U.S. patent application number 14/778887 was filed with the patent office on 2016-02-18 for epinephrine fine particles and methods for use thereof for treatment of conditions responsive to epinephrine.
The applicant listed for this patent is NOVA SOUTHEASTERN UNIVERSITY, Ousama RACHID, Estelle SIMONS, Keith SIMONS. Invention is credited to Ousama RACHID, Mutasem RAWAS-QALAJI, Estelle SIMONS, Keith SIMONS.
Application Number | 20160045457 14/778887 |
Document ID | / |
Family ID | 55301312 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160045457 |
Kind Code |
A1 |
RAWAS-QALAJI; Mutasem ; et
al. |
February 18, 2016 |
EPINEPHRINE FINE PARTICLES AND METHODS FOR USE THEREOF FOR
TREATMENT OF CONDITIONS RESPONSIVE TO EPINEPHRINE
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.
Inventors: |
RAWAS-QALAJI; Mutasem; (Fort
Lauderdale, FL) ; RACHID; Ousama; (Winnipeg, CA)
; SIMONS; Keith; (Winnipeg, CA) ; SIMONS;
Estelle; (Winnipeg, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RACHID; Ousama
SIMONS; Keith
SIMONS; Estelle
NOVA SOUTHEASTERN UNIVERSITY |
Fort Lauderdale |
FL |
US
US
US
US |
|
|
Family ID: |
55301312 |
Appl. No.: |
14/778887 |
Filed: |
March 24, 2014 |
PCT Filed: |
March 24, 2014 |
PCT NO: |
PCT/US14/31579 |
371 Date: |
September 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61804519 |
Mar 22, 2013 |
|
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61804892 |
Mar 25, 2013 |
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Current U.S.
Class: |
424/490 ;
424/489; 514/653; 564/443 |
Current CPC
Class: |
A61K 9/0056 20130101;
A61K 31/137 20130101; A61K 31/00 20130101; A61K 47/26 20130101;
A61K 9/006 20130101; A61K 9/14 20130101; A61K 47/12 20130101 |
International
Class: |
A61K 31/137 20060101
A61K031/137; A61K 9/00 20060101 A61K009/00 |
Claims
1. A pharmaceutical composition comprising epinephrine nanocrystals
or microcrystals, the composition formulated for buccal or
sublingual administration.
2. The pharmaceutical composition in accordance with claim 1,
further comprising at least one of a pharmaceutically-acceptable
carrier, a surfactant, a penetration enhancer, and a
mucoadhesive.
3. (canceled)
4. The pharmaceutical composition in accordance with claim 1,
wherein the composition includes a pharmaceutically-effective dose
of approximately 10 mg to approximately 40 mg epinephrine
nanocrystals or microcrystals.
5. The pharmaceutical composition in accordance with claim 1,
wherein the composition includes a pharmaceutically-effective dose
of approximately 10 mg epinephrine nanocrystals or
microcrystals.
6. The pharmaceutical composition in accordance with claim 2,
further comprising at least one of a taste enhancer and a
sweetening agent and mouthfeel enhancer.
7. The pharmaceutical composition in accordance with claim 6,
further comprising at least one of a filler, a lubricant, and a
disintegrant.
8. The pharmaceutical composition in accordance with claim 6,
wherein the taste enhancer is citric acid and the sweetening agent
and mouthfeel enhancer is mannitol.
9-11. (canceled)
12. A method for treating a condition responsive to epinephrine in
a subject in need thereof, comprising: providing a composition
including epinephrine nanocrystals or microcrystals and at least
one pharmaceutically-acceptable carrier; and administering the
composition to the subject.
13. The method in accordance with claim 12, wherein the composition
includes a pharmaceutically-effective dose of approximately 10 mg
to approximately 40 mg epinephrine nanocrystals or
microcrystals.
14. The method in accordance with claim 12, wherein the composition
includes a pharmaceutically-effective dose of approximately 10 mg
epinephrine nanocrystals or microcrystals.
15. The method in accordance with claim 12, wherein the condition
is a cardiac event or an allergic reaction.
16. The method in accordance with claim 15, wherein the cardiac
event is cardiac arrest and the allergic reaction is anaphylaxis,
asthma, or bronchial asthma.
