DESC:FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

TITLE OF THE INVENTION

“AN IMPROVED AND COMPLIANT PROCESS FOR HUMAN PLACENTAL EXTRACT FOR PHARMACEUTICAL APPLICATION”

APPLICANTS

ALBERT DAVID LIMITED
AN INDIAN COMPANY
“D” BLOCK, 3RD FLOOR, GILLANDER HOUSE
NETAJI SUBHAS ROAD
KOLKATA – 700001
WEST BENGAL, 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 relates to an improved process for production of Human Placental Extract containing bio actives such as nucleotides and peptides responsible for its therapeutic role namely antibiofilm action, wound healing action and tissue regeneration, being obtained from human placenta, a biological waste.

BACKGROUND OF THE INVENTION

It is known from traditional folk knowledge that the placenta, supporting the baby's growth and development in the mother’s womb, contains a wide range of biologically active components. Though a rich source of bioactive components unless recovered, placenta becomes a biomedical waste immediately after childbirth.

Use of human placenta as a therapeutic agent, therefore, in no way hampers ecological balance rather promotes resource recovery from a designated biomedical waste. Since most of the natural products of medicinal value have a vast repertoire of potent biological components, there has been an increasing realization to shun synthetic and semi-synthetic medicines primarily because of their harmful side effects.

Research on human placental extract gained a momentum with the description of the preparation of its extract by Russian ophthalmologist Prof. V.P. Filatov. Prior to his research, there were no documents of therapeutic efficacy of the extract, though its use was popular in Europe and parts of Asia primarily China, Korea and Japan for over centuries. Review of placenta therapy reveals its long usage from the days of distant past.

Placenta serves as a natural storehouse of many biologically active components with significant healing attributes (Tonello et al., 1996). Various extracts of placenta have been described, however, only an aqueous extract of fresh full term human placenta acts as a potent biogenous stimulator and finds importance owing to its ability of curing chronic non-healing wounds including post-surgical dressings and high degree of burn injuries (Wu et al., 2003). Human placental extract systematically helps collagenesis leading to potent healing of wounds (Biswas T. K. et al published in Scientific journal 2001) and also causes lysis of faulty collagen thus helping in remodeling phase of wound healing (P.D. Chakraborty et al 2013). The human placental extract may be useful in suppressing inflammation and platelet aggregation (T.K. Sur et al. 2003). The extract also showed ample antibiofilm actions. On one hand it breaks down preformed bacterial biofilms, it also prevents formation of new biofilms on the other (Goswami S et al 2017).
Wound healing is a complicated interrelated process wherein the injured dermal and epidermal tissue is naturally regenerated. Though well regulated, the process of healing is susceptible to interruption or failure leading to the formation of chronic non-healing wounds. Factors which may contribute to this include diabetes, venous or arterial disease, old age and infection. Placental extract, ever since its usage has been shown to be clinically effective in healing normal as well as infected wounds (V.K. Shukla et al., 2004, P.D. Chakraborty et al., 2009). It is involved in almost every stage of healing. It is also used in wound dressings to speed up the process of recovery. Though clinically well-tested, emphasis is now being laid in understanding the bioactive components involved in various processes of healing.

Encouraged by the properties of the placenta extract multiple methods are already available in the prior art for preparation of extract. One such method for aqueous extraction of the placenta is reported from the present inventors (a)Jain Praveen Kumar; b)Nath Swadhin; c)Sengupta Anup Kumar; d)Saha N. & e) Joardar D.S.) which is as follows:

Collecting fresh human placenta in sterilized containers kept at cold conditions, sealing the containers every time after a sample is collected. Storing the containers in cold conditions at temperature not exceeding 6°C,

subjecting the material to a step of screening for any HIV infection or otherwise affected material - discarding the infected or otherwise affected material,

storing the material in sealed containers in a cold storage at temperature below 6° C for a period of about 4 to 8 days in order to preserve all the natural proteins and other valuable constituents contained in the placenta and to allow any self occurring bio-chemical process to complete,

subjecting the material to a cleansing step to remove any remnants of umbilical chord and fat tissue to obtain a clean placenta under sterile conditions,

quickly mincing said cleaned material at low temperatures not exceeding 6°C using sterilized mechanical placenta to obtain shredded material

thoroughly mixing the same with cold water to enable a dissolution of the water soluble material,

storing the entire material at temperature of less than 100 C in sterile vessels, until complete extraction of all the water soluble material occurs.

subjecting the cold water extract to a heating step to increase the temperature of the contents to around 850C,
maintaining the hot contents at the said temperature of around 850C till a solution is obtained.

subjecting the hot solution to a step of filtration under sterile conditions,

followed by re-heating the filtered solution under sterile conditions to temperatures around 97° C to achieve breaking of any long chain protein,

subjecting the solution of the above step to autoclaving under steam pressure and at temperatures of around 120° C in order to destroy any microbes to obtain a safe sterile solution,

and finally converting the sterile final solution in a conventional manner, under sterile conditions into a desired final product like injectable, gel or cream.

