U.S. patent application number 15/233023 was filed with the patent office on 2017-06-29 for novel formulations for the treatment of vaginal disorders.
This patent application is currently assigned to Mission Pharmacal Company. The applicant listed for this patent is Mission Pharmacal Company, Universite Laval. Invention is credited to Michel G. BERGERON, Jian GAO, Rabeea F. OMAR, Mary Ann WALTER.
Application Number | 20170182006 15/233023 |
Document ID | / |
Family ID | 51257572 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170182006 |
Kind Code |
A1 |
BERGERON; Michel G. ; et
al. |
June 29, 2017 |
NOVEL FORMULATIONS FOR THE TREATMENT OF VAGINAL DISORDERS
Abstract
The present disclosure provides novel formulations suitable for
the intravaginal delivery of tinidazole, as well as methods of
using the same.
Inventors: |
BERGERON; Michel G.;
(Quebec, CA) ; GAO; Jian; (San Antonio, TX)
; OMAR; Rabeea F.; (Quebec, CA) ; WALTER; Mary
Ann; (San Antonio, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mission Pharmacal Company
Universite Laval |
San Antonio
Quebec |
TX |
US
CA |
|
|
Assignee: |
Mission Pharmacal Company
San Antonio
MD
Universite Laval
Quebec
|
Family ID: |
51257572 |
Appl. No.: |
15/233023 |
Filed: |
August 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14314759 |
Jun 25, 2014 |
9446024 |
|
|
15233023 |
|
|
|
|
61839070 |
Jun 25, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4164 20130101;
A61K 47/12 20130101; A61K 47/22 20130101; A61K 9/06 20130101; A61K
9/0034 20130101; A61K 47/34 20130101; A61K 47/10 20130101 |
International
Class: |
A61K 31/4164 20060101
A61K031/4164; A61K 47/34 20060101 A61K047/34; A61K 9/00 20060101
A61K009/00; A61K 47/22 20060101 A61K047/22; A61K 47/12 20060101
A61K047/12; A61K 9/06 20060101 A61K009/06; A61K 47/10 20060101
A61K047/10 |
Claims
1-20. (canceled)
21. A method for treating bacterial vaginosis, trichomoniasis, or
other disease of the vaginal cavity susceptible to tinidazole,
comprising intravaginally administering to a subject in need
thereof a formulation comprising water, tinidazole, a
thermoreversible gelling agent, one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols, a solubility enhancer, and,
optionally, one or more preservatives.
22. The method of claim 21, wherein the thermoreversible gelling
agent is triblock copolymer having a central hydrophobic block
flanked on each side with a hydrophilic block.
23. The method of claim 22, wherein the hydrophobic block is
poly(propylene oxide).
24. The method of claim 23, wherein the hydrophilic block is
poly(ethylene oxide).
25. The method of claim 22, wherein the thermoreversible gelling
agent is poloxamer 407.
26. The method of claim 21, wherein the one or more
pharmaceutically acceptable C.sub.1-C.sub.7 alcohols comprises a
first pharmaceutically acceptable C.sub.1-C.sub.7 alcohol and a
second pharmaceutically acceptable C.sub.1-C.sub.7 alcohol.
27. The method of claim 26, wherein the first and second
pharmaceutically acceptable C.sub.1-C.sub.7 alcohols are selected
from the group consisting of methanol, ethanol, isopropanol,
propylene glycol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, and
combinations thereof.
28. The method of claim 26, wherein the first pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is benzyl alcohol.
29. The method of claim 26, wherein the second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is isopropanol.
30. The method of claim 27, wherein the isopropanol is a 60% (w/w)
solution in water.
31. The method of claim 21, wherein the solubility enhancer is
selected from the group consisting of mono- and di-alkyl ethers of
isosorbide.
32. The method of claim 31, wherein the mono- or di-alkyl ether of
isosorbide is dimethyl isosorbide.
