WO1993010773A1

WO1993010773A1 - Use of neutral endopeptidase inhibitors in the treatment of left ventricular hypertrophy

Info

Publication number: WO1993010773A1
Application number: PCT/EP1991/002338
Authority: WO
Inventors: Angela Monopoli; Ennio Ongini
Original assignee: Schering-Plough S.P.A.
Priority date: 1991-12-06
Filing date: 1991-12-06
Publication date: 1993-06-10

Abstract

Treatment and prevention of left ventricular hypertrophy with neutral endopeptidases such as N-[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine and N-[2-mercaptomethyl-3-(2-methylphenyl)-propioyl]-methionine are disclosed.

Description

USE OF NEUTRAL ENDOPEPTIDASE INHIBITORS IN THE TREATMENT OF LEFT VENTRICULAR HYPERTROPHY

The present invention relates to the treatment and prevention of left ventricular hypertrophy (LVH) by administration of a neutral endopeptidase (NEP) inhibitor.

LVH, characterized by an increase in cardiac mass and growth of abnormal fibrous tissue which compromise cardiac function, is a condition which often occurs in conjunction with high blood pressure associated with essential hypertension. LVH is a primary risk factor associated with heart failure and therefore increases the risk of cardiovascular morbidity and mortality.

Usually, LVH is detected after high blood pressure is diagnosed, although recent reports indicate that LVH may be present before high blood pressure develops, or may be aggravated by a second condition such as atherosclerosis or diabetes mellitus. LVH is diagnosed by several methods, including electrocardiography, wherein enhanced voltage is detected, by chest X-rays, or preferably by echocardiography, which detects increased myocardial wall thickness and mass. The existing therapy for LVH associated with essential hypertension consists of control of arterial blood pressure, for example by administering one or more of a variety of drugs: diuretics such as diazoxide or hydrochlorthiazide; hypotensives such as methyldopa or hydralazine; beta-adrenergic blockers such as propranolol or labetalol; calcium antagonists such as diltiazem or nifedepine; or angiotensin converting enzyme (ACE) inhibitors such as captopril, spirapril or cilazapril. ACE inhibitors and calcium antagonists are known to reduce the mass of the hypertrophied left ventricle, however, many other drugs routinely prescribed to treat essential hypertension, e.g. diuretics and hydralazine, either have no effect on LVH or take a long time to treat it.

NEP (EC 3.4.24.11; enkephalinase; atriopeptidase) is a zinc-containing metalloprotease which cleaves a variety of peptide substrates on the amino terminal side of aromatic amino acids. See Biochem. J.. 241 (1987) p. 237-247. Substrates for this enzyme include, but are not limited to, ANP, brain natriuretic peptide, met and leu enkephalin, bradykinin, neurokinin A, and substance P. It has been previously demonstrated that inhibitors of NEP potentiate the

hypotensive, diuretic, natriuretic and plasma ANP responses to pharmacological injection of ANP in experimental animals. The potentiation of ANP and the consequent use of NEP inhibitors in general to treat volume-dependent hypertension but not angiotensin II- induced hypertension was disclosed in U.S. patent 4,749,688.

We have surprisingly found that NEP inhibitors, in particular N-[2-acetylthiomethyl-3-(2-methylphenyl)-propionyl]- methionine ethyl ester and N-[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxoIan-4-yl)- methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine, reduce LVH without having an effect on high blood pressure resulting from essential hypertension. Therefore, NEP inhibitors can be used to treat LVH when essential hypertension is not present or is not severe enough to require drug therapy, NEP inhibitors can be used in conjunction with antihypertensive drugs which do not themselves treat LVH, or NEP inhibitors can be used in combination with drugs which do treat LVH in order to provide an enhanced effect.

Another aspect of the invention relates to pharmaceutical compositions comprising a combination of an NEP inhibitor and an antihypertensive agent effective to treat or prevent LVH in a

pharmaceutically acceptable carrier.

