ATROPINE SULFATE RAPIDLY-DISINTEGRATING SUBLINGUAL TABLETS, METHODS FOR MANUFACTURE THEREOF, AND METHODS FOR USE THEREOF FOR TREATMENT OF ACUTE ORGANOPHOSPHATE TOXICITY

Abstract

The invention provides atropine sulfate (AS) rapidly-disintegrating sublingual tablets (RDSTs) in a sublingual dosage form and methods for therapeutic use of the AS RDSTs for treatment of organophosphate (OP) exposure and acute toxicity. The AS RDSTs provide an alternative easy-to-use dosage form for the management of organophosphate toxicity. Additionally, the invention provides methods for formulation and quality evaluation of the atropine sulfate rapidly-disintegrating sublingual tablets.

Claims

1. A pharmaceutical composition comprising atropine sulfate and at least one pharmaceutically-acceptable excipient, the composition formulated for rapid disintegration in buccal or sublingual administration.

2. The pharmaceutical composition in accordance with claim 1, wherein the at least one pharmaceutically-acceptable excipient is selected from the group consisting of a filler, a superdisintegrant, and a lubricant.

3. The pharmaceutical composition in accordance with claim 2, comprising a filler, a superdisintegrant, and a lubricant, wherein the filler is microcrystalline cellulose, the superdisintegrant is hydroxypropyl cellulose, and the lubricant is magnesium stearate.

4. The pharmaceutical composition in accordance with claim 3, wherein the hydroxypropyl cellulose is low substituted.

5. The pharmaceutical composition in accordance with claim 3, wherein the composition comprises from about 2 to about 20% of atropine sulfate, from about 20 to about 95% of microcrystalline cellulose, from about 1 to about 15% of hydroxypropyl cellulose, and from about 0.5 to about 3% of magnesium stearate.

6. The pharmaceutical composition in accordance with claim 3, wherein the composition comprises from about 1 to about 20 wt % of atropine sulfate, from about 20 to about 95 wt % of microcrystalline cellulose, from about 1 to about 15 wt % hydroxypropyl cellulose, and from about 0.5 to about 3 wt % magnesium stearate.

7. A method for manufacturing atropine sulfate tablets formulated for rapid disintegration in buccal or sublingual administration, the method comprising: weighing atropine sulfate and pharmaceutically-acceptable excipients; sieving the atropine sulfate and pharmaceutically-acceptable excipients; combining and mixing the atropine sulfate and pharmaceutically-acceptable excipients to form a directly compressible formulation; and directly compressing the formulation to form the atropine sulfate tablets.

8. The method in accordance with claim 7, wherein the formulation is directly compressed using concave punches and dies.

9. The method in accordance with claim 7, wherein the pharmaceutically-acceptable excipients include a filler, a superdisintegrant, and a lubricant.

10. The method in accordance with claim 9, wherein the filler is microcrystalline cellulose, the superdisintegrant is hydroxypropyl cellulose, and the lubricant is magnesium stearate.

11. The method in accordance with claim 10, wherein the hydroxypropyl cellulose is low substituted.

12. The method in accordance with claim 10, wherein the combining and mixing comprises the steps of: a) combining the atropine sulfate with the microcrystalline cellulose and mixing to form a mixture; b) adding two thirds of the hydroxypropyl cellulose to the mixture formed in step a) and mixing for about four minutes to form a mixture; c) mixing the magnesium stearate and the remaining one third of the hydroxypropyl cellulose to form a second mixture; and d) adding the second mixture to the mixture formed in step b) and mixing for about thirty seconds.

13. The method in accordance with claim 12, wherein the formulation is directly compressed using concave punches and dies.

14. Atropine sulfate tablets formulated for rapid disintegration in buccal or sublingual administration produced in accordance with the method of claim 13.

15. A method for treating symptoms of exposure to organophosphates (OPs) in a subject in need thereof, comprising: providing a composition including atropine sulfate and at least one pharmaceutically-acceptable excipient, the composition formulated for rapid disintegration in buccal or sublingual administration; and administering the composition to the subject.

16. The method in accordance with claim 15, wherein a source of the exposure is pesticides or nerve agents.

17. The method in accordance with claim 15, wherein the at least one pharmaceutically-acceptable excipient is selected from the group consisting of a filler, a superdisintegrant, and a lubricant.

18. The method in accordance with claim 17, comprising a filler, a superdisintegrant, and a lubricant, wherein the filler is microcrystalline cellulose, the superdisintegrant is hydroxypropyl cellulose, and the lubricant is magnesium stearate.

