CA1124177A

CA1124177A - Method for preparing an improved hemostatic agent and method of employing the same

Info

Publication number: CA1124177A
Application number: CA327,492A
Authority: CA
Inventors: Philip N. Sawyer
Original assignee: Interface Biomedical Laboratories Corp
Current assignee: Interface Biomedical Laboratories Corp
Priority date: 1978-05-19
Filing date: 1979-05-11
Publication date: 1982-05-25
Also published as: IT7922635A0; US4238480A; GB2051074A; SE7903617L; FR2425860A1; DE2918098A1; CH646607A5; FR2425860B1; GB2051074B; IT1115218B

Abstract

TITLE

A METHOD FOR PREPARING AN IMPROVED HEMOSTATIC
AGENT AND METHOD OF EMPLOYING THE SAME
ABSTRACT

An improved hemostatic agent is made by treating collagen or collagen-like substance to render the surface charge thereof effectively more positive. The thusly modified substance is employed to control or terminate bleeding.

Description

11 ~3~ 7~

FIELD OF INVENTION
This invention relates to hemostatic agents and to method of preparing and using the sameO
. I
BACKGROUND
_____ _ __ In various prior patents, there have been discussions of the modification of the surace charge o vascular systems or substances to be used in a~sociation therewith. Generally~
it has been suggested that such systems be dealt with to make the surface charge thereof more negative to avoid thrombosis and the like.
A variety of hemostatic agents are known such as TM
Gelfoam manufactured by Up-John and disclosed in Patent TM

2,465,351; ~vitene manufactured by Acecon and disclosed TM
in Patent 3,742g955 and Surgicell manufactured by Johnson 1~ and Johnson and disclosed in Patent 3~36~200~
Battista et al in Patent 3,7429955 report that colIagen in various treated or prepared forms is useful in surgery and for the treatmen~ of wounds, and that E. Peacock, Jr. et al An~. Surg. 161,238-479 February~ 1965 teaches that collagen has h~mostatic properties when used as a wo~nd dressing.
Battista et al further report that it has been ~ound that fibrous collagen and ibrous products derive from collagen when properly prepared and when wet with blood will not only demonstrate hemostasis, but also demonstrates an unexpected adhesiveness to severed biological surfaces in warrn blooded animals. They also provide a method of .

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preparing finely divided fibrous collagen and ibrous products derived from collagen which are useful hemostatic agents and have unique adhesive properties in contact with a severed biological surface in a warm blooded animal when w~t with S blood.
Ashton e~ al report in Eatent 3g364,200 that surgical hemostats cons-istîng of conventional gauze pads or similar articles impregnated with a hemostatic material such as ferric chloride~ thrombin or the like~ have been used or many years to arrest bleeding. However, the prior art hemostats are criticized in that they cannot be left -in situ in a closed wound s;nce foreign body tissue reaction would result, this being a serious disadvantage inasmuch as removal of the hemostat from the bleeding sigh~ would disrupt lS any blood clot which has formed to cause renewed bleeding. -Ashton et alobserved5therefore~that a vital need exists for a hemostatic material which could be left in place in : a closed wound without causing serious local tissue reac~ion.
It is also reported that improvement was provided w~en it was discovered that oxidized cellulose not onl~ had hemostatic properties but also was absorbable in ani:mal tissue. Ashton et al provide oxidized cellulose absorbable hemostats having improved stability ~ainst deterioration on storage. The oxidized cellulose is derived from wood pulp~ cotton~ cotton linters, ramie~ jute~ paper and similar materials and regenerated cellulose or rayon produced by either the viscose or Bemberg processes~

