WO2008109953A1 - Method for treating cancer via the mucosal administration of interleukin - Google Patents

Method for treating cancer via the mucosal administration of interleukin Download PDF

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Publication number
WO2008109953A1
WO2008109953A1 PCT/AU2008/000350 AU2008000350W WO2008109953A1 WO 2008109953 A1 WO2008109953 A1 WO 2008109953A1 AU 2008000350 W AU2008000350 W AU 2008000350W WO 2008109953 A1 WO2008109953 A1 WO 2008109953A1
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Prior art keywords
interleukin
cancer
administration
administered
treatment
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PCT/AU2008/000350
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French (fr)
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Sai Ying Ko
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Sai Ying Ko
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Priority claimed from AU2007901333A external-priority patent/AU2007901333A0/en
Application filed by Sai Ying Ko filed Critical Sai Ying Ko
Priority to AU2008226337A priority Critical patent/AU2008226337B2/en
Priority to CN200880013569A priority patent/CN101861162A/en
Publication of WO2008109953A1 publication Critical patent/WO2008109953A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • A61K38/2013IL-2
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods and compositions for use in the treatment of cancer. Specifically the invention relates to treatment involving the mucosal delivery of interleukin, in particular interleukin-2.
  • the World Health Organization has estimated that more than 10 million people developed a malignant tumour in 2002 and more than 6.7 million died from cancer. It is estimated that cancer rates could further increase by 50% to 15 million new cases per year by 2020. Allied to the devastating personal and social effects of cancer is the public health and economic impact; the cost of cancer treatment has been estimated to be growing at 10% to 20% per year.
  • the most common cancers worldwide include lung, breast, prostate, liver, colorectal, bladder and stomach cancers.
  • a number of other cancers are increasing in prevalence including, for example, melanoma, ovarian cancer and cervical cancer.
  • Radiotherapy and chemotherapy is often employed but with mixed results and with numerous adverse side effects including neurotoxicity in the case of a number of chemotherapeutic agents.
  • chemotherapeutic drugs for example approximately 30% of ovarian cancers do not respond
  • some patients that do respond to initial treatment subsequently develop resistance to the drugs and do not respond when their disease relapses.
  • Liver cancer is the predominant cancer in Asia, being of greatest prevalence in China and Japan. Hepatocellular carcinoma is the most frequent malignant liver cancer, with an estimated more than one million new cases and 600,000 deaths worldwide every year. It is a rapidly fatal disease with few effective therapeutic treatments available. Untreated, the average survival is approximately 3 months, with a less than 5% five year survival rate. Surgery including liver resection, transplant or percutaneous therapy may increase survival rates and delay morbidity in some cases but few patients are eligible for such treatment. Tumour ablation and chemotherapy, using drugs including cisplatin, doxorubicin, vincristine, fluorouracil, ifosphamide and etopocide may be employed, but to limited effect. The frequent association of liver cancer with cirrhosis also restricts therapeutic options.
  • IL-2 cytokine interleukin-2
  • IL-2 aldesleukin
  • IL-2 in cancer therapy has been considerably hampered by the significant side effects associated with its administration, due primarily to the intravenous or subcutaneous delivery method required and the associated high doses necessary to achieve any therapeutic benefit. Patients receiving systemic IL-2 therapy often experience flu-like symptoms.
  • hypotension, anaemia, and a decreased platelet count are also associated with the high cumulative doses required for intravenous administration.
  • the most severe toxicities associated with the presently available intravenous or subcutaneous IL-2 administration result from the molecule's ability to increase capillary permeability, which may result in hypotension, ascites, generalized oedema, and pulmonary oedema.
  • Capillary leak syndrome may ultimately lead to severely low blood pressure and reduced blood flow, heart and lung abnormalities, fluid retention, mental changes, kidney abnormalities and/or gastrointestinal abnormalities. These effects may be severe and can result in death.
  • IL-2 stimulates the immune system, it can also exacerbate certain disorders such as arthritis, diabetes, and psoriasis.
  • IL-2 The continued role of IL-2 in cancer therapy is presently being carefully assessed in light of its significant toxicity and relatively modest clinical response rate.
  • the present inventor has now surprisingly found, contrary to expectation, that mucosal delivery of IL-2 offers substantial therapeutic benefits including the ability to achieve tumour reduction with much reduced doses than those required for presently available systemic delivery.
  • the present invention thereby provides a viable new option for the cost-effective, low cost efficacious therapeutic treatment of cancers, including aggressive cancers not readily susceptible to presently available treatments.
  • a method for treating cancer in a subject comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof.
  • the mucosal administration is oral administration, more typically the administration is buccal or sublingual.
  • the interleukin is recombinant human interleukin.
  • the cancer may be selected from, for example, liver, lung, kidney, skin, bladder, stomach, breast and blood cancer. In an embodiment, the cancer is selected from liver, lung, kidney and skin cancer.
  • the interleukin may be administered in any form suitable for oral delivery, typically for sublingual or buccal delivery, such as, for example in solid or liquid unit dosage form.
  • the method may further comprise the administration of one or more anti-cancer agents.
  • agents may be administered by the same route as the interleukin or via a different route.
  • the administration may be sequential or concomitant.
  • compositions for mucosal administration for the treatment of cancer comprising interleukin or a fragment or derivative thereof, optionally together with one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • a third aspect of the present invention there is provided method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of a composition of the second aspect.
  • a fourth aspect of the present invention there is provided use of interleukin or a fragment or derivative thereof for the manufacture of a medicament for treating cancer, wherein the medicament is administered mucosally.
  • the interleukin is selected from the group consisting of: interleukin 2, interleukin 12, interleukin 15 and interleukin 18, or any mixture thereof.
  • the interleukin is interleukin 2 (IL-2).
  • the interleukin may be administered in the form of a polynucleotide encoding the interleukin.
  • the polynucleotide may be located in a genetic construct, operably linked to a promoter.
  • FIG. 1 Tumour mass reduction (%) in mice xenotransplanted with H22 liver tumour cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2. Results of two studies are illustrated (A and B).
  • FIG. 1 Tumour mass reduction (%) in mice xenotransplanted with B16 melanoma cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2. Results of two studies are illustrated (A and B).
  • Figure 4. Tumour mass reduction (%) in mice xenotransplanted with lung tumour cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2.
  • an element means one element or more than one element.
  • therapeutically effective amount includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth.o Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • treating and “treatment” refer to any and all uses which remedy a 5 disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • treatment refers not only to treatment designed to cure or remove symptoms in an individual, but also to ongoing therapy designed to control and suppress the occurrence of symptoms. Treatment may be for a defined period of time, or provided on an ongoing basis 0 depending on the particular circumstances of any given individual.
  • polypeptide means a polymer made up of amino acids linked together by peptide bonds.
