Patent Application
20140050735
This invention is in the field of Crohn's disease. In particular, it relates to the treatment of fistulas in Crohn's disease using anti-IL-13 antibodies. The antibody may be an IgG and in particular may be the anti-IL13 antibody 01951/G12.
Crohn's disease (CD) is a chronic, relapsing/remitting inflammatory disease of the gastrointestinal (GI) tract. The regions of the GI tract most often affected by CD are the small intestine and the colon, including the ano-rectum. The inflammation and ulcerations of CD can extend throughout all layers of the intestinal wall in both the small and large intestines. Common symptoms of CD include diarrhea, abdominal pain, rectal bleeding, and weight loss as well as complications such as intestinal abscesses, fistulas, and intestinal obstructions. CD can become clinically manifest in many different ways including fibrostenotic (stricturing), or nonperforating, nonstricturing (inflammatory), or predominantly perforating (fistulizing) disease. Patients with fistulizing CD tend to have a more aggressive disease course. Fistulas can be either external (enterocutaneous or perianal) or internal, such as entero-enteral or entero-cystic. The cumulative incidence of CD fistulas is 33% and 50% 10 and 20 yrs, respectively, after diagnosis.
Morbidity is greatly increased in patients with fistulizing disease resulting in considerable negative impact on patients' quality of life. Perianal fistulas can lead to fecal incontinence, abscess formation and anal strictures; they may be further associated with pain, abscesses, and drainage. The treatment of fistulas depends on many factors, including location, severity, and previous surgical history.
Overall, CD fistulas are difficult to treat, rarely heal spontaneously and frequently require surgery. Before the introduction of biologic agents, most fistulas required surgical intervention, and the rate of fistula recurrence was estimated to be 30-40% (1-3). The current standard of care is antibiotics (metronidazole/ciprofloxacin—1st line), immunosuppressives (6-MP/azathioprine—2nd line) and biologics (anti-TNFα's—3rd line, or ‘top-down’ 1st line). Calcineurin inhibitors are being tested. Of note, some of the standard-of-care therapies for fistulizing Crohn's disease (e.g. azathioprine and 6-MP) are teratogenic.
The advent of biologics has expanded therapeutic treatment options and changed the practitioners' goal of treatment for fistulas from reduction in fistula drainage to true closure of the fistula tract. However, approximately 50% of patients do not respond to anti-TNFα's and hence, given the risk of incontinence associated with aggressive surgical procedures, there remains an unmet medical need for new and improved therapies for fistula treatment in Crohn's disease.
Pathophysiologically, the transmural inflammation characteristic of CD predisposes patients to the formation of fistulas, and impaired wound healing appears to be involved. While no preclinical animal models are available to further explore this indication, fistulizing CD likely involves dysregulated tissue remodeling processes that occur in the context of chronic transmural inflammation: Tissue repair and consecutive fibrosis result from excessive extracellular matrix (ECM) synthesis due to enhanced myofibroblast activity in conjunction with reduced activity of proteolytic/ECM degrading enzymes, while on the other hand inflammation-induced ulcer formation, i.e. tissue destruction, is driven by oxygen metabolites, activated immune cells, and up-regulated ECM degrading enzymes like matrix metalloproteinases (MMPs) and serine-proteases.
Based on the current understanding of the role of IL-13 in the context of CD (35), IL-13 may have anti-inflammatory properties. Therefore, an anti IL-13 treatment could lead to an exacerbation of the inflammatory activity in CD patients. Anti IL-13 therapy in CD is therefore not normally considered an option.
It has recently been demonstrated that epithelial-to-mesenchymal transition (EMT) related events are present in and around CD-associated fistulas (6). EMT represents the transformation from a differentiated, polarized epithelial cell to a mesenchymal-like cell featuring a myofibroblast phenotype. As a specific characteristic these EMT cells downregulate their intercellular connections and display both, epithelial markers, such as E-cadherin or cytokeratines 8 and 20, as well as mesenchymal markers, such as vimentin or α-SMA (7-8). On a functional level, EMT is essential for embryogenesis, organ development and wound repair, but is also associated with tissue fibrosis as well as with tumour growth and metastasis (7-9).