7~' Correll's Paten-t 2,46S7.;7 relates to a liquid-permeableg water-insoluble9 gelatin sponge havlng general .
physical characteristics of a sponge but being absorbable by animal bodies. The sponge is a porous substance which .~ according to the disclosure9 should be reasonably soft when wet and.have many fine interstices in orde.r to hold a .quan;tity .
.. of a therapeutic agent and to discharge the same slowly or act as an efficient absorbative material for free flowing fluids in a wound area such as blood and exudates. Correll discloses preparing an aqueous solution containing geiatin . . , , ~ . .
and adding a small amount of formalin and thereafter beating the material for an extended period of time to produce a .
fir~ foam of substantially greater than the volume of the -original solution.
The above inventions are of interest in the field of hemostasis~ however~ none of the patents listed above deal with the controlling of the surface electronic or electro-: static charge of the materials involved and consequently did not go to the basis of the hemostasis problem as in accordance .
with the present invention. .
: : ~ .addition to the above, a .discussiorl of collagen ~ .
sponge appears ia Collagen Sponge: Theory and Practice of Medical Applica~ions 3 J. Biomedical Materials Research ~Jo~n W;ley & Sons~ New York) Vol~ 11 No. 5~ September, 1977~ In this article, applications of collagen as a biodegradable material is reviewed inclusive of rate of resorption and antigenicity.
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t7 o~ r ~.
It is an object of the invention to provide an improved hemostatic agentO
Another object of the invention is to provide an improved method for preparing hemostatic agents, Yet another object of the invention is to provide an improved method o~ utilizing hemostatic agents, To achieYe the above and other objects of the invention, there is provided a method comprising modifying one o-P the group consisting of a collagen substance or a collagen-like substance to render the surface charge thereof e~fectively more positive, and applying the thusly modified substance to contr~l bleeding, According to one specific embodiment of the invention, the substance may be modified by non~covalent modification.
Furthermore, this substance is lyophilized, According to another embodiment of the invention, the substance may be modified by covalent modification, This sub-stance is also lyophilized in a preferred modification thereo~, According to the invention, there is provided a hemo-static agent prepared as indicated above, The collagen orcollage~like substance which is employed may preferably be gelatin which is treated with hydrochloric acid, According to a feature o~ the invention, the gelatin may be treated with ethylenediamine.
The invention is particularly directed toward a method of preparing a hemostatic agent comprising modifying one of the groups consisting of collagen substance or a collagen-like sub-stance by dissolving the substance in water and modifying the thusly dissolved substance to render the surface charge thereo~
effectively more positive than prior to modification, aDd freezing and drying the thusly modified substance, The invention is also directed toward a method o~

prepariDg a hemostatic agent comprising modifying one o~ the groups consisting o~ collagen substance or a collagen-llke sub-stance to render the sur~ace charge thereof e~ectively more positive, the substance being modi~ied by covalent modi~ication, The above and other objects, ~eatures and advantages o~ the invention will be ~ound in the detailed description which ~ollows hereina~ter, -4a-. B~IEF DF.SCP~IPl'IOI~ OF DRA~71.NG .
Figs. 1-3 are charts showing the ultraviolet absorption .
spectra OL various embod;.ments of the invertion; .
~ig. 4 is a chart showing the pH o solutions for the cornpolmds of the invention; and Figs.5 and 6 are.charts of the inrared spectra.of embodiments o the inventi.on. .
.
DETAILED DESCRIPTION .
The purpose of this invention is to provide.a .
chemically modified collagen or collagen-like substance as a .
hemostatic agent which is competitive with comrnercially available products and which is superior thereto in certain respects.
. The substance of the inventi.on is a collagen or . .
collagen-like compound, modified with positive moieties~
. which can be used clin;call.y to control bleeding especially .
in non-suturable areas. It is meant to be an adjunct to and not a replacement of con~entional surgical procedures. .
Many factors contribute to the clotting mechanism .
of hemostatic agents~ Some o~ these are: (1) surface chemistry (including biochemical interactions)~ (2) ele trical . or electrostatic-charge characteristics, and (3) micro structure.
In the preliminary stages of synthesizing and evaluating hemostatic agents9 an attempt has been made to recognize .
the importance of each of the above criteria~
The various forms of ~he hemostatic agent provided by the invention are modifications of collagen or a collagen-like compound. Collagen itself demonstrates hemostatic pro-~erties. The modifications which have be~n attempted seek to augment these phenomena both by manipulating the surface charge and micro st~L~ucture.

