COMBINATION OF METHOTREXATE AND TENIDAP FOR THE TREATMENT OF RHEUMATOID ARTHRITIS

Abstract: A combination of tenidap and methotrexate provides an advantageous treatment for rheumatoid arthritis.

Description: Corabi nati on of Methotrexate and Ten idap for the Treatment of Rheumatoid Arthriti s This invention relates to a method of treatment and a pharmaceutical composition comprising tenidap and methotrexate which is an advantageous treatment for rheumatoid arthritis. References Journal references cited herein are listed below. Citations in the text are identified by the author and year of publication. Al-Humidan, A. ., Reilly, K. M„ Leeming, M. R. G, Russell, R. G. G. Tenidap inhibits bone resorption induced by PTH. 1 ,25 Vit D3, IL-1.TNF and PGE2 in vitro by mechanisms independent of inhibition of prostaglandin synthesis. Arthritis Rheum 1991 ;34:S192. Amos, R. S., Constable, T. J., Crockson, R. A., Crockson, A. P., McConkey, B. Rheumatoid arthritis: relation of serum C-reactive protein and erythrocyte sedimentation rates to radiographic changes. Br Med J 1977;1 : 195-197. Arnett, F. C, Edworthy, S. M., Bloch, D. A., McShane, D. J., Fries, J. F., Cooper, N. S., Healey, L A., Kaplan, S. R., Liang, M. H., Luthra, H. S., Medsger, Jr., T. A., Mitchell, D. M., Neustadt, D. H., Pinals, R. S., Schaller, J. G., Sharp, J. T., Wilder, R. L, Hunder, G. G. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31 :315-24. Bloom, E. J., et al., Delayed clearance (CL) of methotrexate (MTX) associated with antibiotics and antiinflammatory agents. Clin Res 1986;34:560A. Carty, T. J., Showell, H. J., Loose, L D., Kadin, S. B. Inhibition of both 5-lipoxygenase (5-LO) and cyclo-oxygenase (CO) pathways of arachidonic acid metabolism by CP-66,248, a novel anti-inflammatory compound. Arthritis Rheum 1988;31 (suppl 4):S89. Cockcroft, D. N., Gault, M. H., Prediction of creatinine clearance from serum creatinine. Nephron 1976,16:31-41. Dawes, P. T., Fowler, P. D, Clarke, S., Fisher, J., Lawton, A., Shadforth, M. F. Rheumatoid arthritis: treatment which controls the C-reactive protein and erythrocyte sedimentation rate reduces radiological progression. Br J Rheumatol 1986;25:44-9. Dingle, J. T., Leeming, M. R. G, Martindale, J. J. Effect of tenidap on cartilage integrity in vitro. Ann Rheum Dis 1993;52:292-9. Emery, P. Methotrexate - its current use in rheumatoid arthritis. J Clin Pharm Ther 1989;14-.239-42, Feison, D. T., Anderson, J. J., Meenan, R. F. The comparative efficacy and toxicity of second-line drugs in rheumatoid arthritis: results of two metaanalyses. Arthritis Rheum 1990;33 (10):1449-61. Gabrielli, A., et al., Methotrexate and non-steroidal anti-inflammatory drugs. Br Med J 1987;294:776. Gardner. M. J., Wilner, K. D., Hansen, R. A, Fouda, H. G., McMahan, G. F. Single and multiple dose pharmacokinetics of tenidap sodium in healthy subjects. Br J Clin Pharmacol 1995; in press. Heinrich, P. O, Castell, J. V., Andus, T. lnterleukin-6 and the acute phase response. Biochem J 1990;265:621-36. Horton, R. C. Methotrexate - an immunomodulator with expanding indications. J Clin Pharm Ther 1990;15:89-95. Kirby, D. S., Loose, L D., Weiner, E. S., Wilhelm, F. E., Shanahan, W. R., Ting, N. Tenidap vs naproxen treatment of rheumatoid arthritis (RA). Arthritis Rheum 1993;36:S112. Kraska, A. R., Wilhelm, F. E., Kirby, D. S., Loose, L D., Ting, N., Shanahan, W. R., Weiner, E. S. Tenidap vs piroxicam vs piroxicam plus hydroxychloroquine in rheumatoid arthritis. Arthritis Rheum 1993;36:S57. Lankelma, J., Poppe, H. Determination of methotrexate in plasma by on-column concentration and ion-exchange chromatography. J Chromatogr 1978;149:587-98. Leeming, M. R. G. A double-blind randomised comparison of tenidap versus auranofin plus diclofenac in early rheumatoid arthritis (RA). Rev Esp Reum 1993;20(1):Th 189. Liftman, B. H., Drury, C. E., Zimmerer, R. O., Stack, C. B., Law, C. G. Rheumatoid arthritis treated with tenidap and piroxicam. Arthritis Rheum 1995; 38:29-37. Maiche, A. G. Acute renal failure due to concomitant action of methotrexate and indomethacin. Lancet 1986;1 :1390. Mallya, R. K., de Beer, F. O, Berr, H., Hamilton, E. D. B., Mace, B. E. W., Pepys, M. Correlation of clinical parameters of disease activity in rheumatoid arthritis with serum concentration of C-reactive protein and erythrocyte sedimentation rate. J Rheumatol 1982;9:3224-8. Moilanen, E., Alanko, J., Asmawi, M. Z., Vapaatalo, H. CP-66,248, a new anti-inflammatory agent, is a potent inhibitor of leukotriene B4 and prostanoid synthesis in human polymorphonuclear leukocytes in vitro. Eicosanoids 1988;1 :35-9. Nordstrom, D. M., West, S. G., Andersen, P. A., Sharp, J. T. Pulse methotrexate therapy in rheumatoid arthritis: a controlled prospective roentgenographic study. Ann Intern Med 1987; 107:797-801. Pazoles, C. J., McNiff, P., Laliberte, R., Gabel, C. A. Tenidap acts in vitro as an antagonist of cytokine-induced functions. Arthritis Rheum 1993;36:S109. Powles AV, Baker BS, Fry L, Valdimarsson H. Cyclosporin toxicity. Lancet 1990;335:610. Pullar, T. [Introduction to supplement] Br J Clin Pharmacol 1995; in press. Singh, R. R., Malariga, A. N., Pondley, J. N., Guleria, J. S. Fatal interaction between methotrexate and naproxen. Lancet 1986;1 :1390. Sipe, J. D., Bartle, L M., Loose, L D. Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response. J Immunol 1992;148:480-4. van Leeuwen, M. A., van der Heijde, D. M. F. M., van Rijswijk, M. H., Houtman, P. M., van Riel, P. L C. M, van de Putte, L B. A., Limburg, P. C. Interrelationship of outcome measures and process variables in early rheumatoid arthritis. A comparison of radiologic damage, physical disability, joint counts, and acute phase reactants. J Rheumatol 1994;21 :425-9. Wilke, W. S., Clough, J. D. Therapy for rheumatoid arthritis: combinations of disease-modifying drugs and new paradigms of treatment. Semin Arthritis Rheum 1991 ;21 :21-34. Wylie, G. A 24 week study of tenidap vs diclofenac in rheumatoid arthritis. Rev Esp Reum 1993;20(1):A306. Background of the Invention Kadin in United States Patent 4,556,672 describes certain 2-oxindole-1- carboxamide compounds with acyl substituents at the 3-position which are inhibitors of cyclo-oxygenase (CO) and lipoxygenase (LO) enzymes. These compounds are useful as analgesic agents in mammals and are useful in ameliorating or eliminating pain, such as pain experienced by patients recovering from surgery or trauma. These compounds are also useful for chronic administration to mammals to alleviate the symptoms of chronic diseases such as the inflammation and pain associated with rheumatoid arthritis and osteoarthritis. Kadin specifically claims a method of eliciting an analgesic response, and also a method of treating an inflammatory disease, in a mammalian subject, which comprises treating said mammalian subject with an effective amount of member selected from a genus of 2-oxindole-1-carboxamides. Tenidap sodium, (2)(Z)-5-chloro-3-(σ-hydroxy-2-thenylidene)-2-oxo-1 -indoline carboxamide, sodium salt is a novel anti-rheumatic drug, the first in a new class of drugs, the oxindoles. It is chemically unrelated to any other anti-rheumatic drug, including nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs) or corticosteroids. It is currently undergoing investigation for the treatment of rheumatoid arthritis (RA). Although tenidap inhibits cyclooxygenase (Carty, et al., 1988; Moilanen, et al., 1988), it is clearly differentiated from NSAIDs by its ability to modulate cytokines (Loose, et al., 1993). Tenidap has been shown to inhibit both the production and activity of cytokines in several in vitro systems. For example, tenidap inhibits the production of interleukin (IL)-1 , IL-6 and tumor necrosis factor alpha (TNF(σ) in cultured human peripheral blood monocytes (Sipe, et al., 1992), while in the same study naproxen demonstrated no effect. The cytokine-mediated production of acute phase proteins (APPs) was inhibited by tenidap in human Hep3B cells (Pazoles, et al., 1993), and tenidap substantially inhibits the IL-1 -mediated catabolic effects on cartilage in porcine cartilage explants (Dingle, et al., 1993). Tenidap inhibits bone resorption in mouse calvaria stimulated by a number of agents including cytokines (Al-Humidan, et al., 1991 ). This evidence suggests that tenidap may affect cytokine-mediated events which underlie the disease processes in RA. Clinical studies have shown that tenidap is effective in the treatment of RA. in studies comparing tenidap 120 mg/day with diclofenac (Wylie 1993), piroxicam (Kraska, et al., 1993), or naproxen (Kirby, et al., 1993), tenidap-treated patients showed significantly greater improvements in clinical and biochemical efficacy parameters than patients treated with NSAIDs. Tenidap has also been shown to be as effective as the DMARD/NSAID combinations of auranofin and diclofenac (Leeming, 1993), and hydroxychloroquine and piroxicam (Kraska, et al., 1993). In all of these studies, tenidap was associated with significant reductions in serum concentrations of the markers of disease activity, acute phase proteins (APPs) C-reactive protein (CRP) and serum amyloid A (SAA). Tenidap is differentiated from NSAID's such as piroxicam by lower levels of acute-phase proteins, ESR and IL-6 after tenidap treatment and these effects of tenidap on acute-phase proteins and IL-6 occur even if patients receive background