19. The pharmaceutical composition in accordance with claim 18, wherein the hydroxypropyl cellulose is low substituted.

20. The method in accordance with claim 15, further comprising administering a cholinesterase re-activator concomitantly with the composition.

21. The method in accordance with claim 20, wherein the cholinesterase re-activator is pralidoxime chloride.

22. A composition comprising atropine sulfate (AS) and at least one pharmaceutically-acceptable excipient for use in the manufacture of a composition for treating symptoms of exposure to organophosphates (OPs).

23. Use in accordance with claim 22, wherein a source of the exposure is pesticides or nerve agents.

24. Use in accordance with claim 22, wherein the at least one pharmaceutically-acceptable excipient is selected from the group consisting of a filler, a superdisintegrant, and a lubricant.

25. A composition comprising atropine sulfate (AS), a filler, a superdisintegrant, and a lubricant for use in the manufacture of a composition for treating symptoms of exposure to organophosphates (OPs).

26. Use in accordance with claim 25, wherein the filler is microcrystalline cellulose, the superdisintegrant is hydroxypropyl cellulose, and the lubricant is magnesium stearate.

27. Use in accordance with claim 26, wherein the hydroxypropyl cellulose is low substituted.

28. A composition comprising atropine sulfate (AS), at least one pharmaceutically-acceptable excipient, and a cholinesterase re-activator for use in the manufacture of a composition for treating symptoms of exposure to organophosphates (OPs).

29. Use in accordance with claim 28, wherein the cholinesterase re-activator is pralidoxime chloride.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] A more complete understanding of the present invention may be obtained by references to the accompanying drawings when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

[0041] FIG. 1 is a schematic illustration of the disintegration apparatus used for carrying out the disintegration test on the rapidly-disintegrating sublingual tablets (RDSTs).

[0042] FIGS. 2A-C are images from the wetting test on the rapidly-disintegrating sublingual tablets: FIG. 2A is an image of a dry tablet prior to the start of the test; FIG. 2B is an image of the tablet placed on a wet paper towel during the test; and FIG. 2C is an image of the wet tablet after completion of the test.

[0043] FIGS. 3A-B are images from the water uptake test on the rapidly-disintegrating sublingual tablets: FIG. 3A shows an image of a dry tablet placed in a test dish prior to the start of the test and FIG. 3B shows an image of a water-saturated tablet after completion of the test.

[0044] FIG. 4 is a graph plotting results from the in vitro diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the cumulative atropine sulfate (AS) diffused per area (μg/cm2) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0045] FIG. 5 is also a graph plotting results from the in vitro diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the atropine sulfate (AS) diffused percent (%) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0046] FIG. 6 is a graph plotting results from the ex vivo diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the cumulative atropine sulfate (AS) diffused per area (μg/cm.sup.2) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0047] FIG. 7 is also a graph plotting results from the ex vivo diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the atropine sulfate (AS) diffused percent (%) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

DETAILED DESCRIPTION OF THE INVENTION

[0048] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments illustrated herein and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modification in the described compositions, formulations, and methods and any further application of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention relates.

[0049] The worldwide use of organophosphates (OPs), as pesticides and nerve agents, has been reported as causing 3 million toxicity cases per year. According to the American Association of Poison Control Centers, almost 7000 toxicity cases were reported in the last two years in the United States alone due to organophosphate use as pesticides. The recommended antidote for organophosphate toxicity is a 2 mg injection of atropine sulfate (AS). Due to the side effects and administration limitations of injections mainly outside of healthcare settings, an alternative easy-to-use dosage form of atropine sulfate would be most beneficial.

[0050] In response to the need for an alternative dosage form for atropine sulfate, the instant inventors formulated and manufactured an exemplary formula for rapidly-disintegrating or dissolving sublingual tablets (RDSTs) of atropine sulfate (AS). They performed several quality control tests to ensure rapid and complete drug release and tested diffusion of atropine sulfate from the AS RDSTs in vitro. These experiments and results are described herein below.

Formulation

Powder Composition

[0051] Six tablet batches of rapidly-disintegrating sublingual tablets (RDSTs) were manufactured by direct compression and evaluated for quality control. The first and second batches were used to manufacture tablets weighing 150 mg and contain 0 mg atropine sulfate (AS) (B0), as placebo, and 2 mg AS (B1). The third to sixth batches were used to manufacture tablets weighing 50 mg and contain 0 mg (B2), as placebo, 2 mg (B3), 4 mg (B4), and 8 mg (B5) atropine sulfate (AS). These formulations are shown below in Table 1.

