DESC:FIELD OF THE INVENTION
The present invention relates to the field of pharmaceuticals, in particular to the field of non-healing and exudating wound management. More particularly, it relates to placental tissue based wound healing pharmaceutical formulations, wherein the tissue is selected from group consisting of amnion and chorion. The present invention relates to an Amchoflo, an advanced topical formulation for exudating wounds.

BACKGROUND OF THE INVENTION
The therapeutic functions of placental membrane in aiding wound healing are widely known and extensively applied in clinical settings. Apart from being immune privileged the abundance of immunomodulatory, tissue remodeling and antimicrobial molecular elements make them ideal for allograft therapies. Importantly, the membrane can be processed and preserved to retain its healing and reparative properties (US20150110850A1). Cryopreserved or dehydrated amnion as well as amnion/chorion composite membrane sheets from various manufacturers (e.g., MiMedex, Osiris) is effectively used in the management of chronic wounds. Micronized dAmCh powder (MiMedx) is well characterized and is more effective in the treatment of ulcerating deep wounds (Lei et al, 2016, EP2884844AG). Excessive wound fluids can however deprive the wound bed from accessing any therapeutic interventions. Referred to as ‘a wounding agent in itself’, chronic wound exudates have elevated protease activity and pro-inflammatory cytokines that leads to considerably reduced levels of growth factors and protease inhibitors (Weigant et al, 2015). Heavily moist wound bed prevents cell migration and maceration of the surrounding skin increasing wound area, whereas less amount of exudation can cause tissue dehydration. Moisture balance is hence critical in promoting healing. Wound exudates are at the risk of microbial infection, if poorly managed recurrent infections leads to progressive increase in fluid volume and malodor (Cutting, 2003). Dressing materials like gauzes, films, hydrogels, hydrocolloids, alginates, hydro fibres foams and super absorbents vary in their ability to manage wound exudates (Weigand, 2015). There is however no single effective treatment modality, but rather a combination of approaches based on assessment strategies across the trajectory of the healing process offer better solutions. Advanced Dressings (ADs) that not only absorbs exudates, but incorporates antimicrobial (silver, polyhexanide, iodine) and deliver bioactive molecules to modulate the biochemical imbalance has improved efficacy. Collagen or alginate based dressings that modulate and sequester protease activity belong to the latter category (White, 2006., Tottoli et al., 2020).

The wound healing properties of sugar is known and applied since ancient times. Direct application of granulated sugar on open wounds exerts a local osmotic effect, promotes tissue granulation and by lowering the pH and ‘water activity (aw) acts as both a bacteriostat and bactericide (Biswas et al., 2010). Due to their biocompatibility, high absorptive nature and poly-functionality several polysaccharides, such as alginates, chitosan, cellulose or their hydrolysates are used in healing formulations that can also serve as trans dermal drug delivery systems (Reibeiro et al,2020).

There are several in vitro and in vivo studies defining the healing properties of low dextrose equivalent (DE) form of starch hydrolysate (Silvetti et al, 1981., Amin et al 2015., Sultan et al, 2016., Salgado et al., 2017/. Multidex®). The Multidex® patent (US6046178A) describes the medication and method of treating open and tunneling wounds with maltodextrin in combination with trace metals, microbiocidals or ascorbic acid.

An adaptation of this method, where the wound is covered with dressing infused with suspension of starch hydrolysates, ascorbic acid, collagen and alpha-tocopherol acetate is presented in the patent, US2004000878A1.

The present invention is an advanced dressing that utilizes the absorptive and antimicrobial properties of maltodextrin, and the polyspecies antiseptic potential of PHMB to manage and prevent excessive fluid formation while making the wound bed conducive to the therapeutic agents in dAmCh membrane. The micronized PHMB impregnated dAmCh membrane is blended in a definite ratio with the powder form of low DE maltodextrin in this formulation. PHMB impregnation of dAmCh is performed as disclosed on EP1473047B1 as well as in de Mattos et al, 2019. The physical nature of the medication is best suited for the irregular and other difficult-to-access wound areas.

