DE3521733A1 - Use of preparations containing interferon-gamma (IFN-gamma) for the systemic treatment of tumours and viral diseases in low dosage - Google Patents

Abstract

The invention relates to the use of interferon-gamma (IFN-gamma) for the prophylaxis and systemic treatment of tumours and viral diseases in a dosage of 20,000 to 2 million international reference units (I.U.) (equivalent to about 2-200 mu g) as daily dose, to be administered at daily to monthly intervals, based on an adult patient weighing about 60 kg and having a body surface area of 1.7 m<2>.

Description

Use of interferon-gamma (IFN-gamma) containing

Preparations for the systemic treatment of tumors and viral diseases in low dosage All known interferons are due of homologies both between the nucleotide sequences of their structural genes and clearly divided into three groups between their amino acid sequences, which are called IFN-alpha; IFN-beta and IFN-gamma are designated.

The terms used follow the latest recommendation of "interferon" Nomenclature Committee, published by J. Vilcek 1983 in Archives of Virology Vol. 77, pp. 283-285. In addition to the main criteria mentioned above can be used to characterize the IFN-gamma group that no immunological Cross-reaction with the other two groups occurs as well as their biological instability at pH 2 in contrast to IFN-alpha and IFN-beta.

IFN-gamma is generated after stimulation of the total population of leukocytes, usually referred to as buffy coats according to the type of preparation, due to mitogens, Antigens or specific antibodies in a complex process together with a a large number of other factors (mediators) such as interleukin 1 (IL 1), interleukin 2 (IL 2), colony-stimulating factor (CSF), migration-inhibiting factor Factor (MIF), Macrophage Activating Factor (MAF), Tumor Necrotizing Factor (TNF) lymphotoxin (LT) and fibroblast growth factor (FGF). Other factors are described by J. A. Georgiades et al. in Came, P. E. and Carter, W.A. (eds.), Interferons and Their Applications, Springer Verlag, 1984, and by Waksman, B. H. in Cohen, S.

et al. (eds.), Biology of the Lymphokines, Academic Press Inc., 1979.

Those factors formed by the subgroup of lymphocytes are commonly referred to as lymphokines. IFN-gamma also belongs to this group calculated, which is mainly produced by T lymphocytes. The synthesis performance the IFN-gamma producing T-lymphocytes will in turn revert to the presence of the factors mentioned above.

There are also established or transformed cell lines that Produce IFN-gamma constitutively, as described by N. Fujii et al. in J. Immunology 130, Pp. 1683-1686, and A. Zlotnik et al.

in J. Immunology 131, pp. 794-800.

A preferred method for obtaining crude human IFN-gamma is described below. Lymphocyte-rich plasma fractions serve as the starting material of blood products, so-called "buffy coats", which are pooled and gently centrifuged plasma residues are removed from 600 - 800 g. The pelleted total leukocytes are suspended in preheated medium with a density of 5 million cells / ml. The cell suspension is aliquots in suitable culture vessels after addition a mitogen (e.g. phythemagglutinin) and phorbol ester (e.g.

Phorbol myristyl acetate (PMA) up to 70 hours at 37 degrees C on one Shaker incubated. The IFN-gamma-containing culture supernatants are purified by centrifugation and can be stored at 4 degrees C until further use. Usually the preparations obtained in this way contain around 10,000 international reference units (IU) IFN-gamma per ml.

The IFN-gamma content is determined by a CPE reduction test ir appropriate Indicator cells (e.g. WISH) with a test virus (e.g. EMC of the mouse) against the reference standard Gg 23-901-530 of the National Institute of Health (USA) as an antiviral activity of the Preparation determined.

Other methods of obtaining IFN-gamma preparations are available by Y.K. Yip et al. in "Infection and Immunity", Oct. 1981, pp. 131-133, vol. 34 and in U.S. Patents 4,376,821 and 4,376,822 and 4,460,685.

A fundamentally different way of obtaining human IFN-gamma preparations is carried out with the introduction of the human structural gene with the help of suitable vectors trodden into heterologous host cells. These recombinant genes become constitutive or expressed by the host cell after the addition of specific inducers.

