WO2019191010A1

WO2019191010A1 - Personalized treatment method for congestive heart failure

Info

Publication number: WO2019191010A1
Application number: PCT/US2019/023946
Authority: WO
Inventors: Zhenhuan Zhang; Tien-Li Lee; Yu-Hsing Tu; James Lee
Original assignee: Aardvark Therapeutics Inc.; Tulex Pharmaceuticals
Priority date: 2018-03-27
Filing date: 2019-03-25
Publication date: 2019-10-03

Abstract

There is disclosed a personalized treatment method of preventing a worsening and treating congestive heart failure, comprising testing for susceptibility of the treatment by determining the presence of SNP (single nucleotide polymorphisms) mutations in at least two of four biomarkers selected from the group consisting of G-protein β-3 (GNB3) subunit genotype, endothelial nitric oxide synthetase (NOS3), glutathione S-transferase μ type 1 (GSTM1), and phosphor diesterase-5 (PDE5) in a particular patient; and administering a sustained release pharmaceutical composition of at least one hydralazine compound or a pharmaceutically acceptable salt thereof. There is further disclosed an extended release formulation of isosorbide dinitrate/mononitrate and hydralazine and excipients to provide sustained blood levels for at least 10 hours to allow bid (twice daily) dosing.

Description

Personalized Treatment Method for Congestive Heart Failure

Technical Field

The present disclosure provides a personalized treatment method of preventing a worsening and treating congestive heart failure, comprising testing for susceptibility of the treatment by determining the presence of SNP (single nucleotide polymorphisms) mutations in at least two of four biomarkers selected from the group consisting of G-protein b-3 (GNB3) subunit genotype, endothelial nitric oxide synthetase (NOS3), glutathione S- transferase m type 1 (GSTMI), and phosphor diesterase-5 (PDE5) in a particular patient; and administering a sustained release pharmaceutical composition of at least one hydralazine compound or a pharmaceutically acceptable salt thereof. The present disclosure further provides an extended release formulation of isosorbide dmitrate/mononi irate and hydralazine and excipients to provide sustained blood levels for at least 10 hours to allow bid (twice daily) dosing.

Background

Congestive heart failure (CHF) is a clinical condition associated with cardiac and peripheral vascular abnormalities that lead to increased morbidity and mortality. Common etiologies leading to CHF include history of myocardial infarction, coronary artery disease, peripheral hypertension, valvular heart disease, cardiac arrhythmias (including atrial fibrillation), and cardiomyopathy. This syndrome is now a leading cause of hospitalization in individuals older than age 65 and affects over 40 million individuals globally.

Clinical observations and studies have revealed that, in general, patients having predominantly African ancestry have a poorer prognosis and proportionally greater risk of morbidity and mortality from CHF than comparable Caucasian (European descent) patients. Notably, it has been shown in general, that patients having predominantly African ancestry with hypertension and/or CHF demonstrate a relatively muted response to pharmaceutical agents (angiotensin converting enzyme (ACE) inhibitors and beta blockers) typically used as first-line treatments for Caucasian patients. Conversely, it has been observed that patients having predominantly African ancestry generally respond more strongly than comparable Caucasian patients when treated with hydralazine and an isosorbide nitrate (typically isosorbide dim irate) contemporaneously. There is currently a commercial pharmaceutical composition that leverages this effect for the treatment of CHF patients that self-identify as '‘black.” However the selection of patients to treat via reliance on racial self-identification is imprecise, given the multiracial composition of world populations, particularly in the U.S., and also incomplete, as non-black patients with certain physiologic characteristics and genotypes could also benefit from a treatment with the same drug combination. Racial self- identification is not a reliable method for discerning biologic and genomic differences among individuals in a population. Therefore, there is a need in the art for more accurate selection of patients that may benefit from a combination formulation of hydralazine and an isosorbide nitrate (dinitrate or mononitrate) based on objective biomarkers instead of racial self- identification.

G-protein b-3 (GNB3) subunit is a component of several G protein complexes, which plays a role in a-adrenergic signaling. The corresponding gene is composed of 11 exons and 10 introns, is located on chromosome 12 and has a common single nucleotide polymorphism at position 825 of the coding sequence (CDS) from the start codon within exon 10 (SNP ID number: rs5443 ), where the two possible nucleotide variations, cytosine (C) or thymine (T), are present to give rise to allele types of CT, TT, and CC. The T allele is associated with enhanced alpha2-adrenergic receptor intracellular signaling and linked to the risk of hypertension and low plasma renin.

Endothelial nitric oxide (NO) synthase (eNOS) is a source of vascular NO. The encoding gene OS3 is located in the 7q35-7q36 region of chromosome 7 and has a co mon T/C SNP at position -813 in the promoter region from the start codon(SNP ID number: r$2070744). The C allele is associated with reduction in the endothelial NO synthesis and affects vasomotility of the epicardial coronary arteries.

Glutathione S-transferases (GSTs) are a family of enzymes that catalyze the conjugation of glutathione with a wide variety of xenobiolies and drugs for detoxification. GST m type 1 (GSTMl), a GTS isoform, is encoded by the GSTM1 gene, which is located on chromosome lpl3.3. In a considerable proportion of different populations, however, the GSTMl gene is homozygously deleted (lack of the GSTMl gene, also called GSTMl null or GSTMl *0). This homozygous deletion polymorphism results in the complete absence of the corresponding enzyme activity, consequently causing the difference in susceptibility to and manifestation of a wide range of diseases, and variability in drug response, as compared to GSTMl gene carriers. NO signaling is a modulator of cardiovascular homeostasis and mainly mediated by cyclic guanosine monophosphate (cGMP) accumulation within the cells as a consequence of increased guanylate cyclase activity. However, cGMP bioavailability is limited by the hydrolytic activity of phosphodiesterases (PDE), most notably PDE5. Tire gene encoding PDE5 ( PDE5A gene) has a length of 4415 base pairs and is located on human chromosome 4 (4q26). At position -1255 from the start codon within the 5’ -flanking regulatory region of PDE5A gene, there is a co on functional G/T polymorphism (SNP ID number: rs3806808), which has been found to he a major contributor to the inhaled NO (iNO) -induced pulmonary' vascular resistance (PVR) decrease in CHF.

Additionally, existing approved combination formulations of hydralazine and isosorbide dinitrate for CHF treatment have a relatively short half-life in vivo, requiring admini tration of the oral drug typically at least 3 times per day. Patient compliance is reduced the more frequently patients are expected to self-administer medications. Hence, there is a need for a composition that reduces the frequency of daily dosing required for CHF patients. The present disclosure describes a sustained release formulation comprising hydralazine and isosorbide nitrates that is designed to be utilized with a genetic test to pre select patients most likely to benefit from such treatment.

Summary

Hie present disclosure provides a personalized treatment method of preventing a worsening and treating congestive heart failure, comprising testing for susceptibility of the treatment by determining the presence of SNP (single nucleotide polymorphisms) mutations in at least two of four biomarkers selected from the group consisting of G-protein b-3 (GNB3) subunit genotype, endothelial nitric oxide synthesase (NOS3), glutathione S- transferase m type 1 (GSTMl ), and phosphor diesterase-5 (PDE5) in a particular patient; and administering a sustained release pharmaceutical composition comprising at least one hydralazine compound or a pharmaceutically acceptable salt thereof. Preferably, the hydralazine compound is hydralazine hydrochloride. Preferably, the sustained release pharmaceutical composition further comprises an isosorbide dinitrate/mononitrate compound. Preferably, the GNB3 SNP is within exon 10 of GNB3 gene with a C-to-T mutation at position 825 from the start codon (SEQ ID NO. 1). Preferably, the NQS3 SNP is in a promoter region of eNOS gene having a T-to-C mutation at position -813 from the start codon (SEQ ID NO. 2) Preferably, the GSTM1 gene (SEQ ID NO. 3) is lacking. Preferably, the PDE5 SNP is at position -1255 from the start codon within the 5 -Hanking regulatory region of PDE5A gene with a G-to-T mutation (SEQ ID NO. 4).

