CA2168263A1

CA2168263A1 - Pharmaceutical compositions

Info

Publication number: CA2168263A1
Application number: CA002168263A
Authority: CA
Inventors: Bernhard Heising; Edelgard Heising; Cathrin Heising; Franziska Heising
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-07-29
Filing date: 1994-07-13
Publication date: 1995-02-09
Also published as: WO1995003812A1; EP0711170A1; JPH08510470A; AU7386094A; EP0711170B1; ATE164764T1

Abstract

The invention relates to a pharmaceutical composition containing T lymphocytes as the active substance, prepared by recovering the leukocytes from mammal whole blood, mammal spleen, or mammal thymus gland, or autoblood, separating the leukocytes on nylon wool, and recovering the concentrated T lymphocyte suspension. Furthermore, the invention relates to a process for preparing said composition and its use for the treatment of viral and bacterial infections.

Description

_ I

Pharmaceutical Composition This invention relates to a pharmaceutical compo-sition which contains T lymphocytes as the active substan-ce, a process for preparing the pharmaceutical composition and the use of same for the preparation of drugs for the treatment of viral and bacterial infections.

Today, infectious diseases caused by bacteria or viruses are largely treated with chemotherapeutics. Anti-bacterial chemotherapeutics are metabolites of microorgan-isms or semi-synthetic derivatives of same (antibiotics) or synthetically prepared compounds (sulfonamides).

Currently, as the chemotherapeutics enabling ther-apy of virus-induced infections by inhibiting the virus production, nucleoside analogues inhibiting the virus-specific enzymes (polymerases) are preferably employed.

A substantial and generally familiar drawback of the chemotherapeutics is that` resistances may occur in prolonged and repeated treatment. In addition, side ef-fects are frequently reported.

So far, in spite of intense efforts, no success has been achieved in providing chemotherapeutics which interfere causally or symptomatically with the virus- or retrovirus-induced events of disease with significantly substantial success. Today, it is not possible to cure certain virus diseases such as, for instance, the acquired immune deficiency syndrome (AIDS), the AIDS-related com-plex (ARC) and their pre-stages, herpes, cytomegaly virus (CMV) infections, influenza and other virus infections or 216826~

to exert favorable influence on the symptoms thereof chemotherapeutically.

Currently, for example, it is almost exclusively 3'-azido-3'-deoxythymidine (AZT) which is available for the treatment of AIDS. However, AZT is characterized by excessively severe toxicities occurring already in the therapeutic range [Hirsch, M.S., J.Infec.Dis. 157, 427-431 (1988)].

Therefore, it is the object of the present inven-tion to develop a therapeutic agent which may be employed in the treatment of viral, particularly retroviral, and bacterial infections, without resistances or side effects occurring in prolonged or repeated treatment and pharma-cologically relevant doses being toxic.

Surprisingly, it has now been found that a pharma-ceutical composition exceedingly simple to prepare and favorable in cost, which contains T lymphocytes from mam-mal blood, spleen or thymus gland or from autoblood as well, is highly suitable for the treatment of viral and bacterial infections and is rapidly effective.

The T lymphocytes contained in the composition are recovered from m~mm~l whole blood, e.g., from rabbits, lambs or pigs, by subjecting the m~mm~l whole blood, to which aqueous trisodium citrate is optionally added as an anticoagulant (0.11 moles/l; 9 parts of blood, 1 part of citrate), to a centrifugation for about 7-8 minutes at about 4000 rpm, and separating the leukocyte boundary layer. By separating the leukocytes on nylon wool, a con-centrated T cell suspension is obtained. Recovery from autoblood is effected in analogous fashion. Preferably, to the autoblood, there is likewise added aqueous trisodium citrate and dextrose.

~3 2168263 Another possibility of recovering leukocytes from m~mm~l whole blood or autoblood consists in passing the whole blood, to which aqueous trisodium citrate (0.11 moles/l; 9 parts of blood, 1 part of citrate) has been optionally added, through a commercially available cellulose filter, washing same with PBS, subsequently centrifuging the obtained leukocyte/thrombocyte suspension for 7-8 minutes at 700 rpm to separate thrombocytes and leukocytes. Again, the separation of leukocytes is effect-ed on nylon wool. Using this process, a T lymphocyte sus-pension particularly high in concentration is obtained.

Similarly, of course, the leukocytes may be sepa-rated from m~mm~l or autoblood using the well-known leuko-pheresis process.

Alternatively, the T lymphocyte suspension of the invention may also be recovered from m~mm~l spleen or from m~mm~ 1 thymus gland by leaching the spleen or gland with PBS (phosphate-buffered physiological sodium chloride solution) optionally admixed with trisodium citrate and dextrose, centrifuging the obtained solution for about 7-8 minutes, and separating the leukocyte layer. Again, by separating the leukocytes on nylon wool, a concentrated T cell suspension is obtained.

