FIELD OF THE INVENTION:

The present invention relates to the field of biotechnology. More specifically, it provides a brain natriuretic peptide liposomal composition and its use in the treatment of congestive heart disease and related conditions.

BACKGROUND AND PRIOR ART:

Brain natriuretic peptide (BNP) also known as B-type natriuretic peptide is an endogenous peptide/a neurohormone that belongs to a family of peptides that are involved in cardiovascular, renal, and endocrine homeostasis. It is released as a pre-proBNP peptide of 134 amino acids and is cleaved into proBNP (108 amino acids) and a signal peptide of 26 amino acids. ProBNP is subsequently cleaved into BNP (32 amino acids) with one disulphide bond and the inactive N-terminal proBNP peptide (NT-proBNP; 76 amino acids). The release of BNP into the circulation is directly proportional to the ventricular expansion and volume overload of the ventricles and therefore reflects the decompensated state of the ventricles. BNP was originally identified in extracts of porcine brain, and was discovered in 1988 (Sudoh, Kangawa et al. 1988), almost a decade after the discovery of atrial natriuretic peptide (ANP).

BNP binds to the natriuretic peptide receptor A (NPR-A), a membrane bound protein on the cell surface and triggers the synthesis of cGMP in the cytosol by guanylate cyclase. It is through this secondary messenger that BNP accomplishes the cardio-vascular, renal, and endocrine effects with which it is associated.

Therapeutic biomolecules such as peptides and proteins are usually characterized by a large size, short plasma half-life, high elimination rate, limited ability to cross cell membranes and poor bioavailability through intestinal administration. Thus, the three major barriers to peptide delivery are the enzymatic barrier sourced from the host luminal and membrane bound peptidases, the immunological cells, and the physical barrier of the epithelial cells. The BNP molecule like other peptides can be produced by several methodologies which include recombinant DNA technology.

Similar problems are encountered with the administration of the rhBNP peptide, as well. The subcutaneous administration of rhBNP leads to an increase in the plasma BNP levels, but the proteolytic degradation of these rhBNP molecules leads to a resultant decrease in the efficacy/bioavailability of the BNP molecules. Native BNP molecule has a relatively short half-life in circulation; and they are hydrolysed by neutral endopeptidases. The circulating half life of BNP is approximately 20 minutes. Previously reported BNPs are administered intravenously, usually by bolus, followed by IV infusion. For most adults and the elderly, a normal dosage is 2 micrograms/kilogram followed by a continuous IV infusion of 0.01 mcg/kg/minute. This may be increased every three hours for a maximum of 0.03 mcg/kg/min. Thus, there is a need for frequent injection of drug over a long therapeutic period. So, there has been a long standing need to increase the half life of BNP to ensure the increased bioavailability of these molecules.

Various approaches have been examined to overcome the delivery problems of proteins and peptides and use of lipid carrier is one approach. Liposomes have been well known as vehicles to carry molecules to desired target sites. A liposome encapsulates the molecules in aqueous solution, inside a hydrophobic membrane; and hence dissolved hydrophilic solutes cannot readily pass through the lipids. Hydrophobic chemicals can be dissolved into the membrane, and in this way liposome can carry both hydrophobic molecules and hydrophilic molecules. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as the cell membrane, thus delivering the liposome contents. By making liposomes in a solution of DNA or peptides or any other molecule (which would normally be unable to diffuse through the membrane) they can be (indiscriminately) delivered past the lipid bilayer.

The unique properties of liposomes are utilized to form efficient drug delivery systems which include improved penetration and diffusion of active ingredients, selective transport of active ingredients, longer release time, greater stability of the encapsulated molecule, reduction of unwanted side effects, and high biocompatibility.

Hence the present invention aims to provide a liposome encapsulated rhBNP that improves the bioavailability of the BNP and imparts a longer half life to circulating BNP molecules.

OBJECT OF THE INVENTION

The object of the invention is to develop a liposome encapsulated rhBNP, wherein the encapsulated BNP molecule exhibits an increased circulating half life, increased bioavailability and a prolonged duration of effect as compared to the unencapsulated BNP molecules.

Brief description of figures;

Figure 1: Chemical structure of N, N-ditetradecyl-N-di-2-hydroxyethylammonium chloride (DTDEAC).

Figure 2: Shows the amino acid sequence of a modified N-terminal growth hormone on the lower line and the wild-type N-terminal on the upper line used as a fusion partner to produce B-type natriuretic peptide.

