CA1066620A - Composition and method of using a protein mixture derived from liver - Google Patents

Abstract

METHOD OF USING A PROTEIN MIXTURE
DERIVED FROM LIVER

ABSTRACT OF THE DISCLOSURE
A method useful in treating certain disease conditions by virtue of a protein mixture having a variety of disparate properties, such as facilitating wound healing and influencing glucose metabolism. The mixture also exhibits pronounced super-oxide dismutase activity. The composition may supplant or be combined with cortico-steroids to provide anti-inflammatory response while facilitating wound healing. It is useful for treating hepatitis, arthritis, and scar tissue.

Description

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BACKGROUND OP THE INY~NTION
This invention relates generally to 8 method of , using a composition of a physiologically active protein ,~ mixture which exhibits a variety of disparate properties, ~;~ such as facilitating wound healing while inhibiting inflamma-tion. It also exhibits pronounced superoxlde dismutase ;~ sctivlty. Some of the properties and uses of the composition have been described and claimed in our U.S. Patent 3,701,768.
Superoxide dismutases are a group of enzymes ~ 10 believed to catalytically ~cavenge the superoxide radical, 'i~` which appears to be an important agent of the toxicity of ~; oxygen. See Fridovich "Superoxide Dismutases'l, 1975 Annual ~; Review of Biochemistry, p. 147.
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'.: . . . . . - ''. .. ~:: ~ : ' -; SUMMARY OF THE INVENTION
In our U.S. Patent 3,701,768, there is disclosed and claimed - a method of extracting a protein mixture from liver and the resulting product. The present application relates to compositions of the product and to the method of using the product there disclosed.
The product of the present invention influences the primary metabolism of carbohydrate and the urea cycle. The product is a biological catalyst present in large amounts in liver. It increases glucose uptake and glycogen synthesis in incubating muscle, increases glucose metabolism in isolated perfused liver and increases glycogen storage in adrena-lectomized animals. It acts in vivo to improve renal functions in intact animals, indicating that it i8 humorally transported and enters peripheral cells or influences their metabolism at the membrane level. The product exhibits pronounced superoxide dismutase activity. It is useful in reducing peripheral blood ammonia; in treating scar tissue, arthritis and hepatitis;
and in facilitating tissue repair at the same time as it inhibits .':
inflammation.
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The wound healing effect of this invention i8 of particular interest because the product also inhibits inflammation. This combination of properties iB unusual. The st widely used anti-inflammatory agents, the cOrtico-steroids, inhibit wount healing, thus limiting the usefulness of these agents. Tissue damage and inflammation frequently occur together, and the use of conventional agents 1ike hydrocortisone actually prolongs the healing duration. There is, therefore, a need for an anti-inflammatory agent which also facilitates wound healing.
By combining the product of this invention with hydro-cortisone, smaller doses of the sometimes toxic cortico-steroids may be used wlth greater anti-inflammatory response then with full doses of cortico-steroids alone. -'~
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I ¦ DESCRIPTION OF THE PREFERRED EMBODIMENT

2 ¦ The biologically active ingredients of the present

3 ¦ invention are conveniently extracted from liver. The process, ¦ .
41 described in our prior U S patent 3,701,768, may be briefly I ~
51 summarized by the following steps: I .
61 (1) treating liver with an aqueous solution of 7 divalent metal activating cation at a pH essentially 81 neutral or slightly alkaline and separating the 9¦ resultant solids from the liquid; ;
lO¦ (2) treating the liquid with heavy metal precipitating lll agent and separating the resultant precipitate from 12 ¦ the liquid;
13 ¦ (3) adjusting the pH of the liquid from step (2) to 14 ¦ a range of between 8.3 and 8.5 and separating the 15 ¦ resultant precipitate from the liquid;
16 ¦ (4) treating the liquid from step (3) with a 17¦ concentrated solution of salting out agent to 18¦ precipitate undesired protein and separating the l9¦ protein precipitate from the liquid; : .
20 ¦ (5) adding solvent to the liquid from step (4) 21¦ in an amount sufficient to give the resultant 22 ¦ mixture a solvent concentration of 20 to 26% by 231 volume and separating the resultant precipitate 241 from the liquid; adding solvent to the liquid in . ~ .
251 an amount sufficient to give the resultant mixture 26 ¦ a solvent concentration to 35 to 40% by volume to :
27 ¦ produce a mixture having three layers; separating 28¦ ~ the upper layer and adding to it in amount sufficient .
29 ¦ to give the resultant mixture a solvent content 70 321 and 77~ by volume whereby protein is precipitated 3.

