U.S. patent application number 13/582346 was filed with the patent office on 2012-12-20 for epinephrine nanoparticles, methods of fabrication thereof, and methods for use thereof for treatment of conditions responsive to epinephrine.
This patent application is currently assigned to UNIVERSITY OF MANITOBA. Invention is credited to Enrique Nieves, Ousama Rachid, Mutasem Rawas-Qalaji, Frances Estelle Reed Simons, Keith John Simons.
Application Number | 20120322884 13/582346 |
Document ID | / |
Family ID | 44542526 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120322884 |
Kind Code |
A1 |
Rawas-Qalaji; Mutasem ; et
al. |
December 20, 2012 |
EPINEPHRINE NANOPARTICLES, METHODS OF FABRICATION THEREOF, AND
METHODS FOR USE THEREOF FOR TREATMENT OF CONDITIONS RESPONSIVE TO
EPINEPHRINE
Abstract
The invention provides a composition including epinephrine
nanoparticles and methods for therapeutic use of the composition in
the treatment of conditions responsive to epinephrine such as a
cardiac event or an allergic reaction, particularly anaphylaxis.
The epinephrine nanoparticles 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. Additionally, the invention provides methods for
fabrication of stabilized epinephrine nanoparticles for use in the
described compositions.
Inventors: |
Rawas-Qalaji; Mutasem; (Fort
Lauderdale, FL) ; Nieves; Enrique; (Fort Lauderdale,
FL) ; Simons; Keith John; (Winnipeg, CA) ;
Reed Simons; Frances Estelle; (Winnipeg, CA) ;
Rachid; Ousama; (Winnipeg, CA) |
Assignee: |
UNIVERSITY OF MANITOBA
Winnipeg, Manitoba
FL
NOVA SOUTHEASTERN UNIVERSITY
Fort Lauderdale
|
Family ID: |
44542526 |
Appl. No.: |
13/582346 |
Filed: |
March 1, 2011 |
PCT Filed: |
March 1, 2011 |
PCT NO: |
PCT/US11/26604 |
371 Date: |
August 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61309136 |
Mar 1, 2010 |
|
|
|
Current U.S.
Class: |
514/653 ;
977/773; 977/788; 977/900; 977/915 |
Current CPC
Class: |
A61K 9/006 20130101;
A61K 9/146 20130101; A61P 11/00 20180101; A61P 11/06 20180101; A61K
31/137 20130101; A61P 37/08 20180101; A61K 9/14 20130101; A61P 9/00
20180101; A61K 9/19 20130101 |
Class at
Publication: |
514/653 ;
977/773; 977/788; 977/900; 977/915 |
International
Class: |
A61K 31/137 20060101
A61K031/137; A61P 37/08 20060101 A61P037/08; A61P 11/00 20060101
A61P011/00; A61P 9/00 20060101 A61P009/00; A61P 11/06 20060101
A61P011/06 |
Claims
1. A pharmaceutical composition comprising stabilized epinephrine
nanoparticles and at least one pharmaceutically-acceptable carrier,
the composition formulated for buccal or sublingual
administration.
2-3. (canceled)
4. A method for fabricating stabilized epinephrine nanoparticles,
comprising: a) combining a pre-determined amount of epinephrine and
a solvent in a reaction chamber to form a mixture; and b) exposing
the mixture to at least one pass at a pre-determined pressure and a
pre-determined temperature.
5. The method in accordance with claim 4, wherein the epinephrine
is an epinephrine base or an epinephrine bitartrate salt.
6. The method in accordance with claim 4, wherein the solvent is
selected from the group consisting of water, isopropyl alcohol
(ISP), methanol, acetonitrile, acetone, hexan, chloroform,
dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate,
phosphoric acid, acetic acid, and sodium metabisulfite.
7. The method in accordance with claim 4, wherein the
pre-determined pressure ranges from about 8,000 psi to 30,000
psi.
8. The method in accordance with claim 4, wherein the
pre-determined temperature ranges from about 8.3 to 43.3.degree.
C.
9. The method in accordance with claim 4, further including
exposing the mixture to a second pass at a different pre-determined
pressure and a different pre-determined temperature from that of
the first pass.
