CA2224451A1 - Method of treating renal disease using an ace inhibitor and an aii antagonist - Google Patents

Abstract

The present invention relates to a method of treating and/or preventing renal disease with the coadministration of an ACE inhibitor and an AII receptor antagonist. The present invention also relates to a method for protection of renal structure and/or renal function with the coadministration of an ACE
inhibitor and an AII receptor antagonist. The combination is also useful in preventing renal injury and protecting glomerular structure.

Description

CA 022244~1 1997-12-11 W O 97102032 PCTrUS96/10942 TITLE OF THE lNVENTION
METHOD OF TREATING RENAL DISEASE USING AN ACE
INHIBITOR AND AN A II ANTAGONIST

5 BAC~KGROUND OF THE INVENTION
Angiotensin II (AII) is a potent vasoconstrictor. Its generation in the renin-angiotensin cascade results from the enzymatic action of renin on a blood plasma, 2-globulin, angiotensinogen, to produce angiotensin I (AI). AI is then converted by angiotensin I () converting enzyme (A~E) to the octapeptide ho~none, AII. AII ha.s been implicated as a causitive agent in hypertension. Therefore, ACE
inhibitiors, which inhibit the production of AII, and and AII receptor antagonists, which inhibit the function of AII, are useful in the treatment of hypertension. The efficacy of these compounds in the 1 5 treatment of heart failure is also being studied.
Pals, et al., Circulation Research~ 29, 673 (1971 ) describe the introduction of a sarcosine residue in position 1 and alanine in position 8 of the endogenous vasoconstrictor hormone AII to yield an (octa)peptide that block.s the effects of AII on the blood pressure of 2 () pithed rats. This analog, [Sarl, Ala~] AII, initially called "P-l 13" and subsequently "Saralasin?" was found to be one of the most potent competitive antagonists of the actions of AII, although, like most of the so-called peptide-AII-antagonists, it also possesses agonistic actions of its own. Saralasin has been demonstrated to lower arterial pressure in 2 5 m~mm~ls and man when the (elevated) pressure is dependent on circulating AII (Pals et ah, Circulation Research, 29, 673 (1971);
Streeten and Anderson, Handbook of Hypertension, Vol. 5, Clinical Phalmacology of Antihypertensive Drugs, A. E. Doyle (Editor), Elsevier Science Publishers B. V., p. 246 (19~s4)). However, due to its 3 () agonistic character, .saralasin generally elicits pressor effects when the pressure is not sustained by AII. Being a peptide, the pharrnacological effects to saralasin are relatively short-lasting and are only manifest after parenteral ~r~ministration, oral doses being ineffective. Although the therapeutic uses of peptide AII-blockers, like saralasin, are severely CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 limited due to their oral ineffectiveness and short duration of action, their major utility is as a pharmaceutical standard.
Sorne known non-peptide antihypertensive agents act by inhibiting an enzyme, called angiotensin converting enzyme (ACE), which is responsible for conversion of angiotensin I to AII. Captopril and enalapril are comrnercially available A~E inhibitors (ACEI's).
Based on experimental and clinical evidence, about 40% of hypertensive patients are non-responsive to treatment with ACEI's. But when a diuretic such as furosemide or hydrochlorothiazide is given together I () with a CEI, the blood pressure of the majority of hypertensive patients is effectively normalized. Diuretic treatment converts the non-renin dependent state in regulating blood pressure to a renin-dependent state.
Although AII antagonist compounds act by a different mech~ni~m, i.e., by blocking the AII receptor rather than by inhibiting the angiotensin converting enzyme, both mech~ni~m~ involve interference with the renin-angiotensin cascade. A combination of the ACEI enalapril maleate and the diruetic hydrochlorothiazide is commercially available under the trademark Vaseretic(~) from Merck & ~o. Publications which relate to the use of diuretics with ACEI's to treat hypertension, in either 2 () a diuretic-first, stepwise approach or in physical combination, include Keeton, T. K. and Campbell, W. B., Pharmacol. Rev., 31:gl (19gl) and Weinberger, M. H., Medical Clinics N. America, 71:979 (19g7).
Diuretic,s have also been :~lmini~tered in combination with saralasin to enhance the antihypertensive effect.
2 5 Losartan potassium (losartan) represents the first antihypertensive in the class of AII receptor antagonists which is disclosed in a U.S. Patent 5,13~s,069 issued on August 11, 1992, and which is assigned to E. I. du Pont de Nemours. Losartan has been demonstrated to be a potent orally active A II antagonist, selective for 3 () the ATl receptor .subtype useful in the treatment of hypertension.
Inhibition of the renin-angiotensin-aldosterone system (~AAS) with angiotensin converting enzyme (ACE) inhibitor and angiotensin II (AII) receptor antagonist therapy has also been sho~vn to prevent and/or ameliorate renal disease of varying etiologies in ~nim~l CA 022244~l l997-l2-ll W O 97/02032 PCTrUS96/10942 models. Considering the differing pharmacodynamic effects of ACE
inhibitors and AII receptor antagonists, i.e. ACE inhibitors (e.g.
captopril, enalapril or lisinopril) inhibit the conversion of angiotensin I
to angiotensin II and potentiate the effects of the kallikrein-kinin system 5 whereas AT1 selective AII receptor antagonists (e.g. losartan) selectively inhibit the function of AII at the receptor site, it is reasonable to suggest that an enhanced beneficial effect might be achieved through the coadministration of compound,s from these therapeutic classes.
1 () The coadmini,stration of an ACE inihibitor with and AII
antagonist has been disclosed in patent applications filed by SmithKline Beecham (WO 92/10097) and Pfizer (WO 91/17771) and have shown the combination to be useful in the treatment of hypertension and cogestive heart failure. Additionally, a patent application filed by l S Merck and INSERM (EP0 62940~) claims enhanced renal blood flow when treating with the combination.