The way in which biological products are produced, controlled and administered requires necessary precautions. Unlike conventional pharmaceutical products, which are normally produced and controlled using reproducible chemical and physical techniques, biological products are manufactured by methods involving biological processes and materials, such as cultivation of cells or extraction of material from living organisms. These processes display inherent variability, so that the range and nature of by-products are also variable.

This variability, commercially termed as batch variation, occurs in products of animal origin so for the manufacture of biological products for direct application to human, full adherence to GMP (Good Manufacturing Practices) is necessary at all production steps, beginning with the starting material from which the active ingredients are produced (WHO, 1992).

The composition of the extracts depends on the method of its preparation and consequently, they show different therapeutic activities. In the earlier disclosed method of the present inventor the removal of debris and insoluble material after autoclaving is problematic and takes longer duration of time to produce extract of desired clarity. Additionally the method is prone to batch-to-batch variation.

Earlier the paper filtration was a crude method and not sufficient enough to filter large molecules besides it was time consuming too.

Thus there exists an urgent need for an improved process with amenable process steps for aqueous extraction of human placenta devoid of batch-to-batch variation and appropriate for strict adherence to the cGMP norms.

OBJECTS OF THE INVENTION

The primary object of the present invention is to provide an improved process comprising amenable process steps for the extraction of therapeutically active peptides and nucleotides from human placenta which is a biological waste.

Another preferred objective of the present invention is to provide said process, which would enable optimum extraction of the placenta components i.e. the bio-active material without compromising on quality and quantity with respect to the existing procedure.

Another object of this invention is to provide said process that would be economical, reliable and having flexibility in operation.

Another objective of the present invention is to provide said process, which would be suitable for strict adherence to the required quality norms.

Yet another object of the present invention is to provide said process which will ensure the quality and quantity of components from the extract that are effective against specific diseases.

Still another object of the present invention is to provide said process, which would enable production of several forms of bioactive therapeutic products e.g. injectable, lotion, gel, cream etc.

How the foregoing objects are achieved will be clear from the following description. In this context it is clarified that the description provided is non-limiting and is only by way of explanation.

SUMMARY OF THE INVENTION

The present invention provides an improved process for the extraction of therapeutically active peptides and nucleotides from human placenta - a biological waste.

It is a validated process, which will be economical, reliable and have flexibility in operation.

The present invention produces several forms of bioactive therapeutic components.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 is a flow diagram showing the process for the preparation of human placental extract according to the present invention.

SOURCE AND GEOGRAPHICAL ORIGIN OF HUMAN PLACENTA - A BIOLOGICAL WASTE

The biological material is obtained from discarded placenta after childbirth. The waste placenta were collected by cold chain process from healthy mothers after delivering live-born babies from various hospitals approved by the Dept of Health and Family Welfare West Bengal, a state in the geographical territory of India.

BRIEF DESCRIPTION OF THE INVENTION

Preparations of extract from human placenta- otherwise a biological waste

Placental extracts can be obtained by three major extraction processes. 1) Aqueous extraction, 2)hydroalcoholic extraction and by 3)acid hydrolysis. The components present in the extract depend on the method of its preparation and are based on solubility of the components in respective solvent of extraction. Thus, an aqueous extract is likely to contain more polar molecules such as peptides/proteins, small organic components like amino acids, nucleotides, polydeoxyribonucleotides (PDRNs), carbohydrates and trace amount of lipids mostly bound to proteins which are comparatively soluble in aqueous medium. Components from aqueous extracts are more related to wound healing and tissue regeneration.