DETAILED DESCRIPTION

The NEP inhibitors suitable for use in this invention include carboxyalkyl dipeptides disclosed in U.S. patent 4,610,816, herein incorporated by reference, having the formula

R₁aCH(COR₂)-NH-CHR₃ ^a-CONH(CH₂)_p ^a-C(R₄ ^aR₅ ^a)-COR₆a wherein preferred compounds are N-[N-[(L)-[1-[(2,2-dimethyl-1 ,3- dioxolan-4-yl)-methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β- alanine and N-[N-[(L)-1-carboxy-2-phenylethyI]-L-phenylalanyl]-β- alanine;

mercaptoacyl amino acids disclosed in U.S. patent 4,801 ,609, herein incorporated by reference, having the formulae

Q^bS-CH₂-CH(-(CH₂)_n ^b-R^1b)-C(O)-NH-CH(R^2b)-C(O)-R^3b lb

Q^bS-CH₂-CH(-(CH₂)_n ^b-R^1ab)-C(O)-NH-CH(R^2ab)-C(O)-R^3b lib

Q^bS-CH₂-CH(-(CH₂)_n ^b-R^1ab)-C(O)-NH-CH(R^2b)-C(O)-R^3ab lllb mercaptoacyl amino acids disclosed in U.S. patent

4,929,641 , herein incorporated by reference, having the formula

Q^cS-CH₂-CH(-(CH₂)_n ^c-R^1c)-C(O)-NH-CH(R^2c)-C(O)-R^3c wherein preferred compounds are N-[2-acetylthiomethyl-3-(2-methyl- phenyl)propionyl]-methionine ethyl ester and N-[2-mercaptomethyl-3-(2- methylphenyl)propionyl]-methionine;

mercaptoacyl amino acids disclosed in PCT/US90/01787, herein incorporated by reference, having the formula

Q^dS-CH₂-CH(-(CH₂)_n ^d-R^1d)-C(O)-NH-CH(R^2d)-CH(R^4d)-(CH2)_t ^d-(CHR^9d)_p ^d-C(O)-R^3d wherein preferred compounds are N-[2(S)-mercaptomethyl-3-(2- methylphenyl)propanoyl]-(S)-isoserine and N-(S)-[3-mercapto-2-(2- methylphenyl)propionyl]-(S)-2-methoxy-β-alanine;

carboxyalkyl dipeptides disclosed in U.S. Serial No.

07/421 ,041 and corresponding PCT/US90/05640, incorporated herein by reference, having the formula

R^1eO-C(O)-CH(R^2e)-NH-C(R^3eR^4e)-C(O)-NH-(CHR^5θ)m^e-(CH₂)_n ^e-CH(R^6e)-C(O)-R^7e wherein preferred compounds are N-[1-[[ 1 (S)-benzyloxycarbonyl-3- phenylpropyl]amino]cycIopentylcarbonyl]-(S)-isoserine and N-[1-[[1 (S)- carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]-(S)-isoserine;

disulfide derivatives of mercaptoacyl amino acids disclosed in U.S. Serial No. 07/525,370, incorporated herein by reference, having the formulae

wherein preferred compounds are 1,1 '-[dithiobis-[2(S)-(2-methylbenzyl)- 1-oxo-3,1-propanediyl]]-bis-(S)-isoserine and 1 ,1'-[dithiobis-[2(S)-(2- methylbenzyl)-1-oxo-3,1-propanediyI]]-bis-(S)-methionine;

mercaptoacyl amino acids disclosed in PCT publication WO90/02117, herein incorporated by reference, having the formula

wherein a preferred compound is N-(3-phenyl-2-(mercaptomethyl)- propionyl)-(S)-4-(methylmercapto)methionine;

mercaptoacyl amino acids disclosed in U.S. patent

4,879,309, incorporated herein by reference, having the formula R₁ ^hS-CH₂-CH(-(CH₂)_n ^h-R₂ ^h)-C(O)-NR₄ ^h-A^h-C(O)-R₃ ^h wherein preferred compounds are N-[2-acetylthiomethyl-3-phenyl- propionyl]-3-aminobenzoic acid and N-[2-mercaptomethyl-3-phenyl- propionyl]-3-aminobenzoic acid;

