Compositions including epinephrine microcrystals
11229613 · 2022-01-25
Assignee
Inventors
- Mutasem Rawas-Qalaji (Fort Lauderdale, FL)
- Ousama Rachid (Winnipeg, CA)
- Keith Simons (Winnipeg, CA)
- Estelle Simons (Winnipeg, CA)
Cpc classification
A61K9/0056
HUMAN NECESSITIES
A61K31/00
HUMAN NECESSITIES
A61K9/006
HUMAN NECESSITIES
A61K9/14
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
International classification
A61K31/00
HUMAN NECESSITIES
A61K9/14
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K31/137
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
Abstract
The invention provides compositions including epinephrine fine particles, including epinephrine nanoparticles or nanocrystals and epinephrine microparticles or microcrystals, and methods for therapeutic use of the compositions for the treatment of conditions responsive to epinephrine such as a cardiac event or an allergic reaction, particularly anaphylaxis. The epinephrine fine particles can be incorporated into orally-disintegrating and fast-disintegrating tablet pharmaceutical formulations and can significantly increase the sublingual bioavailability of epinephrine, and thereby reduce the epinephrine dose required.
Claims
1. A pharmaceutical composition formulated as a tablet for buccal or sublingual administration comprising approximately 10 mg to approximately 40 mg of stabilized epinephrine bitartrate microcrystals, the stabilized epinephrine bitartrate microcrystals having a spherical shape, the spherical shape formed when raw epinephrine bitartrate particles having a rectangular shape undergo a morphological change when reduced in size to form the stabilized epinephrine bitartrate microcrystals of the pharmaceutical composition.
2. The pharmaceutical composition according to claim 1, further comprising at least one of a surfactant, a penetration enhancer, a mucoadhesive, a filler, a lubricant, a disintegrant, a taste enhancer, and a sweetening agent and mouthfeel enhancer.
3. The pharmaceutical composition according to claim 1, wherein the stabilized epinephrine bitartrate microcrystals are formed from raw epinephrine bitartrate particles having been reduced to a particle size of 2.5 μm or less.
4. The pharmaceutical composition according to claim 1, formulated as a tablet for buccal or sublingual administration comprising 20 mg stabilized epinephrine bitartrate microcrystals.
5. The pharmaceutical composition according to claim 2, comprising a filler, a lubricant, and a disintegrant, wherein the filler is microcrystalline cellulose, the lubricant is magnesium stearate, and the disintegrant is a hydroxypropyl ether of cellulose.
6. The pharmaceutical composition according to claim 5, further comprising a taste enhancer and a sweetening agent and mouthfeel enhancer, wherein the taste enhancer is citric acid, and the sweetening agent and mouthfeel enhancer is mannitol.
7. A pharmaceutical composition formulated as a tablet for buccal or sublingual administration having an active ingredient consisting of 10 mg to 40 mg stabilized epinephrine bitartrate microcrystals, the stabilized epinephrine bitartrate microcrystals having a spherical shape, the spherical shape formed when raw epinephrine bitartrate particles undergo a morphological change when reduced in size to form the stabilized epinephrine bitartrate microcrystals of the pharmaceutical composition.
8. The pharmaceutical composition according to claim 7, wherein the stabilized epinephrine bitartrate microcrystals are formed from raw epinephrine bitartrate particles having been reduced to a particle size of 2.5 μm or less.
9. The pharmaceutical composition according to claim 7, further comprising at least one of a surfactant, a penetration enhancer, a mucoadhesive, a filler, a lubricant, a disintegrant, a taste enhancer, and a sweetening agent and mouthfeel enhancer.
10. The pharmaceutical composition according to claim 7, formulated as a tablet for buccal or sublingual administration having an active ingredient consisting of 20 mg stabilized epinephrine bitartrate microcrystals.
11. The pharmaceutical composition according to claim 9, comprising a filler, a lubricant, and a disintegrant, wherein the filler is microcrystalline cellulose, the lubricant is magnesium stearate, and the disintegrant is a hydroxypropyl ether of cellulose.
12. The pharmaceutical composition according to claim 11, further comprising a taste enhancer and a sweetening agent and mouthfeel enhancer, wherein the taste enhancer is citric acid, and the sweetening agent and mouthfeel enhancer is mannitol.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) 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.
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DETAILED DESCRIPTION OF THE INVENTION
(17) 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 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.
(18) Epinephrine (Epi) 0.3 mg IM injection in the thigh is the drug of choice and the only available dosage form for the treatment of anaphylaxis in community sittings. Previously, the instant inventors were able to develop and evaluate rapidly-disintegrating sublingual epinephrine tablets. These studies showed that sublingually administered epinephrine is absorbed and bioequivalent to 0.3 mg IM Injection in a rabbit animal-model.
(19) For the study described herein, it was hypothesized that formulating Epi as nanocrystals (NC) or microcrystals (MC) would significantly enhance its sublingual diffusion. The objectives were to prepare Epi NC or Epi MC and formulate them into rapidly-disintegrating sublingual tablets (ODT) to be tested for their in vitro diffusion, ex vivo diffusion, and in vivo absorption using dialysis membranes, excised sublingual porcine mucosal membranes, and validated rabbit's animal model, respectively.
(20) Epi NC or Epi MC were prepared by top-bottom technique using LV-1 Microfluidizer as described in a previously-filed patent application; U.S. Provisional Patent Application Ser. No. 61/660,273, filed on Jun. 15, 2012. ODTs were manufactured by direct compression using our previously developed and published formulation. The in vitro and ex vivo diffusion of 10, 20, and 40 mg Epi ODT, and 10, 20 mg Epi MC ODT (n=4) were evaluated using static vertical Franz cells. Epi 10 mg solution was used as a control. Mean±SD JAUC.sub.0-90 of diffused Epi, Jmax, and Epi influx (J) from 40 mg Epi ODT and 20 mg Epi MC ODT were not significantly different from each other both in vitro and ex vivo (p>0.05).
(21) The in vivo absorption of 40 mg Epi ODT and 20 mg Epi MC ODT (n=5) were evaluated in a validated rabbits animal-model. Epi 0.3 mg IM injection in the thigh was used as a positive control and placebo ODT was used as a negative control. The mean±SD AUC.sub.0-60 and Cmax from 20 mg Epi MC ODT and 40 mg Epi ODT did not differ significantly (p>0.05) from Epi 0.3 mg IM. However, the mean±SD AUC.sub.0-60 and Cmax of exogenous epinephrine administered through either the sublingual or intramuscular routes differed significantly (p<0.05) from placebo sublingual tablets, endogenous epinephrine.