US13/815,784 relates to tissue grafts composed of at least one membrane, where at least one side of the membrane has micronized placental tissue applied it.

US20200405637 relates to compositions and methods for wound care or the dressing or treatment of wounds in a subject in need thereof. The compositions include an oil - based carrier, a polar solvent comprising one or more polar antimicrobial agents, and collagen or a collagen - based material. In at least some instances, the polar solvent comprising the one or more polar antimicrobial agents and the collagen or collagen - based material are suspended in the oil - based carrier.

US20180126033 relates to compositions containing purified collagen biomaterial derived from tissues, for example, insoluble amnion, soluble amnion, soluble chorion of the human placenta. The collagen compositions can be used to promote wound healing, promote tissue regeneration, prevent or reduce scarring, reduce local inflammation, minimize tissue rejection, promote graft integration. Methods for using the collagen composition as a biomaterial implant for dermal filling, skin grafting, and hair transplantation are also provided.

Wound healing requires a variety of macronutrients and micronutrients, each of which varies according to the stage of healing (Lansdown et al, 1999; Patel, 2005). During the proliferation phase of wound healing, fibroblasts produce collagen fibres, a process dependent upon an adequate availability of dietary nutrients such as iron and copper (Flanagan, 1997). Biological tissues from a variety of sources have been used to treat non-healing wounds. Skin autografts and human skin allografts have been employed extensively for burns and chronic wounds. The therapeutic potential of human amnion/chorion tissue grafts in wound healing has been well established. Early use of fresh amniotic membrane containing both amnion and chorion has been proven to be beneficial in treating ulcers, burns and dermal injuries. The amniotic membrane has a long history of use in wound-healing applications due to the dynamic nature of the tissue and its growth factor/cytokine rich ECM. Human amniotic allograft is also known to be non-immunogenic, reduce inflammation, pain and scarring and provide a matrix for cell colonization as well as a natural biological barrier. Given this functionality, it is not surprising that human amnion allograft containing amnion and chorion (HACM) have also been used therapeutically for cutaneous wounds and appear effective at accelerating wound healing.
Recently, a gentle cleansing and dehydration process was developed to preserve and maintain the biological activities inherent in the native amnion. As shown below, this process retains the natural growth factors and regulatory molecules contained in the amnion and chorion. Dehydrated human amnion/chorion membrane (dHACM) has demonstrated clinical efficacy in the enhancement of wound repair.When compared with standard compression therapy, dHACM contains an array of growth factors, cytokines, and chemokines and it has been shown to promote proliferation and migration of bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (both healthy and diabetic), and hematopoietic stem cells. The treatment of complex, full-thickness wounds may be augmented using a micronized formulation of dHACM (in addition to membrane dHACM) to fill the wound bed and to provide additional bioactive molecules to the site of injury (Lei et al 2017), but lack of antimicrobial activity.

To overcome the problem, there is a need for formulation of micronized dHACM with PHMB and with added Maltodextrin as filler.

OBJECT OF THE INVENTION
It is primary object of the present invention to provide a placental tissue-based formulation with micronized dehydrated human amnion chorion membrane with Polyhexamethylene biguanide, along with maltodextrin as filler.

It is another object of the present invention to provide a powder formulation for treatment of wounds, including the active drug containing dehydrated amnion chorion membrane, PHMB and maltodextrin that are combined to produce a final medicinal product in powder form.

It is another aspect of the present invention to give a placental tissue based topical formulation for exudating wound management attributed to synergistic effect of three components – dehydrated human amnion-chorion (dAmCh) membrane, polyhexamethylene biguanide (PHMB), Maltodextrin.

SUMMARY OF THE INVENTION
One or more of the problems of the conventional prior arts may be overcome by various embodiments of the present invention.

It is a primary aspect of the present invention to provide an advanced wound dressing comprising placental tissue.

It is a primary aspect of the present invention to provide an advanced wound dressing comprising placental tissue, wherein the dressing is topically applied as a powder, gel, film and the like.

