KR20180091035A - Thrombin microcapsule - Google Patents

Abstract

A spray-dried thrombin powder comprising microcapsules, a process for its preparation and its use are provided.

Description

Thrombin microcapsule

The present invention relates to the field of pharmaceuticals, and more particularly to spray-dried thrombin powders comprising microcapsules, a process for their preparation and their use.

Thrombin is a protease that functions multiple times in blood clotting. Thrombin is formed from prothrombin (coagulation factor II), a circulating zymogen precursor protein in plasma. It is cleaved by protein hydrolysis in the coagulation cascade to form thrombin. Thrombin also acts as a serine protease, which not only converts soluble fibrinogen to insoluble strands of fibrin, but also catalyzes many other coagulation-related reactions.

Thrombin is widely used in clinical applications as a clotting factor that converts fibrinogen to fibrin and stops bleeding from the wound. Thrombin is a common component of surgical dressings and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glue, adhesive, and sealant.

Thrombin powders for use in the preparation of pharmaceutical compositions are generally prepared by lyophilization of the solution.

Typically, the term "freeze-drying" refers to a process in which the solution is frozen and subsequently reduced in water concentration, for example by sublimation to a level that does not support biological or chemical reactions.

Freeze drying is a batch process that is limited by the capacity of the freeze dryer and lasts for several days. This puts the entire batch at risk for a significant amount of time until the process is complete. In addition, treating the resulting lyophilized "cake " with the powder can be further reduced, for example by breaking, sieving and / or milling to further reduce particle size . These steps can reduce the efficacy and mass yield of the dry product. Advantageously, using a spray drying process instead of lyophilization provides a cost-effective continuous and high-volume process wherein the dry product is obtained immediately in the form of a dry powder, not as a "cake ".

As used herein, the term " cake "or" solid cake "refers to a porous sponge structure-like composition produced from the lyophilization process.

Spray drying is accomplished by, for example, spraying through a spray nozzle while contacting with an atomizing gas to form atomized particles; The combination of a liquid flow and a gas flow produces a spray, wherein the atomized energy is generated by a two-fluid nozzle atomization provided by a gas flow; Or liquid atomization, such as a solution, suspension or emulsion, by centrifugal atomization in which the solution is delivered to the rotating disk and the spray is generated by the energy produced by the rotation of the disk. Alternatively, the liquid flow can be atomized using a pressure nozzle, wherein the liquid flow is pushed through a small aperture, and a change in pressure converts the liquid flow into a spray of a small droplet.

After formation of the spray particles, drying of the spray in the flow of hot gases (e.g., air and nitrogen) provided by the drying gas conduit is followed. The sprayed particles are quickly dried to powder, which is then separated from the hot air stream in a cyclone apparatus and collected in a vessel, for example a vial. The spray drying process is controlled by several key process parameters including dry air flow rate and temperature, atomizing air flow rate, solution flow rate,

Generally, enzymes such as thrombin are sensitive to the high temperature and shear stress conditions used in prior art spray drying processes, and these conditions usually result in enzyme denaturation and subsequent loss of efficacy. For example, thrombin in solution is sensitive to temperatures above about 45 캜.

The prior art solves this problem by using a relatively low thrombin concentration in the solution used for spray drying or by increasing the concentration of carbohydrates such as trehalose in solution. These conditions result in spray-dried powders containing low concentrations of thrombin (generally less than about 1 IU / mg).

Examples of spray drying processes and their uses in the background art for the preparation of thrombin powders include U.S. Patent Nos. 6,113,948; U.S. Patent Application Publication No. 2012/0315305; International Patent Publication No. WO 92/18164; U.S. Patent No. 6,416,739; European Patent EP0713388B1; U.S. Patent No. 8,846,105; U.S. Patent No. 6,703,047; U.S. Patent Application Publication No. 20120121532; U.S. Patent Application Publication No. 20100249044; International Patent Publication No. WO 14/135689; J Microencapsul. 2013; 30 (7): 624-31. Doi: 10.3109 / 02652048.2013.770097. Epub Mar 14 2013]; Biotechnol Appl Biochem. 1995 Oct; 22 (Pt 2): 203-14; , And Excipient Mediated Biostabilization of Protein Using Spray Drying Technique-Thesis work done by Priyadarsini Pattnayak for a degree of master of technology, Biotechnology and medical engineering, under the guidance of Prof. Gyana Ranjan Satpathy, National Institute of Technology, Rourkela, India. September 2010].

The present invention, in some embodiments thereof, relates to a spray-dried thrombin powder comprising microcapsules, a process for their preparation and their use.

The spray dried thrombin powder can be produced by controlling one or more of the following parameters: the composition of the thrombin solution used for the spray drying process, the spray parameters (e.g., solution flow rate, atomized gas flow rate), dry gas parameters For example, dry gas flow rate, dry gas temperature), and powder collection parameters (e.g., cooling gas flow).

It has been surprisingly found that increasing the rate of the drying gas flow results in a reduction in the water content of the spray dried powder without increasing the temperature used for the drying process.

The introduction of cooling gas downstream of the drying column was further found to lower the temperature of the dried particles below its glass transition temperature. The particles become virtually less adhesive and more elastic, thus tending to less adhere to the inner wall of the spray dryer, leading to an increase in mass yield.

In some embodiments, the method includes, in some embodiments, administering a stabilized protein carrier and a carbohydrate, e. G., A sugar, to a particular aqueous thrombin < RTI ID = 0.0 > ≪ / RTI > For example, U.S. Patent No. 6,416,739 discloses the use of 10 to 20 (% w / v) carrier protein compared to a carrier protein ranging from 0.06 to about 6.0% (w / v) Meanwhile; U.S. Patent No. 8,846,105 discloses 20-30% (w / v) sugar as compared to the range of 0.2-5% (w / v) disclosed herein.

The spray drying method and the use of the aqueous composition described herein have a high level of thrombin activity, i. (In some embodiments, the thrombin content is from about 20 to about 30 IU / mg, such as about 24 IU / mg), and in some embodiments, the water content is less than 5% w / w ), High density (in some embodiments, from about 0.1 to about 1.0 g / ml, such as about 0.46 g / ml thrombin in some embodiments), while high (in some embodiments, greater than 90%) thrombin activity Lt; RTI ID = 0.0 > of thrombin < / RTI >

In some embodiments, the associated density is the conditioned bulk density. In some embodiments, the associated density is a tapped density.

Aspects and embodiments of the present invention are described in the following detailed description and the appended claims.

According to an aspect of some embodiments described herein, there is provided a spray dried thrombin powder comprising microcapsules, wherein the powder comprises thrombin, a carrier protein and a carbohydrate, wherein the thrombin (IU) versus the carrier protein The ratio is about 0.85: 1 to 66,875: 1, and the ratio of thrombin (IU) to carbohydrate (mg) is 0.75: 1 to 26,750: 1.

In some embodiments, the microcapsules comprise thrombin, carrier protein, and carbohydrate, wherein the ratio of thrombin (IU) to carrier protein (mg) is about 0.85: 1 to 66,875: 1 and the thrombin (IU) Is 0.75: 1 to 26,750: 1.

In some embodiments, the ratio of thrombin (IU) to the carrier protein (mg) is from about 110: 1 to about 285: 1, alternatively from about 129: 1 to about 219:

In some embodiments, the ratio of thrombin (IU) to carbohydrate (mg) is from about 40: 1 to about 64: 1, alternatively from about 35: 1 to about 75:

In some embodiments, the carrier protein is selected from the group consisting of albumin, casein, keratin, and combinations thereof.

In some embodiments, the carrier protein comprises albumin, optionally human serum albumin.

In some embodiments, the carrier protein is albumin, optionally human serum albumin.

In some embodiments, the carrier protein comprises casein. In some embodiments, the carrier protein is casein.

In some embodiments, the carrier protein comprises keratin. In some embodiments, the carrier protein is keratin.

In some embodiments, the carbohydrate is a sugar alcohol, optionally selected from the group consisting of glycerol, sorbitol, xylitol, and mannitol. In some embodiments, the sugar alcohol comprises mannitol. In some embodiments, the sugar alcohol is mannitol.

In some embodiments, the carbohydrate is selected from the group consisting of saccharides, such as monosaccharides, disaccharides (optionally selected from the group consisting of sucrose, trehalose, and combinations thereof), oligosaccharides, polysaccharides, and combinations thereof It is a sugar.

In some embodiments, the powder comprises thrombin, albumin and mannitol and the ratio of thrombin (IU) to albumin (mg) is from about 0.85: 1 to 66,875: 1 and the ratio of thrombin (IU) to mannitol From about 0.75: 1 to about 26,750: 1, alternatively from about 35: 1 to about 65: 1, such as from about 50: 1.

In some embodiments, the ratio of thrombin (IU) to albumin (mg) is from about 100: 1 to about 300: 1, from about 110: 1 to about 285: 1, from about 120: 1 to about 240: From about 129: 1 to about 219: 1. In some embodiments, the ratio of thrombin (IU) to albumin (mg) is about 160: 1.