Figure 3: Represents the construction scheme of Pgh

Figure 4: Represents the construction scheme of pGFB-1

Figure 5: Represents the construction scheme of pGFB-2

Figure 6: Shows HPLC Calibration Graph for Aqueous BNP

Figure 7: Shows the cGMP expression levels in HAE Cells treated with naked rhBNP and rhBNP entrapped in formulation 1 and vehicle control DTDEAC

Figure 8: Shows the plasma cGMP expression levels in Wistar Rats (n = 3) at different time intervals after administration of saline, naked rhBNP, liposomal formulation 1 and its vehicle control (pure liposome with no entrapped rhBNP).

Figure 9: shows the plasma BNP levels in wistar rats at different time intervals.

DETAILED DESCRIPTION OF THE INVENTION:

Accordingly, an object of the invention is to develop a liposome encapsulated rhBNP,
wherein the liposome encapsulated rhBNP has an increased circulating half life, increased bioavailability and a prolonged duration effect as compared to the unencapsulated BNP molecules.

The ciculating half life of uncapsulated BNP is approximately 20 mins; and the stability of the same in EDTA at 2-8 °C is for 24 hours.

Another object of the invention is to provide a more effective composition which contains liposome encapsulated rhBNP and methods for treatment of congestive heart failure. More specifically, it is one of the objects of the invention to improve the therapeutic effectiveness of rhBNP by delivering the drug in a liposomal agent.
Contrastingly, the presently described liposome encapsulated rhBNP is stable at 4 °C for one month and the circulating half life is 30 min-3 hr.

These preparations function to protect the BNP against proteolytic enzymes, and thereby permit the effective use of this agent as an agonist of human natriuretic peptide receptor A. As a result of this agonistic activity, there is enhanced production of cGMP stimulating activity relative to the corresponding unconjugated natriuretic compound. The enhanced cGMP stimulating activity is in the range of 50%-80% when compare to unconjugated matriuretic compound.

The BNP molecule to be encapsulated is produced by recombinant DNA technology. Further, another object of the invention is to provide a liposomal formulation, wherein the rhBNP molecule retains its activity of stimulating cGMP and acquires the property of sustained release.

Another object of the invention is to provide a liposomal formulation that comprises of a lipid moiety; and biologically active recombinant human brain natriuretic peptide (rhBNP), wherein the rhBNP comprises of a NPR binding site.
The selection of lipids is generally guided by the consideration of (a) drug-release rate (b) drug entrapment efficiency (c) liposome toxicity (d) bio-distribution and targeting properties.

The important compositional factors of the lipids which have an influence on the in vivo drug-release rates are chain length, degree of unsaturation, and head group charge and side groups in the lipids. The dependence of drug release rate on lipid composition is due in part to differences in the rate of exchange of amphipathic drug with the outer liposome bilayer, and in part to the differing stability of liposomes having different lipid compositions.

Another factor to be considered while selecting a lipid composition is the drug-entrapment efficiency, and it refers to the total amount of drug which can be loaded into liposomes and is expressed as a ratio of drug per mole per liposome molecule. Entrapment efficiency achieved by the method of invention is 50-60% which is quantified by HPLC. High entrapment efficiency is desirable both in terms of preparation costs and for maximizing the amount of drug which can be delivered in liposomal form in a given volume of liposomes.

The lipid moiety for the entrapment of rhBNP to develop a liposome encapsulated rhBNP of the invention is a cationic material namely, N, N-2-hydroxyethyl-N, N-di-n-tetradecylammonium chloride (DTDEAC) and a co-lipid. DTDEAC may be used in pure form or in combination with co-lipid. The co-lipid may be selected from the group comprising sterols, neutral phosphatidyl ethanolamine, and neutral phosphatidyl choline. The co lipid may be Di-oleylphosphatidylethanolamine (DOPE), more preferably, the co-lipid is cholesterol.

The DTDEAC:colipid ratio may be in the range of 1:1 to 4:1, more preferably, 2:1. The rhBNP molecule to be encapsulated may be produced as a physiologically active polypeptide in the form of inclusion bodies, and the same may be recovered in a single step without the involvement of any chaotropic agents.

The therapeutic compound in the liposome composition of the invention is rhBNP. The concentration of the rhBNP in the liposomes is typically between 25-30 mole percent with respect to the total liposome lipids. The ratio of the drug to lipid in terms of moles % is 4.35: 1.

Methods followed for preparing drug-containing liposome suspensions generally are conventional liposome preparation methods such as reviewed Lasic DD, Papahadjopoulos D (1995) Liposomes revisited. Science 267:1275-1276.

