WO2022157552A1

WO2022157552A1 - Composition and processes of preparation of flowable hemostatic matrix

Info

Publication number: WO2022157552A1
Application number: PCT/IB2021/051222
Authority: WO
Inventors: Rajeev Gupta; Piyush Patel; Deepak Patel
Original assignee: Aegis Lifesciences Pvt. Ltd.
Priority date: 2021-01-19
Filing date: 2021-02-13
Publication date: 2022-07-28

Abstract

Described is a processes of making a hemostatic formulation and said processes comprising a preparation containing biocompatible polymer mix suitable for use in hemostasis. The composition includes cross linked amino acid sequence agents of the biocompatible polymer which enhance platelet aggregation and hemostasis. The process also includes other amino acid nanocomposites of the biocompatible polymer which enhance hemostasis, wound healing and excipients which retard degradation of the biocompatible polymer. The above described composition is packed with inert gas, sterilized and readily available for suitable application with saline or any coagulation agent.

Description

PATENT TITLE: Composition and processes of preparation of Flowable hemostatic matrix

INVENTORS: Mr Rajeev Gupta, Mr Piyush Patel, Mr Deepak Patel

APPLICANT: Aegis Life Sciences Pvt LTD, Ahmedabad, Gujarat, India - 382213

Field of Invention:

The present invention relates to composition and processes for making of hemostatic formulation in a storage stable form.

BACK GROUND OF THE INVENTION:

Protein based hemostatic materials such as collagen and gelatine are commercially available in solid and loose or packed powder form for use in Surgical procedures. Mixing of the packed matrix with a fluid such as saline or thrombin solution may from a flowable paste or slurry useful as a ready to use hemostatic composition. It further can be used in cases of diffuse bleeding, particularly from uneven surfaces or hard to reach areas, depending on mixing conditions and relative ratios of the materials. The process of making flowable hemostatic composition and devices for delivery of the same had been described in different patents including WO/2005/016257 A2, WO/2005/016256, WO/ 2013/185776 Al, WO 2011/151400A1.

Floseal Hemostatic matrix (Baxter), Surgiflo Hemostatic matrix (Ethicon) is a kit for producing a hemostatic gelatine paste. The gelatine paste is produced by first making a thrombin solution and then transferring the gelatine matrix- thrombin solution mixture back and forth between two connected syringes for a minimum of five passes. The paste can then be applied to a bleeding site to promote hemostasis.

Gelatine a biopolymer, is generally prepared by thermal denaturalization of collagen, which is available in animal skin and bones in the presence of dilute acids. Gelatine consists of a large number of amino acids with major percentage been glycine, proline, and hydroxy proline.

The mechanism of action of hemostatic devices is supportive and mechanical. Hemostatic devices, when applied directly to bleeding surfaces, arrest bleeding by the formation of an artificial clot and by producing a mechanical matrix that facilitates clotting. Clotting effect is being due to release of thromboplastin from platelets, occurring when platelets entering the gelatine sponge, become damaged by contact with the walls of its myriad of interstices.

At sites of vascular injury, platelets come into contact with gelatine, which triggers their activation and the formation of a hemostatic plug. Besides glycoprotein lb (GPIb) and Alpha and beta 3 integrin, which indirectly interact with gelatine via von Willebrand factor (VWF), several collagen receptors have been identified on platelets, most notably alpha and beta integrin's and the immunoglobulin (Ig) superfamily member GPVI (Glyco Protein IV) It is now recognized that platelet adhesion to gelatine requires prior activation of integrin's through "inside-out" signals generated by GPVI and reinforced by released second-wave mediator's adenosine diphosphate (ADP) and thromboxane A2. These developments have led to revision of the original "2-site, 2-step" model, which now places GPVI in a central position in the complex processes of platelet tethering, activation, adhesion, aggregation, degranulation, and procoagulant activity on gelatine and collagen to initiate hemostasis.

Gelatines and Collagens consist of repeat GXY motifs where G is glycine and X and Y are frequently proline (amino acid code, P) and hydroxyproline (amino acid code, O). The sequence GPO makes up approximately 10% of gelatines. The cross-linking of these monomeric gelatine structures forms fibrillar gelatine, the predominant structure that platelets come into contact with in the ECM. GXY motifs in gelatine are very important to generate signal for GPVI activation and further hemostasis. Apart from GPO sequence the other amino acids like Arginine, Glutamic acid, Methionine though present in less percentage in Gelatines and collagen also play a central role in platelet activation, hemostasis, angiogenesis and wound healing. Branched-chain amino acids (BCAAs), including Tryptophan, Histidine and leucine are also responsible for platelet activation and adhesion that triggers clot formation and arrest of bleeding which are deficient in many gelatines.