I (6) separating the protein precipitate and dispersing 2 it in a solvent as a pH of 7.8 to 8.2;
3 (7) dialyzing the dispersion from step (6);

4 (8) heating the dialyzate from step (7) to a 51 temperature between 46 and 50 C. and separating 6 ¦ the resultant precipitate from the liquid;
7 ¦ (9) subjecting the li~uid from step t8) to 8 1 electrophoresis and recovering a biologically gI active mixture.
~ol The product described in our prior patent is designated ~l as to source by the registered trademark "Acutalyn". For l2¦ simplicity in this application, the term "acutalyn crude" shall ~31 mean the crude product derived from Step 8 above having the six 14 discrete components shown in Figure 2 of the prior U. S. Patent ~51 3,701,768. The designations "acutalyn crude" and "crude product"
l6¦ will be used herein interchangeably to designate the material -~
17 ¦ which contains all of the components of the product of step 8. ;~
18 ¦ As described in our prior patent, one of the components 19¦ is designated Component II. This material makes up about 15-20%
20¦ of the acutalyn crude. For consistency in this application, this¦
2l1 component will be referred to as Fraction A. It appears that 22 ¦ Fraction A is associated with the protein component which gives 231 acutalyn crude its pronounced superoxide dismutase activity. The ;
24 ¦ percent of superoxide dismutase activity varies from about 7 to -25 ¦ 35~ of the acutalyn crude, as determined by direct enzymatic 26 ¦ assay.
271 Also included in acutalyn crude is a biologically 29 ¦ active protein of approximately 25,000 molecular weight which is l described in our prior patent.

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Example 1.
This example illustrates the influence of the present actualyn crude composition on metabolic activity of isolated perfused rabbit livers. A liver perfusion apparatus was used which was a modification of that described by Rink et al. in Anesthesiology, 17:377, 1956. The device is enclosed ~ -in a thermo-regulated cabinet at 37C. and consists èssentially of three components: (1) a pump which maintains the blood flow; (2) an oxygenator which ventilates the blood; and (3) a freshly obtained liver. The system utilizes a closed gas system which per~its a determination of the amount of oxygen utilized by the liver and the amount of carbon dioxide produced.
New Zealand white male rabbits, fasted 24 hours, - were used as liver and blood donors. The perfusate consisted of 200 ml. of heparinized blood collected from two donor animals under light Nembutal anesthesia. The blood was diluted with an equal volume of normal saline solution and 60,000 units of penicillin were added.
The liver was surgically removed from one donor animal, under light Nembutal~ anesthesia. After cannulation of the portal vein, the liver was qsickly perfused with 20 ml.
of a hep~n-sa~l~ solution to prevent clot formation. The time interval between interruption of the portal circulation and establishment of the perfusion circulation ranged from six to ten minutes. The weight range of the livers used in the study was between 80 and 110 grams. The livers were perfused for thirty minutes prior to making any measurements in order to allow for equilibration of the system and to insure adequate oxygen saturation of the perfusate.

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~0666Z0 All samples were obtained from the "venous blood"
immediately after its passage through the liver. Urea nitrogen was determined by the method of Gentzkow and Masen, J. Biol. Chem.
143:531, 1942. Glucose was determined on tungstic acid blood filtrates by the method of Folin and Wu, J. Biol. Chem. 41:367, -1920. Oxygen consumption by the liver was obtained by a direct reading from the graduated oxygen buret after restoring the initial manometric pressure of lcm. water with leveling bulb.
Expired carbon dioxide was absorbed in standard NaOH contained in an alkali trap. The trap was changed every half hour. An excess of BaC12 was added to an aliquot and the C02 precipitated out as BaC03. The residual NaOH was then titrated against a standard O.lN HCl solution. The oxygen used and C02 produced were then corrected to standard conditions and expressed as ml. per 100 grams of liver tissue. The criteria used for a successful experi-ment were the following: -a. A constant perfused flow rate.
b. A steady rate of oxygen utilization.
c. A steady rate of bile formation.
When the acutalyn crude is injected into the perfusate, the rate of synthesis of urea nitrogen by the isolated perfused rabbit liver is increased. The rate of urea nitrogen synthesis of a control study was substantially less than the rate with an injection of acutalyn. It will be noted that the rate of urea nitrogen increase, within 2 hours after injection of the protein, is much greater with actualyn than without. In addition to urea synthesis, there was a marked increase in the rate of production of C02, averaging 35 ml/hour/100 gms. of liver greater than the control. Also, the viability of the liver was greatly prolonged (from about four hours to seven hours or more) as indicated by oxygen utilization, C02 production, urea synthesis and bile flow. - --'" ' ' .