In one preferred method, equimolar amounts of lipids were dissolved in suitable organic solvent or solvent system and dried in vacuo or under an inert gas to a lipid film. The concentration of the drug in the lipid solution may be included in molar excess of the final maximum concentration of drug in the liposomes, to yield maximum drug entrapment in the liposomes. In particular, rhBNP is typically included at a mole ratio, with respect to the other lipid components making up the liposomes of between 25-30 mole percent.

The liposome suspension may be sized by sonication to achieve a selective size distribution of vesicles in a size range of about 100-200 nm measured by dynamic laser light scattering experiment. The sizing serves to eliminate larger liposomes and to produce a defined size range having optimal pharmacokinetic properties.

Free drug, i.e., drug present in the bulk aqueous phase of the medium, is preferably removed to increase the ratio of liposome-entrapped to free drug. Several methods are available for removing free drug from a liposome suspension. One preferred procedure for removing free rhBNP uitilizes gel Alteration using sephadex-50.

EXAMPLES:

The following examples are for the purpose of illustration of the invention and not intended in any way to limit the scope of the invention.

Example 1: Preparation of liposomal formulation

Formulation 1: Di-n-tetradecyl-di-2-hydroxyethylammonium chloride (DTDEAC) is a potent lipofection reagent. "Novel Series of Non-Glycerol-Based Cationic Transfection Lipids for Use in Liposomal Gene Delivery", Rajkumar Banerjee et.al J. Med. Chem., 1999, 42 (21), pp 4292-4299'). To increase the activity of BNP, it was encapsulated in DTDEAC liposomal formulation.

Formulation 1: Equimolar amounts of DTDEAC and Cholesterol (530 and 386 ug, respectively) were dissolved in chloroform and dried under a stream of N2 gas and vacuum-desiccated for 6 h to remove residual organic solvent. The dried lipid film was hydrated in 1 mL of 6 mg/mL aqueous BNP solution for 12 h. The concentration of each of DTDEAC and cholesterol was 1 mM. Liposomes were vortexed to remove any adhering lipid film and bath sonicated for 2 min to form unilamellar vesicles. Liposomally entrapped BNP was separated from any unentrapped BNP by Gel Filtration using sephadex-50. Fractions of two column volume were collected and concentrated using centrifuge. The concentration of liposomally entrapped BNP were measured by analytical HPLC using standard calibration graphs of BNP (Figure 6) constructed using pure aqueous BNP solutions of varying concentrations. The observed entrapment efficiency for this formulation 1 was found to be 50%. The concentration of BNP in Formulation 1 was measured to be 3.0 mg/mL based on quantitative analytical HPLC analysis.

Example 2: Methods for estimating liposomal entrapment efficiencies.

The HPLC calibration graph (Peak Areas vs. Cone, Figure 6) for estimating the liposomal entrapment efficiencies was constructed by injecting aqueous solutions containing 10, 20, 40, 60 and 100 ug/mL BNP using a reversed phase analytical column. The concentrations of BNP in liposomal solutions were directly measured by substituting the observed peak areas for liposomal solutions in this calibration graph (Figure 1). BNP-entrapment efficiencies of the liposomal solution was found to be 50%. HPLC conditions:

Buffer A: Deionized water with 0.1% Triflouro acetic acid Buffer B: Acetonitrile with 0.1% Triflouro acetic acid

Flow rate: lml/min Instrument: Agilent Column: Reverse phase C18 column

Example 3: Invitro activity of cGMP in HAEC cells:

HAEC cells (Human aortic endothelial cells), procured from Lonza, USA, and were cultured in EBM-2 (Endothelial basal media-2) with EBM-2 bullet kit containing various growth factors for HAEC as prescribed by Lonza USA. Approximately, 106 cells per well were seeded in 24 well plate. The next day media was removed. Different concentrations of pure BNP peptide controls, the liposomally entrapped BNP formulation 1 (DTDEAC: Choi) along with the vehicle control (liposome without rhBNP) were added to the cells in serum free media. Incubation at 37 °C was stopped after 60 min by lysing cells using lysis buffer (Assay Designs, Ann Arbor, Michigan). The released intra cellular cGMP was measured using an ELISA based system (Assay Designs, Ann arbour, Michigan) which quantifies cGMP via a competitive immunoassay in 96-well format. Lysates were added to the IgG coated microplate followed by the addition of cGMP conjugated alkaline phosphatase. Plates were incubated for 2 h at room temperature followed by four times washes with washing buffer (detergent containing TBS) supplied with the kit. pNpp (para-nitro phenyl phosphate) substrate solution added to it. The absorbance of the yellow para-nitro phenol formed was measured in an ELISA reader at 405 nm. The color intensity decreased with the increased levels of cGMP secreted by the HAEC. Native rhBNP was tested as positive control in the experiment.

