure 2 were then analyzed using robust linear regression with Q = 1 and the outlier removal (ROUT) coefficient to identify outliers with less potency than expected.

ARBs with ≥3-fold less potency than expected at the CCR2- _AT1R complex compared to potency at _AT1R alone were considered pharmacologically relevant and analyzed by one-sample t-test to assess statistical significance (Figure 3).

Figure 4 shows the effect of a combination of ARBs and CCR2 pathway inhibitors on the coupling of Gαi1 to the CCR2- _AT1R complex as measured by ligand-induced BRET. HEK293FT cells were transiently transfected with plasmids encoding Gαi1-Rluc8 and CCR2-YFP in the presence of _{hemagglutinin} (HA)-tagged AT1R. 48 h after transfection, cells were used to generate agonist-induced BRET signal data in live cells stimulated with 100 nM MCP-1 (CCL2) or 100 nM MCP-1 (CCL2) and 100 nM AngII in the presence of irbesartan, CCR2 pathway inhibitors, or irbesartan and CCR2 pathway inhibitors.

After addition of BRET substrate, BRET detection is performed in living cells by measuring continuous light emission at 400-475 nm and 520-540 nm before and after addition of agonist. The BRET signal is calculated by subtracting the ratio of 520-540 nm emission to 400-475 nm emission from a second aliquot of cells treated with ligand (ligand-induced BRET) from the same ratio of the same cells treated with vehicle. Activation of CCR2 results in a decrease in the BRET ratio, indicating that activation results in a conformational change in the Gαi1 protein preassembled with CCR2. Therefore , data have been presented as 'ligand-induced BRET decrease (% of control'), so the change in BRET signal observed with 100 nM MCP-1 was assigned 100% and no change in BRET signal was assigned 0%.

Potency values for AT1R only were obtained from: IUPHAR Pharmacology Guide Alexander et al., (2021) The Concise Guide to Pharmacology 2021/22 Br J Pharmacol. 2021. 178 Suppl 1:S1-S513 and/or Michel et al. (2013) A Systematic Comparison of the Properties of Clinically Used Angiotensin II Type 1 Receptor Antagonists Pharmacol Rev 65:809–848. These values represent the expected potency of the ARB against the co-stimulated CCR2-AT ₁ R complex. Experimental potency values are reported as pIC50 or pKB values. The pIC50 value is the negative logarithm of the molar concentration of the ARB required to inhibit the BRET change mediated by the co-stimulated CCR2-AT ₁ R complex by 50%. The experimental potency value reported as pKB is the negative logarithm of the molar concentration that would occupy 50% of the receptor at equilibrium in the absence of agonist.

Comparison of ARB potency against _AT1R alone and against the co-stimulated CCR2- _AT1R complex (Figure 2) and Table 4 revealed a good linear regression ^R2 = 0.97, indicating a good correlation between the potency of the ARB against the co-stimulated CCR2-AT1R complex and the potency against _AT1R alone.

This data suggests that most ARBs are within 3.5 times as potent when targeting the CCR2-AT ₁ R complex as ARBs that target only the AT ₁ R receptor. This supports the use of ARBs that are not reduced in potency when targeting the CCR2-AT ₁ R complex for the treatment of disease.

In contrast, outlier analysis revealed that EXP3174 was less potent than expected in inhibiting Gαi protein activation mediated by the co-stimulatory CCR2-AT ₁ R complex, compared to the potency of EXP3147 on AT ₁ R alone.

EX3147 was found to be 4.07-fold less potent against the CCR2-AT ₁ R complex than against AT ₁ R alone and statistically different ( FIG. 3 ; p<0.01, one-sample t test). These data suggest that the potency of ARBs against the CCR2-AT ₁ R complex is not significant and cannot be assumed to be the same as against AT ₁ R alone. ARBs with reduced potency against the CCR2-AT ₁ R complex, such as EXP3174, are not optimal for targeting the CCR2-AT ₁ R complex for treatment of disease and would be expected to have reduced efficacy.

Figure 4 shows the changes in ligand-induced BRET after stimulation of cells expressing CCR2- _AT1R complexes with vehicle, CCL2 (100 nM), or CCL2 (100 nM) and AngII (100 nM). Gαi1 protein activation in cells expressing CCR2- _AT1R co-stimulated with CCL2 (100 nM) and AngII (100 nM) was inhibited by the combination of ARB and CCR2 pathway inhibitor. It is expected that the loss of ARB potency against the CCR2- _AT1R complex will affect the inhibitory effectiveness compared to potency against AT1R alone.