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Example 2 This example illustrates the effect of the acutalyn crude on peripheral blood ammonia in horses. A solution of acutalyn crude in water was injected intravenously into six test horses 12 hours prior to the start of controlled exercise.
Five control horses were not so injected, but were subjected to the same activity. All horses were tested for blood ammonia at rest before the exercise, and then subjected to the same strenuous exercise. Each horse was measured for blood ammonia immediately after exercise; and at 75 minutes and four hours post-exercise. The blood ammonia levels were significantly improved by the administration of acutalyn, as compared to the control group. ~ ~-Similar results in the reduction of blood ammonia were determined with rabbits, where increase in blood ammonia was induced by injection of Metrazol . Disappearance of the blood ammonia proceeded much more rapidly in rabbits pre-viously injected intravenously with acutalyn crude.
Actualyn crude also is useful in facilitating tissue repair., The next two examples relate to the property of enhancing recovery of soft tissue lesions. In horses, fistulous tracts are a chronic condition because they are characteristically lined with a false or adventitious membrane and are exceedingly difficult to heal. These examples show simultaneous anti-inflammatory and wound healing effects of acutalyn.
Example 3 Ten horses with suppurative lesions consisting of fistulous withers, quittor, fistula of the hock, and traumatic podermatitis were selected for treatment. Where necessary, surgical drainage was established and the usual standard manage-ent techniques were applied. Acutalyn crude, equine potency ,. 1 .

(125 mg. pct.), was in~ected intravenously in 5 ml. doses every two to every four days. Total number of injections were two to ten per animal.
Of the ten clinical cases studied, eight had excellent responses to therapy. These responses were ~udged uslng two criteria. The first was the progressive healing of a chronic lesion which had defied all prior attempts to manage the condition. The second was the incidence of relapse dur~ng ~-a two year follow-up period.
The horse in one case, a fistulous withers, was treated early in the course of the condition. No 6urgical dralnage was employed; the lesion was simply aspirated and ~ -~
intravenous acutalyn was administered. Healing was complete, and in this case as in the other cases there was no relapse.
The horses under study all had conditions which are well known for their tendency to recur. A relapse occurred in only one horse, which had responded to therapy with a slow healing period and then had a relapse nine months later. In another case, the treatment of a quittor, acutalyn administra-tion was unsuccessful. There was no apparent response to therapy.
Example 4.
Ten horses which were candidates for slaughter were selected for experimentally induced soft tissue lesion. Each animal recelved 30 cc. of 25X chloral hydrate solution in~ected extraveneously to produce the desired acute lesion. Pour horses, Nos. 1, 3, 9, and 10, received this in~ection intramuscularly in the nec~ region; the remaining six animals were in~ected extraveneously in the area of the ~ugular vein ~
at the usual site of intraveneous in~ections. --Following the injection of chloral hydrate, animal .. : :

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Animals Nos. 4, 5, 6, and 7 received acutalyn crude, the usual equine potency. Animals Nos. 1, 3, 8, 9, and 10 - -received in~ections of 100 units each of Fraction A.
The control experience of incident of development of the typical lesion caused by chloral hydrate in~ected into the tissues surrounding the ~ugular vein has been lOOX. Case Nos. 1 and 2 developed this typical lesion by the sixth day.
The lesion progressed to the necrotic stage and the animal was sacrificed.
The actualyn crude treated animals all demonstrated a significantly lesser degree of inflammation and swelling than the controls. In these treated horses, the slight to moderate leslon which teveloped by the sixth day regressed rapidly.
Complete disappearance of the lesion was observed between the seventh and the twelfth day. Most important was that the lesion did not progress and therefore there was no necrotic or sloughing stage. In Cases Nos. 9 and 10, which received Fraction A, there was an apparent complete protective effect.
There was no evidence whatsoever of any tissue reaction. Case No. 1, which received one in~ection of the acutalyn crude on the seventh day, after the lesion had developed to full size, responded promptly with the necrotic lesion resolving. By the twelfth day healing was complete.