17-38. (canceled)
39. The method in accordance with claim 12, wherein the condition
is a breathing difficulty.
40. The method in accordance with claim 39, wherein the breathing
difficulty is associated with anaphylaxis, asthma, bronchial
asthma, bronchitis, emphysema, or respiratory infections.
41. The method in accordance with claim 12, wherein the treating
includes enhancing sublingual bioavailability of epinephrine.
42. The pharmaceutical composition in accordance with claim 1,
wherein the epinephrine is an epinephrine base or an epinephrine
bitartrate salt.
43. The pharmaceutical composition in accordance with claim 1,
wherein the composition is an oral disintegrating tablet (ODT)
capable of fully dissolving or disintegrating in less than about
one minute.
44. The pharmaceutical composition in accordance with claim 1,
wherein the composition is encapsulated within a polymer.
45. A pharmaceutical composition comprising epinephrine fine
particles, the composition formulated for buccal or sublingual
administration.
46. The pharmaceutical composition in accordance with claim 45,
wherein the epinephrine fine particles range in size from about 2.5
.mu.m or less to about 100 nm.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 61/804,519 (filed on Mar. 22,
2013) and U.S. Provisional Patent Application No. 61/804,892 (filed
on Mar. 25, 2013) the contents each of which is hereby incorporated
by reference.
[0002] This application is related to U.S. Provisional Patent
Application No. 61/660,273, filed on Jun. 15, 2012. This
application is related to U.S. Provisional Patent Application No.
61/550,359, filed on Oct. 21, 2011. This application is related to
International Application No. PCT/US2011/26604, filed on Mar. 1,
2011, which claims the benefit of U.S. Provisional Patent
Application No. 61/309,136, filed on Mar. 1, 2010. 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 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.
[0003] 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
[0004] 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 fine
particles, including epinephrine nanoparticles or nanocrystals and
epinephrine microparticles or microcrystals, for sublingual
administration in treatment of conditions responsive to
epinephrine.
BACKGROUND
[0005] 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.
[0006] 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).
[0007] Likewise, due to high buccal and sublingual vascularity,
buccally- or sublingually-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 or sublingual 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 and
sublingual mucosas have 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).
[0008] The buccal and sublingual mucosas are 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 and sublingual routes could be
useful for drug administration to unconscious patients, patients
undergoing an anaphylactic attack, or patients who sense the onset
of an anaphylactic attack.
[0009] Anaphylaxis is a sudden, severe systemic allergic reaction,
which can be fatal within minutes. Epinephrine (Epi) 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 as an injectable dosage form in ampoules or in
autoinjectors, however these are underused when anaphylaxis occurs
(Simons, F. E. R. J Allergy Clin Immunol 124(4):625-636 2009;
Simons, F. E. R. J Allergy Clin Immunol 125:S161-181 2010). The
drawbacks of Epi auto-injectors include high cost, perceived large
size and bulkiness, limitations on repeated dosing (if required),
fear and anxiety associated with the use of needles (especially in
children), and dosing errors caused by incorrect techniques of
administration (Simons, K. J. et al. Current Opinion in Clinical
Immunology 10:354-361 2010). Furthermore, 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) and thus has limited
shelf-life; approximately one year.
[0010] The sublingual route of administration is a promising
alternative route for epinephrine administration. The formulation
of sublingual tablets of epinephrine would enable the development
of tablets with a range of epinephrine doses to match the
population on an mg/kg basis. Sublingual tablets of epinephrine
would be easy to carry and self-administer eliminating the fear and
anxiety associated with needles used in autoinjectors for young
children, as well as readily providing the capability of multiple
doses. Feasibility studies in humans and animals have shown that
epinephrine 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 epinephrine 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 epinephrine in a suitable
carrier. Based on historical and anecdotal evidence, an
approximately 0.3 mg dose of epinephrine, 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, Epi 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)).
[0011] As stated above, epinephrine (Epi) is typically administered
either subcutaneously (SC) or intramuscularly (IM) by injection.
Thus, Epi injections are the accepted first aid means of delivering
Epi 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 Epi injectors in a convenient place at all
times.
[0012] Given the difficulties associated with manual subcutaneous
(SC) or intramuscular (IM) administration of Epi, such as patient
apprehension related to injections or the burden of an at risk
person having to always maintain an Epi injector close at hand,
there exists a need in the art for more convenient dosage forms
which can provide immediate administration of Epi, particularly to
a person undergoing anaphylaxis wherein the need for injection or
Epi injectors is obviated.
