DK172974B1 - Immunotherapeutic, solid, oral interferon dose form, and a process for producing it - Google Patents

Description

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The present invention generally relates to an improved method of treating diseases of immunopathological etiology in humans using interferon at low oral doses. This invention also relates to the use of interferon in low oral doses to enhance disease-correcting immune responses in humans affected by immune resistance disorders characterized by a seemingly hyperactive or hypoactive immune system function.

"Interferon" is a term that generically encompasses a group of vertebrate glycoproteins and proteins known to have various biological effects, such as antiviral, antiproliferative, and immunomodulatory activity in at least the animal species from which such substances are derived. The following interferon definition has been accepted by an international committee assembled to designate a system for the correct nomenclature of interferons: "To fulfill the conditions of being an interferon, a factor must be a protein that exerts a virus-not -specific antiviral activity at least in homologous cells through cellular metabolic processes involving the synthesis of both RNA and protein ", Journal of Interferon Research, 1, page vi (1980). "Interferon" as used herein to describe the present invention is intended to have this definition.

Since the first descriptions of interferon by Isacs and Lindeman (see Proc. Roy. Soc.

London (Ser. B), Volume 147, page 258 et seq. (1957) and U.S. Patent No. 3,699,222), 20 have been the subject of intensive research worldwide. The literature is full of publications on the synthesis of interferon, its proposed molecular characterizations, its clinical applications, and suggested mechanisms for its antitumor activity, antiviral activity and immune system activity.

Due to the intensity and different origins of research on interferon 25 and its characteristics and applications, there is a significant lack of uniformity in such material as the classification of interferon types. There are also numerous, at times contradictory, theories regarding the mode of action of interferon to induce clinical effects.

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Although interferon was initially isolated from cells of avian origin (chicken allantoin cells), interferon production has been observed in cells of all classes of vertebrates, including mammals, amphibians, birds and reptiles. Interferon production in vertebrate cells is rarely spontaneous, but it is often easily "induced" by treating cells (in vivo or in vitro) with various substances, including viruses, nucleic acids (including those of viral origin and synthetic polynucleotides), lipopolysaccharides and various antigens and mitogens.

Interferons have generally been named based on the species of animal cells producing the substance (e.g., human, murine, or bovine), the type of cell involved (e.g., leukocyte, lymphoblast, fibroblast), and sometimes the type of inducing material responsible for interferon production (eg virus, immune). Interferon has been loosely classified by some researchers according to the mode of induction as either type I or type II, the first class comprising virus-induced and nucleic acid-induced interferon and the latter class comprising the material produced as a lymphokine through induction by antigens and mitogens. Recently, the International Committee, which designates an orderly nomenclature system for interferon, classified interferon into types based on antigenic specificities. In this newer classification, the terms α, β and γ have been used to correspond to the earlier designations of leukocyte, fibroplasty and type II (immune) interferons, respectively. α and β interferons are usually acid stable and correspond to what have been called type I interferons. γ-interferons are usually acid stable and correspond to what have been called type Π interferons. The International Committee nomenclature recommendations apply only to human and murine interferons, Journal of Interferon Research, 1 page vi (1980).

In its earliest applications, interferon was used exclusively as an antiviral agent, 25 and the most successful clinical therapeutic applications to date have been in the treatment of viral or viral disease conditions. However, it became clear that exogenous interferon was sometimes able to cause regression or remission of various metastatic diseases.

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An overview of current clinical trials with interferon as an antiviral and antiproliferative therapeutic agent is provided in Interferon: In Vivo and Clinical Studies, Volume 4, Eds: N.B. Finter and R.K. Oldham, Academic Press, New York, 1985.

The clinical agent currently selected is human leukocyte interferon "mass-produced" by procedures involving the collection and purification of large amounts of human "buffy coat" leukocytes, virus induction, and isolation from culture media.

