DE19544167A1 - Use of interferon-ß for the treatment of bronchial carcinoma in radiation therapy - Google Patents

Abstract

The invention relates to the use of an interferon- beta which is administered during a course of radiation therapy and during the break in therapy. The interferon- beta can be used to produce medicaments for treating bronchial cancer and non-small-cell lung cancer.

Description

The present invention relates to the use of an interferon-β for Manufacture of a medicament for the treatment of bronchial cancer, especially non-small cell lung cancer during and after the irradiation.

State of the art Bronchialkrebs

Every year, about 150,000 patients develop lung cancer in the United States, with most of the advanced patients within die one year after the first diagnosis. Lung cancer is taking in the Number of infested patients too. The histological classification of primary lung neoplasms by the World Health Organization 1977 recommended, should be used in the further. The different Cell types have different natural evolutionary histories and they react differently to therapeutic approaches. Therefore, a correct one histological and etiological classification for the healing efforts necessary. Two major groups of tumors are dominant: Small cell tumors (small-cell carcinoma) and non-small cell tumors (non-small cell tumors) Small-cell carcinoma). The latter group includes the epidermoid form, the Adenocarcinoma and large cell carcinoma. there The group of non-small-cell carcinomas will be treated with both radiotherapy and also treated by surgical means. It has turned out that the non- Small-cell carcinoma is not particularly responsive to chemotherapy.

Cytostatic agents are detailed in BRUHN: cytostatics fibula, Stuttgart: Schattauer 1985 and KUSCHINSKY and LÜLLMANN: Short Textbook of Pharmacology and Toxicology, Stuttgart: Thieme Verlag, 1987, pages 481-490.

The majority of lung tumors are caused by inhaled carcinogens and by Tumor promoters caused by cigarette smoke in the bronchi and the lungs arrive. The bad prognosis for most patients with Lung cancer requires the increased effort, tailored, specific Develop and develop therapeutic approaches. Combinations from under different treatment methods are available, but due to the  poor predictability and the problematic prediction of Therapy success perspective individual treatment methods alone for lead desired goal.

Lung cancer is not considered a disease that is primarily genetic is conditional. The high number of molecular genetic damage in the Tumor cells show that lung cancer, like other cancers of the epithelium tissue, a multi-stage process in which both carcinogens and Tumor promoters are responsible for tumor growth.

In patients with non-small-cell-lung cancer of stages I and II, the one Surgery, the removal of parts of the lungs makes sense. In stage IIIa, in which the tumor is already advanced, should be under Pay attention to the cardiopulmonary functions larger parts of the lungs, parts of the circulatory system and the bronchi.

The use of chemotherapy in Non-Small-Cell-Lung Cancer requires one Careful consideration of the benefits and toxicity of the treatment properly assess. About 30 to 40% of patients can be treated with one Combination of various substances from the group of chemotherapeutic agents treat successfully. However, only in less than 5% of cases is one to observe complete decline of the tumor. Such patients have a legitimate prospect of a significantly increased survival rate compared with the patients who are not on the chemotherapeutic agents react. Basically, however, those who are good at chemo responding patients even without treatment a generally better Forecast.

In particular, patients who are in stages I to III are on the radio suitable for therapy. This treatment is then exclusively turned on sets if surgery is not possible or associated with complications is.

Radiation therapy and radiation break

Radiation therapy can draw on many years of experience. So the radiation field and the dose size in the area of therapy is clear  outlined. DIN 6814 of 3 December 1985 (German Industrial Standard) a well-defined framework for the course of radiation therapy before.

Irradiation therapy thus consists of a large number of each other following irradiations, which closed in itself a therapy unit form.

