IL21979A - Wound-healing compositions and process of production thereof - Google Patents

Description

healing compositions invention relates to compositions and methods of making the It was observed time ago that the healing of wounds of human patients is inhibited or if the patients were at the same time undergoing cortisone It was found further that this inhibition of the healing of the wounds could be overcome by the use of cartilage powder applied It has also been shown that the healing of wounds is accelerated by the use of rather powder of digested bovine tracheal ments were carried out on albino Sherman strain female There was observed an average increase in the rate of healing and in the strength of the healed tissues about over that the control This invention provides a process the manufacture of cartilage preparations of superior wherein cartilage is subjected to a proteolytic to remove protein adhering to the pretreated cartilage ground substantially in the absence of oxygen if the resulting cartilage powder is extracted with aqueous solutions of substances whose aqueous solutions have a low acidity or low alkalinity at the concentrations used for the and the resulting if is It has been found most unexpectedly that cartilage taken om the partly calcified skeletons of very young or newly born or even foetal skeletons is much more in accelerating the healing of wounds than the bovine tracheal powder on which previous observations were Preferably the is not over six months The grinding of the pretreated cartilage in the substantial absence of oxygen is the important feature of the invention and has been substantially to increase the activity of the cartilage She of the aqueous extracting solutions is ably in the pH range to Preferred extraction aids are those which are either volatile and therefore can be readily removed from the extract such for ammonia or ammonium Alternatively they are stances if remaining in the cause no if applied either topically or introduced parenterall sodium sodium formate and the If the extracts thus prepared may be even to dry preparations thus having a substantially unimpaired can be or diluted back to the original strength with saline Concentrating of the extracts ie preferably done in a vacuum or by it is found in accordance with the invention that by subjecting the cartilage or the cartilage powder or the extracts to irradiation by violet light for a short period of the activity of the material is increased to a noticeable tion with ionizing radiation such gamifia rays also increases the activity of the although to a lesser extent than was the case with the It has been found that there exists a surprising synergistic effect in the combination of cacfeilage or according to the invention growth hormone or with tung This effect can be observed both in topical and in parenteral Satisfactory effects also achieved through oral administration of suitably or encapsulated cartilage preparations or cartilage extracts according to the It haa been found that there are very great differences in the activity of the depending on the method used in their the or carriers and in the technique of the cartilage powder as well as the extract are effective if they are absorbed or incorporated with surgical gauze which is then applied to the than they are applied by spraying onto the although spraying salves based on aqueous gels such as those from gum dextran and others are more in applications than the undiluted materials They are also more convenient to apply especially over large areas such as is the case with Cartilage preparations or cartilage extracts ing to the invention suspended in oils such as tung corn olive or linseed may be applied directly to The oil dispersions may then be emulsified in type or water may be emulsified in the oil dispersions forming type The cartilage preparations and cartilage extracts dispersed in aqueous or oil carriers were applied directly to the wounds by by impregnating in bandaging materials or by any other means which it possible to bring the or its extract into contact with the exposed In of parenteral applications the cartilage or cartilage extract preparations may be duced through suppositories introduced into rectal or other Cartilage powders dispersed in oils have been successfully administered It was found that the oil to a large extent protected the cartilage from attack by stomach and allowed its passage into the intestinal tract where it showed a desirable effect the healing of intestinal Cartilage powder be as the form of pellets such as tablets or On the other by incorporating the cartilage powder onto silica gel or other gel forming materials which are capable of coating the stomach the rate of healing of stomach ulcers vas noticeably Unless otherwise the effectiveness of the was established in animal teste as by Baker in on the Acceleration of Wound Healing with Sherman strain albino female rats were employed in the preparation consisted of a centimeter abdominal incision under aseptic conditions and closed interrupted sutures of 000 wound tensile strength at seven days d i t f mercur b a modi ica ion th The rat to be tested is killed by an intracardiac injection of test is prior to the onset of rigor After the sutures have been removed from the a rubber latex prophylactic pouch is inserted into the peritoneal