33. The method of claim 21, wherein the water comprises a mixture
of sterile deionized water and a buffered aqueous solution.
34. The method of claim 33, wherein the buffered aqueous solution
is citrate buffer.
35. The method of claim 21, wherein the formulation has a buffered
pH selected from the group consisting of about 3 to about 5, about
3.5 to about 4.5, and about 4.
36. The method of claim 21, wherein the tinidazole comprises from
about 0.1% to about 2% (w/w) of the formulation.
37. The method of claim 21, wherein the tinidazole comprises about
1%, about 1.25%, or about 1.5% (w/w) of the formulation.
38. The method of claim 21, wherein the subject is a human.
39-43. (canceled)
44. A method for treating a vaginal disorder comprising
intravaginally administering to the subject a formulation
containing about 0.1-2% tinidazole, or an active metabolite
thereof.
45-87. (canceled)
88. The method of claim 44, wherein the tinidazole comprises about
1%, about 1.25%, or about 1.5% (w/w) of the formulation.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 61/839,070 filed Jun. 25, 2013, the entirety of
which is incorporated herein by reference.
NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
[0002] For purposes of 35 U.S.C. 102(c), a joint research agreement
was executed between Mission Pharmacal and Universite Laval in an
invention relating to novel formulations for the treatment of
vaginal disorders.
BACKGROUND OF THE INVENTION
[0003] Bacterial vaginosis (BV) is a common condition that is
related to alterations in the normal vaginal flora. It is the most
common cause of vaginitis in women and has a recurrence rate of
approximately 20%-40% at one month after therapy. In addition to
the general discomfort associated with the condition, BV has been
linked to premature membrane rupture, premature delivery, low birth
weight, acquisition of HIV and other STDs, development of pelvic
inflammatory disease (PID), and post-operative infections following
gynecological procedures.
[0004] Although the precise etiology of BV is unknown, the disease
itself is associated with the decrease or absence of protective
lactobacilli which are normally present in the vagina. Lactobacilli
produce lactic acid from glycogen to maintain the vagina's acidic
pH. This acidic environment inhibits the growth of other bacterial
species typically found in the vagina, albeit at low levels.
Vaginal lactobacilli also produce hydrogen peroxide
(H.sub.2O.sub.2), which is toxic to viruses as well as to bacteria
in vitro. When lactobacilli are sufficiently absent, bacteria such
as Gardnerella vaginalis, Bacteroides spp., Mobiluncus spp.,
Haemophilus spp., peptostreptococci, Mycoplasma hominis,
ureaplasma, and other anaerobes can populate the vaginal tract
without difficulty.
[0005] Currently approved treatments for BV include administering
metronidazole ("MTZ") 500 mg orally twice a day for 7 days;
administering MTZ gel 0.75%, one full applicator (5 g)
intravaginally, once a day for 5 days; administering clindamycin
cream 2%, one full applicator (5 g) intravaginally at bedtime for 7
days; administering tinidazole, 2 g orally once daily for 2 days;
or administering tinidazole, 1 g orally once daily for 5 days.
[0006] Although MTZ remains the primary choice for treating BV in
the United States, tinidazole is particularly useful for treating
MTZ-resistant G. vaginalis, an organism commonly associated with
BV. Despite this utility, tinidazole must be dosed orally, whereas
MTZ can be dose intravaginally. Common side effects associated with
the oral administration of tinidazole include, but are not limited
to, metallic/bitter taste, nausea, anorexia,
dyspepsia/cramps/epigastric discomfort, vomiting, constipation,
tongue discoloration, stomatitis, diarrhea, decreased appetite, and
flatulence.
[0007] Thus, there exists a need for novel tinidazole formulations
suitable for the treatment of BV and other diseases of the vaginal
cavity that respond to or are susceptible to tinidazole (e.g.
trichomoniasis) that reduce or alleviate many of the side effects
commonly associated with the oral form of the drug. The present
disclosure provides such formulations.