In some embodiments, the ratio of thrombin (IU) to mannitol (mg) is from about 35: 1 to about 75: 1, such as from about 40: 1 to about 64: 1.

In some embodiments, the spray dried thrombin powder comprises thrombin at a concentration of from about 0.6 to about 535 IU / mg solids.

In some embodiments, the spray dried thrombin powder comprises a carbohydrate (e.g., mannitol) at a concentration of from about 0.02 to about 0.8 mg / mg solids.

In some embodiments, the spray dried thrombin powder comprises a carrier protein at a concentration of from about 0.008 to about 0.7 mg / mg solids.

In some embodiments, the spray dried thrombin powder comprises albumin at a concentration of from about 0.008 to about 0.7 mg / mg solids.

In some embodiments, the spray dried thrombin powder has a concentration of about 20 to about 40 IU / mg solids of thrombin, a concentration of about 0.1 to about 0.2 mg / mg solids of albumin, and about 0.5 to about 0.6 mg / mg of solids Concentration of mannitol.

In some embodiments, the spray dried thrombin powder has a concentration of thrombin of from about 22 to about 35 IU / mg solids, albumin at a concentration of from about 0.16 to about 0.17 mg / mg solids, and from about 0.55 to about 0.56 mg / mg of solids Concentration of mannitol.

In some embodiments, the spray dried thrombin powder comprises thrombin, casein, and mannitol, wherein the ratio of thrombin (IU) to casein (mg) is from about 0.85: 1 to about 66,875: 1 and the ratio of thrombin (IU) to mannitol ) Is 0.75: 1 to 26,750: 1.

In some embodiments, the spray dried thrombin powder is added at a concentration of from about 0.006 to about 0.115 mg / mg solids, alternatively from about 0.034 to about 0.057 mg / mg solids, alternatively from about 0.044 to about 0.048 mg / mg solids .

In some embodiments, the total salt concentration of the spray dried thrombin powder is from about 0.1 to about 0.4 mg / mg solids, alternatively from about 0.25 to about 0.30 mg / mg solids.

In some embodiments, the spray dried thrombin powder further comprises sodium chloride at a concentration of from about 0.1 to about 0.3 mg / mg solids, alternatively from about 0.19 to about 0.2 mg / mg solids.

In some embodiments, the spray dried thrombin powder further comprises acetate at a concentration of about 0.012 to about 0.086 mg / mg solids.

In some embodiments, the spray dried thrombin powder comprises about 0.001% to about 5% (w / w) of thrombin.

In some embodiments, the spray dried thrombin powder comprises less than about 5% water (w / w), such as less than about 4% water, or less than about 3% water (w / w).

In some embodiments, the spray dried thrombin powder has a D50 of about 5 to about 13 microns and a D90 of about 15 to about 25 microns.

In some embodiments, the powder density of the spray dried thrombin powder is from about 0.1 to about 1.0 g / ml, alternatively from about 0.4 to about 0.6 g / ml.

In some embodiments, spray drying (as measured by moving the blades in an upward lifting mode of displacement using a Freeman Technology FT4 Powder Rheometer), i.e., using a Freeman Technology FT4 powder rheometer, The powdered thrombin powder has a powder flowability of from about 5 to about 15 mJ / g, alternatively from about 9 to about 10 mJ / g.

The resulting spray dried thrombin powder / microcapsules can be stored for a relatively long period of time. After storage, the powder / microcapsules can be used as is, or can be reconstituted by the addition of various volumes of aqueous solutions, such as water for injection. The volume added during reconstitution may be similar to, less than, or greater than the volume of the solution prior to spray drying.

According to a further aspect of some embodiments described herein, there is provided an aqueous composition suitable for use in preparing thrombin microcapsules, the aqueous composition comprising thrombin, a carrier protein and a carbohydrate, wherein the thrombin (IU) The ratio of the carrier protein (mg) is about 1.6: 1 to 5,000: 1 and the ratio of thrombin (IU) to carbohydrate (mg) is 2: 1 to 1,500: 1.

According to a further aspect of some embodiments described herein, a method of making a thrombin microcapsule is provided,

Wherein the ratio of thrombin (IU) to the carrier protein (mg) is about 1.6: 1 to 5000: 1 and the ratio of thrombin (IU) to carbohydrate (mg) is 2: 1 to 1500: 1; And

Spray drying the aqueous composition.

In some embodiments, the spray drying step

Introducing the aqueous composition into a spray drying apparatus;

Producing droplets from the aqueous composition introduced into the apparatus;

By evaporating water from the droplets by dry gas flow,

To prepare thrombin microcapsules.

In some embodiments, generating droplets from an aqueous composition includes using an atomized gas stream.

In some embodiments, the rate of rate of introduction of the aqueous composition into the spray drying apparatus: rate of atomized gas flow is 1: 1000 or more.

In some embodiments, evaporating water from the droplet comprises directing the droplet into a dry gas stream in a drying column at a temperature of about 100 to about 170 < 0 > C.

In some embodiments, the ratio of the rate of introduction of the aqueous composition into the spray drying apparatus to the velocity of the atomizing gas flow versus the velocity of the drying gas flow is 1: (greater than 1000) :( greater than 43,000).

In some embodiments, the streams of aqueous composition flow, atomized gas flow, and dry gas flow are coaxial in the same direction.

In some embodiments, the method further comprises cooling the thrombin microcapsules by exposure to a cold gas flow.

In some embodiments, the rate of introduction of the aqueous composition into the spray drying apparatus is from about 100 to about 1000 ml / hr, alternatively from about 200 to about 800 ml / hr, alternatively from about 300 to about 500 ml / hr.

In some embodiments, the flow rate of the atomized gas stream is from about 3 to about 20 l / min, alternatively from about 5 to about 15 l / min, alternatively from about 7 to about 9 l / min.

In some embodiments, the rate of the dry gas flow is from about 0.1 to about 1.0 m3 / min, alternatively from about 0.3 to about 0.6 m3 / min.

In some embodiments, the aqueous composition comprises about 100 to about 3000 IU / ml thrombin.

In some embodiments, the aqueous composition comprises from about 0.06% to about 6.0% (w / v) of a carrier protein.

In some embodiments, the aqueous composition comprises from about 0.2% to about 5% (w / v) carbohydrates.

In some embodiments of the methods disclosed herein, the carbohydrate is a sugar alcohol, such as mannitol.

In some embodiments of the methods disclosed herein, the carbohydrate comprises a saccharide, such as a monosaccharide, a disaccharide (optionally selected from the group consisting of sucrose, trehalose, and combinations thereof), oligosaccharides, polysaccharides, ≪ / RTI >

In some embodiments of the methods disclosed herein, the carrier protein is selected from the group consisting of albumin, casein, and keratin.

In some embodiments of the methods disclosed herein, the carrier protein comprises albumin.

In some embodiments of the methods disclosed herein, the carrier protein is albumin.

In some embodiments of the methods disclosed herein, the carrier protein comprises casein.

In some embodiments of the methods disclosed herein, the carrier protein is casein.

In some embodiments of the methods disclosed herein, the carrier protein comprises keratin.

In some embodiments of the methods disclosed herein, the carrier protein is keratin.

In some embodiments, the aqueous composition comprises calcium at a concentration of about 5 mM to about 100 mM, optionally about 30 to about 50 mM, alternatively about 38 to about 42 mM.

In some embodiments, the aqueous composition comprises acetate at a concentration of about 7 to about 50 mM, alternatively about 15 to about 25 mM, and about 18 to about 20 mM.

In some embodiments, the aqueous composition comprises sodium chloride at a concentration of from about 100 mM to about 200 mM, alternatively from about 114 mM to about 120 mM.

In some embodiments, the aqueous composition comprises

About 800 to about 1200 IU / ml of thrombin;

About 0.5 to about 0.65% (w / v) of a carrier protein;

About 1.85 to about 2.05% (w / v) carbohydrates;

About 38 to about 42 mM calcium;

About 18.0 to about 20.0 mM acetate; And

About 60 to 240 mM sodium chloride,

The aqueous composition has a density of from about 1.01 to about 1.03 g / ml.

In some embodiments, the composition has a density of from about 1.01 to about 1.03 g / ml.

In some embodiments, the aqueous composition comprises about 1000 IU / ml thrombin.

In some embodiments of the aqueous composition, the carrier protein comprises about 0.06 to about 6.0% (w / v), such as 0.6% (w / v) albumin.

In some embodiments of the aqueous composition, the carbohydrate comprises about 0.2 to about 5% (w / v) mannitol, such as about 2.0% (w / v) mannitol. In some embodiments, the pH range of the aqueous composition is from about 5 to about 8, for example from 6.9 to 7.1.

In some embodiments, the density range of the aqueous composition is from about 1.001 to 1.1 gr / ml.