[0013] Recently, a novel fast-disintegrating tablet suitable for
sublingual (SL) administration of Epi 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. Sublingual doses
ranging from 5 to 40 mg epinephrine as the bitartrate salt were
studied to achieve equivalent plasma concentrations. In an animal
model, it was determined that a 40 mg epinephrine dose administered
sublingually as a bitartrate salt in tablet form resulted in plasma
epinephrine concentrations similar to those achieved by 0.3 mg
epinephrine intramuscular (IM) injection (Rawas-Qalaji et al. J
Allergy Clin Immunol 117:398-403 2006).
[0014] Without being bound by theory, it is thought that
fabrication of epinephrine into fine particles, including
epinephrine nanoparticles or nanocrystals and epinephrine
microparticles or microcrystals, and incorporation of the
epinephrine fine particles 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
[0015] Epinephrine (Epi) is life-saving in the treatment of
anaphylaxis. In community settings, a first-aid dose of epinephrine
in an amount of 0.15 mg or 0.3 mg is injected into the mid-outer
thigh by patients or caregivers using an auto-injector such as an
EpiPen.RTM. (epinephrine auto-injector 0.3 or 0.15 mg, Mylan Inc.,
Basking Ridge, N.J.). Epi auto-injectors are under-used because of
needle phobia, bulky size, and high cost; additionally, there are
only two fixed doses, shelf-life is only 12-18 months, and
unintentional injection and injury sometimes occur.
[0016] The instant invention circumvents the aforementioned
problems by providing a fast-disintegrating epinephrine tablet
formulation for anaphylaxis treatment. Although this formulation
was designed with regard to anaphylaxis, it is equally effective
and contemplated for use in treatment of any condition responsive
to epinephrine such as cardiac events, i.e. cardiac arrest, and
breathing difficulties, i.e. asthma, bronchial asthma, bronchitis,
emphysema, and respiratory infections.
[0017] In a validated rabbit model, this fast-disintegrating
epinephrine tablet formulation resulted in plasma epinephrine
concentrations similar to those achieved after a 0.3 mg epinephrine
intra-muscular injection (Rawas-Qalaji et al. J Allergy Clin
Immunol 117:398-403 2006). Furthermore, epinephrine was stable in
these fast-disintegrating tablets for at least seven years.
[0018] One of the most common approaches to enhance the rate of
drug dissolution and absorption is to significantly reduce its
particle size to the micro- or nano-size range. Drug nanocrystals
(NC) or microcrystals (MC) are advantageous due to the 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.
[0019] The phrase "epinephrine fine particles" refers to
epinephrine particles of about 2.5 .mu.m or less to about 100 nm in
size and includes epinephrine nanoparticles or nanocrystals and
epinephrine microparticles or microcrystals.
[0020] In one aspect, the invention provides epinephrine fine
particles.
[0021] In one aspect, the invention provides epinephrine
nanoparticles. The epinephrine can be either an epinephrine base or
an epinephrine bitartrate salt.
[0022] In another aspect, the invention provides epinephrine
nanocrystals. A nanocrystal is a nanoparticle having a crystalline
structure. The term "nanocrystal" is a more specific term for
describing a nanoparticle. A drug nanocrystal contains almost 100%
pure drug, thus an epinephrine nanocrystal contains almost 100%
pure epinephrine. A drug nanoparticle can include nanocrystals or a
drug encapsulated within a polymer at different ratios. One example
is the epinephrine nanoparticles comprising chitosan and
tripolyphosphate (TPP) described in the previously-filed related
application; U.S. Provisional Patent Application Ser. No.
61/550,359, filed on Oct. 21, 2011.
[0023] In another aspect, the invention provides a composition
including epinephrine nanoparticles or nanocrystals capable of
enhancing the sublingual bioavailability of epinephrine for the
emergency treatment of anaphylaxis.
[0024] In another aspect, the invention provides "oral
disintegrating tablets (ODTs)" including epinephrine nanoparticles
or nanocrystals or epinephrine microparticles or microcrystals.
[0025] 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.
[0026] The invention also provides pharmaceutical compositions
including epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals in ODT form.