~UMMARY OF THE INVENTION
A method of treating and/or preventing renal disease of a 2 () warm-blooded ~nim~l with a therapeutically effective dose amount of a pharmaceutical composition of an ACE inhibitor and an Angiotensin II
receptor antagonist is disclosed. Included within the scope of the term renal disease are diabetic (insulin- and noninsulin-dependent) and non-diabetic nephropathy, including immllnologically- and 2 5 nonimmunologically-based nephropathies and/or glomerulopathies.
Also included within the scope of the invention is a method of protecting renal structure and/or renal function of a m~mm~l with a therapeutically effective amount of a pharmaceutical composition of an ACE inhibitor and an Angiotensin II antagonist. Included within the 3 () scope of the term pharmaceutical composition are a fixed combination and a concomitant therapy of an ACE inhibitor and an AII antagonist.
The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 ~lministrable medicament for the treatment and/or prevention of renal disease.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of treating and/or preventing renal disease with the coadministration, either concomitant therapy or a fixed combination, of an ACE inhibitor and an AII
receptor antagonist. The use of a pharmaceutical composition of an ACE inhibitor and an AII receptor antagonist in the manufacture of an I n orally administrable medicament for the treatment and/or prevention of renal disease. Concomitant therapy would include the sequential ~lministration of members from the two classes of compounds. The term renal disease includes diabetic nephropathy and non-diabetic nephropathy, including immunologically- and nonimmunologically-1 5 based nephropathies and/or glomerulopathies. The term non-diabetic nephropathy includes the condition referred to as human membranou~
glomerular nephritis.
The present invention further relates to a method for protection of renal structure and/or renal function with the 2 () coadministration, either concomitant therapy or a fixed combination therapy, of an ACE inhibitor and an AII receptor antagonist. The combination is also useful in preventing renal injury and protecting glomerular structure.
The angiotensin coverting enzyme inhibitors useful in this 2 5 method of treatment include, but are not limited to: AB-47, alacepril, benazepril, BIBR-277, BIBS39, BMS- 186716, BP 1.137, captopril, ceranopril, cilazapril, delapril, DU-1777, enalapril, fosinopril, FPL-66564, idrapril, imidapril, libenzapril, lisinopril, MDL-100240, moexipril, moveltopril, perindopril, Prentyl, 4uinapril, ramapril, 3 () spirapril, Synecor, S-5590, temocapril, trandolapril, utibapril, zabicipril, and zofenopril. An embodiment of the ACE inhibitor~;
useful in thi,s method of treatment are: captopril, cilazapril, enalapril.
fosinopril, lisinopril, quinapril, ramapril, and zofenopril.

CA 022244~1 1997-12-11 W O 97/0203~ PCT~US96/10942 The angiotensin II antagonists useful in this method of treatment include, AT-1 selective angiotensin II receptor antagonists, as well as non-selective angiotensin II receptor antagonist.s. The specific angiotensin II antagonists within the scope of the invention include, but 5 are not limited to: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS-lg4698, 3-(2'-(tetrazol-5-yl)-1, I '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, BAY106734, BIBR363, CL329167, E4177, EMD73495, HN65021, HR720, HOE720, LRB081, SC5245g, SL910102, UP2696, I () YM[358, EMD66397, ME3221, TAK536, BMS1~469~, CGP42112A, CGP49~70, CP 14~ 130, E41 g~S, EMD666~4, EXP9954, FR 1153332, GA0050, KT3579, LF70156, LRB057, LY266099, LY301~75, PD123177, PD126055, SC51757, SC54629, U96849, UK7777~, WAY 126227, WK 1260, WK 1492, YH 1498, andYM31472. An 1 5 embodiment of the angiotensin II antagonists useful in this method of treatrnent are: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS-184698 and 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine.
2 0 A study was conducted ex~mining the co~lministration of lisinopril (ACE inhibitor) and losartan (ATl-selective AII receptor antagonist) to streptozotocin-induced diabetic rat~. The study results noted a decrea.se in urinary protein excreted by the rats. Further assessmènt of urinary protein data and morphometric assessment of 2 5 renal structure has shown a statistically significant decrease in glomerular area, a further decrease in glomerular basement membrane width and a corresponding decrease in total and high molecular weight urinary protein with losartan-lisinopril coadministration when compared to losartan monotherapy.
3 () Additionally, a twelve-month study was conducted ex~mining the coa~lmini~tration of lisinopril and 3-(2'-(tetrazol-5-yl)-- 1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazoL4,5-b]pyridine to rats with passive Heymann nephritis. This ~nim~l model which manifests in the rat with long lasting proteinuria followed by CA 022244~1 1997-12-11 W O 97/02032 PCT~US96/10942 renal injury (~ JASN 3:624, 1992) is representative of human immunologically-mediated glomerulonepropathy.of renal disease, which also noted enhanced renal protection with the co~lministration of lisinopril and 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-5 dimethyl-2-ethyl-3H-irnidazo[4,5-b]pyridine. Passive Heymann Nephritis manifests in the rat with long lasting proteinuria followed by renal inju~ ee JASN 3:624, 1992. The study demonstrated that coadministration reduced proteinuria and the degree of renal iniury better than either the monotherapy of lisinopril, a.s well as the I () monotherapy of 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-bJpyridine.
The use of the combination of an ACE inhibitor and an Angiotensin II (AII) receptor antagonist has been demonstrated in rat~s to provide a method of treatment for the renally impaired. The 1 5 administration of compounds from these two classes can also be effect in treating renal disease, including diabetic nephropathy insulin- and noninsulin-dependent) and non-diabetic nephropathy including immunologically- and nonimmunologically-mediated nephropathies and/or glomerulopathathies. Within the scope of the term diabetic 2 (1 nephropathy it is understood that the disease state is the result of either non-insulin dependent diabetes mellitus or insulin dependent diabetes mellitus.
Ph~ eutically suitable salts include both the metallic (inorganic) salts and organic salts; a list of which is given in 2 5 Remin~ton's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985). It i.s well known to one skilled in the art that an appropriate salt form is chosen based on physical and chemical stability, flowability, hydro-scopicity and solubility. The preferred salts of this invention include, but are not limited to: potassium, sodium, calcium and ammonium salts 3 () of the ACE inhibitor and/or AII receptor antagonist.
Included within the scope of this invention rs a method of treatment of renal diease using pharrnaceutical compositions comprising an ACE inhibitor, an AII antagonist and a suitable pharmaceutical carrler.

CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 DOSAGE FORMS
The pharmaceutical compositions of this invention can be administered for the treatInent and or prevention of renal disease according to the invention by any means that effects contact of the active 5 ingredient compound with the site of action in the body of a warm-blooded ~nim~l. For example, administration, can be parenteral, i.e., ~subcutaneous, intravenous, intramuscular or intra peritoneal.
Alternatively, or concurrently in some cases ~lministration can be by the oral route.
I () The pharmaceutical composition.s of this invention can be administered by any conventional means available for use in conjunction with pharmaceuticals. The pharmaceutical compositions can be aclministered alone, but are generally ~lmini~tered with a pharnnaceutical carrier selected on the basis of the chosen route of 1 5 administration and standard pharrnaceutical practice.
For the purpose of this disclosure, a warm-blooded ;~nim~l is a member of the ~nim~l kingdom which includes but is not limited to m~mm~ls and birds. The preferred m~mm~l of this invention is human.
The dosage administered will be dependent on the age, 2 () health and weight of the recipient, the extent of disease, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. Usually, a daily dosage of active ingredient compound will be from about 1-500 milligrams per day. Ordinarily, from 10 to 100 milligrams per day in one or more applications is 2 5 effective to obtain desired results.
The active ingredients can be administered orally in solid dosage form,s, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs ~syrups, and suspensions. It can also be ~llministered parenterally, in sterile licluid dosage forms.
3 () Gelatin capsules contain the active ingredient and powdered carrier,s, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured a.s sustained release products to provide for continuous release of CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene gyclols are suitable carriers for parenteral I () .solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such a,s sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In additiion, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propylparaben, and chlorobutanol.
Suitable pharmaceutical carriers are described in Remin~ton',s Pharmaceutical Science.s, A. Osol, a ~standard reference text 2 () in this field.
Useful pharmaceutical dosage-forms for administration of the fixed combination.s of this invention can be illustrated as follow,s:
CAPSULES
2 5 A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsule.s each with a pharmacologically appropriate amount in milligrams of the powdered active ingredients, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligram~
magnesium .stearate .
3 () SOFT GELAT~ CAPSULES
A mixture of active ingredient in a dige.stible oil such soybean oil, cottonseed oil or olive olil is prepared and injected by means of a positive displacement pump into gelatin to forrn ,soft gelatin :
CA 022244~1 1997-12-11 W O 97/02032 PCT~US96/10942 capsules containing a pharmacologically appropriate amount in milligrams of the active ingredient. The capsules are washed and dried.
TABLETS
A large number of tablets are prepared by conventional procedures so that the dosage unit is a pharmacologically appropriate amount in milligrams of the active ingredients, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and I () 9~.~¢ milligrams of lactose. Appropriate coating~ may be applied to increase palatability or delay absorption.

INJECTABLE
A parenteral composition .suitable for administration ~y injection is prepared by stirring a pharmacologically appropriate amount by weight of the active ingredients in 10% by volume propylene gly~ol. The solution is made to volume with water for injection and sterilized.

An aqueous suspension is prepared for oral admini.stration so that each 5 milliliters contain a pharmacologically appropriate amount in milligrams of finely divided active ingredient, 100 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium 2 5 benzoate, 1.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.