10. The method in accordance with claim 4, further including
lyophilizing nanoparticles obtained by carrying out steps a) and
b).
11. A stabilized epinephrine nanoparticle produced in accordance
with the method of claim 10.
12. A pharmaceutical composition comprising the stabilized
epinephrine nanoparticle of claim 11 and at least one of a
pharmaceutically-acceptable carrier, a penetration enhancer, and a
mucoadhesive, the composition formulated for buccal or sublingual
administration.
13-14. (canceled)
15. A method for treating a condition responsive to epinephrine in
a subject in need thereof, comprising: providing a composition
including epinephrine nanoparticles and a
pharmaceutically-acceptable carrier; and administering the
composition to the subject.
16. The method in accordance with claim 15, wherein the condition
is a cardiac event or an allergic reaction.
17. The method in accordance with claim 16, wherein the cardiac
event is cardiac arrest and the allergic reaction is anaphylaxis,
asthma, or bronchial asthma.
18-33. (canceled)
34. The pharmaceutical composition in accordance with claim 1,
further comprising at least one of a penetration enhancer and a
mucoadhesive.
35. The method in accordance with claim 16, wherein the cardiac
event is cardiac arrest.
36. The method in accordance with claim 15, wherein treating
includes enhancing sublingual bioavailability of epinephrine.
37. The method in accordance with claim 15, wherein the condition
is a breathing difficulty.
38. The method in accordance with claim 37, wherein the breathing
difficulty is associated with anaphylaxis, asthma, bronchial
asthma, bronchitis, emphysema, or respiratory infections.
39. The method in accordance with claim 15, wherein the condition
is an allergic emergency.
40. The method in accordance with claim 39, wherein the allergic
emergency is associated with anaphylaxis, asthma, or bronchial
asthma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 61/309,136, filed on Mar. 1,
2010, the content of which is hereby incorporated by reference in
its entirety.
[0002] This application is related to U.S. Provisional Patent
Application No. 60/715,180, filed on Sep. 9, 2005, and U.S.
Provisional Patent Application No. 60/759,039, filed on Jan. 17,
2006. This application is also related to U.S. Utility patent
application Ser. No. 11/672,503, filed on Feb. 7, 2007, now
abandoned, which is a continuation-in-part of U.S. Utility patent
application Ser. No. 11/530,360, filed on Sep. 8, 2006, now
abandoned. The aforementioned provisional and utility applications
are hereby incorporated by reference in their entireties. The
information incorporated is as much a part of the instant
application as filed as if the text was repeated in the
application, and should be treated (the incorporated information)
as part of the text of the application as filed.
FIELD OF THE INVENTION
[0003] The invention generally relates to compositions and methods
for treatment of conditions responsive to epinephrine (also known
as adrenaline), particularly to compositions and methods for
emergency treatment of conditions responsive to epinephrine, and
most particularly to compositions including epinephrine
nanoparticles for sublingual administration in treatment of
conditions responsive to epinephrine.
BACKGROUND
[0004] Tablets that disintegrate or dissolve rapidly in the
patient's mouth without the use of water are convenient for the
elderly, young children, patients with swallowing difficulties, and
in situations where water is not available. For these specially
designed formulations, the small volume of saliva that is available
is sufficient to disintegrate or dissolve a tablet in the oral
cavity. The drug released from these tablets can be absorbed
partially or entirely into the systemic circulation from the buccal
mucosa or sublingual cavity, or can be swallowed as a solution to
be absorbed from the gastrointestinal tract.
[0005] The sublingual route usually produces a faster onset of
action than traditional orally administered tablets and the portion
absorbed through the sublingual blood vessels bypasses the hepatic
first pass metabolic processes (Birudaraj et al., 2004, J Pharm Sci
94; Motwani et al., 1991, Clin Pharmacokinet 21: 83-94; Ishikawa et
al., 2001, Chem Pharm Bull 49: 230-232; Price et al., 1997, Obstet
Gynecol 89: 340-345; Kroboth et al., 1995, J Clin Psychopharmacol
15: 259-262; Cunningham et al., 1994, J Clin Anesth 6: 430-433;
Scavone et al., 1992, Eur J Clin Pharmacol 42: 439-443; Spenard et
al., 1988, Biopharm Drug Dispos 9: 457-464).