(22) These micro-sized Epi ODT improved Epi diffusion by two folds and have the potential to reduce the bioequivalent dose of sublingually administered Epi by 50%. These micro-sized Epi ODT have the potential for the first-aid treatment of anaphylaxis in community settings are suitable for phase I studies in humans.
(23) For the emergency treatment of anaphylaxis, prompt intramuscular injection of epinephrine (Epi) in the thigh muscle is the drug of choice.sup.1-4. Epi auto-injectors such as EpiPen®, EpiPen Jr® (Mylan Inc, Basking Ridge, N.J.), Twinject 0.3 Mg®, and Twinject 0.15® (Shionogi Pharma, Inc. Atlanta, Ga.) are commonly prescribed and the only available dosage form for the first-aid emergency treatment of anaphylaxis in a community setting. However, self-injectable epinephrine is underutilized when anaphylaxis occurs due to several drawbacks.sup.5, 6.
(24) The sublingual route is a promising alternative route for Epi administration. Drugs that can be absorbed sublingually bypass potential metabolic conversion in the gastrointestinal tract and hepatic first-pass metabolism, and reach the systemic circulation in a pharmacologically active form.sup.7-12. Epi is extensively metabolized after oral administration by the catechol-O-methyltransferase in the gastrointestinal tract and by monoamine oxidase in the gastrointestinal tract and in the liver.sup.13.
(25) The high vascularity of the sublingual mucosa and the low molecular weight of Epi facilitate its rapid absorption directly into the venous circulation through the sublingual and frenular veins. The described rapidly-disintegrating sublingual 40 mg Epi tablets, which retain sufficient hardness to withstand shipping and handling and disintegrate to release Epi rapidly (≤30 sec).sup.14-16, have shown to be bioequivalent to the adult dose of Epi IM injection, 0.3 mg, in a validated rabbit model.sup.10, 11. This high dose was essential to create the required concentration gradient that promotes Epi absorption across the sublingual membrane and results in therapeutic plasma drug concentrations.
(26) One of the most common approaches to enhance the rate of drug dissolution and absorption is to significantly reduce its particles size to the micro- or nano-size range. Drug nanocrystals (NC) or microcrystals (MC) are advantageous due to minimal required excipients and almost 100% of the pure drug is produced during the fabrication process.sup.17. Also, the collected dried drug NC or MC can be formulated into various dosage forms.
(27) In designing the experiments described herein, it was hypothesized that using reduced particle size of Epi instead of regular raw Epi crystals will significantly increase Epi dissolution rate and absorption. Also, they would reduce the required bioequivalent dose to Epi 0.3 mg IM injections.
(28) In the study described herein, the in vitro and ex vivo diffusion of epinephrine bitartrate microcrystals (EpiBit MC) against regular epinephrine bitartrate (EpiBit) crystals formulated into our rapidly-disintegrating tablets (ODT) was tested to evaluate the permeability of these micro-sized Epi ODT before performing in vivo studies.
(29) In the in vivo study, the absorption of epinephrine bitartrate microcrystals (EpiBit MC) and regular epinephrine bitartrate (EpiBit) crystals formulated into our rapidly-disintegrating tablets (ODT) was tested against the standard Epi 0.3 mg IM injection in the thigh. The aim was to establish a significantly lower bioequivalent sublingual dose of Epi than the one previously achieved.
(30) These rapidly-disintegrating sublingual epinephrine tablets will have the potential as user-friendly, non-invasive alternative for the first-aid emergency treatment of anaphylaxis in a community setting.
(31) Materials
(32) These materials are useful for the in vitro and ex vivo diffusion studies described below and for the fabrication of epinephrine fine particles and tablets.
(33) (−)-Epinephrine (+) bitartrate was purchased from Sigma-Aldrich (St. Louis, Mo.). Ceolus® PH-301 (microcrystalline cellulose) with a mean particle size of 50 μm was supplied by Asahi Kasei Chemicals Corp (Tokyo, Japan) and low-substituted hydroxypropyl cellulose (LH11) with a mean particle size of 50 μm was supplied by Shin-Etsu Chemical Co (Tokyo, Japan). Magnesium stearate was purchased from Mallinckrodt Baker (Phillipsburg, N.J.). Isopropyl alcohol, 99.5%, was purchased from BDH (VWR, West Chester, Pa.). Spectra/Por® 7 dialysis membranes with 1000 Dalton MWCO were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, Calif.). Potassium phosphate monobasic was purchased from Sigma-Aldrich (St. Louis, Mo.) and sodium hydroxide was purchased from J. T. Baker (Philipsburg, N.J.).
(34) Fabrication and Characterization of Epinephrine Fine Particles Using High Shear Fluid Processor (Microfluidizer)-Homogenization Method
(35) Epinephrine bitartrate fine particles were fabricated, developed, and characterized as described in the previously-filed related application; U.S. Provisional Patent Application Ser. No. 61/660,273, filed on Jun. 15, 2012.
(36) Preparation of Epinephrine Bitartrate Nanocrystals
(37) The EpiBit NC (or EpiBit MC) was prepared by a top-bottom technique using LV-1 High Sheer Fluid Processor “Microfluidizer” (Microfluidics, Newton, Mass.) equipped with G10Z reaction chamber. Briefly, epinephrine bitartrate (2.8 mg/mL), (with and without the use of any excipients), was suspended in 6 mL isopropyl alcohol, sonicated for 30 seconds and injected into the system. The suspension was processed at 30,000 Psi for one cycle. The microfluidizer-receiving coil was immersed in ice to reduce the heat produced during the process. The nanosuspension was centrifuged using Avanti J-25 centrifuge (Beckman Coulter, Inc, Miami, Fla.) at 15,000 rpm and 15° C. for 30 minutes. The upper clear solvent was removed by aspiration and the remaining particles were dried by vacuum concentrator at room temperature.
