US3637641A - Method of treating post-traumatic arthritis - Google Patents

Abstract

THE PAIN AND STRUCTURAL ABNORMALITIES ASSOCIATED WITH POST-TRAUMATIC ARTHRITIS ARE ALLEVIATED BY SYSTEMIC ADMINISTRATION OF ORGOTEIN TO THE PATIENT AFFECTED WITH THE ARTHRITIC CONDITION.

Description

United States Patent Int. Cl. A611; 27/00 US. Cl. 424-177 9 Claims ABSTRACT OF THE DISCLOSURE The pain and structural abnormalities associated with post-traumatic arthritis are alleviated by systemic administration of orgotein to the patient affected with the arthritic condition.

This is a continuation-in-part ofv application S.N. 576,454, filed Aug. 31, 1966, now abandoned, which is a continuation-in-part of application S.N. 494,048, filed Oct. 8, 1965, now abandoned.

BACKGROUND OF INVENTION This invention relates to a method of alleviating the pain and structural abnormalities associated with posttraumatic arthritis, more particularly by the systemic administration of orgotein to a patient suffering from such an ailment.

Painful and crippling modifications of the bony and related ligamentous structure as a result of severe or repeated localized trauma is common in mammals. Such modifications are assigned to variety of names by the medical and veterinary professions, depending on the manner in which the alteration manifests itself and the species of the patient. They can be classified generally as hypertrophic, ostephytic, ligamentous, synovial, membranous, chondro asteitis and/or osteoporosis.

In humans, trauma-related hypertrophic arthritis of osteoarthritis is common. Other examples are chronic bursitis, including olecranon, prepatellar, subdeltoid, radiohumeral, retrocalcaneal, iliopectineal, ischial, infrapatellar, trochanteric and bunion bursitis. Ligamentous sprain also can result in localized osteoarthritic condition.

The American Rheumatism Association, in its classifi cation of arthritis and rheumatism, lists degenerative joint disease (osteoarthritis) and traumatic arthritis (viz, the result of a direct trauma) as types of arthritic disorders of a traumatic origin exhibited by the human body.

Osteoarthritis is a form of hypertrophic arthritis common in humans. This disease condition results in the formation of new bone called asteophytes at the joint surfaces. The condition of osteoarthritis represents the accretion of a series of cumulative insults to the articular tissues.

A common manifestation of osteoarthritis is known as a Hebreden Node, a cartilageinous and bony enlargement of terminal interphalangel joint of the finger. When caused by trauma, the nodes appear shortly after the injury. The immediate epidermal area becomes red, soft swelling sets in and the joint is painful. After a period of several months, the acute swelling subsides and the inflammatory changes are replaced by a hard, sometimes painless enlargement. X-ray studies show an enlargement of the ends of the bones with distortion of joint space and irregularity of of the joint surface. Other joints commonly aliected are the knee, cervical, thoracic and lumbar spine, hip and rib joints.

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Specific arthritic lesions secondary to acute trauma in humans include sprains, bursitis, traumatic synovitis, and hermoarthrosis. Common names for these types of joint or bursal conditions in humans include weavers bottom, policemens heel, pitchers glass arm, housemaids knee, tennis elbow, golfers arm, etc. Shoulder, elbow and wrist affections are frequent in workers using compressed air tools, the repeated vibration eventually causing asplitting the destruction of cartilage.

In animals, especially horses, post-traumatic arthritis, generally known as bony exostosis, is common, especially in race horses and polo ponies. Bony exostosis is an abnormal bone condition in horses generally involving the first, second and third phalanges, as well as the sesamoid bone, cannon bone and carpal joints. In its various clinical manifestions, it is known as asteoarthritis of the carpal joints, splits, osselets, sesamoiditis, ringbone, sidebone and navicular disease.

Osteoarthritis of the carpal joints is hypertrophic arthritis or osteophytic growths of the carpal joints. Splints are osteophytic growths of the splintbones, which are below the carpal joint. .Osselets are osteophytic growths of the fetlock joint, which is between the cannon bone and the first phalanx. Sesamoiditis are osteophytic growths of the proximal sesamoid bones. Ringbone is an osteophytic growth on the anterior or lateral surface of the second phalanx in a circular or ring-like projection. Sidebones are calcification of the bursal and the lateral cartilages, lateral to the third phalanx or coffin bone. Navicular disease begins as a bursitis of the navicular bursa between the deep flexor tendon and the navicular bone. As the disease progresses, osteoporotic changes may occur in the body of the distal sesamoid or navicular bone and spur formations or osteophytic lesions may also appear. Bony exostosis may be the result of several factors, including hereditary predisposition, faulty nutrition and conformation, improper shoeing and traumatism. Physical trauma is the most common cause of bony exostoses. Initial symptoms include lameness and difficulty in locomotion followed by enlargements around the affected joint. In advanced cases, the most dependable method of diagnosis is radiography.

Some of the above bony structural abnormalities are areas of osteoclastic activity.

A four point method for the diagnosis of traumatic arthristis is (a) the trauma suffered must be severe enough to produce an acute inflamation or synovitis as indicated by pain and swelling of the joint, (b) the injured joint is the most severely inflamed, (c) before the injury, joints function was normal, and (d) a progress of articular changes demonstrable radiographically in three to six months.

Trauma to the joint produces an inflammation of the synovial membrane. Hemorrhagic effusion occurs shortly thereafter. Acute bone atrophy, known as painful osteoporosis or causalgia, occurs after traumas to the involved joint. It is characterized by hyperemia in bone, increased vascularity, thinning of bone lamellae and filling of the interl-amellar spaces with fibrous tissue. X-ray studies show a patchy atrophy of bone after approximately four to six weeks. Moderate doses of oral steroids is the preferred therapy if initiated shortly after the onset of the disease. In severe cases, sympathetic ganglion blocks have been used with success. However, ganglionic blocking agents have the disadvantage of allowing progressive damaeg to occur because of the anesthesia produced.

Inflammation of the tendon and its sheath may result from either acute or repeated trauma. This tendinitis may be calcific or non-calcific. Such calcifications have been found in tendons of the elbow, hip and knee. Bursitis, the inflammation of one or more of the bursae, is generally attributed to trauma but there is now increasing evidence that it also may be due to a viral infection alone or in combination with trauma. Current therapy includes cortisone injections and rest for the affected areas.