The present invention provides methods for treating and preventing mortality and morbidity associated with heart failure, quality of life, and exercise tolerance in a genetically select group of patients with CHF. Specifically, a method to analyze blood or serum to detect the presence of biomarkers indicative of likely favorable therapeutic response to the combination formulation. Patients then could receive a therapeutically effective amount of at least one hydralazine compound or a pharmaceutically acceptable salt thereof, and at least one of isosorbide dinitrate and isosorbide mononitrate. The hydralazine compound is preferably hydralazine, or a pharmaceutically acceptable salt thereof, such as hydralazine hydrochloride. The pharmaceutical composition is delivered via an extended release fashion such that twice daily or fewer administrations are sufficient to convey therapeutic efficacy.

The present disclosure further provides am extended release formulation of isosorbide di nitrate/mononitrate and hydralazine and excipients to provide sustained blood levels for at least 10 hours to allow bid (twice daily) dosing. More specifically, the present disclosure provides an extended release formulation comprising from about 2 to about 30% (by weight) of isosorbine dinitrate, from about 4 to about 50% (by weight) of hydrazine hydrochloride, from about 20 to about 70% (by weight) of a polyhydroxylated polymeric material selected from the group consisting of methylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, polyvinyl acetate, microcrystalline cellulose, and combinations thereof. Preferably, the extended release formulation further comprises from about 5% to about 15% (by weight) of lactose. Preferably, the extended release formulation further comprises from about 2% to about 8% (by weight) of magnesium stearate. Preferably, the extended release formulation further comprises from about 5% to about 15% (by weight) of providone.

Preferably, the extended release formulation further comprises from about 2% to about 8% (by weight) of propylene glycol.

Detailed Description

The present disclosure provides a SNP (single nucleotide polymorphism)

identification process that simultaneously detect compilations of the presence of absence of four predictive SNPs. The disclosure provides a PCR detection kit incorporating a SNP compilation that forms a BFC (Binary Footprint Code) that allows for rapid identification of four predictive SNPs based on their SNP footprint. More specifically, the present disclosure provides a quadtyping (4t) method for clonal typing for four predictive SNPs to predict patient benefit for an extended release formulation comprising from about 2 to about 30% (by weight) of isosorbine dinilritrate, from about 4 to about 50% (by weight) of hydrazine hydroichloride, from about 20 to about 70% (by weight) of a polyhydroxylated polymeric material selected from the group consisting of methylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, polyvinyl acetate, microcrystalline cellulose, and combinations thereof; and comprising (a) providing forward primers and reverse primers for four SNPs (single nucleotide polymorphisms) selected from the group consisting of G-protein b-3 (GNB3) subunit genotype, endothelial nitric oxide synthesase (NOS3), glutathione S- transferase m type 1 (GSTM1), and phosphor diesterase-5 (PDE5) in a particular patient, (b) measuring the presence or absence of each SNP, and (c) determining patient likelihood of benefit if at least two of the four mutant SNPs are present.

The present disclosure provides a kit for predicting the presence or absence of four predictive SNPs to predict patient benefit an extended release formulation comprising from about 2 to about 30% (by weight) of isosorbine dinitritrate, from about 4 to about 50% (by weight) of hydrazine hydroichloride from about 20 to about 70% (by weight) of a

polybydroxylated polymeric material selected from the group consisting of methylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, polyvinyl acetate, microcrystalline cellulose, and combinations thereof: comprising providing forward primers and reverse primers for four SNPs (single nucleotide polymorphisms) selected from the group consisting of G-protein b-3 (GNB3) subunit genotype, endothelial nitric oxide synlhesase (NOS3), glutathione S-transferase m type I (GSTM1), and phosphor diesterase-5 (PDE5).

Table 1: Primer sequences and 5x primer mixes for 4-typing reactions

SNP Forward primer/s, 100 V, Reverse primer, 100 mM V, ul Water mM stock mΐ stock to add to 100 L

G-protein b- AGTCTGGGAGTCTG 2.5 GTTGGGAACCAAGG 2.5 95

3 (GNB3) GGACAA (SEQ ID NO. GGTACT (SEQ ID NO.

subunit B) 9)

endothelial CCTCCACTGCTTTTC 2.5 AGCCCACACTCTCA 2.5 95 nitric o ide AGAGG (SEQ ID NO. GTGACC (SEQ ID NO.

synthesase 10) 11)

(NOS3)

glutathione ATGGTTTGCAGGAA 2.5 AACCAGTCAATGCT 2.5 95

S-transferase ACAAGG (SEQ ID NO. GCTCCT (SEQ ID NO.

m type 1 12) 13)

(GSTM1)

phosphor TGGTTGTGGCTACCT 2.5 GCCTTTAACCATTG 2.5 95 diesterase-5 GCATA (SEQ ID NO. CCTCAA (SEQ ID NO.

(PDE5) 14) 15)

SNP-specific primers are designed to identify the selected SNPs at CH clonotyping gene regions, that are suitable for use in alternative isothermal amplification protocols (as well as RT-PCR). One preferred method is a loop-mediated isothermal amplification (LAMP) protocol that includes 2 or 3 layers (depending on the number of primer pairs used) of specificity control. It is also very robust and can use colorimetry (double-stranded DNA dyes) and/or simple turbidity (Mg₂P₂0₇ precipitation) for the reaction read-out. Other isothermal amplification methods include recombinase polymerase amplification (RPA) and helicase- dependent amplification (HAD). Both methods utilize colorimetry for detection, using essentially the same instrumentation platforms as LAMP.

The present disclosure provides methods for the administration of a solid sustained release formulation, which comprises at least one hydralazine compound or a

pharmaceutically acceptable salt thereof, and at least one of isosorbide dinitrate and isosorbide mononitrate in an amount of about 30 milligrams to about 400 milligrams and at least one excipient or carrier. Preferably, the hydralazine compound is hydralazine hydrochloride. More preferably, the amount of hydralazine hydrochloride is 56.25 g.

Preferably, the isosorbide nitrate compound comprised in this formulation is isosorbide dinitrate. More preferably, the amount of isosorbide dinitrate is 30 mg.

The present disclosure utilizes detection of four biomarkers or genetic indicators for guidance in identifying those CHF patients most likely to benefit from a combination of sustained release hydralazine and isosorbide dinitrate.

Hie sequences are:

The wild type sequence of CDS of GNB3 gene is provided as SEQ ID NO. 1. GNB3 subunit homozygous 825TT genotype. The CDS of GNB3 gene having a C to T mutation at position 825 from the start codon is provided as SEQ ID NO. 5.

The wild type sequence of the promoter region of eNOS gene is provided as SEQ ID NO. 2. eNOS C mutant allele (heterozygous or homozygous). The promoter sequence of eNOS gene having a T to C mutation at position -813 from the start codon is provided as SEQ ID NO. 6

The wild type sequence of GSTM1 gene is provided as SEQ ID O. 3. GSTM1 null polymorphism. The GSTM1 gene is lacking.

The wild type sequence of the 5’ -flanking regulatory region of PDE5A gene is provided herein as SEQ ID NO. 4. PDE5A -1255TT genotype. The sequence of the 5’- flanking regulatory region of PDE5A gene having a G to T mutation at position -1255 from the start codon is provided as SEQ ID NO. 7.

A patient with positive presence of at least 2 out of the above-noted 4 mutant SNPs identifies patients likely to benefit from a pharmaceutical composition comprising a combination of hydralazine and isosorbide dinitrate/mononitrate. The following examples are specific formulations of sustained release combination oral hydralazine and isosorbide dinitrate.

Example 1.

Ingredient _ mg/Tablet _ Quantity, kg

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Lactose 100 2

Hydroxypropyl 125 2.52

Methylcellulose K4M

Hydroxypropyl 125 2.52

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Opadry** _ 14.5 _ 0.29 _

Total 453 94)6

** Hydroxypropyl Methylcellulose based film coating system

The Lactose, Hydralazine HC1, Isosorbide Dinitrate, HPMC K4M and HPMC

K100LV are passed through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Next, one adds magnesium stearate to the blend in the V-blender and continues mixing for an additional 5 minutes. The blend is discharged and compressed into tablets with target weight of 453 mg per tablet and hardness in the range of 8 to 20 kp.

Example 2.

Ingredient

Quantity, kg

Core Tablet

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

Povidone K30 10 0.2

Purified Water Appr. Appr

Hydroxypropyl 100 2

Methylcellulose K4M

Hydroxypropyl 150 3

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 85 1.7

Opadry^® ** 15 0.3

Total 462 9.24

*mean particle size = 50 pm

** Hydroxypropyl Methylcellulose based film coating system

The Microcrystallijne, Hydralazine, Isosorbide Dinitrate and PVP K30 are passed through a 20 mesh screen and then loaded to a high shear granulator. Spray appropriate amount of water to the blend and start granulation until the end-point is reached. Remove the granules and place in a fluid bed dryer with inlet temperature of 60 °C and dry until the moisture content is below 2%. Pass the dried granules through Fitzmill equipped with 20 mesh screen. Add the milled granules, HPMC K4M and HPMC K100LV to a 1 cubic foot V- Blender and mix for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 448.5 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® I to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3%.