An irradiation break includes the periods between Irradiation therapies is.

interferon

The nomenclature used in Nature, 286, p 2421 (1980) for the group of Interferons should be used. Two classes of interferons are known. Interferons of class I are small, acid-stable glycoproteins, the cells re resistant to viral infections. Class II interferons are acid labile. Three groups of interferons are known: interferon-α, interferon-β and Interferon-γ. The structure of interferon-β with respect to the DNA sequence and the Amino acid sequence is disclosed in European patent application EP-0 028 033 described. Interferon-β shows biological activity in glycosylated or un glycolized form. (W.E. Stewart et al. (1979) Virology 97: 473-476). Inter Ferron-β is usually not detectable in normal or healthy cells. Only when these cells are exposed to interferon-β inducers, the Interferon-β expressed. Usually, viruses are good interferon-β inducers however, non-viral inducers are also known (S. Baron and F. Dianzani (eds.) (1977) Texas Reports to Biology And Medicine, 35: ("Texas Report") pp 526-540.

Human interferon has long been used successfully in the treatment of some viral infections and cancers.

In addition to the human interferon-β derivatives are known, which are characterized by the Characterize exchange of amino acids. Thus, in the European patent application EP 0 218 825 a recombinant interferon-β described in position 17 has a serine instead of a cysteine. This modification also has biological activity.

Another modified, human interferon-β is known that via a Deletion in position 1, via a substitution in position 17 by  Serine and has a non-existent glycosylation. These too Substance is biologically active.

Combination of interferon-β and radiotherapy

A small number of investigations were made to the combination of interferon-β to combine with the radiation therapy.

DRITSCHILO et al. At the. J. Clinic. Onc. 5: 79 (1982) investigated the effect of Interferon-β on the irradiation reaction of mouse cells 3T3 in tissue cultures. A species-specific amount of radiation kills the 3T3 cells. In this case, mouse L-cells interferon was added to the cultures, whereby the survival rate of the cells was increased. However, the effect was not then convincing when the cells were irradiated in the sub-lethal range.

NAMBA et al. Cancer 54: 2262 (1984) confirmed the results of DRITSCHILO by using the HeLa cells.

GOULD et al., J. Interferon Research 4: 123 (1984) observed an in vitro Irradiation sensitization in human bronchial carcinoma by means of interferon β.

In a combined treatment with radiation and interferon-β obobach NEDERMAN et al. Acta Radiologica Oncology, 21: 231 (1982) a strong Growth decrease in human glioma cell cultures compared to cells, which have been treated either with interferon-β or with irradiation.

MAHALEY et al. J. Bio. Res. Modifiers 3: 19 (1984) reports a Phase I Study in a combined treatment with interferon-β and radiation. He treated nine patients who had an anaplastic glioma tumor possessed. The therapy successes were with the combined treatment with BCNA, a cytostatic, and comparable to radiation. Other reports show an unexpected increase in toxicity when combined Therapy is used.  

Object of the invention

It is an object of the invention, the targeted use of an interferon-β in the context of providing radiation to patients with Bron to treat chial cancer or non-small-cell lung cancer.

Solution of the task

The object is achieved by using at least one interferon-β for the manufacture of a medicament for the treatment of a patient with Bronchial cancer solved,

• (i) wherein the patient is concurrent with radiation therapy
  • a) interferon-β or
  • b) at least one cytostatic or
  • c) a combination of interferon-β and at least one cytostatic
• receives, and
• (ii) wherein the patient additionally
  • (a) during at least part of the period of exposure, or
  • (b) during at least part of the time after the radiation therapy
• Interferon-β receives.

A use according to the invention of at least one is preferred Interferon-β for the manufacture of a medicament for the treatment of a Patients suffering from bronchial cancer form Non-Small-Cell-Lung Cancer is.

Surprisingly, it has been found that in a continuous Treatment of Bronchial Cancer or Non-Small-Cell-Lung Cancer with the help of interferon-β tumor growth was inhibited more effectively than if only during irradiation therapy interferon-β was administered. Apparently, the interferon-β has on tumor cells which have survived a radiation therapy, a more effective effect than at unirradiated cells. The sensitization does not only take place during the Irradiation, but also works after the radiation therapy.  