The same dosage forms can generally be used when the ACE inhibitor compounds and AII antagonist compounds of this 3 () invention are administered in a concomitant fashion. The above dosage forms and route of ~lministration for a fixed combination ACE
inhibitor and AII antagonist should be selected depending on the compatibility of the combined drugs. Suitable dosages, dosage forms and administration routes are illustrated in Tables A and B.

CA 022244F~l 1997-12-ll Table A: Examples of ACE inhibitors that can be combined with the below A II receptor antagonist is useful for the treatment and/or prevention of renal disease Dru~ Dose (mg/day) Formulation Route of Admin.
li.sinopril 5, 10, 20, 40 Tablet Oral enalapril 10-40 Tablet Oral s Table B: Examples of AII receptor antagonists that can be combined with the above ACE inhibitors for the treatment and/or prevention of renal di.sease ~g Dose (mg/day) Formulation Route of Admin.
losartan potassium 25, 50, 100 Tablet Oral l O
The following examples further illustrate the method of tretaing and/or preventinf renal disease using a pharmaceutical composition including the active ingredients of an ACE inhibitor and an AII receptor antagonist and as such, are not to be considered or 1 5 construed as limiting the invention recited in the appended claims.

Study conducted in a Streptozotocin-Induced Diabetic Rat Model using the AII receptor anta~onist, Losartan and the ACE inhibitor. Li.sinopril.
2n Diabetes was induced with intravenous streptozotocin (60 mg/kg) in male Sprague-Dawley rats on study day 1. A daily dose of subcutaneously administered insulin was adjusted on a weekly basis to maintain serum glucose levels between 200 and 400 mg/dl. Losartan 2 5 was administered alone and in combination with lisinopril in the drinking water from study day 5; final dosage levels were 30 and 30/3.5 mg/kg/day, respectively. The effects on renal function and structure were evaluated after one year of treatment. Various parameters were assessed. Those which suggest a potential additive beneficial effect of CA 0222445l l997-l2-ll W O 97/02032 PCTrUS96/10942 losaltan/lisinopril treatment include: sixteen-hour urinary protein excretion [total protein (TUP), high molecular weight protein (HMW)3, histomorphological quantitative assessment of glomerular area (GA) and glomerular basement membrane thickness (GBMT).

Parameter (Units) [n] Control STZ STZ/LOS STZ/LOS/LIS
TUP (mg) [15] 21.8 46.7 * 9.4 6.1 t HMW (mg) ~15] 15.3 31.9 * 2.0 *T 0.5 *t LMVV (mg) rl5] 6.4 14.8 * 7.4 5.6 GA (~Lm2) ~10] 20.7 22.0 21.3 19.5 ~*
GBLT (nm) [5] 332 441 * 359 t 316 t::
[n] = number evaluated per group * = St:~ti~c~lly significantly different from nondiabehc conlrol group t = Stahstically significantly different from STZ diabehc con~ol groups :t = Stahshcally signi~lcantly different from losa~tan-treated STZ diabehc group I O STZ-induced diabetic nephropathy was characterized by statistically significant (p < 0.05) increases in TUP, HMW, low molecular weight protein (LMW), and GBMT with a slight, but non-statistically significant, increase in glomerular area. The latter has been demonstrated to be a precursor to glomerular sclerosis. Losartan treatment, alone and in combination with lisinopril, was clearly protective against diabetic nephropathy. In addition, combination therapy appeared to offer a greater degree of protection. Notably, there was a 5-fold decrease in TUP with losartan monotherapy that wa,s further decreased (p < 0.05) in the losartan/lisinopril treatment group.
2 () Similarly, when compared to the STZ diabetic control group, there wa~
a 16-fold (p < 0.05) decrease in high molecular weight urinary protein in the losartan treatment and a 64-fold decrease (p < 0.05) with lisinopril coadministration. These effects on urinary protein excretion are consistent with the observed decreases in GA and GBMT with 2 5 losartan monotherapy (p < 0.05) and the further decrease (p < 0.05) in these parameters noted with lisinopril coadministration.

U8STITU~E SHE~T ~RULIE ~;~

CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 EX~MPLE 2 Study conducted in a Passive Heymann Nephritic (PHN) Rat Model using the AII receptor antagonist, 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine and the ACE
5 inhibitor. Lisinopril.

Male Sprague-Dawley, CD-COBS rats (Charles River Italia s.p.a., Calco, Italy) with initial body weight of 240-260 g were used.
PHN was induced in non-anesthetized rats by a single i.v. injection of I () 0.5 ml/100 g body wt of rabbit anti-FxlA antibody prepared according to Edgington et al., (1967).

Group 1 PHN rats given daily the AII antagonist, 3-(2'-(tetrazol-5-yl)-(n=8) 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, continuously in the drinking water for 12 months starting at day 7 after PHN induction when ~nim,ql~ have already developed proteinuria. 3-(2'-(Tetrazol-5-yl)- 1, l '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine was ~lmini~tered at the dose of 100mg/1, in the first 6 months of study. Then, due to the low SBP values recorded in some rats the dose was decreased to a dose of 50mg/1.
Group 2 PHN rats were given daily the ACEI, lisinopril (40mg/1) (n=8) continuously in the drinking water for 12 months starting at day 7 after PHN induction.
Group 3 PHN rats were given daily 3-(2'-(tetrazol-5-yl)-1,1'-biphen-(n=8) 4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine and lisinopril in the drinking water for 12 months starting at day 7 after PHN induction.
Group 4 PHN rats were followed for 12 months without any (n=8) treatment.
Group ~ Normal rats with no treatment were followed for 12 months (n=6) and used as control.

