KR100399500B1 - General method of shortening the duration of common colds by application of medicaments to tissues of oral cavity - Google Patents

Abstract

The present invention relates to an interferon composition for contacting a mammalian oral mucosa with an interferon in an amount greater than that administered parenterally to stimulate a host defense mechanism or immune response, or an amount of interferon that stimulates the immune response. And to the use of interferon in the preparation of such oral mucosal compositions.

Description

Interferon composition for oral mucosal administration for stimulating host defense mechanism or immune response {GENERAL METHOD OF SHORTENING THE DURATION OF COMMON COLDS BY APPLICATION OF MEDICAMENTS TO TISSUES OF ORAL CAVITY}

α-interferon can be used for various malignant hematologic malignancies (eg, hairy cell leukemia, chronic myeloid leukemia, lower lymphoma, cutaneous T-cell lymphoma) and solid tumors (eg, renal cell carcinoma, melanoma, carcinoid tumor). And AIDS-related Kaposi's sarcoma, etc.) (see Gutterman JU, Proc. Natl. Acad. Sci. USA , 1994 91 : 1198-1205). Interferon-α (IFN-α) administered by parenteral injection typically exhibits an anti-tumor effect at high doses, often at dosage levels of several tens of millions. Interferon-β (IFN-β) is licensed for clinical use in the treatment of relapsing-remitting multiple sclerosis and chronic hepatitis B and hepatitis C.

Interferon-α and interferon-β are both type I interferons. Type I interferon is one large class of naturally-occurring cytokines comprising about 16 subgroups of IFN-α, IFN-β and IFN-ω. Type I interferons bind to single cell surface receptors and stimulate multiple sequences of signal transduction, ultimately anti-viral, anti-proliferative and other immunomodulatory effects, cytokine induction, and regulation of class I HLA and class II HLA (See Pestka et al. , Annu. Rev. Biochem. , 1987 56 : 727). Individual subtypes of type I IFN vary in activity. The most frequently observed amino acids at each position were identified by injecting multiple allele subtypes of IFN-α, and synthetic type I interferons with consensus sequences were synthesized (Alton et al., The Biology of the Interferon System, "edited by E. de Maeyer and H. Schellekens, Elsevier (1983) 1991-128. Such consensus interferon is commercially available [Infergen; Amgen, Inc., recently proposed to have higher activity (weight / weight) than IFN-α2a or IFN-α2b; Consensus IFN has been suggested to be clinically superior to the IFN of individual native subtypes (see Blatt et al., " J. Interferon and Cytokine Reserch ", 1996 16 : 489-499).

Although many routes of administration are commonly used for the administration of type I interferon, including intravenous injection, subcutaneous injection, intramuscular injection, topical injection and intralesional injection, oral administration has not been generally used, as interferon proteolysis This is because they are considered to be inactivated by enzymes and are poorly absorbed in their natural form in the gastrointestinal tract. Indeed, many studies to detect interferon in blood after oral administration have failed [Cantell and Phyhala, " J. Gen. Virol ." 1973 20 : 97-104; Wills et al., J. IFN Res ., 1984 4 : 399-409; Gilson et al., J. IFN Res ., 1985 5: 403-408.

It is well recognized that interferon should be used at the highest possible dose to obtain the maximum therapeutic effect. Although very high dosage levels can be achieved with recombinants, it has been found that the dosage and duration of treatment of interferon, which can actually be used by the side effects of interferon administration, are quite limited. These side effects include severe malaise and depression, which in some cases even cause suicide. This issue is summarized in a recent article "New England Journal of Medicine" by Hoofnagle (JH) [Hoopnagle and Lau, D., " New Eng. J. Medicine " 1996, 334. ; 1470-1471]. Meta-analysis of the therapeutic effect of interferon-α in patients with hepatitis B antigen-positive chronic hepatitis B results in 5 million IU daily for 3 to 6 months, or 10 million IU per week. Relief rates were 25 to 40% in patients with typical chronic hepatitis B treated three times. However, this results in a lack of treatment if most patients are positive for hepatitis surface antigen and have viral DNA latent when tested by a polymerlace chain reaction. In addition, such doses of interferon are poorly tolerated and 10 to 40% of patients have to reduce the dose due to serious side effects. However, at a good tolerant dose of 1 million IU daily, the remission rate is only 17% (see Perrillo et al., New Eng. J. Medicine ", 1990, 323 : 295-301). In patients with chronic hepatitis C, sustained long-term improvement is associated with a reduction in HCV RNA, which is 10-20% of patients treated three times at a dose of 3 million IU per week for 6 months. (Hoopnagl and Lau, see above citation). In patients with cancer, significant response rates usually appear only at the highest tolerated doses of interferon-α. Thus, in patients with multiple myeloma, for example, the response rate is 50% for patients treated with 20-30 million IU daily, and only 15-20% for patients treated with 3 million IU. However, very few patients can tolerate high-dose care for a period of time (see Ahre et al., Eur. J. Hematol , 1988, 41 : 123-130). Thus, there is a need in the art for methods of administering high doses of interferon without causing serious side effects.

There are many reports of anecdotes of the efficacy of interferon administered at low doses as nasal sprays or oral liquid formulations in the treatment of many viral diseases, especially influenza. However, for most of these reports, the interferon formulations used were relatively crude. According to a placebo-controlled trial of relatively high dose intranasal interferon for the treatment of rhinovirus infection, the treatment was effective but caused significant side effects (Hayden et al., J. Infect. Dis . ", 1983 148 : 914-921". Similarly, a number of studies, including two randomized clinical double blinds (Douglas et al., " New Engl. J. Med. ", 1986 31 4: 65-80; Hayden et al., " New Engl. J. Med. ", 1986 31 4: 71-75, show that nasal administration of high doses of recombinant interferon-α2 is effective in protecting patients exposed to rhinovirus infection. However, these studies did not provide any evidence of systemic effects.

More recently, a series of patent applications include the treatment of infectious rhinobronchiitis (“shipping fever”) in cattle, and the treatment of leukemia in cats; Treatment of other diseases to improve the efficacy of the vaccine; Improving food use efficiency; And the use of oral administration of low doses of interferon of heterologous species origin for the prevention of theileriosis of bovine. See US Pat. No. 4,462,985, Australian Pat. No. 608519, Australian Pat. No. 583332, and US Pat. No. 5,215,741, respectively. U.S. Pat.No. 5,017,371 also discloses the use of interferon in this manner to treat side effects of chemotherapy or radiation therapy of cancer. In these patent specifications, the interferon used was human interferon-α prepared by the method of Cantel and dissolved in phosphate buffered saline at a dose of 0.01-5 IU per pound of body weight. This specification suggests that low doses of interferon administered to the oropharyngeal mucosa, preferably in a form adapted for prolonged contact with the oral pharyngeal mucosa, may be effective in the treatment of a wide variety of diseases, including cancer. Experimental evidence for feline leukemia, canine paravovirus and other diseases other than tyreria is just anecdotal. In particular, in any animal model for human cancer, such treatment has not been attempted with adequate control.

More recent studies have investigated the effect of very low doses of interferon administered orally or oropharyngeal mucosa (Bocci et al. , Clin. Pharmacokinet. , 1991, 21 : 411-417; Critic, Rev. Therap. Drug Carrier Systems , 1992, 9 : 91-133; Cummins and Georgiades, Archivum Immun. Therap. Exp. , 1993, 41 : 169-172. This type of treatment is particularly useful for the treatment of HIV infection and can at least improve the quality of life for AIDS patients (Kaiser et al., "AIDS", 1992 6 : 563-569; Koech et al., Mol. Biol. Ther. , 1990 2 : 91-95]. However, according to other reports, such treatment provides no clinical benefit. Phase I studies have also been reported on the use of oral lozenges containing interferon at low doses to treat hepatitis B (Zielinska et al., Archiv. Immunol. Therap. Exp. ", 1993, 41 : 241-252.