(38) Characteristics of the Epinephrine Bitartrate Nanocrystals
(39) Particle Size and Zeta Potential Measurement
(40) The average particles size (by volume) of EpiBit before processing was measured using laser diffraction technique using Mastersizer (Malvern Instruments Inc, Westborough, Mass.). D (0.1), D (0.5) or median, D (0.9), and D (4, 3) or mean volume are shown in Table 1. Mean±SD particles size distribution (by volume) of EpiBit crystals before processing was 131.8±10.5 μm (n=6). The 10.sup.th percentile (Dv0.1), median (Dv0.5), and 90.sup.th percentile (Dv0.9) were 39.8±3.0 μm, 113.6±9.1 μm, and 254.8±20.1 μm, respectively.
(41) TABLE-US-00001 TABLE 1 Particles Size Distribution (by Volume) of EpiBit Before Processing Before Fabrication (μm) Sample # D (4, 3) D (0.1) D (0.5) D (0.9) 1 147.1 44.4 128.0 282 2 129.5 40.27 111.4 249.6 3 121.6 37.4 105.2 234.7 4 136.0 41.0 117.5 262 5 137.2 40.25 116.1 269.6 6 119.2 35.7 103.3 230.7 Mean 131.8 39.8 113.6 254.8 Standard Deviation 10.5 3.0 9.1 20.1
(42) The Z-average particles size (by intensity) and the average zeta potential of EpiBit after processing were measured using light scattering technique using Zetasizer ZS90 (Malvern Instruments Inc, Westborough, Mass.). Z-average with polydispersity index (Pdi) and zeta potential are shown in Table 2.
(43) Mean (±SD) particles size distribution by intensity and by volume, Pdi, and zeta potential (n=3) of EpiBit crystals after processing using the microfluidizer for one cycle at 30,000 Psi were 2.4±0.4 2.5±0.4 0.185±0.019, and −4.5±1.4 mV, respectively.
(44) The processing of EpiBit results in fine particles with a mean particle size at the low end of the micro-size range but approaching the nano-size range. The particles of this size range were used for diffusion studies and in vivo animal studies.
(45) TABLE-US-00002 TABLE 2 Particles Size Distribution (by intensity) and zeta potential of EpiBit After Processing After Fabrication Sample # Z-average (d .Math. nm) Pdi Z-potential (mV) 1 2649 0.187 −6.0 2 1958 0.165 −3.4 3 2615 0.202 −4.0 Mean 2407.3 0.185 −4.5 Standard Deviation 389.5 0.019 1.4
Fourier Transformation InfraRed (FT-IR)
(46) The processed EpiBit were tested for stability and removal of isopropyl alcohol using FT-IR spectrometer, spectrum 100 (PerkinElmer, Waltham, Mass.) scanned from 4000-650 cm.sup.−1. The FT-IR spectrum of EpiBit before and after processing is shown in
(47) The FT-IR spectrum of isopropyl alcohol and EpiBit after processing is shown in
(48) Differential Scanning Calorimetry (DSC)
(49) Also, the processed EpiBit were tested for purity, stability, and crystallinity changes using Differential Scanning calorimetry (DSC) 4000 (PerkinElmer, Waltham, Mass.) that was calibrated using an indium standard and heated from 30 to 300° C. at rate of 10° C./min and with a nitrogen purge of 20 mL/min. The DSC spectra of EpiBit before and after processing are shown in
(50) Scanning Electron Microscopy (SEM)
(51) The morphologies of EpiBit before and after processing were examined using Quanta 200 Environmental Scanning Electron Microscope (FEI, Hillsboro, Oreg.) operated at an accelerating voltage of 20 kV. Fresh suspension of processed EpiBit and a fresh dispersion of unprocessed EpiBit were deposited on an aluminum stub following the evaporation of isopropyl alcohol and sputter coated with gold using Cressington 108 sputter coater (Cressington Scientific Instruments Ltd, Watford, England). The Scanning Electron Microscopy (SEM) images of EpiBit before and after processing are shown in
(52) Rapidly-Disintegrating Epinephrine Sublingual Tablet Formulation
(53) Rapidly-disintegrating tablets for sublingual administration were developed and evaluated as described in the previously-filed related applications; U.S. Utility patent application Ser. No. 11/672,503, filed on Feb. 7, 2007 and U.S. Utility patent application Ser. No. 11/530,360, filed on Sep. 8, 2006. A range of epinephrine (Epi) doses were formulated as rapidly-disintegrating tablets using equivalent amounts of regular L-epinephrine bitartrate (EpiBit) obtained from Sigma-Aldrich or nanocrystals (NC) or microcrystals (MC) of EpiBit fabricated as previously described. Tablets containing 10, 20, and 40 mg Epi and 10 and 20 mg Epi MC were manufactured using equivalent amounts of EpiBit.
(54) Manufacturing and Quality Control of Tablets for In Vitro and Ex Vivo Diffusion Studies
(55) Five ODT formulations containing EpiBit equivalent to 10 mg, 20 mg, and 40 mg, epinephrine and EpiBit MC equivalent to 10 mg and 20 mg epinephrine were manufactured by direct compression. These tablets were formulated using microcrystalline cellulose, low-substituted hydroxylpropyl cellulose, and magnesium stearate as described in our previous studies.sup.15, 16. The tablet weight was 150 mg. All excipients were used as supplied and kept under low humidity condition before mixing. The mixing process was performed in a nitrogen-preflushed opaque glass container using three-dimensional manual mixer (Inversina, Bioengineering AG, Wald, Switzerland). The powder mixture of the five tablet formulations was compressed right after mixing using 4-stations Colton rotary press (Key Industries, Englishtown, N.J.) at a pre-selected compression force for each tablet formulation, based on our previous results' to ensure sufficient hardness to withstand shipping and handling while maintaining rapid tablet disintegration.
(56) All tablet formulations were tested for quality control as follows:
(57) Dimensions:
(58) Six tablets were randomly selected from each formulation. The diameter and the thickness of rapidly-disintegrating Epi tablets were measured using digital caliper with a range of 0-100 mm and accuracy of 0.02 (Harbor Freight Tools, Camarillo, Calif.). The mean±SD (mm) and RSD % of tablets' diameters and thicknesses are shown in Table 3.
(59) Hardness:
(60) Six tablets were randomly selected from each formulation. The hardness or the breaking force of rapidly-disintegrating Epi tablets was measured using Hardness Tester LIH-3 (Vanguard, Spring, Tex.). The mean±SD (Kgf) and RSD % of hardness for various tablet formulations are shown in Table 3.