[0006] Likewise, due to high buccal vascularity, buccally-delivered
drugs can gain direct access to the systemic circulation and are
not subject to first-pass hepatic metabolism. In addition,
therapeutic agents administered via the buccal route are not
exposed to the acidic environment of the gastrointestinal tract
(Mitra et al., 2002, Encyclopedia of Pharm. Tech., 2081-2095).
Further, the buccal mucosa has low enzymatic activity relative to
the nasal and rectal routes. Thus, the potential for drug
inactivation due to biochemical degradation is less rapid and
extensive than other administration routes (de Varies et al., 1991,
Crit. Rev. Ther. Drug Carr. Syst. 8: 271-303).
[0007] The buccal mucosa is also highly accessible, which allows
for the use of tablets which are painless, easily administered,
easily removed, and easily targeted. Because the oral cavity
consists of a pair of buccal mucosa, tablets, such as fast
disintegrating tablets, can be applied at various sites either on
the same mucosa or, alternatively, on the left or right buccal
mucosa (Mitra et al., 2002, Encyclopedia of Pharm. Tech.,
2081-2095). In addition, the buccal route could be useful for drug
administration to unconscious patients, patients undergoing an
anaphylactic attack, or patients who sense the onset of an
anaphylactic attack.
[0008] Epinephrine (EP) is the drug of choice for the treatment of
anaphylaxis worldwide (Joint Task Force on Practice Parameters,
2005, J Allergy Clin Immunol 115: S483-S523; Lieberman, 2003, Curr
Opin Allergy Clin Immunol 3: 313-318; Simons, 2004, J Allergy Clin
Immunol 113: 837-844). It is available only as an injectable dosage
form in ampoules or in autoinjectors. In aqueous solutions,
epinephrine is unstable in the presence of light, oxygen, heat, and
neutral or alkaline pH values (Connors et al., 1986, in Chemical
Stability of Pharmaceuticals: A Handbook for Pharmacists,
Wiley-Interscience Publication: New York). Feasibility studies in
humans and animals have shown that EP can be absorbed sublingually
(Gu et al., 2002, Biopharm Drug Dispos 23: 213-216; Simons et al.,
2004, J Allergy Clin Immunol 113: 425-438). The recommended dose of
EP for the treatment of anaphylaxis is about 0.01 mg/Kg: usually
about 0.2 mL to about 0.5 mL of a 1:1000 dilution of EP in a
suitable carrier. Based on historical and anecdotal evidence, an
approximately 0.3 mg dose of EP, by subcutaneous (SC) or
intramuscular (IM) injection into the deltoid muscle, has been
agreed upon as the dose required for the emergency treatment of
anaphylaxis. Recent studies have demonstrated that if the
approximately 0.3 mg dose is administered IM into the laterus
vascularis (thigh) muscle, EP plasma concentrations are higher and
occur more quickly than SC or IM administration into the deltoid
muscle. (Joint Task Force on Practice Parameters, 2005, J Allergy
Clin Immunol 115: S483-S523; Lieberman, 2003, Curr Opin Allergy
Clin Immunol 3: 313-318; Simons, 2004, J Allergy Clin Immunol 113:
837-844)).
[0009] As stated above, epinephrine (EP) is typically administered
either subcutaneously or intramuscularly by injection. Thus, EP
injections are the accepted first aid means of delivering EP and
are administered either manually or by automatic injectors. It is
recommended that persons at risk of anaphylaxis, and persons
responsible for children at risk for anaphylaxis, maintain one or
more automatic EP injectors in a convenient place at all times.
[0010] Given the difficulties associated with manual subcutaneous
or intramuscular administration of EP, such as patient apprehension
related to injections or the burden of an at risk person having to
always maintain an EP injector close at hand, there exists a need
in the art for more convenient dosage forms which can provide
immediate administration of EP, particularly to a person undergoing
anaphylaxis wherein the need for injection or EP injectors is
obviated.