In contrast, according to International Patent Application WO95 / 27499 of Brigham and Women's Hospital, interferon-β administered by gastrointestinal intubation, Animal models were able to at least partially inhibit the progression of autoimmune diseases such as type I diabetes, multiple sclerosis and autoimmune arthritis. Interferon-β provided by this route or administered intraperitoneally with an "instander" antigen in the stomach effectively induced tolerability. This suggests that interferon-β is effective in enhancing the induction of oral tolerability rather than in inducing humoral or cellular responses to exogenous antigens.

Australian patent application PN 9765 suggests that low doses of interferon administered to the oropharyngeal cavity by the oral mucosal route are effective in protecting mice against challenge by highly metastatic tumor cells. . Along with the fact that very few substances are active against these highly aggressive tumors, a fairly unusual property of the results is that the administration of interferon to the oropharyngeal cavity is useful for the treatment of cancer. Interferon at oral mucosal doses has also been effective in treating mice injected intraperitoneally with cerebral myocarditis virus (EMCV), which commonly causes rapidly progressing fatal diseases characterized by central nervous system invasion and encephalitis. This system is a very rigorous test for antiviral activity, but the oral mucosal route for interferon administration was more effective than intraperitoneal administration.

Summary of the Invention

The present invention relates to a method of administering an interferon via oral mucosa at a higher dose compared to a dose that causes a pathological response when administered orally (generally homologous interferon-α in humans about 20 × 10 ⁶ IU). Provided are methods for stimulating host defense mechanisms in animals. For another type of interferon, the dose that elicits a pathological response may differ from the dose elicited by homologous interferon-α in humans.

In one embodiment, the present invention is directed to administering an immune stimulus amount (which is greater than that of the same interferon that causes a pathological response when administered parenterally) to the mammal via oral mucosal contact. It may be considered as a method of stimulating an immune response in a mammal.

Alternatively, the present invention provides a method of increasing the therapeutic index of interferon by administering interferon through the oral mucosa.

Oral mucosal administration may comprise administering an effective dose of interferon in a single dose, or the effective dose may be a smaller dose over a period sufficient to elicit an immune stimulus corresponding to a single dose of immune stimulation. Can be administered multiple times. Similarly, the dosage of interferon may be administered continuously over a period of time sufficient to induce an effect corresponding to the effect of a single dosage.

In an applied embodiment, the present invention provides for the use of an autoimmune agent by administering an effective amount of interferon to a mammal via oral mucosal contact, which is an amount greater than that of the same interferon that causes a pathological response when administered parenterally. Provided are methods for treating diseases, mycobacterial diseases, neurodegenerative diseases, tumor diseases and viral infections. In particular, the present invention relates to autoimmune diseases such as arthritis, type I diabetes, lupus and multiple sclerosis, mycobacterial diseases such as leprosy and tuberculosis, spongiform encephalitis and neurodegenerative disorders such as Creutzfeldt-Jakob disease, Parasitic diseases such as malaria and cervical cancer, genital herpes, hepatitis B, hepatitis C, human immunodeficiency virus (HIV), human papilloma virus (HPV), herpes simplex virus (herpes simplex) virus: Provides a method for treating viral diseases such as HSV) -1 and HSV-2.

The present invention also provides for multiple myeloma, hematopoietic leukemia, chronic myeloid leukemia, lower lymphoma, cutaneous T-cell lymphoma, carcinoid tumor, cervical cancer, Kaposi's sarcoma including sarcoma, kidney tumor, renal cell carcinoma, hepatocellular carcinoma, non Methods of treating brain tumors, including carcinomas, including pharyngeal carcinomas, malignant hematologic malignancies, colorectal cancer, glioblastoma, laryngeal papoma, lung cancer, colon cancer, malignant melanoma, and malignant brain tumors. In one embodiment, this method is generally applicable to treating tumors of non-viral etiology.

In another embodiment, the present invention provides an oral cavity comprising a therapeutically effective amount of one or more interferons in a therapeutically effective amount (an amount greater than that which elicits a pathological response when administered parenterally), and a pharmaceutically acceptable carrier. Provided is a pharmaceutical composition for mucosal administration. The composition may be provided as a solution, tablet, lozenge, gel, syrup, paste or controlled release mucosal delivery system. Optionally, the composition may contain buffers, stabilizers, thickeners, absorption and viscosity enhancers, and the like.

In one embodiment, the pharmaceutical composition has about 20 × 10 ⁶ IU to about 1000 × 10 ⁶ IU, preferably about 20 × 10 ⁶ IU to about 500 × 10 ⁶ IU, more preferably about 50 × 10 ⁶ IU to It is presented in unit dosage form containing about 500 × 10 ⁶ IU of interferon.

The method may be implemented as the only therapeutic option, or as an adjuvant to radiotherapy, chemotherapy, or with other cytokines such as interleukin-2, 12 or 15, or with IFN-inducing substances.

The method is preferably performed using type I or type II IFN selected from α, β, γ, ω and consensus interferon, most preferably using recombinant IFN-α.

The present invention provides an interferon composition in contact with the oral mucosa to stimulate host defense mechanisms against pathological diseases in mammals, comprising a greater amount of interferon compared to an amount that can elicit a pathological response when administered parenterally. It is about. In particular, the present invention can be applied to compositions for treating autoimmune diseases, tumor diseases, neurodegenerative diseases, parasitic diseases and viral diseases.

The invention will be described in detail using the following definitions and examples for the purpose of reference only. All patents and publications mentioned herein are expressly incorporated herein by reference.

Justice

“Interferon” herein refers to a type I or type II interferon which usually includes the interferon usually represented by α, β, γ and ω, and mixtures thereof (including consensus sequences). Interferon is available from various commercial sources and is licensed for the treatment of various precursors. Interferons can be obtained from natural sources, but recombinant products are preferred. In the present invention, the term "interferon" refers to a chimeric form of a polypeptide having interferon activity or a fragment thereof and interferon introduced with a sequence change to improve stability without affecting the properties of the biological activity of the interferon, for example. chimeric forms or mutant forms (eg, those disclosed in US Pat. Nos. 5,582,824, 5,593,667, and 5,594,107).

The term “high dose” means a dose that is greater than the maximum dose generally tolerated for the same interferon when administered by parenteral routes such as intravenous or intraperitoneal administration. As currently planned, the high dose of interferon is more than about 20 × 10 ⁶ IU of homologous interferon-α for 70 kg adults. Preferably, the dosage is about 30 × 10 ⁶ IU. In a particularly preferred form of the invention, the total dosage is about 50 × 10 ⁶ IU to about 1000 × 10 ⁶ IU, more preferably about 50 × 10 ⁶ IU to 500 × 10 ⁶ IU. As used herein, “high dose” is generally considered to be a therapeutically effective amount when administered through the oral mucosa, which is a pathology manifested by a surrogate marker of toxicity or the occurrence of unacceptable side effects when given parenterally. May cause an adverse reaction. The definition of high dosage is necessarily variable because the dosage depends, in particular, on the patient's individual sensitivity, body size, weight and age, the nature and neutrality of the disease to be treated, the specific interferon to be used and the specific vehicle for administration. to be. The physician treating a patient with a particular interferon will readily be able to recognize the high dosage range suitable for the patient to be treated.

Optionally, the interferon may be administered jointly with an inducer for interferon synthesis and release. The inducer may be administered with interferon or may be administered separately. Examples of the inducer of interferon include polynucleotides such as poly I: C, and preferably low molecular weight interferon inducers which are orally administrable. Suitable inducers are known in the art and are described, for example, in Tilorone (US Pat. No. 3592819; Albrecht et al. , J. Med. Chem. , 1974, 17 : 1150-1156, and the quinolone derivative Imiquimod (Savage et al., Brit. J. Cancer ", 1996, 74 : 1482-1486.

The methods and compositions of the present invention may optionally be used in conjunction with one or more other therapeutic agents and methods of treatment for a particular disease, and the attending physician or vet will readily be able to select other therapeutic agents and methods of treatment that may be appropriate for the situation.

In one embodiment, the present invention provides a method of treating a tumor disease in a mammal comprising the step of administering interferon as described above. Tumor disease may be metastatic cancer.

