AU716519B2 - Method of treating renal disease using an ace inhibitor and an A II antagonist - Google Patents

Description

WO 97/02032 PCT/US96/10942 -1- TITLE OF THE INVENTION METHOD OF TREATING RENAL DISEASE USING AN ACE INHIBITOR AND AN A II ANTAGONIST BACKGROUND 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 is then converted by angiotensin 1 0 converting enzyme (ACE) to the octapeptide hormone, AII. AII has been implicated as a causitive agent in hypertension. Therefore, ACE inhibitiors, which inhibit the production of All, and and All 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 blocks the effects of AII on the blood pressure of 2 0 pithed rats. This analog, [Sarl, Ala 8 All, initially called "P-113" 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 mammals and man when the (elevated) pressure is dependent on circulating AII (Pals et al., Circulation Research, 29, 673 (1971); Streeten and Anderson, Handbook of Hypertension, Vol. 5, Clinical Pharmacology of Antihypertensive Drugs, A. E. Doyle (Editor), Elsevier Science Publishers B. p. 246 (1984)). However, due to its 3 0 agonistic character, saralasin generally elicits pressor effects when the pressure is not sustained by AII. Being a peptide, the pharmacological effects to saralasin are relatively short-lasting and are only manifest after parenteral administration, oral doses being ineffective. Although the therapeutic uses of peptide AII-blockers, like saralasin, are severely WO 97/02032 PCT/US96/10942 -2limited due to their oral ineffectiveness and short duration of action, their major utility is as a pharmaceutical standard.

Some known non-peptide antihypertensive agents act by inhibiting an enzyme, called angiotensin converting enzyme (ACE), which is responsible for conversion of angiotensin I to All. Captopril and enalapril are commercially available ACE 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 1 0 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 All antagonist compounds act by a different mechanism, i.e., by blocking the AII receptor rather than by inhibiting the angiotensin 1 5 converting enzyme, both mechanisms 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 Co. Publications which relate to the use of diuretics with ACEI's to treat hypertension, in either 2 0 a diuretic-first, stepwise approach or in physical combination, include Keeton, T. K. and Campbell, W. Pharmacol. Rev., 31:81 (1981) and Weinberger, M. Medical Clinics N. America, 71:979 (1987).

Diuretics have also been administered in combination with saralasin to enhance the antihypertensive effect.

2 5 Losartan potassium (losartan) represents the first antihypertensive in the class of All receptor antagonists which is disclosed in a U.S. Patent 5,138,069 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 0 the ATi receptor subtype useful in the treatment of hypertension.

Inhibition of the renin-angiotensin-aldosterone system (RAAS) with angiotensin converting enzyme (ACE) inhibitor and angiotensin II (All) receptor antagonist therapy has also been shown to prevent and/or ameliorate renal disease of varying etiologies in animal models. Considering the differing pharmacodynamic effects of ACE inhibitors and AII receptor antagonists, i.e. ACE inhibitors captopril, enalapril or lisinopril) inhibit the conversion of angiotensin I to angiotensin II and potentiate the effects of the kallikrein-kinin system whereas AT1 selective AII receptor antagonists 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 compounds from these therapeutic classes.

The coadministration of an ACE inhibitor with an 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 congestive heart failure. Additionally, a patent application filed by Merck and INSERM (EPO 629408) claims enhanced renal blood flow when treating with the combination.

Summary of the Invention is A method of treating and/or preventing renal disease of a warm-blooded animal with a therapeutically effective dose amount of a pharmaceutical composition of an ACE inhibitor which is lisinopril and an Angiotensin II receptor antagonist which is losartan is disclosed.

Included within the scope of the term renal disease are diabetic (insulin- and noninsulindependent) and non-diabetic nephropathy, including immunologically- and 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 mammal with a therapeutically effective amount of a pharmaceutical composition of an *ACE inhibitor which is lisinopril and an Angiotensin II antagonist which is losartan.

Included within the scope of the term pharmaceutical composition are a fixed combination 25 and a concomitant therapy of an ACE inhibitor and an AII antagonist.