Example 3.

Ingredient _ mg/Tablet _ Quantity, kg

Core Tablet _

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

Lactose 100 2

Povidone K30 16.15 0.323

Magnesium Stearate 1.6 0.032

Coating Dispersion

Purified Water 188.33 3.77

Propylene Glycol 5 0.1

Polyvinyl Acetate 160 3.2

Dispersion, 30% _

Total 349.6 6.992

*mean particle size = 50 pm

Pass Microcrystalline Cellulose, Hydralazine HC1, Isosorbide Dinitrate, Lactose and PVP K30 through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 304 mg per tablet and hardness in the range of 8 to 20 kp. In a separate container, prepare the spray dispersion by adding the Propylene Glycol into Kollicoat SR^® 30D dispersion and gently mix for 1 hour. Load the tablets into a pan coater and spray the coating dispersion at a controlled target product temperature of 30 °C until the weight gain is 15% w/w.

Example 4

_

Hydralazine HC1 56.25 1.125 Isosorbide Dinitrate 30 0.6

Lactose 100 2 Hydroxypropyl 125 2.52

Methylcellulose K4M

Hydroxypropyl 125 2.52

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 82.2 1.644

_ Opadry** _ 14.5 _ 0.29

Total 453 9.06

** Hydroxypropyl Methylcellulose based film coating system

Pass Lactose, Hydralazine HC1, Isosorbide Dinitrate, HPMC K4M and HPMC K100LV through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 453 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3.3%.

Example 5.

Ingredient

Quantity, kg

Core Tablet

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

PVP K30 10 0.2

Purified Water Appr. Appr

Hydroxypropyl 100 2

Methylcellulose K4M

Hydroxypropyl 150 3

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 85 1.7

Opadry^® ** 15 0.3

Total 462 9.24

*mean particle size = 50 pm

** Hydroxypropyl Methylcellulose based film coating system

Pass Microcrystalline, Hydralazine, Isosorbide Dinitrate and PVP K30 through a 20 mesh screen and load to a high shear granulator. Spray appropriate amount of water to the blend and start granulation until the end point is reached. Remove the granules and place in a fluid bed dryer with inlet temperature of 60 °C and dry until the moisture content is below 2%. Pass the dried granules through Fitzmill equipped with 20 mesh screen. Add the milled granules, HPMC K4M and HPMC K100LV to a 1 cubic foot V-B lender and mix for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 448.5 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® I to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3%.

Example 6

_ Ingredient _ mg/Tablet _ Quantity, kg

Core Tablet _

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

Lactose 100 2

Povidone K30 16.15 0.323

Magnesium Stearate 1.6 0.032

Coating Dispersion

Purified Water 188.33 3.77

Propylene Glycol 5 0.1

Polyvinyl Acetate 160 3.2

_ Dispersion, 30% _

Total 349.6 6.992

*mean particle size = 50 pm

Pass Microcrystalline Cellulose, Hydralazine HC1, Isosorbide Dinitrate, Lactose and PVP K30 through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 304 mg per tablet and hardness in the range of 8 to 20 kp. In a separate container, prepare the spray dispersion by adding the Purified Water, Propylene Glycol into Kollicoat SR^® 30D dispersion and gently mix for 1 hour. Load the tablets into a pan coater and spray the coating dispersion at a controlled target product temperature of 30 °C until the weight gain is 15% w/w.

The following examples are specific formulations of sustained release combination oral hydralazine and isosorbide dinitrate.

Example 1.

Ingredient _ mg/Tablet _ Quantity, kg

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Lactose 100 2

Hydroxypropyl 125 2.52

Methylcellulose K4M Hydroxypropyl 125 2.52

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Opadry** 14.5 0.29

Total 453 9.06

** Hydroxypropyl Methylcellulose based film coating system

The Lactose, Hydralazine HC1, Isosorbide Dinitrate, HPMC K4M and HPMC K100LV are passed through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Next, one adds magnesium stearate to the blend in the V-blender and continues mixing for an additional 5 minutes. The blend is discharged and compressed into tablets with target weight of 453 mg per tablet and hardness in the range of 8 to 20 kp.

Example 2.

Ingredient

Quantity, kg

Core Tablet

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

Povidone K30 10 0.2

Purified Water Appr. Appr

Hydroxypropyl 100 2

Methylcellulose K4M

Hydroxypropyl 150 3

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 85 1.7

Opadry^® ** 15 0.3

Total 462 9.24

*mean particle size = 50 pm

** Hydroxypropyl Methylcellulose based film coating system

The Microcrystallijne, Hydralazine, Isosorbide Dinitrate and PVP K30 are passed through a 20 mesh screen and then loaded to a high shear granulator. Spray appropriate amount of water to the blend and start granulation until the end point is reached. Remove the granules and place in a fluid bed dryer with inlet temperature of 60 °C and dry until the moisture content is below 2%. Pass the dried granules through Fitzmill equipped with 20 mesh screen. Add the milled granules, HPMC K4M and HPMC K100LV to a 1 cubic foot V- Blender and mix for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 448.5 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® I to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3%.

Example 3.

Ingredient _ mg/Tablet _ Quantity, kg

Core Tablet _

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

Lactose 100 2

Povidone K30 16.15 0.323

Magnesium Stearate 1.6 0.032

Coating Dispersion

Purified Water 188.33 3.77

Propylene Glycol 5 0.1

Polyvinyl Acetate 160 3.2

Dispersion, 30% _

Total 349.6 6.992

*mean particle size = 50 pm

Pass Microcrystalline Cellulose, Hydralazine HC1, Isosorbide Dinitrate, Lactose and

PVP K30 through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 304 mg per tablet and hardness in the range of 8 to 20 kp. In a separate container, prepare the spray dispersion by adding the Propylene Glycol into Kollicoat SR^® 30D dispersion and gently mix for 1 hour. Load the tablets into a pan coater and spray the coating dispersion at a controlled target product temperature of 30 °C until the weight gain is 15% w/w.

Example 4

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Lactose 100 2

Hydroxypropyl 125 2.52

Methylcellulose K4M

Hydroxypropyl 125 2.52

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 82.2 1.644

_ Opadry** _ 14.5 0.29

Total 453 9.06

** Hydroxypropyl Methylcellulose based film coating system Pass Lactose, Hydralazine HC1, Isosorbide Dinitrate, HPMC K4M and HPMC K100LV through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 453 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3.3%.

Example 5.

Ingredient

Quantity, kg

Core Tablet

Hydralazine HC1 56.25 1.125

Isosorbide Dinitrate 30 0.6

Microcrystalline Cellulose* 100 2

PVP K30 10 0.2

Purified Water Appr. Appr

Hydroxypropyl 100 2

Methylcellulose K4M

Hydroxypropyl 150 3

Methylcellulose K100LV

Magnesium Stearate 2.25 0.045

Coating Solution

Purified Water 85 1.7

Opadry^® ** 15 0.3

Total 462 9.24

*mean particle size = 50 pm

** Hydroxypropyl Methylcellulose based film coating system

Pass Microcrystalline, Hydralazine, Isosorbide Dinitrate and PVP K30 through a 20 mesh screen and load to a high shear granulator. Spray appropriate amount of water to the blend and start granulation until the end point is reached. Remove the granules and place in a fluid bed dryer with inlet temperature of 60 °C and dry until the moisture content is below 2%. Pass the dried granules through Fitzmill equipped with 20 mesh screen. Add the milled granules, HPMC K4M and HPMC K100LV to a 1 cubic foot V-B lender and mix for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 448.5 mg per tablet and hardness in the range of 8 to 20 kp. Prepare the coating solution in a separate container by adding Opadry^® I to Purified water and mix for 30 minutes. Load the compressed tablets into a tablet coater and spray the coating solution at controlled product temperature of 40 °C until the weight gain is 3%. Example 6

antity, kg

Core Tablet

Hydralazine HC1 56.25 1.125 Isosorbide Dinitrate 30 0.6 Microcrystalline Cellulose* 100 2

Lactose 100 2

Povidone K30 16.15 0.323

Magnesium Stearate 1.6 0.032 Coating Dispersion

Purified Water 188.33 3.77

Propylene Glycol 5 0.1 Polyvinyl Acetate 160 3.2 Dispersion, 30%

Total 349.6 6.992

*mean particle size = 50 pm

Pass Microcrystalline Cellulose, Hydralazine HC1, Isosorbide Dinitrate, Lactose and PVP

K30 through 20 mesh screen and mix in a 1 cubic foot V-Blender for 10 minutes. Add magnesium stearate to the blend in the V-blender and continue mixing for an additional 5 minutes. Discharge the blend and compress into tablets with target weight of 304 mg per tablet and hardness in the range of 8 to 20 kp. In a separate container, prepare the spray dispersion by adding the Purified Water, Propylene Glycol into Kollicoat SR^® 30D dispersion and gently mix for 1 hour. Load the tablets into a pan coater and spray the coating dispersion at a controlled target product temperature of 30 °C until the weight gain is 15% w/w.