(61) Disintegration Time:
(62) Six tablets were randomly selected from each formulation. The disintegration time of rapidly-disintegrating Epi tablets was measured using a previously developed and published method to discriminate between the disintegration times of rapidly-disintegrating tablets or orally disintegrating tablets.sup.15, 16. The mean±SD (Sec) and RSD % of disintegration time for various tablet formulations are shown in Table 3.
(63) USP Weight Variation Test:
(64) Tablet weight variation was measured using the USP methods and criteria.sup.18. The mean±SD (%) and RSD % of weight variation for various tablet formulations are shown in Table 3.
(65) USP Content Uniformity Test:
(66) Tablet drug content uniformity was measured using the USP methods and criteria.sup.18. Drug content was analyzed using a High Performance Liquid Chromatography (HPLC) system with ultraviolet detection (UV) (PerkinElmer, Waltham, Mass.) according to USP.sup.19. The mean±SD (%) and RSD % of content uniformity for various tablet formulations are shown in Table 3.
(67) USP Friability Test:
(68) The friability of rapidly-disintegrating Epi tablets was measured using USP Friability Tester LIC-1 (Vanguard, Spring, Tex.) according to USP methods and criteria.sup.18. The mean tablets weight loss (%) for various tablet formulations are shown in Table 3.
(69) Mean±SD hardness, disintegration time, weight variation, content uniformity, and friability for 10 mg, 20 mg, and 40 mg Epi, and 10 mg and 20 mg Epi MC tablets are shown in Table 3. All tablet formulations were within UDP criteria for weight variation, drug content uniformity, and friability.sup.18, 20.
(70) TABLE-US-00003 TABLE 3 The mean ± SD hardness (n = 6), disintegration time, weight variation, content uniformity, tablet diameter, tablet thickness, and friability for 10 mg, 20 mg, and 40 mg tablet formulations* Tablets Characteristics* Formulations H DT WV (RSD %) CU (RSD %) D T F 10 mg Epi Tablets 1.7 ± 0.3 16.3 ± 0.3 100.0 ± 0.0 (0.0) 100.6 ± 4.0 (4.0) 7.9 ± 0.0 3.5 ± 0.0 0.4 20 mg Epi Tablets 1.6 ± 0.1 15.8 ± 0.4 99.9 ± 0.7 (0.7) 97.7 ± 2.7 (2.7) 7.9 ± 0.0 3.9 ± 0.0 0.5 40 mg Epi Tablets 1.7 ± 0.2 31.3 ± 0.4 100.0 ± 0.6 (0.6) 95.6 ± 2.4 (2.5) 7.9 ± 0.0 3.4 ± 0.0 0.6 10 mg Epi MC Tablets 2.5 ± 0.0 5.5 ± 0.7 99.7 ± 1.2 (1.2) 92.9 ± 0.3 (0.3) 8.0 ± 0.1 3.7 ± 0.0 NA 20 mg Epi MC Tablets 2.5 ± 0.1 8.7 ± 0.3 98.3 ± 1.7 (1.7) 92.2 ± 4.2 (4.5) 8.0 ± 0.1 NA NA *H indicates tablet hardness (kgf); DT, disintegration time (sec); WV, weight variation (%); CU, content uniformity (%); RSD, relative standard deviation (%); D, tablet diameter (mm); T, tablet thickness (mm); F, Friability (%).
Manufacturing and Quality Control of Tablets for In Vivo Absorption Studies
(71) Additionally, five ODT formulations containing EpiBit equivalent to 0 mg and 40 mg Epi and EpiBit MC equivalent to 20 mg Epi were manufactured by direct compression. These tablets were formulated and manufactured using the same excipients and method in our previous studies.sup.15, 16. All tablet formulations were tested for tablet weight variation, drug content uniformity, and friability using the harmonized USP methods and criteria.sup.18, 20. Also, they were tested for disintegration time using a novel in vitro disintegration test developed to simulate the sublingual environment.sup.15, 16 Drug content was analyzed using a high performance liquid chromatography (HPLC) system with ultra violet (UV) detection (PerkinElmer, Waltham, Mass.) according to USP method for Epi injections.sup.19.
(72) These tablets did not contain lactose or bisulfite and met USP standards for tablet weight variation, content uniformity, and friability.sup.18, 20. They also disintegrated in less than 30 seconds.
(73) Methods for In Vitro and Ex Vivo Diffusion Studies
(74) The in vitro and ex vivo diffusion of EpiBit MC and EpiBit formulated into ODT were evaluated using static vertical jacketed Franz Cells with OD of 20 mm and reservoir volume of 20±1 mL (PermeGear Inc., Hellertown, Pa.). For in vitro diffusion studies, 7 Spectra/Por® dialysis membranes with 1000 Dalton MWCO (Spectrum Laboratories, Inc., Rancho Dominguez, Calif.) were used as the diffusion membranes. For ex vivo diffusion studies, sublingual mucosa (floor of the mouth) were excised from pigs and used as the diffusion membranes. Frozen pig's heads were obtained from a local abattoir and defrosted at room temperature. The porcine mucosa were excised by dissecting the sublingual mucosa and removing the underlying connective tissue using a scalpel and fine tweezers using established surgical technique. The excised mucosa were inspected for integrity and then frozen on aluminum foil at −20° C. until used (<4 weeks). The mucosal membranes were defrosted at room temperature before each experiment.
(75) Four ODT containing EpiBit equivalent to 10, 20, and 40 mg Epi or EpiBit MC equivalent to 10, and 20 mg Epi were tested in vitro and ex vivo. EpiBit equivalent to 10 mg Epi was dissolved in 1 mL of the diffusion medium and used as a control (n=4).
(76) The receptor chamber that has a magnetic stirrer was filled with phosphate buffer, pH 5.8 (saliva average pH), as the diffusion medium. Air bubbles were removed after mounting the membrane between the donor and receptor chambers and before the beginning of the experiment. 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 for 30 minutes from both sides before the experiment and were checked for any leaks.
(77) The tested tablet was placed at the center of the donor chamber on the membrane at T.sub.0 and 2 mL of the diffusion medium was added to facilitate tablet disintegration and dissolution. Aliquots, 200 μL, were withdrawn from the receptor chamber using 6 inch-long needles (Popper &Sons, Inc, New Hyde Park, N.Y.) and 1 mL syringes at 5, 10, 15, 20, 30, 45, 60, 75, and 90 min. The withdrawn volumes were replaced with fresh medium. Samples were transferred to HPLC vials for HPLC analysis using UV detector as described below.