TABLE-US-00001 TABLE 1 Atropine Sulfate (AS) Rapidly-Disintegrating Sublingual Tablets (RDSTs). Formulations: No. Ingredient Function B0 B1 B2 B3 B4 B5 1 Atropine Sulfate API 0.00 (0%) 2.00 (1.33%) 0.00 (0%) 2.00 (4%) 4.00 (8%) 8.00 (16%) 2 Microcrystalline Filler 133.65 (89.1%) 130.50 (87%) 44.55 (89.1%) 42.75 (85.5%) 40.95 (81.9%) 37.35 (74.7%) Cellulose (Ceolus ® PH-301) 3 Low-substituted Super- 14.85 (9.9%) 14.50 (9.7%) 4.95 (9.9%) 4.75 (9.5%) 4.55 (9.1%) 4.15 (8.3%) Hydroxypropyl disintegrating Cellulose Agent (L-HPC, LH-11 ®) 4 Magnesium Stearate Lubricant 1.5 (0.5%) 3.00 (2%) 0.50 (1.0%) 0.50 (1%) 0.50 (1%) 0.50 (1%) Total Tablet Weight 150 (100%) 150.0 (100%) 50.0 (100%) 50.0 (100%) 50.0 (100%) 50.0 (100%)

Powder Mixing and Sieving

[0052] All powders were sieved before mixing using sieve number 230 (63 μm). First, atropine sulfate was mixed with microcrystalline cellulose (Asahi Kasei Chemicals Corporation, Tokyo, Japan) by a manual geometric method.

[0053] The mixing procedure was then optimized to achieve both internal and external positioning of the superdisintegrant; i.e. low-substituted hydroxypropyl cellulose. In the method of internal and external positioning, the superdisintegrant is divided into two portions. One portion is added before granule formation (internal) and the remaining portion is added to the granules (external) with mixing prior to compression. When both internal and external methods are used, the extragranular superdisintegrant portion breaks the tablet into granules and the granules further disintegrate to release the active substance by the intragranular disintegrant portion (Kumar G. et al. Journal of Global Pharma Technology 4(02):1-12 2012 and Rawas-Qalaji et al. AAPS PharmSciTech 7:E41 2006).

[0054] Two-thirds of the low-substituted hydroxypropyl cellulose (Shin-Etsu Chemical Company, Tokyo, Japan) was mixed with the powdered mixture of atropine sulfate and microcrystalline cellulose using a three-dimensional mixture (Inversina, Bioengineering AG, Wald, Switzerland) for four minutes. Magnesium stearate and the remaining one-third of the low-substituted hydroxypropyl cellulose was manually mixed and then added to the mixture of atropine sulfate, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose to be mixed for an additional thirty seconds.

Direct Compression

[0055] The mixture was then compressed into tablets via direct compression. In direct compression an active agent, i.e. drug, is blended with a variety of excipients, lubricated, and compressed (Kumar G. et al. Journal of Global Pharma Technology 4(02):1-12 2012). For tablets weighing 150 mg, 5″/16″ concave punches and dies were used and for tablets weighing 50 mg, 3″/16″ concave punches and dies were used (Natoli Engineering Company, Inc. Saint Charles, Mo.).

Quality Control Tests

[0056] The manufactured rapidly-disintegrating sublingual tablets were tested for content uniformity (CU), weight variation (WV), and friability using the United States Pharmacopeia (USP) standard tests and limits. Due to a lack of an accurate USP test that can discriminate small differences between rapidly-disintegrating sublingual tablets (RDSTs), tablet disintegration and dissolution were tested using apparatuses (and procedures) developed by the instant inventors that are capable of detecting small differences between tablets. The wetting time (WT) and water uptake (WU) of the tablets were also tested using procedures modified by the instant inventors. See Shukla, D. et al. Sci Pharma 77:327-341 2009 for an overview of the evaluation techniques for mouth dissolving tablets.

Content Uniformity and Weight Variation Tests

[0057] According to USP, the content uniformity test is performed for tablets that contain less than 25 mg or less than 25% of the active material and be within 90-110% of the labeled amount (USB/NF. Official Monograph: Atropine Sulfate Tablet. In: United States Pharmacopeia. 37/32 ed. Rockville, Md.: United States Pharmacopeia Convention, Inc. 2014, pages 1878-1879). Both content uniformity and weight variation tests were performed according to the USP standards for all AS RDST formulations. Tablet content was analyzed by high-performance liquid chromatography (HPLC) using the standard USP procedures for analysis of atropine sulfate (AS) injection (USB/NF. Physical Tests: <905> Uniformity of Dosage Units. In: United States Pharmacopeia 37/32 ed. Rockville, Md.: United States Pharmacopeia Convention, Inc. 2014, pages 491-494).