[0011] Recently, a novel fast-disintegrating tablet suitable for
sublingual (SL) administration was developed. See related U.S.
applications: U.S. Provisional Patent Application No. 60/715,180;
U.S. Provisional Patent Application No. 60/759,039; U.S. Utility
patent application Ser. No. 11/672,503; and U.S. Utility patent
application Ser. No. 11/530,360. Sublingual administration of 40 mg
epinephrine as the bitartrate salt using these novel tablets
resulted in a rate and an extent of epinephrine absorption similar
to that achieved following intramuscular injections of 0.3 mg
epinephrine in the thigh. SL doses ranging from 5 to 40 mg
epinephrine as the bitartrate salt were studied to achieve
equivalent plasma concentrations.
[0012] Without being bound by theory, it is thought that
fabrication of epinephrine into nanoparticles and incorporation of
the nanoparticles into a tablet formulation with
pharmaceutically-acceptable carriers, penetration enhancers, and
mucoadhesives will significantly increase the absorption of
SL-administered epinephrine and will result in the reduction of SL
epinephrine dose required.
SUMMARY OF THE INVENTION
[0013] The invention provides a composition, including epinephrine
nanoparticles, capable of enhancing the sublingual bioavailability
of epinephrine, particularly in the emergency treatment of
anaphylaxis.
[0014] The invention additionally provides a method for fabrication
of stabilized epinephrine nanoparticles and incorporation of the
fabricated nanoparticles into orally-disintegrating and
fast-disintegrating tablets.
[0015] The invention also provides a pharmaceutical composition
including epinephrine nanoparticles and at least one of a
pharmaceutically-acceptable carrier, penetration enhancers, and
mucoadhesives for buccal or sublingual administration.
[0016] The invention additionally provides a method for treatment
of an allergic emergency comprising the administration of a
pharmaceutical composition including epinephrine nanoparticles to a
patient diagnosed with or suspected of having an allergic
emergency. The allergic emergency can be anaphylaxis, asthma, or
bronchial asthma.
[0017] The invention also provides a method for treatment of a
cardiac event comprising the administration of a pharmaceutical
composition including epinephrine nanoparticles to a patient
diagnosed with or suspected of having a cardiac event. The cardiac
event can be cardiac arrest.
[0018] As described herein, buccal or sublingual oral
disintegrating tablets (ODTs) are distinguished from conventional
sublingual tablets, lozenges, or buccal tablets by the ODTs'
ability to fully dissolve or disintegrate in less than about one
minute in the mouth.
[0019] Other objectives and advantages of this invention will
become apparent from the following description taken in conjunction
with the accompanying drawings, wherein are set forth, by way of
illustration and example, certain embodiments of this invention.
The drawings constitute a part of this specification and include
exemplary embodiments of the present invention and illustrate
various objects and features thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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.
[0021] FIG. 1 is a graph showing mean epinephrine influx
(.mu.g/cm.sup.2/hr) obtained from the tested formulations;
epinephrine nanoparticles suspension (EP-NP Susp) (size 200 nm),
epinephrine solution (Epi-HBCD Sol), epinephrine suspension
(Epi-CMC Susp), and epinephrine bitartrate solution (Epi Bit
Sol).
[0022] FIG. 2A is a Fourier Transform Infrared (FT-IR) spectrum for
epinephrine base nanoparticles after fabrication (processing).
[0023] FIG. 2B is a FT-IR spectrum for epinephrine base
nanoparticles before processing.
[0024] FIG. 3 illustrates particle size distribution of epinephrine
base measured before size reduction (processing) using
Mastersizer.
[0025] FIG. 4 illustrates particle size distribution of epinephrine
base measured after size reduction using NiComp 370.
[0026] FIG. 5 is a FT-IR spectrum for epinephrine bitartrate
nanoparticles before and after processing (nanoparticle
fabrication).
[0027] FIG. 6A is a graph showing the AUC (mean cumulative
epinephrine concentration) (.mu.g/ml) obtained from the four tested
formulations; EP-NP Susp, Epi-CMC Susp, Epi-HBCD Sol, and Epi Bit
Sol.