Here, we demonstrate that TGFβ induces SNAIL1 as well as IL-13 mRNA expression in primary human colonic lamina propria fibroblasts (CLPF) derived from CD patients. High levels of IL-13 and IL-13Rα1 were detected in TC lining the tracts of CD-associated fistulas. In an intestinal epithelial cell (IEC) model of EMT, IL-13 induced SLUG and β6-integrin levels, whereas chronic TGFβ administration resulted in concomitant elevation of SNAIL1 and IL-13 mRNA expression. However, the mediators exerted their effects with opposing kinetics. Our data show that IL-13 is present in CD-associated fistulas and induces the expression of genes associated with invasive cell growth suggesting an important role for the cytokine in the pathogenesis of such fistulas.
We have interpreted these findings to indicate that IL-13 is involved in driving the tissue remodeling that accompanies fistula formation in CD, so that anti IL-13 therapy will be a useful treatment for this patient group.
The IL-13 polypeptide has the below sequence. The N-terminal 34 amino acid residues (in italics) is a signal peptide. The mature cytokine thus has 112 amino acid residues. Anti-IL-13 antibodies will bind to an epitope on the mature polypeptide.
Interleukin 13 amino acid sequence:
In principle any anti-IL-13 antibody which inhibits or neutralizes the activity of IL-13 may be used in the invention. Such antibodies are known in the art, see for example in WO2005/007699, U.S. Pat. No. 6,468,528, WO03007685, WO03034984, US20030143199, US2004028650, US20040242841, US2004023337, US20040248260, US20050054055, US20050065327, WO2006/124451, WO2006/003407, WO2005/062967, WO2006/085938, WO2006/055638, WO2007/036745, WO2007/080174 or WO2007/085815.
In a preferred embodiment, the antibody is 01951/G12 (SEQ ID No. 31 and 33), further described in WO2007/045477.
In one embodiment, the antibodies used in the invention have affinities to IL-13 in the low pM range and inhibit IL-13 induced signalling with an IC50 of about 10 nM. By low pM range we mean 100 pM or less, preferably 500 pM or less, preferably 10 pM or less, more preferably 1 pM or less.
The sequences of the antibodies of the previous tables, including framework regions, are shown below. The full IgG1 antibody light and heavy chain constant regions are also shown below, incorporating, as an example, the variable regions of antibody 01951/G12 (emboldened).
The HC variable amino acid sequence for 01471/G6 is shown in SEQ ID NO: 23 and is encoded by the nucleotide sequence shown in SEQ ID NO: 24
The LC variable amino acid sequence for 01471/G6 is shown in SEQ ID NO: 25 and is encoded by the nucleotide sequence shown in SEQ ID NO: 26
The HC variable amino acid sequence for 03161/H2 is shown in SEQ ID NO: 27 and is encoded by the nucleotide sequence shown in SEQ ID NO: SEQ ID No. 28
The LC variable amino acid sequence for 03161/H2 is shown in SEQ ID NO: 29 and is encoded by the nucleotide sequence shown in SEQ ID NO: 30
The HC variable amino acid sequence for 01951/G12 is shown in SEQ ID NO: 31 and is encoded by the nucleotide sequence shown in SEQ ID NO: 32
The LC variable amino acid sequence for 01951/G12 is shown in SEQ ID NO: 33 and is encoded by the nucleotide sequence shown in SEQ ID NO: 34
The HC variable amino acid sequence for 01771/E10 is shown in SEQ ID NO: 35 and is encoded by the nucleotide sequence shown in SEQ ID NO: 36
The LC variable amino acid sequence for 01771/E10 is shown in SEQ ID NO: 37 and is encoded by the nucleotide sequence shown in SEQ ID NO: 38
The LC amino acid sequence is shown in SEQ ID NO: 39 and is encoded by the nucleotide sequence of SEQ ID NO: 40
CTCCAGGGGA AAGAGCCATC CTCTCCTGCA GGGCCGGTCA GAGTGTTAGC
S Y L V W Y Q Q K P G Q A P R L L
AGTTACTTAG TCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT
CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG
GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT
E D F A V Y Y C Q Q R S S W P P V
GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGCAGCAGCT GGCCTCCGGT
GTACACTTTT GGCCAGGGGA CCAAGCTTGA AATCAAACGA ACTGTGGCTG
The HC amino acid sequence is shown in SEQ ID NO: 41 and is encoded by the nucleotide sequence of SEQ ID NO: 42
R S L R L S C A A S G F T F S S
GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC
Y G M H W V R Q A P G K G L E W V
TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT
A I I W Y D G S N K Y Y A D S V K
GGCAATTATA TGGTATGATG GAAGTAATAA ATACTATGCG GACTCCGTGA
G R F T I S R D N S K N T L Y L
AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG
Q M N S L R A E D T A V Y Y C A R
CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG
L W F G D L D A F D I W G Q G T M
GCTATGGTTC GGGGACTTAG ATGCTTTTGA TATCTGGGGC CAAGGGACAA
TGGTCACCGT CTCCTCAGCC TCCACCAAGG GCCCATCGGT CTTCCCCCTG
It will be readily apparent to the ordinarily skilled artisan that novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein for monoclonal antibodies useful in the present invention.
As used herein, the term “antibody” means a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an epitope, e.g. an epitope found on IL-13, as described above. Thus, the term antibody includes whole antibodies (such as monoclonal, chimeric, humanised and human antibodies), including single-chain whole antibodies, and antigen-binding fragments thereof. The term “antibody” includes antigen-binding antibody fragments, including single-chain antibodies, which can comprise the variable regions alone, or in combination, with all or part of the following polypeptide elements: hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Also included within the definition are any combinations of variable regions and hinge region, CH1, CH2, and CH3 domains. Antibody fragments include, e.g., but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulphide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989; Muyldermans et al., TIBS 24; 230-235, 2001), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). The term “antibody” includes single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger & Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005)). Antigen binding portions of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen binding portions can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).
Preferably, the antibodies used in the invention bind specifically to IL-13. Preferably, the antibodies used in the invention do not cross-react with an antigen other than IL-13.
As used herein, an antibody that “specifically binds to IL-13” is intended to refer to an antibody that binds to IL-13 with a KD of 1×10−8 M or less, 1×10−9 M or less, or 1×10−10 M or less. An antibody that “cross-reacts with an antigen other than IL-13” is intended to refer to an antibody that binds that antigen with a KD of 0.5×10−8 M or less, 5×10−9 M or less, or 2×10−9 M or less.
In an alternative embodiment, the antibody used in the invention is one which cross-blocks one or more of the antibodies recited above. By “cross-blocks” we mean an antibody which interferes with the binding of another antibody to IL-13. Such interference can be detected, for example, using a competition assay using Biacore or ELISA. Such competition assays are described in WO2008/133722.
Included within the scope of the invention are methods of monitoring whether treatment of fistulas in CD patients using an anti-IL-13 antibody have been successful. The simplest method, although not the most accurate, will be whether the subject being treated has noticed an amelioration of symptoms.
Other methods include detecting the expression of various biomarkers such as TGF-β, periostin, eotaxin-1, procollagen type I C-terminal propeptide (PICP) and the N-terminal pro-peptide of collagen type III (PIIINP). IL-4, as well as the degree of phosphorylation of STAT6.
Such methods comprise assessing the level of expression of a chosen biomarker in a subject being treated and comparing said level of expression to a control level (such as the level of expression in the subject prior to treatment or the level in an untreated subject), wherein a level that is different to said control level is indicative of the treated subject responding to treatment.
The method may comprise the steps of:
a) measuring the expression of a biomarker indicative of fistula formation in a patient prior to treatment;
b) treating the patient with an anti-IL-13 antibody;
c) measuring the expression of said biomarker in the patient following treatment;
d) detecting a change in biomarker expression following treatment compared to the pre-treatment expression level, wherein said change in expression indicates a response to anti-IL-13 antibody treatment.