  • polypeptide and “protein” are used interchangeably herein, although for the purposes of the present invention a “polypeptide” may constitute a portion of a full length protein.
  • polynucleotide refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof.
  • the present invention is predicated on the inventor's surprising finding that in a mouse model mucosal administration of recombinant interleukin (in the form of IL-2) results in a substantial and statistically significant reduction in tumour load without evident toxicity. Further, as exemplified herein, this mucosal administration is dose independent. This is in contrast to prior art administration by injection in which higher doses are required to achieve similar therapeutic effect, with the dose to be administered being constrained by the generation of side effects and toxicity.
  • the present invention provides a novel therapeutic treatment option for a variety of cancers with low dosages such that side effects commonly observed with presently available systemic treatments are either obviated or reduced.
  • interleukin therapy for the treatment of cancer has only been attempted intravenously or subcutaneously. Such administration requires high doses of interleukin, at levels considered abnormally high relative to the levels by which cytokines such as IL-2 act to mediate cellular responses naturally. These high doses result in significant side effects to patients. .
  • the inventor's novel finding of therapeutically efficacious mucosal interleukin administration opens the way for the development of cost effective, non toxic therapeutic alternatives to presently available injection therapies.
  • the present invention provides a method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof.
  • the interleukin may be selected from, for example, IL-2, IL-12, IL-15 or IL-18.
  • the interleukin is IL-2.
  • the interleukin (s) used in the methods and compositions of the invention may be natural, s recombinant or synthetic and may be obtained by purification from a suitable source or produced by standard recombinant DNA techniques such as those well known to persons skilled in the art, and described in, for example, Sambrook et al.; Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press (the disclosure of which is incorporated herein by reference).
  • Those skilled in the art will appreciate that the precise sequence of the interleukins to beo employed in accordance with the present invention may vary depending on a number of factors, for example the species and/or the cancer to be treated.
  • interleukin or “interleukins” should be understood as a reference to all forms of this molecule and to functional derivatives, variants and homologues thereof. This includes, for example, any isoforms which arise from alternative splicing of the subject interleukin mRNA or functional mutants ors polymorphic variants of these proteins. Also encompassed within the scope of the invention are homologs or mimetics which possess qualitative biological activity in common with the full-length mature interelukin. Further, the present invention contemplates not only use of the interleukin polypeptide, but also polynucleotides encoding the same.
  • Derivatives of interleukin include analogues, functional fragments, parts, portions or variants from either natural or non-natural sources.
  • Non-natural sources include, for example, recombinant or synthetic sources.
  • recombinant sources is meant that the cellular source from which the subject molecule is harvested has been genetically altered.
  • Aminant sources is meant that the cellular source from which the subject molecule is harvested has been genetically altered.
  • Aminant sources means a polypeptide which is a derivative of interleukin, which derivative comprises addition, deletion,5 substitution of one or more amino acids, such that the polypeptide retains substantially the same function as the native interleukin from which it is derived. Modifications may be made so as to enhance the biological activity or expression level of interleukin or to otherwise increase the effectiveness of the polypeptide to achieve a desired result.
  • amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid witho similar properties within a polypeptide chain (primary sequence of a protein).
  • GIu charged amino acid glutamic acid
  • Amino acid insertional derivatives also include amino and/or carboxyli . c terminal fusions as well as intrasequence insertions of single or multiple amino acids, lnsertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place.
  • “Fragment” refers to a polypeptide that is a constituent of full-length interleukin.
  • the fragment typically possesses qualitative biological activity in common with the full-length interleukin.
  • the fragment may be derived from the full-length interleukin polypeptide or alternatively may be synthesised by some other means, for example chemical synthesis.
  • a "variant" of interleukin means a molecule of substantially similar sequence to the interleukin of which it is a variant and which exhibits at least some of the functional activity of the interleukin of which it is a variant.
  • a variant may take any form and may be naturally or non-naturally occurring.
  • variant polypeptides may share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
  • a "homologue" means that the interleukin is derived from a species other than that which is being treated in accordance with the method of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of interleukin which exhibits similar and suitable functional characteristics to that of the interleukin which is naturally produced by the subject undergoing treatment.
  • the interleukin to be administered is human IL-2.
  • Full length human IL-2 has the amino acid sequence as set forth in SEQ ID NO:1 (precursor form) or SEQ ID NO:2 (mature form) and in its native form is an approximately 15,500 Da glycosylated protein.
  • Embodiments of the present invention also provide for the administration of interleukin in the form of a polynucleotide encoding an interleukin polypeptide as described above.
  • the polynucleotide encodes human IL-2.
  • an IL-2 polynucleotide of the invention may have the nucleotide sequence as set forth in SEQ ID NO:3.
  • the invention also contemplates the use of polynucleotides encoding homologues, fragments and variants thereof.
  • the polynucleotide may be administered in a vector.
  • the vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion and foreign sequences and introduction into eukaryotic cells.
  • the vector is an expression vector capable of directing the transcription of the DNA sequence of the polynucleotide encoding the desired polypeptide into mRNA.
  • the vector may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyodenylation signals and transcription termination sequences.
  • suitable viral expression vectors include for example Epstein-barr virus-, bovine papilloma virus-, adenovirus- and adeno-associated virus-based vectors.
  • the vector may be episomal.
  • the methods and compositions of the present invention are particularly suitable for the treatment of liver cancers, primary and secondary, such as hepatocellular carcinomas.
  • liver cancers primary and secondary, such as hepatocellular carcinomas.
  • the present invention is not so limited and is also applicable to the treatment of any tumour or cancer susceptible to treatment via mucosal administration of an active agent.
  • the methods and compositions of the invention also find application in the treatment of tumours in a variety of organs.
  • the cancer may be a prostate, breast, lung, stomach, intestinal, bladder, ovarian, cervical, oesophageal, pancreatic, renal, or brain cancer, a head or neck tumour, melanoma, or lymphoma.
  • the cancer may be a primary or secondary cancer.
  • the cancer may be a sarcoma, for example a liposarcoma.
  • the present invention provides methods and compositions for the mucosal delivery of interleukin.
  • the mucosal administration of interleukin is oral administration, although other intranasal administration, for example, inhalation, is also contemplated.
  • oral administration comprises sublingual or. buccal administration whereby the composition is placed into contact with the buccal mucosa either under the tongue or in the cheek pouch allowing entry of the active agent directly to the bloodstream by absorption.
  • Suitable forms for oral administration include solid, liquid, emulsion, gel and suspension.
  • a composition of the invention is administered in solid unit dosage form, for example in the form of a tablet, capsule, caplet, or lozenge.
  • the administration may comprise a gel administered to the nasal, buccal or sublingual area.