It was unexpected that the effect of interferon-β sensitivity after the Bestrah lung therapy continues. This suggests a maximum dose of interferon-β. The micrometastases may be interferon-β therapy especially accessible. It was only known that the interferon-β - Treatment increases the radiation sensitivity.

The invention further comprises an interferon-β, which is the human Inter ferron-β or a derivative thereof.

The various modified biologically active interferons show that the Change of one or more amino acids is not necessarily too must result in a failure or a change in the function. Thus, the The term recombinant interferon-β both the sequence and the glycosylation of human interferon-β as well as interferon-β-derivatives. To the derivatives count all allelic modifications that lead to a change in the ami Nosäure sequence, provided that these modifications, the substitutions, the Deletions and / or insertions of up to 15 amino acids. Preferred are deletions, substitutions and / or insertions of up to 10 amino acids, more preferably up to 6 amino acids, most before are the deletions, substitutions and / or insertions of one, two, three, four or five amino acids.

Preferred is a use of an interferon-β, which is a derivative of the is human interferon-β and wherein the derivative

• a) includes all allelic modifications of human interferon which Modifications lead to a change in the amino acid sequence, wherein at least one, at most 15 amino acids substituted, deleted or are inserted without altering the activity of the modified interferon-β to significantly influence compared to the test interferon-β and
• (b) includes all post - translational modifications which do not significantly affect the Activity of the active modified interferon-β compared to the test Interferon-β influence.

More preferred is the use of an interferon-β in which at most Substituted, deleted or inserted 10 amino acids, without the Aktivi the modified interferon-β compared to the test interferon-β essential to influence.  

Very preferred is the use of an interferon-β in which at most 6 amino acids are substituted, deleted or inserted without affecting the activity of the modified interferon-β compared to the test interferon-β substantially to influence.

Even more preferred is the use of an interferon-β in which one, two, three, four or five amino acids are substituted, deleted or inserted, without affecting the activity of the modified interferon-β compared to the test To significantly influence interferon-β.

More preferred is the use of an unglycosylated or glycosylated Interferon-β.

Highly preferred is the use of an interferon-β which is unglycosylated is a substitution in position 17 with serine and a deletion in the Position 1 has. This interferon defines the test interferon-β. A Test method is described in T. TANIGUCHI et al. (1980) 77: 5230-5233 and in D.F. MARK et al. (1984) 81: 5662-5666.

Allelic modifications

Most deletions, insertions, and substitutions do not seem to go through significant change in the characteristics of the protein of the invention result to have. Because it's hard to know the exact effect of a substitution, a dele tion or insertion in advance, the function of the alter interferon-β compared with the function of the modified interferon-β who the. The standard is the modified, recombinant interferon-β (test Inter ferron-β) with the deletion in position 1, the substitution in position 17 by serine and the lack of glycosylation.

The genetic code is degenerate, which means that most amino acids be encoded by more than one codon of three nucleotides. Therefore, feel Some allelic modifications at the nucleotide level do not lead to one Change the amino acid sequence. Therefore, allelic modifika occur These are mainly at the level of DNA and can become secondary to DNA Affect amino acid sequence.  

Amino acids can be substituted as shown in Table 1, without while significantly affecting the function of the protein. In each one Case is to be decided by the activity test, which influence the changes tion on the function of the protein.

The functions or the immunological identity are significantly changed, if substituents are chosen which are less congestive when substituted are more conservative than the amino acids shown in Table 1. Such essential Changes can be made by substituting with amino acids, which differ more in their structure and functional groups. Significant changes have the effect that the dreidimen the structure is changed and / or that, for example, the leaflet structure or the helical structure is affected. Also interactions of the Ladun and the hydrophobic chains are to be considered in the changes.