In some embodiments, there is provided a spray dried thrombin powder comprising microcapsules prepared according to any of the embodiments of the methods disclosed herein.

In some embodiments, a matrix comprising spray dried thrombin powder according to any of the embodiments disclosed herein is provided. In some such embodiments, the high density (e.g., from about 0.1 to about 1.0 g / cm 3, alternatively from about 0.4 to about 0.6 cm 3) of the spray dried thrombin powder disclosed herein can be achieved by lyophilising and milling the thrombin powder , The volume of the powder to be used per 1 cm < 2 > of the matrix can be made smaller, so that the powder height on the matrix is lower and thus adheres more easily to the matrix surface.

In some embodiments, the conditioned bulk density of the spray dried powder is about 0.46 g / cm < 3 >, and the tap density of the spray dried powder is about 0.58 g / cm < 3 >.

In some embodiments, the matrix is a patch, optionally a biodegradable patch.

In some embodiments, the patch comprises a woven fabric.

In some embodiments, the patch comprises a nonwoven.

In some embodiments, the patch comprises a single layer.

In some embodiments, the patches are multilayers that optionally include two, three, or more layers.

In some embodiments, the patch comprises oxidized regenerated cellulose, optionally an oxidized regenerated cellulose matrix, and nonwoven PG910 fibers.

In some embodiments, the patch comprises gelatin.

In some embodiments, the patch may be applied to a bleeding tissue, such as a wound, to achieve a hemostasis time of less than 3 minutes (i. E., A time when hemostasis is completely stopped) when using a porcine spleen model.

In some embodiments, the patch includes any of the embodiments disclosed in WO 2007/117237.

In some embodiments, the matrix further comprises fibrinogen powder.

In some embodiments, the fibrinogen powder is prepared by freeze-drying / freeze-drying. In some such embodiments, the resulting lyophilized cake may be milled.

In some embodiments, a kit is provided that comprises a vessel comprising a spray dried thrombin powder as a first component according to any of the embodiments disclosed herein.

In some embodiments, the kit further comprises a second component selected from the group consisting of fibrinogen, gelatin, collagen, oxidized regenerated cellulose, and combinations thereof.

In some embodiments, the second component is in a form selected from the group consisting of powder, beads, granules, agglomerates.

In some embodiments, the second component is in a form selected from the group consisting of solutions, pastes, gels, and slurries.

In some embodiments, fibrinogen is in powder form.

In some embodiments, the fibrinogen is in the form of a solution, e.g., liquid or frozen.

In some embodiments, the first component and the second component are provided together in the same container.

In some embodiments, the second component is provided in a container separate from the first component.

In some embodiments, the second component is in a form selected from the group consisting of sponges, pads, patches, bandages, and gauze.

The spray dried thrombin powder, matrix or kit as disclosed herein may be used for any therapeutic purpose. The term "any therapeutic purpose" refers to any therapeutic or prophylactic treatment of the subject. Exemplary therapeutic purposes include sealing a borehole formed in a tissue or organ, e.g., a bone; Anastomosis in blood vessels; Connecting tissue parts, for example soft tissue parts; Dura defects such as, for example, treating or preventing tears and leaks, fissures or cracks after dural injection; Treating or preventing bleeding; Treating or preventing an air leak such as after a pulmonary resection; Treating or preventing defects after intestinal perforation; Treating or preventing defects after anastomotic procedures performed in any tissue, such as uterus, esophagus, stomach, pancreas, pancreatic duct, gallbladder, bile duct, bowel (including small intestine and large intestine), and rectum; Treating and preventing post-operative leaks in any tissue, such as uterus, esophagus, stomach, pancreas, pancreatic duct, gallbladder, bile duct, bowel (including small intestine and large intestine), and rectum; For example, by applying a kit component, patch or powder according to the present invention onto at least a portion of a defect, such as a staple / stitch line, to prevent or reduce the occurrence of a post-operative leak in a staple or stitch line; For firmly securing the prosthesis, for example during hernia surgery; For staple / stitch line reinforcement; For preventing or reducing alveolar air leakage; Treating or preventing kidney defects; Treating or preventing a fistula; Treating or preventing a heart defect, for example, a penetrating heart wound; Strengthening the vascular graft prosthesis; And treatment or prevention of cerebrospinal fluid leakage.

In some embodiments, the spray dried thrombin powder, kit or matrix according to any of the embodiments disclosed herein is for providing hemostasis and / or for sealing the leak and / or connecting the structures.

In some embodiments, there is provided a method of providing hemostasis and / or sealing a leak and / or joining structures in a subject in need thereof, the method comprising spray drying the spray dried material according to any of the embodiments disclosed herein Thrombin < / RTI > powder or matrix.

According to a further aspect of some embodiments described herein, there is provided an aqueous composition suitable for use in preparing thrombin microcapsules, the aqueous composition comprising thrombin, a carrier protein and a carbohydrate, wherein the thrombin: The ratio is about 1.6 IU: 1 mg to 5,000 IU: 1 mg, and the ratio of thrombin: carbohydrate is 2 IU: 1 mg to 1500 IU: 1 mg.

According to a further aspect of some embodiments described herein, there is provided a process for preparing thrombin microcapsules comprising mixing thrombin, carrier protein and carbohydrate in an aqueous solution, wherein the ratio of thrombin: carrier protein is about 1.6 IU: 1 mg to 5000 IU: 1 mg, the ratio of thrombin: carbohydrate is 2 IU: 1 mg to 1500 IU: 1 mg; And spray drying the aqueous solution.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, the description, materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

As used herein, the term "microcapsule" refers to particles having an outer shell and an internal volume, the shape of the particles being substantially spherical or cylindrical, the dimensions of the outer shell being in the range of 1 to 1000 micrometers, The particles include at least thrombin and / or one or more excipients. In embodiments where the particles are substantially spherical, the dimensions are diameters. In embodiments where the particles are substantially cylindrical, the dimension is length.

The outer shell may remain intact, rupture, or collapse.

The microcapsules disclosed herein are prepared by drying a droplet containing a solute (e.g., an aqueous thrombin composition).

Solid microparticles can be produced from larger solid particles by micronization (typically by physical impact methods such as fracture, sieving, milling, and / or milling), but such micronization methods can be used to produce microcapsules Inappropriate. Thus, according to some embodiments, microcapsules are not made by micronization of larger particles.

As used herein, the term "powder comprising microcapsules" is intended to encompass, in addition to microcapsules, other particles having a diameter / length in the micrometer range and in the non-micrometer range, such as amorphous microcapsule debris That is, separate shell fragments from microcapsules torn or torn as separate shell fragments). In some embodiments, "powder comprising microcapsule" according to the teachings of the present disclosure means that the microcapsule is at least 50% by weight; In some embodiments, the microcapsules comprise at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt% % Or even 100% by weight, and includes any range between the disclosed percentages. In some embodiments, the outer shell of the microcapsule remains intact.

In some embodiments, the "powder comprising microcapsule" comprises from about 50% to about 100% by weight of microcapsules, such as from 50 to 55% by weight, from 50 to 60% 50 to 95% by weight, 55 to 60% by weight, 55 to 65% by weight, 50 to 75% by weight, 50 to 75% by weight, 50 to 80% 55 to 70 wt%, 55 to 75 wt%, 55 to 80 wt%, 55 to 85 wt%, 55 to 90 wt%, 55 to 95 wt%, 55 to 99 wt%, 60 to 65 wt% 60 to 70 wt%, 60 to 75 wt%, 60 to 80 wt%, 60 to 85 wt%, 60 to 90 wt%, 60 to 95 wt%, 60 to 99 wt%, 65 to 70 wt% 65 to 95 wt.%, 65 to 99 wt.%, 70 to 75 wt.%, 70 to 80 wt.%, 70 to 85 wt.% %, 70 to 90 wt%, 70 to 95 75 to 95%, 75 to 99%, 80 to 85%, 80 to 90% by weight, 70 to 99 wt%, 75 to 80 wt%, 75 to 85 wt%, 75 to 90 wt% 90 to 95% by weight, 95 to 99% by weight, 90 to 99% by weight, 80 to 95% by weight, 80 to 99% by weight, 85 to 90% by weight, 85 to 95% by weight, 85 to 99% To 100% by weight, and 95 to 100% by weight of microcapsules.

In some embodiments, the "powder comprising microcapsules" comprises from about 90 wt% to about 100 wt% of microcapsules.

As used herein, the term "carrier protein" refers to a protein that reversibly interacts with thrombin in aqueous solution, i.e., does not affect thrombin activity or structure.

As used herein, the term " aqueous thrombin composition "or" aqueous composition "refers to a liquid comprising thrombin and one or more additional excipients, wherein the liquid comprises at least 50% water by weight. In a preferred embodiment, the solution comprises not less than 95% by weight of water and not more than 5% by weight of thrombin and other solids dissolved therein, and in a still more preferred embodiment not less than 98% by weight of water and not more than 2% Thrombin and other solids.