Conventional methods of treating post-traumatic arthritis usually include both supportive and chemotherapy. Supportive treatment generally consists of partial or complete rest for the patient followed by whirlpool treatment, cold and astringent applications. Sclerosing agents, usually in conjunction with analgesics, saline irrigation, manipulation and surgery to remove the affected bursa, spur or bony prominence may be employed. Counter-irritants, such as cantharides, are sometimes applied to the affected joint. In animals, if lameness persists, a neurectomy may be performed as a last resort as a usually futile effort to prolong the working life of the animal.

Chemoetherapy often treats traumatic arthritis as if it were an acute inflammation condition, although inflammation generally precedes the condition rather than being associated therewith. Adrenocortical steroids such as cortisone, prednisone, triamcinolone, betamethasone and dexamethasone, when effective, like analgesics, ordinarily produce only interim symptomatic relief. Phenylbutazone, oxyphenbutazone and indomethacin have also been used with only moderate success. The patient often becomes refractory to continued administration of such anti-inflammatory agents. Heretofore, no chemical method of treatment has consistently produced lasting relief from th symtoms of serious traumatic arthritis such as bony exostosis in horses.

If all other treatment fails, it is internationally accepted to fire horses with a hot iron for osteoarthritis of the carpal joints, splints, osselets, sesamoiditis, ringbone, sidebone and navicular disease.

Another method of causing marked hyperemia is with a combination of vegetable oil and iodine injected subcutaneously at intervals about 1 cm. apart.

Following firing or blistering, the entire area is painted with an irritant solution of various kinds and wrapped in bandages.

The result is a severe third degree burn, with marked swelling, inflammation, necrosis, exudation and the abcess heals ultimately with eschar and fibrous tissue with some definite clinical improvement achieved in some cases. The physiologic nature of the procedure is not understood, but there seems to be an international acceptance of it.

Prior methods of therapy for osteoarthritis include resting the affected area because the joints cannot take stress and strain. Excessive exercise results in additional trauma and damage to the involved joints. Notwithstanding the armamentarium of drugs available, no drug is consistently successful in retarding or overcoming the pathological changes of the degenerative joint disease. See Arthritis and Allied Conditions, I. L. Hollander, Lea & Febiger, Philadelphia, Pa. (1966), page 888.

SUMMARY OF THE INVENTION In accordance with this invention, orgotein is administered to a subject (mammal) suffering from posttraumatic arthritis in an amount effective to relieve at least some of the symptoms of the condition.

The clinical use of orgotein is uniquely not painful, even upon repeated administration, does not require such a long period of convalescence, shows a clinical improvement of lameness and manifests smoothing and rounding of osteophytic exostoses sooner and more markedly than is seen after firing and blistering therapy. In many cases there is a resolution of the osteophytic exostoses, demonstrated by a smoothing and rounding of the exostoses within as short a time as 4 weeks from initiation of orgotein therapy given intramuscularly away from the site.

OBJECTS OF THE INVENTION It is an object of this invention to provide a method for treating post-traumatic arthritis.

4 It is another object to provide such a method which relieves the pain usually associated with post-traumatic arthritis and, with continued treatment, at least some of the structural abnormalities associated with the condition. Other objects will be apparent to those skilled in the art to which this invention pertains.

DETAILED DESCRIPTION OF INVENTION The therapeutic agent employed in the method of this invention is orgotein. Orgotein is the non-propriefary name adopted by the United States Adopted Names for an isolated, pure, water-soluble, fairly low molecular weight anti-inflammatory protein metal chelate 'chelated with a divalent metal. (See I.A.M.A., May 26, 1969, volume 208, .No. 8.) In prior filed US. patent application Ser. No. 576,454, filed Aug. 31, 1966, Belgium Pat. 687,828 and British Pat. 1,160,151, there is described a process for the production and isolation of orgotein. An improvement on that process is claimed in US. patent application Ser. No. 657,866, filed Aug. 2, 1967. Stabilizing orgotein with saccharides, preferably sucrose, is the subject of the prior filed US. patent application Ser. No. 657,971, filed Aug. 2, 1967. A method of isolating orgotein completely free from proteinaceous impurities is the subject of the prior filed US. patent application of Wolfgang Huber, Ser. No. 815,175, filed Apr. 10, 1969.

DESCRIPTION OF ORGOTEIN Orgotein is readily soluble (to 50 mg./ml.) in water and common aqueous buffers, pH 6.510.0. The proteins metal ion content and composition are related to its pharmacodynamic activity. Bivalent cations with ionic radii of 0.6-1.0 A are the most effective. A mixture of Cu, Mg, and Zn at a total content of 2-4 gram atoms per mole produces the highest level of physiological activity. The relative amounts of each of the three metals can vary broadly within this total. Most samples also contain trace amounts of Ca, Fe, and Si as the only other metals detectable by emission spectroscopy. All metals can be removed by prolonged dialysis against 10" M EDTA or 10 M orthophenanthroline. At levels below 2 gram atom per mole, the biological effectiveness is diminished, and below 1 gram atom per mole the protein progressively precipitates, losing biological activity. The metal ions thus appear to play a decisive role in maintaining the molecular conformation essential for biological activity, acting as locking pins by producing intra-molecular cross links. Differencesin affinity for buffer anions probably explain why, at identical strength, the new protein is less stable in certain buffers (phosphate, borate) than in others (Tris, maleate, EMTA).

Amino acid composition and physical properties of orgotein isolated from beef liver are listed below. The amino acid composition of congeners isolated from other species is remarkably similar. The data for hard-to-exchange amide hydrogen, the frictional ratio f/f the Sheraga-Mandelkern coefiicient B, and the flat slope of the intrinsic viscosity curve, indicate that the new protein in solution is quite compact. Determination of exposed tyrosyl residues with N-acetyl imidazole showed that only one is titratable. Titration of e-amino groups using 2,4,6-trinitrobenzene sulfonic acid demonstrates the 15-16 e-fll'IliIlO groups are titratable. A single crysteine is exposed in the native molecule as judged by spectrophotometric titration with p-mercuri-benzoate. Digestion with hot orcinol reagent reveals a small amount of carbohydrate. Y

The protein moves in a single, sharp band in the ultracentrifuge, both in free and in sucrose density sedimentation, and emerges as a single, sharp peak from a calibrated gel-filtration column (Sephadex G-200). In gel electrophoresis, the protein exhibits a reproducible, multiband pattern moving anodicailyfrom the origin. Under constant conditions of gel electrophoresis, this pattern is readily and quantitatively reproducible from batch to batch and has served as a valuable tool in the isolation of the protein.