Breaking Force and Friability Tests

[0058] The AS RDSTs were tested (breaking force) according to the USP guidelines (USP/NF. General Chapters: <1217> Tablet Breaking Force. In: United States Pharmacopeia. 37/32 ed. Rockville, Md.: United States Pharmacopeia Convention, Inc. 2014, pages 1146-1148) using Hardness Tester LIH-3 (Vanguard, Spring, Tex.).

[0059] Further, according to the standard USP Friability Test (USP/NF. General Chapters: <1216> Tablet Friability. In: United States Pharmacopeia. 37/32 ed. Rockville, Md.: United States Pharmacopeia Convention, Inc. 2014, pages 1145-1146), sixty five tablets that weighed 6.5 g were tested using the USP Friability Tester (Pharma Test Apparatebau, GmbH, Hainburg, Germany).

Disintegration Test

[0060] The Disintegration Test is one of the standard USP tests. However, the current official USP standard test is unable to discriminate between small formulation differences. Thus, the instant inventors developed their own disintegration test for the AS RDSTs.

Apparatus

[0061] The disintegration apparatus used for carrying out the disintegration test on the rapidly-disintegrating sublingual tablets (RDSTs) is illustrated schematically in FIG. 1 and includes a motor 10 with a rotating shaft 12, a stainless steel round basket 14, a stainless steel wire screen at the base (bottom) and height (walls) of the basket, a glass beaker 16 containing warmed fluid 18, and a water bath 20 equipped with a thermostat 22. Non-limiting exemplary dimensions for the test set up follow. The rotating shaft has a speed rate of 40±10 rpm, a diameter of 8±2 mm, and a length of 220±20 mm. The stainless steel round basket has a diameter of 38.5±1 mm, a height of 23±2 mm, and a welded seam. The stainless wire screen at the bottom (base) of the basket is connected to the rotating shaft at its center and has apertures of 0.36-0.44 mm and a wire diameter of 0.22-0.31 mm. The glass beaker has a diameter of 70±10 mm, a height of 90±10 mm, and a volume of 350 ml. In use, the glass beaker contains 150±5 ml of a suitable warmed fluid; i.e. water at a temperature of 37±2 C°. The fluid level in the water bath has a height of about 100 mm. In use, the basket is partially submerged in the warmed fluid to a depth of about 10±2 mm and then the basket is rotated to facilitate table disintegration.

Example Test Procedure

[0062] 1. Immerse the stainless steel basket to a depth of 10 mm into the warmed fluid.
2. Rotate the basket at a speed of 40 rpm.
3. Randomly select six tablets to test.
4. Drop one tablet (one dosage unit) at a time into the rotating basket.
5. Record the time required (in seconds) for complete disintegration of the tablet.

[0063] Six tablets are selected to test based on the similar number of tablets used in the official USP standard disintegration test. The apparatus can additionally include a built-in stop that starts and stops automatically through connection to an attached sensor. The times can also be recorded using a manual stop watch.

[0064] Disintegration does not necessarily imply complete solution of the tablet. According to the USP standards, complete disintegration is defined as that state in which any residue of the tablet, except fragments of insoluble ingredients and/or coating, remaining on the wire screen is a soft mass having no palpably firm core (U.S. Pharmacopeia, General Chapter 701 “Disintegration” accessed from the internet on Sep. 17, 2014).

Wetting Time (WT)

[0065] The tablet wetting time was measured according to a previously modified non-USP Wetting Test (WT) designed to simulate the low fluid volume and static motion available in the oral cavity and to better discriminate between small differences in formulation in the presence of limited fluid volume (Rawas-Qalaji M. et al. Drug Dev Ind Pharm 33:523-530 2007; Rawas-Qalaji M. et al. AAPS PharmSciTech 7:E41 2006).

[0066] Six tablets were randomly selected and tested. One tablet was placed on double folded tissue paper in a dish having about 6 ml of water. The time for complete wetting of the tablet is recorded as the wetting time (Shukla, D. et al. Sci Pharm 77:327-341 2009). The Wetting Test is illustrated in FIGS. 2A-C: FIG. 2A is an image of a dry tablet prior to the start of the test; FIG. 2B is an image of the tablet placed on a wet paper towel during the test; and FIG. 2C is an image of the wet tablet after completion of the test.

Dissolution Test

[0067] Dissolution of the tablets was measured according to a previously modified non-USP dissolution test designed to simulate the low fluid volume and static motion available in the oral cavity and to better discriminate between small differences in formulation in the presence of limited fluid volume (Rachid O. et al. AAPS PharmSciTech 12:544-552 2011).

[0068] Dissolution testing is a means of identifying and proving the availability of active drug materials in their delivered form. A dissolution test simulates the availability of active substance and allows prediction of the time for complete release of the material from the dosage form (Tablet Dissolution in the section “Analytical Products” accessed from the website of Pharma Test on Sep. 17, 2014).

