US 20130203827A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0203827 A1 Sucharov et al. (43) Pub. Date: Aug. 8, 2013 (54) POLYMORPHISMS IN THE PDE3A GENE Related U.S. Application Data (75) Inventors: Carmen Sucharov, Superior, CO (US); (60) Pygal application No. 61/241,730, filed on Sep. Michael Bristow, Englewood, CO (US); Matthew Taylor, Denver, CO (US); Dobromir Slavov, Aurora, CO (US) Publication Classification (73) Assignee: The Regents of the University of ( 51) Int. Cl. Colorado. a Body, Denver, CO (US) CI2O I/68 (2006.01) (52) U.S. Cl. (21) Appl. No.: 13/395,311 CPC .................................... CI2O I/6883 (2013.01) USPC ............................... 514/392:435/6.11:506/9 (22) PCT Filed: Sep. 13, 2010 (57) ABSTRACT (86). PCT No.: PCT/US10/48629 Embodiments of the invention are directed to identifying or S371 (c)(1), treating a patient that would benefit from phosphodiesterase (2), (4) Date: Nov. 5, 2012 inhibitor therapy. 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SeAe WNAU W9BO Patent Application Publication Aug. 8, 2013 Sheet 8 of 8 US 2013/0203827 A1 s s s SN o Sieme S9. |SeAe WNUlu W9C US 2013/0203827 A1 Aug. 8, 2013 POLYMORPHSMS IN THE PDE3A GENE PDE3A levels and reduced response to treatment, commonly 0001. This application claims benefit of priority to U.S. known as pharmacological tolerance. Provisional Application Ser. No. 61/241,730, filed Sep. 11, 0007 Certain embodiments are directed to methods for 2009, the entire contents of which are hereby incorporated by treating a patient with heart failure comprising treating the reference. patient with an effective amount of a PDE3A inhibitor after the patient is determined to be homozygous for an insertional BACKGROUND OF THE INVENTION polymorphism comprising an Evi-1 binding site in the pro moter of the phosphodiesterase type 3A (PDE3A) gene. The 0002 I. Field of the Invention Evi-1 insertion polymorphism refers to a nucleic acid 0003 Embodiments of this invention are directed gener sequence inserted in the promoter of PDE3A comprising a ally to biology, molecular genetics, and medicine. Certain nucleic acid sequence that is 85,90, 95, 98, to 100% identical embodiments are directed to identifying a patient that would to the nucleic acid sequence of SEQ ID NO:2. In certain benefit from phosphodiesterase inhibitor therapy. aspects the insertional polymorphism comprises an ectotro 0004 II. Background pic viral integration site-1 protein (Evi-1) binding site. In a 0005 Cardiac hypertrophy in response to an increased further aspects the Evi-1 binding site comprises a 5, 10, 15 to workload imposed on the heart is a fundamental adaptive 10, 15, 25, 29 nucleotides or more, including all values and mechanism. It is a specialized process reflecting a quantita ranges there between of the nucleotide sequence of SEQID tive increase in cell size and mass (rather than cell number) as NO:2. the result of any, or a combination of neural, endocrine or 0008. In certain aspects a PDE3A inhibitor is aminone, mechanical stimuli. When heart failure occurs, the left ven cilostazol, milrinone, quaZinone, SiguaZodan, trequinsin, or tricle usually is hypertrophied and dilated and indices of enoximone. In a further aspect the PDE3A inhibitor is enoxi systolic function, such as ejection fraction, are reduced. mone. The PDE3A inhibitor can be administered at a dose of Clearly, the cardiac hypertrophic response is a complex Syn 1, 5, 10, 15.20, 25, 30,35, 40, 45, 50 mg to 40, 50, 60, 70,80, drome and the elucidation of the pathways leading to cardiac 90, 100, or 150 mg, including all values and ranges there hypertrophy and systolic dysfunction will be beneficial in the between. In certain aspects the PDE3A inhibitor is adminis treatment of heart disease resulting from various stimuli. tered 1, 2, 3, 4, 5 or more times a day, a week, or a month, There remains a need for additional treatments for patients including all values and ranges there between. suffering from heart failure. 