carboxyalkylcarbonyl amino acids disclosed in U.S. Serial No. 07/439,765 and corresponding PCT/US90/06655, herein

incorporated by reference, having the formula R^{1 i}O-C(O)-CH(R²ⁱ)-CH₂-C(R³ⁱR⁴ⁱ)-C(O)-NH-(CHR⁵ⁱ)_m ⁱ-(CH₂)_n ⁱ-CH(R⁶ⁱ)-C(O)-R⁷ⁱ wherein a preferred compound is N-[1-(2-carboxy-4-phenylbutyl)- cyclopentanecarbonyl]-(S)-isoserine;

mercaptocycloalkyl amino acids disclosed in U.S. Serial No. 07/455,204 and corresponding PCT/US90/07353, incorporated herein by reference, having the formula

wherein a preferred compound is N-[1-(acetylthiomethyl)cyclopentane- carbonyl]-(S)-methionine ethyl ester;

mercaptoacyl aminolactams disclosed in U.S. Serial No. 07/491 ,148 and corresponding PCT/US91/01420, incorporated herein by reference, having the formula

^

wherein a preferred compound is 3(S)-[2-(acetylthiomethyl)-3-phenyl- propionyl]amino-ε-caprolactam; glutaryl amino acids disclosed in U.S. 4,975,444, herein incorporated by reference, having the formula

glutaryl amino acids disclosed in European Patent Application 274,234, having the formula

wherein A^s completes a 4 to 7 membered carbocyclic ring which may be saturated or mono-unsaturated and which may optionally be fused to a further saturated or unsaturated 5 or 6 membered carbocyclic ring; B^s is (CH₂)_m ^s wherein m^s is an integer of from 1 to 3;

each of R^s and R^4s is independently H, alkyl, benzyl or an alternative biolabile ester-forming group;

R^1s is H oralkyl;

R^2s and R^3s are each independently H, OH, alkyl or alkoxy;

and R^5s is alkyl, alkenyl, alkynyl, arylalkynyl, cycloalkyl, cycloalkenyl, alkoxy, -NR^6sR^7s, -NR^8sCOR^9s, -NR^8sSO₂R^9s or a saturated

heterocyclic group;

or alkyl substituted by one or more substituents chosen from halo, hydroxy, alkoxy, hydroxyalkoxy, alkoxyalkoxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arlyoxyalkoxy, heterocyclyloxy, -NR^6sR^7s, -NR^8sCOR^9s, - NR^8sSO₂R^9s, -CONR^6sR^7s, -SH, -S(O)_p ^sR^10s, -COR^11s or -CO₂R^12s; wherein R^6s and R^7s are each independently H, alkyl, cycloalkyl (optionally substituted by hydroxy or alkoxy), aryl, arylalkyl, alkoxyalkyl or heterocyclyl; or the two groups R^6s and R^7s are taken together with the nitrogen to which they are attached to form a pyrrolidinyl, piperidino, morpholino, piperazinyl or N-alkyl-piperazinyl group;

R^8s is H or alkyl;

R^9s is alkyl, CF₃, aryl, aryl, alkyl, arylalkoxy, heterocyclyl, alkoxy or -NR^6sR^7s wherein R^6s and R^7s are as previously defined;

R^10s is alkyl, aryl, heterocyclyl, or -NR^6sR^7s wherein R^6s and R^7s are as previously defined;

R^{11 s} is alkyl, cycloalkyl, aryl or heterocyclyl;

R^12s is H or alkyl;

and p^s is 0,1 or 2;

and pharmaceutically acceptable salts thereof and bioprecursors therefor; and glutaryl amino acids disclosed in European Patent

Application 343,911 , having the formula

wherein A^u completes a 4 to 7 membered carbocyclic ring which may be saturated or mono-unsaturated and which may optionally be fused to a further saturated or unsaturated 5 or 6 membered carbocyclic ring;

B^u is (CH₂)m^u wherein m^s is an integer of from 1 to 3;

each of R^u and R^4u is independently H, alkyl, benzyl or an alternative biolabile ester-forming group;

R^1u is H or alkyl;