For oral application, from 20 to 50 g of the T cell suspension is added to preferably from 100 to 300 ml of a non-denaturing liquid. In the simplest case, water may serve as the liquid, but also juices or instant soup find application. Use of instant soup offers the great advantage that addition of taste modifiers or sweet-eners for better acceptance of the drug by the patient may be abandoned.

The preparation of an administration form of the pharmaceutical composition of the invention with the aid 216826~3 of, e.g., instant soup is effected by preparing the in-stant soup, cooling same to room temperature, and stirring in the appropriate amount of T cell suspension. Here, preferably, from 20 to 50 g of T cell suspension is dis-solved in 250 ml of instant soup. Preferably, the T cell suspension is stored in frozen condition (in portions) and stirred into the liquid non-thawed. This causes the T cells to break. When using a freshly prepared T cell suspension to prepare the administration form of the in-vention, the suspension must be introduced into the liquid with vigorous stirring (mixer), in order to destroy the T cells. A precondition for the high and rapid effective-ness determined according to the invention is that the T cells are taken up already in broken condition.

The T lymphocyte suspension may also be applied rectally as a suppository or as a clyster. When adminis-tering as a clyster, the T cell suspension preferably is applied undiluted or diluted slightly with water for bet-ter handling.

The administered dose depends on the recipient's age, health and weight.

Conventionally, the daily dose is from 20 to 50 g of T cell suspension to be given in a single application.

A modification of the pharmaceutical composition, according to the invention, also relates to a T cell sus-pension from autoblood prepared in analogous fashion as the T cell suspension from mammal blood, which, however, has to be inhaled using a sprayer. Here, however, applica-tion must be effected using about three portions over a day, where preferably, no more than 10 ml/day should be administered.

~ 2168263 Anaphylactic shocks as observed in the administra-tion of whole blood or whole blood preparations do not occur in oral application of the composition according to the invention.

The pharmaceutical composition of the invention has valuable pharmacological characteristics. It has been found that with bacterial or virus infections, any symp-toms of disease were no longer present as early as after five days of application. In serious cases, primarily with persisting infections, the T cell suspension may be admin-istered for up to 30 days without side effects occurring.
Repeated application of the pharmaceutical composition according to the invention does not give rise to any re-sistances or toxic side effects.

So far, test persons suffering from viral and bacterial infections including those with shingles, facial herpes, Pfeiffer infectious mononucleosis (Epstein-Barr virus), and AIDS, have been treated using the pharmaceuti-cal composition according to the invention prepared from mammal whole blood.

Likewise, a dog fallen sick due to bacterial in-fection was subjected to therapy using the agent according to the invention.

The effect of the formulation according to the invention in retrovirus infections was determined on AIDS
test persons using the Elisa HIV p24 antigen.

Obviously, the composition of the invention inter-feres with retrovirus-induced diseases or symptoms thereof without affecting the regularly working natural body func-tions.

`_ b Therefore, the invention is also directed to the use of the T lymphocyte suspension according to the inven-tion to prepare drugs for the treatment of viral and bac-terial infections, particularly of retroviral infections such as AIDS and ARC (AIDS-related complex).

In the following, the invention will be illustrat-ed in more detail in the embodiments which are not intend-ed to be limiting.

Embodiments Example 1 The pharmaceutical composition according to the invention consisting of 30 g of T cell suspension from rabbit blood in 250 ml of instant soup was repeatedly administered to a 48 year old woman having a virus infec-tion and (4 months later) influenza:

a) Virus Infection - Onset of infection with signs of failing vitality and weakness, severe headache, fever, suppos-itories taken, recuperation without fever after

2 days;

- about 8 days later, still severe headache, return of slight fever, perspiring, shivering, dis-ability, return to recuperation after 4 days;

- visit by physician 13 days after the first signs of infections, for now, severe weakening, slight fever, persistent, hard cough, persistent cold, and hence, sleeplessness and constant tiredness;

~, Physician's diagnosis after blood sampling: Virus infection of an extraordinarily rare virus, with poor liver values;
reatment: Inhalation 3 x daily, nose drops, cough drops, 6 tablets of Wobenzym N (enzyme) 3 x daily, 2 tablets of Gripp-Heel 3 x daily (influenza),

3 drops of Grippe Comp. 2 x daily;
on the 9th and 10th day: total weakness in spite of (after physician's visit) treatment as indicated, unbearable coughing stim-ulus - on the 11th day: administration of first (after physician's visit) composition of the inven-tion - on the 12th day: administration of second composition, slept through the night for the first time, only minor coughing remaining, cold almost gone;

- on the 13th day: administration of third composition, cough and cold completely gone, gen-eral condition regained;

- on the 14th day: administration of fourth composition, no longer complaints; headache gone;
undisturbed sleep at night;

- on the 15th day: administration Of fifth and last composition, no symptoms of sickness any-more.

b) Influenza Four month later, severe influenza recurred which again was subjected to therapy by ingesting the composi-tion of the invention. After only 3 days of ingestion (1 x daily, in the evening), alleviation ensued, and after 10 days the female patient was healthy.