treatment with prednisone, methotrexate, or both (Liftman, et al., 1995). Numerous studies have shown that radiographically assessed disease progression correlates with serum concentrations of CRP, and that DMARDs, but not NSAIDs, reduce the level of CRP in RA patients (Amos, et al., 1977; Dawes, et al., 1986; Mallya, et al., 1982; van Leeuwen, et al., 1994). The effect of tenidap treatment on the serum levels of CRP provides further evidence that it has the ability to modulate cytokines, because cytokines, particularly IL-6, have been found to mediate the release of APPs (Heinrich, et al., 1990). Methotrexate, (2)L-(+)-N-[p[[(2,4-diamino-6-pteridinyl)methyl]methylamino] benzoyljglutamine acid, is an antimetabolite of folic acid, which was initially used as an antineoplastic agent, but has also been used at lower doses for the treatment of several non-malignant disorders, notably psoriasis and rheumatoid arthritis (Horton 1990). A meta-analysis of studies investigating efficacy among DMARDs in the treatment of RA has suggested that methotrexate is equivalent in efficacy to sodium aurothiomalate, sulphasalazine, and penicillamine, while hydroxychloroquine and auranofin are less effective (Felson, et al., 1990). Low-dose methotrexate has been shown to be clinically effective in RA patients (Nordstrom, et al., 1987). Summary of the Invention This invention provides a method of treating rheumatoid arthritis which comprises administering to a patient in need of such treatment an effective amount of tenidap and methotrexate. In another aspect, this invention provides a pharmaceutical composition comprising tenidap and methotrexate. In another aspect, this invention provides a dose pack or treatment pack containing daily dosages of tenidap and methotrexate or tenidap combined with methotrexate to be administered over a defined time period. Detailed Description of the Invention The use of methotrexate in RA has increased significantly over the last few years due to its efficacy, speed of action and safety profile (Emery, 1989). However, there have been reports of methotrexate toxicity when co-administered with NSAIDs, including indomethacin (Maiche, 1986; Gabrielli, et al., 1987), naproxen (Singh, et al., 1986) and diclofenac (Gabrielli, et al., 1987). The mechanism of interaction is unclear, but may result from the displacement of methotrexate from protein-binding sites, or a direct renal effect leading to a reduction in methotrexate clearance. There is evidence that methotrexate may interact with various antibacterial agents; for example, penicillin markedly reduces the clearance of methotrexate (Bloom, et al., 1986). Methotrexate may also result in increased bone-marrow toxicity and immunosuppression when co-administered with antineoplastic or immunosuppressant medication, such as cyclosporin (Powles, et al., 1986). It would seem evident, therefore, that methotrexate has the potential for drug interaction, and consequently its drug-specific interactions must be assessed. Tenidap has very few clinical interactions with other drugs (Pullar, 1995). Nevertheless, as one action of tenidap is the inhibition of cyclooxygenase, and cyclooxygenase inhibitors have been linked with methotrexate toxicity when co-administered, it was necessary to investigate whether tenidap alters methotrexate metabolism. This is particularly important, as new treatment strategies for RA favor the use of polypharmacy (reviewed by Wilke & Clough, 1991), and consequently patients may be treated with a combination of both drugs as part of an integrated program of treatment. When tenidap was coadministered with methotrexate, no dosage adjustment of methotrexate was required. Tenidap has been found to lower levels of stromelysin (STR) mRNA in synovial tissue from rheumatoid arthritis patients. These changes in stromelysin mRNA are significantly correlated with ESR and CRP changes showing that treatment with tenidap will likely result in reduced joint damage. The effects of tenidap on collagenase (COL) are not as profound as its effects on STR. Methotrexate treatment has been shown to reduce COL mRNA expression in synovial tissue and to have no effect on STR mRNA. (Firestein, et al.) The combination of tenidap and methotrexate reduces both COL mRNA and STR mRNA in rheumatoid arthritis patients providing an advantageous treatment resulting in greatly reduced joint damage. Tenidap and methotrexate may be administered separately, for example in individual tablets or in a physical mixture of the two ingredients in a pharmaceutical composition. When used in a human subject, the daily dosage will normally be determined by the prescribing physician but will usually be equal to or