~ I~TITUTE SHEEr (RULE 26 CA 022244~1 1997-12-11 W O 97/02032 PCTrUS96/10942 All ~nim~ls were housed in a constant temperature room with a 12-hour dark 12-hour light cycle and fed a standard diet.
Systolic blood pressure (SBP) was measured before the induction of the disease (basal) and every 2 month~ for 12 months, by the tail cuff 5 method (Pfeffer et al., 1971). At day O (basal) and every two month.
blood sample,s were collected for measurement of plasma creatinine corlcentration. Twenty-four hour urine samples were collected in metabolic cages before PHN induction (basal), at day 7 and every two months for 12 months to measure urinary protein excretion. At the end I () of the study period, all ~nim~ls underwent determination of whole-kidney function (GFAR, as clearance of inulin; RPF, as clearance of p-aminohippuric acid). At sacrifice, blood samples were collected for measurement of plasma AII concentration and the kidneys were removed and processed for histological analysi,s by light microscopy.
I 5 During the study the following mortality was observed: one normal rat died at month 9, two PHN rats treated with 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5 ,7-dimethyl-2-ethyl-3H-imidazo [4,5 -b]pyridine died; one at month 5 and one during renal function studie,s due to anesthesia, one PHN rat treated with lisinopril died at month 9 2 0 and two PHN rats treated with the combined therapy died at month,s 5 and 11, respectively. At autopsy no relevant lesions in kidney and in other organs were detected.
Total food intake was comparable in all PHN and control rats for the entire study period (Table 1). As shown in Table 2, during 2 5 the 12 month study rats with PHN gained weight in a similar manner to normal control rats. Treatment of PHN rat.s with 3-(2'-(tetrazol-5-yl)-1, 1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo~4,5-b]pyridine, lisinopril or the combination of AII receptor antagonist and ACE inhibitor affected ~nim~ls' weight gain; the actual body weights of 3 () the,se ~nim~ls were significantly lower than tho~e of untreated PHN. In the remainder of the study period the differences in weight gain among the rat groups became less evident except than for PHN rats treated with the combined therapy; body weight values for this group remained decreased that still had lower body weight values either at 10 and or 1 CA 022244~l l997-l2-ll W O 97/02032 PCTrUS96/10942 months. At 12 months also body weight.s of PHN rats treated with 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine were lower than those of the untreated control group.
As shown in Table 3, after 4 months of observation untreated PHN rats were normotensive. Six months after disease induction PHN rats exhibited a significant (p<0.05) increase in SBP
compared to normal rats, which persisted over the remainder of the study period. The three groups of PHN given 3-(2'-(tetrazol-5-yl)-1,1'-I ~) biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, lisinopril or the combination, all had SBP values sig~ificantly (p<0.01) lower than those of untreated PHN rats starting from month 2. In addition, these three treatment groups maintained SBP at levels that were even significantly lower than those of normal rats.
1 5 Time course of urinary protein excretion is given in Table 4. PHN rats developed significant (p<0.01) proteinuria as early as 7 days after induction of the disease. Proteinuria progressively increased with time, averaging 702.06+77.02 mg/day at the end of the study. In normal control rats protein excretion rose only to 79.46_15.43 mg/day 2 (1 after 12 months. 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine and lisinopril were both effective in limiting the development of proteinuria of PHN rats. In these treatment groups protein excretion values were significantly (p<0.01) lower than in untreated PHN rats, averaging 51.~4_14.55 and 2 5 148.9~ 61.62 mg/day, respectively at 12 months . More importantly, combined administration of AII receptor antagonist and ACE inhibitor completely blocked the development of proteinuria, which averaged 15.87+1.94 mg/day at the end of the study. Proteinuria values of rats treated with 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-3 () 2-ethyl-3H-imidazo[4,5-b]pyridine + lisinopril were even significantly lower than those of PHN + 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine or PHN +
lisinopriltreated groups during the entire study period and significantly lower than those of control rats starting from month 6.

