Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“A NOVEL CHIMERIC PEPTIDE EXHIBITING THERAPEUTIC EFFECTS AGAINST CARDIAC HYPERTROPHY”

in the name of UNIVERSITY OF MADRAS an Indian National having address at UNIVERSITY OF MADRAS, CHEPAUK, CHENNAI, CHENNAI – 600005, TAMIL NADU, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:

The present invention generally relates to the field of pharmaceuticals. More particularly, the present invention relates to a novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy.

BACKGROUND OF THE INVENTION:

A family of related peptides has been invented that works in concert to achieve salt and water homeostasis in the body. These peptides, termed natriuretic peptides for their role in moderating natriuresis and diuresis, have varying amino acid sequences and originate from different tissues within the body. This family of natriuretic peptides consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), Dendroaspis natriuretic peptide (DNP), and urodilatin (URO, or ularitide). Their tissue-specific distribution is as follows: heart (ANP, BNP, and DNP); brain (ANP, BNP, and CNP); endothelial cells (CNP); plasma (DNP); and kidney (URO). These peptides are constituents of a hormonal system that plays a critical role in maintaining an intricate balance of blood volume/pressure in the human body. For instance, urodilatin, a close analog of ANP secreted by kidney tubular cells, promotes excretion of sodium and water by acting directly on kidney cells in the collecting duct to inhibit sodium and water reabsorption.

There are reports available in the state of arts revealing the existence of natriuretic peptides.

US20060264376A1 discloses the use of a natriuretic peptide, such as urodilatin, for treating a patient suffering from heart failure, such as acute decompensate heart failure. Preferably, a composition comprising an effective amount of urodilatin is intravenously administered to the patient continuously through a time period of at least 24 hours and up to 120 hours, preferably at least 48 hours.

US6407211B1 discloses an isolated and purified peptide fragment of Dendroaspis natriuretic peptide (SEQ ID NO:10), wherein the N-terminus of the peptide fragment does not include the sequence Glu-Val-Lys-Tyr-Asp-Pro-Cys-Phe-Gly-His-Lys-Ile-Asp-Arg-Ile-Asn-His-Val-Ser-Asn-Leu-Gly (SEQ ID NO:11), and wherein said peptide fragment has a biological activity selected from the group consisting of vasodilation, natriuresis diuresis and renin suppression.

DE19951471A1 discloses a the use of urodilatin for treating chronic kidney failure by stimulating the residual kidney function

US20130244937A1 discloses a Therapeutic compositions containing chimeric natriuretic peptides for treating chronic kidney disease alone, heart failure alone, or chronic kidney disease with concomitant heart failure are described. The therapeutic compositions have enhanced stability characteristics to facilitate storage and delivery by provisioning apparatuses under conditions of elevated temperature and mechanical stress. Methods for increasing the stability of therapeutic compositions containing chimeric natriuretic peptides are further described.

Despite widespread natriuretic peptides disclosed in the above prior arts possessing an array of properties that are useful in the diagnosis, prognosis, and treatment of cardiovascular disease and kidney disease, their clinical potential is limited by low bioavailability and unwanted properties such as excessive hypotension and also potential shortfall of having toxic side effects, rapid clearance and lack of local drug delivery. An important challenge is the rapid degradation of natriuretic peptides by peptidases, such as NEP (neprilysin) and IDE (insulin-degrading enzyme), which necessitates intravenous infusion, thus limiting the ability for chronic administration.

Nevertheless, there remains a desperate need for renal-preserving and cardiac enhancing effective therapeutic strategies for an increased heart failure population and in high-risk patients with hypertension.

Hence an attempt has been made to develop a synthetic chimeric peptide to harness the valuable multisystem physiological effects of the native peptides while minimizing or ideally eliminating any deleterious influences overcoming above said drawbacks.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy.

Another object of the present invention is to develop a novel chimeric peptide exhibiting improved receptor activation and stability against peptidases.

Yet another object of the present invention is to develop a novel chimeric peptide comprising of peptide defined by sequence ID no 1 along with C-terminus of DNP of sequence ID no 2coupled to the ring structure of CNP of sequence ID no 3 and N-terminus of ANP of sequence ID no 4coupled to the ring structure of CNP of sequence ID no 3.

Yet another object of the present invention is to develop a novel chimeric peptide to impart natriuretic activity, vascular endopeptidases resistant activity, potent vasorelaxant activity, enhanced diuresis, and reduction in systemic blood pressure.
Yet another object of the present invention is to study NPR-A receptor activity of the novel chimeric peptide.

Further object of the present invention is to utilize the developed novel chimeric peptide for the treatment of cardiac hypertrophy.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts chimeric peptide (EVNP) of the present invention from native natriuretic peptide.

Figure 2 depicts multiple sequence alignments for chimeric and natriuretic peptides (Conserved residues are highlighted in red color).

Figure 3 depicts molecular docking of ANP natriuretic peptide with NPR-A receptor.