  • the interleukin is unstable in a given liquid, this can be overcome by dissolving freeze dried interleukin powder in a 5 diluent, for example water plus honey ⁇ or carboxymethylcellulose, dextran, maltodextrin, gums, albumin, sugars such as dextrose, maltose, mannitol etc.
  • Honey is particularly useful as it contains many antigens which may assist the interleukin immune response.
  • suitable compositions may be prepared according to methods which are known toQ those of ordinary skill in the art and may include' a pharmaceutically acceptable diluent, adjuvant and/or excipient.
  • diluents, adjuvants and excipients must be "acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
  • Examples of pharmaceutically acceptable diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such asQ liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glyco
  • the diluent may be dextran, trehalose, carboxymethylcellulose, albumin, propylene glycol in 99% water.
  • the carrier or carriers will form from 10% to 99.9% by weight of the compositions.
  • the interleukin for example IL-2
  • IL-2 is administered in the form of a solid unit dosage form such as a tablet, capsule or lozenge suitable for oral, most typically sublingual, administration.
  • Suitable solid compositions may comprise a rapidly disintegrating composition comprising interleukin in a pharmaceutically acceptable water soluble or water dispersible carrier material. Such compositions may disintegrate or dissolve in the mouth upon placement under the tongue or insertion into the buccal pouch.
  • Compositions may be formulated for rapid or immediate release of the interleukin or alternatively for delayed or controlled release. Techniques and processes for achieving delayed or controlled release of active agents are well known to those skilled in the art.
  • suitable formulations may be prepared by processes including freeze drying under vacuum, supercritical fluid drying, spray drying using heat, and fluid bed spray drying.
  • a process involving microencapsulation whereby the active ingredient is coated onto granules, tablets or microparticles, typically using organic solvents.
  • One particularly suitable process involves the use of a fluidised bed spray process facilitating the coating onto granules at room temperature of actives including polypeptides with a water solubilising coat, as disclosed in International Patent Application Publication No.
  • Microparticles such as water soluble gel forming particles may be thus coated, or alternatively a blank tablet, lozenge or capsule core may be spray coated.
  • means for the preparation of oral compositions incorporating an effervescent agent as a penetration enhancer to increase the permeability of the active agent across the buccal and sublingual mucosa see for example US Patent No. 6,974,590, the disclosure of which is incorporated herein in its entirety by reference.
  • Other delivery modes contemplated by the present invention include the use of bioadhesives, mucoadhesives and liposomes.
  • compositions of the invention may also be administered in the form of liposomes.
  • Liposomes may be derived from phospholipids, or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are DODMA, synthetic cholesterol, DSPC, PEG-cDMA, DLinDMA, or any other non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes.
  • the compositions in liposome form may contain stabilisers, preservatives and/or excipients.
  • compositions of the invention may also be administered in the form of microparticles.
  • Biodegradable microparticles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and epsilon-caprolactone ( ⁇ -caprolactone) may be used.
  • compositions of the invention may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and an organic solvent or mixture of organic solvents.
  • SAIB sucrose acetate isobutyrate
  • Polymer additives may be added to further increase the viscosity so as to decrease the release rate.
  • Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents.
  • Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol.
  • Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
  • Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.
  • Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
  • Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier.
  • Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid ' paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
  • Suspensions for oral administration may further comprise dispersing agents and/or suspending agents.
  • Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol.
  • Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
  • Emulsions for oral administration may further comprise one or more emulsifying agents.
  • Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
  • the therapeutically effective dose level of a composition of the present invention for any particular patient will depend upon a variety of factors including any one or more of: the type of cancer being treated and the stage of the cancer; the activity of the active agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of sequestration of compounds; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.
  • the preferred dose of interleukin for example IL-2
  • a feature of the present invention is that mucosal administration of interleukin enables significantly lower doses of interleukin to be employed than is possible with prior art approaches whilst retaining therapeutic benefit. Accordingly, the minimum dose of interleukin that may be used in accordance with the invention can be
  • the dose is sufficient to achieve a therapeutic benefit. In terms of the maximum dose, this can also be determined by a person skilled in the art taking into consideration factors such as those discussed herein. For example a dose of up to several million IU per day may be appropriate in some circumstances.
  • the dose of interleukin employed in accordance with the invention is between about 1 IU and about 100,000o IU per day, between about 10 IU and about 50,000 IU per day, between about 100 IU and about 20,000 IU per day, or between about 500 IU and about 10,000 IU per day.
  • the daily dose administered to a patient in need thereof may be in the order of about 100 IU, 200 IU, 300 IU, 400 IU, 500 IU, 600 IU, 700 IU, 800 IU, 900 IU, 1,000 IU, 2,000 IU, 3,000 IU,s 4,000 IU, 5,000 IU, 6,000 IU, 7,000 IU, 8,000 IU, 9,000 IU, 10,000 IU, 11,000 IU, 12,000 IU, 13,000 IU, 14,000 IU, 15,000 IU, 18,000 IU, 20,000 IU, 25,000 IU, 30,000 IU, 35,000 IU, 40,000 IU, 45,000 IU and about 50,000 IU.
  • the optimal quantity and spacing ofo individual dosages will be determined by the nature and extent of the cancer being treated, the form, route and site of administration,, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques known to those skilled in the art. For example, a subject may be administered the desired daily dose in a single unit dosage form once per day, or in two unit dosage forms administered twice a day. 5
  • IL-2 therapy performed in accordance with the present invention may be carried out in conjunction with chemotherapy or radiotherapy or as an adjunct to surgery.
  • IL-2 may also be combined with one or more additional therapeutic agents, including for example other immunomodulatory agents.
  • immunomodulatory agents may include other cytokines or chemokines, such as, for example, interleukins or interferon. Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art.
  • each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired therapeutic effect.
  • the components may be administered by the same route of administration, although it is not necessary for this to be so.
  • the components may be formulated together in a single dosage unit as a combination product.
  • Example 1 Mucosally administered recombinant human IL-2 and liver cancer
  • mice xenotransplanted with a hepatic cancer cell line were used.
  • mice Mature, virulent and active H22 neoplasmic cells were diluted with physiological saline solution to a concentration of 1 ⁇ 10 7 cell/ml. All mice (Kunming Murine Clean type 18 - 22g female mice) were inoculated subcutaneously with 0.2 ml of cells under the arm pit. On day 2 these animals were randomized into ten groups of ten as shown in Table 1 below. Treatment began on the same day after randomization. For mucosal administration each mouse was given 10 ul of liquid twice a day for 9 days. The recombinant human IL-2 and recombinant human IFN- ⁇ 2b were purchased from Beijing SL Pharmaceutical Co., Ltd.
  • mucosally administered recombinant human IL-2 proved effective in reducing the tumour burden in H22 xenotransplanted mice in a dose independent manner at 100 IU, 1 ,000 IU and 10,000 IU. Similar levels of tumour reduction were observed for the group receiving a combination of mucosally administered recombinant human IL-2 at 10,000 IU and mucosally administered recombinant human IFN- ⁇ 2b at 10,000 IU.