It is another aspect of the present invention to provide a topical formulation for treatment of wounds, comprising of:
micronized placental tissue selected from group consisting of dehydrated amnion and chorion membrane (dAmCh) – 2 g;
polyhexamethylene biguanide (PHMB) – 0.5g to 2 g; and
filler – 96g to 97.5g,
wherein the formulation provide synergistic effect with respect to treatment of chronic wound exudates and the formulation is a powder.

It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein each gram of formulation contains 20 mg (2%) of micronized dehydrated amnion and chorion membrane, 5 mg to 20 mg (0.5% to 2%) polyhexamethylene biguanide, and 960 mg to 975 mg (96% to 97.5%) of maltodextrin.

It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein the micronized dehydrated amnion and chorion membrane is impregnated with polyhexamethylene biguanide.

It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein the micronized dehydrated amnion and chorion membrane has a particle size not more than 500 microns.
It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein the filler is maltodextrin, preferably as pharmaceutically acceptable powder.

It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein the dehydrated amnion-chorion layers are derived from human placenta.

It is another aspect of the present invention to provide a method for preparation of topical formulation for treatment of wounds, comprising the step of:
preparing 6.25% w/v PHMB solution;
adding 4 ml of 6.25% of PHMB solution to 1g of dAmCh membrane;
incubating the dAmCh membrane with PHMB for a period of 4 hours in a shaker incubator at a temperature of 37±1 °C;
subjecting the dAmCh membrane with PHMB at a temperature of 39±1 °C for 16 to 24 hours;
cutting the dried membranes into small pieces;
performing the micronization using ball mill;
sieving micronized dAmCh coated PHMB powder and maltodextrin separately using 500-micron sieve;
blending the micronized dAmCh coated PHMB (2.5g to 4g) with maltodextrin (96g to 97.5g) for 60 to 120 mins; and
drying at 39±1 °C for 2 hours,
wherein the formulation provide synergistic effect with respect to treatment of chronic wound exudates and the formulation is a powder.

It is another aspect of the present invention to provide the topical formulation for treatment of wounds, wherein the polyhexamethylene biguanide is composed of repeating basic biguanide units connected by hexamethylene hydrocarbon chains, providing a cationic and amphiphatic structure.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the flowchart of method for preparation of topical pharmaceutical formulation according to the present invention.
Figure 2 illustrates the quantification of cytokine and growth factors eluted from dAmCh powder formulation.
Figure 3 illustrates the effect of extracts of dAmCh powder formulation on human mesenchymal stem cells proliferation.
Figure 4 illustrates the effect of extracts of dAmCh powder formulation on human dermal fibroblast cells proliferation.
Figure 5 illustrates the effect of extracts of dAmCh powder formulation on human macrophages cells proliferation.

DETAILED DESCRIPTION OF THE INVENTION
The present invention pertains to a topical formulation that is a composite powder consisting of synergistically effective amounts of dehydrated Amnion-Chorion membrane (dAmCh), Polyhexamethyne Biguanide (PHMB) and Maltodextrin. It can be referred to as an advanced dressing (AD) with the components, individually or in combination, contributing ‘passively’ and/or ‘actively’ towards healing. Formation of excessive wound fluid is effectively regulated by the absorptive and antimicrobial properties of maltodextrin and the broad-spectrum antiseptic action of PHMB restoring moisture imbalance and preventing tissue deterioration. It reinforces the natural healing process while facilitating the therapeutic action of the endogenous molecules in dAmCh to rebalance the inflammatory milieu. The combination of the three components is more effective, than each used individually, in the treatment of wound with excessive, often purulent and malodorant exudates having corrosive and proliferation inhibiting characteristics. Powder form of the medication improves availability and presentation of bioactive compounds to the site of injury, especially in case of irregular, tunneling and other difficult-to-access wounds.

Context of the invention:
• Non-healing and exudating wound.
• Chronic wound exudates composition is different from that of acute wounds; elevated protease activity, inflammatory cytokines, marked reduction in protease inhibitors and growth factors are characteristics.
• Microbial virulence mechanism leads to progression in exudate volume.
• Prolonged inflammatory state, exudation leads to maceration and excoriation in peri-wound area.