Tests in 30 different enzyme systems, utilizing a broad range of substrates, have failed to reveal any significant activity when the protein was used in lieu of the enzyme in the respective assay system. Included were several each of the oxidoreductases, transferases, hydrolases, lipases and isomerases. Only in the cases of catalyase, peroxidase and snake venom phosphodiesterase were traces, i.e., less than 2% of that of the respective known enzymes run in parallel, of enzymatic activity observed.

The new protein is only weakly antigenic. An extensive immunization regime is required to produce antibodies in the rabbit reliably. Allergenicity tests in guinea pigs, using the classic Landsteiner technique with ten intradermal injections spread over 22 days, followed by two weeks of rest and then a challenge injection, revealed no systemic reaction or other indication of sensitization. Immunoelectrophoretic examination of human sera with fluorescintagged protein failed to reveal the presence of precipitating or non-precipitating antibodies. A similar lack of sensitation was observed in two thoroughbred horses that had received in excess of 100 mg. each of the protein at varying intervals over a period of 12-18 months. At autopsy, the gamma globulin fraction of the horse serum was isolated and purified. It, too, was found to be devoid of antibodies when tested by immunoelectrophoresis.

The new protein is a remarkably effective anti-inflammatory agent. It has been evaluated in several animal models of inflammation viz, carrageenin-induced rat paw edema, cotton pellet-induced rat granuloma, adjuvant-induced rat polyarthritis, and antigen-induced guinea pig skin edema. Within effective dose ranges it exhibits potent anti-inflammatory effects in the antigen-induced guinea pig skin edema and in the cotton pellet-induced rat granuloma test, particularly with adrenalectomized rats. It is moderately effective (about 30% inhibition at 2.5 mg./ kg. using purebred Wistar-derived rats) in the carrageenin-induced rat polyarthritis, when given subcutaneously at 0.2 mg./kg., five days a week for 21 days, the new protein effectively protected the animals against the secondary stages of adjnvant arthritis, which are believed to be immunological in character.

PROPERTIES OF ORGOTEIN Elemental analysis (percent): C, 50.05; H, 7.92; O, 25.15; N, 16.00; S, 1.1; and P, NIL.

AMINO ACID COMPOSITION Ala 20 His 13 Pro 12 Arg 9 Ile 16 Bar 17 Asp 34 Leu 19 Thr 24 Cys/2 8 Lys 21 Try 1 Glu 24 Met 4 Tyr 2 Gly 51 Phe 9 Val 30 Molecular weight:

Gel filtration-34,000i750 Sucrose density gradient-34,000:l000

Osmometry--3 5,3 1000 Amino acid composition-35,000 Sedimentation coefficient:

s. w. sec. (0.85% saline)--3.32 10 Isoelectric point:

Citrate-phosphate bufier5.5i0.2 Isoionic point- .3 5 1:0.05 Absorbence, A l% (glycine buffer, pH 8.5 )2.3i0.2 Intrinsic viscosity:

Partial specific volume:

'y ml./g., observed0.7219

'y ml./g., calculated-0.7224 Frictional ratio:

Wi -1.174; 1.114 Scheraga-Mandelkern coefficient ,8 222x- Hard-to-exchange amide hydrogen-430% Amino acid composition of orgotein congeners AMINO ACID COMPOSITION OF ORGOTEIN CONGENERS FROM VARIOUS SOURCES Red Blood Cells- Liver,

eef Beef Sheep Horse Rabbit 16.4 16.3 17.2 15.9 17.1 4.0 5. 0 4. 5 6. 2 5. 3 5. 2 5. 7 5. 8 4. 7 4. 9 6. 0 5.4 5. 5 5. 9 6.9 6. 6 6. 6 8.1 8. 8 6. 7 1. 2 0. 8 0.6 1.9 0.9 2. 8 2. 5 2. 2 2.9 2. 8 4.0 4.1 4. 8 3. 7 4. 2 5.4 5.3 4. 7 4. 8 5. 7 7. 8 7. 7 6. 2 5. 2 6.6 0.3 0. 2 0.3 0.3 Nil 0.6 0.7 0.6 0.3 0. 2 9. 7 10.0 9.1 89 S). 8

ISOLATION OF ORGOTEIN The process of S.N. 574,454 is a multi-step process which, in its preferred form, isolates orgotein from beef liver by the successive steps of (a) slurrying finely ground fresh beef liver in ice cold water or buffer containing Mn++ ion until substantially all the water soluble proteins are extracted; (b) fractionally precipitating a substantial portion of the dissolved proteins in the cold solution with acetone; (0) dissolving the precipitated portion in cold maleate buifer containing Mn++.,ions; (d) heating the resulting solution at or close to 60 C. for about 20 minutes; (e) cooling the solution and removing the precipitated denatured proteins; (f) precipitating the residual dissolved proteins in the cold solution with ethanol; (g) dissolving the precipitated proteins in cold maleate buffer solution containing Mn++ ions; (h) dialyzing and lyophilizing the buffer solution; (i) dissolving the lyophilized solids in cold tris buifer containing Mg++ ions and fractionally precipitating the proteins from the cold solution With ammonium sulfate; (j) dissolving the fractions insoluble in ammonium sulfate solution at 60 and 75 percent of saturation in cold tris buffer containing Mg++ ions and chromatographing the solution through a column of Sephadex G-lOO dextran resin; and (k) dialyzing against water containing a small amount of Mg++ ions and thereafter 10* M o-phenanthroline to first remove buffer and then excess Mg++ ions.

In the process of my prior filed application S.N. 567,866, filed Aug. 2, 1967, there is disclosed an improved process which uses buffer containing a mixture of Mg, Cu++ and Zn++ ions rather than Mg++ alone. The orgotein isolated by this process contains proportionately less Mg as a chelating metal and correspondingly greater amounts of Cu++ and Zn++.

The orgotein used in this invention is preferably stabilized by lyophilization of a solution of a mixture thereof with sucrose in a proportion such that the orgotein content of the mixture is from 0.01 to 50 preferably 0.01 to 5.0 and more preferably 0.1 to 25.0% as described in application S.N. 657,791. Stabilized sterile lyophilized orgotein is storable for months at room temperature without detectable denaturation of the protein. Aqueous solutions thereof are stable for several weeks at room temperature whereas a solution of non-stabilized protein is rapidly denatured under these conditions.