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- - ~066620 Seven of the eight animals injected with chloral hydrate into the muscle tissues and treated intravenously with acutalyn crude developed a slight to moderate swelling without necrosis which suggests a beneficial protective effect. One horse, Case No.8, showed severe swelling for the first 24 hours which receded rapidly to normal by the seventh day. Two of these horses exhibited complete protection; there was no evidence of any swelling or any local tissue reaction. By the sixth day a typical lesion developed in the two untreated cases and progressed into the necrotic stage. Delayed treatment of Case No. 1 with Fraction A produced a rapid response at that stage and the lesion was completely healed by the twelfth day.
Example 5 A more precise measure of the wound healing effect of acutalyn crude is demonstrated by determing the tensile strength of healing wounds. In this example, a determination was made of -the force required to rupture a surgically induced wound which -has been healed with or without acutalyn crude. A solution of acutalyn crude was prepared of 18 mg. solids per ml. 0.25 ml.
of this solution was injected into 500 - 700 gram guinea pigs each day for 10 days. On the eleventh day, a 1-1/2" wound was -cut on either side and parallel to the spinal column of a female guinea pig. The wound was then sutured according to standard surgical procedures and a test substance was administered subcutaneously once daily for 10 days. On the 11th day the wounded skin was removed en bloc and quick frozen and stored at -92C. Later the same day the tissue specimens were removed from storage, immediately immersed in a water bath at 24C. so as to bring the tissue to ambient temperature, and finally cut into strips 1.6 mm wide with a special cutter. The strips were cut perpendicular to the wound so that the incision was sl:

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positioned in the center of the strip. ~-One end of the strip was attached 1 cm. away from the incision to a Grass FT 0.03 Force Transducer and the other end was attached (also 1 cm. from the incision) to a mechanical device which pulled the specimen at the rate of 0.7 mm/
sec. The force was recorded on a Grass Polygraph 7B. Signifi-cantly greater force was required to rupture wounds healed with the benefit of acutalyn.
Five control animals were treated only with sterile saline solution. Five test animals were treated with acutalyn in a dosage of 0.25 ml/day.
Clinical studies also show the ability of the protein of the present invention to systematically enhance softening of chronic urethral strictures in humans and to cause these strictures to dilate easily without local anesthetic and to remain dilated for periods of one to five or more years without need for further therapy of any kind.
Example 6 The product of thiq invention may be combined with corticosteroids to give a greater anti-inflammatory response than either drug alone. In this test, acutalyn crude was used alone and in combination with hydrocortisone to determine the inhibition of inflammation according to a standard test for carrageenan paw edema using the method described in Winter, E.
Risley, E.A., and Nuss (G.W. PROC. SOC. EXPTL. BIOL. & MED.), 111:544 (1962). Briefly stated, the anti-inflammatory effect of a drug may be measured by injecting a known irritant into the paw of a rat and volumetrically measuring the degree of swelling of the paw. This standardized test gives a comparison between an agent and a control of saline solution and indicates the amount of reduction in swelling resulting from different dosage levels.

` 10666Z0 The effect using acutalyn crude product was measured alone and the effect of hydrocortisone alone was also measured, the latter in its half dosage form and then full dosage. The the inhibition of the two dosage forms in combination with crude product was determined. Table I shows the results. The combined effect was significantly better than either drug alone.
The sometimes undesirable side effects of hydrocortisone, such as inhibiting wound healing, can be minimized by reducing the dosage in half and combining acutalyn crude, with the net result of significantly improved results over even full strength hydro-cortisone. Hydrocortisone was given in standard dose of 1.2 ml per Kg of animal weight of 27.4 mg/ml in 0.5% gum arabic.
Acutalyn crude was given in a dose of 1.2 ml/Kg of animal weight of 15.75 mg of dry solids per ml. -TABLE I.
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% Inhibition of carrageenan ~-induced edema 20 Acutalyn crude 21%

Hydrocortisone - 1/2 dose 50%

Hydrocortisone - standard dose 53% ~-Combined Hydrocortisone (standard dose) 63%
and acutalyn Combined Hydrocortisone (half dose) 65%
and acutalyn . .~ . .