Sequences

SEQ ID NO. 1 GNB3 gene

LENGTH: 1023

TYPE: DNA

ORGANISM: Homo sapiens

(ATG indicates the start codon)

ATGGGGGAGATGGAGCAACTGCGTCAGGAAGCGGAGCAGCTCAAGAAGCAGAT

TGCAGATGCCAGGAAAGCCTGTGCTGACGTTACTCTGGCAGAGCTGGTGTCTGGC

CTAGAGGTGGTGGGACGAGTCCAGATGCGGACGCGGCGGACGTTAAGGGGACA

CCTGGCCAAGATTTACGCCATGCACTGGGCCACTGATTCTAAGCTGCTGGTAAGT

GCCTCGCAAGATGGGAAGCTGATCGTGTGGGACAGCTACACCACCAACAAGGTG

CACGCCATCCCACTGCGCTCCTCCTGGGTCATGACCTGTGCCTATGCCCCATCAG

GGAACTTTGTGGCATGTGGGGGGCTGGACAACATGTGTTCCATCTACAACCTCAA

ATCCCGTGAGGGCAATGTCAAGGTCAGCCGGGAGCTTTCTGCTCACACAGGTTAT

CTCTCCTGCTGCCGCTTCCTGGATGACAACAATATTGTGACCAGCTCGGGGGACA

CCACGTGTGCCTTGTGGGACATTGAGACTGGGCAGCAGAAGACTGTATTTGTGG

GACACACGGGTGACTGCATGAGCCTGGCTGTGTCTCCTGACTTCAATCTCTTCAT

TTCGGGGGCCTGTGATGCCAGTGCCAAGCTCTGGGATGTGCGAGAGGGGACCTG

CCGTCAGACTTTCACTGGCCACGAGTCGGACATCAACGCCATCTGTTTCTTCCCC

AATGGAGAGGCCATCTGCACGGGCTCGGATGACGCTTCCTGCCGCTTGTTTGACC

TGCGGGCAGACCAGGAGCTGATCTGCTTCTCCCACGAGAGCATCATCTGCGGCAT CACGTCCGTGGCCTTCTCCCTCAGTGGCCGCCTACTATTCGCTGGCTACGACGAC

TTCAACTGCAATGTCTGGGACTCCATGAAGTCTGAGCGTGTGGGCATCCTCTCTG GCCACGATAACAGGGTGAGCTGCCTGGGAGTCACAGCTGACGGGATGGCTGTGG CCACAGGTTCCTGGGACAGCTTCCTCAAAATCTGGAACTGA -3’

SEQ ID NO. 2 eNOS

LENGTH: 2506

TYPE: DNA

ORGANISM: Homo sapiens 5’-

GTACCTGCTCTCTAAGAGGGAGGCCTGGGTGGTGCACCTCCAGAGCTGCCCAGG

CTGGGCCTCAAGGAAGAAAAAGATTTTCATTTGTCAGAGGCGGAAGGGAGAGGT

GGAGGGAACAGCACAGCAGCGGCCCAGGGGCAGGGAAGCACAGGACCATTAGG

GAGACACGAGAAAGCCCATTTGTCTAGAACAGAGGATTCAAGCAGTGCACCAAG

GAAAATGAGGGCCAGGCCAATGTGCTGGAGTGGCTTTGTTCTTGGCTGAGGGTTT

TGGGTAGTGCCAAAGCGTAAGGTAAGCCCTGCTTTCCAGAAGAATCTAGCAGAG

TGTGGAGCCCAGATGGGACTGGAAGGCCTGGGAGGGGTCAGGTGGCCACAGGG

ACGGGCCACAGCCAGTGGTGCAGGCAAGAAGACAATGGCCATCCATGGTGGCTC

ACACCTGGAATCCCAGCCCATTGGGAGGTCGAGGCAGGTGGATCACCTGAGGTC

AGGAGTTCGAGACCAGCCTGGTCAACATGGTGAAACCCTGTCTCTAATAAAATT

ATAAAAATTAGCCGGGCGTGGTGGTGGGTACCTGTAATCTCAGCTACTCAGGAG

GCTGGGTCAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTACAGTGAGCTGAG

ATAGCACCATTGCATTCCAGCCTGGACAACAAAAGCGAGACTCTGTCTCAAAAA

AAAAAAAAAATTAGCCAGGCGTGGTGGTGGGTGCCTGTCGTCCTCGGGAGGCTG

AGGCATGAGAATCACTCCGGGAGGCAGAGGTTGCAATGAACCAAGATCACACCA

CTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTAAAAAAAAAAAAAAGA

CAGAAGGATGTCAGCATCTGATGCTGCCTGTCACCTTGACCCTGAGGATGCCAGT

CACAGCTCCATTAACTGGGACCTAGGAAAATGAGTCATCCTTGGTCATGCACATT

TCAAATGGTGGCTTAATATGGAAGCCAGACTTGGGATCTGTTGTCTCCTCCAGCA

TGGTAGAAGATGCCTGAAAAGTAGGGGCTGGATCCCATCCCCTGCCTCACTGGG

AAGGCGAGGTGGTGGGGTGTGGTGGGGCCTCAGGCTTGGGGTCATGGGACAAAG

CCCAGGCTGAATGCCGCCCTTCCATCTCCCTCCTCCTGAGACAGGGGCAGCAGGG

CACACTAGTGTCCAGGAGCAGCTTATGAGGCCCCTTCACCCTCCATCCTCCAAAA

CTGGCAGACCCCACCTTCTTGGTGTGACCCCAGAGCTCTGAGCACAGCCCGTTCC

TTCCGCCTGCCGGCCCCCCACCCAGGCCCACCCCAACCTTATCCTCCACTGCTTTT

CAGAGGAGTCTGGCCAACACAAATCCTCTTGTTTGTTTGTCTGTCTGTCTGCTGCT

CCTAGTCTCTGCCTCTCCCAGTCTCTCAGCTTCCGTTTCTTTCTTAAACTTTCTCTC

AGTCTCTGAGGTCTCGAAATCACGAGGCTTCGACCCCTGTGGACCAGATGCCCAG

CTAGTGGCCTTTCTCCAGCCCCTCAGATGGCACAGAACTACAAACCCCAGCATGC

ACTCTGGCCTGAAGTGCCTGGAGAGTGCTGGTGTACCCCACCTGCATTCTGGGAA

CTGTAGTTTCCCTAGTCCCCCATGCTCCCACCAGGGCATCAAGCTCTTCCCTGGC

TGGCTGACCCTGCCTCAGCCCTAGTCTCTCTGCTGACCTGCGGCCCCGGGAAGCG

TGCGTCACTGAATGACAGGGTGGGGGTGGAGGCACTGGAAGGCAGCTTCCTGCT

CTTTTGTGTCCCCCACTTGAGTCATGGGGGTGTGGGGGTTCCAGGAAATTGGGGC

TGGGAGGGGAAGGGATACCCTAATGTCAGACTCAAGGACAAAAAGTCACTACAT

CCTTGCTGGGCCTCTATCCCCAAGAACCCAAAAGGACTCAAGGGTGGGGATCCA

GGAGTTCTTGTATGTATGGGGGGAGGTGAAGGAGAGAACCTGCATGACCCTAGA

GGTCCCTGTGGTCACTGAGAGTGTGGGCTGCCATCCCCTGCTACAGAAACGGTGC

TCACCTTCTGCCCAACCCTCCAGGGAAAGGCACACAGGGGTGAGGCCGAAGGCC

CTTCCGTCTGGTGCCACATCACAGAAGGACCTTTATGACCCCCTGGTGGCTCTAC

CCTGCCACTCCCCAATGCCCCAGCCCCCATGCTGCAGCCCCAGGGCTCTGCTGGA CACCTGGGCTCCCACTTATCAGCCTCAGTCCTCACAGCGGAACCCAGGCGTCCGG

CCCCCCACCCTTCAGGCCAGCGGGCGTGGAGCTGAGGCTTTAGAGCCTCCCAGCC

GGGCTTGTTCCTGTCCCATTGTGTATGGGATAGGGGCGGGGCGAGGGCCAGCAC

TGGAGAGCCCCCTCCCACTGCCCCCTCCTCTCGGTCCCCTCCCTCTTCCTAAGGAA

AAGGCCAGGGCTCTGCTGGAGCAGGCAGCAGAGTGGACGCACAGTAACATG

(ATG indicates the start codon)