(78) Epinephrine HPLC Analysis
(79) Samples from tablets for content uniformity test and from diffusion studies were analyzed for Epi content according to USP method for Epi injection analysis.sup.19 using HPLC system with UV detection (PerkinElmer, Waltham, Mass.). The calibration curve was linear over the range of 6.25 to 200.0 m/mL with correlation of coefficients (R.sup.2) of >0.99 (n=5). The coefficient of variation (RSD %) of the system reproducibility at concentrations of 6.25 and 200 m/mL (n=5 each) were 1.07% and 0.40%, respectively. The intra- and inter-assay RSD % were 0.40% and 0.70% (n=2) and 2.8% and 1.5% (n=3), respectively.
(80) Data Analysis
(81) The mean±SD cumulative diffused Epi per area (μg/cm.sup.2) and percentage of diffused Epi for each ODT formulation were calculated. The mean±SD Epi influx, J (μg/cm.sup.2/min), and lag time, tL (min), were calculated from the slope and the intercept with the x-axis of each graph (n=4). Also, Epi permeability, P (cm/min), was calculated by dividing J by Epi concentration in the donor chamber at T.sub.0. The area under the curve of diffused Epi per area, JAUC.sub.0-90 (μg/cm.sup.2/min); the maximum Epi diffused, Jmax (μg/cm.sup.2); and the time to reach Jmax, Tmax (min) 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.
(82) Results
(83) 1) The In Vitro Diffusion of Epinephrine Microcrystals Sublingual Tablets
(84) The mean±SD (n=4) cumulative diffused Epi per area and percentage of diffused Epi for each formulation through dialysis membrane are shown in Tables 4 and 5, and illustrated in
(85) TABLE-US-00004 TABLE 4 Mean ± SD (n = 4) cumulative diffused epinephrine per area (μg/cm.sup.2) for each formulation through dialysis membrane. Time (min) 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet 5 62.1 ± 9.3 99.2 ± 30.9 456.1 ± 130.5 735.8 ± 101.0 835.2 ± 107.8 10 183.9 ± 25.0 321.8 ± 153.9 1499.6 ± 694.8 1642.4 ± 370.1 1934.6 ± 391.7 15 329.5 ± 7.6 466.7 ± 123.4 1764.3 ± 337.7 2431.0 ± 659.0 3573.7 ± 240.0 20 436.2 ± 142.4 668.4 ± 262.0 2600.7 ± 996.2 3386.2 ± 770.8 4673.6 ± 833.3 30 606.3 ± 91.4 744.5 ± 223.4 3781.5 ± 1127.9 4112.5 ± 1235.6 5075.7 ± 625.2 45 731.9 ± 90.3 873.4 ± 339.0 3207.6 ± 1180.6 5085.0 ± 698.4 6504.1 ± 105.3 60 683.2 ± 201.9 1198.9 ± 288.5 3739.7 ± 1315.3 5325.4 ± 745.5 6421.7 ± 1041.7 75 876.3 ± 497.1 906.7 ± 364.6 4602.4 ± 857.2 6568.8 ± 755.3 7585.8 ± 1554.4 90 888.1 ± 149.7 1235.3 ± 419.9 4614.7 ± 824.0 6554.1 ± 804.0 7337.4 ± 725.6
(86) TABLE-US-00005 TABLE 5 Mean ± SD (n = 4) percentage of diffused epinephrine (%) for each formulation through dialysis membrane. Time (min) 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet 5 2.0 ± 0.3 3.1 ± 1.0 7.2 ± 2 11.6 ± 1.6 6.6 ± 0.8 10 5.8 ± 0.8 10.1 ± 4.8 23.5 ± 10.9 25.8 ± 5.8 15.2 ± 3.1 15 10.4 ± 2.5 14.7 ± 3.9 27.7 ± 5.3 38.2 ± 10.3 28.1 ± 1.9 20 13.8 ± 4.6 21.0 ± 8.2 40.8 ± 15.6 53.2 ± 12.1 36.7 ± 6.5 30 19.1 ± 2.9 23.4 ± 7.0 59.4 ± 17.7 64.6 ± 19.4 39.8 ± 4.9 45 23.1 ± 2.8 27.4 ± 10.6 50.4 ± 18.5 79.8 ± 11.0 51.1 ± 0.8 60 21.6 ± 6.4 37.6 ± 9.1 58.7 ± 20.7 83.6 ± 11.7 50.4 ± 8.2 75 27.7 ± 15.8 28.5 ± 11.4 72.3 ± 13.5 103.1 ± 11.9 59.5 ± 12.2 90 28.0 ± 4.7 38.8 ± 13.2 72.5 ± 12.9 102.9 ± 12.6 57.6 ± 5.7
(87) The mean (±SD) Epi JAUC.sub.0-90, Jmax, Tmax, J, P, and t.sub.1 are shown in Table 6. Also, Epi J and P for each formulation are illustrated in
(88) The mean (±SD) Epi JAUC.sub.0-90 and Jmax of 40 mg Epi tablets (484184.9±29655.9 μg/cm.sup.2/min and 7508.3±568.7 μg/cm.sup.2, respectively) and 20 mg Epi MC tablets (402852.2±55299 μg/cm.sup.2/min and 6727.2±736.3 μg/cm.sup.2, respectively) were not significantly different (p>0.05) from each other and were significantly higher (p<0.05) than the rest of the formulations (
(89) The mean (±SD) Epi J of 40 mg Epi tablets (234.2±99.6 μg/cm.sup.2/min) and 20 mg Epi MC tablets (172.2±49.8 μg/cm.sup.2/min) were not significantly different (p>0.05) from each other and were significantly higher (p<0.05) than the 10 mg Epi tablets and 10 mg Epi MC tablets (
(90) The mean (±SD) Epi P of 20 mg Epi MC tablets (17.2±5.0 cm/min) was significantly higher (p<0.05) than the rest of the formulations (
(91) TABLE-US-00006 TABLE 6 Mean ± SD (n = 4) of epinephrine JAUC.sub.0-90, Jmax, Tmax, J, P, and t.sub.L for each formulation through dialysis membrane. 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet JAUC.sub.0-90 54604.1 ± 11332.5 72461 ± 21229.2 292089 ± 58875.7 402852.2 ± 55299 484184.9 ± 29655.9 (μg/cm.sup.2/min) Jmax (μg/cm.sup.2) 1070.8 ± 384.2 1297.8 ± 305.3 5093.8 ± 249.5 6727.2 ± 736.3 7508.3 ± 568.7 Tmax (min) 78.8 ± 14.4 82.5 ± 15.0 71.3 ± 28.4 86.3 ± 7.5 82.5 ± 8.7 J (μg/cm.sup.2/min) 22.1 ± 4.1 37.0 ± 13.6 128.6 ± 39.2 172.2 ± 49.8 234.2 ± 99.6 P (cm/min) 4.4 ± 0.8 7.4 ± 2.7 12.9 ± 3.9 17.2 ± 5.0 11.7 ± 5.0 t.sub.L (min) 1.4 ± 0.9 2.0 ± 0.8 0.5 ± 1.0 0.0 ± 0.0 1.6 ± 1.4 JAUC.sub.0-90, area under the curve of diffused Epi per area versus time; Jmax, the maximum Epi diffused; Tmax, the time to reach Jmax; J, Epi influx; P, Epi permeability; t.sub.L, lag time.