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

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Detailed Description of the Invention 30 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

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inhibitor which is lisinopril and an AII receptor antagonist which is losartan. The use of a pharmaceutical composition of an ACE inhibitor and an All receptor antagonist in the manufacture of an orally administrable medicament for the treatment and/or prevention of T renal disease. Concomitant therapy would include the sequential administration of members [R:\LIBAA]07702.doc:tab from the two classes of compounds. The term renal disease includes diabetic nephropathy and non-diabetic nephropathy, including immunologically- and nonimmunologically-based nephropathies and/or glomerulopathies. The term non-diabetic nephropathy includes the condition referred to as human membranous glomerular nephritis.

The present invention further relates to a method for protection of renal structure and/or renal function with the coadministration, either concomitant therapy or a fixed combination therapy, of an ACE inhibitor which is lisinopril and an AII receptor antagonist which is losartan. The combination is also useful in preventing renal injury and protecting glomerular structure.

While the presently claimed invention is directed to the ACE inhibitor being lisinopril the angiotensin converting enzyme inhibitors useful in this method of treatment include, but are not limited to: 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. The ACE inhibitors particularly useful in this method of treatment are: captopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril, ramapril, and zofenopril.

While the presently claimed invention is directed to the angiotensin II inhibitor being losartan, 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 antagonists. These angiotensin II antagonists include, but are not limited to: 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] S 25 pyridine, BAY106734, BIBR363, CL329167, E4177, EMD73495, HN65021. HR720, HOE720, LRBO81, SC52458, SL910102, UP2696, YM358, EMD66397, ME3221, TAK536, BMS184698, CGP42112A, CGP49870, CP148130, E4188, EMD66684, EXP9954, FR1153332, GA0050, KT3579, LF70156, LRBO57, LY266099, LY301875, PD123177, PD126055, SC51757, SC54629, U96849, UK77778, WAY126227, WK1260, WK1492, YH1498, and YM31472. The angiotensin II antagonists particularly useful in this .method of treatment are: candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, BMS-184698 and 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine.

A study was conducted examining the coadministration of lisinopril (ACE inhibitor) and losartan (AT 1 -selective AII receptor antagonist to streptozotocin-induced diabetic rats.

The study results noted a decrease in urinary protein excreted by the rats. Further 1R:\LIBAA]07702.dcc:tab assessment of urinary protein data and morphometric assessment of 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.

Additionally, a twelve-month study was conducted examining the coadministration of lisinopril and 3-(2'-(tetrazol-5-yl)-1,l'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3Hto rats with passive Heymann nephritis. This animal model which manifests in the rat with long lasting proteinuria followed by *o oo*«* *o~ *o 4 ~bo [R:\LIBAA]07702.doc:tab WO 97/02032 PCT/US96/10942 -6renal injury (See JASN 3:624, 1992) is representative of human immunologically-mediated glomerulonepropathy.of renal disease, which also noted enhanced renal protection with the coadministration of lisinopril and 3-(2'-(tetrazol-5-yl)- 1,1'-biphen-4-yl)methyl-5,7dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine. Passive Heymann Nephritis manifests in the rat with long lasting proteinuria followed by renal injury. See JASN 3:624, 1992. The study demonstrated that coadministration reduced proteinuria and the degree of renal injury better than either the monotherapy of lisinopril, as well as the 1 0 monotherapy of 3-(2'-(tetrazol-5-yl)- 1,1'-biphen-4-yl)methyl-5,7dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine.

The use of the combination of an ACE inhibitor and an Angiotensin II (AII) receptor antagonist has been demonstrated in rats 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 0 nephropathy it is understood that the disease state is the result of either non-insulin dependent diabetes mellitus or insulin dependent diabetes mellitus.

Pharmaceutically suitable salts include both the metallic (inorganic) salts and organic salts; a list of which is given in Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985). It is well known to one skilled in the art that an appropriate salt form is chosen based on physical and chemical stability, flowability, hydroscopicity and solubility. The preferred salts of this invention include, but are not limited to: potassium, sodium, calcium and ammonium salts 3 0 of the ACE inhibitor and/or AII receptor antagonist.

Included within the scope of this invention is a method of treatment of renal diease using pharmaceutical compositions comprising an ACE inhibitor, an AII antagonist and a suitable pharmaceutical carrier.

WO 97/02032 PCT/US96/10942 -7- DOSAGE FORMS The pharmaceutical compositions of this invention can be administered for the treatment and or prevention of renal disease according to the invention by any means that effects contact of the active ingredient compound with the site of action in the body of a warmblooded animal. For example, administration, can be parenteral, i.e., subcutaneous, intravenous, intramuscular or intra peritoneal.