W O 97/02032 PCTrUS96/10942 *o ~1 ~ontrol ~* E;~ PHN+Vehicle ~j l* - B3 PHN+Lisinopril PHN+AII compd.
~ PHN+AII compd. +
f~ sinopril o ~ ~ 7DO BVO
proteinuria (mg/day) *p<0-01 vs PHN+vehicle; ~p<0.01 vs control and all PHN groups As shown in Table 5, serum creatinine values of untreated l O PHN rats slightly, although significantly, increased during time as compared to control rats, averaging 0.89+0.04 vs. 0.69+0.02 mg/dl at 12 months. In PHN rats treated with AII receptor antagonist, ACE
inhilbitor or the combination, serum creatinine values were comparable to those of untreated PHN rats up to 8 months; during the last months of 1 5 the study values of treated PHN were numerically lower than those of untreated rats. Because serum creatinine may not be an absolute indicator for CFR, we also measured GFR using inulin clearance at the end of the experimental period. As shown in Table 6, in untreated PHN
rats, GFR decreased significantly (p<0.01) with respect to values 2 0 obtained in normal control rats. Treatment with 3-(2'-(tetrazol-5-yl)-1, 1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, lisinopril or the combination, partially but significantly (p<0.01) prevented the decrease in GFR. RPF as estimated by PAH
clearance was significantly (p<0.01) lower in PHN rats than in controls 2 5 (Table 6). A similar decrease in RPF was observed in PHN rats given the AII receptor antagonist. Administration of lisinopril alone or in combination with 3-(2'-(tetrazol-5-yl)-l,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine resulted in a less decrease of RPF, with values being significantly (p<0.01) higher than those of 3 () PHN untreated rat~s.
The results of morphological analysis by light microscopy on renal biopsies taken at the end of the study are reported in Table 7.
PHN rats showed focal and segmental glomerulosclerosis affecting on average 60.25% of glomeruli. Tubulo-interstitial changes consisted of SiUBSTlTUTE 8H~ET ~P~ULE 26) W O 97/02032 . PCTrUS96/10942 inter.stitial fibrosis and inflammation associated with tubular atrophy and large eosinophilic casts in the tubular lumens.
Limitation of proteinuria in PHN treated rat.s reflected a better preservation of glomerul~ar s~luctural integrity when comp~re(l Lo 5 untreated PHN rats. Thu.s, there were very few segmental sclerotic changes which affected on average 3% of glomeruli in rats given 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, 3.71% in rats given lisinopril and 1.06% in rats receiving the combined therapy. The latter value was comparable with I 0 that of control rats that, with aging, exhibited 1.71% of glomeruli with sclerotic changes. Tubulo-interstitial changes were also significantly limited by the three therapies. Of note, mean scores of tubulo-interstitial damage in rats given AII receptor antagonist + ACE
inhibitor combination were even lower than those of norrnal rats.

Cl Control 70 ~r E;~ PHN+Vehicle 2 0 ~3 PHN+Lisinopril E~ PHN+AII compd.
~I PHN+AII compd. + ~ ~'~~ ~,~
lisinopril O ;,~ ~

o 5_ ~ * *
V ;~ ,~*
*p<0.01 vs PHN+vehicle ~iUBS~T~E SHEE~ ~FLULF 26 W 0 97/02032 PCTtUS96tlO942 ~
, _ .
C c~
V, ~ ,_ ~ ~ C _ ~, _-- _+111 +1 11 -- Cc _ o _ ~ _ o~
00 ~ OG

1-- ~-V, C~, V'l _ ~1 1-- V
' '--' oo +1 11 +1 11+1 11 +1 11 +1 11 -- . O = Vl C _C o C~'I C
_ O '~ --V -- ~--v ~I r~l ~ ~ ~
c _~ _ ~ oC _ ~C_ C~, O ~ G
t +1 11 +111+1 11+1 11 +1 11 o ~ 5 ~, 5 ~ 5 0 5 0 5 ~ V. -- ~''. ~
O ~'I ~ ~ ~'1 ~ ~ -s ~, 00 0 x _00~', Oo ~ x ~ 05 +1 o ~ o +I 11 +111 +

E ~ y ~ _ ~
, ~ G O

~ O 5 + + + _ ~d o _ ~
~ Vl ~ .
o .
o SUB~TUTF SHEE~ (RULE 263 W O 97/02032 PCTrUS96/10942 ~, ~
1 0 ~o ~ O G V'2 Cr~

O ~ O
* -- ~
, ~ . ' ,~_ C O' C ~ ~--l J ~' ~ ~ E ~ ~ ll ~ ~ ~ C g c _ X ~ o X ~

C~ C.~ V~ _ _ ~ _ _ - ~ ~ ll G I ~ C C C C C ~
~ E c ~ ~,c ~c ~ 1l ~r 1l c 11 +1 2 5 0 r~ ~7 _ c ~C

a: ~ q C :1: T C Z Z ~ ~ V V V
0 1~
~ ~ v~

S~Ç3 T~TE SHEET ~ LE 2 W O 97/02032 PCT~US96/10942 - 18,~1---_ V o - ' ~ ~ ~ ~ c ~ v c ~ ~ ~ c - O _ ~ X 0~
-- 5 _ ~ ~n v ~ V, ~l oo ~ ~o c OC c 8 c 2 0 ~ I ~~
~ vOC x 1~ 1-- ~~ E

+'~ 1~ g 1~ n c +1 - h Z

G ~ _ 5 Z Z E Z Z ~ . ~ v v G

::~ S
' 30 ~ E

SUBSTITUTE ~3HEET (PfULE ~6~

W O 97/02032 PCTrUS96/10942 - 19 _ * *
C

~ ~ _ ~. 5 ~ 5 ~ C ~ -r-- i ~ ~
v î C ~i ~o ~ *
~ O --- ~ ,~ +1 ~~+1~~ +~ ~~
I C _ 5 ~ _ ~ ._ ~t ' r~, ~. C O O' 1 0 ~ ~
oc r-- oc ~ _ X 11 X 11 C 11 X 11 +1 1~
~o 5 oo C ~ oo X C
r~ V, -- C ~) ~ ~ ~I
_ ~ ~ ~ ~ ~ ~ ~ _ _ ~ ~ o 1~
- o '~ ~, C ~ C ~G C ~~ C ~ C