less than the doses of tenidap and methotrexate which would be prescribed if used alone. Preferred rates are tenidap: 80 to 120 mg/day and methotrexate: 2.5 to 25 mg/once per week. Tenidap is acidic and forms base salts. All such base salts are within the scope of this invention and they can be prepared by conventional methods. For example, they can be prepared simply by combining the acidic and basic entities, usually in a stoichiometric ratio, in either an aqueous, non-aqueous or partially aqueous medium, as appropriate, or by interconverting one salt with another salt. The salts are recovered either by filtration, by precipitation with another solvent followed by filtration, by evaporation of the solvent, as appropriate, or, in the case of aqueous solutions, by lyophilization. The sodium salt is preferred. As used herein "tenidap" refers to both the free acid and base salts. The combination of tenidap and methotrexate is useful for chronic administration to mammals for the alleviation of the symptoms of rheumatoid arthritis, and the pain therewith. When the combination of tenidap and methotrexate is to be used as either an anti-inflammatory agent or a disease modifying agent, it can be administered to a human subject either alone, or, preferably, in combination with pharmaceutically- acceptable carriers or diluents in a pharmaceutical composition, according to standard pharmaceutical practice. The compound can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical administration. In a pharmaceutical composition comprising tenidap and methotrexate, the weight ratio of carrier to active ingredient will normally be in the range from 1 :4 to 4:1 , and preferably 1 :2 to 2:1. However, in any given case, the ratio chosen will depend on such factors as the solubility of the active component, the dosage contemplated and the precise route of administration. For oral use of tenidap and methotrexate, the combination can be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added. For oral administration in capsule form, useful diluents are lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. When the combination of tenidap and methotrexate is used in a human subject, the daily dosage will normally be determined by the prescribing physician. Moreover, the dosage will vary according to the age, weight and response of the individual patient, as well as the severity of the patient's symptoms and the potency of the particular formulation being administered. On the other hand, it may be necessary to use dosages outside these limits in some cases. For the treatment of rheumatoid arthritis tenidap is usually administered once daily at the rate of 80 to 120 mg/day. Methotrexate is usually administered once or twice a week at the rate of 2.5 to 25 mg/week. The attending physician may, of course, find it desirable to use dosages outside these limits in certain cases. Tenidap and methotrexate may be administered simultaneously in a single dosage or in separate dosage forms. This invention includes dose packs and treatment packs designed to provide dosages of tenidap and methotrexate in amounts required for a discrete period of time, e.g., one week or month. A weekly dose package may contain, for example, 7 doses of tenidap and one or two doses of methotrexate or alternatively, 5 or 6 doses of tenidap and 1 or two doses of tenidap and methotrexate in combination. Tablets and capsules are preferred dosage forms. EXAMPLE 1 METHODS Patients. Male and female RA outpatients older than 21 years were eligible for entry into the study. Patients were included in the study if they met the American Rheumatism Association 1987 Revised Criteria for RA (Arnett, et al., 1988), had active RA ( ≥ 3 months' duration) and had been taking fixed single doses of oral methotrexate of between 7.5 and 15 mg/week for a minimum of three months prior to study entry. If female, they had to be practicing safe contraception for at least three months prior to the beginning of the study. Prior to the study, patients underwent a full medical examination and 12-lead ECG. A full medical history was taken, and a blood sample collected for clinical chemistry and hematological assessments. Patients were excluded if they had any known hematological, renal, hepatic, endocrine, pulmonary, neurologic, psychiatric or cardiovascular disease, or if they had any clinical intolerance to aspirin or other NSAIDs. Patients were also excluded if they had any of the following laboratory test abnormalities at screening: SGOT, SGPT or alkaline phosphatase concentrations greater than 2 x ULN, total bilirubin concentration greater than 10% above ULN, total white blood cell count <3500 cells/mm 3 , poiymorphonuclear