Compositions and methods for the identification and treatment of patients at risk for heart failure

Background of the Invention

Congestive heart failure is a complex clinical syndrome characterized by diminished cardiac contractile 10 function and decreased exercise tolerance. Despite advances in medical therapy, 40% of patients die within one year of diagnosis and 70% are dead at five years.

A number of clinical predictors of survival in heart failure have been identified including age, decreased cardiac 15 output and high blood pressure. However, clinical parameters alone have been of limited value in predicting survival in patients with congestive heart failure. It also appears that the genetic background of the individual plays a role. Accordingly, there is a need to identify genetic markers which 20 are associated with improved or decreased survival in patients with heart failure.

The adenosine monophosphate deaminase 1 (AMPD1) gene is the predominant member of the AMPD multi-gene family expressed in adult cardiac myocytes and adult skeletal muscle.

Approximately 20% of Caucasians and African Americans are heterozygous for a single mutant allele, AMPD1(+/-), which

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- 2 specifies a nonsense mutation in the second coding exon of this gene. This mutation leads to premature peptide chain termination and production of truncated peptide which is catalytically inactive. Deficiency in this enzymatic activity 5 in myocytes resulting from the AMPD1 (+/-) genotype may have one of several metabolic consequences. A marked decrease in AMPD1 activity in myocytes may lower the adenylate energy charge in the myocyte by limiting deamination of AMP. A partial reduction in the activity of this enzyme may cause the 10 myocyte to produce more adenosine following ATP catabolism.

Increased adenosine production may be beneficial as adenosine is known to have potent cardioprotective effects. In patients with congestive heart failure, the residual cardiac myocytes experience greater metabolic demands; therefore, a reduction in 15 AMPD activity may lead to increased adenosine production which could improve cardiac function.

Accordingly, compositions and methods which inhibit AMPD activity are desired. Further, methods of identifying patients who are likely to develop congestive heart failure and 20 identifying those who may benefit from a cardiac transplant are also desired.

Summary of the Invention

AMP deaminase activity has now been found to influence the clinical outcome in patients with heart failure. Patients with a mutant allele of the AMP deaminase 1 gene (AMPD1) have been found to have a significant prolongation of life if they develop congestive heart failure compared to patients without the mutant allele. Further, it has been found that patients who inherit at least one copy of this mutant allele, compared to patients who are homozygous for the wild-type allele, exhibit a longer duration of heart failure symptoms before requiring referral for cardiac transplant. Accordingly, it appears that one or more of the metabolic consequences of reduced AMPD activity provides for improved prognostic and survival advantages for individuals who inherit this AMPD1 mutant allele and subsequently develop heart failure. It is

3A

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• · · · • · · • · • · thus believed that reduction of AMPD activity in individuals suffering from congestive heart failure who are homozygous for the wild-type allele will improve the prognostic and survival advantages in these individuals.

Accordingly, an object of the present invention is to provide compositions and methods for inhibiting the AMPD1 enzyme and related enzymes from the AMPD1 gene family for the treatment of patients with congestive heart failure.

Another object of the present invention is to provide methods of predicting clinical outcome of patients suffering from congestive heart failure. The present invention may also provide a method for identifying those patients most likely to benefit from a cardiac transplant which comprises identifying patients with a mutant allele of the AMPD1 gene.

Accordingly, in one aspect, the present invention provides a method of predicting clinical outcome of patients suffering from congestive heart failure including identifying patients having a mutant allele of an adenosine monophosphate deaminase 1 gene wherein said mutant allele encodes a truncated peptide which is catalytically inactive.

In a further aspect, the present invention provides a method of increasing survival time in patients suffering from congestive heart failure including administering to patients suffering from congestive heart failure a composition which inhibits adenosine monophosphate deaminase 1.

Detailed Description of the Invention

Congestive heart failure (CHF) is a common disorder which is often lethal in a very short period of time. Effective therapeutic options for treatment of this disease have been limited. However, it has now been found that in patients with CHF, the AMPD1 mutant allele is associated with prolonged survival after the onset of CHF symptoms.

Clinical data were collected on 132 consecutive patients (mean age 52.8 + 11.2 years) with advanced congestive heart failure. The patients were evaluated and a clinical database constructed which included medical history, physical examination, assessment of ventricular function and hemodynamics, and measurement of exercise capacity. Left ventricular fraction (LVEF) was determined by standard echocardiaography or gatedblood pool analysis (MUGA). Cardiac output and hemodynamics were determined by the thermodilution technique in the cardiac catheterization

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• 6i

3B laboratory. Exercise capacity was determined by a standard maximal exercise testing protocol with breath-by-breath, on-line expiratory gas analysis. These data were obtained at the time of initial evaluation and all patients were judged to be on optimal medical therapy for heart failure, including angiotensin-

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- 4 converting enzyme inhibitors, diuretics and digoxin at the time of this study. These patients were compared with two different control groups which included 91 and 416 normal volunteers without cardiovascular disease, respectively.