Example 4: Evaluation of the systemic potential of formulation 1 in Wistar rats (n = 3):

The plasma cGMP levels were measured using commercially available ELISA assay kit (Assay Designs, Ann arbour, Michigan, USA). Male Wistar rats (each weighing around 200-250 g, procured from NIN, Hyderabad) were divided into four groups containing three animals in each group. Normal saline was injected (i.v.) to the first group. Native rhBNP proteins (20-50 ug of protein per Kg body weight) were intravenously injected to the second group. Liposomal formulation 1 (DTDEAC: CHOL=l:l, described above) containing entrapped rhBNP was administered (i.v.) to the third group. Vehicle control liposomes (liposome without any entrapped rhBNP) were administered to the fourth group. Blood samples were collected from each animal in pre-loaded heparin eppendorf tubes after 30 min, 1 h, 2 h, 3 h and 4 h. Plasma was collected by centrifuging the blood samples at 4000 rpm for 10 min. 100 uL of each plasma sample was directly added to the immunoglobin coated microplate (96 well plate) followed by the addition of cGMP alkaline phophatase conjugated. Plates were incubated for 2 h at room temperature followed by four washes with washing buffer. Substrate pNPP (p-nitro-phenyl phosphate) was added and absorbance of the liberated p-nitrophenol (405 nm) was measured. Results. The observed cGMP levels at different time intervals are shown in Figure 7. Compared with naked BNP, formulation 1 was found to be more efficacious. Importantly, formulation 1 was capable of producing cGMP for a much longer period in circulation than naked BNP (Figure 8). These results demonstrate therapeutic potential of formulation 1 in treatment of congestive heart failure.

Example 5; Evaluation of the plasma of BNP of formulation 1 in Wistar rats (n = 3): Experimental procedure: The plasma BNP levels were measured using commercially available ELISA assay kit (BIOMEDICA). Male Wistar rats (each weighing around 200-250 g, procured from NIN, Hyderabad) were divided into two groups containing three animals in each group. Native rhBNP proteins (30 mg of protein per Kg body weight) were intravenously injected to the second group. Liposomal formulation 1 (DTDEAC:CHOL=l:l) containing entrapped rhBNP was administered to the second group. Blood samples were collected from each animal in pre-loaded heparin eppendorf tubes after Omin, 30 min, 60 min, 90min, 120min 150min and 180min. Plasma was collected by centrifuging the blood samples at 4000 rpm for 10 min. 100 uL of each plasma sample was directly added to the anti BNP antibody coated microplate (96 well plate) followed by the addition of conjugated BNP. Plates were incubated for 2 h at room
temperature followed by four washes with washing buffer. Substrate was added and
absorbance was measured immediately at 450nm.

Results. The observed plasma BNP levels at different time intervals are shown in Figure 9.

Compared with naked BNP, formulation 1 was found with sustained release of plasma
BNP

Example 6: Expression of rhBNP
a) Construction of by utilizing a DNA sequence encoding the hydrophobic N-terminal 62 amino acids region of growth hormone. The hydrophobic region in the fusion partner promotes the formation of inclusion bodies. The free cysteine in the N-terminal region of fusion partner may be altered to avoid disulfide bond formation with the peptide if there is any disulfide bridge in the peptide.

In the vector of the invention (Figure 2), the N-terminal growth hormone encoding DNA sequence is under the control of a regulatory sequence which is capable of directing expression of the fusion protein in the host cell. The promoter employed may be a T7 promotor or lacZ promoter. A preferred promoter is the E. coli T7 promotor in Escherichia coli. This promoter initiates transcription of the fusion protein encoding DNA sequence in the presence of IPTG in the medium.

b) Synthesis and cloning of a gene encoding a fusion protein to B-type natriuretic peptide: The hBNP gene assembly of synthetic gene was performed from component oligo nucleotides. The hBNP nucleotide sequence (GeneBank Accession No BC025785) was designed based on the E.coli rare codon preference. Factor Xa site upstream to BNP encoding sequence was generated by use of oligonucleotide primers. The following primers were used in PCR reaction.