cell count <1500cells/mm 3 , platelet count < 100,000 cells/mm 3 , hemoglobin <9.5 g/dl for women or <10.5 g/dl for men, creatinine clearance ≤ 60 ml/min (calculated as described by Cockcroft & Gault, 1976), or BUN more than 10% above ULN. Patients were screened before entry into the study for hepatitis B surface antigen, the presence of drugs in the urine and pregnancy in females of childbearing age. They were also required to provide a negative ethanol breath test at the start of the study, and before each hospital admission. Patients were not to have taken any investigational drug (except methotrexate) for at least four weeks prior to entry to the study, and were to provide written informed consent. Protocol. This was an open-label, fixed dose study conducted over a period of 21 days. Patients discontinued any conventional NSAIDs with both short and long half-lives at least 48 hours or one month prior to study entry, respectively. DMARDs (except methotrexate) were discontinued at least two months prior to the start of the study. Each patient continued to take their usual, fixed dose of oral methotrexate, which did not change throughout the study, but also received 120 mg oral tenidap sodium daily. All drugs were administered orally after an overnight fast. Blood samples and 24-hour urine samples were obtained from each patient on days 0 and 21. These were used to evaluate the pharmacokinetics of methotrexate, and the plasma concentrations of tenidap. Patients were confined to the clinical research unit for 24 hours after methotrexate administration on days 0 and 21. Methotrexate pharmacokinetics assessments: Serum. Blood sufficient to provide 4 ml serum for the determination of methotrexate concentrations was collected immediately prior to and 0.5, 1 , 2, 3, 4, 6, 8, 12, 16, 20, 24, 28 and 32 hours after administration of methotrexate on days 0 and 21. The serum was stored at -20° C until analysis of methotrexate concentrations was undertaken by ion-exchange chromatography (Lankelma & Poppe, 1978). The dynamic range of the assay was 2.5-1000 ng/ml. Pooled serum samples taken between I and 4 hours post-dose were used to determine the unbound fraction (fu) of methotrexate. The maximal concentration (C^) in the serum and the time at which C mβx is achieved (t max ) of methotrexate were estimated directly from the experimental data. The terminal phase rate constant (K βl ) was estimated using least squares regression analysis of the concentration-time curve during the terminal log-linear phase. Half-life was calculated as 0.693/K,,. The area under the concentration time-curve from time 0 to time T (the last sampling time with quantifiable methotrexate present)(AUC 0 . τ ) was estimated using linear trapezoidal approximation. The AUC from time T to infinity (AUC-r..,) was estimated as C βst /K βl , where C βst is the estimated concentration at time T. The total area under the curve (AUCo..) was estimated as the sum of AUCo. τ + AUC T ... Methotrexate pharmacokinetics assessments: Urine. Urine samples were collected up to 24 hours after methotrexate administration on days 0 and 21. Urine was collected over three time periods (0-4 hours, 4-8 hours, and 8-24 hours). From each collection, one 50 ml aliquot was withdrawn for the determination of creatinine, uric acid, total protein and albumin concentrations, and another 50 ml aliquot was withdrawn and frozen at -20° C, before analysis for methotrexate concentration by ion-exchange chromatography (Lankelma & Poppe, 1978). Renal clearance (CL,,) was estimated as the ratio of the amount of methotrexate excreted in the urine (XUo. 24 ) to the AUC, j . 24 for the first 24 hours after methotrexate administration. Tenidap plasma concentrations. Blood samples for the determination of plasma tenidap concentrations were collected into heparinized tubes immediately prior to drug administration on days 1 , 2, and 21 , and 24 hours after drug administration on day 21. Plasma (2 ml) was frozen at -20° C until tenidap concentrations were analyzed by HPLC with UV detection (Gardner, et al., 1995). The dynamic range of the assay was 0.1-40 μg/ml. Protein binding. To determine methotrexate protein binding, a 5 ml aliquot of serum, obtained by pooling samples from the 1 , 2, 3, and 4 hour time points taken for serum methotrexate concentration determination, was supplemented with 0.1 μC\ of tritiated methotrexate (specific activity 32.1 mCi/mg; Amersham, USA). These samples were placed in an ultrafiltration device containing a YMT membrane (cut-off size 20,000 kDa) (Amicon) and centrifuged at 2000 x g for 20 minutes at 37° C. Aliquots of the serum (100 μ\) and the ultrafiltrate (100 μ\) were counted in 5 ml of scintillation fluid (Ultima Gold, Packard, USA). The amount of radioactivity was determined in each pooled sample of serum (in triplicate) and in each ultrafiltrate (in duplicate) in a liquid scintillation counter (Packard 2200, Packard, USA). The unbound fraction (fu) of methotrexate was estimated as the ratio of the amount of radioactivity in the ultrafiltrate to the amount of radioactivity in an equal volume of serum. Statistical methodology and evaluation. The values of AUCo.