Although the methods of the invention can be used without being treated simultaneously with other agents, this embodiment of the invention is believed to be particularly useful in the following cases:

a) used as adjuvant therapy following surgery, chemotherapy or radiotherapy provided by standard protocols;

b) For the treatment of interferon-sensitive tumor formation, the methods of the invention are used alone or in combination with conventional chemotherapy or radiotherapy.

c) For the treatment of interferon-resistant tumor formation, the methods of the invention are used alone or most preferably in combination with conventional chemotherapy or radiotherapy.

The method is directed to inducing and / or maintaining the alleviation of the disease. "With other treatments" means that the interferon is administered before, during and / or after using radiotherapy or other chemotherapy. The most suitable protocol depends on various factors as described below.

In particular, the method of the present invention preferably comprises a chemistry using cytostatic agents, one or more other cytokines, which have anticancer activity but different mechanisms from interferons, angiogenesis inhibitors, and agents that enhance the activity of interferon. It will be used in conjunction with one or more other treatment methods selected from the group of treatments. The second cytokine is preferably interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-12 (IL-12) or interleukin-15 (IL-15); The angiogenesis inhibitor is AGM-1470; Preferred interferon-enhancing therapeutics are solid thermogenic or arginine butyrates.

Preferred cytostatic agents that can be administered with interferon include cyclophosphamide, cisplatin, carboplatin, carmustine (BCNU; N, N-bis (2-chloroethyl) -N-nitrosourea) , Methotrexate, adriamycin, α-difluoromethylornithine and 5-fluorouracil.

Tumor diseases affected by the method include cancers that respond to parenteral administration of high doses of IFN-α, such as malignant hematologic malignancies, multiple myeloma, hematopoietic leukemia, chronic myeloid leukemia, lower lymphoma, cutaneous T-cells Lymphomas, and solid tumors (eg, renal cell carcinoma, melanoma, carcinoid tumors and sarcomas of AIDS-associated Kaposi), particularly malignant tumors of nonviral etiology. Viral diseases include, for example, acute or blunt infections such as rhinoviruses, influenza, herpes varicella, herpes zoster, dengue fever, or measles virus encephalitis, Murray Valley Valley) viral encephalitis, including but not limited to encephalitis, Japanese type encephalitis, tick-mediated encephalitis and herpes encephalitis; Hemorrhagic fever such as Ebola virus, Marburg virus, Lassa fever; Hanta viral infections and other viral infections thought to be transmitted from animals to humans, such as morbilliviruses in horses. For many of these diseases, there are no therapies and / or vaccines currently available and helpful therapies may be inadequate. Alternatively, the viral disease may be the result of, for example, hepatitis B, hepatitis C, hepatitis D or other forms of viral hepatitis, and chronic infections such as CMV, HIV, HPV, HSV I and II infections. Hepatitis B and Hepatitis C are both currently treated with parenteral interferon; Long-term interferon treatment for HIV infection progressing to AIDS is under clinical trial.

In a second embodiment, the disease to be treated is malaria and type I or type II interferon is administered as described above. The causative organism of malaria can be Plasmodium malariae , Plasmodium vivax , Plasmodium falciparum or Plasmodium ovale . In particular, the method of the present invention will protect the progression of malaria into the cerebral form.

In a third embodiment, the present invention is directed to an immune system, including HIV, rheumatoid arthritis and multiple sclerosis, or AIDS, irrespective of whether it is a relapse-releasing or chronic progression type, comprising administering interferon as described above. Provided are methods for treating autoimmune disorders such as deficiency.

In addition, the methods and dosage forms of the invention can be used in conjunction with other therapies. For example, for herpes viral infections, acyclovir or gancyclovir can be used. For HIV infection, azidothymidine (dozivudine) or one or more other HIV reverse transcriptase inhibitors, and / or HIV protease inhibitors can be used.

In the preparation of the pharmaceutical compositions of the present invention, various vehicles and excipients for IFN can be used, which will be apparent to those skilled in the art. Representative formulation techniques are particularly taught in Remington, " The Science and Practice of Pharmacy ", 19th Edition, Mack Publishing Co, Easton, PA, 1995 and its preceding editions. IFN formulations may include stability enhancers, such as glycine or alanine, and / or one or more carriers, such as carrier proteins, as described in US Pat. No. 4,496,537. For example, for the treatment of humans, pharmaceutical grade human serum albumin is optionally used, optionally with phosphate-buffered saline as a diluent. If the excipient for IFN is human serum albumin, human serum albumin may be derived from human serum or may be of recombinant origin. When serum albumin is used, it will normally be of homogeneous origin.

IFN will be administered by any means that provides contact of IFN with the oral mucosal cavity of the recipient. Thus, it will be clearly understood that the present invention is not limited to any particular type of formulation. Described herein is the deep administration of IFN into an oral mucosal cavity; This can be accomplished by liquid, solid or aerosol as well as nasal drop or spray. Accordingly, the present invention includes, but is not limited to, liquids, sprays, syrups, lozenges, buccal tablets, and spray formulations. Those skilled in the art will recognize that, for aerosol or spray formulations, the particle size of the formulation may be important and will be well aware of suitable ways to modify such particle size.

In one embodiment, the interferon is administered in a single dose. Alternatively, interferon is administered multiple times over a period of time distributed over smaller doses such that the net effect corresponds to the administration of a single dose. One approach to this mode of delivery is to prepare a sustained release device or controlled release device designed to release interferon over a period of time in an amount equivalent to a single dose, attached to or implanted in the oral mucosa cavity. will be.

Representative formulations of interferon for use via oral mucosa are as follows (all percentages are weight / weight):

The tablets were dextrose BP 45%; Gelatin BP 30%; Wheat starch BP 11%; Cameloose sodium BP 5%; Egg albumin BPC 4%; Leucine USP 3%; Propylene glycol BP 2%; And 50 × 10 ⁶ IU IFN-α2. Tablets may be allowed to slowly dissolve and dissolve in the oral cavity, or may be dissolved in water and maintained in the oral cavity when in contact with the oral mucosa, as desired.

The interferon paste was 50 × 10 ⁶ IU of IFN- from distilled water in an amount such that 45% glycine, 2% sodium CMC, 25% citrate buffer (pH 4.5), 100%, as described in US Pat. No. 4,675,184. It can be made with α2. Interferon paste may be attached to the oral mucosa.

Similarly, gargles or syrups can be prepared by adding the desired amount of interferon to a commercial mouthwash or cough syrup formulation.

Within the specific dosage ranges mentioned above, the optimal treatment in any individual case is based on the nature of the disease involved, the stage of the disease, previous treatment, other treatments that continue, the general state of health of the mammal, the patient's treatment of interferon. It will depend on sensitivity, etc. Therefore, there will be differences in the decision of the doctor or veterinarian with all these circumstances in mind. The duration of treatment will of course depend on the disease to be treated, and the treatment of slow growing cancers, such as prostate cancer, for example, is expected to include a different route of treatment than the treatment of rapidly growing cancers, such as hepatocellular carcinoma. Similarly, acute infections, such as those caused by the Ebola virus, are expected to include different therapeutic routes than chronic diseases such as hepatitis.

The effective dosages disclosed herein can cause a pathological response in mammals when administered orally, but are effective but nontoxic or less toxic when administered via the oral mucosa. Pathological responses may be acute, chronic or cumulative and may be manifested by changes in blood chemistry such as leukopenia, bone marrow degradation or histological variables. Pathological responses herein include side effects such as fever, malaise, or influenza-like symptoms, vascular responses such as phlebitis, and local inflammatory reactions at the injection site. This response will vary considerably between patients as the sensitivity to interferon is individually different.

For many patients, oral mucosal dosages are expected to exceed those known to be tolerated and effective in existing licensed parenteral protocols. In one embodiment, the total dose may be administered several times at lower doses over a period of time, or may be delivered continuously or periodically from a controlled rate of release device attached to or implanted in the oral mucosa.