[0069] Six tablets were randomly selected and tested. Drug released and dissolved from each tablet after 60 seconds was analyzed by High Performance Liquid Chromatography (HPLC) using the standard USP procedure for analyzing atropine sulfate (AS) injections (USP/NF. Official Monograph: Atropine Sulfate Injection. In: United States Pharmacopeia. 37/32 ed. Rockville, Md.: United States Phamacopeial Convention, Inc. 2014, pages 1875-1876).

Water Uptake (WU) Test

[0070] Water uptake was measured in order to test the ability of the tablet to uptake and hold water to swell and dissolve the atropine sulfate (AS) within the tablet. The volume of water up-taken by each tablet was measured by weighing the tablet before and after addition of the water droplets. To test the tablets, water was added dropwise, using a glass pipette, to the top of a pre-weighted tablet placed on an analytical balance (d=0.01 mg) until water starts oozing outside the tablet because the tablet cannot hold any more water. The Water Uptake Test is illustrated in FIGS. 3A-B: FIG. 3A shows an image of a dry tablet placed in a test dish prior to the start of the test and FIG. 3B shows an image of a water-saturated tablet after completion of the test. The percentage of water uptake was determined using the following equation:

Water Uptake %=(weight of wet tablet−weight of dry tablet)×100/weight of dry tablet.

The resulting data obtained from the quality control tests of manufactured AS RDSTs are tabulated below:

TABLE-US-00002 TABLE 2 Quality Control Tests of Atropine Sulfate (AS) Rapidly-Disintegrating Sublingual Tablets (RDSTs): Placebo Tablet Weighing 150 mg (B0); 2 mg AS Tablet Weighing 150 mg (B1); Placebo Tablet Weighing 50 mg (B2); 2 mg AS Tablet Weighing 50 mg (B3); 4 mg AS Tablet Weighing 50 mg (B4); and 8 mg AS Tablet Weighing 50 mg (B5): Tablet B0 B1 B2 B3 B4 B5 Diameter 7.95 ± 0.0 7.95 ± 0.0  4.77 ± 0.006  4.77 ± 0.004  4.76 ± 0.005  4.76 ± 0.004 Content Uniformity (%) N/A  101 ± 5.6 N/A 98.06 ± 2.2  93.17 ± 2.1  99.96 ± 1.5  Breaking Force (KF)  2.94 ± 0.14  2.7 ± 0.19 3.18 ± 0.56  3.28 ± 0.45  2.78 ± 0.12  1.45 ± 0.05 Friability (loss %) 0% 0.70% 0% 0.32% 0.14% 0.09% Disintegration Time (sec) 25.19 ± 0.39 20.67 ± 1.15 15.92 ± 0.92  14.52 ± 1.58 12.14 ± 2.12 13.93 ± 1.29 Drug Dissolved (%).sup.§ N/A 87.8% ± 10  N/A 98.07 ± 4.08 100.0% ± 4.7   88.48 ± 14   Wetting Time (sec) 14.76 ± 4.05 7.56 ± 0.7 7.4 ± 0.2  7.2 ± 0.35 16.7 ± 0.8   11 ± 0.9 Water Uptake (%) 330.3 ± 10.7 296.19 ± 2.8  272.2 ± 13.3  292.6 ± 7.5  275.4 ± 4.3  230 ± 12 * Results are presented as mean ± SD .sup.§Drug dissolution was measured for 60 seconds.

[0071] In a further example, six rapidly-disintegrating sublingual tablets (RDSTs) batches containing 0 mg (placebo), 2 mg, and 4 mg atropine sulfate (AS) were manufactured by direct compression. Tablet weights were 150 mg or 50 mg for placebo and 2 mg AS tablets. The formulation included microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate, and AS. Pre-sieved powders (mesh no. 230) were mixed by three-dimensional mixer and compressed using 5/16″ or 3/16″ bevel punches and dies. Quality control tests including USP friability, weight variation (WV), and content uniformity (CU) tests; and non-USP disintegration, wetting time (WT), water uptake (WU), and dissolution tests were used for all formulated batches (Table 2).

