Methods for treating allergic asthma using descarboethoxyloratadine

Methods utilizing descarboethoxyloratadine (“DCL”), for the treatment of allergic disorders, while avoiding the concomitant liability of adverse side-effects associated with other non-sedating antihistamines. Also included are methods for the treatment of allergic asthma using DCL and either a decongestant or a leukotriene inhibitor, while avoiding the concomitant liability of adverse side-effects associated with other non-sedating antihistamines. The invention also encompasses the administration of DCL in a nasal or oral spray.

5. EXAMPLES 
 5.1 Example 1 
 Preparation of Loratadine and its Metabolites Loratadine can be synthesized by methods disclosed in U.S. Pat. No. 4,282,233. The metabolites are prepared similarly, by reaction steps conventional in the art, as described in U.S. Pat. No. 4,659,716 which is incorporated here by reference in its entirety. One common method of preparing DCL is to reflux loratadine in the presence of sodium hydroxide and ethanol as depicted below. 1 Extraction of Commercially Available Claritin Tablets (600×10 mg): Tablets of loratadine, were diluted with water and chloroform. The mixture was stirred, then filtered through celite, rinsed with chloroform until the filtrate contained no loratadine. The separated aqueous layer was extracted with chloroform twice. The combined organic layer was washed with water, brine and dried over sodium sulfate. The solvent was evaporated to give pure loratadine as a white solid. Saponification of Loratadine: Loratadine (4.0 g) was added to a solution of sodium hydroxide (5.9 g) in 280 mL of absolute ethanol and the mixture was stirred at reflux for four days. The mixture was cooled and concentrated to remove ethanol. The residue was diluted with water and aqueous layer was extracted with methylene chloride five times. The combined organic layer was washed with water, brine and dried over sodium sulfate. The solvent was evaporated to give 2.82 g (87%) of pure loratadine derivative (or metabolite) as a pale-tan solid. 
 5.2 Example 2 Antihistaminic Activity The antihistaminic activity of loratadine and DCL were compared in isolated strips of guinea pig ileum contracted with histamine. This preparation is generally accepted by those skilled in the art as predicative of its efficacy as a peripheral histamine H-1 receptor. Methods: Experiments were performed on pieces of ileum taken from male guinea pigs (Hartley strain, 419-560 grams; Elm Hill Breeding Laboratories, Chelmsford, Mass.). The tissues were suspended in tissue chambers containing 40 ml of Tyrode's solution aerated with 95% oxygen and 5% carbon dioxide at 35° C. The Tyrode's solution contained (in mM) 137 NaCl, 2.7 KCl, 2.2 CaCl 2 , 0.025 MgCl 2 , 0.4 NaHPO 4 , 11.9 NaHCO 3 and 5.5 glucose. Contractions in response to histamine were recorded with isotonic transducers (Model 357, Harvard Apparatus Company, South Natick, Mass.) using an ink-writing polygraph (Model 7, Grass Instrument Company, Quincy, Mass.). A tension of one gram was maintained on all tissues at all times. In each experiment three or four pieces of ileum were removed from a single animal, suspended in individual tissue chambers and allowed to equilibrate with the bathing solution for one hour before the administration of any drugs. In four initial experiments in which tissues were exposed to histamine at concentrations of 1×10 −7 , 1×10 −6 and 1×10 −5 mol/l, histamine at 1×10 −6 mol/l produced strong contractions on the linear portion of the log-concentration-effect curve and this concentration of histamine was chosen for use in all further experiments. For determining the antihistaminic effects of loratadine and DCL, tissues were exposed briefly (about 15 seconds) to 1×10 −6 mol/l of histamine at intervals of 15 minutes. After two successive exposures to histamine produced contractions of approximately the same magnitude, loratadine or DCL, at final concentrations that varied three- or ten-fold, was added to all but one of the tissue chambers, the untreated tissue serving as a control for the treated tissues. After each exposure of drug-treated tissues to histamine, the fluid in the tissue chamber was replaced with fluid free of histamine but containing the same drug at the same concentration. The histamine challenges were made at 5, 20, 35, 50, 65, 80, 95, 110 and 125 minutes of exposure to the drug or at comparable times in the control tissues. Subsequent analyses of the results from each experiment involved (i) normalization of the data from each tissue for differences in inherent contractility by expressing all contractions as a percent of the last predug contraction, (ii) normalization of the data for possible time-related changes in contractility by expressing