cavity through a defect with a Kelly clamp in the apex of the After the rubber pouch is in and the introitus has been snugged firmly a around the tube to the peritoneal the rotary air connected to the pouch is turned on regulating it in such a manner that the air pressure will increase at a rate of 10 of aercury every five The pressure at which the vound splits and pouch extrudes itself or in through the defect is recorded as the tensile strength of the is also a quantitive of the degree of healing or rate of healing achieved in the The following examples illustrate certain present preferred embodiments of the and it is stood that other methods and within the spirit of the invention may be made without departing the scope of the appended Parts and ratios are by weight except as otherwise Example Cartilage tracheae of healthy adult beef cattle were removed within 50 to after the animals were The tracheas were then either with an solution or they were frozen to preserve in which case the previously were then digested for about 3ix hours at in an aqueous solution containing acetic acid and pepsin IX 5500 After digestion tracheal cartilage was removed from the first with water of about than with water of about until the effluent wash water showed no trace of pepsin or acetic acide The cartilage was dried in at The dried tilage was defatted by extracting it with a such as It was then The granulated purified was ground to a fine powder in a laboratory porcelai loaded with flint pebbles in a weight ratio of 1 cartilage to 2 Dry ice was then put on top of the mill charge and the mill was kept open for 5 to allow the to displace the in the lid of the mill wae then clamped on tight and the mill rotated as is customary in the performance of the ing The grinding was carried out at for The ground cartilage was screened through a 400 mesh Cartilage pebble Cartilage obtained from skeleton of old piglet was washed with distilled water immediately after removal the The cartilage was then freed from the matter and then digested with an pepsin solution as described in Example The cartilage was ground and screened in accordance with the method described in Example Bovine traehlal cartilage fluid energy mill n extracted bovine cartilage was washed with distilled and then digested with an pepsin solution in accordance with the procedure described in granulated cartilage was put through fluid energy mill of the having an grinding chamber operated with nitrogen at 100 20 C temperature and at a solid feed rate of 10 The averagle particle of the ground material taken from the collector attached to the mill was 5 microna with 10 microns as the a variety of sources was ground in s fluid energy mill under the conditions indicated aa Particle Size Source Fluid Microns Microns Bovine 3 5 II Air 5 3 l Ai 5 10 Oxygen 7 lt 5 7 Nitrogen 5 7 Argon 5 7 1 day old Nitrogen 5 8 II 2 weeks old rogen 5 8 tl 1 month old 5 8 6 months old 5 8 Calf 1 day old 5 8 2 weeks old ifitrogen 5 8 ft 1 nsonth old 5 8 1 day 5 8 2 weeks old ogen 5 8 aration of cartilage extracts in the pebble mills Extracts of cartilage having high activity were produced as The cartilage was acid pepsin digested as in Example and then without drying was suspended in extracting liquid and then transferred into a pebble mill which was charged to of its with pebbles of average one inch ratio of the cartilage to extracting liquid was kept at liquid suspension charged into mill in sufficient to fill voids of with the top of the pebbles barely covered by the The air was then purged from the with nitrogen and the mill The mill was allowed to run for 6 hours at between and which resulted in a medium grinding of the and in the taneous extraction of the active agents from the At the end of the the mill was the fluid paste strained free the the fluid transferred into a centrifuge operated at 6000 rpm at a temperature of between and After hour the centrifuge was stopped and the supernatant liquid strained through 400 mesh Nylon strained extract was it was returned to the and the repeated until a clear slightly opalescent extract was extracts were stored at with thiiaer sal powder and following extracts were Total Solids of Clear Extract Cartilage Source Extracting Liquid By Bovine trachial Distilled wate Ammonia in water urea in water carbonate in water phosphate in water carbonate in water in water sodium citrate in water II sodium citrate in water aodium formate in wator Piglet 1 day old Isotonic saline solution n 1 ammonia in water ammonium carbonate in water sodium citrate in water one day old Isotonic solution in water Isotonic saline solution plus ammonia to Θ Isotonic saline solution plus ammo ia to pH 10 The isotonic saline solution wae prepared with distilled water and contained cartilage Dry concentrates prepared from cartilage as energy necessary for evaporation of the volatile portions of Instead of ai nitrogen was used for hot A vaned rotating at about was used to the The inlet gas temperature was and the outlet temperature and dryer used as a closed system dryer the exclusion of oxygen to avoid degrading active material during the evaporation of the following dry were thus Appearance of used Sol s Yield Dried Powder yellow II n II Off yellow Off white n yellow II n means percent of solids in the extracting liquid as determined drying at for two