The commercial medical gelatine powder as such compromises a homogenous mix of a prefixed composition of different amino acids which specifically aid in hemostasis. Time to hemostasis and tissue healing are the key performance attributes of different gelatine variants and the central role is mediated by few important cross-linked amino acid sequences described above. In very difficult and critical surgeries, where time is the limiting factor and that could also result in excess loss of blood, there is evident need to reduce the hemostasis time and also tissue healing in critical visceral areas of the surgical sites. There is also necessity to have high biocompatible inactive ingredients (excipients and preservatives) in the gelatine compositions than the conventional ones which can provide utmost safety attributes to the formulations without invoking any undesired effects on longer duration of use.

SUMMARY OF THE INVENTION

The present invention describes is the composition of the flowable hemostatic matrix that will decrease coagulation time and time to hemostasis with better wound healing activity and necessarily contains best biocompatible excipients. The invention also describes the processes of making of the composition of flowable hemostatic matrix and packing therein.

The invention discloses the addition of substantial I quantities of cross-linked non-essential amino acids and nano particles of essential amino acids to the original medical gelatine powder and making a homogeneous mix of the the same. The second part discloses a volume of saline solution with different percentages of lubricants and necessarily containing different amounts of Oxychloro complex or any other antimicrobial preservatives. The second part of the volume is mixed with the first part of the modified gelatine composition at different ratios to make it into a paste or slurry at various predetermined temperatures and humidity conditions.

An inert gas is purged into the slurry to make a homogenous mix and with inert gas dispersed into the slurry or paste. The slurry or paste so prepared can be loaded into Leur syringes or any other applicators or device for dispensing, wherein the unfilled part of the syringe or device is packed with inert gas and the final packing of the components of the kit or devices is again done with inert gas environment within. The above described composition after gamma sterilization, can be readily used for suitable application with saline or any coagulation agent.

DETAILED DESCRIPTION OF THE INVENTION

Both sterilized and unsterilized compositions made by the processes of the invention contain a solid phase containing gelatine powder with preferred particle size of about 100pm to 1000 pm, cross linked non-essential amino acids with preferred particle size of about lOOp to 800pm and nanoparticles of essential amino acids with preferred particle size of about lOOnm to lOOnm, in substantial amounts which are homogeneously mixed. The liquid phase contains normal saline, lubricant and preservative of different rations to make a slurry or paste of predetermined density and peak force of compression. The composition is mixed and dispersed with an inert gas as a gaseous phase within. The composition is packed in nitrogen gas atmosphere wherein the vacant portions of the packing are filled with biocompatible inert gas.

The cross linking of amino acids necessarily (GXY motifs) Glycine (G), Proline (P) and Hydroxy proline (O) ie... GPO sequence is done by conventional Bio conjugation technique. Amino acid profiling for amino acid analysis, cross linked amino acid composition was hydrolysed with 6 N HCI for 24 h at 1200 °C. The resultant mixture was analysed by an Agilent 1260 HPLC system (Agilent, USA) with a fluorescent detector (FLD) after derivatization with OPA (O-phthalaldehyde) (SRL, India). For proline and hydroxyproline identification, 9-fluorenylmethoxycarbonyl (FMOC-CI) (SRL, India) was applied to derivatize the sample. The derivatized sample was loaded (50 pl) onto an HPLC column (ZORBAX-SB- C-18 column (250 x 4.6 m, 5-micron particle size) using sample injector (Agilent 1260). For gradient elution method, the column was eluted using 0.01 M NazHPC buffer and acetonitrile (100%) as a mobile phase solvent system. The flow rate was maintained at 1 ml/min. Data from the system was collected and evaluated using Agilent open LAB control panel software. Amino acid from sample and standard was quantified via comparison to the retention time and absorbance. The amino acid content was expressed as the number of residues/1000 residues.