The aqueous placental extract is prepared by modified Filatov’s procedure holding confidentiality of the proprietary terms as: fresh placentae, stored at 2-4°C, and portions were tested for HIV and HCV antibody and Hepatitis B surface antigen. Cold and hot aqueous extractions were done after incubating dissected and minced placenta. This was followed by sterilization of the extract under saturated steam (pressure 15-lbs/sq inch at 121°C for 40 min). After filtration and addition of 1.5% (v/v) benzyl alcohol as preservative, ampoules were filled and sterilized once again under the said condition for 20 min. In the first sterilization, the extended duration of heat treatment essentially completed precipitation of a number of macromolecules like proteins. The terminal sterilization step was to maintain sterility of the products after they were filled and sealed in ampoules. Each milliliter of the drug was derived from 0.1 g of fresh placenta. A single batch was prepared from the pool of several placentae. As the extract is prepared by repeated sterilization process, it is expected that it may contain macromolecules in their degraded forms together with small bio-organic compounds such as amino acids, peptides, small sized polypeptides, nucleotides, small polynucleotide fragments like PDRNs and NADPH etc. Only those molecules that are heat stable and are able to withstand high sterilization pressure will remain biologically active. Presence of various organic components gives the extract a pale yellowish tinge.

STAGE I
The collected fresh term human placentae are stored in the sealed containers in a cold storage for 4 to 7 days at controlled temperature at 3+10C. This is done to preserve the natural proteins and other valuable constituents in the placenta. Some biochemical changes including breakdown of macro-molecules occurs during this stage yielding smaller biomolecules that are therapeutically active. At the end of this stage, visual inspection of each container is done for further processing.

STAGE-II
In this stage, the contents of each container are cleaned with purified water (at least for 3 times) for removal of blood clots, fat tissue, umbilical cord and other unwanted material and discarded as a waste, to meet biomedical waste disposal requirements.

STAGE-III
The material found suitable for further processing is subjected to mincing operation in a suitable mechanical mincer. This operation is to be very carefully carried out and monitored.

The mincing opens up the entire material and shreds into very small pieces, thus opening, up the surface area and exposing more surface area for further processing.

It is preferably carried out in a sterile condition by sterilizing the mincer and its components as well as the collection tray.

In this stage more of the protein and other valuable component are exposed and to maintain the natural values of these components, it is preferred to carry out the mincing at low temperatures.

The placenta is stored in closed SS drum and weighted 31+0.1% Kg in each drum.

The mincing stage is the crucial stage as this stage forms the basis for further successful operations. At the end of the process, starting material with active biological components for injection was obtained.

STAGE-IV
The starting material is now transferred in a SS 5 jacketed vessel. In the next stage, we immediately collected the entire minced material in cold water and all the water-soluble materials are extracted.

In a preferred example, we used 305 liters at 85 + 30C in SS reactor vessel and cool the WFI to 9+ 10C. In the final stage, the weighted Placenta is transferred in a SS Reactor vessel.

STAGE-V
In the next step, at the controlled temperature condition of 9+10C for a period of 1 hour with intermittent stirring, cold bath treatment on placenta are done to ensure complete extraction of water-soluble material.

STAGE-VI
Following the cold extraction, the entire contents are subjected to a warm extraction by gradually allowing the temperature of the cold extracted matter to warm up using a hot water bath, so that the contents are raised gradually and carefully to a temperature of 86+10C with intermittent stirring (200 RPM for 1 min in every 15 min). It contained all the dissolved biomolecules, proteins and other components of placenta.
The cold and hot extractions are effected in sealed containers.

STAGE-VII
In the next step, the extract is cooled down to 30+10C with intermittent stirring.

STAGE-VIII
The extract now undergoes clarification through nylon cloth (approximately 100 mesh).

STAGE-IX
The extract then undergoes second phase of hot bath treatment for 2 min at 96+10C in a SS Jacketed Reactor vessel with intermittent stirring.

STAGE-X
During the Viral in-activation step, the Placenta extract is exposed in SS Jacketed Reactor vessel at 121+10C for 40+1 minute.

STAGE-XI
In this stage, the aqueous extract is cooled down to 30+10C with slow intermittent stirring.

STAGE-XII
The aqueous extract is now stored for 12-14 hrs in SS Jacketed reactor vessels.

STAGE-XIII
Clarification of the placenta extract is done through multiple polypropylene filter pad by adding purified siliceous earth (celite). The pharmaceutical grade Silica (Celite) will expedite the process of filtration without the loss of components instead of paper filtration.

Objective and Procedure of adding Celite:
Purified siliceous earth (AW Standard Super-Cel USP NF grade) was added during manufacturing to evaluate the efficacy of the extract in terms of bioactive component concentration and also maintain the clarity of the product.