Interferon and Interferon Formulations

Mouse IFN-α / β

Mouse IFN-α / β (Mu IFN-α / β) was prepared and purified from cultures of C243-3 cells induced by Newcastle Disease Virus (NDV) as previously described (Tovey et al., Proc. Soc. Exp. Biol. And Med. ", 1974 146 : 809-815). The formulation used in this study was titrated at 4 × 10 ⁶ international units (IU) / ml and 5 × 10 ⁷ IU per mg protein when assayed for mouse 929 cells challenged with vesicular stomatitis virus (VSV). Had a specific activity (Tovey et al., Proc. Soc. Exp. Biol. And Med. , 1974 146 : 809-815). The formulation was standardized against the National Reference Institute of Murine IFN-α / β (G-002-9004-5411) of the National Institute of Hygiene (NIH).

Human IFN-α-1-8

Recombinant human IFN-α 1-8 [Hu IFN-α 1-8; BDBB Lot No .: CGP 35269-1, Ciba-Geigy, Basel, Switzerland; was prepared and purified as described previously (Meister et al ., J. Gen. Virol .), 1986 67 : 1633-1643. The formulations used in this study had a titer of 70 × 10 ⁶ IU / ml for homologous human WISH cells challenged with VSV, as already described (Torbay et al., Document “ Nature ”, 1977 267 : 455-457). Had a titer of 1 × 10 ⁶ IU / ml for heterologous mouse L929 cells. The formulations were standardized against both the NIH human IFN-α international reference formulation (G-023-901-527) and the NIH murine IFN-α / β standard (G-002-9004-5411). Inactivation of the IFN formulation was 2 × 10 ⁸ IU per mg of protein.

Recombinant murine interferon α

Recombinant murine interferon α was purchased from Life Technologies Inc. The formulation used in this study (lot number: HKK 404) had a titer of 6 × 10 ⁶ IU / mL and an inactivation of 6 × 10 ⁸ IU per mg of protein when assayed for mouse L929 cells challenged with VSV ( Torbay et al., Proc. Seoc. Exp. Biol Med. , 1974, 146 : 406-415.

Recombinant murine interferon β

Recombinant murine interferon β was purchased from R & DSystems Inc. The formulation used in this study (lot number: 1976-01S) had a titer of 3.2 × 10 ⁴ IU / mL and an inactivation of 8 × 10 ⁶ IU per mg of protein when assayed for mouse L929 cells challenged with VSV. (Torbay et al., Proc. Seoc. Exp. Biol Med. , 1974, 146 : 406-415).

Recombinant murine interferon γ

Recombinant murine interferon γ was purchased from R & D Systems Inc. The formulation used in this study (lot number: 2580-03SA) had a titer of 2 × 10 ⁵ IU / mL and inactivation of 1 × 10 ⁷ IUg per mg of protein when assayed for mouse L929 cells challenged with VSV. (Torbay et al., Proc. Seoc. Exp. Biol Med ., 1974, 146 : 406-415).

All interferon preparations were simultaneously titrated in the same assay and standardized against the international reference preparation of murine interferon a / β of the National Institutes of Health (G-002-9004-5411).

Both murine interferon a / β and recombinant murine interferon were placed in Ferrimmune® excipients prior to administration.

Excipient

Interferon formulations were diluted in phosphate buffered saline containing bovine serum albumin (BSA) or in the patented excipients described below. Bovine serum albumin fraction V [RIA grade; Lack of immunoglobulins; List number A7888; Sigma, USA] was dissolved at a final concentration of 100 μg per 1 mL of PBS (pH 7.4), filtered and sterilized [0.2 μ, Millex-GV, Millipore, USA product].

In the experiments described herein, the interferon formulations were diluted in patented excipients. The excipients used were supplied in the form of tablets as follows (from Pharma Pacific, Pharma Pacific):

Weight / wt% mg / tablet Dextrose (Glucose) BP ^** 44.67 ^*** 55.84 Gelatin BP ^** 30.06 37.58 Wheat Starch BP ^** 11.31 14.14 Camelose Sodium BP ^** 4.96 6.20 Egg Albumin BPC ^** 4.03 5.04 Leucine USP 3.00 3.75 Propylene Glycol BP 1.88 2.35 Dextran 40 ^** (as Dextran 40 injection BP) 0.06 0.08 Sodium Phosphate BP 0.03 0.04 Sodium Chloride BP 0.01 0.01 Sodium Acid Phosphate BP 0.01 0.01 sum 100.02 125.04 ** Calculated based on anhydride *** Derived from: Dextrose (glucose) BP (anhydride) 44.64% Glucose BP (as dextran 40 injection BP) 0.03%

Single tablets were dissolved in 1.5 ml phosphate buffered saline, centrifuged at 16,000 g for 15 minutes, then sterile filtration (0.2 μ, Millex-GV, Millipore, USA) and stored at 4 ° C. before use. . Excipients were prepared daily before use.

Interferon delivery system

According to preliminary experiments, 5 μl of crystal violet was applied to each nostril of a normal mouse grown using a P20 Eppendorf micropipette, indicating that the dye was almost immediately distributed over the entire surface of the oropharyngeal cavity. . Staining of the oropharyngeal cavity was still apparent almost 30 minutes after applying the dye. Essentially similar results were obtained using ¹²⁵ I-labeled recombinant human IFN-α 1-8 applied in the same manner. Thus, this method of administration was used in all subsequent experiments.

In the animal experiments described herein, the expression "oral mucosa" or "oral pharynx" or "nasal nasal / oral" or "nasal and oral" or "in / or" relating to the route of administration of IFN is oral mucosal cavity. In other words, it will be clearly understood to mean that the IFN preparation is deeply administered into the nasal cavity so that the IFN preparation is rapidly distributed into contact with the mucous membrane surrounding the cavity.

EMCV (Cerebral Myocarditis Virus)

Placement: Lot number 095001

Expires: December 1997

Preparation: The method previously described [Gresser I. Bourali C, Thomas MT, Falcoff E. Effect of repeated inoculation of interferon preparations on infection of mice with cerebral myocarditis virus. EMCV strain JH was propagated in mouse L-929 cells using Proc Soc Exp Biol Med , February 1968, 127 : 491-6.

Features: The virus stock used in this study had a titer of 5 × 10 ^8.62 TCID ₅₀ on mouse L929 cells.

Storage: Stock EMCV was stored at -70 ° C. Virus titers had to be shifted to temporary preliminary storage at about the same temperature by power down on day 1. The material was always frozen. Viral titer On day 8, the temperature of the -70 ° C freezer was raised to -60 ° C. Diluted EMCV was prepared immediately prior to use and placed in ice or animal refrigerator until use.

Friend Red Leukemia Cells

IFN-α / β-resistant clones of Friend Red Leukemia cells were obtained from E. Affabris, Rome, which is described in detail by Apabris et al., See Virology , 120 : 441-452 (1982). ]. These cells were then passed in vivo for retention. Briefly, DBA / 2 mice were inoculated with intraperitoneal injection of 3C18 cells at about 100LD ₅₀ , and after one week tumor cells were harvested from the abdominal cavity of mice, counted, and the other mice were inoculated again with 100 LD ₅₀ at 3C18 cells. . This procedure was repeated with 60-100 passages. The 3C18 cells used for the 60th to 100th passage were shown to be highly metastatic to liver and spleen (Gresser et al. , Int. J. Cancer 1987 39 : 789-792). Phenotypes of IFN resistance have been commonly identified by in vitro culturing cells passaged in vivo in the presence of IFN-α / β (Belardelli et al. , Int. J. Cancer 1982 30 : 813 -820].

L1210R6 clone and EL4 implantable tumor

L1210R6, an interferon-α / β-resistant clone of L1210 lymphoma cells, was isolated in the laboratory [Graser et al., “ Interferon and cell division ” (1974). IX; Interferon-resistant L1210 cells: “ Characteristics and Origin. J. Nat. Cancer Inst .”, 52: 553-559.

EL4 implantable tumors were derived from mice inoculated with the chemocarcinogen 1,2-dimethyl benzanthrain (Gorer, PA, " Br. J. Cancer ", 1950 4 : 372-381).