Even if the structural gene is a clearly defined one Sequence of nucleotides acts, which are mandatory for the synthesis of a derived therefrom The amino acid sequence is the IFN-gamma molecule of the host cell prepared preparation not necessarily with a naturally occurring one IFN-gamma identical.

This possibility of variation while maintaining the specific effect by "post processing" is extended by the so-called protein design, by that the structural gene through chemical resp.

full biochemical synthesis or modification can be changed.

Bacteria can also be used as host cells to take up human genes (e.g. E. coli as described by G. Simons et al. in Gene 28, pp. 55-64, 1984) also Yeasts (e.g. Saccharomyces cerevisiae, as described by R. Derynck et al. In Nucleic Acids Research 11, pp. 1819-1837, 1983) or eukaryotic cells (e.g. Chinese Hamster Ovary Cells as described by Scahill, S. J. et al. in Proc. Nat. Acad. Sci.

USA 80, pp. 4654-4658, 1983, monkey cells as described by P.W. Gray et al. in Nature 295, pp. 503-508, 1982).

In some of these systems, either IFN-gamma is enriched up to a concentration of more than 100,000 I.U. per ml in the culture liquid or it is enriched in the host itself and provides up to 25 percent of the protein content there of the cell.

The enrichment or purification of the according to the above The IFN-gamma preparations obtained can be processed by the following methods individually or in combination: 1. Controlled Pore Glass (CPG) or silica gel; 2. Gel filtration (e.g. AcA 54 or Sephacel S 200); 3. Ion exchange chromatography (CM-Sepharose or phosphocellulose or DEAE-cellulose); 4. Affinity Chromatography (Con A-Sepharose or Poly-U-Sepharose or Cu-Chelate-Sepharose); 5. Immunoaffinity Chromatography (Anti-IFN-gamma-Sepharose); 6. HPLC (e.g. with reversed phase materials).

Corresponding procedures were carried out by Y.K. Yip et al. "Partial Purification and Characterization of Human Gamma (Immune) Interferon "in Proc. Nat. Acad. Sci. USA, 78, pp. 1601-1605, 1981, or by D. Novick et al., EMBO Journal 2, pp. 1527-1530, 1983, or in DE-PS 3136166 A1 described.

A cleaning up to electrophoretic is possible by suitable combinations Homogeneity possible, e.g. The maximum specific activity achieved is currently at 100 - 200 million I.U., while the described average values at 10 and 50 million I.U.

lie.

The interferons (IFN-alpha, IFN-beta, IFN-gamma) have been used in very different doses and modes of application in patients therapeutically used. Doses up to approximately 10 million administered once or several times per week I.U. are commonly referred to as low, and doses of 20-50 million I.U. as high.

In the early days of the therapeutic use of interferons stood due to the lack of suitable biotechnological manufacturing processes these substances only available in such small quantities that only doses of less than 3 million I.E.

could be administered. The use of higher doses is with IFN-alpha and IFN-beta since the mid-seventies, with IFN-gamma for 1-2 years. Since then, doses of less than 3 million have been used with systemic treatment I.E. usually only in tolerance and toxicity tests as well as in pharmacokinetic tests Studies used.

For IFN-alpha and IFN-beta, the same applies to systemic treatment of tumors and viral diseases doses up to approximately 3 million I.U. either ineffective or higher doses are inferior. Only with local treatment (intratumoral, peritumoral, intraventricular, intrathecal, intralesional, or perilesional intranasal application) show doses of less than 3 million I.U. locally effectiveness comparable to that of systemic application of higher doses.

In acute viral diseases such as herpes zoster, a dose has been added of 0.5 million I.U. IFN-alpha or IFN-beta per kg body weight and day proven, i.e. in adults approx. 30 million I.U. daily for chronic viral diseases like chronic active hepatitis B, 3-10 million I.U. IFN-alpha are usually required or IFN-beta given daily or 3 times a week.

Based on in vitro studies, IFN-gamma is considered to be less antiviral effective as IFN-alpha or IFN-beta. For this reason, IFN-gamma was chosen for systemic Treatment of viral diseases so far not used.