PI ( 5'- CC GGA TCC ATT GAG GGT CGC AGC CCG AAA ATG- 3'), P2 ( 5'-GTT
CAG GGC TCT GGC TGC TTC GGC CGT AAA ATG GAC CGT ATC AGC -3'), P3 (
5'-TCC TCC AGC GGC CTG GGC TGC AAA GTT CTG CGT CGT CAC TAA TAG-
3'), P4(5'- CCT GAA CCA TTT TCG- 3'), P5 ( 5'-TGG AGG AGC TGA TAC-3'), and
P6( 5'- CGG AAT TCC TAT TAG TGA CGA CGC AG- 3'). Equal volumes of ~lmg/ml
were mixed together and diluted with water to a final concentration of ~lng/ul for each
oligonucleotide. The final concentration 0.2ng/ ul for each oligonucleotide was used along with 20mM Tris-HCl (pH8.8),10mM for KC1, 10mM(NH4)2 SO4 , 6mM MgSO4, 0.1% (V/V) triton-Xl00, 0.lmg/ml bovine serum albumin,0.2mM each dNTP and 2.5 U Pfu polymerase. The PCR protocol for gene assembly began with an initial denaturation step for 5 min at 94°C, which is followed by 25 cycles of denaturation for 30 s at 94°C,
annealing for 60s at 56°C, and extension for 60s at 72°C. It is again followed by an
extension step for 10 min at 72°C. hBNP gene was amplified by using lul of the mixture
resulting from the gene assembly as the template and the outer most oligonucleotides PI
and P6 as primers. The hBNP gene with Factor Xa cleavage sequence was PCR amplified
using gene specific primers. The forward primer 5'-
CGGATCCATTGAGGGTCGCAGCCCGAAAATG-3'c ontains BamHl and the reverse primer 5'- CGGAATTCCTATTAGTGACGACGCAG- 3' contains EcoRl site with Stop codon. The PCR amplification cycles are as follows: Initial denaturation at 94°C 5 min; followed by 25 cycles of denaturation at 94°C for 30sec, annealing at 56°C for 1 min, extension at 72°C for 1 min, and final extention at 72°C for l0min with a final hold at 15°C.

Construction of pGFB-1 and expression of hBNP (1-32) fusion protein: The full length growth hormone gene was used as a template to amplify 186 bps growth hormone using gene specific primers. The primers used for amplification contains Ndel site in forward direction and BamHl in reverse primer.

Forward primer F: 5'- CGCATATGTTCCCAACTATTCCACTGAGT-3' and Reverse primer R':5'- CGGGATCCAGGGGTCGGGATACTTTCAGACAAACTCAA-3'. The PCR amplification cycles were as follows: Initial denaturation at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, extension at 72°C for lmin, and a final extention at 72°C for 10 min with a final hold at 10°C. The ampicillin resistent plasmid and PCR product was restriction digested with Ndel and BamHl endonucleases and ligated by using T4 DNA ligase to yield plasmid pGH. The modified plasmid (pGH) containing 7kDa growth hormone encoding tag under the control of T7 promoter was used to clone hBNP gene. The hBNP PCR product and plasmid pGH were digested individually with restriction endonucleases BamHl and EcoRl. The digested samples were ran on agarose gel. The agarose gel electrophoresis purified PCR fragment and plasmid pGH were ligated with T4DNA ligase to yield an expression plasmid pGFB-1. The expression construct with hBNP gene was sequenced by Sanger's Dideoxy method. Escherichia coli strain was transformed with recombinant plasmid containing the hBNP gene. The expression of the hBNP gene was preferably driven by a T7 promoter, which is regulated by inducing the culture with isopropyl -D- thiogalactopyranoside (IPTG) there by allowing high level of expression of the 7kDa GH-hBNP gene. Production of recombinant human B-type natriuretic peptide:

Fermentation and Harvesting:

The seed culture was prepared in LB Media from the Glycerol Stock and incubated for 7-8 hours (O.D of 1.7 to 2.3) at 35°C on rotary shaker. Fermentation was carried out in a Biostat C fermentor (B. BRAUN Germany) in fed-batch mode. Modified LB medium was used for fermentation. The temperature of the fermentation cultures was controlled at 35°C for a period of 5 hours (O.D of 13 to 16) and induced with 0.5 mM IPTG and harvested after 3 hours. The pH was controlled and adjusted to 7.0- 7.2 with 50% Ammonia solution. The rate of agitation was 300 to 700 rpm, and the dissolved oxygen (P02) was maintained above 40%-50% saturation. Foam formation was suppressed by the addition of sterilized 10% silicon based antifoam. The samples for expression analysis were collected and whole cell lysates were analyzed by SDS PAGE. The culture was centrifuged at 6600g for 10 min, the supernatant was removed and the biomass (525g) was determined by weighing the wet pellet. The cells were frozen immediately at -70°C until further analysis was performed. Cell lysis and inclusion bodies (IB) purification:

