CA1249515A - Medicinal extract of thymus glands - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A medicinal extract of whole thymus glands for the treatment of animals suffering from immune related conditions is disclosed. The medicinal extract of thymus glands comprises a saline extract of ground whole thymus glands of chinchilla, bovine or sheep, injectable water and optionally a preservative. If frozen, the medicinal extract would also contain an antibiotic. The process for the preparation of the medicinal extract is disclosed. Additionally, a process of treating animals suffering from immune related conditions is also disclosed.

Description

~r~,,r This invention relates to a medicinal extract made from whole thymus glands of either chinchilla, bovine or sheep. This invention further relates to a method oE preparing such an extract for treating animals suffering from immune related conditions.
The thymus gland is involved with the immune system through its hormones and hormone-like factors including Metcalf's lyl~phocytosis-stimulating Eactor, Miller's competence including factor, homostatic thymus hormone, thymopoeitin I and II and thymosin. Although it is not known if these are the same substances under different names, it is known that individually these thymic substances have lymphocytopoietic effects, and can prevent both wasting disease and fatal virus infections in thymectomized mice. All thymectomized laboratory animals show a significant decrement in the immune response.
In addition, the thymus gland has an indirect effect on the immune system. Many lymphocytes, which are directly involved in the immune system, pass through the thymus. During the time these lymphocytes are in the thymus, the lymphocytes will differentiate and mature and are possibly influenced by thymic hormone-like factors.
Thus, a mixed heterogenous population of thymus cells might control through its final products the hormonally conditioned mature lymphocytes and exert an indirect effect on the immune system.
The thymus gland is active from birth to puberty and then appears to atrophy and slough off its peripheral cells. The thymus gland effects the immune system even after it atrophies. The peripheral cells of the atrophied thymus gland migrate to form some of the reticuloendothelial tissues associated with the development of the immune responses in the adult by producing substances such an antibodies and interferon.
Rach of the isolated thymic hormones and hormone-like factors have a specific function. The thymic medicinal extract herein described '~