In the work described above, interferon has been administered parenterally, viz. intramuscularly and intradermally, with some successful topical 10 and intranasal applications being reported. It has rarely been administered intravenously due to significant side effects caused by "contaminants" in crude and even highly purified isolates.

As discussed above, significant research efforts have been directed toward the development of therapeutic effects of interferon for many different diseases that have an autoimmune pathological basis. Prior to the applicant's first report on the successful oral administration of interferon in its U.S. Patent No. 4,462,985, there was no recognition in the art of the option offered by oral administration of interferon. The general opinion was that interferon could not survive the digestive conditions of the upper digestive tract.

20 Since the applicant's first description of the immunotherapeutic benefit that could be obtained by oral administration of interferon in heterologous mammalian species, this continued investigation into the effectiveness of orally administered interferon. In U.S. Patent No. 4,497,795, Applicant described the use of interferon administered orally or by intravenous administration to stimulate appetite and food efficiency in bovine and porcine species. Recently, in current pending U.S. patent applications, applicants have disclosed the use of interferon in doses that are less than ca. 11IU pr. kg efficiencies and feed utilization in warm-blooded vertebrates, for the prevention and treatment of shipping fever and for increasing vaccine efficacy.

Human α-interferon has been marketed under the trade name Agriferon® by Immuno-modulator Laboratories, Inc. ("IML") from Stafford, Texas for veterinary use in Texas 5 since February 1985. The product is sold for oral administration to cattle to promote growth and food efficiency and to prevent or treat viral respiratory infections.

IML began selling an α-interferon product for horses in 1986. Both products are sold under license from applicant's U.S. Patent No. 4,462,985.

Interferon which is brought into contact with the oral and / or pharyngeal mucosa in amounts of 10 less than 11 IU / kg body weight per day. Today, persistently effective in enhancing disease-correcting immune responses in vertebrates affected by immune resistance disease states is characterized by apparently hyperactive or hypoactive immune system function. Treatment as defined herein has been shown to cause remission of neoplastic disease, hyperallergenicity, immune residual or immunosuppressive viral infections, and autoimmune diseases characterized by chronic tissue degenerative inflammation.

The clinical agent selected for use in the present invention is human leukocyte interferon (human α-interferon) which is "mass-produced" by procedures involving the collection and purification of amounts of human "buffy coat" leukocytes, 20 interferon production with virus and isolation of culture media. (See "Preparation of Human α-Interferon" below). Also acceptable for use in the present invention are human α-interferon products produced by recombinant DNA technology, which are now commercially available from Schering-Plow (as Intron®) and Hoffmann-LaRoche (as Roferon®) and approved by the FDA for the treatment (25) of parenteral hair cell leukemia in humans. Such recombinant interferon products are believed to be particularly effective when used in combination, γ-interferon is also available by recombinant technology and is currently undergoing clinical trials by Genentech and in DK 172974 B1 others. Fibroblast interferon (β-interferon) can be prepared in accordance with Example 1 of applicant US Patent No. 4,462,985, the disclosure of which is incorporated herein by reference.

Human and interferon origin interferon is quantified in the field of art 5 expressed in international units ("IU"). As used herein, a "unit" of interferon (to separate from "IU") means the reciprocal value of a dilution of interferon-containing material which, as determined by analysis, inhibits half the number of plaques of an attack virus, the attack virus being the vesicular stomatitis virus ( "VSV"). Thus quantified, a "unit" of interferon is routinely found to be approx. a tenth of the amount of interferon represented by an "IU". In other words, an entity for the purpose of defining the present invention is approx. = 0.1 IU.

The present invention relates to an improved method of treating immune-resistant disease states with interferon. The present invention is directed to the treatment of human diseases, especially certain diseases which the immune system of 15 species is poorly equipped to handle, shown by either a lack of disease-fighting reaction and / or an apparently flawed immune response that results in a chronic tissue degenerative inflammation state or other physical complications. Although significant research efforts have been directed towards the use of interferon in the treatment of such diseases, the reported results, although a total of 20 positive, are inconsistent. The main reason for such discrepancy is, in light of the applicant's recent research efforts, that previous researchers have failed in defining optimal dose and interferon administration route.