The measuring steps (a) and (c) above may be carried out on tissue samples obtained from the patient. The tissue sample being analysed may be blood, urine, saliva or other tissue from a tissue biopsy.
As an alternative, step (d) may comprise comparing the biomarker expression before and after treatment with control biomarker expression levels, wherein deviation from those control levels indicates a response to treatment with an anti-IL-13 antibody. Such control levels may be from a CD-free patient, a patient treated with placebo, or a patient treated with conventional anti-fistula medication.
Examples of biomarkers that may be measured include, but are not limited to TGF-β, periostin, eotaxin-1, PICP and PIIINP, IL-4, as well as the degree of phosphorylation of STAT6.
The antibodies used in the invention are generally formulated as a composition, e.g., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, formulated together with a pharmaceutically acceptable carrier. For example, a pharmaceutical composition used in the invention can comprise a combination of antibodies that bind to different epitopes of IL-13 or that have complementary activities.
Pharmaceutical compositions used in the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an anti-IL-13 antibody combined with an anti inflammatory agent. Such combinations may be administered simultaneously or sequentially. If administered sequentially, the period between administration of each agent may be a week or less, (e.g. a day or less, 12 hours or less, 6 hours or less, 1 hour or less, 30 minutes or less). The compositions are preferably formulated at physiological pH.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
Such pharmaceutical compositions may also include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, one can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
For administration of the antibody, the dosage ranges from about 0.0001 to about 100 mg/kg, and more usually about 0.01 to about 5 mg/kg, of the host body weight. For example dosages can be about 0.3 mg/kg body weight, about 1 mg/kg body weight, about 3 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight or within the range of about 1- about 30 mg/kg or about 1- about 10 mg/kg. An exemplary treatment regime entails administration about once per week, about once every two weeks, about once every three weeks, about once every four weeks, about once a month, about once every 3 months, about once every three to 6 months, about once every six months or about once a year. Dosage regimens for an anti-IL-13 antibody of the invention include about 1 mg/kg body weight or about 3 mg/kg body weight by intravenous administration, with the antibody being given using one of the following dosing schedules: about every four weeks for six dosages, then about every three months; about every three weeks; about 3 mg/kg body weight once followed by about 1 mg/kg body weight every three weeks.
In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously or sequentially, in which case the dosage of each antibody administered falls within the ranges indicated. The combination could be an anti-IL-13 antibody combined with an anti-IL4 antibody. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months, every six months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1- about 1000 μg/ml and in some methods about 25- about 300 μg/ml.
Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A “therapeutically effective dosage” of an anti-IL-13 antibody of the invention can result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
Compositions used in the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Intravenous and subcutaneous administration are particularly preferred.
Alternatively, an antibody used in the invention can be administered by a nonparenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, the compositions can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which shows an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which shows a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which shows a medication infusion pump for delivering medication at a precise infusion rate, U.S. Pat. No. 4,447,224, which shows a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which shows an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which shows an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.
The invention also provides a kit comprising a first component and a second component wherein the first component is an anti-IL-13 antibody or pharmaceutical composition as described above and the second component is instructions. In one embodiment, said instructions teach of the use of the antibody for treating fistulizing CD. The kit may further include a third component comprising one or more of the following: syringe or other delivery device, adjuvant, or pharmaceutically acceptable formulating solution.
The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The term “about” in relation to a numerical value x means, for example, x±10%. References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2. BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489
Discussion
Interestingly, SNAIL1 mRNA expression was three-fold higher after 7 d IL-13 treatment than in control cells. These observations suggest that SNAIL1 could act as a repressor of SLUG and β6-integrin expression in IEC. This findings could make sense in a way that TGF□/SNAIL1-induced EMT acts as a mechanism of wound healing and tissue regeneration at sites of chronic inflammation and tissue destruction, as present during active CD (33), but does not feature an invasive potential which can be observed in CD fistulae. In contrast, IL-13, which is expressed after chronic exposure of IEC to TGFβ, drives the invasive potential of EMT cells in an autocrine manner. This observation seems to be in contrast of previous findings, since during fibrogenesis, IL-13 acts upstream of TGFβ, whereas in the setting of cell invasion, the cytokine seems to be regulated by the growth factor.