Amino acid profiling of the cross linked amino acids is carried out by HPLC analysis and the results showed higher contents of G, P, and O residues (i.e., 302, 99, and 118 residues per 1000 amino acids residues, respectively). Nano particles of essential amino acids necessarily arginine, Glutamic acid and tryptophan was carried out by interfacial polymerization using poly lactide polymers which resulted in average spherical diameter of 250 nm. Different ratios of the compositions of the cross-linked amino acids and nanoparticle amino acids were used with gelatine powder

The liquid phase is prepared with different volumes of normal saline or Phosphate Buffered Saline (PBS of pH 7.2 to 7.6), glycerol (up to about 30% of weight or from about 5% to 15% based on the weight of liquid phase) and anti-microbial agents either Stablished Oxychloro Complex (SOC, from about 0.001 to 0.0001% on the weight of liquid phase or Benzalkalonium chloride (BKC, from about 0.01% to 0.001 % based on the weight of liquid phase). The gaseous phase comprises necessarily of nitrogen gas for mixing and dispersing in the composition in controlled environment.

The hemostatic composition so formed is a hemostatic paste or slurry with required properties of flowability, extrudability and / or injectability suitable for the application with lesser time of hemostasis than the marked preparations. Composition made by the processes of the present invention may be prepared, filled into medical devices such as syringe or other known applicators used to dispense flowable hemostatic compositions and sterilized by ionizing irradiation well before time of the intended use.

Examples:

Samples were prepared in the examples below and were tested for density, extrudability or peak expression force, invitro Activated coagulation activity using whole blood.

Example 1: Fl

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 125 ml of saline containing 0.005% of BKC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 70 ±5 seconds compared to control time of 85±5 sec

Example 2: F2

1 gram of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 4 ml of saline containing 0.005% of BKC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.6 -0.65gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 11.8 and 16.8 Ibf respectively with invitro activated coagulation time of 75 ±7 seconds compared to control time of 85±5 sec

Example 3: F3

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 125 ml of saline containing 0.001% of SOC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 0.7and 0.75 Ibf respectively with invitro activated coagulation time of 70 ±5 seconds compared to control time of 80±5 sec

Example 4: F4

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with O.lmg of GPO amino acid motifs and 125 ml of saline containing 0.001% of SOC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 65 ±6 seconds compared to control time of 85±5 sec

Example 5: F5

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with O.lmg of GPO amino acid motifs, 0.1 mg of nanocomposite amino acids and 125 ml of saline containing 0.001% of SOC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD Luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 60 ±3 seconds compared to control time of 85±5 sec

Example 6: F6

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 0.1 mg of nanocomposite amino acids and 125 ml of saline containing 0.001% of SOC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 68 ±4seconds compared to control time of 85±5 sec

Example 7: F7

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 0.1 mg of GPO amino acid motifs, 0.1 mg of nanocomposite amino acids and 125 ml of saline containing 0.005% of BKC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 60 ± 5 seconds compared to control time of 85 ± 5 sec

Example 8: F8

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 0.01 mg of GPO amino acid motifs, 0.01 mg of nanocomposite amino acids and 125 ml of saline containing 0.005% of BKC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 68 ± 5 seconds compared to control time of 85 ± 5 sec

Example 9: F9

A total of 30 samples were prepared as follows.

25 grams of Gelatine powder wherein cross linking was carried out at a range of 120° C - 140° C is mixed with 0.01 mg of GPO amino acid motifs, 0.01 mg of nanocomposite amino acids and 125 ml of saline containing 0.001% of SOC and 5 % of Glycerol. The contents loaded into 0.5 pint Donvier mixer fitted with a mixing paddle. A tube connected to a nitrogen source with nitrogen gas was purged into the system for about 20 minutes, for mixing and dispensing into the composition as gaseous phase. The composition was loaded into 60 CC syringe subsequently dispensed into 10 CC BD luer syringes via two way Luer connector. The vacant space and final packing was done with nitrogen atmosphere. The density of the composition was 0.7 -0.75gm/ml. Syringes were capped and sterilized by gamma irradiation at a dose of 25 KGy. The peak expression force of non-sterile and sterile preparation was 16 and 20.7 Ibf respectively with invitro activated coagulation time of 67 ± 3 seconds compared to control time of 85 ± 5 sec

Given the density of the flowable hemostatic composition is an indicator of acceptable mechanical and hemostatic properties of the composition, the density of the flowable hemostatic composition is measured upon completion of gamma sterilization. The invitro activated coagulation time of the compositions is significantly better than the control samples and some marketed preparations as per the literature available in public domain. The accelerated stability studies also indicated no loss of physical properties and invitro activated coagulation activity of the composition.