[0028] FIG. 6B is a graph showing mean epinephrine influx
(.mu.g/cm.sup.2/hr) obtained from the tested formulations; EP-NP
Susp, Epi-CMC Susp, Epi-HBCD Sol, and Epi Bit Sol.
DETAILED DESCRIPTION OF THE INVENTION
[0029] 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.
Summary of In Vitro Diffusion Experiments and Results
[0030] The experiments described herein were carried out to assess
the in vitro diffusion of epinephrine nanoparticles. The use of
epinephrine nanoparticles instead of epinephrine salt was
hypothesized to enhance the sublingual bioavailability of
epinephrine from administration of a fast-disintegrating sublingual
tablet formulation for the emergency treatment of anaphylaxis
and/or treatment of other conditions responsive to epinephrine.
Methods:
[0031] The diffusion of 80 .mu.g epinephrine from four
formulations, epinephrine base nanoparticles suspension (Epi-NP
Susp) (size 200 nm), epinephrine solution (Epi-HBCD Sol);
epinephrine base using hydroxypropyl-.beta.-cyclodetrin as a
solubilizing agent, epinephrine suspension (Epi-CMC Susp);
epinephrine base using 0.3% carboxymethyl cellulose as a suspending
agent, and epinephrine bitartrate solution (Epi Bit Sol), was
studied over 8.5 hours using automated flow-through Franz cell
system (n=6). Cumulative epinephrine concentrations in the donor
cells were measured using HPLC-UV (High Performance Liquid
Chromatography system with an ultraviolet detector). The cumulative
epinephrine concentration versus time (AUC), maximum epinephrine
flux (J.sub.max), time to reach Jmax (Jt.sub.max), and epinephrine
permeation coefficient (Kp) for each formulation were calculated
and statistically analysed using one-way ANOV and Tukey-Kramer
tests, NCSS program, at a level of significance p<0.05.
Results:
[0032] The AUC and Jmax obtained from epinephrine nanoparticles
(Epi-NP Susp), 10.4.+-.1.7 .mu.g/ml/hr and 15.1.+-.1.9
.mu.g/cm.sup.2/hr respectively, were significantly higher than
epinephrine suspension (Epi-CMC Susp), 5.1.+-.1.1 .mu.g/ml/hr and
10.4.+-.1.6 .mu.g/cm.sup.2/hr, epinephrine solution (Epi-HBCD Sol),
5.5.+-.0.5 .mu.g/ml/hr and 8.6.+-.0.3 .mu.g/cm.sup.2/hr, and
epinephrine bitartrate (Epi Bit Sol), 4.6.+-.0.9 .mu.g/ml/hr and
7.9.+-.1.0 .mu.g/cm.sup.2/hr. Jt.sub.max was not significantly
different between the four formulations. The Kp of epinephrine
nanoparticles, 0.19.+-.0.07 cm/hr was significantly higher than
epinephrine suspension, 0.13.+-.0.002 cm/hr, epinephrine solution,
0.11.+-.0.04 cm/hr, and epinephrine bitartrate, 0.10.+-.0.04 cm/hr.
These results are illustrated in the graph of FIG. 1.
Conclusions:
[0033] In these experiments, the permeation of epinephrine
nanoparticles (Epi-NP Susp) was almost 2 folds higher than the
epinephrine bitartrate (Epi Bit Sol) and epinephrine solution
(Epi-HBCD Sol). Epinephrine nanoparticles may have the potential to
enhance the sublingual bioavailability of epinephrine compared to
epinephrine salt in sublingual tablet formulation. Ex vivo and in
vivo studies are contemplated and will be pursued to confirm these
results.
Details of Fabrication Experiments and Results
Fabrication of Nanoparticles
[0034] Nanoparticles were fabricated from epinephrine base and
epinephrine bitartrate (Bit) using high energy fluidization
(microfluidization) techniques. These techniques involve the use of
oversaturated solutions of various solvents, particularly water and
isopropanol, at various temperatures and pressures ranging from
about 8,000 psi to 30,000 psi and to about 8.3.degree. to
43.3.degree. C. under various passes. Particle size was measured
before and after size reduction using a Mastersizer (Malvern)
and/or a NiComp 370 Submicron Particle Sizer (NiComp) and
nano-sized particles were confirmed using laser diffraction
techniques. The particles were lyophilized (freeze-dried) using a
bench top lyophilizer (ART Inc.).