[0027] The invention also provides a pharmaceutical composition
including epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals and a pharmaceutically-acceptable
carrier for buccal or sublingual administration.
[0028] The phrase "pharmaceutically-acceptable carrier" refers to
an inactive and non-toxic substance used in association with an
active substance, i.e. epinephrine, especially for aiding in the
application of the active substance. Non-limiting examples of
pharmaceutically-acceptable carriers are diluents, binders,
disintegrants, flavorings, fillers, and lubricants.
Pharmaceutically-acceptable carriers can have more than one
function, i.e. a filler can also be a disintegrant. Additionally,
pharmaceutically-acceptable carriers may also be referred to as
non-medicinal ingredients (NMIs).
[0029] The invention also provides a pharmaceutical composition,
for buccal or sublingual administration, including epinephrine
nanoparticles or nanocrystals or epinephrine microparticles or
microcrystals and at least one of a pharmaceutically-acceptable
carrier, a surfactant, a penetration enhancer, and a mucoadhesive.
The pharmaceutical composition can further include at least one of
a taste enhancer and a sweetening agent and mouthfeel enhancer. A
non-limiting example of a taste enhancer is citric acid. Citric
acid masks the bitter taste of epinephrine. A non-limiting example
of a sweetening agent and mouthfeel enhancer is mannitol. The
pharmaceutical composition can further include at least one of a
filler, a lubricant, and a disintegrant. Non-limiting examples
include microcrystalline cellulose (filler), magnesium stearate
(lubricant), and hydroxypropyl ethers of cellulose
(disintegrant).
[0030] Additionally, the invention provides a pharmaceutical
composition including epinephrine nanoparticles or nanocrystals or
epinephrine microparticles or microcrystals, in which the bitter
taste of the epinephrine is masked by a taste enhancer. A
non-limiting example of a taste enhancer is citric acid.
[0031] In another aspect, the invention provides a method for
enhancing sublingual bioavailability of epinephrine in a subject in
need thereof including steps for providing a composition including
epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals and at least one
pharmaceutically-acceptable carrier and administering the
composition to the subject. The described fast-disintegrating
epinephrine tablets enhance bioavailability of epinephrine by
releasing epinephrine within sixty seconds of administration.
[0032] In another aspect, the invention provides a method for
treating a condition responsive to epinephrine in a subject in need
thereof including steps for providing a composition including
epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals and at least one
pharmaceutically-acceptable carrier and administering the
composition to the subject. Conditions responsive to epinephrine
react to administration of epinephrine. Non-limiting examples of
conditions responsive to epinephrine include a cardiac event, i.e.
cardiac arrest, or an allergic reaction, i.e. anaphylaxis, asthma,
or bronchial asthma.
[0033] The phrase "effective amount" refers to the amount of a
composition necessary to achieve the composition's intended
function.
[0034] The phrase "pharmaceutically-effective dose" refers to the
amount of a composition necessary to achieve a desired
pharmaceutical effect. It is often desirable to use the smallest
effective dose of a drug. One example of a dose range for the
described epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals is approximately 10 mg to 40 mg
epinephrine nanoparticles or nanocrystals or epinephrine
microparticles or microcrystals.
[0035] The phase "therapeutically-effective amount" refers to the
amount of a composition required to achieve the desired function,
i.e. treatment of the condition responsive to epinephrine.
[0036] In another aspect, the invention provides a method for
treating a breathing difficulty in a subject in need thereof
including steps for providing a composition including epinephrine
nanoparticles or nanocrystals or epinephrine microparticles or
microcrystals and at least one pharmaceutically-acceptable carrier
and administering the composition to the subject. Breathing
difficulties responsive to epinephrine include, but are not limited
to, breathing difficulties associated with anaphylaxis, asthma,
bronchial asthma, bronchitis, emphysema, and respiratory
infections.
[0037] The invention additionally provides a method for treatment
of an allergic emergency in a subject diagnosed with or suspected
of having an allergic emergency including steps for providing a
composition including epinephrine nanoparticles or nanocrystals or
epinephrine microparticles or microcrystals and at least one
pharmaceutically-acceptable carrier and administering the
composition to the subject. Non-limiting examples of allergic
emergencies are anaphylaxis, asthma, and bronchial asthma.