As used herein, the term "yield" in relation to thrombin activity is a percentage of the biological activity of thrombin at reconstitution of the powder as compared to the activity of thrombin in the liquid aqueous thrombin composition prior to spray drying. The terms "thrombin yield" and "thrombin activity recovery" are used interchangeably.

"Thrombin activity" and "Thrombin biological activity" refer to the conversion of a factor VIII to Factor VIIIa as well as the conversion of a protein, such as fibrinogen to fibrin, the conversion of Factor XI to Factor XIa, the conversion of Factor XIII to Factor XIIIa, Mediated transformation of the heterologous substrate comprising the conversion of the factor V to the factor Va. A "heterologous substrate" is a substrate other than thrombin, preferably a protein substrate. In some embodiments, thrombin activity refers to the conversion of fibrinogen to fibrin.

As mentioned above, the microcapsules used to implement the teachings herein are prepared by spray drying an aqueous thrombin solution.

In some embodiments, spray drying, droplet spraying of an aqueous thrombin solution into a drying chamber through an atomizing nozzle (which is referred to as "two-fluid nozzle atomization"), followed by evaporation of water from the droplet by hot dry gas flow To form desired microcapsules. Any suitable droplet size may be used to implement the teachings herein.

In some embodiments, a droplet is formed by the pressure nozzle atomization by which the spray is created by pushing the solution through the opening.

In some embodiments, droplets are formed by centrifugal atomization, in which the spray is generated by transferring the solution to a rotating disk. In some embodiments, the droplets formed have a diameter of less than 1000 micrometers, for example from 1 to 1000 micrometers.

For example, at a temperature of from about 15 째 C to about 30 째 C, for example, at a temperature of from room temperature (18 째 C to 30 째 C), which flows through the atomizing nozzle in the same direction and coaxially with the aqueous thrombin composition as is known in the art, Lt; RTI ID = 0.0 > 25 C) < / RTI > Any suitable inert gas, including air, nitrogen and argon, may be used as the atomizing gas. Preferably, the atomizing gas is dried to contain as little water as possible, and in some embodiments the atomizing gas has a relative humidity of about 30% or less.

As is known in the art, the droplets formed for spray drying can be sprayed and transferred into the drying gas inside the drying chamber. Any suitable gas, including air, nitrogen and argon, may be used as the drying gas at a temperature in the range of about 100 to about 190 < 0 > C. Preferably, the drying gas is dried to contain as little water as possible, e.g., at a relative humidity of about 30% or less. Typically, the dry gas is heated to elevated temperatures as discussed in detail below.

In some embodiments of spray drying, the spray dried particles are recovered from the bottom of the drying chamber and transferred through a cooling chamber where the particles are cooled by contact with a cold gas maintained at a temperature of about 4 to about 24 占 폚. Any suitable gas, including air, nitrogen and argon, may be used as the cold gas. Preferably, the cold gas is dried to contain as little water as possible, e.g., at a relative humidity of about 30% or less. In some embodiments, the water content of the gas is about 0.0001%.

An example of a commercially available spray drying apparatus 10 including a drying column 12 having a drying gas conduit 16 and a spray nozzle 14 is illustrated in FIG.

The spray nozzle 14 has a proximal end 18 and a distal end 20 with a spray nozzle tip 22 at the distal end 20.

A liquid conduit 24 is provided for delivering the liquid to be spray dried to the spray nozzle tip 22, through which liquid passes.

In some embodiments of the apparatus or method described herein, an atomization component 25 is provided at the distal end 20 of the spray nozzle tip 22 and is configured to deliver the liquid to be spray dried to the spray nozzle tip 22 A liquid conduit 24 having a distal end at the spray nozzle tip 22 is provided.

In some embodiments, the atomizing component 25 includes an atomizing gas conduit 26 for directing the flow of atomized gas out through the atomizing gas outlet to contact the liquid exiting the atomizing nozzle tip.

In some embodiments, the liquid is pushed through a small opening located at the distal end of the atomizing component 25 at a high pressure, resulting in a pressure drop, which changes the liquid into a droplet of spray, e.g., a droplet. Thus, in some embodiments, the atomizing component 25 includes an opening located at the distal end of the liquid conduit 24, and the liquid is pushed through the opening to form a spray.

In some embodiments, the atomizing component 25 comprises a rotating disk, wherein a liquid spray is formed by the centrifugal force generated by the rotation of the disk.

The dry gas conduit 16 includes a dry gas outlet 28 for providing a dry gas to dry the spray formed from the liquid as it exits the nozzle tip 22 to form a powder.

The resulting powder is separated from the drying gas by the cyclone unit 30 and collected in the lower part of the cyclone unit 30, for example, in the collection container 32. The path of the separated dry gas and the path of the powder are shown as 35 and 36, respectively.

In some embodiments, a cooling gas having a temperature lower than the drying gas (e.g., below room temperature) is introduced downstream of the drying column 12 by the cooling gas conduit 29.

In some embodiments, a method for preventing thermal damage to thrombin is by preventing the thrombin from overheating once in the liquid conduit 24 and the nozzle tip 22, as shown in FIG. Figure 2 shows a first tube 34a for the introduction of cold water, concentrically arranged around the liquid conduit 24, and a second tube 34b for the introduction of cold water, using a circulating pump (not shown) Which includes a heat exchange mechanism including a second tube 34b for the discharge of cold water wherein the cold water is introduced into the first tube 34a and into the second tube 34b, It circulates from the outside to the tank.

As used herein, the term "atomized gas" refers to a gas that flows in a direction parallel to the flow of a liquid solution (co-current) and forms a low pressure to form a droplet from the liquid stream . In some embodiments, atomized gas is provided in the atomizer gas conduit 26 as shown in FIG.

As used herein, the term "microdrops" refers to droplets that can be dried into powder upon contact with a dry gas stream under certain spray drying conditions, while large droplets are not dried to powder under the same specific conditions But instead sticks to the inner wall of the spray drying apparatus and, if dried, can lead to a loss of process product, i.e. a reduction in mass yield. According to some embodiments, "fine droplets" are less than 1 mm in diameter. According to some embodiments, a "large droplet" refers to a droplet of size greater than 1 mm.

As used herein, the term "dry column" or "drying chamber" refers to a column in which drying of a droplet is effected by the flow of dry gas at a high temperature (e.g., from about 100 to about 170 DEG C). In some embodiments, a drying column or drying chamber is indicated at 12 in FIG.

As used herein, the term "aqueous thrombin composition" refers to a solution comprising thrombin and a solute.

As used herein, the term "dry gas" refers to a gas that contacts a droplet upon contact to dry the droplet into powder / microcapsule. In some embodiments, the drying gas is provided to the conduit 16 and includes a drying gas outlet 28, as shown in FIG.

As used herein, the term "cold gas" refers to a gas that enters a spray dryer and combines with the dry gas flow in contact with the powder flow to reduce the temperature of the dry gas flow and the temperature of the powder. In some embodiments, the cold gas is provided to the conduit 29 as shown in FIG.

As used herein, the term "density" refers to the weight of solids per unit volume. The density can be measured in the form of a liquid, solid (e.g., powder) and can be measured as g / ml or g / cm < 3 >. In the case of powders, the density can be "conditioned bulk density" or "tap density ".

The bulk density of the powder is the ratio of the mass of the untapped powder sample to its volume, including the contribution of intergranular void volume.

As used herein, the term "conditioned bulk density" refers to the amount of powder (e.g., powder) that is used to loosen powder (e.g., using a rotating blade) to remove any precompaction and / Refers to the density of a bulk powder sample that has undergone a conditioning step to displace it.

As used herein, the term "tap density" refers to the increased bulk density obtained after mechanically tapping a vessel (e.g., a vessel) containing a powder sample.

Thrombin microcapsules formed by spray drying can be used as blood coagulants. In some embodiments, the thrombin microcapsules are physically associated (i.e., incorporated or attached) to a solid object, such as a matrix, i.e., a patch of woven or nonwoven.

As used herein, the terms "comprising", "having", "having", and grammatical variants thereof are to be construed as specifying the stated features, integers, steps or components, Does not exclude the addition of features, integers, steps, components or groups. These terms include the terms "consisting" and "consisting essentially of ".

As used herein, "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein, the term " about "refers to +/- 10%.

Fibrinogen and thrombin can optionally be prepared from an initial blood composition. The blood composition may be a whole blood or a blood fraction, i. E., A fraction of whole blood, e. G., Plasma. The origin of fibrinogen and thrombin can be autologous and will be produced from the patient's own blood, pooled blood or fractions. It is also possible that thrombin and fibrinogen are produced by recombinant methods.

In one embodiment of the invention, the fibrinogen is selected from the group consisting of an anti-fibrinolytic agent such as tranexamic acid and / or a stabilizer, such as arginine, lysine, a pharmaceutically acceptable salt thereof, or And a biologically active component (BAC), which is a solution of a protein derived from plasma, further comprising a mixture thereof. BACs are optionally derived from cryoprecipitates, particularly concentrated frozen sediments.