Cell Culture
Patient Samples
Isolation and Culture of Human CLPF
Stimulation of Human CLPF
Immunohistochemistry
Real-Time PCR
Cells were resuspended in RLT-buffer (Qiagen, Valencia, Calif.) and lysed using a 24-gauge needle on a syringe. mRNA was isolated using RNeasy Plus Mini Kit (Qiagen, Valencia, Calif.) using a QIA-Cube (Qiagen) including shredder and DNA was removed by DNase I (Qiagen) according to manufacturer's instructions. RNA concentration was assayed by absorbance at 260 and 280 nm content using NanoDrop ND1000 (Thermo Scientific). cDNA synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif.). Real-time PCR TaqMan Assays and TaqMan Gene Expression Master Mix were obtained from Applied Biosystems. Real-time PCR was performed on a 7900HT Fast Real-Time PCR System using SDS 2.2 Software (Applied Biosystems). Triplicate measurements were performed and human β-actin was used as endogenous control. Results were then analyzed by ΔΔCT-method. The real-time PCR contained 40 cycles.
Preparation of Whole Cell Lysates
Western Blotting
ELISA
SiRNA Transfection
Statistical Analysis
This example shows how the treatment of fistula in CD patients with a particular antibody can be carried out and the effects assessed. It will be obvious to those skilled in the art that other IL-13 antibodies may be used in a similar manner although for example the exact dosing and administration schedule may vary dependent on the particular antibody used.
Efficacy in the proposed trial will require changes in fistulas leading to their closure. The current thinking hypothesizes that high local expression of IL-13 in the cells lining the fistula tracts is a key mediator of tissue remodeling and fibrosis. Thus, to achieve an effect on closure inhibition of local IL-13 in the fistula tissue is necessary. The required amount of 01951/G12 in the fistulas to inhibit IL-13 can be in principle derived from the equation describing the binding equilibrium between 01951/G12 and IL-13. However, the local IL-13 concentrations are not known. In addition, the fistula tissue accessibility for 01951/G12 is unknown, thus local 01951/G12 concentrations can not be predicted from plasma concentrations. As a consequence, sufficiently high doses should be used to drive permeation of 01951/G12 into fistula tissue. To give 01951/G12 the best chance to inhibit local IL-13, it is proposed to dose 10 mg/kg of 01951/G12 every 3 weeks.
01951/G12 150 mg Powder for Solution will be provided in glass vials each containing 150 mg 01951/G12 as a lyophilized cake. The vials contain a 20% overfill to allow a complete withdrawal of the labeled amount of 01951/G12. The manufacturing process for 01951/G12 Powder for Solution consists of standard manufacturing processes: Dilution, mixing/stirring, pre and sterile filtrations, aseptic filling and lyophilization. The drug product is considered to be stable until the date indicated on the drug product label if stored at 2 to 8° C. Based on results of ongoing stability studies the re-test period will be adjusted as appropriate. Immediately prior to administration, Sterile Water for Injection (SWFI) is added to the vial and the powder is dissolved ready for use to yield a single-use Concentrate for Solution for Infusion which needs to be diluted before use. For subsequent intravenous administration, the yielded Concentrate for Solution for Infusion needs to be further diluted to the ready-to-use 01951/G12 Solution for Infusion.