contains most or all of the thymic hormones and hormone-like factors.
The totality of these hormones and factors work to stimulate the immune system to produce the therapeutic results herein described.
An object of the present invention is to provide a medicinal extract made from whole thymus glands of either chinchilla, bovine or sheep which has a therapeutic effect on the immune system that can produce the results herein described.
Another object of the present invention is to provide a meehod of preparing such a medicinal extract of thymus glands.
This new medicinal extract of thymus gland for the treatment of animals c.omprises a saline solution of ground whole thymus glands of chinchilla, bovine or sheep, sterile injectable water and optionally a preservative. The medicinal extract of thymus gland may also contain an antibiotic to prevent bacterial contamination if the medicinal extract is to be frozen for storage. The method of preparing such an extract is claimed herein. Also described is a method of treating animals suffering from immune related conditions with this medicinal extract of thymus gland.
The thymus becomes nonfunctional at puberty. However, I have found that in chinchillas (fur-bearing rodents valued for its pelt), the thymus does not become nonfunctional at puberty but instead remains functional throughout this animal's lifespan. A medicinal extract made from chinchilla thymus will stimulate the immune response even in animals of different species~ Such a medicinal chinchilla thymic extract is more effective than extracts made from other animals when treating animals of a different species. Medicinal extracts made from chinchilla thymus, cattle thymus or sheep thymus will stimulate the immune response when used in treating that same or different species.
Thymic hormones and hormone-like factors each affect the immune system. However, heretofore, an extract from the entire thymus gland has not been used as a therapeutic agent. The thymic extract of the present invention contains most or all of the thymic hormones and factors together. The therapeutic results obtained from the use of this extract made from whole thymus glands are impressive. Use of this thymic extract has resulted in dose related immune responses especially as to the IgG raction when electrophoresis studies have been conducted.
The thymic extract has reduced fever in animals, has caused improvement and complete recovery in animals suffering from demodectic mange, can improve the condition of animals suffering from mycoplasma (which is apparently an immune related disease), has been shown to be effective against stress in cattle, in treating feedlot "knockouts", trea~ing sheep "non-doers" and treating polyarthristis in sheep. Thymic extract has also inhibited mammary tumors in animals and has caused weight gain in animals suffering from low body weight and in young animals suffering from stress.
The medicinal extract can be prepared from either chinchilla, bovine or sheep thymus glands. Basically the method of preparing the medicinal extract comprises homogenizing fresh whole thymus glands in saline, cooling the homogenate to a preselected cooling temperature, centrifuging the homogenate which had been cooled to remove particulate matter, saturating the homogenate in an amount of saline equal to the removed particulate matter, heating and agitating the resulting extract solution at a preselected heating temperature, removing any precipitate formed as a result of the heating, diluting and filtering the extract solution and adding a preservative, if needed.
It is preferred that fresh thymus glands are obtained in a frozen state and allowed to be thawed. The thymus glands are defatted and all extraneous facia and membranes are removed from the glands. The tissue can be ground in a meat grinder and then homogeni7ed in saline e.g. O.lS M NaCl. The preferred ratio of homogenized tissue to saline is 1 to 3 by weight.
The homogenate is cooled to a temperature range of 4 to 6C.
The cooled homogenate is centrifuged to remove particulate matter.
Typically, the cooled homogenate is centrifuged at approximately 2500xg for approximately 15 minutes at approximately 5C. The removed particulate matter is replaced by an approximately equal volume of saline. The resulting solution is allowed to saturate from about 4 to 8 hours, typically 6 hours.
The resulting extract solution is then heated typically at 80C. for approximately 20 minutes to Eorm a precipitate. Acceptable heating temperatures range from 78 to 82C. During heating, the extract solution is agitated until no more precipitate is formed.
While still warm the precipitate is removed. One way to remove the precipitate is by passing the extract solution through a sterile grease filter. The extract solution is thereafter diluted with sterile water. The amount of sterile water added is approximately e~ual to the amount of precipitate removed. The extract is allowed to cool to an acceptable cooling temperature between about 4 to 6C~ After cooling the extract is filtered through 0.45 ~m pore size filter. A
preservative is added if the extract is not to be used immediately. The preferred preservative is 0.75% benzyl alcohol.
The extract can be placed in serum bottles or lyophilized for storage. The medicinal thymic extract may be reconstituted by the addition of nonpyrogenic injectable water up to the original volume.
The medicinal thymic extract may also be frozen for storage.
~efore freezing, antibiotics are added to inhibit bacterial growth. The preferred antibiotics used are 0.25 gm of streptomycin sulfate and 250 I.U. pencillin G. potassium per 500 ml of extract. Alternatively, any acceptable antibiotic to control bacterial growth may be utilized.
After the addition of the antibiotic, the pH of the extract is adjusted s to about 6.8 with HCl or NaCl as needed. The extract is then passed `~ through a millipore filter. The range oE the pore size of the filter is 0.45 ~ to 0.22 ~ . This sterilizes the solution by filtration. The antibiotics and preservatives are precautionary measures against bacterial invasion during storage.
Nonlyophilized extract has a shelf life in excess oE 2 years while lyophilized extract has a shelf life in excess of 4 years.
The amount of extract per dose will vary according to the species treated. Within each species the amount of extract per dose will further vary according to the body weight of the animal treated.
When treating canines the dosage will range from 75 mg. to 200 mg. When treating cattle, the dosage will range from 125 mg. to 350 mg. When treating sheep the dosage will range from 25 mg. to 50 mg. Each dose is administered by an interperitoneal injection and repeated at two week intervals if necessary.
The dose equivalent amounts per injection of raw chinchilla thymus homogenate (C.T.H.) used in the animal studies in examples 1 to 3 was approximately 1.7 mg of chinchilla thymus extract (C.T.e.) in 1/4 cc of physiological saline.
Example 1 A study was conducted to determine the effects of varying doses of interperitoneal injections of bovine thymus extract and chinchilla thymus extract upon rats. No deleterious effects were ascertained. Hemotalogic differential counts did indicate a change in lymphocytes~ Lymphocytes are involed in the immune response. Plasma electrophoresis and immune diffuse electrophoresis showed wide variation according to dose especially as to the IgG fraction which also indicates an immune response. This immune response produced by the thymic gland extract appears to be dose related.