Crude placental extract (with preservative) was collected from manufacturing area of SVP plant without Celite and paper pulp filtration. Weighing of Celite (AW HYFLO SUPER-CEL NF) was done in the laboratory in different amounts resulting in 0.25%, 0.50%, 0.75%, 1%, 1.25% & 1.75% concentrations of placenta extract and waited for 10 mins. Then Shaking has been done thoroughly and vortexed for 10 mins each and then waited for 30 mins. Each sample at first pass through Whatman filter paper(40) then the filtrate again pass through 0.45? filters (by using syringe filter). The resultant filtrate was ready for biomolecule analysis (NADPH, Total peptide & PDRN) and for checking solution clarity.

For this study clarity of the sample could not be measured by Turbidity meter (nephelometer), since this instrument has not that much of sensitivity to measure clarity of the samples corresponding to each concentration of Celite. A sensitive method has been employed by using light scattering phenomena of fluorescence spectroscopy. Where excitation and emission has been set at the same wavelength (i.e. 650 nm) and scattering intensity of each samples were measured to determine turbidity of the solution.

For Control:
1. Crude placenta extract without addition of Celite only filtered through filter paper followed by syringe filter (0.22?)

2. Crude placenta extract (without filtration)

Results:
1. Crude placentae extract :

Conc. of Celite (AW HYFLO SUPER-CEL NF) in Pl. Extract TOTAL PEPTIDE
(µg/ml) NADPH PDRN
Emmax F.I. (µg/ml) (nm) (µg/ml)
0.25% 109.2 427.00 124.17 15.16 617.07 190.44
0.50% 103.4 427.00 124.45 15.19 617.07 212.26
0.75% 115.7 427.00 124.85 15.54 617.07 214.04
1.0% 117.2 427.00 122.50 15.95 617.07 210.90
1.25% 108.4 427.00 122.65 15.90 617.07 209.44
1.50% 111.3 427.00 121.60 14.84 617.07 212.32
1.75% 110.6 427.00 120.54 14.71 617.07 186.05
Crude extract (filtered through 0.22?)* 136.20 427.00 125.50 16.31 617.07 219.53
Crude (without filtration)* 150.32 427.00 125.25 16.05 617.07 222.36

Observation
After performing the study it was concluded that filter aid (Celite) 0.75% to 1% of the extract is sufficient to get desired level of biomolecules as well as clarity of the extract.

STAGE-XIV
A visual inspection of the filtrate is done in this stage. If the clarity of the extract is not satisfactory, then the stage XIII is repeated.

STAGE-XV
To ensure the clarity of the solution and to exclude the presence of any protein, which may lead to protein shock, the Cartridge filter of 3.0 micron nylon filter (and then by PVDF membrane) is being used for filtration of the extract to decrease the bio-burden of the product.

STAGE-XVI
The filtrate aqueous extract is now used to prepare the desired final product.

FINAL PRODUCT:

The clear solution is mixed with sufficient quantity of benzyl alcohol and a conventional preservative is added to the solution. The volume is made up with WFI (Water for Injection) to 310 Lt.

The volume is then under quality-controlled testing and abnormal toxicity. If the test result is satisfactory, the solution is stored in controlled condition at 22.5+2.50C in a SS storage vessel.

The Solution is transferred to injectable compounding vessel and volume is checked up to 300 Lt.

The solution is now further tested for its quality controlling.

The tested solution is filtered through a train of 1.2µ + 0.5µ + 0.45µ pore size cartridge filters. This ensures the sterility of the final product.

The cartridge filtered solution is filled in glass ampoules and sealed. The drug is used as an anti-inflammatory, anti-biofilm, wound healing and tissue regenerative agent.

THERAPEUTIC ACTIVITY OF THE EXTRACT
1. Anti-biofilm activity
2. Ensures comprehensive tissue repair and wound healing
3. Anti- inflammatory activity
4. Immuno-modulatory effect
5. Regenerates cells, tissues and organs
6. Accelerates collagen synthesis, Re-epithelialization
7. Improves immune system against diseases

Key findings of the present invention
• A high degree of batch consistency in respect of the biomolecules has been observed.

• Following biological components with therapeutic activity have been identified to be present in the human Placental Extract and its formulation.