The present invention is based on the applicant's finding that interferon can be used as a sustained effective therapeutic agent for the treatment of diseases having an immunopathological basis, characterized by an insufficient immune response and persistence of the disease, or by a seemingly hyperactive immune response that results in tissue degenerative inflammatory states and related physical manifestations DK 172974 B1 6 stations. Applicant has found that interferon brought into contact with the oral and pharyngeal mucosa in amounts of from approx. 0.022 to approx. 11 IU / kg body weight per Today, persistently effective in treating diseases to which the immune system in humans does not respond effectively.

Disease conditions treated in connection with the present invention apparently include autoimmune diseases characterized by a chronic tissue degenerative inflammatory state. Diseases characterized thus include multiple sclerosis, rheumatoid arthritis, stomatitis and lupus erythematosus. Treatment of such diseases in the context of the present invention comprises administering 10 interferon at a dose of 0.022 to ca. 11 IU / kg per per day in a dosage form adapted to promote contact between this interferon dose and the patient's oral and pharyngeal mucosa. Preferably, the interferon dose is from 0.22 to ca. 8.8 IU / kg per day, more preferably 1.1 to approx. 3.3 IU / kg body weight per day. α-inlerferon derived from tissue culture or from recombinant DNA techniques is a preferred therapeutic agent 15 in connection with this invention.

oc-interferon can be administered alone or in combination with β-interferon or γ-interferon.

It is essential that the interferon is administered in a dosage form adapted to ensure maximum contact between the interferon in the dosage form and the oral and pharyngeal mucosa of the patient undergoing treatment. Contact between interferon and mucous membranes can be increased by maximizing the residence time of the treatment solution in the oral or pharynx. Thus, the best results appear to be achieved in humans when asked to hold the interferon solution in their mouth for a period of time. Contact between interferon and the oral and pharyngeal mucosa and then with the lymphatic system in the patient is undoubtedly the most effective method for administering immunotherapeutic interferon levels.

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Another disease state that responds to treatment in the present invention is neoplastic disease. Thus, the administration of interferon in accordance with the foregoing description, alone or in combination with other drugs or treatments, may assist in the remission of cancers such as malignant lymphoma, melanoma, mesothelioma, Burkitt lymphoma and nasopharyngeal carcinoma and other neoplastic diseases. , especially those known or suspected of viral etiology. Based on the results observed to date, it is believed that the above described method of treatment will at the same time help in the remission of Hodgkin's disease and leukemia.

Other disease states that respond to treatment in accordance with the present invention are infectious diseases of viral origin in humans. What is significant is that a viral infection, which typically exhibits persistent resistance to treatment, has shown a dramatic response to low-dose interferon treatment upon contact with the oral and pharyngeal mucosa in infected patients.

Examples of human viral infections showing remarkable response to treatment in the present invention are infections with human rhinovirus (common cold), herpes simplex I virus (cold sore) and human papovavirus (warts). Based on treatment results to date, low-dose interferon contact with the oral and pharyngeal mucosa is expected to provide effective treatment of acquired immunodeficiency syndrome (AIDS) and disease conditions caused by the herpes simplex II virus. A patient who was in a state of viral myocarditis has responded favorably to the present treatment. Warts often dissolve within 6-8 weeks of initial treatment in accordance with this invention.

Other disorders that respond to low-dose interferon contact are hyper-allergic conditions, such as asthma. One "side effect" noticed by patients treated in accordance with this invention is improved skin appearance. Administration of interferon DK 172974 B1 8 in doses of approx. 0.022 to approx. 11 IU / kg body weight per Thus, day is effective in treating specific acne and generally in improving the human skin appearance.