Table 1 Usual substitution of amino acids in a protein Original amino acid For example, Substitution carried out Ala Gly, Ser bad Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala, Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu mead Leu, Tyr, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

The mutations are compared by the homology (similarity) of two defined proteins. The term homology includes similar ami nosäuren (for example Table 1) and gaps in the sequences of the Ami  nosäuren (homology = similarity). The proteins of the invention have Amino acid sequences that have a homology of at least 80% preferred 90%, more preferably 95%, and most preferably 98% of the erfindungsge may have structures as defined by the sequence of the recombinant modified interferon-β with Ser¹⁷ (test interferon-β) is defined.

Posttranslational modifications

The aforementioned post-translational modifications are understood Changes that occur during or after translation. Tough for that glycosylation, the formation of disulfide bridges, the chemical Modifications of the amino acid, such as sulfation, occurring in the Zu is described in connection with the hirudin. (J.W. Fenton (1989) "Thrombin Interactions with Hirudin ", Seminars in Thrombosis and Hemostasis 15: 265-268).

Glycosylation is an essential function of the endoplasmic reticulum lum and / or Golgi apparatus. The sequences and the ramifications of Oligosaccharides are formed in the endoplasmic reticulum and in changed the Golgi apparatus. The oligosaccharides can be N-linked Oligosaccharides (asparagine-linked) or O-linked oligosaccharides (Serine, threonine or hydroxylysine-linked). The form of glycosidia depends on the producing cell type and the species of which the corresponding cell type is derived. The extent and nature of glycosylation can be influenced by substances, as it is in the European public EP 0 222 313 is described. The variation of glycosylation can be the Change the function of the protein.

Proteins often form covalent bonds within the chains. This di Sulfide bridges are made between two cysteines. This is the Protein specifically folded. The disulfide bridges stabilize the dreidimensio nale structure of the proteins.

Furthermore, the amino acids can be changed as it is in the interna International Publication WO 91/10684. Likewise, the protein be sulfated. This change is related to hirudin ben.  

The invention further includes the use of an interferon-β, the a proportion of biantennary oligosaccharide structures of at least 60%, a proportion of treatennary oligosaccharide structures of at least 15% and a proportion of tetraantennary oligosaccharide structures from 0% to 5% and has a Silainsäuregehalt of at least 80%.

Also very preferred is the use of an interferon-β, the gly is a proportion of biantennären oligosaccharide structures of min at least 60%, a proportion of triatheneic oligosaccharide structures of at least 15% and a proportion of tetraantennary oligosaccharide structures from 0% to 5% and a sialic acid content of at least 80%.

Furthermore, the invention includes the use of an interferon-β, the a proportion of biantennary oligosaccharide structures of at least 60% more preferably 70% and most preferably at least 75% include.

Also, the invention encompasses the use of an interferon-β, the a proportion of triantennary oligosaccharide structures of at least 15%, preferably 20%, and most preferably at least 25%.

An interferon-β is advantageous, the triantennary structures being at least have an N-acetyllactosamine repeat. The triantennary structures may be linked 1-4 and / or 1-6. The tetraantennary fraction can be 0.5% to 3%.

Furthermore, the invention includes the use of an interferon-β, the a sialic acid grade of at least 80%, preferably 85% and most preferably more than 90%. This can be the sialic from N-acetylneuraminic acid and N-glycolylneuraminic acid. there For example, N-acetylneuraminic acid may be 90-100% and N-glycolylneuraminic acid 0-10% of the total sialic acid take.

Furthermore, the invention includes the use of an interferon-β, the a proportion of fucose content of at least 85%, preferably 90% and am most preferably greater than 95%.  

Furthermore, the invention includes the use of an interferon-β, the at least one oligosaccharide structure having one or more of the following Formula include:

In the previous formulas, NeuAc can also be used for N-glycolylneuraminic acid stand.  

Publication of European Patent Application EP 0 529 300 (Filing date 21. 7. 1992 with the application number 92 112 427.7) describes the previously listed forms of glycosylated interferon-β in detail and will cited in the description by the citation and thereby part of Be scription.