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This example relates to the utility of the invention in the treatment of scar tissue, specifically urethral stric-tures.
Prior to the introduction of acutalyn therapy, 44 of the 51 patients in the study had chronic urethral strictures - ~ -requiring routine treatment at regular intervals, some as often as four times monthly, for periods ranging from one year to 42 years. Twenty-four patients had been treated at regular intervals for over 10 years prior to acutalyn therapy; another 14 cases had been treated at regular intervals for two or more years. Many had difficult filiform strictures; many also had required anesthetic for passage of sounds. The addition of acutalyn was the only change in their routine therapy. The acutalyn preparation used in this study contained all the components of acutalyn crude, ampulized into human dosage form containing 50 mg. %. All in~ections were administered either by IV or IM.
Patients were kept under treatment until their prostatltis cleared and 30 French sounds could be passed with-out discomfort or difficulty and without previous local anesthesia. If the improved condition persisted after treat-ments were stoppet for six months, and then for one year, all treatments were dicontinued. The strictures were not considered "curet" until after the lapse of several years in which no therapy was required and there was no evidence of a stricture.
There were favourable responses in 45 cases. 23 cases remained free of urethral strictures without requiring additional acutalyn or other therapy during a long period of follow-up ranging from one to four years. The stricture returned in eight cases after periods of several months to three years. Four cases dap/~

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I i ~-~l had only partial or temporary improvement; they required 21 additional acutalyn therapy. Three cases could not be evaluated.
31 Acutalyn therapy varied from one injection to as many 41 as 53 injections over a period of several years. There were no

5 ¦ adverse effects.
61 Example 8.
7¦ Acutalyn crude has been used effectively in treating 81 chronic and severe hypertrophic, pruritic and painful burn scars.
91 In this example, an adult human male was suffering from deep 10¦ dermal and third degree burns of his right upper arm and elbow.
The patient had been unable to rest and sleep for months because -12¦ of the severe itching on the burn scars. The patient was given - ~ -~3¦ acutalyn crude (50 mg. %) twice weekly by injection either ~41 intravenously or intramuscularly for a total of 11 injections.
15 ¦ The itching subsided and the patient w~s able to rest after the 176~ first injection. The remission was complete.

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:-~" 10666Z0 Example 9.
In this example a young man, age 23, was suffering with an unsightly and pruritic keloid of the left ear. It --covered one-third of the posterior surface of the ear. It was recurrent, beginning 16 years earlier following otoplasty.
It was reslstant to all therapy. Within one to 3 months following surgical excision on five occasions the keloid returned to a more severe degree each time. Acutalyn crude in~ections were initiated at 3-day intervals. After the fourth in~ection, there was a relief in symptoms, including relief of pruritis, itchlng, and tenderness, and reduction in size of the keloid, which continued to improve as long as acutalyn therapy was used. Following 10 additional injections at 3-day intervals, the keloids were excised. Seven acutalyn ln~ections were given at 3-day intervals following surgery and again one month later; no other therapy was given. Acutalyn in~ections were resumed five months later and continued at irregular intervsls. The keloid did not return for ten months. When acutalyn crude was discontinued, kelo~d growth returned, though not as large as previously. The itching and tenderness disappeared completely and keloid flattened out to normal skin condltion. Three years later, the condition remained free of keloid recurrence.
The following four examples relate to the treatment of arthritis and relatet disorders. In the great ma~ority of patients there was a decrease in disability, pain, swelling, cessation of muscular spasm, disappearance of nodules, improve-ment in clinical chemistries, and a return of function in severe disabling cases, as well as in acute cases. Acutalyn therapy enabled patients on long-term corticosteroid, dap~

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-~-'" 1066620 phenylbuta~one, and gold therapy suffering severe side effects to be withdrawn gradually from the toxic therapy with continu-ing improvement of their arthritic symptoms. 50 mg. %
acutalyn crude was used. ~ -Example 10.
This example illustrates how use of acutalyn crude enables chronic arthritis patients suffering severe side effects from long-term corticosteroid therapy to be withdrawn from cortisone therapy without ill-effects, and with dramatic improYement in arthritic symptoms and disappearance of hypercortisonism symptoms. The patient in this example waæ
a 53-year old female with a diagnosis of chronic rheumatoid arthritis, Stage III structural changes, Class III functional impairment, peripheral ~oint involvement, with chronic, advanced, exogenous hypercortisonism, Cushing's syndrome, seven years duration, resulting from long-term corticosteroid therapy. Patient was an opera singer who was unable to contlnue her operatic career or to practice singing because ,~ ;
of these problems. Previous tberapy included prednisolone plus sallcylates.
Examination revealed typical fusiform swelling of -flngers and inablllty to grasp ob~ects; definite swelling of both elbows with limitation of extension; limitation of rotation, flexion, and lateral tilt of neck. Inability to elevate arms fully because of shoulder involvement; both knees swollen, with limitation of flexion. Examinatian also ~ -revealed very typical Cushing's syndrome, with moon face, buffalo hump, depression, considerable hirsutism, and other metabolic problems.