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Furthermore, stimulation of the immune system by oral contact with low-dose interferon is believed to support the body in fighting bacterial infection. Treatment in conjunction with this invention alone or in combination with therapeutic amounts of antibiotics can be particularly effective in suppressing infections of antibiotic-resistant microorganisms.

Preferably, administration of interferon in the present invention is continued until the symptoms of the disease condition being treated subside. This may range from a period of one day, for example, where the disease-causing agent is a human rhinovirus, to a period of up to six months for the treatment of neoplastic disease. Patients with rheumatoid arthritis are pain-free within 2 to 10 days after initiation of treatment in accordance with the present invention. However, treatment of this disease is preferably performed by administering interferon for up to about 15 minutes. three months.

The daily interferon dose may be administered as a single dose or preferably divided and administered in a daily multiple dose regimen. A staggered cure, e.g. one to three days of treatment per day. week or month, can be used as an alternative to continuous daily treatment.

Interferon is administered in accordance with this invention in solid dosage form, such as lozenges adapted to be dissolved by contact with saliva in the mouth with or without assistance in chewing. Such a unit dosage form is formulated to release ca. 1 to approx. 1500IU interferon after solution in the mouth for contact with the oral and pharyngeal mucosa. Thus, a unit dosage form of interferon in accordance with this invention can be prepared by techniques known in the art to form compressed tablets such as chewable vitamins. Similarly, interferon can be incorporated into SFB 1: in DK 172974 B1 9 starch-based gel formulations to form a lozenge that will dissolve and release s interferon into contact with the oral mucosa when held in the mouth. Solid unit dose forms of interferon for use in In accordance with the present invention, dosage formulation techniques known in the art may be prepared.

The pH value of such formulations can range from approx. 4 to approx. 8.5. Of course, when processing such unit dosage forms, heating of a pre-dose formulation after addition of interferon to over ca. 50 ° C.

Preparation of Human Interferon

Human α-interferon can be prepared by the following procedure, commonly referred to as the Cantell procedure. The procedure begins with packages of human leukocytes, in this case obtained from the Gulf Coast Regional Blood Center,

Houston, Texas. The "buffy coats" in these packages are collected in centrifuge bottles and then diluted with 0.83% ammonium chloride. The mixture is incubated for 15 min. with periodic recurrent shaking and then centrifuged for 20 min. at 2000 rpm. The supernatant is discarded and the cell pellet is resuspended with a minimal volume of sterile phosphate buffered saline (PBS). The mixture is then diluted with ammonium chloride and centrifuged. The supernatant is discarded again and the remaining cell pellets resuspended with a minimal volume of tissue culture medium such as Minimal Essential Medium (MEM) obtained from KC Biological. The cell concentration is determined with a Coulter counter.

20 Interferon Induction Occurs in glass or plastic bottles. The induction medium contains MEM, 75 mM Hepes (available from Calbiochem), 75 mM Tricine (available from Sigma Chemical Co.), human γ serum (18 mg / ml) and gentamycin sulfate (from M.A. Bioproducts, 50 µl / πι1). The cells are added to the induction vessels at a final concentration of approx. 5 to 10 million cells per cell ml. The induction vessel is incubated in a 37 ° C water bath and interferon-α is added as a primer.

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After two hours, Sendai virus is added to the induction mixture. This causes α-interferon to be produced in the supernatant of the leukocytes. After an incubation time of 12-18 hours, the induction mixture is centrifuged. The cells are discarded and the supernatant is then purified.

The crude interferon is cooled to 10 ° C or below in an ice bath. Five molar potassium thiocyanate is added to give a final concentration of 0.5 M. This solution is stirred for 15 minutes and then its pH is lowered to 3.3 by the addition of hydrochloric acid. The mixture is then centrifuged at 2800 rpm for 30 minutes and the supernatant discarded.