Benefits of micronized dAmCh-PHMB, maltodextrin powder formulation:
• Individual components of the formulation have significant wound healing properties
a. dAmCh as the source of endogenous molecules with anti -inflammatory, tissue regenerative and remodeling and antimicrobial functions.
b. PHMB is a broad-spectrum antimicrobial biocide, is added owing to its polyspecies antiseptic potential.
c. Maltodextrin absorbs exudates and maintains moisture balance,
source of topical nutrition to wound site, impair microbial growth by
altering water activity (aw) and lowering pH, hygroscopic action
prevents edema, role in promoting collagen turn over.

• In combination the formulation restores moisture balance, is antimicrobial and a source of topical nutrients. By elimination of the tissue deteriorating and proliferation inhibiting activities, the wound bed is made receptive to therapeutic interventions that promote cell proliferation and healing.

• Larger surface area by micronization of dAmCh improves availability and presentation of bioactive compounds to site of injury, especially in case of irregular and deep wounds.

Method of preparation:
Placental membranes collected in aseptic conditions and stringently screened for microbial and infectious agents are processed in a way that preserves its bioactive components. The membrane with intact amnion and chorion layers is gently washed with purified water (mili-Q) / normal saline then clean with placental washing solution (RBC lysis solution containing 18% NaCl) to remove the blood stains/extra layers, at hot air oven 39±1 degree Celsius, and incubated for a standardized time in a defined concentration of PHMB solution, preferably at a concentration of 6.25% w/v. After drying, the PHMB impregnated dehydrated composite membrane is micronized by milling. For the final formulation, the micronized dehydrated AmCh-PHMB is mixed in a definite ratio with pharmaceutically accepted, powder form of the starch hydrolysate, maltodextrin. Gamma irradiation of the final product ensures sterility.

According to an embodiment of the present invention, operational procedure for processing of dehydrated human amnion chorion membrane with epithelial layer are as follows:

Materials used: NaCl (Sodium chloride); NaHCO3 (Sodium bicarbonate); EDTA (Ethylenediaminetetraacetic acid); NH4Cl (Ammonium chloride); PHMB (Polyhexamethylene biguanide).

Reagents used: 1X DPBS without Calcium and Magnesium; 70% IsoPropyl Alcohol; Placental Washing solution (2X RBC lysis solution containing 18% NaCl); 6.25% PHMB solution; Maltodextrin; Water for injection; 1% Hypochlorite; 2X VOA solution.

Equipment used: Biosafety cabinet, weighing balance, shrink wrapping machine, hot air oven, temperature controlled shaker incubator, fume hood, band sealer, vacuum sealer, double cone blender, micropipette, band sealer, oscillating. mixer mill MM400, Sieve shaker, and Double cone blender.

Procedural Steps:
1. AM/CH Processing (Cleaning, Dehydration and Sterilization). Before starting the PHMB soaking, sample were cut appropriately (2*2 size) and subjected to histology analysis Hematoxylin & Eosin (H&E) Staining and Immunohistochemistry staining for type IV collagen.
2. collection of placental tissue;
3. washing in purified water and 2X VOA transfer;
4. subjecting the membranes to microbial and infectious disease screening;
5. cleaning the placental tissue with sterile distilled water;
6. gentle washing the placental tissue in placental washing solution (2X RBC Lysis solution and 18% NaCl) in 100 rpm shaking incubator for 45-60 minutes;
7. washing in sterile distilled water in 100 rpm shaking incubator for 10 – 15 minutes;
8. spreading the membrane on delrin block and keeping the membrane in Tyvek pouch for drying at 40 ± 2 degree Celsius in hot air oven for 16-24 hours;
9. Expected PHMB concentration calculation:
15mg of PHMB in 3g of formulation= 0.5%. Similarly, 7.5mg/30mg of PHMB in 1.5g/6g of formulation yields 0.5%.
10. For 3g formulation, incorporate 15mg of PHMB in 60 mg of AMCH Membrane. For 1.5g/6g formulation, incorporate 7.5mg/30mg of PHMB in
30 mg/120 mg of AMCH membrane.
11. 6.25 % PHMB solution preparation shall be done for the same to make the effective concentration of 100g of final formulation to be 0.5%.
12. Calculate the 6.25% PHMB soaking volume based on weight of the dried membrane (weight * 4ml of 62.5mg/mL) add the PHMB solution to the membrane and parafilm the container. Keep the container for 4 hours in a shaker incubator at 100 RPM (+/-10). Transfer and spread the soaked membrane on a tyvek sheet, where amnion is facing downward and chorion facing upwards. The Chorionic layer is thicker compared to the amnion layer. The amnion layer is comparatively translucent to the chorionic layer. Seal the Tyvek pouch and incubate in a Hot air oven at
39± 1 °C for 16-24 hours