Reconstitution of 01951/G12 150 mg Powder for Solution with 1.0 mL SWFI will produce a Concentrate for Solution for Infusion at a concentration of 150 mg/ml 01951/G12 in a total final volume of 1.2 ml. The Concentrate for Solution for Infusion is available as histidine (pH 6.0±0.5) buffered solution, containing sucrose, glycine and polysorbate 80. The formulation does not contain a preservative as it is to be used for single-dose administration only. This concentrate is subsequently diluted in an infusion bag containing 5% glucose/dextrose solution in accordance with the instructions for use provided below. Since 01951/G12 is a protein, the reconstituted vials may contain a few translucent particles. The Solution for Infusion must therefore be infused through a 0.2 micron in-line filter (see filter supplier requirements under “Materials to be used”).
Note: The vials contain an overfill of 20% of 01951/G12. The dose/volume calculations as described below must be strictly adhered to.
The dose for administration to subjects will be calculated from the individual subjects' body weight as measured at the baseline visit.
Dose levels of 10 mg/kg can be administered.
To obtain the volume of 01951/G12 Concentrate for Solution for Infusion which is needed, the calculated dose is to be divided by the concentration of the Concentrate for Solution for Infusion (i.e. 150 mg/mL)
Subject weight: 65 kg
Dose level: 10 mg/kg
Calculated dose: 650 mg
Calculated volume: 650 mg/150 mg/mL=4.3 mL
Calculated number of vials: 4.3/1.2=4 per dose
The infusion set including the intravenous filter set has to be prepared according to the instructions supplied by the manufacturers (no product reference numbers are given as they might be country-specific):
01951/G12 should be administered as an infusion at a flow rate of about 2 mL/min (total administration time: approximately 120 minutes) using materials specified above (see Preparation of the infusion bags). 01951/G12 infusion can be performed by gravitational way of administration or using infusion pumps (i.e. Colleague CXE volumetric infusion pump if using the Baxter infusion line; Infusomat® fmS volumetric infusion pump if using the B.
Braun Infusomat infusion line, Alaris Gemini infusion pump if using the Alaris Pump module infusion line).
Fistula closure will be clinically assessed by the investigator. Clinical assessment of fistula activity includes assessment and documentation of•Location and appearance of fistula(s) with description of indurations, color and estimation of area of cutaneous fistula opening(s);
Photodocumentation will be utilized during this study to allow for documentation of fistula healing.
MRI is a useful technique to study the pelvis because it offers excellent soft tissue discrimination with a wide field of view, and it is free of radiation hazard. In this study, pelvis MRI will be used to assess the complexity and behavior of perianal fistulas over time. MRI images will be analyzed to produce a score reflecting both anatomical changes and active inflammation around the fistula tracks, as described by (Van Assche, et al 2003).
The purpose of including the Short Inflammatory Bowel Disease Questionnaire (SIBDQ) in this study is to assess disease-specific health related quality of life in subjects with fistulizing Crohn's disease. The SIBDQ is a valid, sensitive and reliable measure used extensively in clinical trials and research. The SIBDQ comprises 10 items, which are grouped into four subscales, including: bowel symptoms, systemic symptoms, emotional function, and social function. Each item is scored on a 7-point Likert scale ranging from 1 (worst) to 7 (best).
Biopsies from the fistula tracts will be obtained endoscopically during screening and 1 week after the first application of 01951/G12 (D8±2 days). Aim is to obtain biopsies from the lining of the fistula tracts via their luminal opening. In case the luminal fistula opening is inaccessible, the investigator will seek sponsor's advice and mutual agreement on a per case basis how to proceed. In such cases it is e.g. conceivable that mucosal biopsies are being obtained from the immediate vicinity of the internal fistula opening.
The choice of endoscopic technique is at the discretion of the investigators; most patients will have to be appropriately analgo-sedated for the biopsy procedures. Under visual control, the investigator will take 1 to 2 standard biopsies from the wall of the fistula tract. One biopsy will be immediately placed into RNA preservation medium and the other biopsy will be placed in a tube containing 10% buffered formalin solution and processed for histological examination. The procedure will be further detailed in a separate guidance sheet.
The samples processed for histology will be examined by a pathologist to evaluate the morphological changes in the wall of the fistula tract and the remaining paraffin block will be saved for further investigations (immunohistochemistry and/or in situ hybridization).