BRIEF SUMMARY OF THE INVENTION
[0008] The present disclosure provides formulations suitable for
the intravaginal delivery of tinidazole to a subject in need
thereof, the formulations comprising water, tinidazole, a
thermoreversible gelling agent, one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols, a solubility enhancer, and,
optionally, one or more preservatives.
[0009] In some embodiments, the thermoreversible gelling agent is
triblock copolymer having a central hydrophobic block flanked on
each side with a hydrophilic block. In certain embodiments, the
hydrophobic block is polypropylene oxide). In some embodiments, the
hydrophilic block is poly(ethylene oxide).
[0010] In some embodiments, the thermoreversible gelling agent is
poloxamer 407.
[0011] In some embodiments, the one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols comprise a first
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol and a second
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol. In some
embodiments, the first and second pharmaceutically acceptable
C.sub.1-C.sub.7 alcohols are independently selected from the group
consisting of methanol, ethanol, isopropanol, propylene glycol,
2-(2-ethoxyethoxy)ethanol, benzyl alcohol, and combinations
thereof.
[0012] In some embodiments, the first pharmaceutically acceptable
C.sub.1-C.sub.7 alcohol is benzyl alcohol.
[0013] In certain embodiments, the second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is isopropanol.
[0014] In some embodiments, the isopropanol is a 60% (w/w) solution
in water.
[0015] In some embodiments, the solubility enhancer is selected
from the group consisting of mono- and di-alkyl ethers of
isosorbide. In some embodiments, the mono- or di-alkyl ether of
isosorbide is dimethyl isosorbide.
[0016] In certain embodiments, the water comprises a mixture of
sterile deionized water and a buffered aqueous solution. In certain
embodiments, the buffered aqueous solution is citrate buffer.
[0017] In some embodiments, the formulation has a buffered pH
selected from the group consisting of about 3 to about 5, about 3.5
to about 4.5, and about 4 to about 4.5.
[0018] In certain embodiments, the tinidazole comprises from about
0.1 to about 2% (w/w) of the formulations and in certain
embodiments, the tinidazole comprises about 1%, about 1.25%, or
about 1.5% (w/w) of the formulations.
[0019] In some embodiments, the formulations comprise about 20%
poloxamer 407 (w/w), about 49.6% citrate buffer (w/w), about 3.1%
benzyl alcohol (w/w), about 12% (w/w) of a 60% (w/w) solution of
isopropanol in water, about 13.8% dimethylisosorbide (w/w), and
about 1.5% tinidazole.
[0020] In other embodiments, the formulations comprise about 19%
poloxamer 407 (w/w), about 53% citrate buffer (w/w), about 2.9%
benzyl alcohol (w/w), about 11% (w/w) of a 60% (w/w) solution of
isopropanol in water, about 12.7% dimethylisosorbide (w/w), and
about 1.25% tinidazole.
[0021] In still further embodiments, the formulations comprise
about 18% poloxamer 407 (w/w), about 57% citrate buffer (w/w),
about 2.5% benzyl alcohol (w/w), about 10% of a 60% (w/w) solution
of isopropanol in water, about 11.5% dimethylisosorbide (w/w), and
about 1% tinidazole.
[0022] In other embodiments, the present disclosure provides
methods for treating bacterial vaginosis, trichomoniasis, or other
diseases of the vaginal cavity susceptible to tinidazole,
comprising intravaginally administering to a subject in need
thereof a formulation comprising water, tinidazole, a
thermoreversible gelling agent, one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols, a solubility enhancer, and,
optionally, one or more preservatives.
[0023] In certain embodiments of the methods described herein, the
thermoreversible gelling agent is triblock copolymer having a
central hydrophobic block flanked on each side with a hydrophilic
block. In certain embodiments of the methods described herein, the
hydrophobic block is polypropylene oxide). In some embodiments of
the methods described herein, the hydrophilic block is
poly(ethylene oxide). In certain embodiments of the methods
described herein, the thermoreversible gelling agent is poloxamer
407.