Pooling, Milling, Sieving, Formulation and Blending:
1. Cut the dried membranes into small pieces using sterile multi blade or single SS scissors inside the LAF (Laminar Air Flow chamber) and transfer 2g into a milling cup.
2. Perform milling -using oscillating Mill MM400 as per below table:
Balls Size Ball No Milling Duration Frequency Purpose Remarks
10 mm 14-16 2 mins 30 Hzs Formulation Coarse Particle

3. After milling, transfer the micronized powder into a 100mL sterile container.
4. After milling 10-30 samples, Clean the milling jar and milling ball and followed by 70% IPA /toluene wash and dry the milling jar and 10 mm balls in a hot air oven.
5. Sieve all the material (AMCH Coated PHMB Powder and Maltodextrin) in
500 micron sieve for 10 mins.
6. Particles less than 500 micron should be taken for further process.
7. Particles more than 500 micron keep it separate.
8. For Formulation (1 Kg), take micronized powder with PHMB and Maltodextrin in a 5 Litre glass bottle / double cone blender.
9. For one Kg batch, take 25g micronized powder with PHMB in sterile 1000ml glass bottles and add 25g maltodextrin mix for 3-5 minutes manually.
10. Followed by adding 50g of maltodextrin and mixing for 3-5 mins manually.
11. Then add 100g of maltodextrin and mix well for 3-5 mins manually.
12. Finally transfer the mixer into a sterilized double cone blender add 800g of maltodextrin in and mix well for 60-120 mins in the double cone blender.
13. Keep the sample in hot air for drying at 39 ±1 oC for 2 hrs.
Formulation:
S. No Total Weight of Formulation (g) Maltodextrin (g) PHMB Coated AMCH Powder (g)
1 1000 975 25
2 2000 1950 50
3 3000 2925 75
4 4000 3900 100
5 5000 4875 125
14. Fill the powdered sample in screw capped polypropylene vials.
15. Packed samples are subjected to gamma sterilization 25 kGy.

The physical and functional characteristics of the AmCh-PHMB membrane that aids in wound healing is validated by established protocols as per the USP monograph, USP 40-NF 35, 6486.

The functional integrity of the components in the formulation, and the overall functional features of the formulation are confirmed through specific in vitro assays.

Characterization of physical properties of dehydrated micronized amnion chorion membrane with PHMB:
Parameter Acceptable limit
3 gm 1.5 gm
Assay PHMB % w/w 0.35-0.55% 0.49 ± 0 0.48 ± 0.005
Moisture % NMT 15% 5.25 ± 0.43 6.3 ± 0.97
Total Protein (w/w %) 0.5%-0.7% w/w 0.60 ± 0.04 0.59 ± 0.06
Tap Density (g/mL) NA 0.586 ± 0.005 0.57 ± 0.01
Bulk Density (g/mL) NA 0.51 ± 0.005 0.52 ± 0.01

Functional assay evaluation report:
The ability of sterile dHACM (dehydrated Human Amnion Chorion Membrane) powder with PHMB formulation were assessed for its competence to modulate inflammatory expression, cell proliferation, migration, angiogenesis, and the presence of various growth factors. These invitro assays represent various imperative processes involved in wound healing.