In our own clinical studies on the therapeutic effectiveness of IFN-gamma patients with various diseases were treated. Here doses of 50,000 I.U. up to 100 million I.U. were used for the first time administered systemically per week to achieve the optimally effective dose of IFN-gamma determine.

In these studies it was surprisingly found that, in contrast to IFN-alpha or IFN-beta the therapeutically most effective dose of IFN-gamma in the very low The range was from 100,000 I.U. to 2 million I.U. or approx. 10-200 pg. Both Patients treated with these low doses was more common found an effect of IFN-gamma than in those who received higher doses. The superiority of the very low over the higher doses was shown in 2 patients particularly impressive: the therapeutic effect of IFN-gamma disappeared when in them the dose was increased (Example 1, 2).

The effectiveness of IFN-gamma in the low dosage of 100,000 I.U. to 2 million I.U. has been shown in diseases as diverse as Hypernephroma: Example No. 1-3, Pseudomyxoma: No. 4, Mastocytosis: No. 5, Immunocytoma: No. 6, melanoma: No. 7, M. Hodgkin: No. 8, Schmincke tumor: No. 9, M. Crohn: No. 10, amyotrophic lateral sclerosis: No. 11, chronically active hepatitis B: No. 12 and Herpes zoster: No. 13, 14.

In particular, the effectiveness in viral diseases was surprising, since IFN-gamma not only in same Dosage like IFN-alpha or IFN-beta, but even in a much lower dosage when the other two types of interferon showed a therapeutic effect (Example 12-14).

For systemic treatment, natural IFN-gamma and recombinant IFN-gamma can be dosed as follows: intravenously as a bolus or over several minutes, Hours, days or weeks, intramuscularly and / or subcutaneously. Even with intratumoral, peritumoral, intralesional or perilesional application is used in addition to the local also achieved a systemic effect. A dose of 20,000 I.U. or approx. 2 pg up to 2 million I.U. or approx. 200 pg can be given once per day or portioned several times.

In the case of administration over several days, the dose can range from 0.02 to 2.0 x 106 I.U.

or about 2-200 pg are administered as follows: a) on consecutive Days b) every 2 to 6 days c) once per week d) every 2 to 4 weeks e) once per Month.

The dose of 0.02-2.0 x 10 IU refers to one patient 60 kg body weight and 170 cm body height, corresponding to 1.74 m2 body surface (Dubois and Dubois, Arch. Intern. Med. 17, 863, 1916). Accordingly, for the the individual dose is determined for each patient.

To increase the effectiveness of interferon gamma preparations the following substances can also be given: a) other interferons and / or others formed by leukocytes or produced by genetic engineering Cell mediators such as IL 1, IL 2, CSF, MIF, MAF, TNF, LT and FGF; b) alkylating agents, Folic acid antagonists, antimetabolites of nucleic acid metabolism, spindle poisons, Antibiotics, immunotherapeutics, pyrimidine analogs, purine nucleosides, amines and triazole nucleosides.

To produce the respective dosage form, the dem EXAMPLE 1 a person skilled in the art used auxiliaries.

Patient: G. S. male Diagnosis: Hypernephroma with bone metastasis Severe pain in the shaft of the right upper arm and poor mobility in the right Poor substance: human IFN-gamma preparation from E. coli Method of application: intramuscular Therapy regimen: 100 pg 4 times a week for 2 weeks, then increasing the dose to 200, 400, 800 and 1 600 pg then 2 100 pg once a week for 4 weeks Result: After the 1st injection of 100 pg the pain was significantly reduced, and the The patient was able to move the arm much better after the 8th injection of 100 pg he had no more pain.

Four days after the 1st injection of 2100 pg, pain returned in the upper arm.

EXAMPLE 2 Patient: P. H. male Diagnosis: Hypernephroma with pulmonary and bone metastasis pain in the pelvic region and the lumbar spine-sacrum area Substance: human IFN-gamma preparation from E. coli Application way: In ~ ramuscular therapy regimen: 100 pg 4 times a week for 2 weeks, then increase the dose to 200 µg Result: After the 2nd injection of 100 µg the pain was gone significantly decreased and decreased further after the 3rd injection of 100-> g. To the 4th injection of. At 200 pg the pain came back increasingly.