[0024] In certain embodiments of the methods described herein, the
one or more pharmaceutically acceptable C.sub.1-C.sub.7 alcohols
comprises a first pharmaceutically acceptable C.sub.1-C.sub.7
alcohol and a second pharmaceutically acceptable C.sub.1-C.sub.7
alcohol. In some embodiments of the methods described herein, the
first and second pharmaceutically acceptable C.sub.1-C.sub.7
alcohols are selected from the group consisting of methanol,
ethanol, isopropanol, propylene glycol, 2-(2-ethoxyethoxy)ethanol,
benzyl alcohol, and combinations thereof. In other embodiments of
the methods described herein, the first pharmaceutically acceptable
C.sub.1-C.sub.7 alcohol is benzyl alcohol. In some embodiments of
the methods described herein, the second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is isopropanol. In certain
embodiments of the methods described herein, the isopropanol is a
60% (w/w) solution in water.
[0025] In some embodiments of the methods described herein, the
solubility enhancer is selected from the group consisting of mono-
and di-alkyl ethers of isosorbide. In some embodiments, the mono-
or di-alkyl ether of isosorbide is dimethyl isosorbide.
[0026] In certain embodiments of the methods described herein, the
water comprises a mixture of sterile deionized water and a buffered
aqueous solution. In certain embodiments of the methods described
herein, the buffered aqueous solution is citrate buffer.
[0027] In some of the embodiments of the methods disclosed herein,
the formulation has a buffered pH selected from the group
consisting of about 3 to about 5, about 3.5 to about 4.5, and about
4.
[0028] In certain embodiments of the methods described herein, the
tinidazole comprises from about 0.1% to about 2% (w/w) of the
formulation. In other embodiments of the methods described herein,
the tinidazole comprises about 1%, about 1.25%, or about 1.5% (w/w)
of the formulation.
[0029] In certain embodiments of the methods described herein, the
subject is a human.
[0030] The present disclosure also relates to a pharmaceutical
composition comprising: about 18-20% poloxamer 407 (w/w), about
49.6-57% citrate buffer (w/w), about 2.5-3.1% benzyl alcohol (w/w),
about 10-12% (w/w) of a 60% (w/w) solution of isopropanol in water,
about 11.5%-13.8% dimethylisosorbide (w/w), and about 1-1.5%
tinidazole (w/w) (and/or an active metabolite thereof), for
treating bacterial vaginosis, trichomoniasis, or other disease(s)
of the vaginal cavity susceptible to tinidazole.
[0031] In certain embodiments the pharmaceutical composition exists
as a viscous liquid at room temperature and gels at or close to
body temperature.
[0032] The present disclosure further relates to the use of the
formulations as described herein for treating bacterial vaginosis,
trichomoniasis, or other disease(s) of the vaginal cavity
susceptible to tinidazole.
[0033] The present disclosure relates also to the use of the
formulations as described herein for the manufacture of a
medicament for the treatment of bacterial vaginosis,
trichomoniasis, or other disease of the vaginal cavity susceptible
to tinidazole.
[0034] An additional aspect of the present disclosure relates to a
method for treating a vaginal infection comprising intravaginally
administering to the subject a formulation containing about 0.1-2%
tinidazole, or an active metabolite thereof.
[0035] An additional aspect of the present disclosure relates to a
method for administering tinidazole to a human subject, comprising
obtaining a formulation containing about 0.1-2% tinidazole, or an
active metabolite thereof, wherein said formulation exists as a
viscous liquid at room temperature; and intravaginally
administering the formulation to a human subject, wherein the
formulation gels at or close to body temperature.
[0036] In a further embodiment, the present disclosure provides a
formulation suitable for the intravaginal delivery of tinidazole to
a human subject in need thereof, the formulation comprising
tinidazole, wherein the formulation has a Franz cell flux of from
about 2 to about 10 (.mu.mol/cm.sup.2)/ t(h).