The orgotein can be in admixture with one or more conventional pharmaceutical carriers or excipients, suitable for intravenous, subcutaneous or intramuscular injections,

usually the latter, e.g., as an aqueous solution, using excipients and carriers conventional for this mode of administration. Usually a non-oily aqueous isotonic solution is preferred for intramuscular injection.

As used herein, the term pharmaceutical carrier denotes a solid or liquid devoid of significant activity in treating post-traumatic arthritic conditions and is composed of a single substance or a number of substances which may be solids, liquids or a combination of solids and liquids, each of which is substantially non-toxic in the amount used in the composition, as measured in the same host animal using the same method of administration, vehicle, etc. Orgotein compositions can, if desired, also contain or be administered in conjunction with other active substances, e.g., anti-inflammatory agents such as, for example, prednisone, prednisolone, cortisone, hydrocortisone and the corresponding A -90c fluoro 16-hydroxy, -16u-methyl and 16,6-methyl substituted steroids, and non-steroids, e.g., acetyl salicylic acid, salieylamide, aminopyrine, chloroquine, hydroxychloriquine, phenylbutazone and indomethacin.

Orgotein is usually administered in multiple successive dosages, spaced as frequently as 6-12 hours apart and as long as six weeks apart. Usually daily doses are administered until symptomatic relief, e.g., pain and stiffness, is obtained from the arthritic condition. Thereafter, doses spaced about 7 days apart are administered until reversal of structural abnormalities occurs. Treatment can be continued over a period of several weeks or months.

The therapeutic amount of orgotein administered is dependent upon several factors, including the species of animal being treated, the condition of the patient prior to orgotein therapy, the degree of progression of the structural abnormalities, and the particular manifestations of post-traumatic arthritis from which the patient is suffering. For horses with osteoarthritis, mg. doses 3 times a week for two weeks, then twice a week for two weeks and more thereafter at weekly intervals or bi-weekly intervals as indicated. For dogs with disc syndrome, 2 mg. daily for 3 to 5 days and later as indicated. For dogs with osteoarthritis: 2 mg. 3 times weekly for two weeks, then 2 times weekly for 2 weeks or as indicated. For humans with osteoarthritis: 1-4 mg, preferably about 2 mg. daily for three days, followed by three times a week for a week or two and maintenance of one or two a week, depending on symptoms. The number of successively spaced doses of orgotein necessary in order to alleviate at least some of the structural abnormalities associated with a post-traumatic or arthritic condition will vary depending on the status of the condition and the above-described factors. In some cases, clinical relief is obtained in as short a time as six hours, or it may take a few days. Chronic conditions require longer periods of therapy of from several weeks up to several months. Acute conditions may respond within six to twelve hours.

Because relief from pain often precedes the more dramatic reversal of any structural deformity associated with the post-traumatic arthritic condition, care must be taken not to terminate orgotein therapy before such reversal is observed radiologically.

Generally, the orgotein can be administered at any convenient site of the body and need not be injected directly into the localized arthritic joint or area.

TREATMENT OF BONY EXOSTOSIS IN HORSES The method of treating bony exostosis according to this invention comprises the administration of successive doses of an effective amount of orgotein, until at least some of thesymptoms of the ailment are relieved. The customary method of administration is by injection, the preferredroute being intramuscular. The frequency of administration is determined by the severity of the condition and can be as often as once or twice a day. The duration of treatment can be from a few days to several weeks or months.

Clinical response to the treatment often is observed shortly after initiation of therapy, usually from 2 to 10 days. Treatment is continued until either recovery has been made or the degree in improvement of the animal has reached a plateau. In younger horses treatment usually can be completed in 2 to 3 weeks in most cases. The orgotein generally is administered 5 mg. 3 times weekly X2, then two times weekly X2, or as indicated.

The amount of orgotein administered in each dose is not critical, e.g., from 0.5 mg. to 20 mg. given from 2-5 times a week. However, the preferred dosage range is from 2.5 to 5 mg. Upon demonstration of clinical response to the treatment, which is usually within 2 to 10 days, the dosage may be decreased, either by reducing the amount of orgotein per injection or the frequency of injections.

Gross observation of osteophytic resolution in the animals affiicated with bony exostosis is difficult to demonstrate clinically. For this reason X-ray diagnosis is advisable in order to monitor the progression of therapy. Within 4 Weeks, X-ray studies usually show abnormal bone formation reduced in size, smoother and more rounded than before treatment. Osteophytes around the joints are arrested, and a rounding off of the existing deposits can be observed. In some cases, the normal progression of the disease and subsequent development of osteophytic changes do not occur.

Clinical treatment was conducted using animals suffering from bony exostosis that had previously been treated extensively with a variety of other drugs. In each instance, prior therapy had not enabled the animals to return to their former usefulness. Each horse was clinically diagnosed to have bony exostosis, which diagnosis included local anesthesia to isolate the involved area and X-rays of the involved area for comparison after conclusion of therapy. In the following examples, orgotein was the only drug administered and the only treatment given to the horse during the observation of the animal.

PREPARATION l.-ORGOTEIN All operations, unless otherwise indicated, are carried out in a cold room (2-5 C.).

Fresh beef liver is ground into a suitable plastic container. Cold distilled water (two liters per kg. of liver) is added with stirring and the mixture is adjusted with 0.1 N sodium hydroxide to pH 7.5 to 7.6. Sufficient 2 N manganese sulfate is added to bring the molarity of the mixture to 0.05. The pH is adjusted to 7.6 and fresh cold water is added to bring the water to three liters per kg. of liver. Thereafter, 50 ml. of toluene per kg. of liver are added and the mixture is stirred in the cold room overnight.

The next morning the suspension is passed through plastic gauze and to the filtrate 1.5 volume of cold acetone (10 C.) is added with gentle stirring. The acetone is added rapidly through a glass tube extending well below the surface of the mixture. The ensuing precipitate is immediately collected by centrifuging and then right away suspended with about 25 percent (v./v.) of 0.05 M maleate-Mn++ buffer, calculated upon the volume of' the filtrate before addition of the acetone. The mixture is stirred in the cold room for several hours, passed through plastic gauze and clarified by centrifuging. I

The supernatant is heated rapidly to about 60 C. With stirring in a stainless steel or glass lined kettle and maintained at or close to 60 C. for about 20 minutes. Thereafter, the mixture is cooled to 5 C. as rapidly as possible and the bulky precipitate is separated by centrifugation or filtration by slow suction over a broad filter surface. The clear filtrate or centrifugate is brought to 2-5 C. and 0.9 volume of denatured ethanol (-10 C.) are added rapidly from a funnel through a glass tube extending well below the surface of the mixture. Effective stirring is essential and the temperature must remain at 2 C. or lower.