SEQ ID NO. 3 GSTM1 gene

LENGTH: 1735

TYPE: DNA

ORGANISM: Homo sapiens 5’-

TCTTACTGAGTGCAGCCCCAGGCGCCCTCTCCCGGGCCTCCAGAATGGCGCCTTT

CGGGTTGTGGCGGGCCGAGGGGCGGGGTCGCAGCAAGGCCCCGCCTGTCCCCTC

TCCGGAGCTCTTATACTCTGAGCCCTGCTCGGTTTAGGCCTGTCTGCGGAATCCG

CACCAACCAGCACCATGCCCATGATACTGGGGTACTGGGACATCCGCGGGGTGA

GCGAGGGTCCGCTGGACGGTGGCGACCTGCGGGCCATCTCTCCCAGCTGGCCCA

CGCCATCCGCCTGCTCCTGGAATACACAGACTCAAGCTATGAGGAAAAGAAGTA

CACGATGGGGGACGGTAATGGCACCCTCGTGTTCGGGCTTTTCACTTCTTCTTCC

CCACCACAGCTCCTGATTATGACAGAAGCCAGTGGCTGAATGAAAAATTCAAGC

TGGGCCTGGACTTTCCCAATGTAGGTGCAGGGGAAGGGGCGGTTTCTTCGCCGGT

TTCCCATCCATCCAGCTGCCCTACTTGATTGATGGGGCTCACAAGATCACCCAGA

GCAACGCCATCTTGTGCTACATTGCCCGCAAGCACAACCTGTGTGAGTGTGGGTG

GCTGCAATGTGTTGAGTCTGTGTTTTGTGGGTGGCAGGTGGGGAGACAGAAGAG

GAGAAGATTCGTGTGGACATTTTGGAGAACCAGACCATGGACAACCATATGCAG

CTGGGCATGATCTGCTACAATCCAGAATTTGTGAGTGTCCCCAGTGAGCTGCATC

TGGTGACAGCTGTTTTCTGCCTCAGGAGAAACTGAAGCCAAAGTACTTGGAGGA

ACTCCCTGAAAAGCTAAAGCTCTACTCAGAGTTTCTGGGGAAGCGGCCATGGTTT

GCAGGAAACAAGGTAAAGGAGGAGTGATATGGGGAATCCCACATATTCTTGGCC

TTCTGCAGATCACTTTTGTAGATTTTCTCGTCTATGATGTCCTTGACCTCCACCGT

ATATTTGAGCCCAAGTGCTTGGACGCCTTCCCAAATCTGAAGGACTTCATCTCCC

GCTTTGAGGTGATGCCCCCATCCTCCTTTCTCTCTGGCCTTATTTTCCCCCCTCTCA

GGGCTTGGAGAAGATCTCTGCCTACATGAAGTCCAGCCGCTTCCTCCCAAGACCT

GTGTTCTCAAAGATGGCTGTCTGGGGCAACAAGTAGGGCCTTGAAGGCCAGGAG

GTGGGAGTGAGGAGCCCATACTCAGCCTGCTGCCCAGGCTGTGCAGCGCAGCTG

GACTCTGCATCCCAGCACCTGCCTCCTCGTTCCTTTCTCCTGTTTATTCCCATCTTT

ACTCCCAAGACTTCATTGTCCCTCTTCACTCCCCCTAAACCCCTGTCCCATGCAGG

CCCTTTGAAGCCTCAGCTACCCACTATCCTTCGTGAACATCCCCTCCCATCATTAC

CCTTCCCTGCACTAAAGCCAGCCTGACCTTCCTTCCTGTTAGTGGTTGTGTCTGCT

TTAAAGGGCCTGCCTGGCCCCTCGCCTGTGGAGCTCAGCCCCGAGCTGTCCCCGT

GTTGCATGAAGGAGCAGCATTGACTGGTTTACAGGCCCTGCTCCTGCAGCATGGT

CCCTGCCTTAGGCCTACCTGATGGAAGTAAAGCCTCAACCACATTTGCTGTGTGT

CTTGTCTTATTTGCTCCTGGCCATCTACCCAGACTGTCTGTCTGTCTGTCACTGCC

TCTTCCAAGGGACTGGCTGGTGATCCTGGCAG -3’