R^2u and R^3u are each independently H, OH, alkyl or alkoxy, or R^2u and

R^3u are linked together and are (CH₂)r^u wherein r^u is an integer from 1 to 4;

Y^u is an optional alkylene group of from 1 to 6 carbon atoms which may be straight or branched-chain; and R^5u is R^6uCONR^9u-, R^6uSO₂NR^9u-, R^6uCO₂-, R^6uCO-, R^6uSO_q ^u-, R^7uNR^9uSO₂-, or R^7uOCO-;

wherein R^6u is a group of the formula

R¹⁰u R^10u

| |

R^8u- (C-CONR^9u)_nu-C- | |

R^11u R^11u

R^7u is a group of the formula

R^10u

|

R^12u-C- |

R^11u

and R^9u is H, alkyl, aryl, cycloalkyl, heterocyclyl, arylalkyl, or

hererocyclylalkyl;

wherein R^8u is R^9uCONR^9u-, R^9uSo₂NR^9u- ,R^13uR^14uN-(CH₂)_pu-, or R^9uO-, wherein each R^9u is as previously defined;

R^10u and R^11u are each independently H or alkyl; or R^10u is H and R^11u is alkyl which is substituted by OH, SH, SCH₃, NH₂, arylalkyl-OCONH-,

NH₂CO-, CO₂H, guanidino, aryl or heterocyclyl; or the two groups R^10u and R^11u are joined together to form, with the carbon atom to which they are attached, a 5 or 6 membered carbocyclic ring which may be saturated or mono-unsaturated and which may optionally be substituted by alkyl or fused to a further 5 or 6 membered saturated or unsaturated carbocyclic ring;

or R^10u is H, n^u is 0 and R^8u and R^11u are linked to form a 2-(N-COR^9u- 4-aminopyrrolidinyl) group;

R^12u is R^13uR^14uNCO-, R^9uOCH₂- or heterocyclyl, wherein R^9u is as previously defined;

R^13u and R^14u are each independently H, alkyl, cycloalkyl, aryl, arylalkyl, alkoxyalkyl, aminoalkyl, heterocyclyl or heterocyclylalkyl; or the two groups R^13u and R^14u are taken together to form, with the nitrogen to which they are attached, a pyrrolidinyl, piperidino,

morpholino, piperazinyl, N-alkylpiperazinyl, pyrrolyl, imidazolyl, pyrazolyl or triazolyl group;

n^u is 0 or 1;

p^u is 0 or an integer of from 1 to 6; and q^u is 0, 1 or 2;

and pharmaceutically acceptable salts thereof and bioprecursors therefor.

The above descriptions of NEP inhibitors suitable for use in the present invention were taken from the noted patents or applications.

Reference should be made to such patents and applications for their full disclosures of such classes and specific compounds within those classes, and as to any typographical errors or the like which may have occurred in transcription. Also, in describing such suitable NEP inhibitors, the superscript letters a-i, k, q-s and u were included to distinguish among the various classes of compounds and the variable substituent groups thereof.

Other suitable NEP inhibitors include SQ 28603 (N-[2-

(mercaptomethyl)-1-oxo-3-phenylpropyI]-β-alanine), disclosed in South African Patent Application 84/0670; UK 69578 (cis-4-[[[1 -[2-carboxy-3-

(2-methoxyethoxy)propyl]-cyclopentyI]carbonyl]amino]- cyclohexanecarboxylic acid) and its active enantiomer(s); thiorphan and its enantiomers; retro-thiorphan; phosphoramidon; and SQ 29072 (7-

[[2-(mercaptomethyl)-1-oxo-3-phenylpropyI]amino]-heptanoic acid). Also suitable for use are any pro-drug forms of the above-listed NEP inhibitors, e.g., compounds in which one or more carboxylic acid groups are esterified.

Of the above NEP inhibitors, most preferred are N-[2- acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl ester, especially the S,S isomer thereof, and N-[N-[(L)-[1 -[(2,2-dimethyl-1 ,3- dioxolan-4-yl)-methoxy]carbonyI]-2-phenylethyl]-L-phenylalanyl]-β- alanine.