' C ~, -- , X ~_ oo C
~ ~ ~ ~ _ c ~ c o * c ~ * c v~vJ _ _ ~
Z E O ~ ~ ~ ~

~ ~ ~ ~ ~ ~ ~ c ~ c ~ c ~ ~ E E
c . c -- . c u,l 1-- ~ c ~o ~ ~

~ . ~ ~ ~ v ~ ~ _ ~ + _ ~ V V ~c v ~ ~ a~ ~ :c ~e SU~TITUTE SHEEr ~RULE 26~

CA 0222445l l997-l2-ll W O 97/02032 PCTrUS96/10942 ,~ ~ E ~ ~ ~, C oc ~ oc ~' x . 0 , ~
-- D E~tY, ~ ~, _ ~
1 0 ;~ l ,"~ ~ ~ ~ , X

D ~ ~ ~~ ~~l ~~ cr~ X ~r) oc ,~~
v; (-; ~ I ~ _ C ~o ~ 5 ~ _ ~ o ~ ~ ~ * * ~
~ ~ ~ * r-- '~
o ~ G ,_ ~, X X ~

~ ~ c ~ _ c ~ ~ E

~ ~So 5 ~ .E z T ~ O ~ -- X ~ 1 ~1 11 ¦ 11 +¦ 11 C C ~ C

~ E E ~~ ~ ~ ~ ~ ~ ~ _ ~
2 5 ~ ~ T E~ - ~.' ~ O T ~ T

o ~ ~ C oo + + + _ C~ V ' V V V

o ,C .~C ~ ~ * ~ o oC
3 ~ ~ ~

SU~I~UTE SHE~ (RULE :26) W O 97/02032 PCT~US96/10942 ~ ~ ~ ~ * ~ G o ~ ' O ~

C ~ ~ ~ ~, X X X
~ ~ V~

10, ~ o ~I .~

' ~ o ,_ C o ~ ", C C~ C

_ ô o 15~ ~ ~ ~ *
~ 3 o ~ o ~' ~ -~ ~ a ~~ 'C G ~ C ~ o C

2 0~ C G o * 5 ~", I

T O ~ a +l 11 +1 11 ~Cl 11 _ 11 ~ 1-- C G G G
25c E E ~ ~ ~ ~ G G ~ ~ o C ~ C ~ ~ + + ~ C ~ V--30~ E s o c~ V ~ ~ a& Z Z & ~ & '~ ~& '~ov o ~ ~ oo ,. . ~

SU~;TI~UTE SHEi~ ~RULE 5~6~

W O 97/02032 PCTrUS96/10942 _ i21 -o ,_ ~ ~ô
~, o ~ C ~~ o ~~ C~~
~ ~ - ~ - ~ _ ~ _ ~ _ - C' C C C o ~ ~ *
--' ~C c +1 1' +1 1' +1 C +l ~ +I c -- C C O G
1 0~ ''I --* ~ *

_ r~ +I c +I c +I c +I c +I c ~, ~ O C' O O C

, o ~~ +1 1l +1 1l +I c +I c +; C
, O ~ O C O
._ h ~ +l C +l C +l C +l C +l C
- G C o o o ~o 00 0 X O X ~ 00 0 X
~ - '~ ''' +I 11 +I c +I c +I c +I c G 5 ~1; G G ~5 ~~ G J --'~

~ E ~ ~ +I 1l +I c +I c +I c +I c c o ~ .5 c c C o o E
~~ r ' ~~ ~ G C CL

~ 30 E - ~ G + + + - ~ V V V
~ ~, . U~ ~C Z~ Z Z Z ~ C~ . * ~I

SUBS i iTlJTE SttEET ~RULE ~6~

W O 97102032 PCTrUS96/10942 j j ~' .~ ~ ~ o ~ o ~ o G o ~ o E +l ~ +l ll +l il +l ll +
~ ~ o o, ~s5 ~o C~
~ ' E ~
~o . , ,_ m ~ _ ~ ~ ~ ~ O~' ~

. E G - ~ Z

O + + + . ~ V e ~ . _ Z ~
Q~ . -o ~
~!
o ~,; ..
~ , r ~
~ ' ~
~ ~

~ 5 ' SUBSTITU~E SHEET (PUI E 26) W O 97102032 PCT~US96/10942 ~ , ~ E ~ c O _ ~ _ o _ o O o - O g ~' ~- . ~ E
~ _ o ~ C
m 3 ' ~ ~ a~

v~ o ~ V~ ~ ~ ~ a o c -- ~ o o o 8 o '-- o o o c Z

~ ~- E E -o o ~ O + + +
3 ~ g ~ , C Cl~ ~ +

c .~, , .

' 30 SUBSTITUTE SHtET fflULE 26~1

Claims (28)

WHAT IS CLAIMED IS:

1. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for the treatment and/or prevention of renal disease.