the contractions recorded during drug-exposure as a percent of the corresponding value for the untreated tissue, and finally (iii) calculation of the drug-related percent reduction of each contraction. The resultant sets of data for drug concentration and corresponding percent reduction in histamine-response were then used to estimate for each experiment the concentration of drug that would have produced a 50 percent reduction in the histamine response, the IC 50 . This was done by fitting straight lines to the data using the method of least squares and calculating the ICs from the equation of the line. The mean &plus;/− standard error of the values for the experiments on each drug were calculated, and differences between the drugs was examined using the Kruskal Wallis 1-way analysis of variance by ranks. A summary of the results are shown in the following two tables. The percentages of reduction of histamine-induced contractions of the isolated guinea pig ileum produced by exposure for 125 minutes to various concentrations of each drug are set forth below: 1 TABLE 1 Reduction of Histamine-induced Guinea Pig Ileum Contractions (Percent) Expt Concentration of drug (mol/l) Drug No. 3 × 10 -10 1 × 10 -9 3 × 10 -9 1 × 10 -8 3 × 10 -8 1 × 10 -7 Loratadine 1 — 19.05 — 13.33 — 88.57 2 — — — 28.32 54.42 98.66 3 — — — 39.64 44.68 93.38 4 — — — 55.86 45.83 86.46 DCL 1 11.93 73.12 2 38.91 38.81 56.71 3 40.00 62.69 76.21 4 35.43 44.13 76.43 2 TABLE 2 Reduction of Histamine-induced Guinea pig Icum Contractions (IC 50 ) Drug Expt IC 50 (M) Loratadine 1 1.90 × 10 −8 2 2.21 × 10 −8 3 2.10 × 10 −8 4 1.22 × 10 −8 Mean 1.86 × 10 −8 S.E. 0.22 DCL 1 6.36 × 10 −10 2 19.2 × 10 −10 3 5.26 × 10 −10 4 8.66 × 10 −10 Mean 9.75 × 10 −10 S.E. 3.20 Note: There is a statistically significant drug-related difference in IC 50 values (P &equals; 0.0209). These results indicate that DCL is approximately 20 fold more potent at the histamine receptor than loratadine. 
 5.3 Example 3 Receptor Binding Studies Receptor binding studies on the binding affinities of loratadine and DCL at histamine H-1 receptors were performed. The methods described by Dini et al., which is hereby incorporated by reference herein (Agents and Actions, 33: 181-184, 1991), were used for these binding studies. Guinea pig cerebella membranes were incubated with 0.5 nM 3H-pyrilamine for 10 min at 25° C. Following incubation, the assays were rapidly filtered under vacuum through GF/B glass fiber filters (Whatman) and washed several times with ice-cold buffer using a Brandel Cell Harvester. Bound radioactivity was determined with a liquid scintillation counter (LS 6000, Beckman) using a liquid scintillation cocktail (Formula 989, DuPont NEN). IC 50 values were determined for compounds tested and pyrilamine at the H-1 histamine receptor: 3 TABLE 3 Inhibition of Pyrilamine Binding at H-1 Receptor H-1 receptor Compound IC 50 (nM) (nH) Loratadine 721 (1.55) DCL 51.1 (1.12) Pyrilamine 1.4 (0.98) As shown above, DCL was found to have a 14 fold greater affinity than loratadine for histamine H-1 receptors. These results are consistent with the findings demonstrating a higher potency of DCL over loratadine for inhibition of histamine-induced contractions of guinea pig ileum. These studies confirm that-DCL has a higher potency for histamine receptors than loratadine. 
 5.4 Example 4 Tumor Promoting Activity Inhibition of lymphocyte mitogenesis was used to screen the potencies of loratadine and DCL as tumor promoting agents. Mitogenesis Studies: Fresh spleen cells (5×10 5 ) obtained from 5-week old BALB/c mice (Charles River, ST. Constant, PQ) were suspended in RPMI 1640 medium containing 2% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) seeded into replicate microwell plates (Nunc) to which concanavalin (Con) A (2 &mgr;g/ml; Sigma Chemical Co., St. Louis, Mo.) was added and incubated (37° C., 95% air, 5% CO 2 ) in the absence or presence of increasing concentrations of the test agents dissolved in saline or other vehicles. Forty-three hours after the addition of Con A, 0.25 nmol 3 H-thymidine (6.7 Ci/nmol; ICN Radiopharmaceuticals, Montreal, PQ) was added to each well. After an additional 5-hour incubation, the cells were washed from the wells onto filter papers employing an automated cell sorter. The filters were placed into vials containing 5 ml scintillation fluid (Readysafe; Beckman), and radioactivity incorporated into DNA at 48 hours was determined (n&equals;3). IC 50 values for inhibition of mitogenesis were determined over wide range of concentrations (0.1 to 10 &mgr;M). 4 TABLE 4 Inhibition of Concanavalin A Induced Stimulation of Lymphocytes (IC 50 ) Loratadine 1.0 &mgr;M DCL 5.6 &mgr;M These results indicate that DCL is 5-7 fold less active than loratadine at promoting tumor growth. 