0009 Certain embodiments are directed to methods for evaluating a heart failure patient comprising analyzing a bio SUMMARY OF THE INVENTION logical sample from the patient for the presence of an ecto 0006 Chronic activation of the f-Adrenergic Receptor tropic viral integration site-1 protein (Evi-1) binding site (B-AR) can have deleterious effects on the heart, and animal deletion or insertional polymorphism in the promoter of one models over-expressing the B-AR develop heart failure. In the or more phosphodiesterase type 3A (PDE3A) genes of the classical B-AR pathway, activation of the receptor results in patient, wherein a patient determined to be homozygous for increased cyclic AMP (cAMP) levels. However, B-ARs are the insertional polymorphism is responsive to PDE3A inhibi desensitized in the failing heart and cAMP levels are tor therapy. decreased. Phosphodiesterase 3A (PDE3A) hydrolyzes 0010. In certain aspects the methods described herein can cAMP in certain Subcellular compartments in cardiac myo further comprise obtaining a biological sample from the cytes, regulating cAMP levels and Subsequent protein kinase patient. A biological sample can be a blood sample, a buccal A mediated cell signaling. Since cAMP is reduced in certain Smear, a tissue sample, or a primary culture of Somatic cells important cardiac myocyte Subcellular compartments such as from the patient. In certain aspects analyzing the sample the microdomain occupied by phospholamban and SR Ca2+ comprises performing nucleic acid sequencing, restriction ATPase, it has been postulated that PDE3A inhibition will digestion, allele-specific nucleic acid amplification, single benefit the heart failure clinical syndrome by restoring cAMP Stranded conformational polymorphism analysis, or allele levels in the phospholamban microdomain/suncellular com specific hybridization analysis. The methods described partment. However, a recently published large clinical trial herein can further comprising preparing a report containing showed benefits of a PDE3 inhibitor on only a subset of information regarding the genotype of one or more PDE3A patients. Here we show that the promoter region of PDE3A genes of the patient. In a further aspect the patient has symp contains a 29 nucleotide insertional polymorphism (SEQID toms of or has been diagnosed with heart failure. In still a NO:2) that renders the PDE3A promoter non-responsive to further aspect the patient is determined to be homozygous for increased cAMP levels. The inventors demonstrate that the insertional polymorphism in the PDE3A gene and is sub increased cAMP levels in the cardiac cell, mediated by iso sequently treated with a PDE3A inhibitor. proterenol, a cAMP analogue or PDE3A inhibition, results in 0011. In certain embodiments the patient is determined increased promoter activity when the promoter construct not to be homozygous for the insertional polymorphism in the lacks the 29 nucleotide sequence. However, promoteractivity PDE3A gene and is subsequently treated with a non-PDE3A does not go up when the sequence is present. Similarly, inhibitor treatment. mRNA levels for heart failure patients containing the inser 0012. Further embodiments are directed to methods for tion polymorphism were not up-regulated in response to evaluating a patient having or at risk of developing heart PDE3A inhibition treatment while mRNA levels for patients failure for responsiveness to phosphodiesterase inhibitor lacking this region were up-regulated 2-fold in response to therapy comprising obtaining a determination of the patients treatment. These results indicate that only patients who are genotype for an insertional polymorphism in a phosphodi homozygous for the insertion should be treated with a esterase type 3A (PDE3A) gene, wherein homozygosity for PDE3A inhibition. Inhibition of PDE3A in other patients will the insertional polymorphism is predictive of responsiveness create a positive feedback loop that will result in increased to phosphodiesterase inhibitor therapy for treating heart fail US 2013/0203827 A1 Aug. 8, 2013 ure in the patient. In certain aspects the nucleic acid sequence TGACTAGCTG). A second primer can comprise the nucleic insertion comprises an ectotropic viral integration site-1 pro acid sequence of SEQID NO:7 (GCCAAAAGGAGATCCT tein (Evi-1) binding site. In a further aspect the Evi-1 binding TGAGAT). site has a nucleic acid sequence that is 85,90, 95, 98, or 100% 0016 Certain embodiments are directed to a nucleic acid identical to the nucleic acid sequence of SEQID NO:2. The probe that specifically hybridizes to a phosphodiesterase type methods can include obtaining a patient history. In certain 3A nucleic acid comprising nucleotides 274 to 302 of SEQID aspects this includes determination of the patient’s genotype NO:3 or a nucleic acid that is 85,90, 95, 98, or 1005 identical for an insertional polymorphism in a phosphodiesterase type to SEQID NO:2.