SEQ ID NO. 4

LENGTH: 3805

TYPE: DNA

ORGANISM: Homo sapiens 5’-

AAGCTTGCTGAATCACCTCTTAATTCTTGTAGTTGCTTTGTGCATTCCTTTGGGTA

TTCCTCATAGATACTCATGTCTGCAAATGGAGAATGTTTACTTTTTCATTTTTATG

CCTTATATTTCTTTTTTGTGTTTTTGCTTTGTTGCATTTGTTTTTTCTATTTGTATGA

CCAAAATCTTTAGCAGTACAGGTAAGTAACAACCAAATAATGTAGAACCCCATA AGCCACGTTACAGAGTTTGAATTTTATTTTAGCACAGTGGGAATACATTGAAGGT

CTTTAGTTAAGCTGTTGCTCATGAGCAACAAATGAGCAATGACATATATGTATGT

AT AT AC AC AT AT AT ATC ATTG ATT AT AT AT AT AT AT AT AT AT AT AT AT AT ATCTAT

CTTAGTCCACTTGTGTTGCAATAACAAAATACCACAGACTGGGTCATTTACAAAA

ATTAAATATATATATACATATACACACATATATATATCATACATATACACATACA

TACATCATTGCTCATTTGTTTGTTATAAATAGCATTAACAGCATTTTTCAAGTTAT

ATCCTGGGAGTGTTTATGATTTACTTATTCTTCAACTAATTCCATAACAAGATTTG

AGGTGCTTAGAACAATTCATGCCAAGTTAAAACAAAATAATTGGGCAAATTGGG

ATAAAGAATAAAATGGAGTTGAAAAACAAGAGGCCCAGGTAATGTCAGTTCAAA

ATATGCTTACCTTTAACTACTTTAAATTTACAGGAGGTATAGTTACACATTTTGGC

TGAATCTCCCAGAGACTAGAACTGTTTGAGACACTTCTGTTCCCCAATCCCTTGT

GAT ATGTTTCTC AGGT A AT AGGCCTTC AC AGT AACTCCC A A ACT ATC AT AT AT AC

CACACAGACTTGAGATTCACTATTGAGAGAATCTATGTACTGTTTTTCTTTTTTTT

TCTTTTTTGTTATAGAGCCGGGGGTCTTACACTGTCACTGAGGCTGAAGTGCAAT

GGCACGATCATGGCTCACTGCAGCCTTGACCTCCTGGGCTCAATCCTCTTGCCTC

AGCCTCTCGAATAACTAGGATTACAGGTGTGTTCCCCCATGCCTGGCTAATTTTT

AAAAATTTTGTGTAGAGATGGGGTCATGCCATGTGGCCCAGGCTGGTTCAAACTC

CTGAGCTCAAGTATCCTTCTACCTCTCCCTCCCAAAGTTCTGAGATTACAGGAAT

GAGCCACTGTGCCCAGCCTATAGATTGTTTTTCTTGAAGCAATTTTTCAGAAACC

TTCCTGGTTTCTGATAATTTAACCCTTTCAGGTTAGGAGAGAAAAATGAACATTT

TGATATTACCCACTGTCTTAGTCCATTTGTGTTGCTGTAATAAAATATCACAGACT

GGGATATTTATAAACAATAGAAATTAATTTCTCTCAGTTCTGGAGGCTGGAAACT

CCAAAATCAAAGTGCCAGCAGATTTGGCAACTGGTGAGGGCTGCTCTTTGCTTAC

AAAATGGCACCTTGTTGCTGCATCCTCAGCAAGGGTCAGTGCTGTGTCTTCACAT

AGTGGAAAGAATAGAAGGGGCCAACTGTCTCCTTTGGGCCTTTTTTTAAAAAGGC

ACTAATGCATTCACAAAGGCAGAGCCCTAATGGTCTAATCACCACTTAAAGGCA

CCTCCTCTTAATACTGTTGAATTAGGGATTAAGTTTCAACATGAATTTTGGAGGG

AATACAAACATTGAAATGATTATACGTGTTTATTTAATCAAGTATCCAACAAAAG

CAAATAATTCAAGCCCCAAATTCACTGCATCTTTAGTAGATAAGCAGAGTTTTAA

ATTACGATTGATCTCCTGTTAGGAGGAATGCATGGATTTCCACAAGAAAAAACTG

TACTGAGGAGAAACTTTCCACAGTAATGTGCCACTTTTCAGTCAACGACAGACCA

CATATATGAGTCCCATAAGATAATACTATATTTTTACTGTACCTTTTCTATGTTTA

GATATGTTTAGACACACAAATATCATTGCATTACAATTGCCTACAGTATTCAGTA

CAGTAATATGCTGTATAGATTTGTGGTCTAGGAGCAATAGCCTAAGTGTGTAGTA

GGCTGAGCCATCTATTTTGTGTTAGTACACTGTGATGTTCAGAGAAGGATGAAAT

TGCCTAAGGATACATTTCTCAGAATGTATCCTGTTGTTCGGTGACGCATGACTGT

ATTCCATGAGCACTATAATCACTATCATAGTAACACATTAGGAGAGAATTCTCAT

TTCTAAATCCAATATAATTTATCACCCATTAGTTCATACTCTACTGCTTTGATTGC

TTTTCTTTGGTTGTGGCTACCTGCATACAGCAGTAAAGTTTCAGAAAAACTGAAG

TCGCAAAAGGTCAATTACTCAATGAAGGAAAGATAAACCATTGCATTGGGGGAC

TAGAAGATACTTTTAAAAGTTCTCAGATTATCAATTTAATGATGTGTTTCTATGTA

GTGAATAATGCCTTAAATTCTTGCCAAGAGTATTTAGAAGGAAGTTGTCAGAAGT

ATATCAGCGAACTCATTTTTTTTTATATCACTGCTAATGGTGTCATTCACACATTG

TGCAACCCATAATTCCAGATTTAATTCTACCAAAAAATATAGGTCATTGCAAAAT

GCCATATTAAAACTGCCAATGCATGACAGGAAGATGGGGATGCAGACAAAGCAA

AGGATGACACCAATTCCTTTTTTAAAGAAGCAAGATAGGGATTGGACAAAAAGG

CTGAGCCATTTTTAATGGATACTTTTGAGGGAGTGTTAATTCCAATTTAATTAAA

ATGATGCATTAATTTAAAATTGGGATAACTGGTTGCCCTCGACTGCACCTGGGTT

GCGCCAGTGCTCTCGGATTAACCTAATTGTACAGAGGTGCCCTTGTTTTCTAACTT

CATGCACAAAGCATTGGAAATTATTTGTTTGCTTTTTCTTTTCCAAGTAAATCTTT TTCCAGTTATGCAAAAGGGAAGTTTGAGGCAATGGTTAAAGGCACTTAAGTTATA

ATTATTGCTGTTATCATTAACATTAAGCACGGGTATGGCTTTGTTGCAAGTTACCC

ACCTACACCTGCAAATCTCTCTTGCTAGCACACGCCCCAGCTCTCTCCACCCGCA

GTGGTCCGTGGCTGGACCGCTTTAAGTCACTGAGCGGGCTGGGCTCTGAAGGAG

GTCGGTCCCGCTCCTCCCAGACCCAAGCGTAGGGCTAGGGAAAAGCTAGGCGGG

AAGGTCATTGCACTCCCAGGCCCCAGGAAAAGGGCCCAGGGTCTCATCATCTCTT

ACTTTCGGGCAAAACTTCCCACATCGCGACCTTCCCTCCCTGGGGCACTCTGAGA

ACACACCCAGTCACCTAGCGCGCTCCCCAGAAGTCGGCTTGGCACACAGCGCAC

CCCAGCGGCCGCGCGGCCTCCTTCCAGCCGCCGCCACTTGGCTTCCGGAGAGCTC

GCCGGGCGCTGCCGCCGCCGCCGCCGCCGCCTCCTGGGAACCAGGGGACTGAAG

AGCCTGCGAGAGCGGAACACTGCCGGACCCCGGGTGGGGGGGCGCAGCAGCTG

CGCCTGGCCCCGCCCACCACACCTGGGCGCCCGTAGAACCGCGCGGGGCGGGGC

GGGGCAGGAGGCTGGCCTGGCGCTCCGGCCGCTTTGTCGAAAGCCGGCCCGACT

GGAGCAGGACGAAGGGGGAGGGTCTCGAGGCCGAGTCCTGTTCTTCTGAGGGAC

GGACCCCAGCTGGGGTGGAAAAGCAGTACCAGAGAGCCTCCGAGGCGCGCGGT

GCCAACCATG -3’

SEQ ID NO. 5 GNB3 gene having a C to T mutation at position 825 from the start codon

LENGTH: 1023

TYPE: DNA

ORGANISM: Homo sapiens 5’-

ATGGGGGAGATGGAGCAACTGCGTCAGGAAGCGGAGCAGCTCAAGAAGCAGAT

TGCAGATGCCAGGAAAGCCTGTGCTGACGTTACTCTGGCAGAGCTGGTGTCTGGC

CTAGAGGTGGTGGGACGAGTCCAGATGCGGACGCGGCGGACGTTAAGGGGACA

CCTGGCCAAGATTTACGCCATGCACTGGGCCACTGATTCTAAGCTGCTGGTAAGT

GCCTCGCAAGATGGGAAGCTGATCGTGTGGGACAGCTACACCACCAACAAGGTG

CACGCCATCCCACTGCGCTCCTCCTGGGTCATGACCTGTGCCTATGCCCCATCAG

GGAACTTTGTGGCATGTGGGGGGCTGGACAACATGTGTTCCATCTACAACCTCAA

ATCCCGTGAGGGCAATGTCAAGGTCAGCCGGGAGCTTTCTGCTCACACAGGTTAT

CTCTCCTGCTGCCGCTTCCTGGATGACAACAATATTGTGACCAGCTCGGGGGACA

CCACGTGTGCCTTGTGGGACATTGAGACTGGGCAGCAGAAGACTGTATTTGTGG

GACACACGGGTGACTGCATGAGCCTGGCTGTGTCTCCTGACTTCAATCTCTTCAT

TTCGGGGGCCTGTGATGCCAGTGCCAAGCTCTGGGATGTGCGAGAGGGGACCTG

CCGTCAGACTTTCACTGGCCACGAGTCGGACATCAACGCCATCTGTTTCTTCCCC

AATGGAGAGGCCATCTGCACGGGCTCGGATGACGCTTCCTGCCGCTTGTTTGACC

TGCGGGCAGACCAGGAGCTGATCTGCTTCTCCCACGAGAGCATCATCTGCGGCAT

CACGTCTGTGGCCTTCTCCCTCAGTGGCCGCCTACTATTCGCTGGCTACGACGAC

TTCAACTGCAATGTCTGGGACTCCATGAAGTCTGAGCGTGTGGGCATCCTCTCTG GCCACGATAACAGGGTGAGCTGCCTGGGAGTCACAGCTGACGGGATGGCTGTGG CCACAGGTTCCTGGGACAGCTTCCTCAAAATCTGGAACTGA -3

SEQ ID NO. 6 eNOS gene having a T to C mutation at position -813 from the start codon