Quantification of cytokines and growth factors in dHACM with PHMB:
Quantification of cytokines and growth factors from dHACM with PHMB samples (n=3) was performed using Enzyme linked immunosorbent assay (ELISA). 100 mg powder incubated in lysis buffer for 24 hours, homogenized to prepare cytokine extract which was centrifuged at 4500 g for 10 minutes; supernatant was collected in separate tubes and stored at -80 degree Celsius for further analysis. Overall, 3 cytokines (TGFb1, EGF, bFGF) and growth factors quantification was done.

dHACM with PHMB extract preparation:
1 ml of 1X lysis buffer containing 1x protease inhibitor cocktail was added into the powder sample (dHACM with PHMB powder) and incubated at 2-8°C for 24 h. Powder were homogenized using glass homogenizer and centrifuged at 4500g for 10 min to remove residues. The supernatant obtained was collected as extract and stored at -80°C until the assay was performed. The concentration of growth factor / cytokines was calculated from the standard curve generated from known concentration. The concentration was converted from pg/cm2 to pg/mg by dividing with weight of 1×1 cm2.

Figure 2 illustrates the cytokines for dHACM powder formulation with PHMB. These results demonstrated that processed powder formulation of the present invention retain the native composition of ECM and signaling molecules normally present in amnion and chorion membrane, it preserves bioactivity that could potentially support wound healing process.

Effect of sterile dHCAM-PHMB powder on mesenchymal stem cells proliferation:
Gamma sterilized dHACM – PHMB powder were assessed for its effect on proliferation of human umbilical cord derived Mesenchymal stem cells (HUCMSCs). 3 gm powder taken and put in 1 ml MEM (Minimum essential medium). Incubate in 4-8 degree Celsius and allowed to soak for 24 hours. Elute obtained after 24 hours and aliquoted stored at -80 degree Celsius for further experiments. For BrdU assay, triplicate wells were set up for each sterile dHCAM – PHMB product with one donor MSC cultured cells. AMEM (MEM) without any growth factors was used as negative control. 5% complete media with growth factors was used as positive control.

Figure 3 illustrates Fold change of dHACM powder with PHMB from Negative control on MSCs. In all tested samples, the medium containing elute from sterile dHCAM-PHMB powder demonstrated significant increase in mesenchymal stem cell proliferation when compared to negative media control.

Effect of sterile dHCAM-PHMB powder on human dermal fibroblast cell proliferation:
Gamma sterilized dHACM powder were assessed for its effect on proliferation of human dermal fibroblasts. 3 gm powder taken and put in 1ml MEM. Incubate in 4-8 degree Celsius and allowed to soak for 24 hours. Elute obtained after 24 hours and aliquoted stored at -80 degree Celsius for further experimentation. For BrdU assay, triplicate wells were set up for each sterile dHACM – PHMB product with DF cultured cells. MEM without any growth factors was used as negative control. 5% complete media with growth factors was used as positive control.

Figure 4 illustrates effect of sterile dHCAM-PHMB powder on Human dermal fibroblast relative to media. In all tested samples, the medium containing elute from sterile dHACM – PHMB product demonstrated significant increase in dermal fibroblast proliferation when compared to negative media control.

Effect of sterile dHACM-PHMB powder on human macrophage cell proliferation:
Gamma sterilized dHACM – PHMB powder were assessed for its effect on proliferation of macrophage cells. Extract of powdered samples were prepared by mixing 3 gm powder in 1 ml MEM (Minimum essential medium); incubating at 4-8 degree Celsius and allowed to soak for 24 hours.

Figure 5 illustrates the effect of sterile dHCAM -PHMB on human macrophage cell proliferation relative to media. In all tested samples, the medium containing elute from sterile dHACM – PHMB product demonstrated significant increase in Dermal fibroblast proliferation when compared to negative Media control. The results from the in vitro study clearly establish that Sterile dHCAM – PHMB powder contains one or more soluble factors capable of stimulating Dermal Fibroblast. The fold response of soluble extract of Sterile dHACM – PHMB product when normalized to response observed with complete media is consistent across Dermal Fibroblast for each sample.