means the dry solids percent obtained from liquid by the drying The powders were stored in tightly closed glass jars in refrigerator at of cartilage A laboratory vacuum ahelf dryer was extracts were refrigerated to The shelf temperature was The vapor pressure was about 800 microns of was determined at Extract weed Solids Yield Appearance of Dried Material Off white coarse powder ft tt n 5 tt tt n tt it II n tt tt n tt tt it n tt n materials were stored in tightly closed glass jars in a refrigerator at Example Cartilage applied to wounds by with a hand atomiser about 30 the edges of a abdominal incision of the Cartilage of Wound Control 100 Example 1 125 2 135 3 130 100 110 120 105 130 135 130 140 140 135 130 140 140 142 n 135 of related to loo means expressed of pressure required to rupture the healed tissue of the wound as compared the pressure Example Cartilage extracts applied to by swabbing to longitudinal midline of Cartilage Rate of Heali g Isotonic control 100 100 125 125 125 125 115 150 ft 130 130 135 140 140 II 130 II 135 140 145 150 II 130 effect of cartilage extracts on t healing of In case 5 of the extract was injected into subcutaneous the within 24 hours after the abdominal Cartilage Isotonic saline control loo 105 125 155 135 140 demonstrates the effect of cartilage extracts combined with a bovine In of extract was mixed with 10 of a bovine growth distributed by the Endocrinology Study Section of the National Institute of Health through pituitary distribution Approximate assay of prolactin International oxytocin Test animals and wound are as stated in Rate of Healing Hone saline control 100 Isotonic saline with growth control 108 with growth hormone no 130 135 135 145 148 12 This example demonstrates value of tuted and extracts in the healing of extracts were dissolved either in water or in isotonic depending on salt content of the original The solutions were adjusted to correspond the content of the tracts from w the dried were The appled by parenteral injections into rats as in Dried Extract Solvent Rate of Wound Healing Rone Isotonic Saline control 100 Water 115 Example 120 Isotonic saline 125 130 Water 130 n 135 8 10 Example 15 example demonstrates the value of intravenous injections of cartilage extracts or solutions of dried extracts or solutions of dried extracts in the healing of were on dogs circular Wounds not sutured but protected only with sterile The rate of measured by observing the degree of Extract Rate of Wound Healing Isotonic saline control 100 120 135 rt 140 140 145 14 This example demonstrates the use of cartilage powder on open Powders were held layers of porous surgical and held through bandages to the ests were made on The rate of healing estimated by observing the degree of Cartilage powder Rate of Wound dry gauze Control 100 Example 2 135 135 145 145 Example This example demonstrates the effect of applying liquid cartilage extracts on open Porous surgical gauze was extracts and applied to open and unsut red while still Tests were on The rate of healing measured by the observed degree of of Wound Isotonic saline control 100 Example 125 140 140 150 16 This example demonstrates effect of applying dried cartilage extract on open Porous surgical saturated with the dried to o a moisture content of about at C and at a pressure of 50 e dried gauze was applied to the open and unsutured and observations were as in Example Cartilage of Wound Healing None Extract control 100 Example 120 135 135 150 Example 17 testa involved the intravenous injection were on In carrying out tests 100 blood was taken from the animal treated as The blood was mixed with 10 extract ad reinfected into the plasma was obtained from the mixed with 10 cartilage extract and reinjected into the same animal from which the blood was blood was replaced by an equal volume of an isotonic aqueous saline solution of polyvinylpyrrolidone viscosity grade and 10 of a cartilage The control were treated as Control 100 blood taken and then reinjected the same Control The blood taken in case of Control the plasma separated and reinjected the same Control blood was taken from the animal as above in case of Control and replaced with a saline solution of polyvinylpyrrolidone viscosity All blood samples taken citrated to prevent The rate of heating was measured by observed degree of extract as per o Control 1 100 Control 2 90 Control 3 85 I 130 II 125 120 I 150 145 tt 130 I 150 II II 145 II 135 The animals used in these experiments not less than Cartilage Ointments prepared with of cartilage powder and ointment The ointment a applied on surgical gavise to the open and red wounds of rate of healing was measured by the degree of Cartilage of Wound Healing Control Corn oil 100 125 Control oil 105 Example 135 None Control Sung oil emulsion 100 Example Control 100 135 Example Suppositories were prepared wit cartilage powder suppository suppository base was prepared accordance with XVI pages rinated Gelatin suppositories were 2 size and were administered rectally to The surface of the rectum was removed for 1 from the rectal about one hour before the insertion of the Λ suppository was introduced every six The rate of healing determined by visual powder None Control 100 Suppository base Control 105 130 135 Cartilage powder tablets and were orally The tablets were without and with enteric The capsules