  • Example 2 Sublingual administration of recombinant human IL-2 and liver cancer
  • Example 1 Further to the experiments described in Example 1 above, an additional experiment was carried out using the same protocol comparing the efficacy of sublingual delivery of recombinant human IL-2 (performed as per Example 1 above) with subcutaneous delivery of recombinant human IL-2 in a BSA/PBS solution. Treatment groups and doses are shown in Table 3 below. Other parameters are as for Example 1. Table 3
  • human IL-2 (1 IU to 500 IU) administered sublingually
  • Example 3 Sublingual administration of recombinant human IL-2 and kidney cancer
  • mice 20 inoculation, well-grown renal cancer clumps were removed and isolated into single cell suspension (10 7 cells/ml) in physiological saline. All mice were inoculated with 2x10 6 cancer cells (0.2ml) by subcutaneous injection into the dorsum region. On the second day these animals were grouped randomly (10 mice per treatment regime) into 7 groups for treatment with: 1 IU, 10 IU, 100 IU, or 500 IU IL-2 by sublingual administration; 100 IU IL-2 by subcutaneous injection; no
  • mice 25 treatment negative control; and placebo treatment with vehicle only.
  • Each mouse was given 10 ⁇ l of liquid twice a day for 15 days. On day 21, mice were sacrificed. Body weight and the extracted tumor weight were recorded. The tumor suppression percentage was calculated as described in Example 1.
  • Example 4 Sublingual administration of recombinant human IL-2 and skin and lung cancers
  • Example 5 Compositions for treatment
  • IL-2 is typically administered in the form of a pharmaceutical- composition suitable for oral, most typically sublingual administration.
  • a composition in accordance with the invention iso outlined below. The following is to be construed as merely an illustrative example and not as a limitation of the scope of the present invention in any way. 2 ' 1
  • Example 5A Composition for oral administration in table form
  • a composition comprising IL-2 in the form of a tablet may be prepared by incorporating the IL-2 into a film comprising one or more of gelatin, maltodextrin, carboxylmethyl cellulose, glucose, carbomer and coating the film onto a blank tablet core made up of known pharmaceutically acceptable ingredients selected from starch, calcium phosphates, carboxymethylethyl cellulose, mannitol, maltose, talc and magnesium stearate.
  • Example 5B - Composition for oral administration in capsule form A composition comprising IL-2 in the form of a capsule may be prepared by filling a standard two- piece hard gelatin capsule with IL-2, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.

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Abstract

The invention provides a method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof.

Description

METHOD FOR TREATING CANCER VIA THE MUCOSAL ADMINISTRATION OF INTERLEUKIN
Technical Field
The present invention relates to methods and compositions for use in the treatment of cancer. Specifically the invention relates to treatment involving the mucosal delivery of interleukin, in particular interleukin-2.
Background Art
The World Health Organisation has estimated that more than 10 million people developed a malignant tumour in 2002 and more than 6.7 million died from cancer. It is estimated that cancer rates could further increase by 50% to 15 million new cases per year by 2020. Allied to the devastating personal and social effects of cancer is the public health and economic impact; the cost of cancer treatment has been estimated to be growing at 10% to 20% per year.
The most common cancers worldwide include lung, breast, prostate, liver, colorectal, bladder and stomach cancers. A number of other cancers are increasing in prevalence including, for example, melanoma, ovarian cancer and cervical cancer. For most if not all of these and other cancers there are few effective treatment options currently available. Radiotherapy and chemotherapy is often employed but with mixed results and with numerous adverse side effects including neurotoxicity in the case of a number of chemotherapeutic agents. Further, not all patients respond equally to chemotherapeutic drugs (for example approximately 30% of ovarian cancers do not respond) and some patients that do respond to initial treatment subsequently develop resistance to the drugs and do not respond when their disease relapses.
Liver cancer is the predominant cancer in Asia, being of greatest prevalence in China and Japan. Hepatocellular carcinoma is the most frequent malignant liver cancer, with an estimated more than one million new cases and 600,000 deaths worldwide every year. It is a rapidly fatal disease with few effective therapeutic treatments available. Untreated, the average survival is approximately 3 months, with a less than 5% five year survival rate. Surgery including liver resection, transplant or percutaneous therapy may increase survival rates and delay morbidity in some cases but few patients are eligible for such treatment. Tumour ablation and chemotherapy, using drugs including cisplatin, doxorubicin, vincristine, fluorouracil, ifosphamide and etopocide may be employed, but to limited effect. The frequent association of liver cancer with cirrhosis also restricts therapeutic options.
Accordingly, there is an urgent need for the development of new therapeutic and management strategies for not only liver cancers, but cancer in general.
One agent that has been considered promising for the treatment of some infectious diseases, immune disorders and cancer types, is the cytokine interleukin-2 (IL-2). A recombinant form of interleukin-2, aldesleukin (Proleukin®) has received FDA approval for the treatment of metastatic melanoma and metastatic renal cell carcinoma via injection.
However to date the use of IL-2 in cancer therapy has been considerably hampered by the significant side effects associated with its administration, due primarily to the intravenous or subcutaneous delivery method required and the associated high doses necessary to achieve any therapeutic benefit. Patients receiving systemic IL-2 therapy often experience flu-like symptoms.
Hypotension, anaemia, and a decreased platelet count are also associated with the high cumulative doses required for intravenous administration. The most severe toxicities associated with the presently available intravenous or subcutaneous IL-2 administration result from the molecule's ability to increase capillary permeability, which may result in hypotension, ascites, generalized oedema, and pulmonary oedema. Capillary leak syndrome may ultimately lead to severely low blood pressure and reduced blood flow, heart and lung abnormalities, fluid retention, mental changes, kidney abnormalities and/or gastrointestinal abnormalities. These effects may be severe and can result in death. As IL-2 stimulates the immune system, it can also exacerbate certain disorders such as arthritis, diabetes, and psoriasis.
The continued role of IL-2 in cancer therapy is presently being carefully assessed in light of its significant toxicity and relatively modest clinical response rate. However the present inventor has now surprisingly found, contrary to expectation, that mucosal delivery of IL-2 offers substantial therapeutic benefits including the ability to achieve tumour reduction with much reduced doses than those required for presently available systemic delivery. The present invention thereby provides a viable new option for the cost-effective, low cost efficacious therapeutic treatment of cancers, including aggressive cancers not readily susceptible to presently available treatments.
Summary of the Invention
According to a first aspect of the present invention there is provided a method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof.
Typically the mucosal administration is oral administration, more typically the administration is buccal or sublingual. Typically the interleukin is recombinant human interleukin.