When NEP inhibitors are administered in combination with other antihypertensive agents, preferred antihypertensives are ACE inhibitors and calcium antagonists. Preferred ACE inhibitors are spirapril, enalapril, ramipril, perindopril, indolapril, lysinopril, quinapril, pentopril, cilazapril, captopril, zofenopril, pivalopril and fosinopril.

Preferred calcium antagonists are diltiazem, nifedipine, verapamil, nicardipine and nimodipine The effectiveness of NEP inhibitors in treating LVH in an animal model can be demonstrated according to the following procedures for measuring the change in heart weight and the degree of fibrosis:

Animals and treatment regimen:

Adult (16-week-old) male spontaneously hypertensive rats (SHRs) supplied by Charles River (Calco, Italy) are used. They are housed in individual cages for one week before starting the experiment, with free access to food and water. The animals are selected for stable baseline arterial pressure and assigned randomly to four groups of 12 each (first experiment). The NEP inhibitor is administered for 4 weeks at 3,10, 30, or 100 mg/kg orally twice daily (at 9:00 a.m. and 4:00 p.m.), with carboxymethylcellulose (CMC 0.5%) used as vehicle and as a control.

A second experiment involves administering an NEP inhibitor at 100 mg/kg or the vehicle as a control to two groups of SHRs twice a day for 4 weeks . Water intake, urine volume and sodium excretion are monitored by placing the rats in metabolic cages over a 16-hour period from the last daily administration of the drug to the first dosage of the following day.

In each experiment, additional animals were treated with spirapril at 1 mg/kg as a positive control.

Blood pressure and heart rate measurement:

Systolic blood pressure (SBP) and heart rate are

measured in conscious animals by the tail-cuff method using a pulse detector (ll TC Instruments, Woodland Hills, CA, USA) connected to a computer (Basis Trade, Verona, Italy). Animals are maintained at a temperature of 26 ± 1°C during blood pressure recording sessions and before starting the experiment, the animals undergo a week of training to stay in restraining holders. Recordings are taken once before treatment and at weekly intervals within 3 hours after the morning dose. Heart rate (HR) is determined from blood pressure tracings. Measurement of Ventricular Hypertrophy:

At the end of the experimental period, SHRs of the first set of experiments are sacrificed by cervical dislocation, the hearts are rapidly excised, fixed in formalin 4% and the weights of the right ventricle and left ventricle inclusive of the septum are recorded.

In the second set of experiments, SHRs are anesthetized with fentanyl citrate (50μg/kg) and droperidol (250 μg/kg; Leptofen, Farmitalia Carlo Erba, Milan, Italy) and the hearts fixed by perfusion as follows. The abdominal aorta below the renal arteries is cannulated with a catheter (PE 200) filled with phosphate buffer (0.2 M, pH 7.4) and heparin (100 lU/ml), the catheter is connected to a perfusion apparatus, and perfusion is adjusted to diastolic arterial pressure measured in vivo. The heart is then arrested in diastole by an intravenous injection of 1 ml of KCI (1 meq/ml) through the jugular vein, the thorax is opened and the vena cava is cut to allow drainage of blood and perfusate. The coronary vasculature is then perfused with a glutaraldehyde-formaldehyde mixture diluted 1 :1 with phosphate buffer. The heart is excised, the inner longitudinal diameter measured, and the right and left ventricle inclusive of the septum dissected from the atria and their weights recorded separately.

The left ventricle is transversely cut into 10-12 rings perpendicular to the longitudinal axis of the heart. The thickness of the left ventricular free wall and septum and the transverse luminal diameter of the ventricular chamber are measured in the intermediate slice with a stereomicroscope at a calibrated magnification of 16X, having an ocular micrometer accurate to 0.01 mm. Five to ten equally spaced

measurements of the free wall and four to six of the septum are collected and their values averaged. The minimal and maximal transverse chamber diameters are measured and their geometric mean computed. The longitudinal and transverse diameters are used to compute chamber volume. The apical slice of each left ventricle is used for dry weight determinations. Light microscopic morphometry of the left ventricle - amount of replacement fibrosis:

The four middle slices of the free wall of each ventricle are radially cut to obtain 28-30 tissue blocks extending from the

endocardium to the epicardium. The specimens were postfixed in OsO₄ (1%) dehydrated in acetone and infiltrated and embedded in a fixative agent, e.g. an epoxy resin such as Araldite (Ciba Geigy). From each block, 1μm thick sections were obtained and stained with methylene blue and safranin for the morphometric evaluation of foci of replacement fibrosis. Ten consecutive fields from each endocardium,

midmyocardium and epicardium in each animal are examined at a calibrated magnification of 250X with a reticle containing 42 sampling points. This reticle defines an uncompressed tissue area of 144,000 μm², which is used to determine the number of lesions represented by foci of fibrosis per unit area of myocardium. The number of points overlying these foci is also counted to compute the volume fraction of fibrosis in the myocardium and the average cross sectional area of the foci profiles.

Results of tests run according to the above procedures using N-[2(S)-acetylthiomethyl-3-(2-methylphenyl)propionyl]-(S)- methionine ethyl ester (Compound A) at 3 and 30 mg/kg, N-[N-[(L)-[1- [(2,2-dimethyl-1 ,3-dioxoIan-4-yl)-methoxy]carbonyl]-2-phenylethyl]-L- phenylalanyl]-β-alanine (Compound B) at 10 and 100 mg/kg and spirapril (SPIR) (1 mg/kg) are as follows (Data are expressed as means ± standard error, and analysis of variance and appropri ate

comparisons for each parameter were used as statistical tests.

Differences were considered significant at p< 0.05).

Baseline blood pressure ranged from 189±4 to 197±2 mmHg. 4 weeks of treatment with either Compound A or Compound B did not significantly affect arterial pressure, even at the highest dose level tested: 189±4 mmHg before, 195±4 mmHg after 4 weeks of a 30 mg/kg dose of Compound A; 196±4 mmHg before, 190+6 mmHg after 4 weeks of a 100 mg/kg dose of Compound B. The SPIR group showed a significant reduction in systolic pressure during the treatment period: 197±2 mmHg before, 181 ±3 mmHg after 4 weeks of a 1 mg/kg dose of SPIR (p< 0.05). In all groups tested, heart rates were not significantly different from those observed in the vehicle controls.

Both Compound A and Compound B significantly reduced left ventricular weight at the highest dose level compared to the vehicle control: compared to a control weight of 1036.7± 11.2 mg, the 30 mg/kg dose level of Compound A reduced the left ventricular weight to

969.8±11.9 mg (p< 0.01), while the 100 mg/kg dose level of Compound B reduced the left ventricular weight to 983.5±11.3 mg tøx 0.05). SPIR at 1 mg/kg reduced the left ventricular weight to 948.5±11.5 (p< 0.01 ). The body weight gain was similar in all groups, indicating that the treatments did not affect SHR growth.

The extent of structural damage in the left ventricle, evaluated by morphometric analysis as the amount of fibrotic tissue, was significantly reduced by treatment with Compound B: the volume fraction of replacement connective tissue was decreased by 42% in the rats treated with Compound B compared with the vehicle control (p< 0.01).

A variety of pharmaceutical dosage forms are suitable for NEP administration, preferably for oral or parenteral administration, although mechanical delivery systems such as transdermal dosage forms are also contemplated.

The typical daily dosage of the NEP inhibitor for treatment or prevention of LVH is about 0.3 mg/kg to about 100 mg/kg of mammalian weight per day administered in single or divided doses. The exact dose of any NEP inhibitor to be administered is determined by the attending clinician and is dependent on the potency of the compound administered, the age, weight, condition and response of the patient.

Generally, in treating humans suffering from LVH or in preventing LVH, the NEP inhibitors of this invention can be administered in dosage ranges of about 10 to about 1000 mg NEP inhibitor per dose given 1 to 4 times a day.

Typical oral formulations for drugs used in this invention include tablets, capsules, syrups, elixirs and suspensions. Typical injectable formulations for drugs used in this invention include solutions and suspensions.

