Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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
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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
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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.
331
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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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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
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(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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No. l received no immediate therapy; Animal No. 2 received
chymotrypsin intramuscularly, Animals Nos. 3 throughlO all
received acutalyn crude intraveneously. No additional acutalyn
crude was administered to the horses after the first day,
except Animal No. ~ which received one intravenous injection
taily for five days and Animal No. l which received one
intravenous in~ection on the seventh day with the hope of
stopping or preventing the necrosis and slough of tissue.
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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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
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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.
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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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Example 7.
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
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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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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,
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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
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and finger with no relief. Examination revealed both hands
severely swollen, red, painful nodes, distal phalanges
(Herbenden's nodes), painful right shoulder, weak grip right
hand. A total of eight 5.0 ml. in~ections of acutalyn crude,
50 mg. %, were given i.v., one weekly for four weeks, then
bi-weekly for eight weeks. Symptoms began to improve following
the first in~ection, with decrease in pain. Following the
third in~ection, the nodes on fingers were softer, following
the fourth in~ection there was marked improvement in both
swelling and pain. The pain disappeared following the sixth
inJection and swelling of hands and nodes continued to tecrease.
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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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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10666Z~ ~
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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- 10666Z0
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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1066620
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.
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~ 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.
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1066620
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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