(92) The JAUC, Jmax, J, P for 20 mg Epi MC tablets was not significantly different (p>0.05) from 40 mg Epi tablets in vitro. The reduction of EpiBit particles size close to the nano-size range increased EpiBit influx two folds, which presents a great potential for these reduced-sized Epi ODT to reduce the required Epi sublingual dose by half.
(93) 2) The Ex Vivo Diffusion of Epinephrine Microcrystals Sublingual Tablets
(94) The mean±SD (n=4) cumulative diffused Epi per area and percentage of diffused Epi for each formulation through sublingual mucosa are shown in Tables 7 and 8, and illustrated in
(95) TABLE-US-00007 TABLE 7 Mean ± SD (n = 4) cumulative diffused epinephrine per sublingual mucosa area (μg/cm.sup.2) for each formulation through sublingual mucosa. Time (min) 10 mg Epi Solution 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet 5 24.5 ± 8.7 16.8 ± 12.7 32.5 ± 27.4 40.2 ± 44.9 176.1 ± 128.7 156.6 ± 159.4 10 80.3 ± 26.5 72.5 ± 50.5 161.5 ± 80.8 124.5 ± 123.1 639.1 ± 469.1 622.5 ± 559.3 15 143.0 ± 40.5 182.3 ± 104.3 296.7 ± 110.0 232.5 ± 217.1 1211.1 ± 808.0 1147.4 ± 1023.4 20 198.9 ± 56.5 248.0 ± 116.9 401.2 ± 110.1 341.7 ± 302.1 1588.9 ± 998.6 1689.4 ± 1437.7 30 219.8 ± 70.2 288.7 ± 88.7 465.5 ± 101.1 525.7 ± 444.6 2161.7 ± 1285.2 2415.0 ± 1834.7 45 273.8 ± 96.2 341.0 ± 37.6 499.0 ± 88.7 664.9 ± 501.1 2628.4 ± 1496.8 3311.4 ± 2321.8 60 248.7 ± 60.5 364.3 ± 75.9 488.9 ± 86.8 898.1 ± 643.1 3037.6 ± 1574.1 3989.8 ± 2648.3 75 266.1 ± 73.4 390.0 ± 47.8 479.5 ± 80.0 1072.8 ± 733.2 3435.1 ± 1828.8 4464.8 ± 2928.8 90 277.2 ± 80.8 430.1 ± 100.1 478.4 ± 58.9 1263.1 ± 807.6 3496.3 ± 1722.8 4795.7 ± 2988.2
(96) TABLE-US-00008 TABLE 8 Mean ± SD (n = 4) percentage of diffused epinephrine (%) for each formulation through sublingual mucosa. Time (min) 10 mg Epi Solution 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet 5 1.1 ± 0.7 0.5 ± 0.4 1.0 ± 0.9 0.6 ± 0.7 2.8 ± 2.0 1.2 ± 1.3 10 3.1 ± 1.2 2.3 ± 1.6 5.1 ± 2.5 2.0 ± 1.9 10.0 ± 7.4 4.9 ± 4.4 15 5.0 ± 1.5 5.7 ± 3.3 9.3 ± 3.5 3.7 ± 3.4 19.0 ± 12.7 9.0 ± 8.0 20 6.5 ± 1.8 7.8 ± 3.7 12.6 ± 3.5 5.4 ± 4.7 24.9 ± 15.7 13.3 ± 11.3 30 7.1 ± 2.3 9.1 ± 2.8 14.6 ± 3.2 8.3 ± 7.0 33.9 ± 20.2 19.0 ± 14.4 45 8.7 ± 3.0 10.7 ± 1.2 15.7 ± 2.8 10.4 ± 7.9 41.3 ± 23.5 26.0 ± 18.2 60 8.0 ± 2.0 11.4 ± 2.4 15.4 ± 2.7 14.1 ± 10.1 47.7 ± 24.7 31.3 ± 20.8 75 8.5 ± 2.4 12.2 ± 1.5 15.1 ± 2.5 16.8 ± 11.5 53.9 ± 28.7 35.0 ± 23.0 90 8.6 ± 2.5 13.5 ± 3.1 15.0 ± 1.8 19.8 ± 12.7 54.9 ± 27.0 37.6 ± 23.5
(97) The mean (±SD) Epi JAUC.sub.0-90, Jmax, Tmax, J, P, and t.sub.L are shown in Table 9. Also, Epi J and P for each formulation are illustrated in
(98) The mean Epi JAUC.sub.0-90 and Jmax of 40 mg Epi tablets (264556.4±182820.3 μg/cm.sup.2/min and 4795.7±2988.2 μg/cm.sup.2, respectively) and 20 mg Epi MC tablets (211368.5±116025.1 μg/cm.sup.2/min and 3526.8±1754.6 μg/cm.sup.2, respectively) were not significantly different (p>0.05) from each other and 40 mg Epi tablets was significantly higher (p<0.05) than the rest of the formulations (
(99) The Epi J of 40 mg Epi tablets (106.0±82.4 μg/cm.sup.2/min) and 20 mg Epi MC tablets (91.1±54.6 μg/cm.sup.2/min) were not significantly different (p>0.05) from each other but due to the high variability there were not significantly different (p>0.05) form 20 mg Epi tablets (19.9±16.0 μg/cm.sup.2/min) and 10 mg Epi tablets (24.8±6.5 μg/cm.sup.2/min) as well (
(100) The Epi P of 20 mg Epi MC tablets (9.1±5.5 cm/min) and 40 mg Epi tablets (5.3±4.1 cm/min) were not significantly different (p>0.05) from each other and 20 mg Epi MC tablets was significantly higher (p<0.05) than 20 mg Epi tablets (2.0±1.6 cm/min) (
(101) All the diffusion parameters for both 10 mg Epi solution and 10 mg Epi ODT (Table 9) were not significantly different (p>0.05) from each other.