• a) Peptides: Containing Fibronectin type III like peptide, Ubiquitin like peptide and CRF peptide showed marked anti-biofilm activities. They prevented the formation of biofilm and broke down pre-formed biofilms. One of the mechanism noted was interference of Quorum Sensing.

• Fibronectin type III peptide showed debridement action through remodeling of protease activity, cell adhesion, proliferation/wound healing.

• Ubiquitin peptide exhibited direct collagenase action, tissue regeneration and remodeling with minimal fibrotic changes.

• CRF peptide exhibited anti-inflammatory action.

• b) Polydeoxyribonucleotides (PDRNs – facilitates anti-inflammatory/wound healing).

• c) Free & bound NADPH (facilitates wound healing)

• d) Amino acids – with specific reference to Glutamate that is found to induces cell migration in/wound healing.

• Lipid Fraction (induces cell migration/wound healing)
• In vitro Nitric Oxide (NO) induction, cell adhesion and antimicrobial properties have also been demonstrated.

Standardization of the manufacturing process of the drug
Isolating active components present in the drug ‘Placentrex’ is a natural propagation towards characterization of the drug. The contents present in the drug depend on the method of its preparation. The drug ‘Placentrex’ contains NADPH, amino acids, peptides, polydeoxyribonucleotide fragments (PDRNs). Some of them can be chemically or biologically synthesized but many of them can only be obtained by isolating them from a natural source.

Major findings with its constituents include demonstration of minimal batch variation of the extract using conventional spectroscopic and chromatographic techniques like UV-VIS absorption spectra, TLC, HPTLC and HPLC and also by the method for fingerprinting of multi-component drugs using fluorescence Excitation-Emission-Matrix (EEM) plots. Since by EEM a wide excitation and emission zones are covered in a single profile, there is no scope of missing any newly emerged fluorophore in a batch. A high degree of consistency between different batches of the drug was observed from EEM. Validity of these results from EEM was crosschecked by different spectroscopic and chromatographic modes. This consistency reflects standardization of the manufacturing process of the drug and also adherence of uniform placental composition irrespective of the nutritional status of the mothers.

Product : PLACENTREX INJECTION SPECIFICATION

Sl. No. Parameters
(Quality characteristics) Acceptance limit

1. Description Colourless to pale yellow coloured clear liquid.
2. Identification 2.1. Glutamic acid Present
2.2. Polydeoxyribonucleotides Present
3. Extractable Volume 2.0 mL to 2.3 mL
4. pH 6.0 to 7.5
5. Abnormal toxicity Should pass the test
6. Sterility Should pass the test
7. HIV Antibody,
HBS Antigen &
HCV Antibody Absent
8. Assay 8.1. NADPH : Not less than 10µg/mL
8.2. Total peptides : Not less than 90µg/mL
8.3. Total Nitrogen : Not more than 0.08 % (w/v)
9. Benzyl alcohol (as preservative) 1.35 – 1.65% (v/v)
10. Bacterial Endotoxin Not more than 40 EU/mL

Results of stability studies of Placentrex Injection

Components present in Placentrex and their mechanism as anti-inflammatory and wound healer

1. NADPH

The drug ‘Placentrex’ contains NADPH, a nucleotide that helps in wound repair. Normal human keratinocytes contain a constitutive (natural) enzyme Nitric Oxide (N O) synthase. This enzyme is cytosolic and requires the presence of Ca+, NADPH (reduced form of NADP – nicotinamide – adenine dinucleotide phosphate) for its functioning. This helps in NO mediated wound healing. NO helps in 1) Killing Bacteria, 2) Debrides Necrotic Tissues & 3) Helps in re-epithelialisation (Datta and Bhattacharyya 2004).

It also regulates capillary permeability and maintains adequate tissue perfusion both in topical and systemic administration. The wound healing and tissue growth promoting activity of the drug has been confirmed.
When excited at 340 nm, Placentrex results in fluorescence emission having maxima around 436 nm, which is fairly specific for nicotinamide adenine dinucleotide phosphate, reduced form (NADPH). The excitation spectra, having emission at 440 nm, show patterns comparable to this nucleotides. Thin layer chromatography and reversed-phase (RP) HPLC confirm presence of NADPH in the extract. The emission and excitation patterns of NADPH purified after HPLC resemble exactly with the reference compound.
2. PEPTIDEs