The cancer may be selected from, for example, liver, lung, kidney, skin, bladder, stomach, breast and blood cancer. In an embodiment, the cancer is selected from liver, lung, kidney and skin cancer.
The interleukin may be administered in any form suitable for oral delivery, typically for sublingual or buccal delivery, such as, for example in solid or liquid unit dosage form.
The method may further comprise the administration of one or more anti-cancer agents. Such agents may be administered by the same route as the interleukin or via a different route. The administration may be sequential or concomitant.
According to a second aspect of the present invention there is provided a pharmaceutical composition for mucosal administration for the treatment of cancer, the composition comprising interleukin or a fragment or derivative thereof, optionally together with one or more pharmaceutically acceptable carriers, excipients and/or diluents.
According to a third aspect of the present invention there is provided method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of a composition of the second aspect. According to a fourth aspect of the present invention there is provided use of interleukin or a fragment or derivative thereof for the manufacture of a medicament for treating cancer, wherein the medicament is administered mucosally.
In an embodiment in accordance with any one of the first to fourth aspects of the invention, the interleukin is selected from the group consisting of: interleukin 2, interleukin 12, interleukin 15 and interleukin 18, or any mixture thereof. In a particular embodiment, the interleukin is interleukin 2 (IL-2).
According to any one of the first to the fourth aspects, the interleukin may be administered in the form of a polynucleotide encoding the interleukin. The polynucleotide may be located in a genetic construct, operably linked to a promoter.
Brief Description of the Drawings
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings.
Figure 1. Tumour mass reduction (%) in mice xenotransplanted with H22 liver tumour cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2. Results of two studies are illustrated (A and B).
Figure 2. Tumour mass reduction (%) in mjce xenotransplanted with kidney tumour cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2. Results of two studies are illustrated (A and B).
Figure 3. Tumour mass reduction (%) in mice xenotransplanted with B16 melanoma cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2. Results of two studies are illustrated (A and B). Figure 4. Tumour mass reduction (%) in mice xenotransplanted with lung tumour cells following parenteral (INJ) or sublingual (ORA) administration of human IL-2.
Detailed Description of the Invention
s Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 0
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. s As used herein the term "therapeutically effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth.o Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.
As used herein the terms "treating" and "treatment" refer to any and all uses which remedy a5 disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Thus, "treatment" refers not only to treatment designed to cure or remove symptoms in an individual, but also to ongoing therapy designed to control and suppress the occurrence of symptoms. Treatment may be for a defined period of time, or provided on an ongoing basis0 depending on the particular circumstances of any given individual. As used herein the term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds. The terms "polypeptide" and "protein" are used interchangeably herein, although for the purposes of the present invention a "polypeptide" may constitute a portion of a full length protein. The term "polynucleotide" as used herein refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof.
The present invention is predicated on the inventor's surprising finding that in a mouse model mucosal administration of recombinant interleukin (in the form of IL-2) results in a substantial and statistically significant reduction in tumour load without evident toxicity. Further, as exemplified herein, this mucosal administration is dose independent. This is in contrast to prior art administration by injection in which higher doses are required to achieve similar therapeutic effect, with the dose to be administered being constrained by the generation of side effects and toxicity.
Accordingly, the present invention provides a novel therapeutic treatment option for a variety of cancers with low dosages such that side effects commonly observed with presently available systemic treatments are either obviated or reduced. To date, interleukin therapy for the treatment of cancer has only been attempted intravenously or subcutaneously. Such administration requires high doses of interleukin, at levels considered abnormally high relative to the levels by which cytokines such as IL-2 act to mediate cellular responses naturally. These high doses result in significant side effects to patients. .
The inventor's novel finding of therapeutically efficacious mucosal interleukin administration opens the way for the development of cost effective, non toxic therapeutic alternatives to presently available injection therapies.
Accordingly, in one aspect the present invention provides a method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof. For use in accordance with the present invention the interleukin may be selected from, for example, IL-2, IL-12, IL-15 or IL-18. In one embodiment of the invention, the interleukin is IL-2.
The interleukin (s) used in the methods and compositions of the invention may be natural, s recombinant or synthetic and may be obtained by purification from a suitable source or produced by standard recombinant DNA techniques such as those well known to persons skilled in the art, and described in, for example, Sambrook et al.; Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press (the disclosure of which is incorporated herein by reference). Those skilled in the art will appreciate that the precise sequence of the interleukins to beo employed in accordance with the present invention may vary depending on a number of factors, for example the species and/or the cancer to be treated. Reference to "interleukin" or "interleukins" should be understood as a reference to all forms of this molecule and to functional derivatives, variants and homologues thereof. This includes, for example, any isoforms which arise from alternative splicing of the subject interleukin mRNA or functional mutants ors polymorphic variants of these proteins. Also encompassed within the scope of the invention are homologs or mimetics which possess qualitative biological activity in common with the full-length mature interelukin. Further, the present invention contemplates not only use of the interleukin polypeptide, but also polynucleotides encoding the same. 0 "Derivatives" of interleukin include analogues, functional fragments, parts, portions or variants from either natural or non-natural sources. Non-natural sources include, for example, recombinant or synthetic sources. By "recombinant sources" is meant that the cellular source from which the subject molecule is harvested has been genetically altered. "Analogue" means a polypeptide which is a derivative of interleukin, which derivative comprises addition, deletion,5 substitution of one or more amino acids, such that the polypeptide retains substantially the same function as the native interleukin from which it is derived. Modifications may be made so as to enhance the biological activity or expression level of interleukin or to otherwise increase the effectiveness of the polypeptide to achieve a desired result. The term "conservative amino acid substitution" refers to a substitution or replacement of one amino acid for another amino acid witho similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (GIu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution. Amino acid insertional derivatives also include amino and/or carboxyli.c terminal fusions as well as intrasequence insertions of single or multiple amino acids, lnsertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place.
"Fragment" refers to a polypeptide that is a constituent of full-length interleukin. The fragment typically possesses qualitative biological activity in common with the full-length interleukin. The fragment may be derived from the full-length interleukin polypeptide or alternatively may be synthesised by some other means, for example chemical synthesis. As used herein a "variant" of interleukin means a molecule of substantially similar sequence to the interleukin of which it is a variant and which exhibits at least some of the functional activity of the interleukin of which it is a variant. A variant may take any form and may be naturally or non-naturally occurring. Generally, variant polypeptides may share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
As used herein a "homologue" means that the interleukin is derived from a species other than that which is being treated in accordance with the method of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of interleukin which exhibits similar and suitable functional characteristics to that of the interleukin which is naturally produced by the subject undergoing treatment.
In accordance with an embodiment of the present invention, the interleukin to be administered is human IL-2. Full length human IL-2 has the amino acid sequence as set forth in SEQ ID NO:1 (precursor form) or SEQ ID NO:2 (mature form) and in its native form is an approximately 15,500 Da glycosylated protein.