The term "frozen precipitate" refers to a blood component obtained from frozen plasma prepared from a component plasma collected from whole blood, recovered plasma or plasmapheresis. The frozen precipitate is optionally obtained when the frozen plasma is slowly thawed at low temperature, typically at a temperature of 0 to 4 캜 to form a precipitate containing fibrinogen and factor XIII. The precipitate can be collected, for example, by centrifugation and dissolved in a buffer containing a suitable buffer, for example 120 mM sodium chloride, 10 mM trisodium citrate, 120 mM glycine, 95 mM arginine hydrochloride, 1 mM calcium chloride . The solution of BAC may optionally be in the form of a solution, for example as described in U.S. Patent No. 6,121,232 and International Patent Publication WO9833533, such as Factor VIII, fibronectin, von Willebrand factor (vWF), vitronectin, Contains additional arguments. The composition of BAC optionally comprises stabilizers, such as tranexamic acid and arginine hydrochloride. The amount of tranexamic acid in the solution of BAC optionally ranges from about 80 to about 110 mg / ml. The amount of arginine hydrochloride optionally ranges from about 15 to about 25 mg / ml.

Optionally, the fibrinogen solution is buffered to a physiologically compatible pH value. Buffering agents include glycine, sodium citrate, sodium chloride, calcium chloride and water for injection as vehicles. Glycine is optionally present in the composition at a concentration ranging from about 6 to about 10 mg / ml; Sodium citrate is optionally present in a concentration ranging from about 1 to about 5 mg / ml; Sodium chloride is optionally present in a concentration ranging from about 5 to about 9 mg / ml; The calcium chloride is optionally present in a concentration ranging from about 0.1 to about 0.2 mg / ml.

In one embodiment of the invention, the fibrinogen is derived from blood, for example a BAC composition. In another embodiment of the present invention, the concentration of plasminogen and / or plasmin in the blood-derived component is lowered. Removal of plasmin and plasminogen from blood-derived components can be performed as described in U.S. Patent No. 7,125,569 and International Patent Publication No. WO02095019.

Some embodiments of the present invention are described in the present invention with reference to the accompanying drawings. The detailed description together with the drawings will be apparent to persons skilled in the art how some embodiments of the invention may be practiced. The drawings are for purposes of example discussion and are not an attempt to provide structural details of embodiments in more detail than are necessary for a basic understanding of the invention. For clarity, some of the items shown in the figures are not shown to scale.
In the drawing:
1 (Prior Art) is a schematic view of a spray drying apparatus;
2 (Prior Art) is a schematic view of a spray nozzle;
Figure 3 is a plot showing the effect of thrombin solution composition on percent recovery of thrombin activity of spray dried thrombin powder;
Figure 4 is a line graph showing the effect of inlet gas temperature on the thrombin activity recovery rate (%) and water content (%) of spray dried thrombin powder;
5 is a line graph showing the effect of the inlet gas flow rate on the thrombin activity recovery rate (%) and water content (%) of the spray dried thrombin powder.

The present invention, in some embodiments thereof, relates to a spray-dried thrombin powder comprising microcapsules, a process for their preparation and their use.

The principles, use, and practice of the teachings of the present invention may be better understood with reference to the following detailed description. Having read the detailed description, those skilled in the art will be able to practice the invention without undue effort or experimentation.

Before describing at least one embodiment in detail, it will be understood that the invention is not necessarily limited in its application to the details of the structure and / or arrangement of components set forth in the following description. The invention is capable of other embodiments, of being practiced or of being carried out in various ways. The terms and terminology used herein are for the purpose of description and should not be regarded as limiting.

As demonstrated in the following results section (see Examples 1, 2 and 3), embodiments of the reconstituted thrombin compositions disclosed herein, including both salt and carrier proteins, have relatively high thrombin activity-recovery rates About 80%, composition 6). The thrombin activity-recovery rate after spray drying is higher (about 85%, Composition 7) for reconstituted thrombin compositions further comprising carbohydrates, especially sugar alcohols such as mannitol, and the reconstituted thrombin composition is relatively high in calcium chloride (About 95%, composition 8). If all are the same, higher thrombin activity-recovery rates are achieved by compositions with relatively higher carrier protein / thrombin ratio (see Table 2B and Table 3).

Embodiments of the reconstituted thrombin compositions disclosed herein, including both carrier-protein (e.g., albumin) and carbohydrates (e.g., sugar alcohols such as mannitol) as demonstrated in the following results section Has surprisingly high thrombin activity-recovery rates (see Example 2 and Example 4), surprisingly even at high thrombin concentrations (see Example 6 and Example 6). While not wishing to be bound to any one theory, it is presently believed that the combination of carrier protein and carbohydrate, particularly by providing thermal protection, helps to maintain thrombin structure and efficacy.

The thrombin activity recovery rate provided by the embodiment of the teachings herein is superior to that provided by the prior art. For example, U.S. Patent Application Publication No. 2012/0315305 discloses spray-dried thrombin having a concentration of 25 to 1000 IU / g. Comparable Example 6 shows that the thrombin composition according to the teachings herein comprising carrier protein provides substantially higher rates of thrombin activity recovery.

Some of the advantages of some of the embodiments according to the teachings herein are believed to be the result of the spray-drying conditions used, which yield active thrombin powders at high mass and potency yields and at high concentrations. In some embodiments, a high level of thrombin activity recovery rate is achieved by one or more of the following:

A dry gas (e.g., dry air or nitrogen) at a temperature of 190 占 폚 or less; 1: 1000 or more Thrombin solution flow rate: When spraying the ratio of atomized gas flow rate, that is, 1 ml of thrombin solution per minute, the flow rate of atomized gas is preferably 1 l / min or more;

1: More than 43,000 thrombin solution flow rate: The dry gas flow rate is preferably greater than or equal to 43 l / min when the ratio of dry gas flow rate, ie, 1 ml thrombin per minute is dried.

Specifically, the dry gas temperature at 190 占 폚 in Example 8a yielded a spray dried thrombin powder having a water content as low as 1.5%. In Example 9, it is shown that it is desirable to set the inlet gas temperature to about 170 占 폚 or less in order to obtain a spray dried thrombin powder having a high thrombin activity recovery rate and a low water content. In some embodiments, to reduce the water content while maintaining the thrombin activity recovery rate, the dry gas flow rate is increased rather than the dry gas temperature.

Surprisingly, it has been found, in some embodiments, that despite increasing the retention time of thrombin in the drying column, an increase in the dry gas flow rate leads to a decrease in the water content of the resulting dry thrombin composition.

The increased dry gas flow rate is believed to reduce the pressure on the dry particles, thereby increasing evaporation. While not wishing to be bound by any one theory, when the powder reaches the bottom of the drying column, the powder tends to adhere to the inner parts of the tube that are hot and the particles transfer the powder flow from the drying column and the drying column to the cyclone , It is noted that the mass yield is reduced. This adhesion will occur when the powder is at a temperature above its glass transition temperature and is expected to cause the surface of the particles to be substantially " rubbery " or fired. Thus, introducing cold gas to the bottom of the drying column reduces the temperature at which the particles flow, thus reducing the surface temperature of the particles below its glass transition temperature so that the particles are in a vitreous or elastic state, It is expected that the mass yield will increase as a result.

As shown in Example 7, particularly embodiments with low drying temperature (140 占 폚) and high dry gas flow rate (thrombin solution: dry gas flow rate 1: 900000) provided dry spray dried thrombin powder and had a narrow particle size Distribution, and had a high thrombin activity recovery rate.

Embodiments of spray dried thrombin compositions according to the teachings of the present invention having relatively high thrombin concentrations provide a number of advantages. For example, when a fibrin patch comprising such a composition is used, a higher thrombin concentration is formed in the formed fibrin clot, allowing more fibrin fiber to be present in the coagulum, Leads to faster coagulation (time to hemostasis (TTH), see Example 11) as well as adhesion to tissue (see Example 10).

Embodiments of spray dried thrombin compositions according to the teachings herein may be stored at room temperature for extended periods of time without requiring storage in a cold (i.e., 2-8 占 폚) or frozen (i.e., less than -18 占 폚) state.

The flow properties of the spray dried thrombin compositions according to the teachings herein are shown in Example 12. As used herein, the term "powder flowability" refers to the ability of a blade to move in an upward lifting displacement mode and, when measured with a powder rheometer (FT4 powder rheometer, Freeman Technology, Gloucestershire, UK) This refers to the ease with which the powder will flow. Examples of such conditions include the pressure on the powder, the roughness of the particle surface, the humidity of the air around the powder, and the device through which the powder passes or flows out. The fluidity is expressed as the energy required to displace the powder composition divided by the weight of the powder being displaced. The proven low specific energy of the composition means that lower energy is needed to displace the composition. In practice, during the manufacturing process, the low specific energy composition is less likely to cause interlocking and friction which can block the machinery used to deliver the composition (e.g., auger dozer).