a. Fibronectin Type III like peptide

The peptide 7.4 kDa has been purified from the drug ‘Placentrex’. A partial amino acid sequence, derived from mass spectrometric analysis, revealed its homology with human fibronectin type-III, which is a component of the extracellular matrix (ECM)(Chakraborty PD and Bhattacharya D). The importance of fibronectin in cutaneous wound healing is well documented as a general cell adhesion molecule by promoting the spread of platelets at the site of injury. It also helps in the adhesion and migration of neutrophils, monocytes, fibroblasts and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue.
Considering the importance of peptides in wound healing, the drug was further characterized in terms of regulation of some enzyme activities related to repair mechanism. Investigation revealed that Placentrex stabilizes serine proteases against their autodigestion by reversibly inactivating them, which enhances the efficiency of proteolytic enzymes thereby facilitates wound healing. Rayleigh scattering, size-exclusion HPLC, fluorescence resonance energy transfer, and surface plasmon resonance show that fibronectin type III-like peptide present in the extract interacts with trypsin. The peptide–trypsin complex is dissociated in presence of high concentration of substrates. Thus, regulation of trypsin activity, which is reversible in nature.by ‘Placentrex’, is evident (De D, Chakraborty PD et al 2011).

Binding of fibronectin peptides can augment integrin signalling and activate cell-to-cell adhesion in some cell types, facilitating wound healing. It has also been reported that placental fibronectin is involved in endothelial cell proliferation, promoting wound healing more efficiently than normal fibronectin. The fibronectin type-III like peptide of Placentrex helps physiological debridement by serine protease regulation, cell signaling and cell adhesion thus helps in wound healing.

b. Ubiquitin like peptide

‘Placentrex’ itself displayed distinct proteolytic activity, and the component responsible for the proteolytic activity was identified to be an ubiquitin-like peptide. It is already known that ubiquitin and ubiquitin-like proteins (Ubls), collectively known as ‘ubiquitons’, perform a host of important functions such as protein degradation, antigen processing, apoptosis, biogenesis of organelles, cell cycle and division, fertilization and gametogenesis, DNA transcription and repair, differentiation and development, signaling, immune response and repair.
Subsequent studies reported that an ubiquitin-like component from human placental extract exhibits distinct collagenase activity (De D, Chakraborty P D 2013).They cause lysis of faulty collagen tissue responsible for hypertrophied scar or keloid formation. These activities ensure that the drug causes healing with minimal scar formation.

Though it is well known that blood proteases are quite specific about their substrates, the question of stability of the drug components remains an important issue from clinical point of view. It has been demonstrated by size exclusion HPLC that the peptide fraction of the drug remains unaffected in presence of plasma and serum proteases from human blood. In reverse, it has also been demonstrated by gelatin zymography that the blood proteases remain unaffected after incubation for 48 hrs in presence of the peptide fraction of Placentrex.

2. PDRNs:
These potentiate anti-inflammatory responses as they inhibit plasma mediated inflammatory mediators from the literature it has been known that PDRN exerts an anti-inflammatory action working on purinergic A2-receptors. It suppresses the cytokines and pro-inflammatory chemicals from plasma and cells.

PDRNs identified in Placentrex are capable of suppressing the chemical mediators of inflammation thus exerting an antinflammtory action as reported earlier by Bianchini et al.

PDRN also stimulate Growth factors like FGF, PIGF AND TGF-beta that helps in tissue repair and healing. Results both in vitro and in vivo are in agreement, suggesting a clinical value for PDRN as healing factor acting at both the purinergic and metabolic levels.

3. GLUTAMATE

Rapid migration of neutrophils to the wound site is a prerequisite to the wound healing process. Gel ?ltration analysis of placental extract gave the initial cue about the presence of migration-promoting factor of the extract. HPLC analysis of the extract revealed glutamate to be the predominant free amino acid. Recent studies show that glutamate induced phenotypic neutrophil chemotaxis, as seen in the time lapse- and transwell assays. Glutamate was also found to induce chemokinesis of the neutrophil, though the stimulation of chemotaxis was more pronounced (Gupta R et al 2009).

Glutamate identified in Placentrex at an optimum concentration induced phenotypic neutrophil chemotaxis, as seen in the time lapse- and trans-well assays. The glutamate-induced chemotaxis was accompanied by polarization of the actin cytoskeleton, and by polymerization of F-actin. These data indicate that glutamate has a strong chemotactic functionality in the neutrophil, which could be of interest both therapeutically and in further investigation of the molecular basis of chemotaxis(S -Palchowdhuri 2013).
All these changes have produced clinical benefits to the patients without causing any serious side effects even after prolonged use either as an injection or as topical agents.