Diffusion Studies

In Vitro and Ex Vivo Diffusion of Atropine Sulfate

[0072] Evaluation of the in vitro and ex vivo diffusion of atropine sulfate (AS) from the AS RDSTs was performed using static vertical jacketed Franz cells with an OD of 20 mm and a reservoir volume of 20±1 ml (PermeGear Inc. Hellertown, Pa.). For the in vitro studies, a cellulose dialysis membrane, 7 Spectra/Por® with a 1000 Dalton MWCO (Spectrum Laboratories Inc. Rancho Dominguez, Calif.) was used as the diffusional membrane. For the ex vivo studies, an excised porcine sublingual membrane (PSM) was used as the diffusional membrane. PSM was documented to be structurally and physiologically similar to human sublingual membrane (Rawas-Qalaji M M, Simons F E R, Simons K J. Fast-disintegrating sublingual tablets: Effect of epinephrine load on tablet characteristics. AAPS PharmSciTech. 2006: 7(2):E72-E8; Collins P, Laffoon J, Squier C, editors. Comparative structure of porcine oral epithelium. Journal of Dental Research; 1981; Birudaraj R, Berner B, Shen S, Li X. Buccal permeation of buspirone: mechanistic studies on transport pathways. Journal Of Pharmaceutical Sciences. 2005; 94(1):70-8; Ong C M Y, Heard C M. Permeation of quinine across sublingual mucosa, in vitro. International Journal of Pharmaceutics. 2009; 366(1-2):58-64). Frozen lower jaws of male adult domestic pigs (Sus domesticus) were obtained from a local butcher shop and thawed at room temperature then dissected to separate the epithelial sublingual membrane at the floor of the mouth from underneath connective tissues. The dissected membranes were inspected visually for integrity and frozen again at −20° C. Membranes were thawed at room temperature to be used before each experiment. A receptor chamber with a magnetic stirrer, filled with phosphate buffer (ranging in pH from 5.0 to about 6.5), was used as the diffusion medium. This test simulates sublingual conditions as pH 5.8 is the average pH of saliva.

[0073] Before beginning each experiment air bubbles were removed after mounting the membrane between the donor and receptor chambers. The water bath was set at 37° C. and water was circulated in the jacketed Franz cells. The mounted membranes were equilibrated with the diffusion medium from both sides for 30 minutes and were checked for leaks.

[0074] Each tablet tested was at the center of the donor chamber on the membrane at TO and then 2 ml of the diffusion medium was added to facilitate tablet disintegration and dissolution. Aliquots of 200 μl were withdrawn from the receptor chamber using 6-inch needles (Popper & Sons, Inc. New Hyde Park, N.Y.) and 1 ml syringes at 5, 10, 15, 20, 30, 45, 60, 75, and 90 minute time intervals. The volumes withdrawn were replenished with fresh medium. Samples were transferred to High Performance Liquid Chromatography (HPLC) vials for HPLC analysis using an ultra violet (UV) detector according to the standard USP method for analyzing atropine sulfate injections (USP/NF. Official Monograph: Atropine Sulfate Injection. In: United States Pharmacopeia. 37/32 ed. Rockville, Md.: United States Phamacopeial Convention, Inc. 2014, pages 1875-1876). A 2 ml solution, 1 mg/ml, of atropine sulfate was used as a reference.

Statistical Analysis

[0075] The mean±SD cumulative diffused atropine sulfate (AS) per area (μg/cm.sup.2) and percentage of diffused AS for each rapidly-disintegrating sublingual (RDST) tablet formulation were calculated. The mean±SD AS influx, J (μg/cm.sup.2/minute), and lag time, tL (minutes) were calculated from the slope and the intercept with the x-axis of each graph (n=4). Also, AS permeability, P (cm/minute), was calculated by dividing J by AS concentration in the donor chamber at T.sub.0. The area under the curve of diffused AS per area (cumulative diffused AS per area versus time), JAUC.sub.0-90 (n/cm.sup.2/minute); the maximum AS diffused, Jmax (μg/cm.sup.2); and the time to reach Jmax, Tmax (minutes) were calculated using WinNonlin software (Pharsight Mountain View, Calif.). Data were statistically compared by one-way ANOVA and Tukey-Kramer tests using NCSS statistical software (NCSS Kaysville, Utah). Differences were considered to be statistically significant at p<0.05.

In Vitro and Ex Vivo Diffusion Study Results

[0076] The cumulative atropine sulfate (AS) diffused in vitro per area (μg/cm.sup.2) versus time and percentage of in vitro diffused AS for each rapidly-disintegrating sublingual tablet (RDST) formulation are shown in FIGS. 4 and 5, respectively.

[0077] FIG. 4 is a graph plotting results from the in vitro diffusion studies on the tablets. The graph shows the cumulative atropine sulfate (AS) diffused per area (μg/cm2) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0078] FIG. 5 is also a graph plotting results from the in vitro diffusion studies on the tablets. The graph shows the atropine sulfate (AS) diffused percent (%) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0079] The cumulative atropine sulfate (AS) diffused ex vivo per area (μg/cm.sup.2) versus time and percentage of ex vivo diffused AS for each rapidly-disintegrating sublingual tablet (RDST) formulation are shown in FIGS. 6 and 7, respectively.