Embodiments of the present invention also provide for the administration of interleukin in the form of a polynucleotide encoding an interleukin polypeptide as described above. Typically the polynucleotide encodes human IL-2. For example an IL-2 polynucleotide of the invention may have the nucleotide sequence as set forth in SEQ ID NO:3. In addition to polynucleotides encoding the full-length human polypeptide, the invention also contemplates the use of polynucleotides encoding homologues, fragments and variants thereof.
In particular embodiments of the invention the polynucleotide may be administered in a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion and foreign sequences and introduction into eukaryotic cells. Typically the vector is an expression vector capable of directing the transcription of the DNA sequence of the polynucleotide encoding the desired polypeptide into mRNA. The vector may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyodenylation signals and transcription termination sequences. Examples of suitable viral expression vectors include for example Epstein-barr virus-, bovine papilloma virus-, adenovirus- and adeno-associated virus-based vectors. The vector may be episomal.
In one embodiment, the methods and compositions of the present invention are particularly suitable for the treatment of liver cancers, primary and secondary, such as hepatocellular carcinomas. However, those skilled in the art will readily appreciate that the present invention is not so limited and is also applicable to the treatment of any tumour or cancer susceptible to treatment via mucosal administration of an active agent. For example the methods and compositions of the invention also find application in the treatment of tumours in a variety of organs. For example the cancer may be a prostate, breast, lung, stomach, intestinal, bladder, ovarian, cervical, oesophageal, pancreatic, renal, or brain cancer, a head or neck tumour, melanoma, or lymphoma. The cancer may be a primary or secondary cancer. The cancer may be a sarcoma, for example a liposarcoma.
The present invention provides methods and compositions for the mucosal delivery of interleukin. Preferably the mucosal administration of interleukin is oral administration, although other intranasal administration, for example, inhalation, is also contemplated. Typically oral administration comprises sublingual or. buccal administration whereby the composition is placed into contact with the buccal mucosa either under the tongue or in the cheek pouch allowing entry of the active agent directly to the bloodstream by absorption. Suitable forms for oral administration include solid, liquid, emulsion, gel and suspension. In a particular embodiment, a composition of the invention is administered in solid unit dosage form, for example in the form of a tablet, capsule, caplet, or lozenge. In one embodiment, the administration may comprise a gel administered to the nasal, buccal or sublingual area. In instances where the interleukin is unstable in a given liquid, this can be overcome by dissolving freeze dried interleukin powder in a 5 diluent, for example water plus honey^ or carboxymethylcellulose, dextran, maltodextrin, gums, albumin, sugars such as dextrose, maltose, mannitol etc. Honey is particularly useful as it contains many antigens which may assist the interleukin immune response.
In general, suitable compositions may be prepared according to methods which are known toQ those of ordinary skill in the art and may include' a pharmaceutically acceptable diluent, adjuvant and/or excipient. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. s Examples of pharmaceutically acceptable diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such asQ liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid5 esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. The diluent may be dextran, trehalose, carboxymethylcellulose, albumin, propylene glycol in 99% water. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions. o In particular embodiments of the present invention the interleukin, for example IL-2, is administered in the form of a solid unit dosage form such as a tablet, capsule or lozenge suitable for oral, most typically sublingual, administration. Suitable solid compositions may comprise a rapidly disintegrating composition comprising interleukin in a pharmaceutically acceptable water soluble or water dispersible carrier material. Such compositions may disintegrate or dissolve in the mouth upon placement under the tongue or insertion into the buccal pouch. Compositions may be formulated for rapid or immediate release of the interleukin or alternatively for delayed or controlled release. Techniques and processes for achieving delayed or controlled release of active agents are well known to those skilled in the art.
A broad range of processes for the preparation of such dosage forms are well known to those skilled in the art and are contemplated by the present invention. For example, suitable formulations may be prepared by processes including freeze drying under vacuum, supercritical fluid drying, spray drying using heat, and fluid bed spray drying. Of application in the context of particular embodiments of the present invention is a process involving microencapsulation whereby the active ingredient is coated onto granules, tablets or microparticles, typically using organic solvents. One particularly suitable process involves the use of a fluidised bed spray process facilitating the coating onto granules at room temperature of actives including polypeptides with a water solubilising coat, as disclosed in International Patent Application Publication No. WO 02/058735 (the disclosure of which is incorporated herein in its entirety by reference). Microparticles such as water soluble gel forming particles may be thus coated, or alternatively a blank tablet, lozenge or capsule core may be spray coated. Also known in the art are means for the preparation of oral compositions incorporating an effervescent agent as a penetration enhancer to increase the permeability of the active agent across the buccal and sublingual mucosa (see for example US Patent No. 6,974,590, the disclosure of which is incorporated herein in its entirety by reference). Other delivery modes contemplated by the present invention include the use of bioadhesives, mucoadhesives and liposomes.
The compositions of the invention may also be administered in the form of liposomes. Liposomes may be derived from phospholipids, or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are DODMA, synthetic cholesterol, DSPC, PEG-cDMA, DLinDMA, or any other non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes. The compositions in liposome form may contain stabilisers, preservatives and/or excipients. Methods for preparing liposomes are well known in the art, for example see Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 ff., the contents of which are incorporated herein by reference.
The compositions of the invention may also be administered in the form of microparticles. Biodegradable microparticles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and epsilon-caprolactone (έ-caprolactone) may be used.
The compositions of the invention may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and an organic solvent or mixture of organic solvents. Polymer additives may be added to further increase the viscosity so as to decrease the release rate.
Those skilled in the art will readily appreciate that a number of suitable processes and techniques exist for the manufacture of suitable oral compositions in accordance with the present invention and that the invention is not limited by reference to any one particular process or technique.
Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid 'paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
The therapeutically effective dose level of a composition of the present invention for any particular patient will depend upon a variety of factors including any one or more of: the type of cancer being treated and the stage of the cancer; the activity of the active agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of sequestration of compounds; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.
One skilled in the art would be able, by routine experimentation, to determine an effective, nontoxic dosage which would be required to treat applicable tumours. These will most often be determined on a case-by-case basis.