The pellets are resuspended in 95% ethanol and stirred for 15 min. This suspension is centrifuged at 2800 rpm for 20 minutes and discarded. The pH of the supernatant is then adjusted to 5.8 with sodium hydroxide. The mixture is stirred for 10 min. and then centrifuged at 2800 rpm for 20 min. Pellets are discarded. The pH of the supernatant is then adjusted to 8 with sodium hydroxide. This solution is stirred for 10 min, followed by centrifugation at 2800 rpm for 20 min. The supernatant is discarded and pellets are resuspended with 0.5 M potassium thiocyanate in a 0.1 M sodium phosphate buffer. This suspension is stirred at 4 ° C.

The suspension is then centrifuged at 2800 rpm for 20 minutes and discarded. The pH of the supernatant is adjusted to 5.3 with hydrochloric acid. After stirring for 10 min. and centrifugation, adjust the supernatant pH to 2.8 with hydrochloric acid, followed by a further stirring for 20 minutes. This mixture is centrifuged at 2800 rpm and the resulting pellet is purified human α-interferon.

The pellet is resuspended with 0.5 M potassium thiocyanate in 0.1 M sodium phosphate buffer having a pH of 8.0. It is then dialyzed against PBS at 4 ° C with two shifts of PBS.

This mixture is then centrifuged and the precipitate discarded. The remaining purified 25 α-interferon is sterilized by filtration through a 0.2 μτη filter. A human α-interferon is manufactured in accordance with this procedure by Immuno Modulators Laboratory, Stafford, Texas and sold under the trade name Agriferon® for use in cattle and Equiferon® for use in horses.

Other methods known to those of skill in the art are available for the preparation of interferons such as human α-interferon and human γ-interferon.

For example, U.S. Patent Nos. 4,376,821 and 4,460,685 disclose methods for producing human γ-interferon. A process for producing bovine fibroplast (P) interferon is described in Applicant's U.S. Patent No. 4,462,985.

Clinical studies

Human treatment with exogenous human cc-interferon 10 Human patients were treated with human α-interferon in the treatment of acute rheumatoid arthritis, multiple sclerosis, asthma, acne, malignant lymphoma, mesothelioma and stomatitis aphthosa. The treatment consisted of oral administration of 1.5 IU per day. kg body weight twice daily, once in the morning and once in the evening. None of the patients noted any fever or anorexia associated with the administration of α-interferon.

Interferon was administered in a buffered solution at such a concentration that a single dose could be administered in a volume of approx. 1 to approx. 20 ml of liquid. Each patient generally maintained the oral interferon solution for a period of up to ca. 1 minute. After this time, the solution was either swallowed or discharged from the patient's mouth.

Two patients suffering from rheumatoid arthritis were treated - a white male of 44 years and 20 a white female of 44 years. The male patient was pain free within 7 days and the female was pain free within 10 days. They both continued on oral interferon therapy for a total of 21 days and remained without symptoms.

It has been found that re-emergence of a treated arthritic condition can be minimized if the treatment in question is continued over a period of up to ca. 3 months.

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A 30-year-old white female nurse suffering from multiple sclerosis who had undergone a comprehensive neurological examination at the City of Hope Hospital in Los Angeles received the treatment for 21 days. The patient has had no recurrence of the neurological symptoms for the past nine months.

5 A 42-year-old white man who was diagnosed with a malignant lymphoma had complete chemotherapy with poor outcomes and was considered to be in the last position. He was treated for three weeks with oral interferon. Six months after starting treatment, he was released from his oncologist as free of the disease.

An 82-year-old white woman was diagnosed with mesothelioma. Currently, there is no effective treatment for this disease, and an average survival rate of only 9 months is predicted. During her treatment with human α-interferon, she received thoracocentesis on two occasions for pleural depletion. Otherwise, the patient has been active and has survived for 43 months.

A 32-year-old Asian male with stomatitis aphthosa was treated for two weeks with 15 human α-interferon in accordance with the present invention. There has been no recurrence of ulcers in the past six months after termination of treatment.