L1210 lymphoma cells were retained by serial passage in vivo in DBA / 2 mice without specific pathogens.

EL4 tumors were retained by serial passage in vivo in C57BL / 6 mice without specific pathogens.

B16 melanoma

B16 melanoma is a transplantable tumor of natural origin derived from C57BL / 6 mice (Fidler IJ and Kriple, ML, " Science 197 ", 1977 893-897). B16 melanoma is a melanogenic tumor that grows rapidly, is very degenerative, and metastasizes mainly to the lungs. B16 melanoma is considered a good model of a rapidly growing, highly aggressive human tumor.

B16 melanoma cells were retained in vivo serial passage in C57BL / 6 mice without specific-pathogens.

animal

Mice used in this study were obtained from colonies devoid of specific pathogens (IFFA CREDO, France). They were bred in a specific pathogen-free animal breeding unit at the Institut Federatif CNRS of Villejuif, according to the EEC standard method.

Biological Assay of Interferon

Interferon was assayed according to conventional methods. In summary, a sample (20 μL) was taken from Eagle's Minimal Essential Medium (MEM) containing 2% heat-inactivated fetal calf serum (FCS) (Gibco, France) (France Diluted in 80 μl of Gibco® and added to each well of a microtiter plate (Falcon, List No .: 3072) using a multi-tube micropipette (Finnpipette, Labsystem, 50-300 μl). WISH or L929 cells (2 × 10 ⁴ cells / well) were added to 100 μl of MEM containing 2% FCS and incubated overnight at 37 ° C. under 5% CO ₂ atmosphere in air [Forma 3029 CO ₂ constant temperature Incubator]. Cells were then examined for any indication of toxicity using an Olympus IM GLDW inversion microscope equipped with a 10-fold objective. Samples that did not exhibit detectable toxicity were then placed in a microplate containing 100 μl of fresh Eagle's medium containing 2% FCS per well in a microplate containing 100 μl of diluted material by a multi-tube micropipette, thereby yielding 2% FCS. Two-fold dilutions were taken starting from the first 1:10 dilution ratio in a total volume of 200 μl of Eagle's contained MEM. Appropriate serial 2-fold dilutions of NIH human IFN-α reference standards (G-023-901-527) or NIH Mu IFN-α / β reference standards (G-002-9004-5411) were also prepared. Then either WISH or L929 (2 × 10 ⁴ cells / well) in 100 μl of Eagle's MEM containing 2% FCS was added to each appropriate plate and incubated overnight at 37 ° C. under an atmosphere of 5% CO ₂ in air. Subsequently, the cell monolayer was examined for any signs of toxicity and for no manifest toxicity, the cultures were aspirated and 100 TCID _{50 of} VSV (2 × 10 ⁻⁴ VSV for WISH cells). ₂₃ , or 10 ⁻⁵ VSV ₂₃ ) for L929 cells were replaced with 200 μl of Eagle's MEM containing 2% FCS. The plates were then incubated overnight at 37 ° C. in an atmosphere of 5% CO ₂ in air. Cell monolayers were then examined for specific viral cytopathic effects using an Olympus IM ULWD inverted microscope. Interferon titers were determined from dilution inverses that protect 50% against certain viral cytopathic effects, expressed in international standard units / ml (IU / ml).

Example 1 Effect of High Dose Interferon on Survival After Lethal Antigen Administration by EMCV (Cerebral Myocarditis Virus)

The effect of IFN-α doses of 1,000, 10,000 and 100,000 IU provided by the oral mucosal route was tested in male and female mice injected with lethal doses of EMCV. Different types of IFN-α were tested and the effects of administration by the oral mucosal route were compared with those administered by the intraperitoneal (ip) route. In addition to monitoring survival after lethal challenge, the toxicity of IFN treatment was monitored using various clinical chemistry and hematological parameters.

At the start of post-viral infection treatment, mice were treated with 10 ⁵ IU of IFN-α once daily for 4 days via the oral mucosal route and were fully protected in all animals. IFN treated animals survived 100% and were good on day 100 after being infected with the lethal dose of EMCV (100 LD ₅₀ ) under conditions in which all virus-infected untreated control animals died within 7 days.

By weight, the treatment of mice at 10 ⁵ IU by the oral mucosal route is comparable to the 240 million IU dose for humans, which, to the knowledge, is considerably higher than the dose administered in humans.

Treatment of mice with 10 ⁵ IU of IFN-α by the nasal / oral route results in greater protection (more viral infections, or more protection than animals treated with 10 ⁴ IU of INF-α). Effects on higher amounts of tumor) will be obtained by higher doses of IFN-α. So far, no plateaus have been observed in the dose-response curves.

The results also show that high or very high doses of IFN by the nasal / oral route have very protective antiviral effects, and 10 ⁵ IU of IFN-α provided by the route is dependent on the dose of EMCV used. Demonstrates complete healing. The dose used was very high on a weight basis (equivalent to 240 × 10 ⁶ IU) compared to that administered to humans, but no clinical, biochemical and hematological evidence of toxicity was observed. In contrast, the maximum tolerated dose of parenteral IFN in clinical practice ranges from 20 to 30 × 10 ⁶ IU per day.

Example 2 Effect of High Dose IFN-α on Mice Inoculated by High Metastatic Tumor Cells

Groups of 10 6-week-old DBA / 2 mice were challenged intravenously with 10 ⁵ friend erythroleukemia cells of interferon-resistant clone 3CI8 or 10 ⁵ L1210 lymphoma cells (interferon-resistant L1210R cells) on day 0. After inoculation, mice were not treated or treated twice daily by the nasal / oral route for 20 days with 10 ⁵ IU of mouse IFN-α / β in a volume of 10 μl of excipient, or 10 μl of excipient alone (control). .

Animals treated with IFN by the nasal / oral route survived 50% at 100 days after inoculation by highly metastatic Friend Leukemia cells and were good. Animals treated with IFN by the nasal / oral route survived 30% and were good at 100 days after challenge with L1210 lymphoma cells. Clinical trials suggest that all IFN-treated animals will survive their normal life unless they survive the 100th day. Histological examination of the organ showed no residual tumor. In contrast, all untreated and control animals died within 13 days of antigen challenge by Friend erythroleukemic cells and within 14 days of challenge with L1210 lymphoma cells, respectively.

These results are very significant because both tumor cell lines used are very aggressive and the dosing dose used corresponds to an LD ₅₀ of about 20,000 fold. Moreover, friend red leukemia and L1210 lymphoma are quite different tumor types, with friend red leukemia cells carrying the friend leukemia virus, a retrovirus, whereas L1210 lymphoma is not associated with any known viral etiology. The results obtained by the nasal / oral IFN-α treatment of animals inoculated with L1210 lymphoma have been shown to be the same as or even superior to the results obtained by the systemic IFN-α treatment in this model (Gracer's unpublished results). ). In a previous study reported in Australian Provisional Patent Application No. PN 9765, none of the mice inoculated with Friend Red Leukemia cells and treated with 100 or 1,000 IU of IFN-α survived at a dose of 10,000 IU. Only 10-20% of animals were considered treated.

Example 3 Effect of High Dose IFN-α on Mice Immunized with Highly Metastatic B16 Melanoma Cells or EL4 Tumor Cells

Groups of 10 6-week-old C57B1 / 6 mice were challenged intravenously with 10 ⁵ B16 melanoma cells, or 10 ⁵ EL4 tumor cells. After inoculation, mice were not treated or treated twice daily by the nasal / oral route for 20 days with 10 ⁵ IU of mouse IFN-α / β in a volume of 10 μl of excipient, or 10 μl of excipient alone (control). .

Animals treated with IFN by the nasal / oral route survived 30% and were good at 100 days of inoculation with highly metastatic B16 melanoma cells or EL4 tumor cells. In contrast, all untreated and control animals died within 20 days of challenge with B16 melanoma cells and within 22 days of challenge with L4 tumor cells. Clinical trials suggest that all IFN-treated animals survived on day 100 will survive if they do not kill even if they stop interferon treatment on day 20. The histological examination of the organs revealed no residual tumors in the interferon treated animals killed at 100 days.