The sample intended for gene expression profiling will be processed for RNA microarray analysis.
TGF-β, periostin, eotaxin-1, PICP and PIIINP, IL-4
Serum and plasma samples will be collected to evaluate downstream biomarkers of the IL-13 pathway or in relation to other fibrotic mechanisms. The final biomarker panel will include, but will not be limited to TGF-β, periostin, eotaxin-1, PICP, PIIINP and IL-4. To be able to evaluate further biomarkers like IL-13 receptors depends on the availability of related assays.
The potential relationship between biomarker and clinical responses will be explored.
The question of whether any specific biomarkers (or combination of them) at certain levels are more important predictors of clinical response to treatment than others will be investigated to allow better definition of the target population. Additional blood for plasma (4 mL per time point) is taken for potential further investigation.
A single 14 ml blood sample will be drawn to ensure 9 ml serum.
All blood samples will be taken by either direct venipuncture or an indwelling cannula inserted in a forearm vein and collected into a sterile tube. After blood collection, the blood sample is allowed to clot for 30 min at room temperature. The tube must then be placed on ice. Samples should be then centrifuged immediately at 2000×g for 10 min at 4° C. After centrifugation, the supernatant is transferred to a new sterile polypropylene tube and gently mixed by inversion.
Serum-samples are aliquoted at 250 μl in 0.5 ml polypropylene cryovials (Sarstedt No. 72.730.006 or equivalent) and frozen immediately at least at −20° C. (=−70° C. is the preferred temperature, however, samples should be treated equally) within 45 minutes of venipuncture. Shipment must be performed on dry ice on the same day as collection. Upon arrival in the central lab and the site of analysis, samples should be stored at=−70° C.
Except for clotting, leaving samples at room temperature during processing (even if only for a few minutes) should be avoided. Leaving samples on ice (2-4° C.) during processing should not exceed a total of 45 minutes. Samples should be shipped as specified in the laboratory manual.
A single 4 ml venous blood sample should be collected in an EDTA tube to ensure 2 mL of plasma.
Immediately after each tube of blood is drawn, it should be inverted gently several times to ensure the mixing of tube contents (e.g., anticoagulant). Avoid prolonged sample contact with the rubber stopper. Place the tube upright in a test tube rack surrounded by ice until centrifugation. Within 30 minutes, centrifuge the sample at between 3 and 5° C. for 10 minutes at approximately 2500 g (or sufficient settings to achieve a clear plasma layer). Immediately after centrifugation transfer the supernatant plasma to 250 μl aliquots in 0.5 ml polypropylene cryovials (Sarstedt No. 72.730.006 or equivalent) and frozen immediately at least at −20° C. (−70° C. is the preferred temperature, however, samples should be treated equally) within 45 minutes of venipuncture. Shipment must be performed on dry ice on the same day as collection. Upon arrival in the central lab and the site of analysis, samples should be stored at −70° C.
Fecal calprotectin and lactoferrin levels are broadly used biomarkers for the assessment and follow-up of the Crohn's Disease activity and correlate with endoscopic findings and will provide a non invasive, inflammatory disease marker.
For each scheduled sampling time point, two fecal samples (each approx. 5 g) will be collected into two 30 mL stool collection tubes, which are immediately stored at −18° C. to −20° C. The samples can be shipped on dry ice to the Central Lab with the next available shipment.
Changes in levels of fecal calprotectin and lactoferrin as non-invasive, inflammatory disease markers by ELISA will be explored with regard to their relationship to clinical efficacy.
A preliminary assessment of biological responses to the study treatment described above, showed that a patient with fistula responded to treatment with the 01951/G12 IL-13 antibody. The overall fistula activity in the patient decreased; specific improvements observed included pain reduction and cessation of mucupurulent discharge from fistula. The conclusion is that the IL-13 antibody had a positive clinical effect, reducing the fistula activity.
It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB12/50699 | 2/15/2012 | WO | 00 | 10/30/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61443829 | Feb 2011 | US |