Effect of sterile dHCAM – PHMB powder on human endothelial cells tube formation:
Gamma sterilized dHCAM-PHMB were assessed for their effect on tube formation of human umbilical cord vein endothelial cells (HUVECs). 3 gm sample was taken and put in 1 mL Endothelial Growth Medium, incubate in 2-8 °C incubator and allow to soak for 24 hours. Elute obtained after 24 hours. Elute was collected and aliquoted and stored at -80°C for further experiments. For Tube formation assay, single well was set up for each Sterile dHCAM-PHMB product with a single donor HUVEC cultured cells.

There was significant production of tube in all samples in comparison to control Endothelial basal medium. In all cases, Sterile dHCAM-PHMB powder has shown to support the formation of new blood vessels, and this is further substantiated with proteins quantified in previous experiments. There are soluble signals in dHCAM-PHMB powder also stimulated human microvascular endothelial cells to proliferate in vitro, and further, to increase production of several endogenous growth factors, cytokines, and receptors related to angiogenesis. Our findings strongly support dHCAM-PHMB powder exerting therapeutic actions both directly and indirectly by activating multiple signaling pathways that promote angiogenesis within healing wounds.

Advantages of the present invention:
An advanced dressing formulation that utilizes the absorptive and antimicrobial properties of maltodextrin, and the polyspecies antiseptic potential of PHMB to manage and prevent excessive fluid formation while making the wound bed conducive to the therapeutic agents in dAmCh membrane. Restores moisture balance, is anti- microbial, and have regenerative properties.
,CLAIMS:WE CLAIM:
1. A topical formulation for treatment of wounds, comprising of:
micronized placental tissue selected from group consisting of dehydrated amnion and chorion membrane (dAmCh) – 2 g;
polyhexamethylene biguanide (PHMB) – 0.5g to 2 g; and
filler – 96g to 97.5g,
wherein the formulation provide synergistic effect with respect to treatment of chronic wound exudates and the formulation is a powder.

2. The topical formulation for treatment of wounds as claimed in claim 1, wherein each gram of formulation contains 20 mg (2%) of micronized dehydrated amnion and chorion membrane, 5 mg to 20 mg (0.5% to 2%) polyhexamethylene biguanide, and 960 mg to 975 mg (96% to 97.5%) of maltodextrin.

3. The topical formulation for treatment of wounds as claimed in claim 1, wherein the micronized dehydrated amnion and chorion membrane is impregnated with polyhexamethylene biguanide.

4. The topical formulation for treatment of wounds as claimed in claim 1, wherein the micronized dehydrated amnion and chorion membrane has a particle size not more than 500 microns.

5. The topical formulation for treatment of wounds as claimed in claim 1, wherein the filler is maltodextrin, preferably as pharmaceutically acceptable powder.

6. The topical formulation for treatment of wounds as claimed in claim 1, wherein the dehydrated amnion-chorion layers are derived from human placenta.

7. A method for preparation of topical formulation for treatment of wounds, comprising the step of:
preparing 6.25% w/v PHMB solution;
adding 4 ml of 6.25% of PHMB solution to 1g of dAmCh membrane;
incubating the dAmCh membrane with PHMB for a period of 4 hours in a shaker incubator at a temperature of 37±1 °C;
subjecting the dAmCh membrane with PHMB at a temperature of 39±1 °C for 16 to 24 hours;
cutting the dried membranes into small pieces;
performing the micronization using ball mill;
sieving micronized dAmCh coated PHMB powder and maltodextrin separately using 500-micron sieve;
blending the micronized dAmCh coated PHMB (2.5g to 4g) with maltodextrin (96g to 97.5g) for 60 to 120 mins; and
drying at 39±1 °C for 2 hours,
wherein the formulation provide synergistic effect with respect to treatment of chronic wound exudates and the formulation is a powder.