[0080] FIG. 6 is a graph plotting results from the ex vivo diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the cumulative atropine sulfate (AS) diffused per area (μg/cm.sup.2) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0081] FIG. 7 is also a graph plotting results from the ex vivo diffusion studies on the rapidly-disintegrating sublingual tablets. The graph shows the cumulative atropine sulfate (AS) diffused percent (%) versus time from the rapidly-disintegrating sublingual tablets (RDSTs) (n=4). The reference is 2 ml of 1 mg/ml atropine sulfate (AS) solution.

[0082] Four formulations and the reference sample of atropine sulfate were tested (Table 3). The four formulations were as follows: B1, 2 mg AS with 150 mg tablet weight; B3, 2 mg AS with a 50 mg tablet weight; B4, 4 mg AS with a 50 mg tablet weight; and B5, 8 mg AS with a 50 mg tablet weight.

[0083] For in vitro diffusion, mean±SD JAUC.sub.0-90 of diffused AS and influx (J) of B5 (47485.9±7846.7 μg/cm.sup.2/min, 20.0±6.48 μg/cm.sup.2/min, respectively) were significantly higher (p<0.05) than B4 (30103.8±4248.9 μg/cm.sup.2/min, 10.3±3.5 μg/cm.sup.2/min, respectively), which were also significantly higher (p<0.05) than B3 (9488.0±1907.5 μg/cm.sup.2/min, 4.2±1.3 μg/cm.sup.2/min, respectively), B1 (8063.8±1211.39 μg/cm.sup.2/min, 2.5±0.5 μg/cm.sup.2/min, respectively), and the reference (11387.8±468.5 μg/cm.sup.2/min, 3.3±0.3 μg/cm.sup.2/min, respectively). However, JAUC.sub.0-90 and J for B3 were not significantly different (p>0.5) from the reference. Results are shown below in Table 3.

TABLE-US-00003 TABLE 3 In Vitro Diffusion of Atropine Sulfate (AS) Rapidly-Disintegrating Tablets (RDSTs); 2 mg AS Tablet Weighing 150 mg (B1); 2 mg AS Tablet Weighing 50 mg (B3); 4 mg AS Tablet Weighing 50 mg (B4); 8 mg AS Tablet Weighing 50 mg (B5); and reference, 2 ml of 1 mg/ml solution B1 B3 B4 B5 Reference JAUC.sub.0-90 7867.5 ± 1251.3 9488.0 ± 1907.5 .sup. 30103.8 ± 4248.9.sup.¥ 47485.9 ± 7846.7.sup.§   11387.8 ± 468.5  (μg/cm.sup.2/min) J (μg/cm.sup.2/min) 2.5 ± 0.5 .sup. 4.1 ± 1.3.sup.£ .sup. 10.3 ± 3.5.sup.¥ .sup.  20 ± 6.48.sup.§ 3.3 ± 0.3 P (cm/min) 1.3 ± 0.3 2.1 ± 0.7  2.6 ± 0.9 2.5 ± 0.81 1.63 ± 0.15 t.sub.L (min) 1.8 ± 2.1 0.5 ± 1.0 0.25 ± 0.5 2.0 ± 2.82 1.81 ± 0.85 *Results are presented as mean ± SD, JAUC.sub.0-90, J: influx, P: permeability, t.sub.L: lag time .sup.§p < 0.05 .sup.¥p < 0.05, from B3 and B1 .sup.£p < 0.05, from B1

[0084] For ex vivo diffusion, mean±SD JAUC.sub.0-90 of diffused AS and influx (J) of B5 (8849±680 μg/cm.sup.2/min, 3.89±1.38 μg/cm.sup.2/min) were significantly higher (p=<0.05) than B4 (4557±761.3 μg/cm.sup.2/min, 1.57±0.69 μg/cm.sup.2/min), B3 (2296±761.3 μg/cm.sup.2/min, 1.22±0.54 μg/cm.sup.2/min), B1 (2473.5±894.5 μg/cm.sup.2/min, 0.953±0.36 μg/cm.sup.2/min), and the reference solution (2292±761.3 μg/cm.sup.2/min, 0.92±0.28 μg/cm.sup.2/min). Results are shown below in Table 4.