The cell pellet was thawed on ice, suspended in 787.5ml of Buffer A (50mM Tris and 5mM EDTA pH 8.0) on ice, and homogenized for 2-3 min at maximum speed. Cell disruption was done in dynamill (miltech Switzerland) with 0.5mm of Glass beads up to 80% of the total chamber volume. Cell lysate was passed at a flow rate of lOOml/min which takes about 10 mins for each cycle and the process was optimized to 3 cycles. The lysate at the inlet was maintained at 10-12°C and the sample collected at out let was maintained 15- 20°C. The IB fraction was collected by centrifugation at 15,000 g for 20 min at 4°C. The crude IB preparation was initially washed with Buffer A, then with 0.1% deoxycholate (sodium salt) in Buffer A and finally with Buffer A plus 2 M urea. Traces of deoxycholate or urea were removed by a final wash with the Buffer B (20mM Tris pH 7.5). IB pellet was collected by centrifugation at 12,000 g for 20 min at 4 °C and the IB pellet (57g) was stored at-20°C.

Solubilization of inclusion bodies:

The inclusion body was suspended in 570ml solublization buffer (20mM Tris PH 7.5,
100mM Nacl, 0.3% Sodium dodecyl sulfate (SDS)) and dissolved by stirring overnight at 4°C on a magnetic stirrer. The solution was centrifuged at 12,000g at 4 °C for 10 min. The protein concentration was measured according to the method of Lowry et al. 1975. The supernatant was then diluted with 20mM Tris PH 7.5, to adjust 0.3% SDS to less than 0.1% of final concentration.

Cleavage of GH-hBNP with Factor Xa

The fusion protein (GH-Factor Xa-hBNP) at concentration of 3-5 mg/ml is found to be
optimum for cleavage with Factor Xa at 25°C. The bovine Factor Xa (serine protease)
specifically recognizes and cleaves at the C-terminal of linker site and release the rhBNP
without any N-terminal modification. The digestion showed greater than 90% cleavage
results into 7kD GH and 3.4kD hBNP.

Purification of rhBNP by reverse phase chromatography (RPC):

GH-BNP digested mixture was further fractionated on source 30 RPC column to separate
pure peptide from other contaminants like residual 1 lkDa, 7kDa GH and other E.coli host proteins. The peptide was eluted with a linear gradient of acetonitrile. The elution profile showed two peaks. The first (I) peak contained the rhBNP and second peak contained contaminants. rhBNP peak collected was assessed by SDS- PAGE and analytical RP for purity (fig 4). SDS-PAGE analysis showed a single band with both the pools (a) and (b) where as the pool (a) was found to be 60-70 % pure when analyzed by analytical HPLC over C18 column. The 60-70% pure indicating the presence of modified forms like oxidized and deamidated forms. The pool b fractions were highly pure rhBNP which is greater than 99%. These fractions proved to be pure were dialyzed against water injection and next 20mM citric acid buffer pH 4.2. The final yield obtained by this method was approximately 200mg of pure rhBNP /liter cell culture.

Example 7:

a) Construction of Expression Vector:

The expression vector is constructed by utilizing a DNA sequence encoding the hydrophobic N-terminal 62 amino acids region of growth hormone. The hydrophobic region in the fusion partner promotes the formation inclusion bodies. The free cysteine in the N-terminal region of fusion partner may be altered to avoid disulfide bond formation with the peptide if, there is any disulfide bridge in the peptide. In the vector of the invention (Figure 2), the N-terminal growth hormone encoding DNA sequence is under the control of a regulatory sequence which is capable of directing expression of the fusion protein in the host cell. The promoter employed is a T7 promotor or lacZ promoter. A preferred promoter is the E. coli T7 promotor in Escherichia coli. This promoter initiates transcription of the fusion protein encoding DNA sequence in the presence of IPTG in the medium.

b) Synthesis and cloning of a gene encoding a fusion protein to B-type natriuretic peptide:.

The hBNP gene assembly of synthetic gene was performed from component oligo
nucleotides. The hBNP nucleotide sequence (GeneBank Accession No BC025785) was
designed based on the E.coli rare codon preference. Acid cleavage dipeptide site upstream to BNP encoding sequence was generated by use of oligonucleotide primers. The following primers were used in PCR reaction.