[0038] In an additional aspect, the invention provides a method for
treatment of a cardiac event in a subject diagnosed with or
suspected of having a cardiac event including steps for providing a
composition including epinephrine nanoparticles or nanocrystals or
epinephrine microparticles or microcrystals and at least one
pharmaceutically-acceptable carrier and administering the
composition to the subject. A non-limiting example of a cardiac
event is cardiac arrest.
[0039] Any of the above-described epinephrine fine particles
(including epinephrine nanoparticles or nanocrystals and
epinephrine microparticles or microcrystals), compositions, and
pharmaceutical compositions can be formulated for buccal or
sublingual administration, particularly those epinephrine fine
particles (including epinephrine nanoparticles or nanocrystals and
epinephrine microparticles or microcrystals), compositions, and
pharmaceutical compositions intended for use in emergency
situations.
[0040] In another aspect, any of the above-described epinephrine
fine particles (including epinephrine nanoparticles or nanocrystals
and epinephrine microparticles or microcrystals) can be used in the
manufacture of any of the above-described compositions and
pharmaceutical compositions.
[0041] 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
[0042] 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.
[0043] FIG. 1 is an FTIR spectra of epinephrine bitartrate dried
particles before and after processing of a 2.8 mg/mL sample
processed at 30 KPsi for 1 pass (cycle).
[0044] FIG. 2 is an FTIR spectra of epinephrine bitartrate dried
particles after processing of a 2.8 mg/mL sample processed at 30
KPsi for 1 pass (cycle) and isopropyl alcohol.
[0045] FIG. 3 is a Differential Scanning Calorimetry (DSC) spectrum
of epinephrine bitartrate (EpiBit) before processing.
[0046] FIG. 4 is a Differential Scanning Calorimetry (DSC) spectrum
of epinephrine bitartrate (EpiBit) after processing.
[0047] FIGS. 5A-D: FIG. 5A is another view of the DSC spectrum of
epinephrine bitartrate (EpiBit) before processing. FIG. 5B is
another view of the DSC spectrum of epinephrine bitartrate (EpiBit)
after processing. FIG. 5C is a Scanning Electron Microscopy (SEM)
image of epinephrine bitartrate (EpiBit) before processing. FIG. 5D
is a Scanning Electron Microscopy (SEM) image of epinephrine
bitartrate (EpiBit) after processing.
[0048] FIG. 6 shows the mean.+-.SD (n=4) cumulative diffused
epinephrine per dialysis membrane area versus time.
[0049] FIG. 7 shows the mean.+-.SD (n=4) percentage of diffused
epinephrine through dialysis membrane versus time.
[0050] FIG. 8 shows the mean.+-.SD (n=4) of epinephrine influx (J)
through dialysis membrane.
[0051] FIG. 9 shows the mean.+-.SD (n=4) of epinephrine
permeability (P) through dialysis membrane.
[0052] FIG. 10 shows the mean.+-.SD (n=4) cumulative diffused
epinephrine per sublingual mucosa area versus time.
[0053] FIG. 11 shows the mean.+-.SD (n=4) percentage of diffused
epinephrine through sublingual mucosa versus time.
[0054] FIG. 12 shows the mean.+-.SD (n=4) of epinephrine influx (J)
through sublingual mucosa.
[0055] FIG. 13 shows the mean.+-.SD (n=4) of epinephrine
permeability (P) through sublingual mucosa.
[0056] FIG. 14 shows the mean.+-.SD plasma epinephrine
concentration versus time plots (n=5) after administration of
epinephrine by intramuscular (IM) injection, epinephrine
microcrystals sublingual (SL) tablets, epinephrine sublingual (SL)
tablets, or placebo sublingual tablets.
[0057] FIG. 15 shows the correlation between the cumulative
diffused epinephrine per area through dialysis and excised
sublingual membranes.
DETAILED DESCRIPTION OF THE INVENTION
[0058] 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.
[0059] 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.
[0060] 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 aborption
using dialysis membranes, excised sublingual porcine mucosal
membranes, and validated rabbit's animal model, respectively.
[0061] 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).
[0062] 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.
[0063] 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.
[0064] 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.RTM.,
EpiPen Jr.RTM. (Mylan Inc, Basking Ridge, N.J.), Twinject 0.3
mg.RTM., and Twinject 0.15.RTM. (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.