Alternatively, or concurrently in some cases administration can be by the oral route.

1 The pharmaceutical compositions of this invention can be administered by any conventional means available for use in conjunction with pharmaceuticals. The pharmaceutical compositions can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of 1 5 administration and standard pharmaceutical practice.

For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom which includes but is not limited to mammals and birds. The preferred mammal of this invention is human.

The dosage administered will be dependent on the age, 2 0 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 effective to obtain desired results.

The active ingredients can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and powdered carriers, 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 as sustained release products to provide for continuous release of WO 97/02032 PCT/US96/10942 -8medication 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 1 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 as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and 1 5 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 Remington's Pharmaceutical Sciences, A. Osol, a standard reference text 2 in this field.

Useful pharmaceutical dosage-forms for administration of the fixed combinations of this invention can be illustrated as follows:

CAPSULES

A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with a pharmacologically appropriate amount in milligrams of the powdered active ingredients, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.

3 0 SOFT GELATIN CAPSULES A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive olil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin WO 97/02032 PCT/US96/10942 -9capsules 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 1 (0 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

INJECTABLE

A parenteral composition suitable for administration by 1 5 injection is prepared by stirring a pharmacologically appropriate amount by weight of the active ingredients in 10% by volume propylene glycol. The solution is made to volume with water for injection and sterilized.

SUSPENSION

An aqueous suspension is prepared for oral administration 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 benzoate, 1.0 grams of sorbitol solution, 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 0 invention are administered in a concomitant fashion. The above dosage forms and route of administration 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.

WO 97/02032 PCT/US96/10942 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 Drug Dose (mg/day) Formulation Route of Admin.

lisinopril 5, 10, 20, 40 Tablet Oral enalapril 10-40 Tablet Oral Table B: Examples of AII receptor antagonists that can be combined with the above ACE inhibitors for the treatment and/or prevention of renal disease Drug Dose (mg/day) Formulation Route of Admin.

losartan potassium 25, 50, 100 Tablet Oral 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.

EXAMPLE 1 Study conducted in a Streptozotocin-Induced Diabetic Rat Model using the AII receptor antagonist, Losartan and the ACE inhibitor, Lisinopril.

Diabetes was induced with intravenous streptozotocin 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 WO 97/02032 PCTIUS96/10942 11 losartan/lisinopril treatment include: sixteen-hour urinary protein excretion [total protein (TUP), high molecular weight protein (HMW)], histomorphological quantitative assessment of glomerular area (GA) and glomerular basement membrane thickness (GBMT).

Parameter (Units) Control STZ STZ/LOS STZ/LOS/LIS TUP (mg) [151 21.8 46.7 9.4 6.1 t HMW (mg) [15] 15.3 31.9 2.0 *t 0.5 *t LMW (mg) [15] 6.4 14.8 7.4 5.6 GA (m 2 [10] 20.7 22.0 21.3 19.5 t' GBLT(nm) 332 441 359 t 316 t# number evaluated per group Statistically significantly different from nondiabetic control group t Statistically significantly different from STZ diabetic control groups T Statistically significantly different from losartan-treated STZ diabetic group 1 0 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 nonstatistically significant, increase in glomerular area. The latter has been demonstrated to be a precursor to glomerular sclerosis. Losartan 1 5 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 was further decreased (p 0.05) in the losartan/lisinopril treatment group.

Similarly, when compared to the STZ diabetic control group, there was 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.

SUBSTITUTE SHEET (RULE 26) WO 97/02032 PCT/US96/10942 12- EXAMPLE 2 Study conducted in a Passive Heymann Nephritic (PHN) Rat Model using the AII receptor antagonist, 3-(2'-(tetrazol-5-yl)- ,l'-biphen-4yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine and the ACE inhibitor, Lisinopril.

Male Sprague-Dawley, CD-COBS rats (Charles River Italia 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 1 0 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 All antagonist, 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3Hcontinuously in the drinking water for 12 months starting at day 7 after PHN induction when animals have already developed proteinuria. 3-(2'-(Tetrazol- 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3Hwas administered 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 Group 2 PHN rats were given daily the ACEI, lisinopril 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)-l,l'-biphen- 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 treatment.

Group 5 Normal rats with no treatment were followed for 12 months and used as control.