EXAMPLE 3 Patient: W. R. male Diagnosis: Hypernephroma with cutaneous, pulmonary and bone metastases psoriasis vulgaris Substance: human IFN-gamma preparation from E.coli and difluoromethylornithine (DFMO) Application: IFN-gamma intravenous, DFMO oral therapy scheme: IFN-gamma initially 50 µg daily for 10 days, then daily 100 µg for 7 days, DFMO 3 x 4 g daily Result: On the 7th day the pain subsided back, on the 14th day the patient was completely free of pain. Formed from the 12th day the psoriasis vulgaris receded and had almost completely disappeared by the end of therapy.

When the patient was discharged after 17 days of therapy, there was a partial Recognition of the regression of the cutaneous and liver metastases. The general condition the patient had improved significantly.

-BE SHEI- 4 Patient: F.D. female Diagnosis: Pseudomyxoma peritonei with 4 fistulas in the abdominal wall Substance: human IFN-gamma preparation from E. coli Method of application: intramuscular therapy regimen: 100 pg 4 times a week for 2 weeks Result: After the 3rd injection of 100 pg, 2 of 4 fistulas had closed.

EXAMPLE 5 Patient: K. W.

Diagnosis: malignant mastocytosis with diffuse bone marrow and liver infiltration and extramedullary blood formation Substance: human IFN-gamma preparation from E. coli Method of application: intravenous Therapy regimen: 50 pg 5 times a week for 5 weeks Result: After 5 weeks of therapy there was a decrease in volume of the enlarged Liver and a drop in alkaline phosphatase from 1,700 U / 1 to 1,000 U / 1 (normal range up to approx. 190 E / 1).

BEIrPw 6 patient: U. F.

Diagnosis: progressive polymorphic cell immunocytoma Substance: human IFN-gamma preparation from E. coli Method of application: intravenous Therapy scheme: 50 pg 5 times a week for 4 weeks Result: The disease was over after 2 weeks of therapy no longer progressive, after a further 2 weeks the lymph node swellings subsided return.

EXAMPLE 7 Patient: H. E.

Diagnosis: skin metastasis the size of a fist from a malignant melanoma, primary tumor surgically removed Substance: human IFN-gamma preparation from E. coli Method of application: intravenous therapy regimen: 100 pg 3-Sx / week for 5 weeks Result: after 3 weeks Therapy had reduced the metastasis by 90% and then remained stable.

-BESTEL 8 patient: G. K. male diagnosis: M. Hodgkin stage IVb, Metastases in the liver, lungs, bone marrow and abdominal lymph nodes Substance: human IFN-gamma preparation from human leukocytes Method of application: initially subcutaneously, then intravenous therapy scheme: daily for 3 weeks, dose from 0.01x106 to 1.0x10 I.E. increasing Result: At the end of therapy, there was a significant reduction in the size of the liver metastases ascertain. In addition, the B symptoms had improved.

EXAMPLE 9 Patient: G. P. male Diagnosis: Schmincke tumor with Recurrence of the lung metastases Substance: human IFN-gamma preparation from E. coli Application: 0.5x106 I.U. daily except on Saturdays for 12 weeks Result: After 12 weeks of therapy there was a partial remission of the extensive relapse of lung metastases.

The patient could be discharged from the clinic.

B S EL íC Patient: I. B. male Diagnosis: M. Crohn Substance: human IFN-gamma preparation from E. coli Application: intramuscular or intravenous Therapy regimen: 3-4 times a week for 8 weeks, dose of 0.02 up. 0.5 x 106 I.U. Increasing result: The Crohnin.dex had fallen from 290 to 59 as a result of the therapy. (Several factors go into the Crohn index, including

Number of stools, abdominal pain, general well-being, weight. These are assessed with points: the higher the number of points, the more pronounced it is the disease.) EXAMPLE 11 Patient: W. W. male Diagnosis: Amyotrophic lateral sclerosis Substance: human IFN-gamma preparation from E. coli Method of application: intramuscular Therapy regimen: 3 times a week for 4 weeks, dose from 0.1 x 106 to 1 x 106 I.U. increasing.