[0065] 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.
[0066] 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 (.ltoreq.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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
Materials
[0072] 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.
[0073] (-)-Epinephrine (+) bitartrate was purchased from
Sigma-Aldrich (St. Louis, Mo.). Ceolus.RTM. PH-301
(microcrystalline cellulose) with a mean particle size of 50 .mu.m
was supplied by Asahi Kasei Chemicals Corp (Tokyo, Japan) and
low-substituted hydroxypropyl cellulose (LH11) with a mean particle
size of 50 .mu.m 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.RTM.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.).
Fabrication and Characterization of Epinephrine Fine Particles
Using High Shear Fluid Processor (Microfluidizer)-Homogenization
Method
[0074] 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.
Preparation of Epinephrine Bitartrate Nanocrystals
[0075] 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.degree. C. for 30 minutes. The
upper clear solvent was removed by aspiration and the remaining
particles were dried by vacuum concentrator at room
temperature.
Characteristics of the Epinephrine Bitartrate Nanocrystals
Particle Size and Zeta Potential Measurement
[0076] 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.
[0077] Mean.+-.SD particles size distribution (by volume) of EpiBit
crystals before processing was 131.8.+-.10.5 .mu.m (n=6). The
10.sup.th percentile (Dv0.1), median (Dv0.5), and 90.sup.th
percentile (Dv0.9) were 39.8.+-.3.0 .mu.m, 113.6.+-.9.1 .mu.m, and
254.8.+-.20.1 .mu.m, respectively.
TABLE-US-00001 TABLE 1 Particles Size Distribution (by Volume) of
EpiBit Before Processing Before Fabrication (.mu.m) 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
[0078] 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.
[0079] 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 .mu.m, 2.5.+-.0.4 .mu.m, 0.185.+-.0.019, and
-4.5.+-.1.4 mV, respectively.
[0080] 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.
TABLE-US-00002 TABLE 2 Particles Size Distribution (by intensity)
and zeta potential of EpiBit After Processing After Fabrication
Sample # Z-average (d 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)
[0081] 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
FIG. 1. There was no evidence of EpiBit degradation after
processing as the spectra before and after processing were
similar.
[0082] The FT-IR spectrum of isopropyl alcohol and EpiBit after
processing is shown in FIG. 2. The isopropyl alcohol peaks are
missing, which indicates successful removal of the isopropyl
alcohol. Thus, there was no evidence of isopropyl alcohol remaining
in the EpiBit particles after drying as shown in the spectrum of
processed EpiBit.
Differential Scanning Calorimetry (DSC)
[0083] 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.degree. C. at rate of 10.degree. C./min and with a nitrogen
purge of 20 mL/min. The DSC spectra of EpiBit before and after
processing are shown in FIGS. 3 and 4, respectively. There was no
evidence of EpiBit degradation or crystallinity change after
processing. FIG. 5A shows another view of a DSC spectrum of EpiBit
before processing and FIG. 5B shows another view of a DSC spectrum
after processing. These spectra (FIGS. 5A and 5B) are similar
before and after processing.
Scanning Electron Microscopy (SEM)
[0084] 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 FIGS. 5C and 5D, respectively. There was a
morphological change in the EpiBit crystals from a rectangular
shape before processing to a smaller, spherical shape after
processing.
Rapidly-Disintegrating Epinephrine Sublingual Tablet
Formulation
[0085] 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.
Manufacturing and Quality Control of Tablets for In Vitro and Ex
Vivo Diffusion Studies
[0086] 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.sup.16 to ensure sufficient hardness to withstand
shipping and handling while maintaining rapid tablet
disintegration.
[0087] All tablet formulations were tested for quality control as
follows:
[0088] Dimensions: 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.
[0089] Hardness: 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.
[0090] Disintegration Time: 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.
[0091] USP Weight Variation Test: 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.
[0092] USP Content Uniformity Test: 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.
[0093] USP Friability Test: 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.
[0094] 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.
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
[0095] 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.
[0096] These tablets did not contain lactose or bisulfite and met
USP standards for tablet weight variation, content uniformity, and
friability.sup.8,20. They also disintegrated in less than 30
seconds.