Genomic DNA was prepared from peripheral white blood cells from each subject. AMPDl genotype was determined using a PCR based assay allele-specific oligonucleotide detection assay. In brief, genomic DNA was extracted from whole blood by rapid lysis technique and PCR-amplified in IX PCR buffer and

1.25 U Tag polymerase.

PCR primers were as follows :

AMP 5'

GCAATCTACATGTGTCTACC

ID

NO:

1) and

AMP

3'ATAGCCATGTTTCTGAATTA (SEQ ID NO: 2).

Thirty cycles seconds (denaturation), 55°C for 30

72°C for 30 seconds (extension) were performed. PCR products were denatured and slot blotted in duplicate onto a nylon membrane along with positive and negative controls. The filter was hybridized with kinase labeled oligonucleotides representing the normal or the mutant sequence at nucleotide 34 of the cDNA. DNA from individuals heterozygous for the mutation at nucleotide 34 hybridized with both normal and mutant oligonucleotides.

The frequencies of the AMPDl (+/+), AMPDl (+/-) and AMPDl (-/-) genotypes in patients with CHF and control groups were compared. Genotype frequencies were compared using Fischer's exact test between cases and controls. The mean and median duration of heart failure symptoms between the AMPDl (+/+) and AMPDl (+/-)(-/-) genotypes were determined. Analyses were performed to evaluate whether the duration of disease differed between the AMPDl ( + / + ) and the AMPDl ( + /-) (-/-) genotypes using survival analysis techniques. Both KaplanMeier and Proportional Hazards models were fitted (Daniel, W.W. Applied Non-parametric Statistic, Second Edition. Boston: PWSKent Publishing Company (1990); and Shapiro, S.S. and Wilk, M.B. An Analysis of Variance Test for Normality (Complete Subjects). Biometrika 1965; 52:591-611) . Only one CHF subject was homozygous for the mutant AMPDl allele. For purposes of this study, the single AMPDl (-/-) individual was included in

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- 5 the groups of heterozygous for the mutant allele; exclusion of this individual from the analysis did not alter the results of any statistical analyses. All continuous variable were calculated as mean + one standard deviation (S.D.)

The onset of heart failure symptoms was defined by the first hospital admission for the diagnosis of CHF. This date was established and verified by either retrospective chart review, direct patient interviews, and/or discussion with the referring physician by a single investigator blinded to the genotype of the patient. For the survival analyses, heart failure duration was defined as the duration of disease symptoms from first hospital admission for CHF to time of either cardiac transplantation, death, or the end of the study observation period. In addition, total disease duration was adjusted for age and sex prior to proportional hazards of Kaplan-Meier analysis by linear regression. Significance was established if the null hypothesis could be rejected at p value &lt; 0.05.

At the time of referral for cardiac transplantation evaluation, the mean age of the 132 CHF patients was 52.8 + 11.2 years. The mean LVEF was 19.7% + 6.7% and the mean peak VO₂ max was 13.9 + 4.9 ml/kg/minute. These values are consistent with New York Heart Association functional class IIIV heart failure symptomatology. Etiologies of CHF included: 1) coronary artery disease (n=69); 2) idiopathic cardiomyopathy (n=48); and 3) other (N=15, 4 valvular; 2 infiltrative; 3 congenital; 2 myocarditis; 4 alcohol). This distribution of clinical characteristics is very similar to most referral populations evaluated for cardiac transplantation (Loh et al. Card. Rev. 1, 218-227 (1993). No Asians were among these patients, an important exclusion since the AMPD1 mutant allele does not appear to be present in this population (Morisaki et al. Proc. Natl Acad. Sci. USA 1992 89, 6457-6461). Of the 132 patients enrolled in the study, ill were homozygous for the wild-type allele (+/+), 20 were heterozygous (+/-), and one individual was homozygous for the mutant allele (-/-) .

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- 6 At the time of the initial evaluation, all patients exhibited severe, end-stage heart failure physiology (see Table 1). LVEF, cardiac index, pulmonary capillary wedge pressure, VO₂ max and pulmonary vascular resistance were not significantly different between CHF patients with the (+/+) versus ( + /-) (-/-) genotypes.