BKC is a 29 year old white woman and KKJ is a 20 year old white woman. Both suffer from acne-like skin spots at the time of their monthly menstrual cycle.

Oral human α-interferon given with approx. 2.2 units / kg body weight for 3 days before their cycle time reduces the severity and number of skin spots.

Treatment of Warts in Men.With Bovine Interferon MAH, a 38-year-old white woman, had 7 warts on the middle finger of the right hand. After 9 months, medical treatment was sought and liquid nitrogen was applied by a dermatologist. Only one wart on his finger went back after treatment. Three warts collapsed to form a large warty area, which over the next year acquired a square shape of approx. 12 mm. Oral treatment with bovine α-interferon was started at a dose of 6 ml daily on 6 consecutive days. The α-interferon concentration 5 was 30 units / ml. It resulted from nasal secretions of cattle infected with infectious bovine rhinotracheitis virus. All of the warts recurred completely within 6 weeks of the first interferon dose.

Interferon dose formulations il) Pastil.

A starch gel-based lozenge containing interferon is prepared by combining 150 g of sucrose, 550 ml of phosphate-buffered saline and 250 g of a cold water-soluble starch such as that described in U.S. Patent No. 4,465,702 to heat this mixture with stirring. a temperature of approx. 75 ° C, cool the mixture to approx. 30 ° C and then in the paste-like mass mix 50 ml of phosphate-buffered saline PBS 15 containing human α-interferon at a concentration of 250 IU / ml. The mixture is then formed into several portions of approx. 5 to approx. 10 g each, which cures on standing under drying conditions to a starch scandal gel-like texture. The lozenges made thereby can be administered to a patient alone or in combination. The patient is instructed to hold the lozenge in the mouth until completely dissolved to release the interferon component for contact with the oral mucosa.

A chewable vitamin formulation is prepared e.g. in accordance with the specification of U.S. Patent No. 3,857,939 by coating one or more components thereof prior to tableting with an interferon solution in an amount sufficient to provide about 1 to approx. 1500 units of interferon in each chewable vitamin tablet.

Claims (9)

1. Improved solid, immunotherapeutic dosage form of interferon for human use, characterized in that the dosage form comprises approx. 0.022 to approx. 11 IU / kg body weight 5 and is formulated to be dissolved by contact with saliva in the mouth to provide interferon in saliva solution for contact with the oral and pharyngeal mucosa to stimulate in an immunotherapeutic response. A dosage form according to claim 1, characterized in that the interferon is α-interferon or β-interferon. The dosage form according to claim 1 or 2, characterized in that the dosage form comprises from ca. 0.22 to approx. 8.8 IU / kg α-interferon and a saliva-soluble carrier. The dosage form according to claim 1, characterized in that the interferon is human α-interferon and the dosage form comprises from ca. 1.1 to approx. 3.3 IU / kg of interferon and a saliva-soluble carrier. Interferon dosage form according to claim 1 or 2, characterized in that it is in the form of a lozenge adapted to be dissolved by contact with saliva in the mouth to provide interferon in saliva solution, which lozenge comprises approx. 1 to approx. 1500 IU α-interferon or β-interferon. Chewable tablet according to claim 1 or 2. An immunotherapeutic dosage form according to claim 1, characterized in that the interferon is α-interferon and that the dosage form is formulated to provide from ca. 10 to approx. 1500 IU α-interferon per dose for contact with the oral and pharyngeal mucosa. DK 172974 B1 Process for the preparation of an agent according to any one of the preceding claims, characterized by combining interferon with a pharmaceutically acceptable saliva-soluble carrier to form the interferon agent. Method according to claim 8, characterized in that the pharmaceutically acceptable carrier is selected such that the agent is adapted to be held in the patient's mouth for a sufficient period of time to form a salivary solution of the interferon and the carrier and to enable contact between the dissolved interferon and the patient's oral and pharyngeal mucosa. 10