In addition, drying of embodiments of the aqueous thrombin composition feed according to the teachings herein using embodiments of the spray drying process in accordance with the teachings herein to produce spray dried thrombin microcapsule compositions can be performed at elevated Resulting in the production of a concentrated thrombin composition. As compared to a conditioned bulk density of 0.37 g / ml and a tap density of 0.48 gr / ml for a comparable lyophilized composition, as shown in Table 17 (Example 12), according to the embodiment of the teachings herein The conditioned bulk density of the spray dried thrombin composition (i.e., the air gap between particles, e.g., the powder density not removed by tapping) was 0.46 g / ml and the tap density The powder density after being removed by tapping) was 0.58 g / ml.

Example

Materials and methods

material

Thrombin (NDC No. 63713-460, manufactured by Omrix Biopharmaceuticals Ltd., Israel). In the following experiment, thrombin components were used, such as in EVICEL (R) fibrin sealant (manufactured by Omics Biopharmaceuticals Ltd., Israel).

Human serum albumin (Plasbumin 25, 25% sterile solution of albumin in aqueous diluent, NDC No. 13533-692-20) was obtained from Grifols USA, Research Triangle Park, North Carolina.

D-mannitol (Cat. No. 443907), sodium acetate trihydrate (Cat. No. 1370121000) and calcium chloride dihydrate (1371015000) were obtained from Merck Milipore (Villarrica, MA).

Until use, thrombin was stored in a standard laboratory freezer at a temperature of about -18 ° C, and human serum albumin and all solid materials were stored at room temperature (about 24 ° C) not exceeding 30 ° C.

equipment

Spray dryer. A 4M8-TriX spray dryer was used (ProCepT nv from Gel. Zeit in Belgium).

Way

Preparation of thrombin solution:

For each experiment, the desired amount of frozen thrombin solution (Omics Biopharmaceuticals Ltd., Israel) was allowed to warm to room temperature in a sealed bottle. The thrombin solution was prepared by dissolving the warmed thrombin in triple-distilled water to the desired concentration (see table below).

Spray Drying:

The prepared thrombin solution was aspirated into the syringe and placed in the syringe pump of the spray dryer. With the supply valve closed, the syringe pump was set to the desired flow rate.

With the syringe pump supply valve closed, the spray dryer was operated and the desired atomizing gas flow rate, drying gas flow rate, drying gas temperature, cooling gas flow rate and cyclone gas flow rate were set.

The cooling gas flow rate and temperature were selected to prevent the powder from sticking to the glass part, for example, by reducing the gas flow temperature below the glass transition temperature of the composition without interfering with laminar flow in the drying column.

The spray dryer was turned on until the actual measured value of the parameter reached the set level and reached a steady state where it remained stable. The feed valve was then opened to cause the thrombin solution to flow through the feed inlet to the spray nozzle and atomize into small droplets by the flow of atomized gas, followed by drying in a drying column. Spray dried thrombin powder was formed which was collected at the powder outlet of the cyclone in the spray dryer.

The spray dried thrombin powder recovered from the cyclone was weighed in a dehumidifier with a relative humidity of less than 30% and divided into 100-200 mg samples. Until evaluation, each sample was individually sealed in a stoppered test tube and sealed with Parafilm (Bemis, Oshkosh, WI).

Characterization of spray-dried thrombin powder

Determine the thrombin activity, water content, particle size distribution, total protein and coagulable protein, density (conditioned bulk density and tap density), flowability and mass yield, including thrombin activity recovery rate (relative to thrombin activity prior to spray drying) To thereby characterize the spray dried thrombin powder.

Thrombin activity determination

The thrombin activity of the aqueous thrombin solution was determined using a coagulation time assay, by measuring thrombin coagulation activity in solution according to the modified European Pharmacopeia Assay (0903/1997) procedure. Briefly, thrombin standards (Fibri-Prest (R) 2 by Beijing Stago Diagnosis Trading Co. Ltd., Beijing, China) were mixed with various different thrombin concentrations Were mixed with 0.1% fibrinogen solution (OMRIX Biopharmaceuticals Ltd., Israel) in a total volume (4, 6, 8, and 10 IU / ml) and analyzed using a coagulation measuring device (Beijing Stegan Diagnostics Trading Company Limited, A calibration curve of thrombin concentration versus solidification time was prepared by measuring the time it took for each sample to solidify using STart (TM)).

For each assay, a sample of approximately 35 mg of spray dried thrombin was dissolved in 1 ml of double distilled water (providing a thrombin solution with a thrombin activity of about 1000 IU / ml). This solution was diluted 1: 200 to produce a solution with an efficacy of about 5 IU / ml. 40 μl of diluted thrombin solution was mixed with 160 μl of 0.1% fibrinogen solution in a coagulation measuring device. The clotting time of the sample was determined and the thrombin activity was extrapolated with reference to the calibration curve.

Determination of water content

Determination of water content was carried out using a volumetric Karl Fischer titration (KFT) based on US Pharmacopoeia analysis (USP 27, <921>, P. 2398-2399). Before titration, water was extracted from the spray dried thrombin powder by adding about 10 ml of dry methanol to about 100 to about 200 mg of spray dried thrombin powder.

Determine particle size distribution

The size distribution of the suspended particles in the liquid is measured using the light scattering principle. The measurement was carried out using a particle size analyzer LS 13 320 (Beckman Coulter Inc., Pasadena, CA). The pattern measured by LS 13 320 is the sum of the patterns scattered by each constituent particle in the sample. The LS 13 320 consists of an optical bench and a Micro Liquid Module (MLM), which includes a chemical resistant liquid cell and a stir bar to maintain the suspended particles homogeneously mixed. The system is designed to operate with a small amount of suspended fluid (12 ml). A powder sample of 100 to 200 mg of spray-dried thrombin was suspended in 10 ml of a hydrofluoroether (HFE-7000, manufactured by 3M Company, Tuverhabs, MN, USA). The sample was placed in a micro-liquid module (MLM) to determine the particle size distribution.

Total protein and coagulable protein

The total protein test was used to determine the total amount of protein in the fibrin sealant patch sample. The powder was extracted from the fibrin sealant patch and dissolved in a solubilization solution capable of dissolving the protein in the sample. The protein concentration is determined by the Lowry method for the calibration curve.

Friability

The friability of the patches containing fibrinogen and thrombin powder was to determine the degree to which the powder flaked or crumbled from the fibrin sealant patch after applying a force to the sample of the patch. To determine friability, the patch was cut to a specific size and the weight of the sample was determined. The sample was placed in a vial and dropped through a fixed-height tube onto a rubber stopper to allow the sample to undergo a defined impact force. The patch samples were weighed to determine the amount of powder loss and the percent weight loss of the sample was calculated.

Tissue exfoliation

The adhesive strength of the fibrin sealant patch to the calf corium tissue substrate was measured using a 5000 series Instron Tensiometer (Instron Corp., Norwood, Mass.) . To measure the adhesive strength, a fibrin patch was applied to the surface of the dermis and wetted with brine. The average force required to perform the 90 degree peel test of the fibrin patch from the calf dermis was then determined by measuring the integral under the force vs. distance curve.

The tensile strength

The tensile strength of a fibrin patch sample in the shape of a dogbone having a width at the end of 25 mm, a width at the middle of 15 mm and a length of 102 mm was determined. The samples were placed between Instron grips (5000 series Instron tensiometer, Instron Corporation, Norwood, Mass.). The crosshead was moved at a predetermined constant elongation speed of 305 mm / min. This test was terminated when a peak load drop of 30% was achieved, indicating the occurrence of a rupture.

Density (conditioned bulk density, tap density)

The conditioned bulk density, tap density and specific energy were measured using a powder rheometer (FT4 powder rheometer, Freeman Technologies, Gloucestershire, UK). The test was carried out in a 25 mm bar with 23.5 mm blades in 250 taps.

The term "density" refers to the weight of solids per unit volume. Density was measured in liquid, solid (e.g., powder) or gas form. In the case of powders, the density can be "conditioned bulk density" or "tap density ". The bulk density of the powder is the ratio of the mass of the un-tapped powder sample to its volume, including the contribution of the interparticle void volume.

As used herein, the term "bulk density" refers to the density of a bulk powder sample of known weight of known weight.

As used herein, the term "conditioned bulk density" refers to the volume of a bulk powder sample that has undergone a conditioning step that smoothly displaces the powder using a rotating blade to loosen the powder to remove any pre-consolidation or excess air Density.

As used herein, the term "tap density" refers to the density measured after tapping a conditioned bulk density powder sample a certain number of times.

Fluidity (non-energy)

Using a FT4 powder rheometer (FT4 powder rheometer, Freeman Technology, Gloucestershire, UK), the resistance (resistance of the powder to flow) applied by the powder to the rheometer blade was measured and the torque (radial resistance) and force Vertical resistance).