Then 0.5 x 106 IU once a week for 4 weeks.

Result: Under -herat'ie the functions of the upper ones improved Extremity. The hands could be bent and straightened again, and they were also moderate Extensions and flexions as well as slight rotations in the elbow joint are possible. A slight elevation was again possible in the shoulder joint.

EXAMPLE 12 Patient: M.K. female Diagnosis: chronic active hepatitis B Substance: human IFN-gamma preparation from E. coli Method of application: intravenous Therapy scheme: 5 times a week for 10 weeks, dose from 0.05 x 106 to 2.0 x 106 I.E. increasing result: During the therapy, the DNAP decreased from 17,422 to 5,546.

EXAMPLE 13 Patient: E. S. female Diagnosis: Herpes zoster oticus with hearing impairment and facial paralysis Substance: human IFN-gamma preparation from leukocytes Method of application: intramuscular therapy regimen: 0.5 x 106 I.U. 2 times per day for 5 days Result: After 3 days of therapy, the herpes zoster and the function of the nerve had almost completely healed had normalized.

EXAMPLE 14 Patient: T. S.

Diagnosis: Herpes zoster oticus with facial nerve palsy Substance: human IFN-gamma preparation from E.coli Application: intramuscular Therapy scheme: 100 µg twice a day Result over 5 days: After 5 days of therapy, the zoster had almost healed, and the nerve had normalized.

Claims (17)

Claims 1. Use of preparations containing interferon-gamma for the systemic treatment of tumors and viral diseases in the dosage of 20,000 to 2 million international reference units (I.U.) (corresponds to approx. 2-200 pg) as a daily dose to be administered at intervals of daily up to monthly, based on an adult patient of about 60 kg body weight and 1.7 m2 body surface. 2. Use according to claim 1 for the treatment of solid tumors and hemoblastosis. 3. Use according to claim 1 for the prevention of recurrence of solid tumors and hemoblastosis. 4. Use according to claim 1 for the treatment of acute viral diseases. 5. Use according to claim 1 for the treatment of chronic viral diseases. 6. Use according to claim 1 for the prophylaxis of acute viral diseases. 7. Use according to claim 1 for the prophylaxis of chronic viral diseases. 8. Use according to claim 1 for the treatment of amyotrophic lateral sclerosis. 9. Use according to claim 1 for the treatment of Crohn's disease and Pseudomyxoma. 10. Use according to claim 1 to 9, characterized in that the interferon-gamma from human cells or by genetic engineering was obtained. 11. Use according to claim 1 to 10, characterized in that the preparation containing interferon-gamma additionally other interferons and / or others formed by leukocytes or produced by genetic engineering processes Contains cell mediators. 12. Use according to claim 1 to 11, characterized in that in addition, others previously used in the therapy of tumors and viral diseases Substances such as alkylating agents, folic acid antagonists, antimetabolites of nucleic acid metabolism, Spindle poisons, antibiotics, immunotherapeutics, pyrimidine analogues, purine nucleosides, Amines and triazole nucleosides are administered. 13. Use of preparations containing interferon-gamma one of claims 1 to 12, characterized in that the preparation in one administration form to be administered intravenously, intramuscularly or subcutaneously. 14. Use according to claim 13, characterized in that the dem Depot forms known to those skilled in the art for polypeptide therapeutic agents are administered. 15. Single dose as a lyophilisate or in aqueous form, if appropriate with the usual auxiliaries and carriers for the systemic treatment of tumors and Viral diseases, characterized in that they contain interferon-gamma in one dosage Contains from 20,000 to 2 million International Reference Units (IU). 16. Single dose according to claim 15, characterized in that it in the form of a nasal spray, inhalation spray or sublingual tablet. 17. Multiple dose according to claims 15-16, characterized in that that it is in a production form that is a multiple of 20,000 to 2 Contains millions of international reference units.