[0037] In some embodiments, the Franz cell flux is from about 6 to
about 9 (.mu.mol/cm.sup.2)/ t(h).
[0038] In another embodiment, the present disclosure provides a
formulation suitable for the intravaginal delivery of tinidazole to
a human subject in need thereof, the formulation comprising
tinidazole, wherein the formulation has a mean flux rate in
EpiVaginal.TM. tissue of from about 15 nmol/cm.sup.2/min to about
30 nmol/cm.sup.2/min.
[0039] In certain embodiments, the mean flux rate in EpiVaginal.TM.
tissue is from about 18 nmol/cm.sup.2/min to about 22.5
nmol/cm.sup.2/min.
[0040] The present disclosure also provides a formulation suitable
for the intravaginal delivery of tinidazole to a human subject in
need thereof, the formulations comprising tinidazole, wherein the
formulation has a mean tinidazole receiver fluid concentration at 1
hour of from about 0.5 mM to about 2.5 mM.
[0041] In some embodiments, the formulation has a mean tinidazole
receiver fluid concentration at 1 hour of from about 1.14 mM to
about 1.74 mM.
[0042] The present disclosure also provides a tinidazole gel
formulation suitable for the intravaginal delivery of tinidazole to
a subject in need thereof, the formulation comprising water,
tinidazole, a thermoreversible gelling agent, one or more
pharmaceutically acceptable C.sub.1-C.sub.7 alcohols, a solubility
enhancer, and, optionally, one or more preservatives, the
tinidazole gel formulation prepared by the process of a) dissolving
the thermoreversible gelling agent in the water to form a first
solution; b) combining the one or more pharmaceutically acceptable
C.sub.1-C.sub.7 alcohols, the solubility enhancer, and the
tinidazole to form a second solution; and c) adding the second
solution to the first solution.
[0043] In certain embodiments, the thermoreversible gelling agent
is triblock copolymer having a central hydrophobic block flanked on
each side with a hydrophilic block.
[0044] In certain embodiments, the hydrophobic block is
polypropylene oxide).
[0045] In certain embodiments, the hydrophilic block is
poly(ethylene oxide).
[0046] In certain embodiments, the thermoreversible gelling agent
is poloxamer 407.
[0047] In some embodiments, the one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols comprise a first
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol and a second
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol.
[0048] In some embodiments, the first and second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols are independently selected from
the group consisting of methanol, ethanol, isopropanol, propylene
glycol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, and combinations
thereof.
[0049] In certain embodiments, the first pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is benzyl alcohol.
[0050] In some embodiments, the second pharmaceutically acceptable
C.sub.1-C.sub.7 alcohol is isopropanol. In certain embodiments, the
isopropanol is a 60% (w/w) solution in water.
[0051] In some embodiments, the solubility enhancer is selected
from the group consisting of mono- and di-alkyl ethers of
isosorbide. In certain embodiments, the mono- or di-alkyl ether of
isosorbide is dimethyl isosorbide.
[0052] In some embodiments, the water comprises a mixture of
deionized water and a buffered aqueous solution. In particular
embodiments, the buffered aqueous solution is citrate buffer.
[0053] In certain embodiments, the formulation has a pH of from
about 3 to about 5. In other embodiments, the formulation has a pH
of from about 3.5 to about 4.5.
[0054] The present disclosure further provides a process for the
preparation of a tinidazole gel formulation suitable for the
intravaginal delivery of tinidazole to a human subject in need
thereof, the process comprising a) dissolving a thermoreversible
gelling agent in water to form a first solution; b) combining one
or more pharmaceutically acceptable C.sub.1-C.sub.7 alcohols, a
solubility enhancer, and tinidazole to form a second solution; and
c) adding the second solution to the first solution.
[0055] In certain embodiments, the thermoreversible gelling agent
is triblock copolymer having a central hydrophobic block flanked on
each side with a hydrophilic block.