(102) TABLE-US-00009 TABLE 9 Mean ± SD (n = 4) of epinephrine JAUC.sub.0-90, Jmax, Tmax, J, P, and t.sub.L for each formulation through sublingual mucosa. 10 mg Epi Solution 10 mg Epi Tablet 10 mg Epi MC Tablet 20 mg Epi Tablet 20 mg Epi MC Tablet 40 mg Epi Tablet JAUC.sub.0-90 19325.8 ± 5599.3 26441.6 ± 5651.6 36799.7 ± 7226.5 60031.0 ± 43809.8 211368.5 ± 116025.1 264556.4 ± 182820.3 (μg/cm.sup.2/min) Jmax (μg/cm.sup.2) 236.4 ± 101.9 436.7 ± 96.9 507.2 ± 81.4 1263.1 ± 807.6 3526.8 ± 1754.6 4795.7 ± 2988.2 Tmax (min) 75.0 ± 21.2 86.3 ± 7.5 48.8 ± 18.9 90.0 ± 0.0 82.5 ± 8.7 90.0 ± 0.0 J (μg/cm.sup.2/min) 11.7 ± 3.2 17.1 ± 6.7 24.8 ± 6.5 19.9 ± 16.0 91.1 ± 54.6 106.0 ± 82.4 P (cm/min) 2.3 ± 0.6 3.4 ± 1.3 5.0 ± 1.3 2.0 ± 1.6 9.1 ± 5.5 5.3 ± 4.1 t.sub.L (min) 2.9 ± 0.4 5.8 ± 2.0 3.6 ± 1.5 5.1 ± 2.8 3.0 ± 2.4 5.2 ± 2.3 JAUC.sub.0-90, area under the curve of diffused Epi per area versus time; Jmax, the maximum Epi diffused; Tmax, the time to reach Jmax; J, Epi influx; P, Epi permeability; t.sub.L, lag time.
(103) The JAUC, Jmax, J, P for 20 mg Epi MC tablets was not significantly different (p>0.05) from 40 mg Epi tablets. The reduction of EpiBit particles size close to the nano-size range increased EpiBit influx two folds, which presents a great potential for these reduced-sized Epi ODT to reduce the required Epi sublingual dose by half.
(104) In Vivo Absorption Studies
(105) The research was conducted according to current guidelines published by the Canadian Council on Animal Care.sup.21 and was approved by the University of Manitoba Protocol Management and Review Committee.
(106) Methods
(107) Using a prospective, placebo-controlled, randomized, crossover study design, six New Zealand female white rabbits (mean±SD weight 3.6±0.1 Kg) were investigated on different study days at least four weeks apart, using a protocol described previously.sup.10, 11. Each rabbit received sublingually either Epi 40 mg, Epi MC 20 mg ODT, or placebo ODT (as a negative control). Epi 0.3 mg IM injection was given in the rabbit's thigh muscle from an EpiPen® as a positive control.
(108) For the sublingual administration of tablets, the rabbit's mouth was opened using speculum and the tablet was placed underneath the tongue using a pair of flat forceps. A 0.1-0.2 mL volume of water was administered immediately after dosing to facilitate tablet disintegration. The rabbit's tongue was gently pressed for 2 minutes to prevent the rabbit from chewing or swallowing the tablet. At the end of the 2-minute immobilization time, the mouth was rinsed with 30-40 mL of water, in order to remove any insoluble tablet residue from the oral cavity.
(109) Epi 0.3 mg was injected IM in the thigh using an EpiPen®, after which the solution remaining in the EpiPen® was evacuated into a plastic tube and frozen at −20° C., to be analyzed for Epi content using a reverse phase high performance liquid chromatography (HPLC) system (Waters Corp., Milford, Mass.) with ultra violet detection (UV) according USP method.sup.19.
(110) Measurement of Plasma Epinephrine Concentrations
(111) An indwelling catheter (22 G 1″, BD, Ontario, Canada) was inserted into an ear artery at least 30 minutes before dosing. A 2 mL blood sample was withdrawn immediately before dosing and at 5, 10, 15, 20, 30, 40, and 60 minutes afterwards.
(112) All collected blood samples were transferred into Vacutainer plasma separation tubes containing EDTA (BD, Ontario, Canada), refrigerated within 1 hour of sampling, and centrifuged at 1600 g, 4° C. Plasma were transferred into appropriately labeled polypropylene tubes, and stored at −20° C. until analysis. Before analysis, plasma was thawed at room temperature and Epi was extracted by a solid-liquid extraction process, with an efficiency of 78%-83%. Epi concentrations were measured using HPLC system (Waters Corp., Milford, Mass.) with electrochemical detection (EC).sup.22-24. Two calibration curves with two different Epi concentration ranges were prepared. The low range calibration curve was linear over the range of 0.1 to 1.0 ng/ml with a coefficient of variation of 0.4% at 0.1 ng/ml and 0.1% at 1.0 ng/ml. The high range calibration curve was linear over the range of 1.0 to 10.0 ng/ml with a coefficient of variation of 0.1% at 1.0 ng/ml and 0.1% at 10.0 ng/ml.
(113) Data Analysis
(114) The maximum plasma Epi concentration (C.sub.max), the time at which C.sub.max was achieved (T.sub.max), and the area under the plasma concentration versus time curves (AUC) were calculated from the plasma Epi concentration versus time plots of each individual rabbit using WinNonlin® 5.3 (Pharsight, Mountain View, Calif.). The AUC, C.sub.max, and T.sub.max values for each rabbit were compared using ANOVA, ANCOVA and Tukey-Kramer multiple comparison tests using NCSS Statistical Analysis Software (NCSS, Kaysville, Utah). Differences were considered to be significant at p<0.05.
(115) Results
(116) The mean (±SD) of Epi dose injected using EpiPen® auto-injectors was 0.29±0.02 mg as calculated by multiplying the Epi concentration, measured in the solution remaining in the EpiPen® after injection, by the stated injected volume (0.3 mL).