In particular embodiments of the present invention the preferred dose of interleukin, for example IL-2, is in the order of about 1 IU to about 1 million IU per day, although doses above and below this range are also contemplated. ' A feature of the present invention is that mucosal administration of interleukin enables significantly lower doses of interleukin to be employed than is possible with prior art approaches whilst retaining therapeutic benefit. Accordingly, the minimum dose of interleukin that may be used in accordance with the invention can be
5 determined by those skilled in the art, provided that the dose is sufficient to achieve a therapeutic benefit. In terms of the maximum dose, this can also be determined by a person skilled in the art taking into consideration factors such as those discussed herein. For example a dose of up to several million IU per day may be appropriate in some circumstances. Typically the dose of interleukin employed in accordance with the invention is between about 1 IU and about 100,000o IU per day, between about 10 IU and about 50,000 IU per day, between about 100 IU and about 20,000 IU per day, or between about 500 IU and about 10,000 IU per day. Depending on a variety of factors as described herein, including the nature and severity of the cancer to be treated, the daily dose administered to a patient in need thereof may be in the order of about 100 IU, 200 IU, 300 IU, 400 IU, 500 IU, 600 IU, 700 IU, 800 IU, 900 IU, 1,000 IU, 2,000 IU, 3,000 IU,s 4,000 IU, 5,000 IU, 6,000 IU, 7,000 IU, 8,000 IU, 9,000 IU, 10,000 IU, 11,000 IU, 12,000 IU, 13,000 IU, 14,000 IU, 15,000 IU, 18,000 IU, 20,000 IU, 25,000 IU, 30,000 IU, 35,000 IU, 40,000 IU, 45,000 IU and about 50,000 IU.
It will also be apparent to one of ordinary skill in the art that the optimal quantity and spacing ofo individual dosages will be determined by the nature and extent of the cancer being treated, the form, route and site of administration,, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques known to those skilled in the art. For example, a subject may be administered the desired daily dose in a single unit dosage form once per day, or in two unit dosage forms administered twice a day. 5
It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests. o Methods and compositions of the present invention may be employed in combination with other therapies for the treatment of cancers. For example, IL-2 therapy performed in accordance with the present invention may be carried out in conjunction with chemotherapy or radiotherapy or as an adjunct to surgery. IL-2 may also be combined with one or more additional therapeutic agents, including for example other immunomodulatory agents. Such immunomodulatory agents may include other cytokines or chemokines, such as, for example, interleukins or interferon. Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art.
For such combination therapies, each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired therapeutic effect. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Alternatively, the components may be formulated together in a single dosage unit as a combination product.
All publications mentioned in this specification are herein incorporated by reference. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention. Examples
Example 1 - Mucosally administered recombinant human IL-2 and liver cancer
To determine the efficacy of mucosal administration of IL-2 in the treatment of cancer, mice xenotransplanted with a hepatic cancer cell line were used.
Mature, virulent and active H22 neoplasmic cells were diluted with physiological saline solution to a concentration of 1χ107 cell/ml. All mice (Kunming Murine Clean type 18 - 22g female mice) were inoculated subcutaneously with 0.2 ml of cells under the arm pit. On day 2 these animals were randomized into ten groups of ten as shown in Table 1 below. Treatment began on the same day after randomization. For mucosal administration each mouse was given 10 ul of liquid twice a day for 9 days. The recombinant human IL-2 and recombinant human IFN-α2b were purchased from Beijing SL Pharmaceutical Co., Ltd. (Beijing, People's Republic of China) and Tianjin Hualida Biotechnology Co., Ltd. (Tianjin, People's Republic of China) respectively. These were stored as freeze dried materials in vials before being diluted to the concentration required prior to use. For intraperitoneal administration, the rHulFNoώb is diluted with bovine serum albumin in phosphate buffer solution (0.5% w/w BSA/PBS). On day ten, mice were sacrificed. Body weight and the weight of the extracted tumour were recorded. The tumour suppression percentage was calculated as follows.
(Mean tumour weight of control group - mean tumour weight of treatment group )
Mean tumour weight of control X 100 %
Results are shown as % tumour reduction in Table 1. Table 1
Figure imgf000018_0001
1. Expressed as IU on a per day basis
2. In the placebo group the extracted tumour weight was 3g
As shown above, mucosally administered recombinant human IL-2 proved effective in reducing the tumour burden in H22 xenotransplanted mice in a dose independent manner at 100 IU, 1 ,000 IU and 10,000 IU. Similar levels of tumour reduction were observed for the group receiving a combination of mucosally administered recombinant human IL-2 at 10,000 IU and mucosally administered recombinant human IFN-α2b at 10,000 IU.
No evidence of toxicity was observed for the groups receiving mucosally administered IL-2 alone or in combination with IFN-α2b (data not shown). Further, as shown in Table 2 below, animals in each treatment group gained weight over the course of the treatment period at similar rates to those animals in the placebo group and those receiving no treatment. Table 2
Figure imgf000019_0001
Example 2 - Sublingual administration of recombinant human IL-2 and liver cancer
Further to the experiments described in Example 1 above, an additional experiment was carried out using the same protocol comparing the efficacy of sublingual delivery of recombinant human IL-2 (performed as per Example 1 above) with subcutaneous delivery of recombinant human IL-2 in a BSA/PBS solution. Treatment groups and doses are shown in Table 3 below. Other parameters are as for Example 1. Table 3
Figure imgf000020_0001
As shown in Table 3 above, sublingual delivery of recombinant human IL-2 administered at 1,000 5 IU per day resulted in a 60% reduction in tumour burden compared with only a 43% reduction for the same dosage of subcutaneously administered recombinant human IL-2.
Subsequent experiments were carried out (as described above) using lower concentrations of human IL-2 (1 IU to 500 IU) administered sublingually, For sublingual administration mice were
10 administered two doses per day, each containing, half of the daily dose of 1, 10, 100 or 500 IU. Exemplary data from two such experiments are shown in Figures 1A and 1B. It can be seen that sublingual administration of 10 IU resulted in greater tumour mass reduction than parenteral injection of 10-fold higher concentration (100 IU) of IL-2. Even greater tumour mass reduction is observed following sublingual administration of 100 IU IL-2.
I5
Example 3 - Sublingual administration of recombinant human IL-2 and kidney cancer
Adopting the protocols described above in Examples 1 and 2, Kunming Murine Clean type 18 - 22g female mice were xenotransplanted with Renca kidney tumour cells and the effect of recombinant human IL-2 administration of subsequent tumour mass was determined. Before
20 inoculation, well-grown renal cancer clumps were removed and isolated into single cell suspension (107 cells/ml) in physiological saline. All mice were inoculated with 2x106 cancer cells (0.2ml) by subcutaneous injection into the dorsum region. On the second day these animals were grouped randomly (10 mice per treatment regime) into 7 groups for treatment with: 1 IU, 10 IU, 100 IU, or 500 IU IL-2 by sublingual administration; 100 IU IL-2 by subcutaneous injection; no
25 treatment negative control; and placebo treatment with vehicle only. Each mouse was given 10 μl of liquid twice a day for 15 days. On day 21, mice were sacrificed. Body weight and the extracted tumor weight were recorded. The tumor suppression percentage was calculated as described in Example 1.