TABLE 1

Clinical Characteristics of AMPD1 (+/+) vs AMPD1 (+/-)(-/-)

Patients with Heart Failure

<td></td><td> AMPDl (+/-)(-/-) (n=21)</td><td> AMPDl (+/+) (n=lll)</td>

<td> Age (yrs)</td><td> 56.8 + 7.1</td><td> 52.1+11.6</td>

<td> Gender Male</td><td> 16 (79%)</td><td> 97 (89%)</td>

<td> Female</td><td> 5 (21%)</td><td> 14 (11%)</td>

<td> Etiology CAD</td><td> 8 (38%)</td><td> 55(50%)</td>

<td> IDCMP</td><td> 11 (52%)</td><td> 38 (34%)</td>

<td> Other</td><td> 2 (10%)</td><td> 18 (16%)</td>

<td> LVEF (%)</td><td> 18.9 + 7.4</td><td> 19.9+6.9</td>

<td> Cardiac index (l/min/m²⁾</td><td> 1.8 + 0.6</td><td> 1.8 + 0.8</td>

<td> PCWP (mmHg)</td><td> 21.6 + 10.9</td><td> 22.0 + 9.0</td>

<td> PVR (Wood units)</td><td> 2.9+2.1</td><td> 2.9 + 2.4</td>

<td> V0₂ max (ml/kg/min)</td><td> 15.1+6.7</td><td> 13.7 + 4.6</td>

<td> Symptom duration (yrs)</td><td> 7.6+6.5</td><td> 3.2 + 3.6*</td>

*p &lt; 0.001 = AMPDl ( + /-) (-/-) vs AMPDl ¢ + / + ) all other comparison p&gt;0.05 all values - mean + S.D.

<td> AMPDl</td><td> (+/ + )</td>

<td> AMPDl</td><td> ( + /-)</td>

<td> AMPDl</td><td> (-/-)</td>

<td> CAD</td><td></td>

<td> IDCMP</td><td></td>

<td> PCWP</td><td></td>

<td> PVR</td><td></td>

<td> vo₂</td><td></td>

homozygous for wild-type allele heterozygous for mutant allele homozygous for mutant allele : coronary artery disease : idiopathic dilated cardiomyopathy : pulmonary capillary wedge pressure : pulmonary vascular resistance : maximal oxygen consumption

The time from the first hospital admission for CHF symptoms to the clinical endpoint of death or cardiac

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- 7 transplantation was markedly different between AMPDl ( + /-) (-/-) and AMPDl (+/+) patients, i.e. 7.6+6.5 years in AMPDl (+/-) or (-/-) versus 3.2 + 3.6 years in AMPDl ( + / + ) (p&lt;0.0001). There was a trend for patients with a mutant AMPDl allele to be older than the AMPD1 (+/+) homozygotes at the time of initial referral for cardiac transplantation evaluation. The mean age of AMPDl (+/-) (-/-) patients was 56.8 + 7.1 years while the mean age of AMPDl ( + / + ) patients was 52.1 + 11.6 years (p=0.07; t-test assuming unequal variances).

The difference in time or progression to end-stage CHF symptomatology and referral for transplantation evaluation is not explained by differences in etiology of heart failure or in gender between the genotypes (see Table 1). Further blinded chart reviews detected no differences in access to medical care, prescribed medical regimens or patient compliance.

However, Kaplan Meier analysis demonstrated that individuals with one or two mutant AMPDl alleles live without transplantation for a significantly longer time after the first hospitalization for CHF symptoms than do patients homozygous for the wild-type allele. The proportional hazards model indicates a risk ratio of 4.65 (95% confidence interval: 1.67, 12.99) for age and sex-adjusted disease durations.

The frequency of AMPDl genotype in heart failure patients was also determined. CHF patients were divided into two groups, those presenting within 5 years of the first hospitalization for CHF symptoms, and those presenting greater than 5 years after the first hospitalization for CHF symptoms. For the group that presented within 5 years from the first hospitalization for CHF, 7 of the 97 (16.7%) inherited the mutant AMPDl allele. In the group that presented 5 years or later after the first hospitalization for CHF, 14 of the 35 inherited the mutant AMPDl allele (43%). Using the MantelHaenstel method of analysis, a patient carrying the AMPDl mutant allele has an 8.6 odds ratio, relative to an individual homozygous for the wild-type allele, of living 5 or more years with CHF before requiring cardiac transplantation or dying.

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- 8 When the AMPD1 genotype frequency of these two subgroups of CHF patients was compared to the group of normal volunteers, the CHF patients who presented less than 5 years after hospitalization for CHF were found to have a lower frequency of the mutant allele (16.7%) compared to the control population (25.3%). In contrast, CHF patients who presented 5 years or longer after the initial hospitalization for CHF demonstrated a trend towards a higher frequency of the mutant allele (43%) compared to the control population (25.3%). This finding is consistent with the AMPD1 mutant allele being a marker which identifies patients have a milder or more prolonged course of heart failure before requiring referral for consideration of cardiac transplantation.