The present invention has been described with reference to some preferred embodiment purely for the sake of understanding and not by way of any limitation and the present invention includes all legitimate developments within the scope of what has been described herein before.
,CLAIMS:We claim

1. A process for manufacture of aqueous extract of human placenta (a biological waste) based pharmaceutically active preparation including therapeutically active peptides and nucleotides.
i) providing human placenta (a biological waste) material in sealed containers in cold storage at temperature of 3+10C for a period of 4 to 7 days;
ii) subjecting duly cleansed placenta material to mincing for shredding into small pieces and opening its surface area for further processing;
iii) carrying out cold extraction for obtaining water soluble bio active components from said minced placenta material in cold water maintaining temperature condition of 9+10C for a period of 1 hour to ensure complete extraction of water soluble materials;
iv) after said cold extraction subjecting to hot extraction by gradually increasing the temperature of cold extracted placenta material to hot bath in the temperature range of 86+10C for a period of 3 minutes.
v) cooling the thus obtained water soluble extracts of placenta material and filtering it through nylon cloth for removal of tissue debris;
vi) subjecting said filtered extract for second phase of hot bath treatment for 2 min at 96+10C with intermittent stirring;
vii) in-activating the microbial load, if any, by exposing the said Placenta at 121+10C for 40+1 minutes;
viii) cooling the thus obtained water soluble extracts of placenta material followed by clarification and filtration selectively including siliceous earth to obtain said extracts of placenta material as a clear placenta material extract solution including said therapeutically active peptides and nucleotides.

2. The process as claimed in claim 1 comprising carrying out said clarification and filtration step involving purified siliceous earth (celite) preferably at a selective concentration of 0.75% to 1% with respect to the extract for obtaining filtrate (filtrated extract) with desired biomolecules as well as clarity.

3. The process as claimed in any one of the claims 1 and 2 comprising mixing to said clear placenta material extract solution with aromatic alcohol preferably benzyl alcohol as preservative.

4. The process as claimed in any one of the claims 1 to 3 comprising:
i) providing freshly collected and screened human placenta (a biological waste) material in sealed containers in cold storage at temperature of 3+10C for a period of 4 to 7 days;
ii) subjecting duly cleansed placenta material obtained of said stored human placenta (a biological waste) material by cleansing with purified water for removal of blood clots, fat tissues, umbilical cord and other unwanted material, to mincing for shredding into small pieces and opening its surface area for further processing;
iii) carrying out cold extraction for extracting water soluble bio-active materials from said minced placenta in water preferably by cooling to 9+10C, maintaining temperature condition for a period of 1 hour to ensure complete extraction of water soluble materials;
iv) after said cold extraction subjecting to hot extraction by gradually increasing temperature of cold extracted placenta material to hot bath temperature in the range of 86+10C for a period of 3 minutes.
v) cooling the thus obtained water soluble extract of placenta material and clarifying through nylon cloth facilitating cleaning up of the insoluble biomaterials as well as placenta residues and tissue debris;
vi) subjecting said clarified extract for second phase of hot bath treatment for 2 min at 96+10C with intermittent stirring (around 200 RPM for 1 min in every 15 min interval) to breakdown proteins with tertiary or quaternary protein structure;
vii) inactivating the viral load if any by exposing the said Placenta extract at 121+10C for 40+1 minutes;
viii) cooling the thus obtained water soluble extract of placenta material to 30+10C and then stored overnight and taking for clarification and filtration by passing through multiple polypropylene filter pad by adding purified siliceous earth (celite) for expediting the process and cartridge filter of 3.0 micron nylon filter and then by PVDF membrane to thereby obtain said clear placenta extract solution including therapeutically active components comprising of
a) peptides containing Fibronectin type III like moiety, ubiquitin peptide like moiety, CRF like peptide, b) Polydeoxyribonucleotides, c) Free & bound NADPH d) Amino acids.
5. The process as claimed in any one of the claims 1 to 4 wherein the obtained water-soluble extract after first heat treatment is cooled to 30+10C and then it was clarified and filtered by passing through a series of 1.2µ + 0.5µ + 0.45µ pore size cartridge filters.

Dated this 14th day of January 2018.



To (MONAJSAHA)
The Controller of Patents IN/PA-1884
The Patent Office for S.S. Datta & Associates
Kolkata Applicants' Agent