TABLE-US-00004 TABLE 4 Ex Vivo Permeation of Atropine Sulfate (AS) Rapidly-Disintegrating Sublingual Tablets (RDSTs): 2 mg AS Tablet Weighing 150 mg (B1); 2 mg AS Tablet Weighing 50 mg (B3); 4 mg AS Tablet Weighing 50 mg (B4); 8 mg AS Tablet Weighing 50 mg (B5); and reference, 2 ml of 1 mg/ml solution. B1 B3 B4 B5 Reference JAUC.sub.0-90 2473.53 ± 894.54   2296 ± 761.3 .sup.  4557 ± 761.3.sup.¥ .sup.  8849 ± 680.9.sup.§  2292 ± 761.3 (μg/cm.sup.2/min) Diffusion (%) 6.29 ± 2.45 10.26 ± 4.57  4.58 ± 1.22 8.95 ± 3.8  10.39 ± 4.1  J (μg/cm.sup.2/min) 0.9525 ± 0.36  1.22 ± 0.54 1.57 ± 0.69 3.894 ± 1.38.sup.§  0.9191 ± 0.28  P (cm/min) 0.47 ± 0.18 0.61 ± 0.27 0.39 ± 0.17 0.49 ± 0.17 0.4 ± 0.1 t.sub.L (min) 0 ± 0 5.75 ± 3.3  7.25 ± 6.75  7.8 ± 6.25 0 ± 0 *Results are presented as mean ± SD, JAUC.sub.0-90, J: influx, P: permeability, t.sub.L: lag time .sup.§p < 0.05 .sup.¥p < 0.05, from B3 and B1

Effect of Tablet Dimension and Weight on the Influx of Atropine Sulfate

[0085] The effect of tablet dimension and weight on the influx of atropine sulfate (AS) from the rapidly-disintegrating sublingual tablets (RDSTs) was also investigated.

[0086] Two batches of AS 2 mg RDSTs were manufactured by direct compression (Table 1). The first AS RDSTs batch was compressed using 3/16″ bevel punches and dies to make 50 mg tablet weight. The second AS RDSTs batch was compressed using 5/16″ bevel punches and dies to make 150 mg tablet weight. The in vitro diffusion of AS 2 mg RDSTs weighted 50 mg (n=4) and 150 mg (n=8) were performed using Franz cells through cellulose membrane (MWCO 1000D). Phosphate buffer, pH 6.5, was used as the diffusion media. AS RDST was placed on the cellulose membrane in the donor chamber, which contained 2 ml of phosphate buffer. Aliquot samples, 200 μl, were withdrawn from the receptor chamber at preset time intervals.

[0087] Mean±SD thickness and diameter of AS 2 mg RDSTs weighted 50 mg and 150 mg were 3.1±0.0 mm and 4.77±0.00 mm, and 4.0±0.0 mm and 7.95±0.0 mm, respectively. Mean±SD AS influx (J) from RDSTs weighted 50 mg (4.1±1.3 μg/cm2/minute) was significantly higher (p<0.05) than RDSTs weighted 150 mg (2.5±0.5 μg/cm2/minute). However, AS permeability was not significantly different (p>0.05) between RDSTs weighted 50 mg and 150 mg, 2.1±0.7 cm/minute and 1.3±0.3 cm/minute, respectively. Thus, the tablet dimensions and weight of atropine sulfate (AS) 2 mg RDSTs can affect the influx of AS.

CONCLUSION

[0088] In conclusion, the formulation of atropine sulfate (AS) as rapidly-disintegrating sublingual tablets (RDSTs) was shown to be successful. AS RDSTs disintegrated and dissolved in <30 seconds and 60 seconds without compromising hardness to pass the USP friability test.

[0089] The diffusion of atropine sulfate from the RDSTs was similar to its diffusion from solution formulation of the same concentration, which indicates that diffusion of atropine sulfate was not delayed by the RDST formulation. Increasing the atropine sulfate dose significantly increases its diffusion. Furthermore, the tablet dimension and weight of 2 mg AS RDSTs was shown to affect influx of atropine sulfate.

[0090] The herein described atropine sulfate (AS) rapidly-disintegrating sublingual tablets (RDSTs) provide a non-invasive sublingual drug delivery of atropine sulfate for the treatment of acute organophosphate (OP) toxicity as a potential alternative (to the conventional parenteral delivery), patient-friendly, convenient, and cost-effective dosage form.

[0091] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. It is to be understood that while a certain form of the invention is illustrated, it is not intended to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions, rapidly-disintegrating sublingual tablets (RDSTs), atropine sulfate rapidly-disintegrating sublingual tablets (AS RDSTs), therapeutic compositions and methods, pharmaceutical tablets, methods, procedures, and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention. Although the invention has been described in connection with specific, preferred embodiments, it should be understood that the invention as ultimately claimed should not be unduly limited to such specific embodiments. Indeed various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the invention.