SUBSTITUTE SHEET (RULE 26 WO 97/02032 PCT/US96/10942 13 All animals 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 months for 12 months, by the tail cuff method (Pfeffer et al., 1971). At day 0 (basal) and every two months blood samples were collected for measurement of plasma creatinine concentration. 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 0 of the study period, all animals underwent determination of wholekidney function (GFAR, as clearance of inulin; RPF, as clearance of paminohippuric acid). At sacrifice, blood samples were collected for measurement of plasma All concentration and the kidneys were removed and processed for histological analysis by light microscopy.

1 5 During the study the following mortality was observed: one normal rat died at month 9, two PHN rats treated with 3-(2'-(tetrazol-5yl)-1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5b]pyridine died; one at month 5 and one during renal function studies due to anesthesia, one PHN rat treated with lisinopril died at month 9 and two PHN rats treated with the combined therapy died at months 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 As shown in Table 2, during the 12 month study rats with PHN gained weight in a similar manner to normal control rats. Treatment of PHN rats with 3 1,1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5b]pyridine, lisinopril or the combination of All receptor antagonist and ACE inhibitor affected animals' weight gain; the actual body weights of 3 0 these animals were significantly lower than those 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 12 WO 97/02032 PCT/US96/10942 14months. At 12 months also body weights of PHN rats treated with 3- (2'-(tetrazol-5-yl)-1, 1 '-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3Hwere 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'- 1 0 biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, lisinopril or the combination, all had SBP values significantly (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 0 after 12 months. 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7dimethyl-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 51.84+14.55 and 148.98+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 0 2-ethyl-3H-imidazo[4,5-b]pyridine lisinopril were even significantly lower than those of PHN 3-(2'-(tetrazol-5-yl)-1,l'-biphen-4yl)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.

WO 97/02032 PCT/US96/10942 we 3 Control PHN+Vehicle a PHN+Lisinopril a PHN+All compd.

PHN+A1 compd. lisinopril 0 0 1d/ 60d0 76 B!ow 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 1 0 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 inhibitor 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 GFR, 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[4,5b]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 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)-1,1 '-biphen-4-yl)methyl-5,7dimethyl-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 0 PHN untreated rats.

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 SUBSTITUTE SHEET (RULE 26) WO 97/02032 PCT/US96/10942 16interstitial fibrosis and inflammation associated with tubular atrophy and large eosinophilic casts in the tubular lumens.

Limitation of proteinuria in PHN treated rats reflected a better preservation of glomerular structural integrity when compared to untreated PHN rats. Thus, there were very few segmental sclerotic changes which affected on average 3% of glomeruli in rats given (tetrazol-5-yl)-1,1'-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H- 3.71% in rats given lisinopril and 1.06% in rats receiving the combined therapy. The latter value was comparable with 1 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 tubulointerstitial damage in rats given AII receptor antagonist ACE inhibitor combination were even lower than those of normal rats.

E] Control 3 PHN+Vehicle so 0 PHN+Lisinopril E PHN+AIl compd. PHN+All compd. lisinopril p<0.01 vs PHN+vehicle p<O.Ol vs PHN+vehicle SUBSTITUTE SHEET (RULE 26) TABLE I Food intak~e of control rats. PHN rats untreated or treated with 3-(2'-(tetazol-5-yl)- 1,1'-biphen-4-y) methyl1-5.7 -dim ethyl- 2-ethyl-3 H- (referred to in the Tables as All compd), lisinopril or 3-(2'-(tetrazol-5-yl)-1,1'-biphen-4-yl)methiyl-5,7-dimethyl-2-ethvl- 3H-imidazo-[4.5-bloyridine, p2lus lisinopril at months 0. 4, 8 and 12 FOOD INTAKE (gr/24 hi) Months 0 4 8 12 Control 26.25 +0.96 22.25 1.82 23.00 2,35 18.28 2.20 (n 8) (n (n (n =7) PHN 26.75 1.41 25.25 1.68 24.25 2.11 22.00 1.60 (n (n (n (1 8) PHN All compd. 25.00 2.26 21.25 1.96 21.71 2.52 18.00 2.09 (11=89) (n (n (n =7) PHN Lisinopril 23.50 1.29 23.50 +2.02 24.50 1.17 19.42 2.16 (n (n (Dn=8) (n =7) P1-N All compd.+ 26.50 1.11 24.50 +2.06 23.42 3.25 19.001+ 1.52 Lisinopril (n 8) (n 8) (n 7) (n 6) Data are expressed as mean SE Un 4