The invention includes the use of an interferon-β, the phar Makologische excipients and carriers, which are physiologically compatible, includes.

Pharmacological excipients and carriers are described in Remington's Pharmaceutical Science, 15 ^th ed. Mack Publishing Company, Easton Pennsylvania (1980).

The weight ratio of the agent according to the invention can be used in the application be varied for the described indication within wide limits.

The invention further includes the use of at least one inter feron-β, where the patient is in the total time after an irradiation pie interferon-β receives.

Also, the invention includes the use of at least one Inter feron-β, where the patient is in the total time between the irradiation receives interferon-β.

The interferon-β is usually until the onset of tumor recurrence or administered about two years after the last radiation therapy. Individual modifications are made.

The invention further provides

• (i) a method for the treatment of bronchial cancer, in particular non- Small-Cell-Lung Cancer, which procedure involves administration of a Substance amount of at least one interferon-β according to the Invention, wherein the amount suppresses the disease, and wherein the amount of substance is given to a patient who is one Drug required and wherein the patient interferon-β during the Irradiation therapy and additionally during at least one part the radiation break or during at least part of the time receives after the radiation therapy;  
• (ii) a pharmaceutical composition for the treatment of Bron chialcancer, especially non-small-cell-lung cancer, which treat ment an inventive interferon-β and at least one pharma ceutically acceptable carrier and additive, wherein the patient Inter feron-β during radiation therapy and additionally during at least part of the radiation break or during at least part of the time after the radiation therapy.

For this therapeutic effect, the appropriate dose is different and depends, for example, on the interferon-β, the host, the mode of administration and the nature and severity of the conditions to be treated.

In general, however, in larger mammals, for example Men satisfactory results can be expected with daily doses of 10⁴ up to 10⁸ units of interferon-β.

Preference is given to values of 10⁵ to 4 × 10⁷ units (interferon-β) per 48 hours more preferably 8 × 10⁵ to 2 × 10⁷ units (interferon-β) per 48 hours and most preferably 4 x 10⁶ to 8 x 10⁶ units (interferon-β) per 48 Hours.

The active ingredients can be added with the usual additives in galenic pharmacy Carrier substances and / or flavoring agents according to known Methods are processed to the usual application forms.

For oral administration in particular tablets, dragees, capsules, Pills, suspensions or solutions in question.

For parenteral administration are oily solutions such. B. sesame oil or Ri zinuso solutions, suitable. To increase the solubility can be solutions mediators, such as Benzyl benzoate or benzyl alcohol.

Interferon-β can be used in systemic treatment by any usual route be administered, in particular injection solutions or suspensions the appropriate forms for administration.

The use of the modified interferon-β is the most preferred Combination.  

Even more preferred is the use of a human interferon-β, which a proportion of at least 60% of biantennary oligoscopic structures, a proportion of treatennary oligosaccharide structures of at least 15% and a proportion of tetraantennary oligosaccharide structures from 0% to 5% and has a Silainsäuregehalt of at least 80%.

These two substances are used, for example, in larger mammals, z. As humans, administered in the manner previously illustrated. The infusion solution as a continuous infusion in conventional aqueous solvents, for. B. physiological Saline is the preferred administration for systemic treatment chung form.  

Examples

The interferon-β can be used effectively in the following tests:
Composition of an Interferon β Injection 9.6 x 10⁶ units of recombinant modified interferon-β (Met ^-1 ; Ser¹⁷ and unclycosylated) corresponding to 0.3 mg of the active substance are taken up in 0.54% saline. 15 mg of human albumin and 15 mg of dextrose are added. Per 0.1 kg of body weight, 1.2 x 10⁴ units are administered per 48 hours. The injectable amounts are taken up by adding the small concentrated daily dose to a solution of 0.54% saline with 15 mg human albumin and 15 mg dextrose to maintain injectable units of 0.2 to 0.5 ml ,

radiotherapy

The irradiation is carried out according to standard methods, such as by the AECL-Theratron 80, Varian Clinac 4 or Varian Clinac. The maximum area which is irradiated at a time, should not be larger than 300 cm² be. A useful dose is up to 60 Gy, with the specific Dose must be modified according to the body parts.