Figure 5 and Table 4 show the expected potency of the combination of ARB and the CCR2 pathway inhibitor rapamycin to inhibit Gαi1 protein activation in cells expressing CCR2- _AT1R co-stimulated with CCL2 (100 nM) and AngII (100 nM). Table 4 : Inhibitory potency or affinity ( _pIC50 /pKb) of ARBs against _AT1R alone or against co-stimulated _AT1R +CCR2 complex ARB AT ₁ R only AT ₁ R-CCR2 AT ₁ R+CCR2 +Rippergermanium Potency reduction factor Potency ¹ (pIC ₅₀ /pKb) Potency reduction factor Potency ² (pIC ₅₀ /pKb) Potency reduction factor Potency ³ (pIC ₅₀ /pKb) Azisartan 1.00 9.9 0.85 9.97 0.56 10.15 Candesartan 1.00 10 1.41 9.85 1.95 9.71 Candesartan Celexiba - - - 8.72 - 8.63 Eprosartan 1.00 8.8 0.44 9.16 0.66 8.98 EXP3174 1.00 9.62 4.07** 9.01 5.62** 8.87 Irbesartan 1.00 8.52 1.00 8.52 1.55 8.33 Losartan 1.00 8.05 2.45 7.66 1.91 7.77 Olmesartan 1.00 9.9 2.34 9.53 2.57 9.49 Olmesartan M - - - 9.73 - 9.56 Spasentan 1.00 9.1 2.19 8.76# 1.51 8.92 Telmisartan 1.00 8.61 1.95 8.32 0.93 8.64 Valsartan 1.00 9.26 0.56 9.51 0.76 9.38 Fimasartan 1.00 8.82 ^$ 0.93 8.85 1.05 8.80 1. From IUPHAR Pharmacology Guidelines and/or Michel et al. (2013). 2. Potency of ARBs in inhibiting dissociation of Gα-protein from CCR2/Luc-AT1R complex co-stimulated with AngII and CCL2 (pIC ₅₀ ). 3. Potency of predictive ARBs in inhibiting dissociation of Gα-protein from CCR2/Luc-AT1R complex co-stimulated with AngII and CCL2 in the presence of rapamycin. ^; Fimasartan data are predictive. $, data from Choung et al., 2018 Discovery of the bifunctional modulator of angiotensin II type 1 receptor (AT1R) and PPARγ derived from the AT1R antagonist, Fimasartan. Bioorganic & Medicinal Chemistry Letters (28), 19:3155-3160 # Affinity of sparsentan for AT1R-CCR2 complex (pKb). **, p<0.01 single sample t test, significantly reduced compared to the potency against AT1R alone.

(without)

Other features of the invention will be more fully described in the following description of several non-limiting embodiments. This description is included for the purpose of illustrating the invention only. It should not be construed as limiting the broad generalization, disclosure or description of the invention set forth above. The description will be made with reference to the accompanying figures, in which: Figure 1 is a graph of the effect of different doses of rapamyr on therapeutic efficacy. The efficacy of the rapamyr doses exhibits a "bell-shaped" inhibition curve. Figure 2 is a graph comparing the potency of ARBs against _AT1R alone and the potency of inhibiting the dissociation of the G protein self-co-stimulated CCR2- _AT1R complex. Cand; Candesartan, CandC; Candesartan cilexetil, Epro; Eprosartan, EXP; EXP3174. Irb; irbesartan, Fim; fimasartan*, Los; losartan, Olm; olmesartan, OlmM; olmesartan methoate, Spar; sparsentan, Tel; telmisartan, Val; valsartan. Figure 3 is a graph showing the statistical analysis of the potency (pIC50) of EXP3174 on AT ₁ R alone and the potency of inhibiting the dissociation of G protein from the co-stimulated CCR2-AT ₁ R complex. **, p<0.01, determined by one-sample t test. Figure 4 is a graph showing the effects of ARB, CCR2 or ARB+CCR2 inhibition on the dissociation of G protein (Gαi1-luciferase) from the co-stimulated CCR2-AT ₁ R complex. Figure 5 is a graph comparing the potency of ARBs against _AT1R alone and the potency to inhibit G protein dissociation from the co-stimulated CCR2- _AT1R complex in the presence of rapamycin*. Cand; Candesartan, CandC; Candesartan cilexetil, Epro; Eprosartan, EXP; EXP3174, Fim; Fimasartan*, Irb; Irbesartan, Los; Losartan, Olm; Olmesartan, OlmM; Olmesartan methoate, Spar; Sparsentan, Tel; Telmisartan, Val; Valsartan.