Solubility Studies
[0035] In order to determine suitable vehicles to suspend
epinephrine base and epinephrine bitartrate (Bit) for nanoparticle
fabrication, solubility studies were carried out.
TABLE-US-00001 TABLE 1 Solubility Amount Sample Name Dissolved %
Epinephrine Base solubility in water 2.67 Epinephrine Bit
solubility in methanol 10.45 Epinephrine Bit solubility in
isopropyl alcohol 0.62 Epinephrine Bit solubility in acetonitrile
0.77 Epinephrine Bit solubility in acetone 1.56 Epinephrine Bit
solubility in hexane 0.03 Epinephrine Bit solubility in choloroform
0.09 Epinephrine Bit solubility in dichloromethane (DCM) 0.00
Epinephrine Bit solubility in tetrahydrofuran (THF) 7.76
Epinephrine Bit solubility in ethyl acetate 0.63
Fabrication of nanoparticles was first attempted using epinephrine
base.
Fabrication: Epinephrine Base
TABLE-US-00002 [0036] TABLE 2 Epinephrine Base Pressure Particle
Size Sample Sample Concentration (psi) Distribution Temperature
Color after Sample Solvent (mg/ml) (#passes) (nm) (NiComp)
(.degree. C.) Processing 1 water 0.3 30,000 273.9 43.3 brown 2
water 0.308 29,000 334 18.3 brown (1) 3 0.1% phosphoric 1.03 15,000
335 36.8 first pass brown acid (2) 41.1 second pass 4 1M acetic
Acid 1.55 8,500 392 36.6 first pass brown (1) 38.4 second 15,000
(1) pass 5 water 4.03 15,000 905.9 10 brown 6 0.1 mM sodium 4.02
15,000 903.1 8.3 brown metabisulfite in water 7 0.1 mM sodium 12.02
15,000 903.1 8.3 pink metabisulfite in Note: 111.5 nm 0.1M
perchloric (80)%) and 2.2 nm acid (20%) using Zetasizer machine 8
water 10.0 8,000 447 (1) 15,000 (1)
[0037] Nanoparticles of epinephrine base in various sizes were
produced ranging in diameter from about 273.9 to 905.9 nm.
First Sample
[0038] The sample consisted of 30 mg epinephrine in 100 ml of
distilled water. One pass at 30,000 psi was applied and a
temperature of 43.3.degree. C. was measured after the process. The
sample was processed using a M-110P High Energy Fluidizer.TM.
(Microfluidics). The particles were lyophilized using bench top
lyophilizer (ART Inc.). The mean particle size obtained was 273.9
nm using the NiComp 370 Submicron Particle Size Analyzer. The
sample was stored in the refrigerator.
Second Sample
[0039] This sample consisted of 30 mg epinephrine in 100 ml of
distilled water. One pass at 29,000 psi was applied and a
temperature of 18.3.degree. C. was measured after the process. The
homogenizer was setup using the cooling coil. Ice packs and tap
water were used to cool the pressurized sample to 14.degree. C. The
mean particle size obtained was 334.3 nm using the NiComp 370
Submicron Particle Size Analyzer. The sample was stored in the
refrigerator.
Third Sample
[0040] This sample was prepared in 0.1% phosphoric acid. The
phosphoric acid solution was prepared by diluting 0.5 ml of
phosphoric acid 85% (Mallinckrodt Chemicals, LOT H39A04, Exp. Sep.
30, 2011) in 500 ml of distilled water. The epinephrine sample was
prepared by weighing 103 mg of epinephrine base into 100 ml of 0.1%
phosphoric acid solution prior to sample passes. Two passes at
15,000 psi were applied to the sample. In the first pass a
temperature of 36.8.degree. C. was measured after the process and
in the second pass a temperature of 41.1.degree. C. was obtained.
The mean particle size obtained was 334.6 nm using the NiComp 370
Submicron Particle Size Analyzer. The sample was stored in the
refrigerator.