were either or enteric material such as Both the tabids and the capsules contained 2 gnu cartilage powder The test animals were dogs and the wounds were circular dosage was cartilage body weight six The rate of healing was determined by visual Powder Form Coating of Wound Control 100 Tablet Hone 115 Enteric 120 Gelatin 115 Capsule 115 125 130 Cartilage Cartilage was irradiated with ultra violet radiation produced by a pressure mercury arc tube type The thickness of the powder layer exposed to radiation was 5 the radiant source 15 from the cartilage The exposure time was one experiment carried out in a nitrogen The irradiated were tested on as in case of Example Cartilage Rate of Wound Control 100 Example not irradiated Control 150 In addition to pebble mill and fluid satisfactory powders may obtained by ball milling in inert While ball or pebble milling the cartilage with extracting liquid gives satisfactory other such as cartilage powders in the with a high high closed turbine or passing the Example of emulsion ointment base used in Examples and The emulsion is composed as illed Sodium Methyl cellulose Com oil or tung oil he methyl cellulose is with of boiling The hot methyl mixture is added to 400 of cold water while mixing vigorously with a high speed homogenising When methyl cellulose been completely in a clear the sodium sulfate dissolved in 150 of water is added thereto under vigorous the oil is added and the mixture is stirred until a homogeneous and uniformly fine grained emulsion is cartilage preparation is then added to the resulting ointment base and mixed therewith until a uniform and homogeneous dispersion ie Example Preparation of the emulsion base used in and emulsion is composed as oil Laurie acid Sodium chloride Distilled water acid is dissolved in the tung oil by warming and resulting salt solution is gradually added to the oil under constant and vigorous mixing until a homogeneous emulsion is Preferably the cartilage preparation is added to the salt solution and the is while the cartilage is still mixing and dispersing equipment used in of the emulsions and in the dispersion of cartilage preparations was a model Mixe manufactured and supplied by Barrington of Example Enteric coating for tablets of Example The enteric coating is composed as Butyl stearate bleached shellac The components thoroughly mixed until the shellac is completely tablets are given first two subcoatings of gelating solution followed by seven costings of the shellac using talc as the running The finished enteric coatings are tested with hydrochloric acid for their ability the action of gastric 900 of aluminum hydroxide gel and 100 of cartilage powder obtained according to Example are intimately mixed with each li gel 75 of bismuth 75 and 750 of distilled water are intimately and thoroughly mixed with each 100 of cartilage extract obtained according to are added thereto mixture hyl 60 of methyl cellulose and 840 of thoroughly stirred with and 100 of powder obtained according to Example then admixed thereto to a While isotonic saline is an effective extraction more complete extraction with higher healing vity is obtained the is raised with Salts other than provide more effective as ehown in Example An inert atmosphere during the extraction in extracts of greater potency than when the tion is carried out in the presence of The use of suitable may carrying out the extraction in the presence of air without serious of bovine tracheal cartilage from mature a year old or is purposes satisfactory with substantially greater potency is obtained from the of very young highest potency material is obtained animals less than one month Cartilage froa foetal skeletons is often most Cartilage from other in to and and is effective in healing wounds in accordance with the present so with such as cartilage of birds and cartilage may be particularly effective in view of the extraordinary ability of the reptiles to regenerate their tissues and even their When dry cartilage extract is is but is satisfactory in inert drying is satisfactory when oxygen is excluded and temperature of the liquid is held below The cartilage may be dusted on the or It may also be applied in the form of ointment on the as exemplified The may be applied directly to the wound by spraying on the swabbing it or brushing it Both the powder and the extract may be applied first to an absorbent medium which is then applied to wound and held on by a bandage or adhesive or other suitable The cartilage or the extract may be incorporated into capsules or suppositories and applied or in vaginal or Test results indicated above for have been surprisingly The invention may also be successfully practiced in healing human particularly special as for example in cases involving cortisone Cartilage extracts may be or The dried may be used as powders or may reconstituted and used as the The wounds to which the active materials are applied be sutured or be left open without affecting the rate of The active materials may be administered preferably within the first 24 hours of the incisio or be applied before the incision or they may be applied in several applications in he use of growth hormone in oombination with cartilage or extract increases the rate of ing cartilage powder with ultraviolet radiation in absence of oxygen increases its insufficientOCRQuality