-B

0D TABLE 2 Body weight of control rats. P1-N rats untreated or treated with 3-(2'-(tetrazol-5-yl)- 1.1 -hiplhen-4-yl)thttyl- 5.7 -di meth yl-2-ethyl-3 H-imidazo [4.5 -bjpyri dine, lisinopril or 3-(2'-(tetrazol-5-y1)-1,1 I-biphen-4-yl)methyl- 5,7-dimethyl-2-ethyl-3H-imidazol4,5-blpvridine plus lisinopril at months 0, 2, 4, 6, 8. 10 and 12 BODY WEIGHT (gramns) MONTHS 0 2 4 6 Control 251.50 4.13 601.50 21.46 701.75 20.89 764.37 24.72 (n (n (n (n =8) PHN 253.25 2.56 570.00 21.77 658.37 19.06 747.87 31.03 (n (n (n (n =8) PHN All 254.00 3.38 517.50 23.88. 583.37 30.83* 606.71 31.20*A compd. (n1= 8) (n (n (n =7) PHN Lisinopril 251.25 2.72 540.00 11.58. 612.25 16.31* 659.62 21.65* (n (n (n (n =8) PHN All 253.00 3.02 530.50 21.15- 595.00 27.74. 604.14 31.59*A compd. (n (n (n (n =7) Lisinopril Data are expressed as mean SE p<0.

0 *p<0.01 vs control at corresponding time A p<0.05 vs PHN at corresponding time TABLE 2 (CONT'D) -4 Body weight of control rats. PHN rats untreated or treated with 3-(2'-(tetrazol-5-yl)-l.l-biphien-4-yl)methyl-5,7-dimethiyl-2-ethyl-3H-imidazot4.5-blpyridine, lisinopril or 3-(2'-(tetrazol-5-vl)- 1.1 -biphen-4- Yfl methvl-5,7-dimethvl-2-ethyl-3H-imidazo[4.5-blovridine plus lisinopri! at months 0, 8. 10 and 12 BODY WEIGHT (grams) MONTHS 8 10 12 Control 806.25 30.07 828.28 34.12 852.14 42.38 (n (n (n =7) PHN 795.00 32,78 824.37 29.75 800.00 27.66 (n (n (n =8) PHN All 654.28 32.14-A 737.00 45.35 717.57 31.91compd. (n (n 7) (n 7) PHN Lisinopril 716.87 25.62. 784.28 23.04 811.42 22.48 (n (n (n =7) PHN All 644.85 43.03-A 664.00 46.75-A 668.83 29.70*A compd. (n (n (n =6) Lisinopril Data are expressed as mean SE P<0.05 *p<0.0l vs control at corresponding time A p<0.05 vs PHN at corresponding time 0

'C

-4 0 fr.~ 0 TABLE 3 Systolic blood pressure of control rats, PH N rats untreated or treated with 3-(2'-(telrazol-5-yl)- 1.1'-biphien-4-y I) meth yl-5,7dimethyl-2-ethyl-3H-imidazo[4.5-bJpyridine, lisinopril or 3-(2'-(tetrazol-5-v1)- 1,1 -biphen-4-yl)methivl-5.7-dii-nethyl-2-etliyl-3Himidazo14.5-blpyridine plus lisinopril at months 0. 2. 4, 6. 8, 10, 12 BLOOD PRESSURE (mmiHg) Months Control PH N PHN All compd.

PHN Lisinoprit 0 139.82 2.87 (n 8) 133.26 4.69 (n =8) 132.46 6,70 (n 8) 135.12 2.36 (n1 8) 135.61 2.64 (11 8) 2 132.93 6.00 (n =8) 140.61 4.48 (n =8) 110. 13 4.16*.

(n 8) 106.85 2.49*.

(n =8) 109.98 3.19*.

(n =8) 4 127.46+ 5.74 (n =8) 137.17 4.84 (n =8) 98.07 3.40*A (n1 8) 112.01+ 2.tO0*A 01 8) 101.21 3.65 *A (n 8) 129.30 6.22 (n =8) 149.27 6.15.