[0056] In some embodiments, the hydrophobic block is polypropylene
oxide).
[0057] In some embodiments, the hydrophilic block is poly(ethylene
oxide).
[0058] In some embodiments, the thermoreversible gelling agent is
poloxamer 407.
[0059] In some embodiments, the one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols comprise a first
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol and a second
pharmaceutically acceptable C.sub.1-C.sub.7 alcohol.
[0060] In some embodiments, the first and second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols are independently selected from
the group consisting of methanol, ethanol, isopropanol, propylene
glycol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, and combinations
thereof.
[0061] In some embodiments, the first pharmaceutically acceptable
C.sub.1-C.sub.7 alcohol is benzyl alcohol.
[0062] In certain embodiments, the second pharmaceutically
acceptable C.sub.1-C.sub.7 alcohol is isopropanol. In some
embodiments, the isopropanol is a 60% (w/w) solution in water.
[0063] In certain embodiments, the solubility enhancer is selected
from the group consisting of mono- and di-alkyl ethers of
isosorbide. In some embodiments, the mono- or di-alkyl ether of
isosorbide is dimethyl isosorbide.
[0064] In some embodiments, the water comprises a mixture of
deionized water and a buffered aqueous solution. In certain
embodiments, the buffered aqueous solution is citrate buffer.
[0065] In some embodiments, the formulation has a mean tinidazole
receiver fluid concentration at 2 hours of from about 1.5 mM to
about 3.5 mM.
[0066] In other embodiments, the formulation has a mean tinidazole
receiver fluid concentration at 4 hours of from about 3 mM to about
6 mM.
[0067] In other embodiments, the formulation has a mean tinidazole
receiver fluid concentration at 2 hours of from about 2.3 mM to
about 2.6 mM.
[0068] In some embodiments, the formulation has a mean tinidazole
receiver fluid concentration at 4 hours of from about 4 mM to about
5.5 mM.
[0069] The present disclosure further provides a tinidazole gel
formulation prepared by any of the processes described herein.
[0070] The present disclosure further provides a tinidazole gel
formulation that does not inhibit or only minimally inhibits
lactobacillus strains at a tinidazole concentration sufficient to
inhibit Gardnerella vaginalis strains associated with bacterial
vaginosis.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0071] The foregoing summary, as well as the following detailed
description of the embodiments, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustration, the drawings may describe the use of specific
embodiments. It should be understood, however, that the
formulations described herein are not limited to the precise
embodiments discussed or described in the figures.
[0072] FIG. 1 describes the standard for visually evaluating rabbit
vaginal tissue exposed to the formulations described herein.
[0073] FIG. 2 describes the observed effects of vehicle formulation
V1 on external rabbit vaginal tissue.
[0074] FIG. 3 describes the observed effects of vehicle formulation
V2 on external rabbit vaginal tissue.
[0075] FIG. 4 describes the observed effects of vehicle formulation
V3 on external rabbit vaginal tissue.
[0076] FIG. 5 describes the observed effects of formulation F1 on
external rabbit vaginal tissue.
[0077] FIG. 6 describes the observed effects of formulation F2 on
external rabbit vaginal tissue.
[0078] FIG. 7 describes the observed effects of formulation F3 on
external rabbit vaginal tissue.
[0079] FIG. 8 describes the evaluation standard for
histopathological analysis of explanted rabbit vaginal tissue.
[0080] FIGS. 9A and 9B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with vehicle formulation V1.
[0081] FIGS. 10A and 10B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with vehicle formulation V2.
[0082] FIGS. 11A and 11B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with vehicle formulation V3.
[0083] FIGS. 12A and 12B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with formulation F1.
[0084] FIGS. 13A and 13B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with formulation F2.
[0085] FIGS. 14A and 14B describe the observation and results of
histopathological evaluation of rabbit vaginal explants previously
treated with formulation F3.
[0086] FIG. 15 is a graphical representation of the mean.+-.SD of
tinidazole released from formulations F1, F2, and F3 in Franz cell
studies.