Claims (25)

- 29 - 21979/3 CLAIMS.

1. A process for producing wound-healing compositions, wherein cartilage is subjected to a proteolytic pretreatment to remove protein adhering to the cartilage, the pretreated cartilage is ground substantially in the absence of oxygen and, if desired, the resulting cartilage powder is extracted with aqueous solutions of substances ("extraction aids") whose at aqueous solutions have low acidity or low alkalinity and the concentrations used for the extraction, and the resulting extract, if desired, is concentrated.

2. A process according to Claim 1, wherein the cartilage powder is extracted with an isotonic salt solution.

3. A process according to Claim 1 or 2, wherein the extraction aid is ammonia, urea, ammonium carbonate, sodium citrate, disodium phosphate, trisodium phosphate, or sodium formate, and the solution has preferably a concentration in the ea»e range from 0.9 to 3.0$>.

4. « A process according to Claim 1, 2 or 3» wherein the aqueous extraction solution has a pH in the range of 6.5 to 10.

5. A process according to any of Claims 1 to 4, wherein the cartilage is ground to a powder having a particle, size not exceeding 40 microns, preferably not above 10 microns.

6. A process according to any of Claims 1 to 5, wherein the extract from the powdered cartilage is concentrated by evaporation under reduced pressure at a temperature below 40°C, by spray-drying, or by freeze-drying.

7. A process according to^Claims 1 to 6, characterized by the use of cartilage from an animal less than one year old, preferably not more than six montlis old, preferably less than one month old} or from a foetal skeleton. - 30 - 21979/3

8. A process according to any of Clai&s 1 to 7, wherein the ground cartilage, or the extract prepatred therefrom, is subjected to irradiation for increasing its wound-healing activity, e.g. with ultra-violet radiation.

9. Processes for producing wound-healing compositions, substantially as described herein with reference to the Examples.

10. Wound-healing compositions when prepared by a process as claimed in any one of Claims 1 to 9.

11. * A wound-healing composition comprising growth hormone or tung oil admixed with cartilage powder prepared by a process according to any one of Claims 1 to 9*

12. Surgical gauae having absorbed thereon a composition as claimed in Claim 10 or 11.

13. An ointment or salve based on an aqueous gel and comprising a composition as claimed in Claim 10 or 11.

14. A ointment or salve as claimed in Claim 13» wherein in the aqueous gel is an alginate, tragacanth, gelatin, gluten, casein, dextran, or polyvinyl pyrrolidone.

15. An ointment or salve as claimed in Claim 13 and substantially as described in an one of Examples 18, 19 or 25 to 27.

16. An oil-in^water or water-in-oll emulsion of a composition as claimed in Claim 10 or 11.

17. Am emulsion as claimed in Claim 16 and substantially as described in xample 22or 23.

18. A suppository comprising a composition as claimed in Claim 10.

19. suppository as claimed in Claim 18 and substantially as described in Example 19. - 31 - 21979/3

20. An orally administrable composition comprising a composition as claimed in Claim 10 or 11 in the form of oil-coated cartilage particles suspended in an oil, a capsule, or an enterically coated tablet or pellet, or in admixture with a gel forming material capable of coating the walls of the stomach,

21. An orally administrable composition as claimed in Claim 20, wherein the gel forming material is silica gel.

22. An orally administrable composition as claimed in Claim 20 and substantially as described in any one of Examples 24 to 27.

23. An isotonic saline solution of a cartilage preparation as claimed in Claim 10.

24. « A parenteraliy administrable suspension of a cartilage preparation as claimed in Claim 10 or 11 in an injectable oil or in blood, blood plasma, or blood plasma substitute.

25. A parenteraliy administrable solution as claimed in Claim 24 and substantially as described in Example 17. For the Applicant RC/rb