After the addition of the alcohol has been completed, the mixture is kept in the cold room just long enough to permit the precipitate to compact and to settle. The precipitate is recovered by centrifugation or filtration at low vacuum and immediately dissolved in cold 0.001 M maleate-Mn++ buffer, pH 7.0. The amount of buffer is approximately 4 v./wt. The solution is clarified by centrifuging, the supernatant decanted, the precipitate re-extracted using small amounts of cold buffer, the supernatants combined and lyophilized. Prior dialysis to remove buffer ions while possible, is not necessary at this point. The resultant powder is stable for several months at room temperature but preferentially is kept in the cold room. It represents a mixture of the desired protein, arginase and other enzymes, albumin and other non-essential proteins.

This powder is dissolved in about twelve times the volume of cold 0.2 M tris-0.001 M Mg++ buffer, pH 7.8. This solution is treated with cold saturated ammonium sulfate solution, 0.001 M in Mg++. Five increments of 375 ml. each are added per 1,000 ml. of buffer solution. The respective states of saturation achieved by this technique are 15 percent, 30 percent, 45 percent, 60 percent and 75 percent. In each instance the addition of the ammonium sulfate solution is carried out slowly at -5 C. with stirring. Stirring is continued for another 30 minutes and the resulting precipitate is collected by centrifuging at 4500 r.p.m. for thirty minutes at 0 C.

Of the five precipitates obtained, the first one (A) is discarded. It represents high-molecular-weight protein impurities. The second and third precipitates (B and C) are combined. They represent arginase and other enzymes which can be processed separately for the isolation of these products. The fourth and fifth (D and E) are also combined. They contain the desired protein, in a still crude state, contaminated with albumin and various other proteins both of lower and higher molecular weight. The final supernatant is discarded. It contains lower molecular proteins and other undesirable impurities.

Precipitates D and E are dissolved in 0.03 M tris-glycine 0.001 M Mg++ buffer, pH 7.8 at a concentration as close to percent (w./v.) as possible and dialyzed against cold buffer until negative to sulfate ion. The dialyzed solution is clarified by centrifuging and the supernatant is passed through a Millipore filter. The filtrate is applied directly to the head of chromatography columns (3 X 18 inches) filled with Sephadex G-100 (epichlorohydrin cross-linked dextran resin; Pharmacia, Sweden). The Sephadex has been swelled, refined and washed by standard techniques described in literature of the manufacturer. The packed columns are equilibrated with 0.03 M tris-glycine 0.001 Mg++ buffer, pH 7.8 and adjusted to a flow rate of about ml. per hour.

After application to the column, the sample is permitted to equilibrate within the first few cm. of the resin bed for approximately -45 minutes when fractionation is started. Individual fractions of up to 10 ml. are collected. The emergence of peaks is determined by measuring the protein concentration by the absorbence at 280 millimicron.

Two and sometimes three peaks emerge from the column prior to the emergence of the desired protein. They represent albumin and other undesirable protein impurities of similar or larger molecular weight. Fractions representing these peaks are discarded. The desired protein generally emerges in the range of 100-150 ml. of total eluate. These fractions from the columns are combined for further processing. Residual, lower molecular weight protein impurities emerge from the column on further elution, particularly on increasing the ionic strength of the buffer. They are removed to clear the column for a subsequent run.

The combined fractions containing the desired protein are dialyzed against deionized H O-0.001 M Mg++ until they contain less than 10- M tris buffer. Thereafter, dialysis is continued against deionized water containing 1.5 10 M ortho-phenanthroline or ethylenediamine tetraacetic acid salts until the concentration of Mg++ has been reduced to less than 10 M. If a protein chelate is desired whose predominant metal is other than magnesium, e.g., calcium, copper, iron or zinc, expose the protein after dialysis for about a day to a soluble salt of the metal of choice of a molarity which maintains the protein in solution, and then remove excess metal ion in the manner described above. The resultant solution is clarified by centrifuging.

75 kg. of fresh beef liver (22.5 kg. dry weight) yields about 200 grams (1 percent) of the Mn++ protein chelate intermediate from which 12.5-17.5 grams (0.06-0.08 percent) of the desired protein is obtained from the combined D and E ammonium sulfate fractions. On Sephadex Chromatography, these amounts of D and E fractions yield 2.4 to 2.9 grams of the desired protein, equivalent to an over-all yield of 0.011 to 0.014 percent calculated on the dry weight of the liver.

PREPARATION 2.-ISOLATION OF ORGOTEIN FROM BEEF BLOOD Fresh beef blood was centrifuged at 2,600XG for 10 minutes at 0 C. and the plasma decanted. The red cells were then washed repeatedly with 2 to 3 volumes of 0.9 percent saline solution. The washed red cells were hemolyzed by mixing with 1.1 volumes of cold deionized water containing 0.02 percent detergent (Saponin). After a minimum of 30 minutes at 4 C., 025 volume (per volume of hemolysate) of ethyl alcohol at -15 C. was slowly added with stirring followed by 0.31 volume (per volume of hemolysate) of chloroform, also at 15 C. Stirring was continued for about 15 minutes at 5 C. or below, at which time the mixture was a thick paste. The hemoglobin precipitation was carried out in a cold bath which was kept at below 10 'C. After the paste had stood for a further 15 minutes at 4 C., 0.2 volume of cold 0.15 M NaCl solution was added, giving an easily poured suspension. The precipitate and excess chloroform were removed by centrifuging at 20,000XG at about 10 C. for 10 minutes. The supernatant liquid was filtered and dialyzed against cold-deionized water. The dialyzed solution was lyophilized.

The alcohol-chloroform precipitate was re-extracted with deionized water by blending the precipitate and water in a blender and centrifuging. Usually, a volume of water equal to that of the volume of starting red blood cell is needed. The re-extraction solution was dialyzed and lyophilized. Re-extraction of the precipitated hemoglobin often yields 30-50 percent of protein mixture present in the original supernatant. An additional re-extraction may give an additional 10-15 percent.