(n =8) 101.61 3.46*A (n =7) 106.06 2.98*A (n =8) 88.55 5.28*A (n 7) 136.68 5.85 (11 8) 159.88 6.67- (n 8) 112.30 2.88*A (n 7) 107.88 4.43*A (n =8) 10 13 1.22 6.00 (n1 7) 157.73 7.14.

(n =8) 104.0t6 4.46*A (n 7) 108.55 4.45*A (11 7) 12 132.12 6.61 (D 7) 160.50 6.76A (11 8) 111.51 4.72*.

(n1= 7) 110.28 3.67*.

(a 7) 105.73 4.08*.

(11 6) PHN All compd. Lisinopril 95.58 5.61 *A 99.62 5.68*A (n 7) (n =7) Data are expressed as mean SE *p<0.01 vs PHN at corresponding time -p< 0 0 Ap<t).0l vs control at corresponding time TABLE 4 Proteinurla of controled rats, PHN rats untreated or treated with 3-(2'-(tetrazol-5-yl)- 1,1I'-biphen-4-yl)methyl- 5.7-dimethyl-2-ethyl-3H-imidazo[4.5-b]pyridine, lisinopril or 3-(2'-(tetrazol-5-yl)- 1,1 '-biphen-4-yl)methyl- 5,7-dimethyl-2-ethyl-3H-imidazol4,5-bipyridine plus lisinopril at days 0,7 (before treatment) and at months 2.

4, 6, 8, 10 and 12 Proteinuria (mng/day) 0 day 7 month 2 month 4 Control 15.11 1.38 24.21 2.71 33.85 4.54 34.94 +4.51 (n 8) (n 8) PHN 16.83 +3.30 110. 74 +20.54* 455.39 +82.26* 495.23 +95.32* (n 8) (n 8) (n 8) PHN All compd. 15.47 2.29 157.54 5 1.95* 60.37 9.12-A 39.27 8.30A (n (n (n (n =8) PHN Lisinopril 13.25 1.27 59.35 6.88* 125.18 5.39-A 101.51 36.97A (n (11= 8) (n (n =8) PHN All compd. 16.91 2.27 97.58 17.57* 33.84 2.08AO 23.73 1.91AO Lisinopril (n (n (n (n =8) Data are expressed as mean SE p<O.O I -p<0.05 vs control at corresponding time Ap<0.01 vs pHN at corresponding time Op<O.0l. 0.05 0 lp<O.0l vs PHN+ lisinopril and PHN All compd. at corresponding time 01n TABLE 4 (CONT'D) Proteinurla of controled rats, PHN rats untreated or treated with 3-(2'-(tetrazol-5-yl)- 1,1 '-biphien-4-yl)methyl- 5,7-dimethyl-2-ethyl-3H-irnidazof4,5-b]pyridine, lisinopril or 3-(2'-(tetrazol-5-yl)- 1,1'-biphen-4-yl)methyl-5,7di-methyl-2-ethyl-3H-imidazo[4,5-blpyridine plus lisinopril at days 0,7 (before treatment) and at months 2, 4, 6. 8. 10 and 12 Proteinuria (mg/day) Control P1-N month 6 52.96 +7.96 (n 8) 787.14 116.50* (n 8) 28.45 6.96A (n 7) 74.41+ 16.03A (n 8) month 8 51.57 7.53 (n 8) 838.45 108.57* (n 8) 36.97 9.72A (n =7) 101.74 24.52A (n =8) PHN All compd.

PHN Lisinopril month 10 72.57 +8.30 (n 7) 900.61 120.36* (n 8) 55.48 13.02A (n =7) 122.31 44.05A (n 7) 17.82 2.13A 0 (n =7) month 12 79.46 15.43 (n 7) 702.06 77.02* (n1 8) 51.84 14.55A (n 7) 148.98 61.62A (n 7) 15.87 l.94A*o (n 6) PHN All 16.27 2.37AO* 18.

compd. (n =7) Lisinopril Data are expressed as mean SE p<O.OlI -p<0.05 vs control at corresponding time 49 2.94AO* (n 7) Ap<0.O 1 vs pHN at corresponding time Op<O.0l. 0.05 0 p<O.OI vs PHN+ lisinopril and PHN All compd. at corresponding time (-Il 0 -4 0 0 w TABLE 5 Serum creatini ne of control rats, PHN rats untreated or treated with 3-(2'-(tetrazol-5-yl)- 1.1 -biphien-4-vI)mieth~yl-5.7-dimetlhyl-2-ethl.