~ '7 7 The modification o~ a compound charge density can be achieved by twv distlnct methods; (1) non-covalent modificatlon of dissolved bone gelatin (Baker U.S,P.) using positlve groups, such as provided by HCl, (2~ covalent attachment o~ a variety of ligands to the peptide chain of gelatin.
The preparation o~ multiple ~orms of a positively charged hemostatic agent are synthesized as shown below based by way of example on the following initial preparation of a collagen or collagen like substance or compound: -By way o~ example, one liter of 1% (or 10 grams ~or 1 liter) solution of gelatin (Baker U,SOP. crude) is dissolv~d in distilled and deionized water at room temperature with con-stant stirring. From this stock solution, 200 ml, aliquots are withdrawn and used in the various techniques given below.
A. Non-Covalent Modi~ication .
The 200 ml, aliquot of protein solution is adjusted to desired pH (e.g. 9 p~=2.5~ with a 1% gel (low density) HCl (LDHCl). For pH-3~0, a 5% gel (high density) HCl (~CHCl) is used, To the sQlution is added lN HCl, which is diluted from a concentrated HCl (Fisher reagent grade). This is done with constant stirring to insure hemogenicity and to minimize any denaturation, The gelatin-HCl solution is then stirred ~or two hou~s at room temperature, ~iltered through a Whatman No. 4 ~ilter into a 600 ml Virtus flask, The flask is then immersed in a dry-ice acetone bath (-40C) and, with constant swirling of the ~lask, th~ proteln solution inside becomes shel~ frozenO
This material is then placed on a Virtus lyophilizer (Research Equipment, N,Y,) and dried until the ~olution has a oam-like character, The material is then removed from the Virtus flnsk and placed into desiccator glass or plastic bottles~ Alter-natively, shelf-~reezing techniques are employed.

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.'' A second modification is the addition of CaCl~ 2H20 (Fisher reagellt gracle) to the purifi.ed gel~.tin in inal Ca~ concentrations of 0.001M~ 0.01M~ 0.10M or 0.25M (Table 13.
B. Covalent_Modif~cation The covalent attachment is obtained utilizing ~he structure of collagen or gelatin as a support media.(con-sidering it to be, for example9 similar to a Sephrose matrix wi.th its free-carboxylic acid end groups) and binding the ligand to this.matrix through a peptide bond created between the end ~ COOH groups of the gelatin and the free amino groups of the various ligandsO
This peptide bond formation occurs easily at pH
4.75 with the use of the condensing agen~ 1 ethyl - 3 (3 dimethylaminopropyl) carbodiimi.de - HCl (E.D.C. purchased from Sigma Corp.) - .
This bond fonmation is explained since the bone gelatin used is assumed to be similar in amino acid composition to bovine bone collagen. Bovine bone collagen possesses 44 aspartic acid groups, and 77 glutamic acid groups or, in other words~ 121 COOH per 1000 residues. Based on the .~ above analysis, it can be ass~med tha~ the gelatin in this experiment contains 100/1000 free carboxylic acid groups.
Thus~ 100 mg/l gm of gelatin should be modified if the . modifying ligand is in large excess. All other modifications were carried out analogously.
ExamE~ To a 200 ml aliquot of protein solution was added enough ligand (1 molar) to be in 5X excess of the possible binding sites~ The solution was adjusted to a pH of 4.75 with the use of an appropriate ac;d (HCl) or base (NaOH).
To this stirring solution was added 5gms. solid EDC
~minimum carbodiimide required to make a final concen-tration of .lM~ The solution was then stirre~ for 2 hours. The reaction was followed by measurement with a Leeds~Northrop pH meter. There was a chan~e in p~l (i.e. to pH = 3) which was compensated for by the addition of ~ase. The sample was then stirred for 24 hours, in order to insure complete reaction of all possible binding sites.
The protein was then dialysed with the use oE
running water for 6 hours and again against 4 liters of distilled and deionized water for 2 hours repeated four times~ This was to assure removal of all unreacted ligand and condensing agent. The material was then filtered and handled identic,ally to the non-covalently modified material (shell frozen and lyophilized).

X~MPLES
CODE NO. GELATIN LIGAND EDC BINDIN&
. 1. 1% - - -' , .
2. 1% HCl - non covalent

3. - 1% N~4Cl Sgms covalen~

4. 1% - 5gms Internal

5. 1% Ekhylenediamine 5gms CovaIent

6. 1% AlCl3 ~ Covalent 8. 5%2 _ _ Cova1entl 9. 5%2 HCl non-covalent 10. 1% AlC13~ Urea 5gms covalent l) The covalent nature of this binding has yet to be established quantitatively.
2) The change to 5% (high density) was indicated when evaluating the 1% foams as these examples were ext~emely hydroscopic and dissolved quickly in profuse bleeding.