The lyophilized material was redissolved in 0.025 M tris-glycine buffer containing 0.001 M Mn++ at pH 7.5 (usually to a concentration of 20 mg./ml.). The solution was heated at or close to 65 C. for about 15 minutes. This step removes carbonic anhydrase and other heat labile enzymes from the solution. After heating, the solution was immediately cooled in an ice bath to 5 C. The solution was then centrifuged at 20,000 G at 0 C. for 10 minutes to remove the precipitate. The supernatant was dialyzed against deionized water to remove excess metal ions and buffer and then lyophilized. The resulting solid is rich in orgotein.

(a) Gel filtration Sephadex G-75 is slowly added to warm deionized water (approximately 60 C.) with continuous stirring. The beaker containing the mixture is then placed in a 60 C. water bath for five hours and 45 minutes, removed and allowed to stand for one hour at room temperature. The supernatant and fines are decanted by suction. Buffer is added to the swollen Sephadex gel at four to five times its volume. The Sephadex gel is stirred, allowed to settle, and the fines and supernatant removed by suction. Fresh buffer is again added to the swollen gel, and the above process repeated four times. The final suspension is chilled to 4 C. and then deaerated under reduced pressure before use.

A recirculating column made of polymethacrylate was used. The column is 1050 mm. long and has an internal diameter of 32 mm. In filling the column with degassed buffer, special care is taken to insure that no air bubbles are trapped near the filter and on the sides of the column. The buffer filled column is then moved into the cold room and clamped into a vertical position with the aid of a carpenters level. After equilibration in the cold room, the gel slurry is poured into a funnel connected to the top of the column with continued mechanical stirring. When a layer of Sephadex a few centimeters thick has formed on the bottom of the column, the outlet at the bottom of the column is opened to allow an even flow. During the packing, a rising horizontal surface of gel in the tube indicates proper uniformity in packing. After approximately 95 cm. of gel has settled, the excess gel and buffer are removed. After the top surface of the gel has completely settled, the top of the column is closed with a plunger fitted with a filter disc. Buffer is then circulated through the column for two days in order to stabilize the bed. Flow rate is maintained at ml. per hour. Final bed volume, V=1rI' /Z=(3.14)(1.6 cm.) (96.5 cm.)=775.7

The lyophilizate from the heating step is dissolved in buffer mg./ml.). Insolubles, if present, are removed by centrifugation followed by Millipore filtration. The clear solution is loaded on the column using an LKB selector valve (Model No. 4911B).

All column runs are performed at 4 C. The buffer used is 0.05 m tris-HCl, pH 7.5, 0.15 M in KCl and 0.005 M in glycine, containing 10- M Mg++, 10- Cu++ and 10 Zn++.

The protein solution is loaded from the bottom. Ascending buffer flow rate is maintained at 10 ml. per hour. Protein content of fractions is determined by absorbence at 280 mg.

The elution volume for each protein can be monitored both volumetrically and gravimetrically.

If prior processing has proceeded normally, the first peak which emerges from the column is orgotein. This generally emerges in the range of 300-400 ml. of total eluate. These fractions are combined for further processing. Following the main, well defined peak is sometimes a shoulder which contains orgotein mixed with a small amount of a minor impurity. It can, but need not, be separated by collecting the eluate fractions separately When their protein content drops precipitously from the maximum concentration. Lower molecular weight protein impurities emerge from the column substantially later, upon further elution. They are removed to clear the column for a subsequent run.

(b) Buffer and excess metal ion removal The orgotein solution obtained from the gel filtration is filtered through a column of mixed bed resin Amberlite MB-l Monobed gel-type Ion Exchange Resin (Rohm & Haas), a styrene-divinyl benzene strongly acidic (SO -H strongly basic (-+N(CH CH OHOH-) group-containing mixed copolymer which reduces buffer and unbound Mg++, Cu++ and Zn++ ion concentrations to less than 10- M. Alternatively, this can be done by dialysis.

A column 1.45 x inches is half filled with demineralized water from which all air bubbles have been removed. A slurry of the resin in air-free demineralized water is poured gently into the column and allowed to settle. The column is then back-Washed several times with demineralized water to constant pH (ca. 7.0) and ionic strength (conductance about 1.0 mho.) of the efiluent. The final bed height is 33 inches, giving a bed volume of 58.3 cubic inches (957 milliliters) and total exchange capacity of 440 milliequivalents, based on a factor of 0.46 given by the manufacturer for this resin.

The eluate from the gel filtration step containing the orgotein is concentrated, if necessary, to a protein content of 8-10 percent. This solution is carefully loaded onto the top of the column of ion exchange resin and thereafter developed with demineralized water. The flow rate is adjusted to about 20 milliliters per minute and the appearance of the protein in the eluate is followed by ultraviolet absorption (A The eluate is collected in 25 to 50 milliliter fractions. The desired protein generally appears in the fourth to twelfth fractions. Buffer-Me concentration drops well below 10* M, as indicated by a drop of conductivity from 4,000 to 5,000 mho. before column filtration to 1.5-2.5 mho. thereafter.

PREPARATION 3: STABILIZED ORGOTEIN (A) Lyophilization stabilization Fifteen parts of the isolated orgotein obtained as in Preparation 1 or 2 and thirt parts of sucrose are weighed and mixed. The mixture is dissolved in 30 parts of demineralized water that has been adjusted to pH 9.4 by gaseous ammonia. The solution is then filtered with slight vacuum through an 045 pre-wetted Millipore filter. The volume of filtrate is measured and the weight of protein therein calculated as follows: 2 m1. of the filtrate is mixed with 3 ml. biuret reagent and the mixture incubated for 15 minutes at 37 C. Absorbence at 555 mp. of the mixture is measured against a water (buffer) blank. Concentration in mg./ml. is determined by multiplying absorbence at 555 mn by 9.1. This conversion factor was obtained by plotting the following data obtained from samples of known concentration on a graph:

Protein conc. Absorbence mg./ml. A 3.7 0.407 1.8 0.197 0.9 0.100 0.45 0.050 0.22 0.024 0 0 Dry weight (ca. 10 percent less than wet weight).

The sample is then shell-frozen, and thereafter lyophilized material is reconstituted to approximately the initial concentration by adding the appropriate volume of demineralized water brought to pH 9.4 with ammonia. The solution is checked, whether it is clear or not, then filtered through a pre-wetted 0.45/1. millipore filter and its absorbence at 280 mu measured. The A /mg. is determined. The weight of protein lost is expressed by the difference between the protein concentration calculated from the weight of protein and volume of solution before lyophilization and the Weight of protein and volume of solution after lyophilization.