5 The results of two independent studies are shown in Figure 2. In the first study, the tumor growth inhibition rate of mucosal administration of 10IU, 100IU, 500IU rhlL-2, were 37.05%, 44.68% and 32.60%, respectively. Mucosal administration of 100IU rhlL-2 showed better tumor growth inhibition efficacy than subcutaneous administration (inhibition rate 34.48%). In the second study, the tumor growth inhibition rate of mucosal administration of 10IU, 100IU, 500IU rhlL-2, wereo 40.89%, 47.66% and 17.85%, respectively. Mucosal administration of 100IU and 500IU rhlL-2, twice a day for 15 days showed better tumor growth inhibition efficacy than subcutaneous administration (inhibition rate 32.32%). The results indicate that rhlL-2 significantly inhibits transplanted mouse renal cancer growth.
s Example 4 - Sublingual administration of recombinant human IL-2 and skin and lung cancers
Adopting the protocols described above in Examples 1-3, Kunming Murine Clean type 18 — 22g female mice were xenotransplanted with either B'16 melanoma cells or Lewis lung tumour cells and the effect of recombinant human IL-2 administration of subsequent tumour mass was0 determined.
As shown in Figures 3 (melanoma) and 4 (lung), sublingual administration of daily doses of 10 IU and 100 IU IL-2 resulted in greater tumour mass reduction than was achieved with parenteral injection of 100 IU IL-2. 5
Example 5 - Compositions for treatment
In accordance with embodiments of the present invention as disclosed herein IL-2 is typically administered in the form of a pharmaceutical- composition suitable for oral, most typically sublingual administration. An example of a composition in accordance with the invention iso outlined below. The following is to be construed as merely an illustrative example and not as a limitation of the scope of the present invention in any way. 2'1
Example 5A - Composition for oral administration in table form
A composition comprising IL-2 in the form of a tablet may be prepared by incorporating the IL-2 into a film comprising one or more of gelatin, maltodextrin, carboxylmethyl cellulose, glucose, carbomer and coating the film onto a blank tablet core made up of known pharmaceutically acceptable ingredients selected from starch, calcium phosphates, carboxymethylethyl cellulose, mannitol, maltose, talc and magnesium stearate.
Example 5B - Composition for oral administration in capsule form A composition comprising IL-2 in the form of a capsule may be prepared by filling a standard two- piece hard gelatin capsule with IL-2, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.

Claims

Claims:
1. A method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of interleukin or a fragment or derivative thereof.
2. The method of claim 1 wherein the mucosal administration is oral administration.
3. The method of claim 1 or 2 wherein the administration is buccal or sublingual.
4. The method of any one of claims 1 to 3 wherein the interleukin is recombinant human interleukin.
5. The method of any one of claims 1 to 4 wherein the interleukin is IL-2.
6. The method of any one of claims 1 to 5 wherein the cancer is selected from liver, lung, kidney, skin, bladder, stomach, breast and blood cancer.
7. The method of claim 6 wherein, the cancer is selected from liver, lung, kidney and skin cancer.
8. The method of any one of claims 1 to 7 wherein the interleukin is administered in the form of a polynucleotide encoding the interleukin, optionally located in a genetic construct, operably linked to a promoter.
9. The method of any one of claims 1 to 8 further comprising the administration of one or more anti-cancer agents.
10. The method of any one of claims 1 to 9 wherein the interleukin is administered at a dose of between about 1 IU and 100,000 IU per day.
11. The method of any one of claims 1 to 10 wherein the interleukin is administered at a dose of between about 1 IU and 10,000 IU per day.
12. The method of any one of claims 1 to 11 wherein the interleukin is administered at a dose of between about 100 IU and 10,000 IU per day.
13. A pharmaceutical composition for mucosal administration for the treatment of cancer, the composition comprising interleukin or a fragment or derivative thereof, optionally together with one or more pharmaceutically acceptable carriers, excipients and/or diluents.
14. The composition of claim 13 wherein the interleukin is IL-2.
15. A method for treating cancer in a subject, the method comprising mucosally administering to the subject an effective amount of a composition according to claim 13 or claim 14.
16. The method of claim 15 wherein the interleukin is IL-2.
17. Use of interleukin or a fragment or derivative thereof for the manufacture of a medicament for treating cancer, wherein the medicament is administered mucosally.
18. Use according to claim 17 wherein the interleukin is IL-2.
PCT/AU2008/000350 2007-03-14 2008-03-13 Method for treating cancer via the mucosal administration of interleukin WO2008109953A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012065212A1 (en) 2010-11-19 2012-05-24 Sai Ying Ko Anti-inflammatory compositions
US12006354B2 (en) 2017-05-24 2024-06-11 Novartis Ag Antibody-IL2 engrafted proteins and methods of use in the treatment of cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679647A (en) * 1993-08-26 1997-10-21 The Regents Of The University Of California Methods and devices for immunizing a host against tumor-associated antigens through administration of naked polynucleotides which encode tumor-associated antigenic peptides
WO1999018992A1 (en) * 1997-10-10 1999-04-22 Pharma Pacific Pty. Ltd. Oromucosal cytokine compositions and uses thereof
CN1533807A (en) * 2003-03-27 2004-10-06 谭述军 High dosage recombination human alpha interferon oral tablet and its manufacturing method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679647A (en) * 1993-08-26 1997-10-21 The Regents Of The University Of California Methods and devices for immunizing a host against tumor-associated antigens through administration of naked polynucleotides which encode tumor-associated antigenic peptides
WO1999018992A1 (en) * 1997-10-10 1999-04-22 Pharma Pacific Pty. Ltd. Oromucosal cytokine compositions and uses thereof
CN1533807A (en) * 2003-03-27 2004-10-06 谭述军 High dosage recombination human alpha interferon oral tablet and its manufacturing method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 200507, Derwent World Patents Index; Class B04, AN 2005-058488 *
TOVEY M.G. ET AL.: "Mucosal Cytokine Therapy: Marked Antiviral and Antitumor Activity", JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, vol. 19, no. 8, 1999, pages 911 - 921, XP000998238 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012065212A1 (en) 2010-11-19 2012-05-24 Sai Ying Ko Anti-inflammatory compositions
JP2013544244A (en) * 2010-11-19 2013-12-12 イン コ,サイ Anti-inflammatory composition
KR20140009262A (en) * 2010-11-19 2014-01-22 잉 코 사이 Anti-inflammatory compositions
AU2011331901B2 (en) * 2010-11-19 2015-01-22 BioLingus IP II GmbH Anti-inflammatory compositions
US9289493B2 (en) 2010-11-19 2016-03-22 Biolingus Ip Llc Method for treating inflammatory conditions with mucosally administered interleukin-2
KR101989201B1 (en) * 2010-11-19 2019-06-13 바이오링거스 아이피 엘엘씨 Anti-inflammatory compositions
US12006354B2 (en) 2017-05-24 2024-06-11 Novartis Ag Antibody-IL2 engrafted proteins and methods of use in the treatment of cancer

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