Methods for In Vitro and Ex Vivo Diffusion Studies
[0097] 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.RTM. 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.degree. C. until used (<4 weeks). The mucosal membranes were
defrosted at room temperature before each experiment.
[0098] 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).
[0099] 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.degree. 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.
[0100] 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 .mu.L, 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.
Epinephrine HPLC Analysis
[0101] 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 .mu.g/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 .mu.g/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.
Data Analysis
[0102] The mean.+-.SD cumulative diffused Epi per area
(.mu.g/cm.sup.2) and percentage of diffused Epi for each ODT
formulation were calculated. The mean.+-.SD Epi influx, J
(.mu.g/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
(.mu.g/cm.sup.2/min); the maximum Epi diffused, Jmax
(.mu.g/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.
Results
1) The In Vitro Diffusion of Epinephrine Microcrystals Subligual
Tablets
[0103] 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 FIGS. 6
and 7, respectively.
TABLE-US-00004 TABLE 4 Mean .+-. SD (n = 4) cumulative diffused
epinephrine per area (.mu.g/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
TABLE-US-00005 TABLE 5 Mean .+-. SD (n = 4) percentage of diffused
epinephrine (%) for each formulation through dialysis membrane. 10
mg 10 mg Epi 20 mg 20 mg 40 mg Time (min) Epi Tablet MC Tablet Epi
Tablet Epi MC Tablet 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
[0104] 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 FIGS. 8 and 9, respectively.
[0105] The mean (.+-.SD) Epi JAUC.sub.0-90 and Jmax of 40 mg Epi
tablets (484184.9.+-.29655.9 .mu.g/cm.sup.2/min and 7508.3.+-.568.7
.mu.g/cm.sup.2, respectively) and 20 mg Epi MC tablets
(402852.2.+-.55299 .mu.g/cm.sup.2/min and 6727.2.+-.736.3
.mu.g/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 (FIG. 6 and Table 6).
The Epi Tmax was not significantly different (p>0.05) between
all formulations (Table 6).
[0106] The mean (.+-.SD) Epi J of 40 mg Epi tablets (234.2.+-.99.6
.mu.g/cm.sup.2/min) and 20 mg Epi MC tablets (172.2.+-.49.8
.mu.g/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 (FIG. 8 and Table 6).
The Epi t.sub.L was not significantly different (p>0.05) between
all formulations (Table 6).
[0107] 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 (FIGS. 7 and 9, and Table 6).
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
(.mu.g/cm.sup.2/min) 54604.1 .+-. 11332.5 72461 .+-. 21229.2 292089
.+-. 58875.7 402852.2 .+-. 55299 484184.9 .+-. 29655.9 Jmax
(.mu.g/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
(.mu.g/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.
[0108] 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.
2) The Ex Vivo Diffusion of Epinephrine Microcrystals Sublingual
Tablets
[0109] 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 FIGS. 10 and
11, respectively.
TABLE-US-00007 TABLE 7 Mean .+-. SD (n = 4) cumulative diffused
epinephrine per sublingual mucosa area (.mu.g/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
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
[0110] 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 FIGS. 12 and 13, respectively.
[0111] The mean Epi JAUC.sub.0-90 and Jmax of 40 mg Epi tablets
(264556.4.+-.182820.3 .mu.g/cm.sup.2/min and 4795.7.+-.2988.2
.mu.g/cm.sup.2, respectively) and 20 mg Epi MC tablets
(211368.5.+-.116025.1 .mu.g/cm.sup.2/min and 3526.8.+-.1754.6
.mu.g/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 (FIG. 10 and
Table 9).
[0112] The Epi J of 40 mg Epi tablets (106.0.+-.82.4
.mu.g/cm.sup.2/min) and 20 mg Epi MC tablets (91.1.+-.54.6
.mu.g/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 .mu.g/cm.sup.2min) and 10 mg Epi tablets (24.8.+-.6.5
.mu.g/cm.sup.2/min) as well (FIG. 12 and Table 9). The Epi J of 40
mg Epi tablets was only significantly higher (p<0.05) than the
10 mg Epi solution (11.7.+-.3.2 .mu.g/cm.sup.2/min) and 10 mg Epi
tablets (17.1.+-.6.7 .mu.g/cm.sup.2/min) (FIG. 12 and Table 9). The
Epi t.sub.L was not significantly different (p>0.05) between all
formulations (Table 9).