Accordingly, in an embodiment of the present invention, a method for the identification of patients at risk for heart failure is provided. In this method, patients are tested to determine whether they carry the mutant AMPD1 allele. Those having a mutant allele of the AMPD1 gene have a significant prolongation of life if they develop congestive heart failure. Thus, those patients not carrying this marker are at a higher risk for heart failure-and may require closer monitoring and more aggressive clinical interventions that those carrying the mutant allele. By identifying a patient's AMPD1 genotype, it is possible to select the most appropriate clinical treatment plan. Patients at the highest risk for heart failure can be identified and treated accordingly. The present invention thus provides a means for predicting and managing clinical outcome for patients with heart failure.

The present invention also provides a method and compositions for the treatment and, more specifically, the prolongation of survival in patients suffering from congestive heart failure who are homozygous for wild-type AMPD1. In humans, AMPD1 is the dominant member of a multigene family expressed in cardiac myocytes. Cardiac muscle has a very low level of AMPD enzyme activity; normal cardiac muscle has only 2% of the activity found in skeletal muscle. Prior studies have demonstrated that skeletal muscle from patients homozygous

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- 9 for the AMPD1 (-/-) allele have residual AMPD activity comparable to that found in normal heart. This low level of residual AMPD activity is associated with increased adenosine production following ATP catabolism in skeletal muscle.

Adenosine is a potent cardioprotective agent that enhances coronary flow, suppresses arrhythmias, induces the ischemic preconditioning response and suppresses cytokine production. These cardioprotective effects of enhanced adenosine production are believed to lead to increased survival in heart failure patients who carry at least a single copy of the mutant AMPD1 allele. Compositions and methods which enhance local adenosine production as a result of reduced AMPD1 activity would therefore be useful to patients with heart failure. Accordingly, it is believed that inhibitors of the AMPD enzyme will ..be useful for the treatment. and more specifically in the prolongation of survival in patients suffering from CHF. Further, even as little as a 50% reduction in the activity of the enzyme is expected to be beneficial.

Adenosine itself has not proven desirable for treatment of heart failure due to its very short in vivo half life (seconds). Further, adenosine must be administered by selective infusion into the coronary artery and often results in angina. Accordingly, inhibitors of the AMPD enzyme are preferred for the treatment of heart failure. The present invention provides compositions and methods for inhibiting the AMPD enzyme for the treatment of heart failure.

In accordance with the present invention, an effective amount of an AMPD inhibitor is administered to a patient with heart failure. By effective amount it is meant a concentration of a composition of the present invention which reduces the activity of the AMPD enzyme by at least 50%. Reduction of enzyme levels to 50% appears to be non-toxic based upon the general good health enjoyed by individuals heterozygous for the mutant AMPD1 allele. Concentrations of inhibitors to be administered can be routinely determined by those of skill in the art based upon the present disclosure and

V what is well known in the art. The inhibitors of the present invention may be administered in a pharmaceutically acceptable carrier. Suitable carriers are well known in the art and are selected in accordance with the intended route of administration and the standard pharmaceutical practice. For example, 5 formulations for intravenous administration may include sterile aqueous solutions.

The sequence of AMPD1 is described in Sabina et al, Characterization of the human and rat myoadenylate deaminase genes, J. Biol. Chem. 265 (lb) 9423-9433(1990) the disclosure of which is incorporated herein by reference.

Throughout this specification the word comprise, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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- 11 SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Trustees of the University of Pennsylvania,

Edward Holmes, Judith Swain (ii) TITLE OF INVENTION: Compositions and Methods for the Identification and Treatment of Patients at Risk for Heart Failure (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Jane Massey Licata, Esq.

(B) STREET: 66 E. Main Street (C) CITY; Marlton (D) STATE: NJ (E) COUNTRY: USA (F) ZIP: 08053 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE (B) COMPUTER: IBM 486 (C) OPERATING SYSTEM: WINDOWS FOR WORKGROUPS (D) SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: not assigned (B) FILING DATE: Herewith (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/027,582 (B) FILING DATE: October 3, 1996 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Jane Massey Licata (B) REGISTRATION NUMBER: 32,257

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PCT/US97/17986 (C) REFERENCE/DOCKET NUMBER: PENN-0556 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (609) 779-2400 (B) TELEFAX: (609) 810-1454 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

GCAATCTACA TGTGTCTACC 2 0 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

<td> (A)</td><td> LENGTH: 20</td><td></td>

<td> (B)</td><td> TYPE: Nucleic</td><td> Acid</td>

<td> (C)</td><td> STRANDEDNESS :</td><td> Single</td>

<td> (D)</td><td colspan="2"> TOPOLOGY: Linear</td>

(iv) ANTI-SENSE: No (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATAGCCATGT TTCTGAATTA