Where NEP inhibitors are administered in combination with other antihypertensive agents, including ACE inhibitors, calcium antagonists, diuretics and beta-adrenergic blockers, the combinations can be administered from a single pharmaceutical composition which combines the actives in a pharmaceutically acceptable carrier, or the drugs may be administered separately. That is, a patient can undergo parallel courses of treatment with the two different actives; simultaneous administration of dosage forms is not required. Since the methods of this invention relating to the combinations comprise administering two different drugs, any suitable combination of dosage forms can be used, e.g. oral NEP inhibitor / oral antihypertensive agent or injectable NEP inhibitor / oral antihypertensive agent.

Since the present invention relates to methods of treating or preventing LVH with a combination of active ingredients, i.e. an NEP inhibitor and an antihypertensive agent, wherein said active ingredients may be administered separately, the invention also relates to combining separate pharmaceutical compositions in kit form. That is, a kit which combines two separate units, an NEP pharmaceutical composition and an antihypertensive composition (particularly an ACE inhibitor or a calcium antagonist composition), in one package is contemplated. The kit form is particularly advantageous when the separate components must be administered in different dosage forms (e.g. oral and parenteral) or are administered at different dosage intervals.

Claims

1. A method of treating left ventricular hypertrophy comprising administering an effective amount of a neutral endopeptidase inhibitor to a mammal in need of such treatment.

2. A method of claim 1 wherein the neutral endopeptidase inhibitor is selected from the group consisting of:

N-[N-[(L)-[1-[(2,2-dimethyl-1 ,3-dioxolan-4-yI)-methoxy]carbonyl]- 2-phenylethyl]-L-phenylalanyl]-β-alanine;

N-[N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyl]-β-alanine; N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl ester;

N-[2-mercaptomethyl-3-(2-methylphenyl)propionyl]-methionine; N-[2(S)-mercaptomethyl-3-(2-methylphenyl)propanoyl]-(S)- isoserine;

N-(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy- β-alanine;

N-[1-[[1 (S)-benzyloxycarbonyl-3-phenylpropyl]amino]cyclopentyl- carbonyl]-(S)-isoserine;

N-[1-[[1 (S)-carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]- (S)-isoserine;

1,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis- (S)-isoserine;

1,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis-

(S)-methionine;

N-(3-phenyl-2-(mercaptomethyl)-propionyl)-(S)-4-(methyl- mercapto)methionine;

N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid; N-[2-mercaptomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;

N-[1-(2-carboxy-4-phenylbutyl)-cyclopentanecarbonyl]-(S)- isoserine;

N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl ester; 3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-ε- caprolactam;

SQ 28603; UK 69578; thioφhan; retro-thiorphan;

phosphoramidon; and SQ 29072; or a pharmaceutically acceptable ester thereof.

3. A method of claim 1 wherein the neutral endopeptidase inhibitor is N-[2-acetylthiomethyl-3-(2-methylphenyl)-propionyl]- methionine ethyl ester or N-[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yI)- methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine.

4. A method of preventing left ventricular hypertrophy comprising administering an effective amount of a neutral

endopeptidase inhibitor to said mammal in need of such treatment.

5. A method of claim 4 wherein the neutral endopeptidase inhibitor is selected from the group consisting of

N-[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]carbonyl]- 2-phenylethyl]-L-phenylalanyl]-β-alanine;

N-[N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyl]-β-alanine;

N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyI]-methionine ethyl ester;

N-[2-mercaptomethyl-3-(2-methylphenyl)propionyI]-methionine;

N-[2(S)-mercaptomethyl-3-(2-methylphenyl)propanoyl]-(S)- isoserine;

N-(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy- β-alanine;

N-[1-[[1(S)-benzyloxycarbonyl-3-phenylpropyI]amino]cyclopentyl- carbonyl]-(S)-isoserine;

N-[1-[[1(S)-carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]-

(S)-isoserine;

1,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis- (S)-isoserine; 1 ,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis- (S)-methionine;

N-(3-phenyl-2-(mercaptomethyl)-propionyl)-(S)-4-(methyl- mercapto)methionine;

N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;

N-[2-mercaptomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;

N-[1-(2-carboxy-4-phenylbutyl)-cyclopentanecarbonyI]-(S)- isoserine;

N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl ester;

3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-ε- caprolactam;

SQ 28603; UK 69578; thiorphan; retro-thiorphan;

phosphoramidon; and SQ 29072; or a pharmaceutically acceptable ester thereof.