EXAMPLE 1 A 6-year old thoroughbred gelding in training for polo was observed to have a large lemon-sized medial splint inside of the left foreleg and a small walnut-sized splint inside the right foreleg. The splints were believed to be caused by a congenital misalignment of the carpal joints. The animal was very lame and stumbled while walking.

Prior treatment consisted of phenylbutazone administration and the animal was rested for a period of 30 days. Upon resumption of exercise the animal again became lame. Phenylbutazone therapy provided no lasting benefit.

5 mg. of orgotein were administered in the form of a saline solution (1 mg. of orgotein per ml. of saline) twice a week for two months and then 5 mg. once a week for 11 months, after which treatment was discontinued. During this treatment the animal was exercised. No other therapy was given.

During the 11-month period, gross and X-ray observation confirmed the reduction in size of the splints on both legs. The smaller of the two disappeared altogether. At the end of the period of therapy the horse was returned to full use and able to play polo without any sign of recurrent lameness.

1 EXAMPLE 2 A 6 year old quarter horse racer with sesmoiditis was severely and permanently lame, unable to race, and was to be sent to abattoir. The animal was obtained from the owner for experimentation. X-ray findings showed extensive bony degeneration and calcification about the entire right fore fetlock joint and a downshift of the sesamoid bones due to strains on suspensory ligaments.

Prior treatment: 1 /2 yearsorthodox procedures with no benefit. Whirlpool treatments early. Blistered several times; the fetlock joint continued to have severe pain, mobility of the joint gradually was lost, all but for a very slight percentage of the normal degree of movement.

Orgotein treatment: mg. in 5 ml. saline, twice weekly for three monts with no other treatment administered. Horse was exercised throughout treatment.

Results of orgotein treatment: after three weeks horse was clinically improved and only slightly lame. After 7 weeks, horse was clinically sound even after hard work and showed no more than a trace of lameness. X-ray findings showed some smoothing and rounding off, and a slight reduction of the bony degeneration.

EXAMPLE 3 A 5-year old thoroughbred mare and ex-racehorse in have a navicular right front contracted foot. Prior treatment with phenylbutazone produced only temporary relief. Over a 3-day period, orgotein was administered by intra-muscular injections of 5 mg. daily. The horse was exercised. No other drugs were administered. At the end of the third day of therapy the horse was no longer lame. It was sold as sound.

EXAMPLE 4 A 5-year old thoroughbred mare and ex-race horse in training for polo was diagnosed by X-ray studies to have a medial fetlock injury at the left forefoot. New bone growth was found about the injured area and also at the attachment sites of the collateral ligaments. Without corrective therapy, such an injury often gives rise to ring bone formation and continuous lameness upon strenuous use. The initial observation indicated that the animal was quite lame, which lameness markedly increased when the horse was turned on the hard ground.

Prior treatment included the application of Gelocast bandages to the involved leg twice weekly, along with a stall rest. Improvement was fair, but the degree of lameness increased proportionately to the amount of exercise given the animal.

Intramuscular injections of 5 mg. of orgotein in 5 ml. saline solution were given two times on the first day and once on each of the next two days. Therapy was stopped for 3 months, then one injection was given weekly for 3 weeks. The horse was exercised throughout treatment.

X-ray studies indicate smoothing of new bone growth in the injured area. No evidence of the bony exostosis extending towards the joint surface. Gross observations showed no indication of lameness and the mare was examined and found sound about two months after cessation of therapy. 1 I

EXAMPLE 5 Sixteen horses with traumatic arthritis were given two I.M. injections of 0.50 mg. of orgotein as in isotonic saline solution. An observable improvement was noted in as short a period as four days.

EXAMPLE 6 Ten horses in unusable condition with trauma contusion and cardinal signs of inflammation were given intra-muscular injections of 0.16 to 1.0 mg. of orgotein. Marked improvement was noted in less than 24 hours,

with decreased inflammation and tenderness. The horses were in condition for return to use in 1 to 3 days.

EXAMPLE 7 An Arabian mare saddle horse, 30 years old, was very lame and not safe to ride. X-ray findings showed advanced stages of arthritis. The joint spaces between the radial carpal bones, the intermediate carpal bones, the first, second, and third carpal bones and the metacarpal bones II and III were completely lost with fusion. There was extensive pen'articular bony growth with bony projections protruding into the soft tissues.

Prior treatment was orthodox, including butazolidine. No lasting benefit. Various linaments were used and massaged over the carpal areas for several extended periods, going into many weeks.

Orgotein treatment consisted of 5 mg. in 5 ml. saline doses twice weekly administered intramuscularly for six weeks. No other treatment was given.

After six weeks of orgotein treatment, there was marked clinical improvement and the horse was fully usable for riding.

At three months, X-ray findings after cessation of treatment showed a decrease in density of bone production and the bony projections. The exostoses were smaller and more rounded off.

After eight months, clinical improvement was even more marked, even though treatment ceased 8 months ago.

EXAMPLE 8 A 6-year old thoroughbred gelding, an ex-racehorse, now in training for polo, had a large lemon-sized medial splint inside the left foreleg, a small walnut-sized splint on the inside right foreleg, a scar on the right fore pastern joint, and splints caused by congenital misalignment of carpal joints. The animal was very lame and stumbled while walking. It was considered completely unusable. As the splints continued to enlarge, periostitis and bony callus formation put considerable pressure upon the suspensory ligament in this area, producing the lameness.

Prior treatment was orthodox, including butazolidine, but it had no lasting benefit. Rest for periods of 30 days and repeated work-splint and lameness condition again got worse. Then leg paints and blisters were applied. There was a slight improvement until work was started again.

Then, orgotein treatment (5 mg. in 5 ml. saline) was administered intra-rnuscularly twice weekly for two months; once weekly thereafter for almost one year. No other treatment was administered. The horse was exercised throughout treatment.

After 6 Weeks, the large medial splint on left foreleg was considerably reduced in size, the small splint on right foreleg nearly disappeared, as observed both grossly and by X-ray. The horses gait greatly improved, and travel was free and normal.

After 6 months, definite improvement continued. The left splint further reduced in size, right splint had disappeared, as judged both grossly and by X-ray. The horse was considered sound and was able to canter, trot downhill, play two chukkers of poloall without any sign of lameness.