Figure 4 depicts ANP natriuretic peptide.

Figure 5 depicts molecular interaction between EVNP and NPR-A receptor.

Figure 6 depicts cGMP quantification of novel chimeric peptides (CP) in control and experimental group of H9c2 cells.

Figure 7 depicts cGMP quantification of only novel chimeric peptides (CP) treated cells.

SUMMARY OF THE INVENTION:

The present invention discloses a novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy. The novel chimeric peptide of the present invention comprising of peptide defined by sequence ID no 1.

The present invention also discloses formulation exhibiting therapeutic effect against cardiac hypertrophy comprises of peptide defined by sequence ID no 1 and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy. The novel chimeric peptide of the present invention comprising of peptide defined by sequence ID no 1.

The novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy of the present invention comprising of C-terminus of DNP of sequence ID no 2at Proline 24th position coupled with the ring structure of CNP of sequence ID no 3 to cysteine at 23rd position and N-terminus of ANP of sequence ID no 4at Serine 6th position coupled with the ring structure of CNP of sequence ID no 3to cysteine at 7th position to impart natriuretic activity, vascular endopeptidases resistant activity, potent vasorelaxant activity, enhanced diuresis, and reduction in systemic blood pressure.

The developed chimeric peptide is then subjected to in silico, in vitro and in vivo studies to ascertain its efficiency.

In silico computational approaches for the rational design of designer natriuretic peptides

A prototype designer natriuretic peptide is mentioned in Figure 1, wherein the ring structure of CNP is attached to the N-terminus of ANP and C-terminus (15 aa) of DNP. The CNP is more susceptible to proteolytic digestion and lacks the natriuretic property due to the absence of the C-terminus. In the chimeric peptide of the present invention, the C-terminus of DNP is attached to the ring structure of CNP, to impart natriuretic property while making the chimeric peptide resistant to vascular endopeptidases. In addition, the ring structure of CNP and the N-terminus of ANP, would impart preferential binding to the NPR-B and NPR-A receptors, respectively, resulting in potent vasorelaxant activity, enhanced diuresis, and subsequent reduction in systemic blood pressure.

Sequence Alignment: The modeled Natriuretic peptides (NPs) [ANP, BNP, DNP] and Chimeric peptides (EVNP) [CP, CNP and CDNP] was performed multiple sequence alignments. It was observed that the chimeric peptide (EVNP) has conserved residues CFG-DRI-S-LGC which is also similar to other natriuretic peptides (Figure 2).

Molecular Protein-Protein Docking (PPD):

The binding interactions analysis of chimeric peptide (EVNP) with NPR-A receptor and ANP peptide with NPR-A then carried out for comparison of activation mechanisms.

From the PPD results, the total binding energy of ANP natriuretic peptide has-314.68 kcal/mol and it molecular interactions showed in Table 1. ANP peptide forms four hydrogen bond interactions with Phe188:A, Glu119:A, Arg174:B and Asp160:B homo dimer of NPR A receptor. In the case of EVNP chimeric peptide of the present invention have the binding energy of 490.17 kcal/mol and it found to have eleven hydrogen bond interactions such as Asp118:A, Met173:A, His148:A, Phe425:A, Pro421:A, Arg30:A, Cys423:A, Cys60:B, Gly159:B, Leu158:B and Glu187:B, respectively. From the above molecular docking results, the chimeric EVNP peptide shows better binding free energetic and favorable interactions compare than ANP peptide. These results indicate that newly designed chimeric peptide has potential binding affinity with NPR-A receptor and it activates the mechanisms. The detailed molecular interactions are shown in Figure 3, 4 & 5.

Table 1: Binding free energy and molecular interaction of NPR-A receptor with ANP and EVNP peptides

Peptide Complexes
?G Total binding free energy
(kcal \ mol)
NPR-A - ANP -314.68
NPR-A - EVNP -490.17

In vitro experimental Approach:

To determine potency and downstream signaling of chimeric peptides (EVNP) in vitro culture studies are performed on adult cardiac myocytes cells isolated from the constricted rat hearts. NPR-GC A/B receptor specificity and occupancy studies are done using HEK293 cells over expressing either NPR-GC A or NPR-GC B receptors. The key downstream signalling genes/proteins is studied using ELISA, while mRNA and protein levels are measured by RT-RT-PCR and Western blotting, respectively. The control myocytes is stimulated with a hypertrophic agonist Isoproterenol (ISO) or native peptides or chimeric peptides (EVNP). In addition, hypertrophy markers gene expressions such as c-fos, c-myc, ß- myosin heavy chain (ß-MHC) and skeletal actin is analyzed to confirm the induction of cardiac hypertrophy. In the second set of experiments, normal and aortic constricted animals are administered with chimeric peptides (EVNP), and the NPR’s expression at the mRNA and protein levels are measured by RT-PCR, and Western blotting, respectively.