Example 4 Effect of Oral Mucosal Interferon on Antivesicular Stomatitis Virus

Groups of 10 6-week-old mice from sarcoma colonies devoid of specific pathogens were intranasally infected with 100 LD ₅₀ of 10 μl volume of excipient 10 Vl excipient [Torbay et al., Proc. Soc. Exp. Biol. Med . ", 1974, 146 : 406-415. Seven hours after viral infection, mice were not treated, or at the prescribed dose of murine interferon a / β in 10 μl volume of excipient, or by the nasal / oral route for 4 days with only 10 μl excipient (control). One treatment per day.

Treatment of mature mice with murine interferon α / β resulted in a significant increase in the percentage of animals surviving by infection with VSV at the lethal dose. Thus, animals treated with interferon a / β10,000 IU survived 30% on day 21 after being infected with VSV at the lethal dose under conditions where all untreated or excipient control treated virus-infected animals died on day 10. Clinical observations suggest that most of the surviving interferon-treated animals survived on day 21.

Example 5 Effect of Oral Mucosal Interferon on Expression of Cellular Proteins

IFN-α is known to induce expression of many cellular proteins after binding the protein to its protein cell surface receptors. These proteins are thought to provide useful markers of IFN action.

Applicant has applied IFN- administered via the nasal / oral route for expression of three IFN-derived proteins, MHC class I antigen, Ly 6A / E antigen and 2'-5'-oligoadenylate synthetase. The influence of α was evaluated.

Mu 2 <20,000 IU in the nasal / oral route, under conditions in which as little as 20 IU administered intraperitoneally increases the expression of antigen (H-2-K ^d ) in both peripheral blood monocytes and granulocytes. Treatment of DBA-2 mice (H-2K ^d ) with IFN-α did not significantly increase H-2-K ^d expression in peripheral blood lymphocytes, monocytes, or granulocytes.

Similarly, treatment of mice with IFN-α of 20,000 IU or less by the nasal / oral route did not significantly affect the expression of Ly6 A / E antigens, but its expression varied after parenteral treatment of type I IFN. Significantly increased on the surface of lymphoid cells (see Dumont et al ., J. Immunol , 1986 137 : 201-210). Similar results were obtained with 200 or 20,000 IU of Mu IFN-α or Hu IFN-α administered by the nasal / oral route.

Intraperitoneal injection of Mu IFN-α in as little as 20 IU into Swiss or DBA / 2 mice markedly increased 2 ', 5'-oligoadenylate synthetase activity in both peripheral blood mononuclear cells and spleen cells. In contrast, the expression of 2 ', 5'-oligoadenylate synthetase was not significantly increased in the same experiment in mice treated with Mu IFN-α of 20,000 IU or less by the nasal / oral route. Furthermore, when treated with 200 or 20,000 IU of Mu IFN-α or Hu IFN-α 1-8 by the nasal / oral route, 2 ', 5'- at any point in time tested up to 10 days after initiation of IFN treatment There was no significant effect on oligoadenylate synthetase activity.

Example 6 Bioavailability of Interferon After Oral Mucosal Administration

To investigate the bioavailability and pharmacodynamics of IFNs, mice with the most desirable drug-blood dose ratio for this study were treated with high doses of recombinant IFN-α labeled up to the highest specific radioactivity possible at ¹²⁵ I.

A pure formulation of 70 × 10 ⁶ IU of Hu IFN-α 1-8 was dissolved in 1.4 ml of PBS and chlor disclosed in Hunter and Greenwood, “ Nature ”, 1962 194 : 495-496. A modified method of the amine-T method was used to iodide as disclosed in Mogensen et al., " Int. J. Cancer ", 1981 28 : 575-582.

¹²⁵ I-labeled Hu IFN-α 1-8 (lot no .: CGP35269-1) was subjected to 2 × 10 ⁷ IU / mL of biological activity and VSV-administered mice when analyzed in human WISH cells challenged with VSV Analysis on L929 cells showed a biological activity of 1 × 10 ⁶ IU / ml.

Female Swiss mice aged 6 to 7 weeks were injected intravenously, intraperitoneally, or intranasally / orally with 2 × 10 ⁷ IU of ¹²⁵ I Hu IFN-α 1-8 (corresponding to 1 × 10 ⁶ murine IU) ( 1.0369 x 10 ⁷ cpm / mouse). At defined times, three mice per group were killed and blood was collected to determine dose. Kidney, liver, lung, spleen and stomach / esophagus were extracted, blotted and weighed with a precision of ± 1.0 μg. Radioactivity of each sample was measured individually using a gamma counter. The whole blood was then separated by centrifugation (800 gx 10 min, 4 ° C.), serum was harvested, counted and frozen at −80 ° C. Serum IFN content was then analyzed using standard biopsies on human WISH cells and mouse L929 cells as described above. The radioactive material present in the serum samples was then separated by affinity immunoprecipitation and analyzed by SDS-PAGE.

5 minutes after injection of ¹²⁵ I-labeled Hu IFN-α 1-8 with 1.0369 x 10 ⁷ cpm / mouse with intravenous bolus, a very large amount of radioactivity (> 2 x 10 ⁶ cpm / ml) in the peripheral blood of the animal ) Was detected. The amount of radioactivity present in whole blood then gradually decreased at 15 and 30 minutes. Five minutes after intraperitoneal injection of ¹²⁵ I Hu IFN-α 1-8 at 1.0369 x 10 ⁷ cpm, the amount of radioactivity detected in the peripheral blood of the animal was about 20 times less than the amount detected after the injection of intravenous test. The amount of radioactivity then increased gradually 15 and 30 minutes after injection. ^The amount of radioactivity detected in the blood of animals 5, 10 or 15 minutes after nasal / oral administration of ¹²⁵ I IFN-α 1-8 was greater than the amount detected at a given time after intraperitoneal injection of the same amount of radiolabeled IFN. It was quite small. In all three routes of administration, a greater amount of radioactivity was detected in serum than in whole blood following nasal / oral administration of ¹²⁵ I-labeled IFN-α 1-8. The smaller amount of detected radioactivity per ml of whole blood compared to the same dose of serum means that the calculated dose of serum after removal of the cellular component of whole blood is actually greater.

Serum samples from all mice in this experiment were tested for the presence of biologically active IFNs using standard biopsies as described above and at the time of testing all ¹²⁵ I Hu IFN-α 1-8 intravenously or intraperitoneally Easily detectable amounts of biologically active IFNs were found in the sera of all animals injected. In contrast, after intranasal / oral administration of IFNs, despite the presence of relatively large amounts of radioactivity in the serum of these animals, no biologically active IFN was detected in the serum of any animal at any time tested.

To determine whether the radioactive material detected in the serum of an animal treated with ¹²⁵ I Hu IFN-α 1-8 actually exhibits a unique IFN, the sample is immunoprecipitated with protein AG agarose and the immunity present in the sample To precipitate globulin, it was treated with affinity purified polyclonal anti-IFN-α antibody and further immunoprecipitated. The sample was then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described above.

^125. Hu IFN-α 1-8 by the I intravenous or intraperitoneal injection and then the same electric and if the radioactive material is not injected with the result, ¹²⁵ I Hu IFN-α 1-8 by SDS-PAGE analysis in the serum electrophoretic mobility One uniform band is shown as it moves. The apparent molecular weight of the material was estimated to be about 20000 Daltons, which corresponds exactly to the molecular weight of the unique Hu IFN-α 1-8. In contrast, serum samples obtained from mice nasal / oral treated with ¹²⁵ I IFN-α 1-8 had no apparent IFN-like apparent molecular weight, even if the same amount of radioactive material was loaded on each gel. It did not contain any material.