After 10 months, the horses left foreleg medial splint continued to reduce in size and the horse was restored to full use and plays hard polo regularly without any sign of recurrent lameness.

The bony exostoses were greatly reduced in size as evidenced by X-ray data. Horse was restored from complete uselessness to full use as a polo pony.

EXAMPLE 9 A thoroughbred mare, Argentine bred polo pony, 15 years old, was severely lame with osselets and not usable. X-ray findings typical of degenerative involvement of fetlock joint of both forelegs. Also, evidence of prior lip- 15 ping fracture, with a small piece of bony growth on the dorsal surface of fetlock joint. Joint space very narrow with rough surface areas with some calcification. Prognosis: very poor. Manipulation of the fetlock joints of both forelegs caused severe pain.

Prior treatment was orthodox, including several years butazolidine, surgical removal of fractured splint bone, due to a polo injury, when the fetlock joints became sore and stiff and stumbling occurred.

Orgotein treatment consisted of mg. in 5 ml. saline intramuscularly administered twice weekly for over a year. Horse exercised throughout treatment and no other treatment was received.

After 6 weeks of orgotein treatment, the horse was sound and playing polo. X-ray findings indicated no significant changes, although some smoothing and rounding of joint surface and calcification were apparent.

This aged, arthritic mare continued to play tournament polo actively and was very useful in spite of the original poor prognosis.

EXAMPLE 10 An Arabian gelding, 18 years old, both forelegs lame with ringbone for over one year. Previous therepy was rest with no exercise for one year. Extensive, chronic bony exostosis very evident on both fore. pasterns.

Orgotein therapy: 5 mg. every other day for 8 weeks, then twice weekly for 4 weeks. In two weeks the horse was only slightly lame and more comfortable. After 12 weeks on medication, the horse was no longer lame and sound on severe exercise. One month after cessation of exercise, horse remained sound.

EXAMPLE 1 l A 6-year old horse, a jumper, was lame and unusable. X-ray findings showed side bone formation on lateral side of hoof on both right and left forelegs, accompanied by extensive calcification on these areas.

Prior treatment: orthodox, including butazolidine to which the horse became refractory. Ointments were applied.

Orgotein treatment: 5 mg. in 5 ml. saline, twice weekly for two months. Horse was exercised throughout treatment. No other treatment was administered.

Results of orgotein treatment: after two months, lameness completely disappeared. Horse returned to owner and back in full use. X-ray findings showed definite improvement.

EXAMPLE 12 A 9-year old gelding horse was afiiicted in both forelegs with severe chronic navicular disease for over one year, becoming progressively more severe in the last six months. Classic spur development on tip of navicular. Previous therapy was butazolidine with rest and shoeing with elevated heels.

Orgotein therapy: 5 mg. every other day for two weeks, then twice weekly for two weeks. Improvement was marked. Horse normal and sound at end of therapy. One month and three months after therapy, horse was playing polo and sound.

EXAMPLE 13 A 12-year old polo pony, A; thoroughbred mare had hip involvement which was confirmed diagnoses by local nerve blocking that ruled out other lamenesses. The horse had the true symptoms of hip joint lameness. It was lame and useless. Its age and occupation as a polo horse gave a poor prognosis.

Prior treatment: butazolidine gave temporary relief. Rest gave a little temporary improvement only.

Orgotein treatment: 1 mg. in 2 cc. intra-muscularly about every other day for five weeks (20 doses). The only other treatment was exercise. The horse originally was stumbling. In two weeks it was ridden with no stumbling lameness. It played polo with excellent performance. Long after treament, the animal remained sound.

EXAMPLE 14 In humans with post-traumatic arthritis (osteoarthritis) the usual dose is three daily injections of 2 mg. each intramuscularly, followed by 2 mg. three times weekly for three weeks and thereafter as indicated. Orgotein is effective as demonstrated by reduction of pain, by X-ray changes, by reduction in ring size and similar tests and by increased performance capabilities.

EXAMPLE 15 In dogs with disc syndrome, where surgery is not indicated (about of cases), increased performance and mobility is achieved by injection of orgotein intramuscularly 2 mg. daily for three to five doses, then repeated as necessary. Usually the beneficial effects last for several months.

In dogs with osteoarthritis, increased performance and lack of pain, stiffness and soreness are noted. Dose 2 mg. three times weekly for two weeks. Then, twice weekly for two weeks or as indicated.

What is claimed is:

1. A method of treating post-traumatic arthritis in mammals which comprises administering systemically successive spaced doses of orgotein to the mammal affected with the arthritic condition in amounts effective to alleviate at least some of the clinical symptoms of the condition.

2. A method according to claim 1, wherein the mammal is a horse and the post-traumatic arthritic condition is boney exostosis.

3. A method according to claim 2, wherein the amount of orgotein administered is from about 0.5 to 20 mg. per dose.

4. A method according to claim 3, wherein the orgotein is administered by intramuscular injection.

5. A method according to claim 4, wherein the amount of orgotein administered is about 2.5 to 5.0 mg. per injection.

6. A method according to claim 1, wherein the patient is human and the arthritic condition is hypertrophic.

7. A method according to claim 6 wherein the orgotein is administered by intramuscular injection.

8. A method according to claim 7, wherein the amount of orgotein administered is from 1 to 4 mg. per injection.

9. A method according to claim 7, wherein the treatment is continued for a period of at least two weeks.

No references cited.

RICHARD L. HUFF, Primary Examiner y V UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3. 537 641 Dated January 25, 1972 n Wolfgang Huber and Thomas L. Schulte It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the Specification:

Col. 1, line 42, asteitis" should be --osteitis---;

Col. 1, line 56, "asteophytes" should be ---osteophytesv Col. 2, line'l6, "asteoarthritis" should be ---osteoarthritis--;

Col. 2, line 46, arthristis" should be ---arthritis---;

Example 3 Col. 13 line 29, delete "A 5-year old thoroughbred Inare and ex-racehorse in" and substitute therefor ---A 6-year old quarter horse gelding Was diagnosed to---.

Signed and sealed this 20th day of June 1972.

Attest:

EDWARD M.FLETCHER, JR.. ROBERT GOTTSCHALKv Attesting Officer Commissioner of Patents- FORM PO-lOSO (10-69) USCOMM-DC 037s-p59 h u.s, GOVERNMENT PRINTING OFFICE: I989 0-366334