Cell culture: Adult cardiac myocytes cells are grown in DMEM-F12 medium supplemented with 15% fetal calf serum. The cultures are maintained at 37°C in an atmosphere of 5% CO2/95% O2. Cardiomyocytes cell cultured in 24-well plates at a 2 x 104 cells/well density. Control and treated myocytes will be stimulated with increasing concentrations of agonist (isoproterenol) alone or combined with ANP/Chimeric peptides (EVNP) and VP (valporic acid). After 24 or 48 h, mRNA expression studies and Western analysis is then carried out. The expression results of ANP, BNP, ß-MHC, and a -SK will be normalized with GAPDH. Reverse transcriptase-PCR: To measure the Npr1, Npr2, and Npr3 mRNA expression, RT-PCR assay will be performed using the Superscript one-step RT-PCR kit from Invitrogen.

EVNP co-treatment with ANP improves the intracellular cGMP levels in ISO-treated H9c2 cells: Figure 6 shows the ELISA analysis of intracellular cGMP levels in control; ISO, ISO+ ANP, ISO+ANP +VA, ISO+EVNP, and ISO+VA+EVNP treated cells. Figure 7 shows the ELISA analysis of intracellular cGMP levels in EVNP treated cells. The cGMP levels were drastically reduced in ISO-treated cells as compared with control group of cells. ANP co-treatment failed to enhance the cGMP levels in ISO-treated cells. Further, the ISO-induced altered cGMP levels was found to be reverted upon EVNP treated cells. Interestingly, the intracellular cGMP was further elevated in the presence of VP (valproic acid) treated cells as compared with ISO-treated cells respectively.
Thus, the from the above studies it is concluded that the novel chimeric peptide (EVNP) of the present invention be a suitable alternative to the existing peptides in achieving a high success rate of restoring heart function with lesser side effects as well with minimal invasiveness. Moreover, it has the combined beneficial properties of 2 or 3 distinct natriuretic peptides with a biological profile that the native peptides and with improved bioavailability.

In one of the preferred embodiments, the present invention shall disclose a novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy. The novel chimeric peptide of the present invention comprising of peptide defined by sequence ID no 1.

As per the invention, the novel chimeric peptide of the present invention exhibiting therapeutic effect against cardiac hypertrophy comprising of C-terminus of DNP of sequence ID no 2at Proline 24th position coupled with the ring structure of CNP of sequence ID no 3 to cysteine at 23rd position and N-terminus of ANP of sequence ID no 4at Serine 6th position coupled with the ring structure of CNP of sequence ID no 3to cysteine at 7th position to impart natriuretic activity, vascular endopeptidases resistant activity, potent vasorelaxant activity, enhanced diuresis, and reduction in systemic blood pressure.

In further preferred embodiment, the present invention shall disclose a formulation exhibiting therapeutic effect against cardiac hypertrophy comprises of peptide defined by sequence ID no 1 and a pharmaceutically acceptable carrier.

Abbreviations:
Npr1/ Nppa: Natriuretic peptide receptor 1
Npr2/ Nppb: Natriuretic peptide receptor 2
GC-A: Guanylyl Cyclase-A
GC-B: Guanylyl Cyclase-A
ANP: Atrial Natriuretic Peptide
BNP: Brain Natriuretic Peptide
CNP: C-type Natriuretic Peptide
GAPDH: Glyceraldehyde phosphate dehydrogenase
Sequence ID no 1: NH2-Ser-Leu-Arg-Ser-Ser-Cys-Phe-Gly-Leu-Lys-Lys-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys-Pro-Ser-Leu-Arg-Asp-Pro-Arg-Pro-Gln-Ala-Ser-Thr-Ser-Ala-COOH
Sequence ID no 2: EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA
Sequence ID no 3: YKGANKKGLSKGCFGLKLDRIGSMSGLGC
Sequence ID no 4: SLRRSSCFGGRMDRIGAQSGLGCNSFRY
Although the invention has now been described in terms of certain preferred embodiments and exemplified with respect thereto, one skilled in art can readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the scope of the following claims.
, Claims:WE CLAIM:

1. A novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy, comprising of peptide defined by sequence ID no 1.
2. The novel chimeric peptide exhibiting therapeutic effect against cardiac hypertrophy of claim 1 comprising of C-terminus of DNP of sequence ID no 2 at Proline 24th position coupled with the ring structure of CNP of sequence ID no 3 to cysteine at 23rd position and N-terminus of ANP of sequence ID no 4 at Serine 6th position coupled with the ring structure of CNP of sequence ID no 3 to cysteine at 7th position to impart natriuretic activity, vascular endopeptidases resistant activity, potent vasorelaxant activity, enhanced diuresis, and reduction in systemic blood pressure.
3. A formulation exhibiting therapeutic effect against cardiac hypertrophy comprises of peptide defined by sequence ID no 1 and a pharmaceutically acceptable carrier.

Dated this 19th day of DEC 2023

For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
IN/PA 1477