The tissue distribution of the radiolabelled material showed very high radioactivity in the kidneys of animals and high radioactivity in the liver, lungs and spleen 5 minutes after intravenous injection of ¹²⁵ I IFN-α 1-8. The amount of radioactivity present in each of these four organs was found to gradually decrease after 15 and 30 minutes. In contrast, gastrointestinal radiation levels gradually increased at 15 and 30 minutes, reaching levels comparable to those present in animal serum at 30 minutes after intravenous bolus injection.

Intraperitoneally administered ¹²⁵ I IFN-α 1-8 showed the highest levels of radioactivity within 15 minutes of administration in all tissues examined and decreased after 30 minutes. Similarly, nasal / oral administration of ¹²⁵ I Hu IFN-α 1-8 showed the highest levels of radioactivity in all tissues tested 15 minutes after administration, with a slight decrease in the levels of radiation present after 30 minutes. The amount of radioactivity present in the gastrointestinal / esophagus is more within one digit range than the amount detected in any other organ after nasal / oral administration of ¹²⁵ I-labeled IFN-α 1-8, which is intravenous or After parenteral administration of the same amount of radiolabeled Hu IFN-α 1-8 by the intraperitoneal route, it is significantly greater than the amount present in these tissues.

Example 7 Pharmacodynamics of Interferon After Nasal / Oral Oral Administration

To precisely measure the pharmacodynamics of Hu IFN-α 1-8, ¹²⁵ I-labeled Hu IFN-α 1-8 1.0369 x 10 ⁷ cpm / mouse were administered intravenously, intraperitoneally or nasal / orally to mice, The level of radioactivity present in both whole blood and serum was measured at a series of time points for 24 hours.

The pharmacodynamic profile of ¹²⁵ I-labeled Hu IFN-α 1-8 present in the blood of mice after intravenous bolus administration closely followed the algebraic clearance curve. This is in contrast to previous results performed in mice using the highly related molecule recombinant human α A / D (Bgl) (see Bohoslawed et al., J. IFN Res. , 1986 6 : 207-213) . Matched. The amount of bioavailable material calculated from the area under the curve of concentration vs. time was also similar to that of human α A / D. ^A biphasic time-consuming clearance curve was observed following the administration of ¹²⁵ I IFN-α 1-8 intravenous bolus, which is a characteristic of the substance removed through the kidneys and the results of Example 6 Matched. After intraperitoneal injection of ¹²⁵ I-labeled IFN-α 1-8, the pharmacodynamics of this material were very similar to those previously reported for intramuscularly administered IFNs.

Easily detectable amounts of biologically active IFN were present in the serum of all animals following injection of ¹²⁵ I-labeled IFN-α 1-8 intravenously or intraperitoneally.

1. Discussion of antitumor activity

Because Friend Red Leukemia is very malignant and metastasizes to both the liver and spleen when injected intravenously, the Friend Red Leukemia model has been selected as a very rigorous preclinical trial of antitumor activity. Indeed, the results obtained using this model have become the acceptance criteria for parenteral injection of IFN-α for the treatment of human cancer. In all the tests performed in the experiments, both untreated animals and animals treated with the control formulation died within 10 to 11 days. If not treated, injecting only 4 or 5 FLC cells will kill the mouse. In contrast, some of the animals treated with murine IFN-α by the oral mucosal route are still alive over 100 days after injection of 10 ⁵ FLC and can be considered treated.

Indeed, from previous studies, IFN-α administered by the oral mucosal route, parenteral administration of cyclophosphamide, 5-fluorouracil or metoke, which increased survival time only for a few days in animals injected with FLC It has been shown to be more effective than lexate (see Greser et al., J. Natl. Cancer Inst. , 1988 80 : 126-131). Other drugs such as cisplatin, vincristine, doxorubicin, bleomycin or etoposide are ineffective in this tumor (see Greser et al., J. Natl. Cancer Inst ., 1988 80 : 126-131).

Similarly, IFN-α administered by the oral mucosal route has been shown to be more effective against FLC than other cytokines such as systemically administered IL-1β, IL-2 and TNF-α (systemic administered IL-1β, IL-2 And TNF-α showed little activity in this model).

Previous studies have shown that parenterally administered IFN is one of the most active anti-tumor drugs in this model, and IFN treatment is effective even when starting after tumor metastasis already exists in the liver (Grecer et al., " Intl J. Cancer ", 1987 39 : 789-792). The results indicate that IFN administered by the oral mucosal route is equivalent to or more effective than parenteral administration.

Daily injections of IFN-α provided with a single dose of cyclophosphamide markedly increased survival of lymphoma-containing AKR mice compared to animals treated with only one of the agents (starting treatment after lymphoma diagnosis) (Graser Et al., Eur. J. Cancer , 1978 14 : 97-99. Successful combinations with IFN-αZβ and BCNU, cis-DDP (cisplatin), methotrexate, adriamycin and α-difluoromethyl ornithine Therapies have also been reported in various preclinical animal tumor models: Combination treatment with 5- (fluorouracil) (5-FU) and IFN has also been reported to be beneficial for the treatment of metastatic colon cancer in humans [Ernstoff et al. , " Journal of Clinical Oncology ", 1989 7 : 1764-1765. However, treatment of IFN with cyclophosphamide (Marquet et al. , Int. J. Cancer , 1983 31 : 223-226; Lee et al., " Biochem, Pharmacol. ", 1 984 33 : 4339-3443], Adriamycin (Blackwill et al . , Cancer Res. , 1984 44 : 904-908), or 5-FU (Market et al. , Int. J. Cancer) , 1985 109 : 156-158). That is, other experiments have reported that antitumor activity is reduced when combined with IFN treatment of drugs that have been shown to have a beneficial effect in combination with parenteral IFN treatment. Combinations of IFNs with other chemotherapeutic agents can be readily tested using the methods disclosed herein.

Combined interleukin-1 (IL-1) and IFN-α / β treatment increase antitumor effect in mice injected with FLC (Belardelli et al., Int. J. Cancer, 1991 49: 274-278]. The same treatment also exerts significant antitumor effect against metastatic variant (p11-R-Eb) of Eb lymphoma, whereas only one of these agents is ineffective (Gabriele et al., Invasion Metastasis ). ", 1993 13 : 13 : 147-162]. Of all the cytokines tested, IL-1 has been found to be most effective when combined with parenteral I type IFN treatment.

Angiogenesis inhibitor AGM-1470 [(chloroacetyl) -carbamic acid (3R- (3α, 4α (2R ^* , 3R ^* ), 5β, 6β))-5-methoxy-4- provided with IFN-α / β Combination therapy with (2-methyl-3- (3-methoxy-2-butenyl) oxyranyl) -1-oxaspiro (2.5) oct-6-yl ester] The effect was significantly increased (see Brem et al., J. Pediatric Surgery , 1993 28 : 1253-1257).

Overtemperature has been shown to enhance the antitumor action of IFN-α / β on Lewis lung cancer (see Yerushalmi et al., Proc. Soc. Exp. Biol. Med. , 1982 169 : 413-415). ]. Arginine butyrate has also been shown to enhance the antitumor action of IFN-α (see Chany and Cerutti, Int. J. Cancer , 1982 30 : 489-493). Comparison of the level of protection obtained when administering a given type and dose of IFN by the oral mucosal route with the results obtained after systemic administration (intraperitoneal injection) shows that parenteral administration of IFN is in some cases slightly more effective than oral mucosal administration. In other cases, it is not more effective.

2. Discussion of antiviral activity

Antiviral activity was not detected in the serum of animals after nasal / oral administration of ¹²⁵ I IFN-α 1-8, Mu IFN-α / β and Mu IFN-α, but statistically for infection by EMCV at lethal doses. Was significantly protected in these animals (Table 1).