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Example 2 The effects of varying doses of interperitoneal injections of chinchilla thymus extract upon rats that had DMBA (7,12-Dimethybenz [ ~]-anthracene) induced mammary tumors were studied. The spontaneous occurence of tumors in the absence DMBA in rats of the age used is approximately 50% as shown by others. The pre-treatment of chinchilla thymus extract on older rats resulted in a more pronounced response.
Additional injections of chinchilla thymus extract after exposure to DMBA demonstrated a further protection against DMBA induced mammary tumors. This study has demonstrated that thymus extract has a dose related effect upon the inhibition of mammary tumors in rats.

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Chinchilla thymic extract was demonstrated to have effects upon leghargic mice as shown by the recorded 0% mortality rate as compared to the normal mortality rate of 81%.

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However, w~en subsequently comparing the effects of chinchilla and bovine thymic extracts on lethargic mice, it was found that chinchilla thymic extract resulted in more pronounced regrowth of the thymus upon post mortum examination and a better weight gain response than the bovine thymic extract. In this case, the chinchilla thymic extract was preferred.
~ xample ~
A licensed veterinarian was treating a calf suffering Erom an unknown ailment for three weeks without apparent success. The veterinarian suspected the cause of the calf's illness was of an immune nature. The calf was then treated with 200 mg. of chinchilla thymus gland extract prepared four years earlier. The extract was administered in 20 cc of injectable water in one interperitoneal injection. The calf received no further treatment of any kind. Within three days of the medicinal thymus extract treatment, the veterinarian pronounced the calf well.
Example S
A calf suffering from high fever complicated by nasal discharge of pulmonary origin had been treated by a licensed veterinarian for three days without success. The calf was administered one interperitoneal injection of 175 mg of lyophilized bovine thymus gland extract (prepared four years earlier) in 25 cc of injectable water.
Within twenty four hours, the calf' 9 temperature was normal and the nasal discharge had ceased. At seven days post treatment the calf had reestablished normal eating behavior and appeared normal in all respects.
Example 6 A Dachshund puppy was ~uffering from immune related demodectic mange. Lesions covered 2/3 of the dog's body. In the past, medication has not been effective in the treatment of this condition resulting in ~2~

the extermination oE most affected animals. The dog was given one interperitoneal injection every two weeks of 75-10~ mg. of chinchilla thymus extract (prepared several years earlier) in 15 cc. of injectable water. Within two weeks, the veterinarian pronounced the dog better.
After six weeks the veterinarian claimed the prognosis for complete recovery looked good. The lesions were healing and new hair was growing.
Treatment stopped after the third injection since no more chinchilla thymus extract was available. After a six months post treatment follow-up, the veterinarian pronounced the dog completely cured.
Example 7 A 65 lb. Doberman Pincer dog diagnosed as suffering from demodictic mange did not respond to any medication. Lesions covered most of the dog's body. The dog was treated with one interperitoneal injection every two weeks for the next six weeks. The injection was 25 cc of freshly prepared chinchilla thymus extract in solution containing approximately 190 mg of thymus protein. After two weeks. the lesions were healed. By the fourth week, new hair had grown over the affected areas. After a six month post treatment follow-up the dog was completely recovered.
Example 8 A 125 lb. great dane dog was suffering from immune related demodectic mange and other immune related disorders. Approximately 2/3 of the dog's body was affected. Several weeks of conventional medical treatment had produced no results. Although the owner wanted to destroy the dog, the dog was referred to experimental treatment. The dog was administered one interperitoneal injection of 25 cc (190 mg thymus protein) of chinchilla thymus gland extract. After two weeks, the dog was somewhat improved. Due to the amount of scratching by the dog, the veterinarian increased the dosage to 1/4 cc. per poud of body weight.
~0 After an additional week, the dog's condition was markedly improved.