Claims (29)

A therapeutic formulation adapted to deliver a daily dose of 200 mg to 280 mg of repagermanium or propagermanium. A therapeutic formulation suitable for delivering a daily dose of a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. A therapeutic formulation suitable for delivering a daily dose of: a) a therapeutically effective amount of rapamycin or propamycin; and b) a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. The formulation of claim 3, wherein the daily dose of rapamycin or propamycin is 200 mg to 280 mg. The formulation of claim 2 or 3, wherein the _AT1R blocker is selected from the list consisting of candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, fimasartan and sparsentan. The formulation of claim 2 or 3, wherein the daily dose of the AT ₁ R blocker is 2 mg to 800 mg. A dosing regimen comprising administering 200 mg to 280 mg daily of rapamyr or propamyr. A dosing regimen comprising administering a daily dose of a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone. A dosing regimen comprising administering a daily dose of: a) a therapeutically effective amount of rapagent or propagent; and b) a therapeutically effective amount of an AT ₁ R blocker having an affinity for the CCR2-AT ₁ R complex that is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone. A method for treating, ameliorating or preventing kidney disease, the method comprising the step of administering to an individual: a) A therapeutic formulation suitable for delivering 200 mg to 280 mg of rapamyr or propamyr daily. A method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: a) a therapeutically effective amount of an AT ₁ R blocker, wherein the affinity of the AT 1 R blocker to the CCR2-AT ₁ R complex is equal to or greater than the affinity of the AT ₁ R blocker to AT ₁ R alone. A method for treating, improving or preventing kidney disease, comprising the step of administering to an individual: a) a therapeutically effective amount of rapamyr or propamyr daily; and b) a therapeutically effective amount of an _AT1R blocker, wherein the affinity of the AT1R blocker for the CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. Use of 200 mg to 280 mg daily dose of rapamyr or propamyr for manufacturing a formulation for treating, ameliorating or preventing kidney disease in an individual. A use of 200 mg to 280 mg daily dose of rapamycin or propamycin for treating, ameliorating or preventing kidney disease in an individual. A use of a therapeutically effective amount of an AT ₁ R blocker for the manufacture of a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for the CCR2-AT ₁ R complex is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone. A use of a therapeutically effective amount of an AT ₁ R blocker for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for the CCR2-AT ₁ R complex is equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone. A use of (a) a therapeutically effective amount of rapamycin or propamycin and (b) a therapeutically effective amount of an _AT1R blocker for the manufacture of a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for AT1R alone. A use of (a) a therapeutically effective amount of rapamycin or propamycin and (b) a therapeutically effective amount of an _AT1R blocker for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. A therapeutically effective amount of rapamycin or propamycin for use in a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the rapamycin is administered to the individual simultaneously or sequentially with a therapeutically effective amount of an _AT1R blocker having an affinity for the CCR2- _AT1R complex that is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone. A therapeutically effective amount of an _AT1R blocker for use in a formulation for treating, ameliorating or preventing a kidney disease in an individual, wherein the affinity of the blocker for CCR2- _AT1R complex is equal to or greater than the affinity of the _AT1R blocker for _AT1R alone, wherein the at least one _AT1R blocker is administered to the individual simultaneously or sequentially with rapamycin or propamycin. A kit for treating or preventing kidney disease in an individual, the kit comprising: a) 200 mg to 280 mg daily of rapamyr or propamyr; and b) instructions for use. A kit for treating or preventing kidney disease in an individual, the kit comprising: a) a therapeutically effective amount of an AT ₁ R blocker, the affinity of the blocker for the CCR2-AT ₁ R complex being equal to or greater than the affinity of the AT ₁ R blocker for AT ₁ R alone; b) instructions for use. A kit for treating or preventing kidney disease in an individual, the kit comprising: a) a therapeutically effective amount of rapamycin or propamycin; b) a therapeutically effective amount of an _AT1R blocker, the affinity of the blocker for the CCR2- _AT1R complex being equal to or greater than the affinity of the _AT1R blocker for _AT1R alone; and c) instructions for use. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the rapamyr or propamyr is provided in a daily dose of 200 mg to 280 mg of rapamyr or propamyr. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the AT ₁ R blocker is selected from the list consisting of candesartan, eprosartan, fimasartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, fimasartan and sparsentan. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the subject to be treated is a human mammal. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the kidney disease is a kidney disease associated with proteinuria. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the renal disease is selected from the list comprising: focal segmental glomerulosclerosis (FSGS; including idiopathic (primary) FSGS, secondary FSGS, hereditary FSGS and any Other causes of FSGS), renal fibrosis, immunoglobulin A nephropathy (IgAN), Alport syndrome; chronic kidney disease, including chronic kidney disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic); and renal failure diseases, including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, primary nephrotic proteinuria and renovascular hypertension. The dosing regimen of any one of claims 7 to 9, the method of any one of claims 10 to 12, the use of any one of claims 13 to 20, or the kit of any one of claims 21 to 23, wherein the AT ₁ R blocker is not EXP3174.