6. A method of claim 4 wherein the neutral endopeptidase inhibitor is N-[2-acetylthiomethyl-3-(2-methylphenyl)-propionyl]- methionine ethyl ester or N-[N-[(L)-[1-[(2,2-dimethyl-1 ,3-dioxolan-4-yl)- methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine.

7. A method of treating or preventing left ventricular

hypertrophy comprising administering to a mammal in need of such treatment an effective amount of a combination of an neutral

endopeptidase inhibitor and an antihypertensive agent.

8. A method of claim 7 wherein the antihypertensive agent is an angiotensin converting enzyme inhibitor or a calcium channel blocker.

9. A method of claim 8 wherein the neutral endopeptidase inhibitor is selected from the group consisting of

N-[N-[(L)-[1-[(2,2-dimethyl-1 ,3-dioxolan-4-yl)-methoxy]carbonyl]- 2-phenylethyl]-L-phenylalanyI]-β-alanine; N-[N-[(L)-1-carboxy-2-phenylethyi]-L-phenylalanyl]-β-alanine; N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl ester;

N-(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy- β-alanine;

N-[1-[[1(S)-carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]- (S)-isoserine;

1,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis-

(S)-methionine;

N-(3-phenyI-2-(mercaptomethyl)-propionyl)-(S)-4-(methyl- mercapto)methionine;

N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid; N-[2-mercaptomethyl-3-phenyl-propionyI]-3-aminobenzoic acid;

N-[1-(2-carboxy-4-phenylbutyl)-cyclopentanecarbonyl]-(S)- isoserine;

N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl ester;

3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-ε- caprolactam;

SQ 28603; UK 69578; thiorphan; retro-thiorphan; phosphoramidon; and SQ 29072; or a pharmaceutically acceptable ester thereof.

10. A pharmaceutical composition comprising a combination of an antihypertensive agent and a neutral endopeptidase inhibitor in an amount effective to treat or prevent left ventricular hypertrophy in a pharmaceutically acceptable carrier.

11. A composition of claim 10 wherein the antihypertensive agent is an angiotensin converting enzyme inhibitor or a calcium antagonist and the neutral endopeptidase inhibitor is selected from the group consisting of

N-[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]carbonyl]-

2-phenylethyl]-L-phenyialanyl]-β-alanine;

N-[N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyI]-β-alanine;

N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl ester;

N-[2-mercaptomethyl-3-(2-methylphenyl)propionyl]-methionine;

N-[2(S)-mercaptomethyl-3-(2-methylphenyl)propanoyl]-(S)- isoserine;

N-(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy- β-alanine;

N-[1-[[1 (S)-benzyloxycarbonyI-3-phenylpropyl]amino]cyclopentyl- carbonyl]-(S)-isoserine;

1 ,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis- (S)-isoserine;

1 ,1'-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis- (S)-methionine;

N-(3-phenyl-2-(mercaptomethyl)-propionyl)-(S)-4-(methyl- mercapto)methionine;

N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;

N-[2-mercaptomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;

N-[1-(2-carboxy-4-phenylbutyl)-cyclopentanecaιtjonyl]-(S)- isoserine;

N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl ester;

3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-ε- caprolactam;

SQ 28603; UK 69578; thioφhan; retro-thiorphan; phosphoramidon; and SQ 29072; or a pharmaceutically acceptable ester thereof.

12. A kit comprising in separate containers in a single package pharmaceutical compositions for use in combination to treat left ventricular hypertrophy in a mammal which comprises in one container a pharmaceutical composition comprising an antihypertensive agent and in a second container a pharmaceutical composition comprising a neutral endopeptidase inhibitor.