Fourth Sample
[0041] This sample was prepared in 1M acetic acid. The 1M acetic
acid solution was prepared by diluting 27.5 ml of glacial acetic
acid (BDH Aristar, ACS, USP, FCC grade, LOT 200929924) in 500 ml of
distilled water. The epinephrine sample was prepared by weighing
155 mg of epinephrine base into 100 ml of 1M acetic acid solution.
The M-110p was flushed with distilled water, followed by acetic
acid solution prior to sample passes. Two passes were applied to
the sample, in the first pass a pressure of 8,500 psi was applied
and a temperature of 36.6.degree. C. was measured in the collected
sample. In the second pass a pressure of 15,000 psi was applied and
a temperature of 38.4.degree. C. was measured in the collected
sample. The mean particle size obtained was 392.0 nm using the
NiComp 370 Submicron Particle Size Analyzer. The sample was stored
in the refrigerator.
Fifth, Sixth, and Seventh Samples
[0042] These samples were prepared in a dark room to avoid light.
The homogenizer was setup using the cooling coil. Ice packs and tap
water were used to cool the pressurized samples. Higher drug
concentration was used in the seventh sample since the acidic
solvent tends to dissolve more drug than the other previously-used
solvents.
Visual Observations
[0043] The main problem was discoloration (a brown color formed)
due to degradation. All samples were discolored to a pinkish color
and then became dark brownish after processing, indicating
epinephrine instability. The seventh sample (water+0.1 mM sodium
metabisulfite+0.1 M perchloric acid) discolored to a slightly
pinkish color. 0.1 mM sodium metabisulfite+0.1 M perchloric acid
usually provided optimum stability for epinephrine for several
months.
[0044] The FT-IR spectrum for epinephrine base before (FIG. 2B) is
different from the FT-IR spectrum after processing (FIG. 2A), which
reflects the degradation that occurs during processing. The
epinephrine base required stabilization with acetic acid or
phosphoric acid (in the suspension media) and cooling of the
reaction chamber to minimize degradation.
Sizing
[0045] The first sample (epinephrine in water) was used.
TABLE-US-00003 TABLE 3 Sizes of Epinephrine Base Before and After
Processing After Fabrication Sample Before Fabrication (nm) (110
F., 30 Kpsi) (nm) 1 33030 273.9 2 32530 3 33160 Mean 32900 Standard
Error 192.04 Standard Deviation 332.62 179
[0046] The epinephrine particle size reduction to nanosize was
successful. The mean.+-.SD size was reduced from 32.91.+-.0.33
.mu.m (FIG. 3) to 273.9.+-.179.0 nm (FIG. 4).
Fabrication: Epinephrine Bitartrate (Bit)
[0047] In light of the instability associated with the epinephrine
base particles, fabrication using the epinephrine salt, epinephrine
bitartrate, was pursued.
[0048] Isopropyl alcohol (IPO) was selected as a suspending vehicle
based on its safety profile and the solubility study previously
performed (see above) for several solvents.
TABLE-US-00004 TABLE 4 Epinephrine Bitartrate (Bit) Concentration
Pressure (psi) PSD nm Sample Solvent (mg/ml) (# passes) (NiComp) 1
IPO 7.0 15,000 (1) 43,000 2 IPO 3.5 25,000 (1) 8,766 25,000 (1)
3,879 3 IPO 0.875 25,000 (1) 3,971 4 IPO 0.70 25,000 (6) 2,368
25,000 (16) 1,203
Observations
[0049] Nanoparticles of epinephrine bitartrate in various sizes
were produced ranging in diameter from about 43,000 to 1,203
nm.
[0050] The first sample, a suspension of 7.0 mg/ml, was used as a
stock suspension and was used to prepare the other dilutions. Thus,
the passes are additive and each (pass) represents an additional
pass to the previous dilution.
[0051] After ten passes in the last run, includes samples one, two,
three, and the first pass of sample 4, the particle size
distribution (PSD) did not change (no effect after ten passes)
according to NiComp readings.
[0052] The fourth sample was processed six times (6 passes in one
step) followed by an additional ten passes (for a total of sixteen
passes continuously).
[0053] The epinephrine bitartrate (salt form of epinephrine) was
more stable than the epinephrine base, did not show any
discoloration, and tolerated the fabrication conditions
(nanomilling).