[0113] 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) (FIGS. 11 and 13, and Table 9).
[0114] 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.
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 (.mu.g/cm.sup.2/min) Jmax (.mu.g/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 (.mu.g/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.
[0115] 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
In Vivo Absorption Studies
[0116] 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.
Methods
[0117] 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
previouslyl.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.RTM. as a positive control.
[0118] 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.
[0119] Epi 0.3 mg was injected IM in the thigh using an
EpiPen.RTM., after which the solution remaining in the EpiPen.RTM.
was evacuated into a plastic tube and frozen at -20.degree. 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.
Measurement of Plasma Epinephrine Concentrations
[0120] An indwelling catheter (22G 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.
[0121] 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.degree. C. Plasma were transferred into appropriately labeled
polypropylene tubes, and stored at -20.degree. 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.
Data Analysis
[0122] 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.RTM. 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.
Results
[0123] The mean (.+-.SD) of Epi dose injected using EpiPen.RTM.
auto-injectors was 0.29.+-.0.02 mg as calculated by multiplying the
Epi concentration, measured in the solution remaining in the
EpiPen.RTM. after injection, by the stated injected volume (0.3
mL).
[0124] 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.RTM. are shown in FIG. 14 . Mean (.+-.SD) AUC,
C.sub.baseline (endogenous E), C.sub.max, and T.sub.max values
after the sublingual administration of placebo ODT, Epi 40 mg ODT,
and Epi MC 20 mg ODT, and Epi 0.3 mg IM injection are shown in
Table 10. No adverse effects were observed.
[0125] 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)
[0126] 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).
[0127] 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.
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 .RTM. Epinephrine dose (mg) 0 40.0 20.0 0.3 AUC
(ng/ml/min) 220.1 .+-. 78.0 678.0 .+-. 149.0.dagger. 942.0 .+-.
243.7.dagger. 592.0 .+-. 122.3.dagger. C.sub.baseline (ng/ml) 1.1
.+-. 1.2 5.0 .+-. 3.0 2.9 .+-. 1.6 5.6 .+-. 1.9.dagger-dbl.
C.sub.max (ng/ml) 7.5 .+-. 3.0 31.7 .+-. 10.1.dagger. 38.0 .+-.
9.9.dagger. 27.6 .+-. 7.0.dagger. T.sub.max (min) 33.3 .+-. 17.5
20.0 .+-. 7.1 28.0 .+-. 29.3 30.0 .+-. 0.0 *n = 5 .dagger.p <
0.05 from placebo tablet but not from each others. .dagger-dbl.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
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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 (FIG. 1). Also, the
drying step to obtain the reduced-sized EpiBit crystals was very
efficient and no evidence in the FT-IR spectrum for any remaining
isopropyl alcohol, which was used as a carrier to process EpiBit
(FIG. 2).
[0132] The DSC spectra of EpiBit before and after processing were
also similar with a single endothermic peak around 157.degree. C.
that indicates for the absence of any change in the purity and
crystallinity of EpiBit (FIGS. 5A-5B).
[0133] The Scanning Electron Microscopy (SEM) images (FIGS. 5C-5D)
of EpiBit before and after processing demonstrate clearly the
change in EpiBit crystalline morphology from rectangular to
spherical crystals with much smaller size.
[0134] 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.
[0135] Results from both in vitro and ex vivo experiments were
highly correlated, (R.sup.2.gtoreq.87) (FIG. 15) and demonstrated
that the percentage of Epi diffused from 20 mg Epi MC ODT was
significantly higher than the rest of the formulations including 40
mg Epi ODT (FIGS. 7 and 11). This resulted in similar
JAUC.sub.0-90, Jmax, and influx (J) for both 20 mg Epi MC ODT and
40 mg Epi ODT, despite of the non-statistically different
permeability, although higher, for 20 mg Epi MC ODT (Tables 6 and
9). Also, formulating EpiBit into the ODT tablet formulation did
not pose any delay nor influenced EpiBit diffusion as shown from
comparing the 10 mg Epi diffusion from solution and ODT (Table
9).
[0136] 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.
[0137] 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%.
[0138] 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
microcrystals, pharmaceutical tablets, pharamaceutically-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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