„ and C max were normalized to a 12.5 mg dose of methotrexate. These values were then natural log-transformed and the ratio between means on day 21 and day 0 was analyzed with 90% confidence intervals. Differences between untransformed mean values on day 21 compared with day 0 for t max , K,,., and CL,, were analyzed with 90% confidence intervals. Safety. The following laboratory tests were performed at screening, and prior to tenidap administration on days 1 , 7, 14, and 21 , and 24 hours after dosing on day 21 : CBC with differential and platelet count, urinaiysis with microscopic examination, and clinical chemistries. A 12-lead ECG was obtained on all subjects at screening and prior to drug administration on days 1 and 21. Patients who had abnormal ECGs were not allowed to enter or continue in the study. Patients were monitored for side effects throughout the trial. RESULTS Patients. Of the ten patients (three male and seven female) that entered the study, eight patients completed it. The baseline demographic characteristics of these patients are shown in Table I. All patients had RA at stage I or II according to ARA classification. Two patients discontinued from the study, one after 9 days of tenidap treatment because of treatment-related side effects (abdominal pain, nausea and vomiting), and the other on day 20 of the study as an intravenous line could not be established. Pharmacokinetics parameters. The mean values for methotrexate pharmacokinetics parameters are shown in Table II. The mean C max , normalized to a 12.5 mg dose, was 364.3 ng/ml on day 0 compared with 305.6 ng/ml on day 21 , representing a decrease of approximately 16%. Mean t max was 1.3 hours on day 0 and 1.1 hours on day 21. The mean K„. of methotrexate was 0.2169/hr on day 0, compared with 0.1475/hr on day 21. This decrease corresponded with an increase in half-life from 3.2 hours on day 0 to 4.7 hours on day 21. The mean AUC^ ^ , normalized to a 12.5 mg dose of methotrexate, increased by approximately 6% between days 0 and 21. There was a decrease of approximately 24% in the mean CL,, of methotrexate, from 115.3 ml/min on day 0 to 88.0 ml/min on day 21. There was also an increase of approximately 26% in the mean percentage of unbound methotrexate in serum from 33% on day 0 to 42% on day 21. Plasma tenidap concessions. The mean predose concentrations of tenidap in plasma increased from 6.0 μg/ml on day 2 to 9.0 μg/ml on day 21. Visual inspection of the predose plasma concentrations indicated that steady-state had been achieved by day 21. Safety. Three patients had treatment-related side effects (see Table III). One of these patients experienced severe nausea, with abdominal pain and intermittent vomiting, and discontinued study treatment. All other side effects were mild or moderate in severity. Of the other two patients affected, one subject had mild nausea and moderate dyspepsia, and the other subject had mild dyspepsia. All reported side effects were considered to be treatment-related. There were no laboratory test abnormalities which were considered to be related to treatment. In addition, no apparent trends or consistent changes in ECGs were observed during the study. EXAMPLE 2 Clinical Study Design: A double-blind, multicenter, twelve week, crossover study was conducted comparing the within patient biochemical and clinical effects of six weeks of piroxicam (20 mg/day) with six weeks of tenidap (120 mg/day) treatment. Details of protocol design and the results of this study, based on 49 completed patients, have been published elsewhere (Liftman, 1995). Synovial biopsies were performed in a subset of ten of these 49 completed patients. As in the overall study, these biopsied patients had active disease and CRP >.1.5 mg/dl. In addition they had active knee synovitis and agreed to undergo three percutaneous needle synovial biopsies over the 12 weeks of the study. The baseline demographic data on these 10 patients are listed in Table IV. Patients were instructed to discontinue their current NSAID therapy after the last dose on the day prior to the baseline visit. At baseline patients were randomly assigned to double-blind therapy with either tenidap or piroxicam. After treatment in