First Sample
[0054] The particle size distribution (PSD) of epinephrine
bitartrate after processing (fabrication) using Zetasizer was 5000
nm (60%) and 500-1000 nm (30-40%). The yield of fabricated
epinephrine bitartrate after drying was 68%. The Fourier
Transformation Infrared (FT-IR) spectrums are similar in both
epinephrine bitartrate before and after processing (FIG. 5).
Details of In Vitro Diffusion Experiments and Results
[0055] Epinephrine diffusion was evaluated using an automated, flow
through cell system (n=6) under the following parameters:
[0056] Flow rate: 50 .mu.l/minute
[0057] Donor cell orifice area: 0.2 cm.sup.2
[0058] Sample volume added to donor cell: 200 .mu.l
[0059] Medium in receptor cells: phosphate buffer (pH=5.8)
[0060] Membrane: 7 Spectra/Por.RTM. dialysis membranes (1000 MWt
cutoff).
[0061] Epinephrine, base or salt equivalent to 400 .mu.g/ml
epinephrine base, in the following four different formulations were
used:
[0062] 1) Epinephrine base nanoparticles suspension (Epi-NP
Susp).
[0063] 2) Epinephrine base suspension using 0.3% carboxymethyl
cellulose as a suspending agent (Epi-CMC Susp).
[0064] 3) Epinephrine base solution using
hydroxypropyl-f3-cyclodetrin as a solubilizing agent (Epi-HBD
Sol).
[0065] 4) Epinephrine bitartrate solution (Epi Bit Sol).
[0066] 200 .mu.l from each of the four formulations was spiked into
the donor cells. Samples were collected every 30 minutes for 8.5
hours and analyzed by High Performance Liquid Chromatography (HPLC)
for epinephrine concentration.
HPLC Analysis
[0067] HPLC analysis was performed under the following
parameters:
[0068] PerkinElmer HPLC system with ultraviolet (UV) detector
[0069] Column: Econspher (Alltech), C.sub.18 4.6.times.150 mm, 3
.mu.m
[0070] Mobile Phase: USP 26.sup.th Edition, 2003
[0071] Flow Rate: 1 ml/minute
[0072] Detection Wavelength: 280 nm
[0073] Retention Time: epinephrine 4.8 minutes
Statistical Analysis of Results
[0074] Results were statistically analyzed using one-way ANOV and
Tukey-Kramer tests, NCSS program, at a level of significance
p<0.05.
[0075] Mean.+-.SD values of cumulative epinephrine concentration
versus time (AUC), maximum epinephrine flux (JMax), time to reach
JMax (tJMax), and epinephrine permeation coefficient (Kp) for each
formulation was calculated.
Results
[0076] Mean.+-.SD values of cumulative epinephrine concentration
versus time (AUC), maximum epinephrine flux (JMax), and epinephrine
permeation coefficient (Kp) obtained from EP-NP Susp were
significantly higher than Epi-CMC Susp, Epi-HBCD Sol, and Epi Bit
Sol (p<0.05). The time to reach JMax (tJMax) was not
significantly different between the four formulations. These
results are illustrated in the graphs of FIGS. 6A-B.
TABLE-US-00005 TABLE 5 In Vitro Diffusion Data Formulation: Epi-NP
Susp Epi-CMC Susp Epi-HBCD Sol Epi Bit Sol AUC (.mu.g/ml/hr) 10.4
.+-. 1.7* 5.1 .+-. 1.1 5.5 .+-. 0.5 4.6 .+-. 0.9 JMax
(.mu.g/cm.sup.2/hr) 15.1 .+-. 1.9* 10.4 .+-. 1.6 8.6 .+-. 0.3 7.9
.+-. 1.0 t.sub.Jmax (hr) 9.41 .+-. 0.26 9.41 .+-. 0.50 10.17 .+-.
0.10 10.12 .+-. 0.09 Kp (cm/hr) 0.19 .+-. 0.07* 0.13 .+-. 0.002
0.11 .+-. 0.04 0.10 .+-. 0.04
[0077] 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 nanoparticles, 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.
* * * * *