2. The use as recited in Claim 1, wherein the renal disease is diabetic nephropathy or non-diabetic nephropathy.

3. The use as recited in Claim 1, wherein the pharmaceutical composition consists of a fixed combination of an ACE
inhibitor and an AII receptor antagonist and a pharmaceutically acceptable carrier.

4. The use as recited in Claim 3, wherein the renal disease is diabetic nephropathy.

5. The use as recited in Claim 1, wherein the composition consists of the concomitant administration of an ACE
inhibitor and an AII receptor antagonist.

6. The use as recited in Claim 5, wherein the renal disease is diabetic nephropathy.

7. The use as recited in claim 1, wherein the ACE
inhibitor is selected from the group consisting of: AB-47, alacepril, benazepril, BIBR-277, BIBS39, BMS-186716, BP1.137, captopril, ceranopril, cilazapril, delapril, DU-1777, enalapril, fosinopril, FPL-66564, idrapril, imidapril, libenzapril, lisinopril, MDL-100240, moexipril, moveltopril, perindopril, Prentyl, quinapril, ramapril, spirapril, Synecor, S-5590, temocapril, trandolapril, utibapril, zabicipril, and zofenopril.

8. The use as recited in claim 7, wherein the ACE
inhibitor is selected from the group consisting of: captopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril, ramapril, and zofenopril.

9. The use as recited in claim 1, wherein the AII
receptor antagonist is selected from the group consisting of: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS - 184698, 3 -(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl )methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, BAY 106734, BIBR363, CL329167, E4177, EMD73495, HN65021, HR720, HOE720, LRB081, SC52458, SL910102, UP2696, YM358, EMD66397, ME3221, TAK536, BMS 184698, CGP42112A, CGP49870, CP148130, E4188, EMD66684, EXP9954, FR1153332, GA0050, KT3579, LF70156, LRB057, LY266099, LY301875, PD123177, PD126055, SC51757, SC54629, U96849, UK77778, WAY126227, WK1260, WK1492, YH 1498, and Y M31472.

10. The use as recited in claim 9, wherein the AII
receptor antagonist is selected from the group consisting of: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS- 184698 and 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5 -b]pyridine.

11. The use as recited in claim 10, wherein the ACE
inhibitor is captopril, enalapril or lisinopril and the AII receptor antagonist is losartan or 3 -(2'-(tetrazol-5 -yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo [4,5-b]pyridine.

12. The use as recited in claim 11, wherein the ACE
inhibitor is enalapril and the AII receptor antagonist is losartan.

13. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for the protection renal structure.

14. The use as recited in Claim 13, wherein the the renal structure is the glomerular structure.

15. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for the prevention of renal injury.

16. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for the protection of renal function.

17. The use as recited in Claim 16, wherein the pharmaceutical composition consists of a fixed combination of an ACE
inhibitor and an AII receptor antagonist and a pharmaceutically acceptable carrier.

18. The use as recited in Claim 16, wherein the pharmaceutical composition consists of a concomitant administration of an ACE inhibitor and an AII receptor antagonist.

19. The method as recited in claim 16, wherein the ACE
inhibitor is selected from the group consisting of: AB-47, alacepril, benazepril, BIBR-277, BIBS39, BMS- 186716, BP 1.137, captopril, ceranopril, cilazapril, delapril, DU-1777, enalapril, fosinopril, FPL-66564, idrapril, imidapril, libenzapril, lisinopril, MDL-100240, moexipril, moveltopril, perindopril, Prentyl, quinapril, ramapril, spirapril, Synecor, S-5590, temocapril, trandolapril, utibapril, zabicipril, and zofenopril.

20. The use as recited in claim 19, wherein the ACE
inhibitor is selected from the group consisting of: captopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril, ramapril, and zofenopril.

21. The use as recited in claim 16, wherein the AII
receptor antagonist is selected from the group consisting of:
candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS- 184698, 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, BAY106734, BIBR363, CL329167, E4177, EMD73495, HN65021, HR720, HOE720, LRB081, SC52458, SL910102, UP2696, YM358, EMD66397, ME3221, TAK536, BMS184698, CGP42112A, CGP49870, CP148130, E4188, EMD66684, EXP9954, FR1153332, GA0050, KT3579, LF70156, LRB057, LY266099, LY301875, PD 123177, PD 126055, SC51757, SC54629, U96849, UK77778, WAY 126227, WK 1260, WK; 1492, YH 1498, andYM31472.

22. The use as recited in claim 20, wherein the AII
receptor antagonist is selected from the group consisting of:candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS- 184698 and 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine.

23. The use as recited in claim 22, wherein the ACE
inhibitor is captopril, enalapril or lisinopril and the AII receptor antagonist is losartan or 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo [4,5-b]pyridine.

24. The use as recited in claim 23, wherein the ACE
inhibitor is enalapril and the AII receptor antagonist is losartan.

25. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for reducing proteinuria.

26. The use as recited in claim 25, wherein the ACE
inhibitor is enalapril or lisinopril and the AII receptor antagonist is losartan.

27. The use of a pharmaceutical composition of an ACE
inhibitor and an AII receptor antagonist in the manufacture of an orally administrable medicament for the treatment of membranous glomerular nephritis.

28. The use as recited in claim 27, wherein the ACE
inhibitor is enalapril or lisinopril and the AII receptor antagonist is losartan.