However, the owner decided to have the dog destroyed even though the veterinarian felt that the dog would recover completely given time.
Ex3mple 9 A chihuahua dog was diagnosed as suffering from demodectic mange that was resistant to conventional medication. The dog was treated with one interperitoneal injection of 1/4 cc. per pound of body weight (1.9 mg of thymus protein per pound) of chinchilla thymus gland extract. After two weeks there was a marked improvement. A second in~jection was administered. The owner never returned the dog for additional treatments.
Example 10 A 13 lb. toy poodle dog was suffering from re-occurring mammnary tumors. These tumors had for several years been removed surgically approximately every six months by a veterinarian. The dog was treated with one interperitoneal injection of 13 cc. (approximately 98 mg) of chinchilla thymus extract after the surgical removal of the mammary tumors. One year later the dog's owner had not brought the dog back for any further removal of mammary tumors.
Example 11 A 13 yr. old miniature female schnauzer dog had 1 0-15 mammary tumors of various sizes. These tumors reoccurred periodically and had to be surgically removed each time. The veterinarian advised against continued surgery due to the age of the dog. The dog was treated by injecting about 2 cc. of chinchilla thymus gland extract inter-peritoneally and by injecting the extract into and about each tumor.
The dose was varied according to the size of each tumor. Several treatments were administered at two week intervals. After several weeks the small tumors totally regressed while the large tumors regressed only partially.

Example 12 Two goats were suffering from mycoplasma, which is of an immune related nature. The symptoms of mycoplasma are swelling of the joints, general arthritis, and chronic respiratory involvement. ~ach goat received one interperitoneal injection of 30 cc~ (approximately 25 mg) of bovine thymus gland extract. As a result, the swelling of the joints regressed and the goats appeared normal for about six months. It is felt that a series of such treatments would cause greater improvement.
Example 13 This experiment was conducted on "knock-out" animals. As used herein, knock-out animals are defined as animals that are hauled a great distance, given a series of medications, kept in crowded confinement, branded and often de-horned and castrated~ and as a result do not eat very well and demonstrate a weight loss. These animals usually get sick and are removed to the sick pen and given a broad spectrum of antibiotics. This happens to animals in feed lots. Knock-outs usually never go back on feed or do well in the feed lots. The majority of them die. As the animals were purchased from the feed lots, they were given a grade according to their condition, 0 to 4.
Grade 0 - no apparent illness, not eating well l - showing signs of stress, will not eat well

2 - noticeably sick, becoming thin

3 - sick, thin, will not eat, slight fever, beginning to demonstrate some respiratory trouble

4 - very sick, fever, thin, will not eat, mucous running from the nose, respiratory trouble, unsteady on feet ~eventy-eight calves were purchased with the following grades of 9 ickness:
6 - Grade 4 l9 - Grade 3 12 - Grade 2 9 - Grade l 32 - Grade 0 (11 of grade 0 were not treated) 78 - Total The 78 calves were given between 40 and 60 cc. of bovine thymus gland extract interperitoneally, depending on body weight (1/8 cc. per pound). There was marked improvement in all the calves on about the 7th to 9th day post-treatment. They began to consume feed and have a steady weight gain. After a few months on Eeed, they were sold at the regular auction sale barn. Eleven animals out the group died, of which three calves had hardware disease and three calves had some other malady unrelated to the immune system.
It was concluded that the animals that are placed in a sick pen at the feed lot are given such massive doses of antibiotics that they are almost beyond help. However, the bovine thymus gland extract was highly successful in treating approximately 80% of the animals purchased. It would have been preferred to give the bovine thymus gland extract to these animals at the onset of the stress condition without the use of antibiotics.
Example 14 This exper;ment was conducted on non-doer animals. As used herein~ ~Inon-doers~ are defined as lambs on feed that do not eat very well, do not gain weight and are stunted compared to their peers~ They eventually die or are culled out of the feed lot program. This represents quite a monetary loss to the owner of the animals. The purpose of this experiment is to stimulate the animals' immune systems in such a manner so as to restore their emmaciated condition and cause thesn to go back on feed.
Each lamb was given 5 cc. of sheep thymus gland extract interperitoneally. The animals weighed between 30 to 60 pounds.
Within three days the animals started to improve and begin to eat and drink, to show healthy activity, and had bright eyes. Within a week, the animals appeared normal. Within two weeks they demonstrated a marked increase in weight gain over their peers.
~xample 15 Five non-doer lambs were each given 5 cc. of sheep thymus gland extract interperitoneally. Five normal lambs were used as controls and received no treatment. Both groups were observed for weight gain. After three days, the non-doer lambs began to eat normally and gain weight. By the second week the non-doers' weight gain equaled the controls' weight gain. By the third week, the non-doer animals exceeded the control group in weight gain. All animals were then returned to the regular feed lot pens.
Example 16 Fifteen non-doer sheep were treated with interperitoneal injections of 5 cc. of sheep thymus gland extract. Three days after treatments the non-doer sheep were eating and drinking. Three weeks later these animals were apparently normal, were eating and gaining weight and had glossy coats.
Example 17 This experiment was conducted on young calves suffering from stress. These animals are stressed from being weaned too young, hauling, crowded conditions and su~ner heat. Often the death loss is tremendous within a ~ew months due to this stress condition. The following experiment was conducted on such young stressed calves:
15 - control animals, no treatment 16 - animals injected with 25 cc. of sheep thymus gland extract interperitoneally 15 - animals injected with 25 cc. of bovine thymus gland extract interperitoneally After 30 days, several of the control group appeared severely stressed and died. This degree of loss was expected. The treatment groups were eating and gaining weight. Only one animal was lost from tne treatment groups. This animal was emmaciated and weak at the time oE treatment. All the other 30 animals in the treatment groups survived.
~xample 18 In a pre-conditioning feed lot, four calves were down from trauma. These animals suffered from trauma due to being hauled for some distance, unloaded, run through a chute, branded, castrated, given shots and sometimes dehorned, all in one operation. Animals suffering from such trauma often do not recover. These four calves were treated with one interperitoneal injection of 200 mg. of bovine thymus gland extract (prepared four years previously) in 25 cc. of injectable water.
The next day the calves were up and eating. Within ten days, they were back on the regular feed lot regime and appeared completely normal.
Example 19 Five sheep suffering from polyarthritis were each given S cc.
of sheep thymus gland extract interperitoneally. Five healthy sheep were used as controls and were treated identically. The day following treatment the five poly-arthritic sheep were on their Eeet ~previously they could not get up) and were eating normally. l'he control sheep also appeared completely normal. Three weeks later the sheep were put in the fattening sheep feed lot.