 5.5 Example 5 Cardiovascular Effects The effects of DCL on cardiac potassium currents were studied. Methods: Single ventricular myocytes of the guinea-pig and the rabbit were dissociated by enzymatic dispersion (see Carmeliet, J. PharMacol. Exper. Ther., 1992, 262, 809-817 which is incorporated herein by reference in its entirety). The single suction patch electrode, with a resistance of 2 to 5 M&OHgr; was used for voltage clamp (Axoclamp 200A). P-clamp software (Axon Instruments) was used to generate voltage-clamp protocols and to record and analyze data. The standard solution contained in mM: NaCl 137.6, KCl 5.4, CaCl 2 1.8, MgCl 2 0.5, HEPES 11.6 and glucose 5, and NaOH was added to pH 7.4. The intracellular solution contained KCl120, MgCl 2 6, CaCl 2 0.154, Na 2 ATP 5, EGTA 5, and HEPES 10, with KOH added until pH 7.2. Effect on the Delayed Rectifying K &plus; Current, (I kr ) in Rabbit Ventricular Myocytes: The voltage clamp protocol consisted of clamps from a holding potential of −50 mV to &plus;10 mV for a duration of 4 sec. The change in tail current was measured as a function of the drug concentration. This concentration was changed between 10 −7 and 10 −5 M in five steps. Exposure to each concentration lasted 15 min. At the end, washout was attempted during 30 min. Effect on the Inward Rectifier Current in Guinea-pig Myocytes: The inward rectifier was measured by applying ramp voltage clamps starting from −50 mV and hyperpolarizing the membrane to −120 mV at a speed of 10 mV/sec. The starting concentration was the 50% efficiency concentration, determined in the preceding experiments. Higher concentrations were applied if this initial concentration was without effect. Effect on IK 5 in Guinea-pig Ventricular Myocytes: Tail currents were measured following depolarizing clamps of 2-sec duration to potentials between −30 mV and &plus;60 mV; holding potential −50 mV. The results from these studies indicate that DCL is less active than terfenadine in inhibiting the cardiac delayed rectifier and thus has no potential for cardiac side-effects. Thus, the methods of the present invention are less toxic than methods which use other non-sedating antihistamines. 
 5.6 Example 6 Inhibition of Cytochrome P450 This study is conducted to determine the extent that loratadine and DCL inhibit human cytochrome P4503A4 (CYP3A4). CYP3A4 is involved in many drug-drug interactions and quantitation of inhibition of CYP3A4 by loratadine or DCL indicates the potential of such drug-drug interactions. Inhibition is measured using the model substrate testosterone and cDNA-derived CYP3A4 in microsomes prepared from a human lymphoblastoid cell line designated h3A4v3. Study Design: The inhibition study consists of the determination of the 50% inhibitory concentration (IC 50 ) for the test substance. A single testosterone concentration (120 &mgr;M, approximately twice the apparent Km) and ten test substance concentrations, separated by approximately ½ log, are tested in duplicate. Testosterone metabolism is assayed by the production of the 6(&bgr;)-hydroxytestosterone metabolite. This metabolite is readily quantitated via HPLC separation with absorbance detection. Storage/Preparation of the Test Substances and Addition to the Incubations: The test substances will be stored at room temperature. The test substances will be dissolved in ethanol for addition to the incubations. The solvent concentration will be constant for all concentrations of the test substance. IC 50 Determination: Final test substance concentrations will be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0 &mgr;M. Each test concentration will be tested in duplicate incubations in accordance with the method below: Method: A 0.5 ml reaction mixture containing 0.7 mg/ml protein, 1.3 mM NADP&plus;, 3.3 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride and 120 &mgr;M testosterone in 100 mM potassium phosphate (pH 7.4) will be incubated at 37° C. for 30 min. A known quantity of 11(&bgr;)-hydroxytestosterone will be added as an internal standard to correct for recovery during extraction. The reaction mixture will be extracted with 1 ml methylene chloride. The extract will be dried over anhydrous magnesium sulfate and evaporated under vacuum. The sample will be dissolved in methanol and injected into a 4.6×250 mm 5u C18 HPLC column and separated at 50° C. with a mobile phase methanol/water at a flow rate of 1 ml per min. The retention times are approximately 6 min for the 6(&bgr;)-hydroxy, 8 min for 11(˜)-hydroxy and 12 min for testosterone. The product and internal standard are detected by their absorbance at 254 nm and quantitated by correcting for the extraction efficiency using the absorbance of the 11(&bgr;)-hydroxy peak and comparing to the absorbance of a standard curve for 6(&bgr;)-hydroxytestosterone. Data Reporting: For each test substance, the concentration of 6(&bgr;)-hydroxytestosterone metabolite in each replicate incubation is determined and the percentage inhibition relative to solvent control is calculated. The IC 50 is calculated by linear interpolation. Useful pharmaceutical dosage forms for administration of the compounds used in the methods of the present invention can be illustrated as follows: 
 5.7. Example 7 Capsules A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 0.1 to 10 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate. 
 5.8. Example 8 Soft Gelatin Capsules A mixture of active ingredient in a digestible oil such as soybean oil, lecithin, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 0.1 to 10 milligrams of the active ingredient. The capsules are washed and dried. 
 5.9 Example 9 Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit was 0.1 to 10 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The foregoing disclosure includes all the information deemed essential to enable those skilled in the art to practice the claimed invention. Because the cited patents or publications may provide further useful information these cited materials are hereby incorporated by reference in their entireties.