(117) Mean (±SD) plasma Epi concentration versus time plots after the sublingual administration of placebo ODT, Epi 40 mg ODT, and Epi MC 20 mg ODT, and the IM injection of Epi 0.3 mg using EpiPen® are shown in
(118) Mean (±SD) AUC after the administration of Epi MC 20 mg ODT (942.0±243.7 ng/ml/min), Epi 40 mg ODT (678.0±149.0 ng/ml/min), and Epi 0.3 mg IM (592.0±122.3 ng/ml/min) did not differ significantly, but were significantly higher than after placebo ODT (220.1±78.0 ng/ml/min).
(119) Mean (±SD) C.sub.max values after Epi MC 20 mg ODT (38.0±9.9 ng/ml), Epi 40 mg ODT (31.7±10.1 ng/ml) and Epi 0.3 mg IM (27.6±7.0 ng/ml) did not differ significantly, but were significantly higher than after placebo ODT (7.5±3.0 ng/ml).
(120) Mean (±SD) T.sub.max after the sublingual administration of placebo ODT (33.3±17.5 min), Epi MC 20 mg ODT (28.0±29.3 min), and Epi 40 mg ODT (20.0±7.1 min), and IM injection of Epi 0.3 mg (30.0±0.0 min) did not differ significantly.
(121) TABLE-US-00010 TABLE 10 Epinephrine bioavailability after sublingual administration of placebo, epinephrine and epinephrine nanocrystals tablets and epinephrine intramuscular injection in the thigh. Sublingual ODT IM Injection Mean ± SD* Placebo 40 mg Epi 20 mg Epi MC EpiPen ® Epinephrine dose (mg) 0 40.0 20.0 0.3 AUC (ng/ml/min) 220.1 ± 78.0 678.0 ± 149.0† 942.0 ± 243.7† 592.0 ± 122.3† C.sub.baseline (ng/ml) 1.1 ± 1.2 5.0 ± 3.0 2.9 ± 1.6 5.6 ± 1.9‡ C.sub.max (ng/ml) 7.5 ± 3.0 31.7 ± 10.1† 38.0 ± 9.9† 27.6 ± 7.0† T.sub.max (min) 33.3 ± 17.5 20.0 ± 7.1 28.0 ± 29.3 30.0 ± 0.0 *n = 5 †p < 0.05 from placebo tablet but not from each others. ‡p < 0.05 from placebo tablet but not from others. AUC: area under the plasma concentration versus time curve; C.sub.baseline: Baseline plasma concentration (endogenous epinephrine); C.sub.max: maximum plasma concentration (mean ± SD of individual C.sub.max values from each rabbit, regardless of the time at which C.sub.max was achieved); T.sub.max: time at which maximum plasma epinephrine concentration was achieved (mean ± SD of individual T.sub.max values from each rabbit).
Discussion of Experiments
(122) Previously, the Epi was delivered sublingually using rabbit's animal model. It was determined that 40 mg Epi, using EpiBit, is the bioequivalent sublingual dose using the novel ODT tablets.sup.15, 16 to the recommended IM injection of 0.3 mg Epi given in the thigh muscle for adults.sup.10, 11. Also, the ODT formulations were developed to taste mask the bitter taste of Epi.sup.25 and this ODT formulation was evaluated using electronic tongue.sup.14. This new taste-masked, sublingually administered 40 mg Epi ODT formulation was bioequivalent to 0.3 mg Epi IM injection as well.sup.26.
(123) In order to enhance the sublingual bioavailability of Epi, the particles size of EpiBit crystals were reduced up to 55 folds. Significant reduction in the drug particles' size results in increasing the saturation solubility, which increases the concentration gradients that promotes absorption, and dissolution rate of the drug that will ultimately increase its bioavailability, thus, resulting in a significant reduction in the required dose and any associated side effects.sup.17, 23. This is particularly important for the sublingual drug delivery due to the small saliva volume available for drug dissolution and the short sublingual residence time compared to the GIT.
(124) Despite that the aim was to reduce the particles size of EpiBit to the nano-size (1000 nm or less), the size was reduced to a range that is very close to the nano-size range. It was very challenging to reach to a nanoosize range while not using a surfactant, which may need to be evaluated later, and by processing EpiBit for only one cycle to reduce any potential stress on EpiBit that can influence its stability.sup.28. The concentration of EpiBit suspension, the pressure applied, and the number of cycles were optimized to obtain the smallest particle size range with the lowest possible number of cycles.
(125) The FT-IR spectra of EpiBit before and after processing for one cycle using Microfluidizer, LV-1, were similar, which indicates for the stability of the EpiBit during the particles size reduction process under these processing conditions (
(126) The DSC spectra of EpiBit before and after processing were also similar with a single endothermic peak around 157° C. that indicates for the absence of any change in the purity and crystallinity of EpiBit (
(127) The Scanning Electron Microscopy (SEM) images (
(128) The diffusion studies were conducted using dialysis membranes initially and then by using excised porcine sublingual mucosal membranes. It has been already established that the sublingual mucosa of pigs and rabbits are very similar to the human sublingual mucosa and were previously used for similar studies.sup.29, 30. Therefore, pigs' sublingual mucosa was selected for these diffusion studies and rabbits were always been selected in our previous studies for in vivo studies.sup.10, 11, 26. The sublingual mucosa of pigs has bigger surface area that is easy to be excised surgically for ex vivo studies and rabbits are easier to handle and house for in vivo studies.
(129) Results from both in vitro and ex vivo experiments were highly correlated, (R.sup.2≥87) (
(130) The significant reduction of the particles size of EpiBit increased its influx two folds, which presents a great potential for these micro-sized Epi ODT to reduce the required Epi sublingual dosed by half. Animal studies in rabbits have shown similar results.
(131) This study demonstrates that reducing the particles size of EpiBit to almost to the nano-size range improved its diffusion from rapidly-disintegrating tablet formulation (ODT) by two folds. These micro-sized Epi ODT tablets have the potential to reduce the bioequivalent dose of sublingually administered Epi by 50%.
(132) 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, epinephrine fine particles, epinephrine nanoparticles, epinephrine nanocrystals, epinephrine microparticles, epinephrine microcrystal s, pharmaceutical tablets, pharmaceutically-effective doses of epinephrine nanoparticles or nanocrystals or epinephrine microparticles or microcrystals, 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.
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