Table 1:

Density range, Average peak expression force and Invitro activated clotting time of different formulations of the composition after sterilization

Claims

Claims We claim

1. The composition of the flowable hemostatic matrix comprising a solid phase biocompatible hemostatic agent or mix of hemostatic agents constituting additional cross-linked amino acid sequences and nanoparticle composite amino acids.

Introducing a liquid phase of volume of liquid solution with different percentages of lubricants, wetting agents and containing different amounts of preservatives. Mixing of the liquid phase with the solid phase at different ratios using biocompatible gas to mix and disperse the slurry or paste at different temperature and humidity conditions. Biocompatible gas is filled in vacant volume of the devices or applicator of dispensing in the final packing at prefixed atmospheric conditions for radiation sterilization. The composition so prepared is readily available for suitable application with saline or any coagulation agent.

2. The composition of claim 1 wherein said the hemostatic agent may be protein, cellulose or synthetic based polymer containing gelatine, collagen, beeswax, cellulose, modified cellulose, polymers of alcohols, aldehydes, acetals, lactides, glycolides

3. The composition of claim 1 wherein said hemostatic agent is from different sources, types and compositions

4. The composition of claim 1 wherein said the amino acids are essential, non-essential, semi essential, conditional or branched amino acids from different sources

5. The amino acids of claim 4 are cross-linked by different methods of linking including physical, chemical, thermal, biological or bio conjugation

6. The nano particle amino acids of claim 1 wherein said can be prepared by different methods including plasma method, chemical vapour deposition, co-precipitation, hydrothermal synthesis, inert gas condensation, inter facial polymerization, ion sputtering scattering, micro emulsion, microwave, pulse laser ablation, sol-gel, sonochemical, spark discharge, template synthesis, biological synthesis and gamma irradiation.

7. The nanoparticles in claim 6 wherein said the nanoparticles prepared with polymers consisting of natural or synthetic source

8. The process of claim 1 wherein said mixing of the hemostatic matrix and cross linked amino acids and nanoparticle amino acids consisting of different methods including of paddle, turbines, tumblers and static mixers

9. The composition of claim 1 wherein said liquid is aqueous.

9 The composition of claim 9 wherein said liquid comprises sterile saline at different temperatures The composition of claim 1 wherein said the lubricants and wetting agents can be solids are liquids. The composition of claim 11 wherein said the lubricants comprises of water soluble and water insoluble agents The composition of claim 12 wherein said the lubricant comprises of glycerol, magnesium stearate, sodium benzoate, Sodium oleate and sodium lauryl sulphate. The composition of claim 1 wherein said the preservatives are antimicrobial agents, antioxidants and chelating agents The composition of claim 14 wherein said the preservatives are natural or synthetic. The composition of claim 14 wherein said antimicrobial preservative comprises sodium benzoate, Benzalkonium chloride, Stabilized oxychloro complex. The process of claim 1 wherein said the Biocompatible gases are inert gases from the group consisting of nitrogen, argon, Xenon. The composition of claim 1 wherein said the average diameter of said hemostatic agent is from about 10pm to 1000pm The composition of claim 7 wherein said the average diameter of the said nanoparticle composites is from about lOnm to lOOOnm The composition of claim 1 wherein said added nanoparticle composites are from about 0.1 mg to 0.01 mg The composition of claim 1 wherein said cross linked amino acid sequence motifs are from about 100pm to 800pm. The composition of claim 1 wherein said added cross linked amino acid sequence motifs are from about 0.1 mg to 0.01 mg The process of claim 1 wherein said solid particles, said liquid, said gas are present in said flowable hemostatic composition at a ratio of from about 1:2:1 to about 1:20:16 based on g:ml:ml The process of claim 1 wherein said the density of said flowable hemostatic composition is from about O.lg/ml to about l.Og/ml The process of claim 1 wherein said peak expression force of said flowable hemostatic composition is from about 15 to 25 Ibf. The composition of claim 1 wherein said composition is sterilized by ionizing radiations. The composition of claim 1 wherein said composition can be suitably filled in Leur syringes, other medical devices or applicators for dispensing The composition of claim 1 wherein said composition can readily be used with saline or any coagulation agent for hemostasis and wound healing.

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