LENGTH: 2506

TYPE: DNA

ORGANISM: Homo sapiens 5’-

GTACCTGCTCTCTAAGAGGGAGGCCTGGGTGGTGCACCTCCAGAGCTGCCCAGG

CTGGGCCTCAAGGAAGAAAAAGATTTTCATTTGTCAGAGGCGGAAGGGAGAGGT

GGAGGGAACAGCACAGCAGCGGCCCAGGGGCAGGGAAGCACAGGACCATTAGG

GAGACACGAGAAAGCCCATTTGTCTAGAACAGAGGATTCAAGCAGTGCACCAAG

GAAAATGAGGGCCAGGCCAATGTGCTGGAGTGGCTTTGTTCTTGGCTGAGGGTTT

TGGGTAGTGCCAAAGCGTAAGGTAAGCCCTGCTTTCCAGAAGAATCTAGCAGAG

TGTGGAGCCCAGATGGGACTGGAAGGCCTGGGAGGGGTCAGGTGGCCACAGGG ACGGGCCACAGCCAGTGGTGCAGGCAAGAAGACAATGGCCATCCATGGTGGCTC

ACACCTGGAATCCCAGCCCATTGGGAGGTCGAGGCAGGTGGATCACCTGAGGTC

AGGAGTTCGAGACCAGCCTGGTCAACATGGTGAAACCCTGTCTCTAATAAAATT

ATAAAAATTAGCCGGGCGTGGTGGTGGGTACCTGTAATCTCAGCTACTCAGGAG

GCTGGGTCAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTACAGTGAGCTGAG

ATAGCACCATTGCATTCCAGCCTGGACAACAAAAGCGAGACTCTGTCTCAAAAA

AAAAAAAAAATTAGCCAGGCGTGGTGGTGGGTGCCTGTCGTCCTCGGGAGGCTG

AGGCATGAGAATCACTCCGGGAGGCAGAGGTTGCAATGAACCAAGATCACACCA

CTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTAAAAAAAAAAAAAAGA

CAGAAGGATGTCAGCATCTGATGCTGCCTGTCACCTTGACCCTGAGGATGCCAGT

CACAGCTCCATTAACTGGGACCTAGGAAAATGAGTCATCCTTGGTCATGCACATT

TCAAATGGTGGCTTAATATGGAAGCCAGACTTGGGATCTGTTGTCTCCTCCAGCA

TGGTAGAAGATGCCTGAAAAGTAGGGGCTGGATCCCATCCCCTGCCTCACTGGG

AAGGCGAGGTGGTGGGGTGTGGTGGGGCCTCAGGCTTGGGGTCATGGGACAAAG

CCCAGGCTGAATGCCGCCCTTCCATCTCCCTCCTCCTGAGACAGGGGCAGCAGGG

CACACTAGTGTCCAGGAGCAGCTTATGAGGCCCCTTCACCCTCCATCCTCCAAAA

CTGGCAGACCCCACCTTCTTGGTGTGACCCCAGAGCTCTGAGCACAGCCCGTTCC

TTCCGCCTGCCGGCCCCCCACCCAGGCCCACCCCAACCTTATCCTCCACTGCTTTT

CAGAGGAGTCTGGCCAACACAAATCCTCTTGTTTGTTTGTCTGTCTGTCTGCTGCT

CCTAGTCTCTGCCTCTCCCAGTCTCTCAGCTTCCGTTTCTTTCTTAAACTTTCTCTC

AGTCTCTGAGGTCTCGAAATCACGAGGCTTCGACCCCTGTGGACCAGATGCCCAG

CTAGTGGCCTTTCTCCAGCCCCTCAGATGGCACAGAACTACAAACCCCAGCATGC

ACTCTGGCCTGAAGTGCCTGGAGAGTGCTGGTGTACCCCACCTGCATTCTGGGAA

CTGTAGTTTCCCTAGTCCCCCATGCTCCCACCAGGGCATCAAGCTCTTCCCTGGC

CGGCTGACCCTGCCTCAGCCCTAGTCTCTCTGCTGACCTGCGGCCCCGGGAAGC

GTGCGTCACTGAATGACAGGGTGGGGGTGGAGGCACTGGAAGGCAGCTTCCTGC

TCTTTTGTGTCCCCCACTTGAGTCATGGGGGTGTGGGGGTTCCAGGAAATTGGGG

CTGGGAGGGGAAGGGATACCCTAATGTCAGACTCAAGGACAAAAAGTCACTACA

TCCTTGCTGGGCCTCTATCCCCAAGAACCCAAAAGGACTCAAGGGTGGGGATCC

AGGAGTTCTTGTATGTATGGGGGGAGGTGAAGGAGAGAACCTGCATGACCCTAG

AGGTCCCTGTGGTCACTGAGAGTGTGGGCTGCCATCCCCTGCTACAGAAACGGTG

CTCACCTTCTGCCCAACCCTCCAGGGAAAGGCACACAGGGGTGAGGCCGAAGGC

CCTTCCGTCTGGTGCCACATCACAGAAGGACCTTTATGACCCCCTGGTGGCTCTA

CCCTGCCACTCCCCAATGCCCCAGCCCCCATGCTGCAGCCCCAGGGCTCTGCTGG

ACACCTGGGCTCCCACTTATCAGCCTCAGTCCTCACAGCGGAACCCAGGCGTCCG

GCCCCCCACCCTTCAGGCCAGCGGGCGTGGAGCTGAGGCTTTAGAGCCTCCCAG

CCGGGCTTGTTCCTGTCCCATTGTGTATGGGATAGGGGCGGGGCGAGGGCCAGC

ACTGGAGAGCCCCCTCCCACTGCCCCCTCCTCTCGGTCCCCTCCCTCTTCCTAAGG

AAAAGGCCAGGGCTCTGCTGGAGCAGGCAGCAGAGTGGACGCACAGTAACATG

-3’

SEQ ID NO. 7 PDE5A -1255TT genotype. The sequence of the 5’-flanking regulatory region of PDE5A gene having a G to mutation at position -1255 from the start codon