Fl ( 5'- CC GGA TCC GAC AGC CCG AAA ATG- 3'), P2 ( 5'-GTT CAG GGC TCT GGC TGC TTC GGC CGT AAA ATG GAC CGT ATC AGC -3'), P3 ( 5'-TCC TCC AGC GGC CTG GGC TGC AAA GTT CTG CGT CGT CAC TAA TAG- 3'), P4(5'- CCT GAA CCA TTT TCG- 3'), P5 ( 5'-TGG AGG AGC TGA TAC-3'), and P6( 5'- CGG AAT TCC TAT TAG TGA CGA CGC AG- 3')- Equal volumes of ~lmg/ml were mixed together and diluted with water to a final concentration of ~lng/ul for each oligonucleotide. The final concentration 0.2ng/ ul for each oligonucleotide was used along with 20mM Tris-HCl(pH8.8),10mM for KCl, 10mM(NH4)2 S04 , 6mM MgS04,0.1 % (V/V) triton -XI00, O.lmg/ml bovine serum albumin,0.2mM each dNTP and 2.5 U Pfu polymerase. The PCR protocol for gene assembly began with an initial denaturation step for 5 min at 94°C, which is followed by 25 cycles of denaturation for 30 s at 94°C, annealing for 60s at 56° C, and extension for 60s at 72° C. It is again followed by an extension step for 10 min at 72°C. hBNP gene was amplified by using lul of the mixture resulting from the gene assembly as the template and the outermost oligonucleotides Fl and P6 as primers. The hBNP gene with acid cleavage dipeptide sequence was PCR amplified using gene specific primers. The forward primer 5'- CGGATCC GACAGCCCGAAAATG-3' contains BamHl and the reverse primer 5'- CGGAATTCCTATTAGTGACGACGCAG- 3' contains EcoRl site with Stop codon. The PCR amplification cycles are as follows: Initial denaturation at 94° C 5 min; followed by 25 cycles of denaturation at 94° C for 30sec, annealing at 56° C for 1 min, extension at 72°C for 1 min, and final extention at 72°C for lOmin with a final holdatl5°C.

Construction of pGFB-2 and expression of hBNP (1-321 fusion protein: The full length growth hormone gene was used as a template to amplify 186 bps growth hormone using gene specific primers. The primers used for amplification contains Ndel site in forward direction and BamHl in reverse primer.

Forward primer F': 5'- CGCATATGTTCCCAACTATTCCACTGAGT-3' and Reverse primer R':5'- CGGGATCCAGGGGTCGGGATACTTTCAGACAAACTCAA-3'. The PCR amplification cycles were as follows: Initial denaturation at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, extension at 72°C for lmin, and a final extention at 72°C for 10 min with a final hold at 10°C. The ampicillin resistent plasmid and PCR product was restriction digested with Ndel and BamHl endonucleases and ligated by using T4 DNA ligase to yield plasmid pGH. The modified plasmid (pGH) containing 7kDa growth hormone encoding tag under the control of T7 promoter was used to clone hBNP gene. The hBNP PCR product and plasmid pGH were digested individually with restriction endonucleases BamHl and EcoRl. The digested samples were ran on agarose gel. The agarose gel electrophoresis purified PCR fragment and plasmid pGH were ligated with T4DNA ligase to yield an expression plasmid pGFB-2. The expression construct with hBNP gene was sequenced by Sanger's Dideoxy method. Escherichia coli strain was transformed with recombinant plasmid containing the hBNP gene. The expression of the hBNP gene was preferably driven by a T7 promoter, which is regulated by inducing the culture with isopropyl -D- thiogalactopyranoside (IPTG) there by allowing high level of expression of the 7kDa GH-hBNP gene. Fermentation and harvesting:

The seed culture was prepared in LB Media from the Glycerol Stock and incubated for 7-8 hours (O.D of 1.7 to2.3) at 35°C on rotary shaker. Fermentation was carried out in a Biostat C fermentor (B. BRAUN Germany) in fed-batch mode. Modified LB medium was used for fermentation. The temperature of the fermentation cultures was controlled at 35°C for a period of 5 hours (O.D of 13 tol6) and induced with 1 mM IPTG and harvested after 3 hours. The pH was controlled and adjusted to 7.0- 7.2 with 50% Ammonia solution. The rate of agitation was 300 to 700 rpm, and the dissolved oxygen (PO2) was maintained above 40%-50% saturation. Foam formation was suppressed by the addition of sterilized 10% silicon based antifoam. The samples for expression analysis were collected and whole cell lysates were analyzed by SDS PAGE. The culture was centrifuged at 6600g for 10 min, the supernatant was removed and the biomass (482g) was determined by weighing the wet pellet. The cells were frozen immediately at -70°C until further analysis was performed.