the first 6 week period (weeks 1-6), patients were switched, without a washout, to treatment in the second 6 week period (weeks 7-12) with the other agent. Patients were evaluated at baseline and at weeks 1 , 3, 6, 7, 9 and 12. Laboratory safety studies, standard clinical measurements, ESR, CRP and SAA were determined at each visit and plasma for cytokine determinations was obtained at all but the week 1 and 7 visits. Synovial biopsies were performed at baseline, at the end of the first treatment (six weeks) and at the end of the second treatment (12 weeks). Each biopsy was obtained from the same knee for individual patients. Synovial biopsy results and their relationship to systemic markers of disease activity are discussed below. Statistical Methods: The changes in mRNA expression during tenidap and during piroxicam were calculated and the percent change from pretreatment levels was determined for each patient. However, because this was a crossover study with no washout periods, the change in mRNA expression during each treatment is presented without any additional analysis and the primary statistical analysis was based on a within patient comparison of the final value on tenidap compared to the final value on piroxicam (2-tailed paired T-test). There were no significant period effects on mRNA expression. Using the within patient treatment difference data, the relationships between synovial tissue mRNA and the ESR, CRP and plasma cytokine levels were evaluated by a least squares regression analysis. Lastly, using data from 10 patients over the first six weeks of treatment (to avoid using data from each patient more than once), and a least squares regression analysis, we also explored correlations of changes in metalloproteinase and TMP mRNA expression with changes in cytokines, CRP and the ESR. For all regression analyses, the significance of the slope of the regression line (different from zero) was determined. C-reactive protein (CRP) and Serum Amyloid A (SAA): CRP was determined on serum samples by a central laboratory (SmithKline Bio-Science Laboratories) using a rate nephelometry assay. The lower limit of the assay was 0.1 mg/dl and values less than this were considered to be 0.09 mg/dl. Westergren Erythrocyte Sedimentation Rate (ESR): The ESR was determined locally at the time of the patient's visit. Cytokine Assays: ELISA assays for IL-6, IL1 ? and TNFσ were performed as previously described (Liftman, 1995). All cytokine assay methods were validated first by confirming that cytokine readings were fully neutralized using specific antibody, that the assay could detect 100% of cytokine spiked into RA plasma and that all readings were in the linear portion of the standard curve. All plasma samples were freshly obtained by separation in a refrigerated centrifuge (4°C) and stored frozen at -70 °C until tested. All samples from a given patient were assayed together. The range of detection for IL-6 and IL-1 7 was 1 to 190 pg/ml. Values below 1 pg/ml were treated as if they were 0.9 pg/ml. The range for the TNFσ assay was 4 to 150 pg/ml and values below 4 pg/ml were treated as if they were 3.9 pg/ml. Synovial Tissue: Percutaneous synovial biopsies were performed on the same knee in each patient using a 14-gauge Parker-Pearson needle (5-10 pieces per procedure). Tissues were snap frozen in 2-methylbutane and liquid nitrogen and stored at -70° C until used. Frozen sections (4μm) were fixed in 4% paraformaldehyde and duplicate or triplicate sections and sections from adjacent areas of the joint (other pieces obtained at the same procedure) were processed together. In addition, biopsies taken at different times from the same patient were also processed together (7). in situ hybridization: Biopsy slides from each patient were blinded as to sequence and processed together. In situ hybridization was performed as previously described (Liftman, 1995) using labeled RNA antisense and sense probes for collagenase (COL), stromelysin (STR) and the tissue inhibitor of metalloproteinase-1 (TMP). Similarly, as a control, hybridization with a riboprobe specific for actin (ACT) mRNA was performed. Photographic emulsion was applied and slides were developed. After autoradiography was completed, slides were counterstained with hematoxylin. Computer-assisted image analysis was used to quantify the area of synovial lining covered by grains as previously described (Liftman, 1995). For each biopsy a total of five representative regions of the synovial intimal lining from 3-4 individual tissue samples were analyzed and the mean was calculated. Results were normalized by expressing them as the ratio of mean COL, STR or TMP grain area divided by the mean ACT grain