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In an fort to quantify what chemical modifications of the colla"en were actually occurring" the foL1owing .
analyticcl procedures were used:
1.) polyacrylamide disc electrophoresi.s (P.A.G.E.) 2.) binding studies 3.) circular dichromism studies P.~.G.E. is a widely used technique for visuali~ing puri-ty, mass and charge of a protein. Proteins migrate .through a medium on the basis of charge/mass (e/m) ratios.
Since the migration of-protein is due to this ratio7 it is possible to use this technique to evaluate either criteria.
More conventionallyg the criterion evaluated by thi.s techni~ue is mass (S.D.S. denaturing gel electrophoresis) .. though, with some slight modifications, the banding pattern of a protein could be used to discriminate between identical mass proteins.with modified charge . characteristics. The technique can also be used to detect the quantity of any charge alteration due to modifylno collagen, without resorting to the more diffIcultg expensive, . through conventional isoelectric focusing.
As to binding-studies, it is apparent that binding studies must be.used to determine the type and quantity.of modifications made. The covalent modification used in this procedure creates.in essence pseudo lysine residues. ~.ny technique which can differentiate free NH2 groups on an intact.
protein can be used to determine the amount of binding by comparing the number of NH2 groups both pre and post treatment.
Such techniques include ninhydrin assay, and/or fluorescamine as~ay (Purcell et al)~

Finally, the conormation of a protein determines to some extent its chemical properties. It is therefore helpful to rnonitor any alteration of chemical conformation caused by a modification by means of circular dichromism studies both before and after treatment. Ultimately~ correla-tion snoulcl be established between alterations of conformation with clotting properties.
To evaluate the materials synthesized9 there have been utilized acute in vivo animal e-xperiments (dog) and in vitro TRT (thrombin recalcification time~ analysis~
The evaluation of the hemostatic agents of the invention and comparison thereof consisted of:
1) Subcutaneous implantation of a variety of agents in a given an`imal for a period ranging from one day, 2 days~ 7 days and 2 weeks, (2) bleedi~g time and semi-quantitative analysis of the weight of blood loss in two separate anatomical locations (skin and spleed). From these tests, there was obtained infor-mation on ~1~ relative clottability (efficacy) of each of the agents9 (2) information on the physi al structural integrity of these materials both before and after contact with blood, (3~ gross indications of toxicity and histology, (4) different types of fibrin formed ~hen exposed to blood from different areas which is dependent on both the properties of the agent and the properties of the blood involved~ (5)handling characteristics of each agent under operating room conditions,and (6) clinician's subjective opinions of each agent as it relates to handling~
clottability and interaction with surrounding tissue.

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Implantation studies were accompllshed by creating a pocket in the museularis o~ -the chest (dog) and placing each agent in individual pocl~ets, and suturing them closed with silk, The samples were excised prior to sacrifice, ~ixed in ~ormalin-glutaraldehyde and marked ~or histological and microscopic e~amination, The samples obtained are as ~ollows:
DATE No. o~ DQGS ORDERCOMMENTS

2/1/77 D~:45 NoO 07I,III,II,1,2, NoO 5 = wet and slipping 3,5,4 No. 4 = crystalline 2/l/77 D6 53 No. 6,I,III,II,1,2, No. 3 = was wet &
3,5,4crystalline No. 4 = caused abscess No. 6 = turned blood brown 2/1/77 45:131 No. 4,0,I,III,II,l, No. lO on right side 2,3,5,10,9 2/23/78 C6:18 No. 8,11,12,13,14 Key. O = control no agent I = Gelfoam Surgicell IXI = Avitene 1 = gelatin foam 1%
2 = gelatin foam 1% = H~l 3 = gelatin foam 1% + N~ Cl + EDC
4 = gelatin foam 1% ~EDC4 5 = gelatin ~oam 1% +EDA + EDC
6 = gelatin foam 1% + AlCl *7 = gelatin -foam 1% ( 2 week implantation ln neck) 8 = gelatin foam 5%
9 = gelatin ~oam 5% + HCl 10 = gelatin foam 1% + AlCl + Urea + EDC
11 = gelatin foam 5% + ~,OO~M CaC~2 12 = gelatin foam 5% + .OlOM CaCl2 13 = gelatin foam 5% + ,lOM CaC12 14 = gelatin foam 5% + ,25M CaC12 In addition, photographic evaluations were made of all sites at the l, 2 and 7 day interval.
The results of these implantation studies and struc~urnl investigation are summarized in Tables 1 and 2.