Subsequent to this second ingestion, the patient's health condition had improved in such way that so far, she did not have any viral or bacterial infect for 1.5 years.

Example 2 A 43 year old male patient suffering from influen-za received the composition of the invention according to Example 1.

Mr. R.H. complained about general feebleness, limb pain, lack of appetite and slight dizziness when changing position. On the next day, shivering appeared as well as febrile temperatures to axillary 38.8C.

Results:

- Good nutritional state and reduced general condition; unhealthy appearance; skin pale, warm, slightly sweating; sufficient blood flow in mucous membranes; head freely movable, minor headache over both maxillary sinuses; tongue wet, slightly furred;
rear throat wall slightly reddened.
- Heart: nothing abnormal detected - Lungs: bilaterally aerated, discretely in-tensified breathing noise, at both sides paravertebrally.
- Abdomen: nothing abnormal detected - Extremities: freely movable - Neurology: nothing abnormal detected.

Subsequent to administering the first composition according to the invention, there was strong sweating the next night. On the next morning, the patient already felt much more comfortable. After administration of the second composition according to the invention, there was further improvement, and after administration of the third compo-sition on the next day, the above mentioned symptoms had disappeared except for a slight coughing.

Example 3 ~ m;n;stration of the composition according to the invention as a clyster.

A dog fallen ill with a bacteria-induced snow gastritis was cured by a 14 day administration of 20 g of T cell suspension according to the invention daily. Appli-cation was effected enterally using a syringe.

Example 4 A~m;n;stration of the composition according to the invention in the case of shingles.

A patient fallen ill with shingles was cured suc-cessfully by a 10 day oral administration of the composi-tion of the invention according to Example 1.

Example 5 A~m;n;stration of the composition according to the invention in the case of facial herpes.

A female patient with a large area of facial herpes induced by the Herpes Simplex Virus was cured by a 5 day administration of the composition of the invention according to Example 1. When ingesting once per day, the inflammation declined as early as on the third day, and on the fifth day the inflammatory area was completed healed.

Example 6 Administration of the composition according to the invention to a female patient with Pfeiffer infectious mononucleosis (Epstein-Barr virus).

The composition of the invention was administered to a female patient having Pfeiffer infectious mononucleo-sis once per day. As early as after the second ingestion, the fever declined considerably, and the patient felt better. On the third day, the patient was free of fever, and after another three days she was able to step out of her house again.

Example 7 A~m;n;stration of the composition according to the invention to an AIDS patient.

The composition of the invention according to Example 1 was administered to an AIDS patient (HIV-l) once per day for thirty days.

While in the HIV p24 antigen test (measuring range 5-500 pg/ml) conducted prior to onset of therapy p24 anti-Il 2168263 gen was unambiguously detected, p24 antigen could nolonger be detected after completion of the therapy. This reveals that obviously, inhibition of virus replication had occurred.

Claims

C L A I M S

1. A pharmaceutical composition containing T lym-phocytes as the active substance, prepared by re-covering the leukocytes from mammal whole blood, mam-mal spleen, or mammal thymus gland, or autoblood, separating the leukocytes on nylon wool, and recover-ing the concentrated T lymphocyte suspension.

2. The pharmaceutical composition according to claim 1, prepared by recovering the leukocytes by centrifugation of mammal blood or autoblood and sepa-rating the leukocyte boundary layer.

3. The pharmaceutical composition according to claim 1, prepared by recovering the leukocytes by filtration of mamml whole blood or autoblood, to which aqueous trisodium citrate solution has been optionally added as an anticoagulant, on a cellulose filter, washing the filter with PBS, centrifuging the obtained leukocyte/thrombocyte suspension, and sepa-rating the leukocyte boundary layer.

4. The pharmaceutical composition according to claim 1, prepared by recovering the leukocytes from mammal whole blood or autoblood by means of leuko-pheresis.

5. The pharmaceutical composition according to claim 1, prepared by recovering the leukocytes by leaching mammal spleen or mammal thymus gland with PBS, to which trisodium citrate has been optionally added, centrifuging the obtained solution, and sepa-rating the leukocyte boundary layer.

6. A process for the preparation of a pharmaceu-tical composition containing T lymphocytes as the active substance by recovering the leukocytes from mammal whole blood, mammal spleen, or mammal thymus gland, or autoblood, separating the leukocytes on nylon wool, and recovering the concentrated T lympho-cyte suspension.

7. Use of a T lymphocyte suspension for the prep-aration of drugs for the treatment of viral and bacte-rial infections, which is prepared by recovering the leukocytes from mammal whole blood, mammal spleen, or mammal thymus gland, or autoblood, separating the leukocytes on nylon wool, and recovering the concen-trated T lymphocyte suspension.

8. The use of a T lymphocyte suspension according to claim 7 for the preparation of drugs for the treat-ment of AIDS and ARC.