Energy (fluidity) = [distance x (torque + force)] / split mass

Here, the distance refers to the distance the blade has moved in the upward lifting displacement mode.

As used herein, the term "dispense mass" refers to the mass of powder displaced during the measurement.

In the examples described below, the test conditions were as follows: -5 ° helix and 100 mm / sec blade tip speed. A sample volume of 25 ml was used.

Example 1: Effect of thrombin solution composition on maintaining thrombin activity during spray drying

Various thrombin solutions (including different salts and / or carbohydrates and / or proteins) were prepared to test the effect of excipient (s) in solution on maintaining thrombin activity during spray drying.

Eight different such thrombin solutions were prepared as shown in Table 1:

[Table 1]

Composition 1 to Composition 8 were spray dried as described above with the following parameters:

One. Thrombin Sample flow rate: 400 ml / h;

2. Atomized gas flow rate: 7 l / min;

3. Dry gas (dry air) flow rate: 600 l / min;

4. Drying gas (dry air) temperature: 140 캜;

5. Cooling gas (dry air) Flow rate: 100 l / min;

6. Cyclone gas (dry air) flow rate: 150 l / min.

After recovery from the cyclone, each spray-dried thrombin composition (dried thrombin with excipients) was reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was compared to the thrombin activity of each mother solution. The relevant activity is shown in the graph of FIG.

As shown in Figure 3, the reconstituted compositions containing only salts (Composition 1 to Composition 3) had a relatively low recovery rate (less than 25%) of thrombin activity after spray drying. Reconstituted compositions with salts and sugar alcohols (e.g., mannitol) usually had a recovery rate of thrombin activity after spray drying (about 50%, Composition 4, Composition 5). Reconstituted compositions with salts and proteins (e. G., Human serum albumin, HSA) had relatively high rates of recovery of thrombin activity after spray drying (about 80%, Composition 6). The reconstituted composition having a salt, a sugar alcohol (e.g., mannitol) and a protein (e.g., human serum albumin, HSA) had a very high recovery rate of thrombin activity after spray drying (about 85%, composition 7) Was much higher when it contained a relatively large amount of calcium chloride along with the salt, sugar alcohol and protein (about 95% thrombin activity recovery rate, Composition 8).

Example 2 Effect of Thrombin and Human Serum Albumin (Carrier Protein) Concentration on Maintaining Thrombin Activity During Spray Drying of Thrombin Solution

Thrombin, and albumin, which acts as a carrier protein, at different concentrations was prepared as shown in Table 2:

[Table 2]

Composition 9 to Composition 23 were spray dried as described above. As discussed above, after spray drying, three 35 mg samples of each spray dried thrombin composition were reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was measured twice and compared to the thrombin activity of each mother solution, giving a total of six measurements for each solution. The average thrombin activity recovery rate is shown in Table 2.

As can be seen in Table 2b, additional parameters indicating the ratio of albumin weight to thrombin activity in mother liquor and its effect on the recovery rate of thrombin activity after spray drying were calculated (albumin / thrombin (g / IU) ratio Followed by low to high). The indicated thrombin activity recovery rate represents the mean ± standard deviation.

[Table 2b]

The results indicate that the greatest thrombin activity recovery rate is achieved in a composition having relatively high levels of albumin to thrombin, at which time only Composition 21 becomes outliers. (Table 2b). However, as can be seen by the large thrombin activity recovery rate variation obtained in composition 11 and composition 12 and the relatively low thrombin activity recovery rate in composition 22 and composition 23, a high ratio of albumin to thrombin ensures a high rate of thrombin activity recovery after spray drying I do not.

Example 3: Effect of casein (carrier protein) concentration on maintaining thrombin activity during spray drying

Four compositions of thrombin solutions with different concentrations of casein (carrier protein) were prepared as shown in Table 3:

[Table 3]

Solutions 24 to 27 were spray dried as described above. As discussed above, after spray drying, three 35 mg samples of each spray dried thrombin composition were reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was measured twice and compared to the thrombin activity of each mother solution, giving a total of six measurements for each solution. The recovery rates of thrombin activity obtained are shown in Table 3. The results achieved using casein are similar to those achieved using albumin, which results in a high thrombin activity recovery rate when the ratio of carrier protein to thrombin is high, but at a high variance of thrombin activity recovery rate in composition 27 As can be seen, the high ratio of carrier protein to thrombin does not guarantee a high thrombin activity recovery rate.

Example 4: Effect of carbohydrate type and concentration on maintaining thrombin activity during spray drying

25 thrombin solutions with different thrombin concentrations, carbohydrate types and carbohydrate concentrations with or without carrier protein were prepared as shown in Tables 4 to 8:

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

Composition 28 to Composition 52 were spray dried. Samples were recovered and reconstituted in water as described above and the thrombin activity of each reconstituted solution was compared to the thrombin activity of each mother solution. The relevant activities are shown in Tables 4 to 8, respectively.

The result shows:

Table 4: Addition of only sugar alcohol (mannitol) to thrombin solution was not sufficient to maintain thrombin activity during spray drying.

Table 5 and Table 6: Addition of only disaccharide (trehalose in Table 5, sucrose in Table 6) to the thrombin solution was not sufficient to maintain thrombin activity during spray drying. However, when the disaccharide was added together with the carrier protein (albumin), the thrombin activity recovery rate was substantially increased.

Table 7 and Table 8: Addition of only polysaccharides (starch in Table 7, maltodextrin in Table 8) to the thrombin solution was not sufficient to maintain thrombin activity during spray drying.

Example 5: Effect of thrombin concentration with fixed molar ratio of thrombin to human serum albumin (carrier protein) for maintaining thrombin activity during spray drying

Three thrombin compositions with different thrombin and albumin concentrations were prepared as shown in Table 9, wherein the molar ratio of thrombin to albumin was 1: 6.4 in all three solutions:

[Table 9]

Samples of Composition 53 to Composition 55 were spray dried. Samples were recovered and reconstituted in water as described above and the thrombin activity of each reconstituted solution was compared to the thrombin activity of each mother solution. The thrombin activity recovery rate is shown in Table 9. It can be seen that the thrombin activities of the three reconstituted solutions of the low thrombin activity recovery rates (Composition 53 and Composition 55) or of the highly dispersed thrombin activity recovery (Composition 54) are approximately the same.

Example 6: Effect of trehalose (disaccharide) on maintaining thrombin activity during spray drying

United States Patent Application Publication No. 2012/0315305 discloses spray drying of a thrombin composition comprising trehalose and reports 97% recovery of thrombin activity.

Solution 56 and Solution 57, similar to those described in U.S. Patent Application Publication No. 2012/0315305 (except that the sources of thrombin were different and the spray dryer was of a different type) were prepared as shown in Table 10. In addition, a novel thrombin solution 58 comprising trehalose and a protein (human serum albumin) was prepared.

[Table 10]

Composition 56 to Composition 58 were spray dried under similar spray drying conditions according to the disclosure above. Samples were recovered and reconstituted in water and the thrombin activity of each reconstituted solution was compared to the thrombin activity of each mother solution. The thrombin activity recovery rate is shown in Table 10.

The results indicate that Solution 56 and Solution 57 exhibited less than 65% recovery of thrombin activity, as reported in US Patent Application Publication No. 2012/0315305. However, the addition of carrier protein (human serum albumin) in composition 58 according to some embodiments of the teachings herein resulted in a significant increase in thrombin activity recovery rate.

Example 7: Spray drying process and product characterization of the preferred thrombin composition

Based on Composition 8 of Example 1, 3 liters of Composition 59 consisting of thrombin, albumin, mannitol, acetate, calcium and sodium chloride in water as shown in Table 11 was prepared.

[Table 11]

The parameters of the spray dryer were set as follows:

Thrombin Sample flow rate: 400 ml / h; Atomized gas flow rate: 7 l / min; Dry gas flow rate: 600 l / min; Drying gas temperature: 140 占 폚; Cooling gas flow rate: 100 l / min; Cyclone gas flow rate: 130 l / min; Tip diameter of 2-fluid nozzle: 0.4 mm.

Three liters of composition 59 was spray dried as described above. During spray drying, the actual values of various spray drying parameters were recorded every minute. 63.25 g of spray dried composition was collected in a 200 ml glass bottle designated Bottle # 1 and 48.41 g of spray dried composition was collected in a 200 ml glass bottle designated Bottle # 2 when Bottle # 1 was full. After filling with the dried composition, the bottle was immediately capped and sealed with Parafilm (TM) until analysis was performed.

The average measured spray drying parameters as well as the spray dried thrombin composition properties were as shown in Table 12:

[Table 12]

The resulting spray-dried thrombin powder was dried with a narrow particle size distribution, and most importantly, it maintained the thrombin activity of the parent solution.