Cell lysis and inclusion bodies (IB) purification:

The cell pellet was thawed on ice, suspended in 1446 ml of Buffer A (50mM Tris and 5mM EDTA pH 8.0) on ice, and homogenized for 2-3 min at maximum speed. Cell disruption was done in frenchpress (miltech Switzerland) with a applied pressure of 1000 bars. Cell lysate was passed at a flow rate of lOOml/min which takes about 10 mins for each cycle and the process was optimized to 3 cycles. The lysate at the inlet was maintained at 20-22°C and the sample collected at out let was maintained 18-20°C. The IB fraction was collected by centrifugation at 12,000 g for 20 min at 4°C. The crude IB preparation was initially washed with Buffer A, then with 0.1% deoxycholate (sodium salt) in Buffer A next with Buffer A plus 2 M urea. Traces of deoxycholate or urea were removed by a wash with the Buffer B (20mM Tris pH 8.0) and finally the pellet was washed with double distilled water. IB pellet was collected by centrifugation at 12,000 g for 20 min at 4°C and the IB pellet (52g) was stored at -20 °C.

Cleavage of GH-hbnp inclusion bodies with dilute HCL at high temperature
The Inclusion bodies were diluted tol% solution with 0.015NHCL, adjusted the pH tol.7-
1.9 and fusion protein (GH-D-hBNP) concentration of 1-1.5 mg/ml was optimum for
cleavage at 85°C for 3h with a supply of inert gas and homogenous stirring of the solution throughout the reaction. The low concentration of HCL at high temperature specifically recognizes (D-S) dipetide amino acid and cleaves at the C-terminal of linker site at a reaction rate of IX and release the rhBNP without any N-terminal modification. The digestion showed greater than 60% cleavage results into 7kD GH and 3.4kD hBNP. Purification of rhBNP by reverse phase chromatography (RPQ:

GH-BNP digested mixture was further fractionated on source 30 RPC columns to separate pure peptide from other contaminants like residual 1 IkDa, 7kDa GH and other E.coli host proteins. The peptide was eluted with a linear gradient of acetonitrile. The elution profile showed two peaks. The first (I) peak contained the rhBNP and second peak contained contaminants. rhBNP peak collected was assessed by SDS- PAGE and analytical RP for purity. SDS- PAGE analysis showed a single band with peakl the peptide fractions were lyophilized and suspended in lOmM citric acid buffer pH 4.2 and the pH was adjusted to neutral 0. IN NaOH and kept for refolding at 25°C for 3h. The mixture of folded, unfolded and modified forms were separated by repurification by RPHPLC and analyzed by analytical HPLC over CI8 column. The rhBNP peptide fractions were greater than 99%pure. These fractions proved to be pure were dialyzed against water injection and next 20mM citric acid buffer pH 4.2. The final yield obtained by this method was approximately 50-60mg of pure rhBNP /liter culture.

We claim:

1. A liposomal formulation comprising a lipid moiety and a biologically active recombinant human Brain Natriuretic Peptide (rhBNP) for the treatment of cardiovascular diseases.

2. The liposomal formulation as claimed in claim 1, wherein the lipid moiety comprises a cationic lipid material namely, N,N-2-hydroxyethyl-N,N-di-n-tetradecylammonium chloride (DTDEAC) and optionally a co- lipid.

3. The liposomal formulation as claimed in claim 2, wherein the co lipid is selected from a group comprising of sterols, neutral phosphatidyl ethanolamine and neutral phosphatidyl choline.

4. The liposomal formulation as claimed in claim 2, wherein co-lipid is cholesterol.

5. The liposomal formulation as claimed in claim 2, wherein co-lipid is Di-oleylphosphatidylethanolamine (DOPE).

6. The liposomal formulation as claimed in claim 2, where in the molar ratio of DTDEAC to co-lipid used is in the range of 1:1 to 4:1.

7. The liposomal formulation as claimed in claim 5, where in the preferred molar ratio of DTDEAC to co lipid is 2:1.

8. The liposomal formulation as claimed in claim 1, wherein the said formulation is administered intravenous, intramuscular, subcutaneous or intraperitonial mode.

9. The liposomal formulation as claimed in claim 1, wherein the said formulation comprises amount of entrapped peptide in the range of 30-100 ug/kg body weight of the mammal per dose.

10. The liposomal formulation as claimed in claim 1, wherein entrapment of rhBNP is more than 50%

11. The liposomal formulation as claimed in claim 1 wherein the resulting cGMP stimulating activity is 50%-80% higher relative to the corresponding unconjugated natriuretic compound.

12. The liposomal formulation as claimed in claim 1, wherein the cardiovascular diseases is selected from the group comprising of congestive heart failure condition and pulmonary embolism