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. : , . , Injections of acutalyn crude, 50 mg. %, i.v. were given twice weekly for 9 weeks and the patient was tapered off cortico-steroids gradually over an eight-day period. Following the third in~ection, patient noted more flexibility of all ~oints and ability to clench fists. Following seventh injec-tion, Cushing's syndrome was gone and skin texture was soft and appeared much more youthful. Following ninth in~ection, the patient was able to ambulate , turn head easily, and had full extension of both arms as well as ability to raise arms above head. Her appetite was much improved. Six weeks after the first in~ection, the patient was able to resume singing practice. During the period of steroid withdrawal patlent had superficlal infections and was given antibiotics. Final examination at the end of the ninth week revealed complete restoration of all ~oint mobility, rheumatic involvement completely disappeared; freedom from all pain; complete dis-appearance of classical Cushing's syndrome and chronic exogenous hypercortisonism; feeling of well-being, resumption of operatic career and normal household duties; full and complete reversal to normalcy. There were no toxic reactions or hematological abnormalities. During the course of treatment, patient lost 17-1/2 lbs. Final laboratory findings: Hb.
13.8 gms.; erythrocytes 4,510,000 per cu. mm.; leukocytes 9,500 per cu.mm.; eosionophiles 2%; normal differential; corrected setimentation rate 22 mm./hr.
Example 11.
The patient in this case was a female, age 39, with a diagnosis of Osteoarthriti6, 6tage II structural change~, class II functional impairment, three years duration. Previous therapy included in~ections of hydrocortisone into shoulder dap/~
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Followlng the seventh ln~ection, the patient was asymptomatic and there were no terminal phalangeal swellings or nodes.
Example 12.
The patient in this example was a male, age 59, with -a diagnosis of chronic gouty arthritis, severe, two years duration. Patient had not been able to work at his ~ob for the past year (foreman of large construction firm). Previous therapy included steroids for one year without relief of symptoms. Prlor to acutalyn therapy, both feet were bulging with shoes loosely laced; patient unable to tie shoe laces, etc., because of large noduiar deformed fingers and wrists;
unable to write name or clench fists; both knee ~oints badly swollen with much crepitus and ~oint mica; unable to elevate arms above head due to pain in shouders; facial expression that of a chronically ill person suffering from severe pain.
Laboratory blood chemistries: Serum Uric Acid 6.2 mgm. X, Sedimentation rate 38 mm./hr.

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^-` 10666Z0 A total of four 5.0 cc. injections of acutalyn crude, 50 mg. %; i.v., were given over an 18-day period. Following the first injection, swelling in both feet and pain completely disappeared overnight; the patient's shoes were loose and he could move about with ease, extend his arms over his head, and write with a pen. After each of the next three in~ections.
the patient showed marked improvement. He could move about easily, write, clench his fists, and all nodules were dis-appearing gradually. Following the last injection, the patient was able to resume his occupation as construction foreman.

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Example 13.
The patient was a female, age 35, with a diagnosis of recurrent, severe, calcific subdeltoid bursitis, 3 years duration, complete disability. Previous therapy included -numerous intrabursal injections of hydrocortisone with slight relief followed by aggravation of symptoms~5 also salicylates, -and other conventional therapy. Examination revealed calcific ~ -subdeltoid bursitis, left shoulder, with complete restriction of motion of arm; arm in sling; marked tenderness, swelling, tip of left shoulder; patient unable to sleep because of constant pain day and night; patient sick looking and tired. X-ray of left shoulder revealed l x 2 cc. diameter calcium deposit in soft tissues adjacent to the superior lateral margin head of humerus. Seven 5.0 ml. injections of acutalyn crude, 50 mg.%, were given over a period of ten days. Patient began to respond following the third injection and was able to move arm slightly.
Following the fourth injection, the sling was no longer required and the patient was able to sleep. Following the sixth injection, the patient was asymptomatic, with full range of motion in left arm, all angles, and she reported complete relief from pain and a feeling of well-being.