The amount of extract per dose will vary according to the species treated, and the body weight of the animal treated.
Dosage Range Canine:
Chinchilla or bovine extract -71 to 100 mg. - up to 15 pounds body weight 125 mg. - 15 - 25 pounds body ueight 150 mg. - 25 - 50 pounds body weight 200 mg. - over 50 pounds body weight Administered interperitoneally, every two weeks, if necessary.
Bovine:
Chinchilla, bovine or sheep extract -125 mg. - up to 100 pounds body weight 200 mg. - 100 - 400 pounds body weight 250 mg. - 400 - 500 pounds body weight 350 mg. - over 600 pounds body weight Administered interperitoneally, every two weeks, if necessary.
Sheep:
Chinchilla, bovine or sheep extract -25 mg. - up to 50 pounds body weight 35 mg. - 50 - 100 pounds body weight 50 mg. - over 100 pounds body weight Administered interperitoneally, every two weeks, if necessary.

Claims (5)

The embodiments of the invention, in which an exclusive property or privilege is claimed are defined as follows:

1. A medicinal extract of thymus gland for the treatment of animals comprising an effective amount of a saline extract of ground whole thymus gland of chinchilla free of heat precipitable impurities, and injectable water.

2. A medicinal extract of thymus gland as recited in claim 1 further comprising a preservative.

3. A medicinal extract of thymus gland as recited in claim 1 further comprising an antibiotic.

4. A process for the preparation of the medicinal extract of thymus gland as recited in claim 1 comprising:
homogenizing fresh ground whole thymus gland of chinchilla in saline;
cooling the homogenate to a cooling temperature of 4°-6°C.;
centrifuging the cooled homogenate to remove particulate matter;
saturating the homogenate in an amount of saline equal to the removed particulate matter;
saturating the homogenate in an amount of saline equal to the removed particulate matter;
heating and agitating the resulting extract solution at a heating temperature of 78°-82°C.;
removing any precipitate formed;
diluting the extract solution;
filtering the extract solution;

adding an antibiotic; and adjusting the pH or the resulting extract solution.

5. An injectable preparation containing from about 25 to 350 milligrams of the medicinal extract of thymus gland of chinchilla, as recited in claim 1.