3H-imidazo[4.5-blpyridinie. lisinopril or 3-(2'-(tetr-azol-5-y1)- 1.1 -biphen-4-y1)methyI-5,7-dimethv1-2-ethy1-3H-imidazo[4.5..

b1lpyridine plus lisinopril at months 0, 2.4. 6. 8. 10, 12 SERUM Creafinine (mng/dI) Months 0 2 4 6 8 10 12 Control 0.61 0.01 0.62 0.01 0.61 0.01 0.61 0.01 0.67 0.01 0.68 0.01 0.69 +0.02 (n 8) 8) (n (n1 8) (n 7) (n 7) PHN 0.62 0.01 0.67+ 0.02 0.70 +0.02 0.72+±0.02. 0.82 0.02* 0.87 +0.04. 0.89 ±t 0.04.

(1 8) (n (n (n (n 01n=8) (n =8) PF{N All compd. 0.62 0.01 0.65+ 0.01 0.71 0.02 0.75 0.74 0.01J *A 0.72 0.OIA 0.72 0.OIA (n 8) 8) (n 8) (n (n 7) (n 7) (n =7) PHN Lisinopril 0.63 0.01 0.65 0.02 0.71 0.02 0.75± 0.02* 0.76 0.01 0.71 0.O1A (1.74 0.03A (n1= 8) (n (n (n (n (n (n =7) PHN All compd. 0.59 0.02 0.70+ 0.02 0.71 0.02 0.78 0.04* 0.81 0.0.3 0.79 +0.02* 0.79 +0.02.

Lisinopril (n (n (n (n (n (n 7) (n =6) Data re expressed as mean SE p<0.05 *p<O.O1 vs control at corresponding timre A p<0.05 vs pHN at corresponding time

I

CD

t.j TABLE 6 GFR and RPF of control rats, PHN rats untreated or treated with 3-(2'-(tetrazol-5-yl)-l,1'-biphen4-yl)metiyl-5,7-dimethyl-2ethyl-3H-imidazojj4.5-bjpyridine, lisinopril or 3 -(2'-(tetrazol -5 -yl) 1,1 -biphen-4- yl) methyl -5,7 -dirnethyl-2 -ethyl -3H imid oP 5-b]Dvridine DIUS lisinonril at month 12 GFR and RPF (mi/min) GFR RPF (mi/mmn) (mi/min) Control 2.49 +0.07 7.51 0.20 (n=7) PHN 1. 18 0.05* 5.04 0.20* 01i=8) (n=8) PHN All compd. 1.67 0.04*A 5.02 0.12A (n=6) PHN Lisinopril 1.76 0. 1I0*A 6.64 0.50A (n=7) PHN All compd. 1.79 0.12*A 6.02 0.13A Lisinopril (n=6) Data are expressed as mean SE p<0.01, vs control at corresponding time A p<0.0 I vs PH-N at corresponding time TABLE 7 Pathological changes in PHN rats after 12 months of treatment with 3-(2'-(tetrazol-5-yl)-l,1'-biphen -4-yI) methyl -5.7 dimethyl-2-ethyl-3H-imidazo[4,5-blpyridine, lisinopril or 3-(2'-(tetrazol-5-yl)- 1,1 -biphen-4-yI)methyl-5,7-dimethyl-2ethyl -31--imidazo [4.5 -blpvridine plus lisinopril Month 12 Groups Glomeruli with Interstitial damage Tubular damage sclerotic change (score) (score) Control 1.71 0.57 0.57 (0-1) PHN 60.25 2.62* 2.62* (10-90) (1-3) PHN All compd. 3.00A 0.7 1-A 0.71A (0-1) PHN Lisinopril 3.71IA 0.71LA 0.71LA (0-2) PHN All compd. 1.06A 0.50A 0.33A Lisinopril (0-1) Data are expressed as mean percentage and mean score.

p<0.01. vs control A p<0.01 vs PHN Range is in parenthesis.

Claims (39)

1. The use of pharmaceutical composition of an ACE inhibitor which is lisinopril and an AII receptor antagonist which is losartan 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 lisinopril and losartan 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 lisinopril and losartan.