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Exhibit 14.
The following exa~ple illustrates the utility of acutalyn crude for the treatment of cirrhosis of the liver. The patient was a 56-year old male, in hepatic coma for four days, with a diagnosis of advanced cirrhosis of the liver, resulting from regular intake of alcohol. Prior therapy initiated upon his entry into the hospital nine days earller included transfusions, i.v. fluids, multiple vitamins, mucuhydrin, digitsli~. Patient's condition continued to deteriorate and three days after hospitalization the patient went into hepatic coma. Arglnine i.v.
was added to his conventional therapy starting the fifth day, but his condltion continued to deteriorate, the coma deepened, spasticity and abnormal neurological changes developed, the patient suffered a cerebral vasculsr accident, and on the seventh day, the patient was moribund.
Upon examination, the patient was unconscious, with severe ~aundice, ascites, and edema. The liver was severely enlarged, Babinski positive on rlght, right arm and leg spastic, and there were additional abnormal neurological changes. LaboratDry tests revealed esophageal varices; ECG
borderline abnormal; SGOT 74; cephalin flocc. 4~; serum bilirubin direct 5.4, total 9.8; BSP 41%; Hgb 9.9 gr.; WBC normal; anisocytosis; serum proteln 5.8. Beginning the ninth day when the patient was morlbund, 5.0 ml. in~ections of acutalyn crude, 50 mg. X, were given I.M. daily for six days. The patient began to respond after the first in~ection.
Two days later he was awake, cooperative, hallucinating; bilirubin dropped to 2.6 direct, total 4.4; icterus index dropped to 18. The following day the patient was walking. Acutalyn crude injections, I.M. or I.V., were resumed on the eigthth day and given daily or every two days for six weeks. The patient continued to improye, bilirubin dropped to 1.6 direct, total 3.1; thymol turbidity 12, and the patient was released from the hospital.

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Two years later the patient was hospitalized again with severe prostatic obstruction, rising azotemia, impending clinical uremia, extremely poor renal function, elevated blood ammonia 310 mcg., and hepatic coma. Because of hepatic disease, surgical procedure for the prostatic obstruction was not performed. Arginine i.v.
was administered. His condition worsened, and he was threatened with terminal hepatic failure and not expected to live through ~
the night. An injection of acutalyn crude, 50 mg.% was given on -~ -an emergency basis and the patient again recovered from the hepatic coma. Nine additional injections were given and the patient was discharged from the hospital under the care of his -regular physicians.

22.
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: . ~ . .. . ~ :. :

~r -~ 1066620 Example 15 The following example illustrates the utility of acutalyn crude for the treatment of hepatitis in humans. The patient was a male, age 58, with a diagnosis of chronic serum hepatitis. (Patient had trans urethral resection five months earlier and was given two transfusions because of severe hemorrhaging.) All accepted conventional methods of therapy were tried, but the patient continued a downward course. The patient was jaundiced, with bile in urine; the liver was tender and enlarged, three to four finger breadths below right costal margin. Jaundice had been increasing in severity and the patient was weak and tremendously fatigued, very sick, and apprehensive.
Cephalin flocc. was 2+. Patient was given 5.0 ml. injections of acutalyn crude, 50 mg.%, once a week for two weeks, then two injections one month later. At the time of the fourth injection the patient was completely asymptomatic, able to work, and to perform his usual duties. Cephalin flocc. was l+ and bilirubin ;~
reduced to 0.62.

~;, :., ~'' "' ' 23.
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It is therefore apparent that the drug of this invention has great utility for a wide variety of disease conditions. The method of treating these and other conditions with acutalyn -.
crude accomplishes these purposes. The precise composition of acutalyn crude is not known, but its biological activity apparently is due in part to the superoxide dismutuse activity of the protein mixture.

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a4.

Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An anti-inflammatory composition comprising a biologically effective amount of acutalyn crude, an inflammation inhibiting amount of a corticosteroid and a pharmaceutically acceptable excepient.

2. The composition according to claim 1 wherein the corticosteroid is hydrocortisone.