* N,B, No. 7 and No. 1 are identical compounds 2 ~1~7 . Skin Incision Healin~Time ___________._________ __ _ A 3 cm incision was made into the left or right torso of the dog, which penetrated the muscularis. Then the hemostatic agent was added and the incision was allowed to clot. There was no pressure placed on any of the agents.
The time to clot was obtained via stop watch~
In one set of experiments3 under each incision was placed a pre-weighed 4x4 bandage or sponge and the blood was collected and weighedO The weight of the blood was obtained by subtracting the weight of the clean dry 4x4 sponge from the weight of the blood soaked 4x4 bandage or sponge~ The results are tabulated in Table 2.
O~gan Bleedin~ Time In order to obtain information on the relative hemostatic ability of each of the agentY on a non-suturable organ such as the spleen or liver, incisions were made on the spleen and the bleeding times were obtained. The procedure was essentially analogous to the forementioned skin test. A 3 cm incision was made in the lateral aspect of the spleen~ the hemostatic agents were placed on the injury and the bleeding time and weight o blood were obtained. There was some difficulty noted in evaluating this data as, with different degrees of injury (i.eO cut arteries3 etc.)g there were differential bleed;ng rates. This is noted ~hen necessary on the results. The spleen was then excised~ fixed and marked for histologic evaluation.
In Vitro Ana~ysis _~
An i ~ itro analysis consisting of standard T~T
(thrombin recalcification time) substituting dissolved hemostatic agents at the same concentration for normal saline was made.

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RESULTS
~_~_ The results indicate that a number of varieties of the hemostatic agent of the invention are viable alternatives to those hemostatic agents commercially available (Avitene9 Surgicell and Gel~o~n~ in both hemostatic abilit~T and-histo-. logical evaluationO These are H.D. HCl ~high density 5% HCl treated), L.D. HCl (low density 1% HCl treated)~
and LD O(low density 1% gelatin). Furthel~ore~ the investigators and clinicians involved in this preliminary study listed the order of preference of hemostatic agents . as H.D. HCl~ L.D~ HCl, Avitene, L.D.~., Surgicell and , Gelfoam.
The results indicated above are tabulated in the fol win~ Tables 1 and 2~

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2~7~d TABL~ 2 BLEEDING TIME AND BLOOD LOSS IN SKIN AND SPLEEN INCISIONS
~ * Per cent *Per cent **Per cent **Per Cent Change in Change ~n Change Change in Bleeding Weight o~ ~leeding Total Time vs Total Time vs Blood Loss Control Blood Loss Control vs Control vs Control (gm,) _ _ -Gelfoam -24,8 -73,0 ~65,9 -70,0 Surgicell -26.0 -78,8 -78/5 -68,7 Avitene -2197 +15,3 -79~5 -48,7 Gelatin Foam 1% -42,6 ~80.3 -87.7 -62,5 Gelatin Foam 1% +
~Cl -61,5 -86O5 -85.7 -67 3 5 Gelatin Foam 1% +
N~4Cl + EDC +01,0 -85,3 -78.2 -68,0 Gelatin Foam 1% +
EDC -03,0 -82O3 -61.9 -64,0 Gelatin Foam 1% +
EDA + EDC -16.6 -78,0 -72,1 -60,7 Gelatin Foam 1% +
AlC13 +73,0 +93.8 -~2,1 -35~5 Gelatin Foam 5% -39.1 -75.0 -90,4 65.5 Gelatin Foam 5% +
HCl -56,4 -81.0 -91,1 -69,5 Gelatin Foam 1% +
AlC13 + Urea + EDC +98,7 +56,9 - -Gelatin Foam 5% +
0,001M CaC12 -50,0 -88.5 -91.1 -90,7 Gelatin Foam 5% +
.010M CaC12-56.2 -94,6 -93,8 -12.5 Gelatin Foam 5% +
,10M CaC12-57~5 -72,3 -93,8 - 0~4 Gelatin Foam 5% +
,25M CaC12-56.2 ~92,1 -94.5 - 0.1 - = decrease * SKIN
+ = increase ** SPLEEN