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

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

8. The use as recited in claim 7, wherein the renal structure is the glomerular structure.

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

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

11. The use as recited in claim 10, wherein the pharmaceutical composition consists 9 25 of a fixed combination of lisinopril and losartan and a pharmaceutically acceptable carrier.

12. The use as recited in claim 10, wherein the pharmaceutical composition consists of a concomitant administration of lisinopril and losartan.

13. The use of a pharmaceutical composition of an ACE inhibitor which is lisinopril and an AII receptor antagonist which is losartan in the manufacture of an orally 30 administrable medicament for reducing proteinuria.

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

15. A method for the treatment or prophylaxis of renal disease in a mammal a requiring said treatment or prophylaxis, which method includes or consists of administering [R:\LIBAA]07702.doc:tab to said mammal an effective amount of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan.

16. The method as recited in claim 15, wherein the renal disease is diabetic nephropathy or non-diabetic nephropathy.

17. The method as recited in claim 15, wherein a pharmaceutical composition consisting of a fixed combination of lisinopril and losartan and a pharmaceutically acceptable carrier is administered.

18. The method as recited in claim 17, wherein the renal disease is diabetic nephropathy.

19. The method as recited in claim 15, wherein the method consists of the concomitant administration of lisinopril and losartan.

The method as recited in claim 19, wherein the renal disease is diabetic nephropathy.

21. A method for the protection renal structure in a mammal requiring said protection, which method includes or consists of administering to said mammal an effective amount of at least one ACE inhibitor which is lisinopril and at least one All receptor antagonist which is losartan.

22. The method as recited in claim 21, wherein the renal structure is the glomerular structure.

23. A method for the prevention of renal injury in a mammal requiring said prevention, which method includes or consists of administering to said mammal an effective amount of at least one ACE inhibitor which is lisinopril and at least one AII receptor Santagonist which is losartan.

24. A method for the protection of renal function in a mammal requiring said 25 protection, which method includes or consists of administering to said mammal an effective Samount of at least one ACE inhibitor which is lisinopril and at least one AII receptor S. antagonist which is losartan.

25. The method as recited in claim 24, wherein a pharmaceutical composition consisting of a fixed combination of lisinopril and losartan and an acceptable carrier is 30 administered.

26. The method as recited in claim 24, wherein the method consists of a concomitant administration of lisinopril and losartan.

27. A method for reducing proteinuria in a mammal requiring reduction of proteinuria, which method includes or consists of administering to said mammal an effective amount of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan. [R:\LIBAA]07702.doc:tab

28. A method for the treatment or prophylaxis of membranous glomerular nephritis in a mammal requiring said treatment or prophylaxis, which method includes or consists of administering to said mammal an effective amount of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan.

29. A pharmaceutical composition of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan when used for the treatment or prophylaxis of renal disease in a mammal.

The composition as recited in claim 29, wherein the renal disease is diabetic nephropathy or non-diabetic nephropathy.

31. The composition as recited in claim 29, wherein the pharmaceutical composition consists of a fixed combination of lisinopril and losartan and a pharmaceutically acceptable carrier.

32. The composition as recited in claim 31, wherein the renal disease is diabetic nephropathy.

33. A pharmaceutical composition of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan when used for the protection of renal structure in a mammal.

34. The composition as recited in claim 33, wherein the renal structure is the glomerular structure.

35. A pharmaceutical composition of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan when used for the prevention of renal injury in a mammal.

36. A pharmaceutical composition of at least one ACE inhibitor which is lisinopril and at least one AII receptor antagonist which is losartan when used for the protection of •25 renal function in a mammal.

S37. The composition as recited in claim 36, wherein a pharmaceutical composition consisting of a fixed combination of lisinopril and losartan and an acceptable carrier is administered.

38. The composition as recited in claim 36, wherein the method consists of a S 30 concomitant administration of lisinopril and losartan. o

39. A pharmaceutical composition of at least one ACE inhibitor which is lisinopril S and at least one AII receptor antagonist which is losartan when used for reducing proteinuria a in a mammal. 0 [R:\LIBAA]07702.doc:tab A pharmaceutical composition of at least one ACE inhibitor which is lisinopril and at least one All receptor antagonist which is losartan when used for the treatment or prophylaxis of membranous glomerular nephritis. Dated 10 November, 1999 Merck Co., Inc. Laboratoires Merck Sharp Dohme-Chibret SNC Giuseppi Remuzzi Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\L1BAAj07702.doc~tab