Experimental Procedure patient clientele

Patients who are suitable for the experiment, can be out be chosen. Histologically, a tumor must be identifiable. there it should be clarified that the tumor is stable.

Two different populations are formed.

• a) patients who are irradiated with a defined dose and during receive this treatment time with irradiation interferon-β, and
• b) patients who are irradiated with a defined dose and during and after this treatment time with irradiation, interferon-β was obtained. The Interferon-β is thus administered continuously in this group.

Formulation and application of the test substance

The test substance interferon-β is in the form of (6-9) · 10⁶ international Units injected three days a week. In this case, 0.3 ml should be subcutaneous be sprayed.

The irradiation is 60 Gy. It takes place over 6 weeks with a daily Load of 2 Gy.

After radiation therapy, interferon-β becomes 6 · 10⁶ international Units sprayed once a week. Interferon-β therapy lasts preferably 2 years.

evaluation

Mean values ± SE of tumor areas, tumor weights and, if necessary Organ weights are determined. The growth course of the tumors becomes shown graphically.

The statistical evaluation is done with the Dunnett test.

The observed tumor growth inhibition or remission of the tumors at Treatment with the agent according to the invention during the Bestrah Breakthrough pauses are those that occur only during the radiation treatment Inter have received feron-β, superior. That patients under point b) have exhibit a growth inhibition against the patients under a).

Claims (9)

1. Use of at least one interferon-β for the manufacture of a medicament for the treatment of a patient with bronchial cancer

• (i) wherein the patient is concurrent with radiation therapy
  • a) interferon-β or
  • b) at least one cytostatic or
  • c) a combination of interferon-β and at least one cytostatic
• receives, and
• (ii) wherein the patient additionally
  • (a) during at least part of the period of exposure, or
  • (b) during at least part of the time after the radiation therapy
• Interferon-β receives.

2. Use of at least one interferon-β for the preparation of a Medicament according to claim 1 for the treatment of a patient, in which the bronchial cancer is the form Non-Small-Cell-Lung Cancer. 3. Use of an interferon-β surface according to one of the preceding Ansprü, wherein the interferon-β is a derivative of human interferon-β and wherein the derivative

• a) all allelic modifications of human interferon-β, which modifications result in a change of the amino acid sequence, wherein at least one, at most 15 amino acids are substituted, deleted or inserted, without affecting the activity of the modified interferon-β compared to the test Interferon-β significantly affect and
• b) includes all post-translational modifications that do not significantly affect the activity of the active modified interferon-β compared to the test interferon-β.

4. Use of an interferon-β according to one of the preceding Ansprü che, wherein the interferon-β is unglycosylated or glycosylated. 5. Use of an interferon-β according to claim 4, which unglycosylated is a substitution in position 17 with serine and a deletion in has the position 1.   6. Use of an interferon-β according to claim 4, which comprises a portion at biantennären Oligosccharid structures of at least 60%, one Proportion of treatennary oligosaccharide structures of at least 15% and a proportion of tetraantennary oligosaccharide structures of 0% to 5% and a Silainsäuregehalt of at least 80%. 7. Use of an interferon-β according to one of the preceding Ansprü where the interferon-β is pharmacological auxiliaries and excipients, which are physiologically compatible. 8. Use of an interferon-β according to one of the preceding Ansprü the patient in the entire time after an irradiation therapy receives interferon-β. 9. Use of an interferon-β according to one of the preceding Ansprü che 1 to 7, wherein the patient in the entire time between the Be radiation therapy interferon-β.