area on adjacent sections. Inflammation Scores: Synovial inflammation was analyzed as previously described (Liftman, 1995). Briefly, the inflammation score is the sum of three components: synovial lining thickness (1-2 cells = 0, 2 -4 cells = 1 +, 5 - 8 cells •= 2+ and >8 cells = 3+), subintimal mononuclear cell infiltration (<5% of the field = 0, 5- 33%=1 +, 33-67%=2+ and >67%=3+) and lymphoid aggregates (absent = 0 and present = 1 +). Each tissue was read twice and the mean of the 2 scores was used as the final inflammation score. RESULTS Changes in COL, STR and TMP mRNA Expression: Adequate biopsies before and after 6 weeks of treatment were available from 10 patients during tenidap and from 7 patients during piroxicam. The changes in the actin-normalized values for STR, COL and TMP mRNA on each treatment for each subject were expressed as a percentage of the pretreatment value. STR mRNA decreased in 7 of 10 patients during tenidap treatment and in 1 of 7 patients during piroxicam treatment. Decreases on tenidap were in the range of 23% to 96% although three patients had large increases. During piroxicam all but one patient had increased levels of STR mRNA. Changes in COL and TMP mRNA were more variable between patients with no consistent treatment pattern. mRNA Expression: Within Patient Comparison: Treatment differences between the effects of tenidap and piroxicam on mRNA expression could be evaluated in the 7 patients who had adequate biopsies after both treatments. A negative value indicates mRNA expression was lower after tenidap compared to after piroxicam in that patient. The ACT-normalized results for STR, COL and TMP are given in Table V. Note that both the mean and median treatment difference for STR mRNA are about -2.9 while the treatment differences for COL and TMP are close to zero. The values after tenidap were also significantly lower (p=0.037, 2-tailed paired t-test) compared to the values after piroxicam. To be sure that this finding was not due to systematic changes in ACT mRNA levels, the STR mRNA was also normalized by TMP mRNA. Again treatment differences were negative for 6 of the 7 patients as only one patient had lower STR/TMP after piroxicam. Thus the major difference between the effects of tenidap and piroxicam was on the level of STR mRNA. Correlation of Percent Changes From Baseline: Synovial mRNA changes from baseline to 6 weeks could be evaluated (ignoring treatment) in the 10 subjects who had at least the first two biopsies. STR mRNA changes were significantly correlated with the change in ESR (R=0.853, p=0.0035) and the change in CRP (R=0.734, p=0.016). No significant associations of changes in synovial STR mRNA with cytokine levels or of COL or TMP mRNA expression with acute phase and cytokine levels were found. Correlation of Treatment Differences: The treatment differences for COL mRNA and plasma TNFσ were significantly correlated (R=0.877, p=0.022). There were no significant correlations of treatment differences for COL, STR or TMP with treatment differences for other cytokines, the ESR or CRP. 10 ι 15 10 15 20 EXAMPLE 3 Oral Capsule Dosage Form Containing the sodium of tenidap and methotrexate. The following ingredients are blended, wet granulated with 875 mL of water and finally dried to 5% water by Karl Fischer: (550 g tenidap free acid) The dried, wet granulated powder was then further blended with: Sodium starch glycolate 210.00 g (Explotab) Magnesium stearate 42.00 g Sodium lauryl sulfate 21.00 g Soft gelatin capsules containing 100 mg tenidap and 10 mg methotrexate are prepared on a conventional capsule filling machine, using a fill weight of 385 mg of the finished blend.

Claims: CLAIMS 1. A method of treating rheumatoid arthritis comprising administering to a patient in need of such treatment an effective amount of tenidap and methotrexate. 2. A pharmaceutical composition comprising tenidap and methotrexate. 3. A dose pack or treatment pack comprising dosages of tenidap and methotrexate to be administered over a defined time period. 4. A dose pack or treatment pack of claim 3 wherein said defined time period is one week. 5. A dose pack or treatment pack of claim 4 comprising seven doses of tenidap and one or two doses of methotrexate. 6. A dose pack or treatment pack comprising dosages of tenidap and dosages of a pharmaceutical composition of claim 2. 7. A dose pack or treatment pack of claim 6 comprising five or six doses of tenidap and one or two doses comprising tenidap and methotrexate.

Inventors: LITTMAN BRUCE H, LITTMAN, BRUCE, H.
Applicants: LITTMAN BRUCE H, PFIZER, LITTMAN, BRUCE, H., PFIZER INC.
Application year: 1995
Main classification: A61K31
IPC codes: A61K31, A61P29, A61P43