Effect of Monotherapy with ¹²⁵ I-IFN-α on Survival of Swiss Mice Injected with EMCV Number of surviving animals per group (date after injection) Treatment group Dosage Route 3 4 5 6 7 8 9 10 15 30 Untreated 6/6 4/6 2/6 0/6 Hu IFN α 1-8 1.4 × 10 ⁵ IU in / or 6/6 5/6 4/6 3/6 1/6 1/6 1/6 1/6 1/6 1/6 Mu IFN α / β 6.4 × 10 ⁴ IU in / or 6/6 5/6 5/6 4/6 3/6 2/6 2/6 2/6 1/6 1/6

Effect of single treatment (once daily for 4 days) with Mu IFN-α on survival of Swiss mice injected with EMCV Number of surviving animals per group (date after injection) Treatment group Dosage Route 3 4 5 6 7 8 9 10 15 30 Untreated 10/10 9/10 4/10 0/10 Mu IFN α 2 × 10 ⁴ IU in / or 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 7/10 7/10

The results obtained in a well-defined preclinical model of acute viral infections provided clear evidence for the use of high doses of IFN through the oral mucosa for the treatment of acute systemic viral infections in humans. , Multiple IFN-α subtypes and single recombinant IFN-α isotypes (such as Mu IFN-α) indicate that they have statistical significance for antiviral activity in this model. Natural Mu IFN-α / β and Hu IFN-α 1-8 appear equally effective when administered to oral mucosa. Recombinant Mu IFN-β and Mu IFN-γ show similar antiviral activity.

When the degree of protection obtained when a given dose and type of IFN is administered via the oral mucosal route is compared with the degree of protection obtained after systemic administration (intraperitoneal injection), parenteral administration of IFN is in some cases compared to oral mucosal administration. Was marginally more effective and in other cases it was not effective.

3. General Discussion

Biomarker pilot experiments showed that any of the three biomarkers tested (MHC class I antigen, Lys6 A / E antigen, and 2 ', 5'-oligoadenylate synthetase activity) were routed to the oral mucosal route. It is very clearly shown that it does not properly contribute to the very significant antitumor activity exhibited by the administered IFN-α.

All experiments taken after intraperitoneal injection of as little as 20 IU resulted in a significant increase in the expression of all three IFN-induced proteins and after administration of IFN-α up to 20,000 IU by the oral mucosal route. Surprisingly, there was no possible impact.

Applicants cannot exclude the possibility that an effect on one biomarker or other biomarker will be observed in the early or mid-term, but since IFN acts on the transcription of genes encoding these proteins, several hours after IFN treatment It is unlikely that this would be possible because the effects on any of these biomarkers could not be predicted so far.

Once again, the possibility of observing the systemic effects on one of many other IFN-induced proteins following IFN-α treatment with the oral mucosal pathway cannot be ruled out, but this is due to the specific IFN-induced gene expression. It seems unlikely because it involves differential control. However, there is a possibility that after administration of IFN-α by the oral mucosal route, the effect on IFN biomarkers can be observed locally, for example in nasal lymphocytes.

Consistent with no detectable effect on the tested biomarkers, even in animals treated with IFN-α up to 20,000 IU, consistent effects were not observed on any of the hematological or hemochemical variables observed during oral mucosal IFN treatment. Did.

The results of the pharmacokinetic-bioactivity studies showed that the radiolabeled Hu IFN-α 1-8 under conditions where no circulation of IFN in peripheral blood could be detected using a sensing method that was approximately several times more sensitive than previously used. It is quite clear that a statistically significant antiviral effect can be obtained by administration through a single dose of oral mucosa. Consistent with these results, the degree of antiviral activity exerted by IFN administered via the oral mucosa appears to follow the classic dose-response relationship.

After oral mucosal administration of ¹²⁵ I-labeled IFN-α 1-8, an easily observable amount of radiolabelled material was found in both whole blood and serum of the animal. This result was compared with previous test results that failed to detect IFN in the serum of animals even after oral administration of large amounts of unlabeled IFN. However, radioactive substances detected in both whole blood and serum after oral mucosal administration were biologically inactive. Furthermore, the results of SDS-PAGE analysis indicated that the material had a low molecular weight, presumably indicating that the digested product of IFN digested in the gastrointestinal and small intestine was absorbed. Tissue distribution test of radiolabelled material after oral mucosal administration showed significantly more radioactivity in the stomach than any of the other organs tested. These experimental results show very clearly that the treatment shows statistically significant antitumor activity in vivo, even if no biologically active IFN is observed after oral mucosal administration.

While not wishing to be bound by any mechanism proposed for the observed beneficial effects, test results have not been established to date, in which oral mucosal administered IFNs do not involve direct action of externally administered IFNs or induction of endogenous IFNs. It is suggested that new mechanisms affect tumor cells. The fact that the three biomarkers or circulating IFNs tested were not present in detectable amounts, supports the above proposal. This mechanism appears to be able to at least partially act on the stimulation of the rich lymphoid tissue surrounding the nasopharynx and esophagus. Applicants have shown that oral mucosal IFNs can be at least comparable in efficacy to systemically administered IFNs, and the test results strongly support the need for administering IFNs by the oral mucosal route in the treatment of tumor diseases. This may be an important indication for the clinical use of IFN.

Although the invention has been described in some detail for clarity and understanding, those skilled in the art will recognize that various changes can be made to the aspects and methods described herein without departing from the scope of the inventive concept disclosed herein. will be.

Claims (17)

Comprising interferon in an amount greater than 20 × 10 ⁶ IU to 1000 × 10 ⁶ IU, an amount greater than that which can elicit a pathological response upon parenteral administration, An interferon composition for oral mucosal administration for stimulating a host defense mechanism or immune response in a mammal suffering from a tumor disease, autoimmune disease, chronic viral disease or acute viral disease, or tuberculosis. The method of claim 1, A composition comprising 50 × 10 ⁶ IU to 500 × 10 ⁶ IU interferon. The method of claim 1, Compositions suitable for treating tumor diseases susceptible to interferon treatment. The method of claim 3, wherein Multiple myeloma, follicular cell leukemia, chronic myeloid leukemia, low lymphoma, cell lymphoma, carcinoid tumor, kidney tumor, renal cell carcinoma, hepatocellular carcinoma, carcinoma, colorectal cancer, glioblastoma, lung cancer, colon cancer, And an interferon composition for oral mucosal administration for stimulating a host defense mechanism or an immune response in a mammal having a tumor disease selected from the group consisting of malignant brain tumors. The method of claim 1, A composition suitable for treating acute viral infections or chronic viral infections that are sensitive to interferon treatment. The method of claim 5, Influenza virus, herpes varicella, herpes zoster, dengue fever, viral encephalitis (e.g. measles virus encephalitis, Murray Valley encephalitis, Japanese encephalitis B, Tick-mediated encephalitis and herpes encephalitis), hemorrhagic fever (eg Ebola virus, Marburg virus, Lassa fever and Hanta viral infection), and from animals to humans Oral mucosa to stimulate host defense mechanisms or immune responses in mammals with acute viral infections, or chronic viral infections, selected from the group consisting of other viral infections of interest (e.g., morbillivirus in horses). Interferon composition for administration. The method of claim 6, Compositions suitable for treating herpes varicella (chickenpox). The method of claim 1, Compositions suitable for treating tuberculosis. The method of claim 1, A composition further comprising an interferon-inducing substance selected from the group consisting of poly I: C, tirone and imiquimod in addition to interferon. The method of claim 1, A composition further comprising a therapeutic agent selected from the group consisting of cytostatic agents, anticancer agents, angiogenesis inhibitors and antiviral agents. The method of claim 1, Wherein the interferon is type I interferon. The method of claim 11, Type I interferon is selected from the group consisting of interferon (IFN) -α, IFN-β, IFN-ω, consensus IFN, recombinants and mixtures thereof. The method of claim 1, Wherein the interferon is type II interferon. The method of claim 13, The composition of type II interferon is IFN-γ or a recombinant substance. The method according to any one of claims 1 to 9 and 10 to 14, A composition wherein an effective amount of interferon is administered in a single dose. The method according to any one of claims 1 to 9 and 10 to 14, An effective amount of interferon is administered in multiple doses divided into small doses over a period sufficient to elicit the same therapeutic response as when administered in a single dose. The method according to any one of claims 1 to 9 and 10 to 14, An effective amount of interferon is administered continuously over a period of time sufficient to elicit the same therapeutic response as when administered in a single dose.