Example 8 Effect of Drying Gas Temperature on Spray-Dried Thrombin Properties

The desired thrombin composition (Composition 8) was spray dried with several spray drying runs with only varying dry gas temperatures per run:

Thrombin composition flow rate: 7 ml / min

Flow rate of atomizing gas: 7 l / min

Dry gas flow rate: 300 l / min

Dry gas temperature: 100, 110, 120, 130, 140, 150, 160, 170,

Cooling gas flow rate: 150 l / min

Cyclone gas flow rate: 300 l / min

Dried samples of spray dried thrombin compositions with different drying gas temperatures were collected from the cyclone and the rate of thrombin activity recovery of each reconstituted solution was determined as described above. The results of thrombin activity are shown in Fig. 4 together with the water content of the sample.

As shown in Fig. 4, lower thrombin activity was measured at a drying gas temperature of 170 DEG C or higher. At lower drying gas temperatures, for example below 170 ° C, high thrombin activity was observed. As the drying gas temperature increased, the water content of the spray dried thrombin composition decreased. At a temperature of 100 캜, a water content of about 4.5% was obtained. Increasing the temperature to 190 [deg.] C yielded spray dried thrombin powders with water contents as low as 1.5%.

Example 9: Effect of dry gas flow rate on spray dried thrombin properties

The desired thrombin composition (composition 8) was spray dried with several spray drying runs with only varying dry gas flow rates per run:

Thrombin composition flow rate: 7 ml / min

Flow rate of atomizing gas: 7 l / min

Dry gas flow rates: 300, 350, 400, 450, 500 and 550 l / min

Drying gas temperature: 155 ° C

Cyclone gas flow rate: A flow rate that replenishes the spray dryer with a total flow rate of 600 l / min (ie 300, 250, 200, 150, 100 and 50 l / min).

Samples of dry spray dried thrombin were collected from the cyclone and the thrombin activity recovery rate of each reconstituted solution was measured as described above. The results of thrombin activity are shown in FIG.

As shown in Figure 5, at all tested dry gas flow rates, high thrombin activity was observed. As the dry gas flow rate increased, the water content of spray dried thrombin decreased. At a gas flow rate of 300 l / min, a water content of about 3.5% was obtained. Increasing the dry gas flow to 550 l / min yielded spray dried thrombin powders with water content as low as 2%. Taken together, the results indicate that it is desirable to increase the inlet gas temperature to only a certain extent, for example to about 170 ° C, in order to obtain a spray dried thrombin powder having a high thrombin activity recovery rate and a low water content. Surprisingly, it is recommended to increase the dry gas flow rate first before increasing the dry gas temperature, in order to reduce the water content of the spray dried powder, without compromising the rate of spray dried thrombin activity recovery.

Example 10: Preparation of a fibrin patch with lyophilized-milled BAC2 and spray-dried thrombin

The thrombin composition 8 described in Example 1 was spray dried under the conditions described in Example 1. The resulting spray-dried thrombin powder recovered from the cyclone was used with lyophilized-milled BAC2 to produce a fibrin patch as described in WO2007117237.

The characteristics of the fibrin patches were analyzed as shown in Table 13:

[Table 13]

The characteristics of the fibrin sealant patch were analyzed as shown in Table 14:

[Table 14]

A fibrin sealant patch made from spray dried thrombin powder was found to have low water content (2.1%) and high thrombin activity (36 ± 2.7 IU / cm 2).

In the tissue exfoliation test, the adhesive strength of the wet fibrin sealant patch to the dermal tissue substrate was determined by determining the force required to exfoliate the patch from the tissue. The patches made from spray dried thrombin compositions showed high adhesion.

Example 11 In Vivo Efficacy of Fibrin Sealant Patches

The hemostasis time (TTH) of the fibrin sealant patch described in Example 10 was tested.

Two pigs of similar size and weight were anesthetized during the entire surgical procedure and the spleen was surgically exposed. Five, depth 3 mm and length 15 mm wounds were made on each one of the two spleens using a suitable blade and fibrin patches were applied to each one of the wounds. The results after 3 minutes are shown in Table 15, and the results after 10 minutes are shown in Table 16, and "check" indicates complete hemostasis.

[Table 15]

[Table 16]

As can be seen in Table 15 and Table 16, the patch achieved 100% hemostasis within 3 minutes and no rebleeding was observed after 10 minutes.

Example 12: Characterization of spray-dried thrombin compositions

The thrombin composition 8 of Example 1 was spray dried or lyophilized under the conditions described in Example 1. The two dried thrombin powders produced were analyzed for bulk density, tap density and specific energy (Particle Technology Labs, USA), as shown in Table 17.

[Table 17]

The results presented in Table 17 show that the spray dried thrombin powder is more dense than the lyophilized thrombin powder. The results in Table 17 also indicate that the spray dried thrombin powder has a lower specific energy for fluidity than the lyophilized thrombin powder, which means that the spray dried thrombin easily flows. In practice, spray dried thrombin powders are less likely to cause mechanical engagement and friction in machinery and, for example, reduce the risk of clogging of delivery devices, delivery mechanisms and tubing.

Claims (19)

A spray-dried thrombin powder comprising microcapsules,
Wherein the powder comprises thrombin, carrier protein and carbohydrate, the ratio of thrombin: carrier protein is about 0.85 IU: 1 mg to 66,875 IU: 1 mg, the ratio of thrombin: carbohydrate is 0.75 IU: 1 mg to 26,750 IU: 1 mg of spray dried thrombin powder. 2. The spray dried thrombin powder according to claim 1, wherein the ratio of thrombin: carrier protein is from about 110 IU: 1 mg to about 285 IU: 1 mg, alternatively from about 129 IU: 1 mg to about 219 IU: 1 mg. 3. The spray-dried thrombin powder of claim 1 or 2, wherein the carrier protein comprises albumin. 4. The spray-dried thrombin powder according to any one of claims 1 to 3, wherein the carbohydrate is sugar alcohol or saccharide. 5. The method of any one of claims 1 to 4, wherein the carrier protein is albumin, the carbohydrate is mannitol, the ratio of thrombin to albumin is about 0.85 IU: 1 mg to 66,875 IU: 1 mg, thrombin: mannitol Wherein the ratio is 0.75 IU: 1 mg to 26,750 IU: 1 mg. 6. The spray dried thrombin powder according to claim 5, wherein the ratio of thrombin: albumin is from about 110 IU: 1 mg to about 285 IU: 1 mg, alternatively from about 129 IU: 1 mg to about 219 IU: 1 mg. The spray-dried thrombin powder according to claim 5 or 6, wherein the ratio of thrombin: mannitol is about 40 IU: 1 mg to about 64 IU: 1 mg. 8. A spray dried thrombin powder according to any one of claims 1 to 7 comprising from about 0.001% to about 5% w / w of thrombin. 9. A spray dried thrombin powder according to any one of claims 1 to 8, comprising less than about 3% w / w water. 10. The spray dried thrombin powder of any one of claims 1 to 9 having a particle size distribution of D50 from about 5 to about 13 microns and D90 from about 15 to about 25 microns. As aqueous compositions suitable for use in preparing thrombin microcapsules,
Wherein the ratio of thrombin: carbohydrate is from about 1.6 IU: 1 mg to 5,000 IU: 1 mg, and the ratio of thrombin: carbohydrate is from 2 IU: 1 mg to 1500 IU: 1 mg. &Lt; / RTI &gt; A method for producing thrombin microcapsules,
The method comprises mixing thrombin, carrier protein and carbohydrate in an aqueous solution, wherein the ratio of thrombin: carrier protein is about 1.6 IU: 1 mg to 5000 IU: 1 mg, the ratio of thrombin: carbohydrate is 2 IU: 1500 IU: 1 mg; And
Spray-drying the aqueous solution
&Lt; / RTI &gt; 13. The method of claim 12, wherein the spray drying step
Introducing said aqueous solution into a spray drying apparatus;
Using an atomizing gas flow to produce a droplet from the aqueous solution introduced into the apparatus;
Evaporating water from the droplets by a dry gas flow,
A method for producing thrombin microcapsules, comprising preparing thrombin microcapsules. 14. The method according to claim 12 or 13,
About 800 to about 1200 IU / ml of thrombin;
About 0.5 to about 0.65% w / v of a carrier protein;
About 1.85 to about 2.05% w / v carbohydrate;
About 38 to about 42 mM calcium;
About 18.0 to about 20.0 mM acetate; And
About 60 to 240 mM sodium chloride,
Wherein the solution has a density of about 1.01 to about 1.03 g / ml. A spray-dried thrombin microcapsule prepared according to the method of any one of claims 12 to 14. A matrix comprising the spray dried thrombin powder of any one of claims 1 to 10 and / or the spray dried thrombin microcapsules of claim 15. 17. The matrix of claim 16, further comprising a fibrinogen powder. 18. The matrix of claim 17, wherein the fibrinogen powder is produced by freeze-drying. A kit comprising a spray-dried thrombin powder of any one of claims 1 to 10 and / or a spray-dried thrombin microcapsule of claim 15.

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