LENGTH: 3805

TYPE: DNA

ORGANISM: Homo sapiens 5’-

AAGCTTGCTGAATCACCTCTTAATTCTTGTAGTTGCTTTGTGCATTCCTTTGGGTA

TTCCTCATAGATACTCATGTCTGCAAATGGAGAATGTTTACTTTTTCATTTTTATG

CCTTATATTTCTTTTTTGTGTTTTTGCTTTGTTGCATTTGTTTTTTCTATTTGTATGA

CCAAAATCTTTAGCAGTACAGGTAAGTAACAACCAAATAATGTAGAACCCCATA

AGCCACGTTACAGAGTTTGAATTTTATTTTAGCACAGTGGGAATACATTGAAGGT CTTTAGTTAAGCTGTTGCTCATGAGCAACAAATGAGCAATGACATATATGTATGT

AT AT AC AC AT AT AT ATC ATTG ATT AT AT AT AT AT AT AT AT AT AT AT AT AT ATCTAT

CTTAGTCCACTTGTGTTGCAATAACAAAATACCACAGACTGGGTCATTTACAAAA

ATTAAATATATATATACATATACACACATATATATATCATACATATACACATACA

TACATCATTGCTCATTTGTTTGTTATAAATAGCATTAACAGCATTTTTCAAGTTAT

ATCCTGGGAGTGTTTATGATTTACTTATTCTTCAACTAATTCCATAACAAGATTTG

AGGTGCTTAGAACAATTCATGCCAAGTTAAAACAAAATAATTGGGCAAATTGGG

ATAAAGAATAAAATGGAGTTGAAAAACAAGAGGCCCAGGTAATGTCAGTTCAAA

ATATGCTTACCTTTAACTACTTTAAATTTACAGGAGGTATAGTTACACATTTTGGC

TGAATCTCCCAGAGACTAGAACTGTTTGAGACACTTCTGTTCCCCAATCCCTTGT

GAT ATGTTTCTC AGGT A AT AGGCCTTC AC AGT AACTCCC A A ACT ATC AT AT AT AC

CACACAGACTTGAGATTCACTATTGAGAGAATCTATGTACTGTTTTTCTTTTTTTT

TCTTTTTTGTTATAGAGCCGGGGGTCTTACACTGTCACTGAGGCTGAAGTGCAAT

GGCACGATCATGGCTCACTGCAGCCTTGACCTCCTGGGCTCAATCCTCTTGCCTC

AGCCTCTCGAATAACTAGGATTACAGGTGTGTTCCCCCATGCCTGGCTAATTTTT

AAAAATTTTGTGTAGAGATGGGGTCATGCCATGTGGCCCAGGCTGGTTCAAACTC

CTGAGCTCAAGTATCCTTCTACCTCTCCCTCCCAAAGTTCTGAGATTACAGGAAT

GAGCCACTGTGCCCAGCCTATAGATTGTTTTTCTTGAAGCAATTTTTCAGAAACC

TTCCTGGTTTCTGATAATTTAACCCTTTCAGGTTAGGAGAGAAAAATGAACATTT

TGATATTACCCACTGTCTTAGTCCATTTGTGTTGCTGTAATAAAATATCACAGACT

GGGATATTTATAAACAATAGAAATTAATTTCTCTCAGTTCTGGAGGCTGGAAACT

CCAAAATCAAAGTGCCAGCAGATTTGGCAACTGGTGAGGGCTGCTCTTTGCTTAC

AAAATGGCACCTTGTTGCTGCATCCTCAGCAAGGGTCAGTGCTGTGTCTTCACAT

AGTGGAAAGAATAGAAGGGGCCAACTGTCTCCTTTGGGCCTTTTTTTAAAAAGGC

ACTAATGCATTCACAAAGGCAGAGCCCTAATGGTCTAATCACCACTTAAAGGCA

CCTCCTCTTAATACTGTTGAATTAGGGATTAAGTTTCAACATGAATTTTGGAGGG

AATACAAACATTGAAATGATTATACGTGTTTATTTAATCAAGTATCCAACAAAAG

CAAATAATTCAAGCCCCAAATTCACTGCATCTTTAGTAGATAAGCAGAGTTTTAA

ATTACGATTGATCTCCTGTTAGGAGGAATGCATGGATTTCCACAAGAAAAAACTG

TACTGAGGAGAAACTTTCCACAGTAATGTGCCACTTTTCAGTCAACGACAGACCA

CATATATGAGTCCCATAAGATAATACTATATTTTTACTGTACCTTTTCTATGTTTA

GATATGTTTAGACACACAAATATCATTGCATTACAATTGCCTACAGTATTCAGTA

CAGTAATATGCTGTATAGATTTGTGGTCTAGGAGCAATAGCCTAAGTGTGTAGTA

GGCTGAGCCATCTATTTTGTGTTAGTACACTGTGATGTTCAGAGAAGGATGAAAT

TGCCTAAGGATACATTTCTCAGAATGTATCCTGTTGTTCGGTGACGCATGACTGT

ATTCCATGAGCACTATAATCACTATCATAGTAACACATTAGGAGAGAATTCTCAT

TTCTAAATCCAATATAATTTATCACCCATTAGTTCATACTCTACTGCTTTGATTGC

TTTTCTTTGGTTGTGGCTACCTGCATACAGCAGTAAAGTTTCAGAAAAACTGAAG

TCGCAAAAGGTCAATTACTCAATGAAGGAAAGATAAACCATTGCATTGGGGGAC

TAGAAGATACTTTTAAAAGTTCTCAGATTATCAATTTAATGATGTGTTTCTATGTA

GTGAATAATGCCTTAAATTCTTGCCAAGAGTATTTAGAAGGAAGTTGTCAGAAGT

ATATCAGCTAACTCATTTTTTTTTATATCACTGCTAATGGTGTCATTCACACATTG

TGC A ACCCATA ATTCCAGATTTA ATTCT ACCA A A A A AT ATAGGTCATTGC A A A AT

GCCATATTAAAACTGCCAATGCATGACAGGAAGATGGGGATGCAGACAAAGCAA

AGGATGACACCAATTCCTTTTTTAAAGAAGCAAGATAGGGATTGGACAAAAAGG

CTGAGCCATTTTTAATGGATACTTTTGAGGGAGTGTTAATTCCAATTTAATTAAA

ATGATGCATTAATTTAAAATTGGGATAACTGGTTGCCCTCGACTGCACCTGGGTT

GCGCCAGTGCTCTCGGATTAACCTAATTGTACAGAGGTGCCCTTGTTTTCTAACTT

CATGCACAAAGCATTGGAAATTATTTGTTTGCTTTTTCTTTTCCAAGTAAATCTTT

TTCCAGTTATGCAAAAGGGAAGTTTGAGGCAATGGTTAAAGGCACTTAAGTTATA ATTATTGCTGTTATCATTAACATTAAGCACGGGTATGGCTTTGTTGCAAGTTACCC ACCTACACCTGCAAATCTCTCTTGCTAGCACACGCCCCAGCTCTCTCCACCCGCA GTGGTCCGTGGCTGGACCGCTTTAAGTCACTGAGCGGGCTGGGCTCTGAAGGAG GTCGGTCCCGCTCCTCCCAGACCCAAGCGTAGGGCTAGGGAAAAGCTAGGCGGG AAGGTCATTGCACTCCCAGGCCCCAGGAAAAGGGCCCAGGGTCTCATCATCTCTT ACTTTCGGGCAAAACTTCCCACATCGCGACCTTCCCTCCCTGGGGCACTCTGAGA ACACACCCAGTCACCTAGCGCGCTCCCCAGAAGTCGGCTTGGCACACAGCGCAC CCCAGCGGCCGCGCGGCCTCCTTCCAGCCGCCGCCACTTGGCTTCCGGAGAGCTC GCCGGGCGCTGCCGCCGCCGCCGCCGCCGCCTCCTGGGAACCAGGGGACTGAAG AGCCTGCGAGAGCGGAACACTGCCGGACCCCGGGTGGGGGGGCGCAGCAGCTG CGCCTGGCCCCGCCCACCACACCTGGGCGCCCGTAGAACCGCGCGGGGCGGGGC GGGGCAGGAGGCTGGCCTGGCGCTCCGGCCGCTTTGTCGAAAGCCGGCCCGACT GGAGCAGGACGAAGGGGGAGGGTCTCGAGGCCGAGTCCTGTTCTTCTGAGGGAC GGACCCCAGCTGGGGTGGAAAAGCAGTACCAGAGAGCCTCCGAGGCGCGCGGT GCCAACCATG -3’

Claims

We claim:

1. A personalized treatment method for preventing a worsening of and treating congestive heart failure, comprising testing for susceptibility of the treatment by determining a presence of SNPs (single nucleotide polymorphisms) mutations in at least two of four biomarkers selected from the group consisting of G -protein b-3 (GNB3) subunit genotype, endothelial nitric oxide synthetase (NOS3), glutathione S-transferase m type 1 (GSTMl), and phosphor diesterase-5 (PDE5) in a particular patient: and administering a sustained release pharmaceutical composition of at least one hydralazine compound or a pharmaceutically acceptable salt thereof.

2. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1, wherein the hydralazine compound is hydralazine hydrochloride.

3. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1, wherein the sustained release pharmaceutical composition further comprises an isosorbide dinitrate/mononitrate compound.

4. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1, wherein the GNB3 gene having a C to T mutation at position 825 from the start codon is SEQ ID NO. 5.

5. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1, wherein the NOS3 SNP is in a promoter sequence of eNOS gene having a T-to-C mutation at position -786 (SEQ ID NO. 6).

6. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1 , wherein the wiki type sequence of GSTMl gene (SEQ ID NO. 3) is lacking.

7. The personalized treatment method for preventing a worsening of and treating congestive heart failure of claim 1, wherein the phosphor diesterase-5 (PDE5) SNP the 5’- flankmg regulatory region of PDE5A gene having a G to T mutation at position -1255 from the start codon is provided as SEQ ID NO. 7.

8. An extended release formulation comprising from about 2 to about 30% (by weight) of isosorbine dinitrate, from about 4 to about 50% (by weight) of hydrazine hydrochloride, from about 20 to about 70% (by weight) of a polyhydroxylated polymeric material selected from the group consisting of methy!eeliulose, hydroxyethylcellulose, hydroxypropyl methylcell ulose, poly vinyl acetate, microcrystalline cellulose, and combina ti on s thereof.

9. The extended release formulation of claim 8, wherein the extended release formulation further comprises from about 5% to about 15% (by weight) of lactose.

10. The extended release formulation of claim 8, wherein the extended release formulation further comprises from about 2% to about 8% (by weight) of magnesium stearate.

11. The extended release formulation of claim 8, wherein the extended release formulation further comprises fro about 5% to about 15% (by weight) of providone.

12. The extended release formulation of claim 8, wherein the extended release formulation further comprises from about 2% to about 8 (by weight) of propylene glycol.