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Figs. 1-3 show U.V. absorption spectra of various formu-lations according to the invention. The basic compound alone or with CaC12 added does not absorb at 280 mll ~ indicating that there is no protein contamination. Addition of bovine serum albumin (BSA) 3 however, results in an absorption peak at 280m~. The decrease in absorption with increasing proportions, relative to protein9 of CaC12 is seen in Fig. 3~ probably as a result of dilution of the protein. All samples were made up as 1% solutions and then diluted 10-fold before measurement.
The percentages shown in Figs. 1-3 refer to the concentrations before lyophilization.
Fig. 4 shows the pH of 10 ml of 0.1% Super Stat solutions with additions of 100 ~ liters of O.OlN NaOH or OoOlN HCl.
The pH cl-rve for distilled waterg which has almost no buffering capacity~ is also sho-~n. With increasing proportion of CaC12 relative to protein~ there is a decrease in buffering capacity9 probably as a result of dilution of the protein~
A1so, additions of base result in a greater change in pH than do addit;ons of acid. I`his difference is expected since:
(1) The pH of the novel compound in distilled water ~pH=5~5) is acidicy approximately 5.9, and (2) the net negative charge on the ~el molecules would act to neutralize H+ more efficiently than OH O
Figs. 5 and 6 show the infrared spectra of the novel compounds. Absorption peaks for N-H and C=O are indicated.
Increasing the proportion of CaC12 relative to protein results in a decrease in the two absorption peaks~ 25 would be expectedg but does not affect the shape of the peaks.

~p~

From the above, it will be seen that the lnvention provides the improved hemostatic agent as well as a method for producing the same, The invention also provides an improved method of controlling bleeding, Although lyophilization techniques are known, the ~ollowing steps may be used relative to the above disclosure:
1, Dispense 50 ml amounts into plastic lOOmm petri dishes, 2, Shelf-freeze in lyophilizer (e,g, Virtus model 100 SRC-7) at minus 30 to minus 50C for 3 to 5 hours or until eutectic point has been determined, 3, Set condenser for one to two hours; be~in vacuum with no heat for 3 hours, 4. Set shelf heat to plus 30C and continue for 48 hours, The following may be used for Sterilization:
; 1. Place in gas sterilization envelope and s~al with indicator inside, 2. Gas sterilize with ethylene oxide through normal cycle4 3, Aerate thoroughly following exposure to ethylene oxi de .
There will now be obvious to those skilled in the art many modifications and variations of the above embodlments, These modifications and variations will not depart ~rom the scope o e the invention if defined b~ the following claims.

Claims

The embodiments of the invention in which an exclu-sive property of privilege is claimed are defined as follows:

1. A method of preparing a hemostatic agent com-prising modifying one of the groups consisting of collagen substance or a collagen-like substance by dissolving the subs-tance in water and modifying the thusly dissolved substance to render the surface charge thereof effectively more positive than prior to modification, and freezing and drying the thus-ly modified substance.

2. A method as claimed in claim 1 wherein the subs-tance is modified by non-covalent modification.

A method as claimed in claim 2 wherein the subs-tance is lyophilized

4 A method of preparing a hemostatic agent com-prising modifying one of the groups consisting of collagen substance or a collagen-like substance to render the surface charge thereof effectively more positive, the substance being modified by covalent modification.

A method as claimed in claim 4 wherein the subs-tance is lyophilized.

6. A hemostatic agent prepared as claimed in claim 1.

7. A hemostatic agent prepared as claimed in claim 3.

8. A hemostatic agent prepared as claimed in claim 5

9. A method as claimed in claim 3 wherein the subs-tance is gelatin which is treated with hydrochloric acid.

A method as claimed in claim 5 wherein the substance is gelatin which is treated with ethylenediamine.

11. A method as claimed in claim 5 wherein the substance is gelatin which is treated with NH4Cl.

12. A method as claimed in claim 5 wherein the substance is gelatin which is treated with HC1.

130 A method as claimed in claim 5 wherein the substance is gelatin which is treated with calcium,

14. A method as claimed ill claim 13 wherein the calcium is used in the form of calcium chloride,