[0087] FIG. 16 is a graphical representation of a culture well and
insert used in the EpiVaginal.TM. tissue studies described
herein.
[0088] FIG. 17 is an example of a multi-well culture plate
representative of the types of multi-well culture plates that can
be used in the EpiVaginal.TM. tissue studies described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The articles "a," "an," and "the" are used herein to refer
to one or to more than one (i.e., to at least one) of the
grammatical object of the article. By way of example, "an element"
means one element or more than one element.
[0090] The phrase "substantially free of" as used herein means less
than 5%, less than 2.5%, less than 1%, less than 0.1%, less than
0.01%, or less than 0.001% of a given component in the formulations
described herein.
[0091] As used herein, "administration" or "administering" to a
subject includes, but is not limited to, the act of a physician or
other medical professional prescribing a pharmaceutical composition
of the invention for a subject.
[0092] Throughout the specification, the present disclosure
provides certain ranges and certain sub-ranges within those ranges.
The identification of certain sub-ranges notwithstanding, the
present disclosure should be read to include and disclose all
sub-ranges within a given range (or sub-range). For example, if a
range of 1 to 20 is described herein and a sub-range of 5 to 15 is
also described, the present disclosure should be understood to
include and disclose all other sub-ranges within the originally
defined range and sub-range, e.g., 1 to 5, 7 to 12, 15 to 20, 2 to
18, etc.
[0093] The present disclosure provides novel aqueous tinidazole
formulations suitable for intravaginal administration to a subject
in need thereof. In particular embodiments, the formulations can
comprise water, tinidazole or an active metabolite thereof, a
thermoreversible gelling agent, one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols, a solubility enhancer, and,
optionally, one or more preservatives. In certain embodiments, the
formulations can further include a polyol, such as, but not limited
to polyethylene glycol, an optional penetration enhancer, or an
optional excipient such as HPMC. In particular embodiments, the
formulations can be completely or substantially free of boric acid
and/or ethylenediaminetetraacetic acid (EDTA). In certain
embodiments, the formulations can be free or substantially free of
a second active ingredient, such that tinidazole is the only active
ingredient in the formulations.
[0094] An important characteristic of the formulations described
herein are their thermoreversible nature. In particular
embodiments, the formulations exist as a viscous liquid at room
temperature (i.e. about 20.degree. C. to about 23.degree. C.). At
higher temperatures, though, the formulations can exist as a gel.
In particular embodiments, the formulations can be a gel at or
close to body temperature (i.e. about 35.degree. C. to about
37.degree. C.). Without wishing to be bound to any particular
theory, it is believed that gel formation at higher temperature is
a useful property because it allows the formulations to flow into
the vaginal cavity and to reach the smallest irregularities of the
mucosal surface where they subsequently gel and adhere to
biological surfaces such as the vaginal epithelia.
[0095] Although using thermoreversible gels for the prevention of
STDs was previously disclosed, see, e.g. WO 97/42962 and WO
99/53897 (both of which are hereby incorporated by reference in
their entirety), neither publication teaches the use of tinidazole.
Moreover it has now been unexpectedly discovered that the
percentage of tinidazole in a formulation including a
thermoreversible gelling agent can be significantly increased using
the formulations disclosed herein.
[0096] In certain embodiments, tinidazole can be used in the
formulations described herein at any suitable concentration. In
other embodiments, the concentration of tinidazole can be about
0.1% to about 2% (w/w), about 0.5% to about 2% (w/w), or about
0.75% to about 2% (w/w). In particular embodiments, the
concentration of tinidazole can be about 0.75 to about 1.5% (w/w).
In specific embodiments, the concentration of tinidazole can be
about 1%, about 1.25%, or about 1.5% (w/w). The formulations can
also include an active metabolite of tinidazole at any suitable
concentration. Known active metabolites of tinidazole include, but
are not limited to
1-(2-(ethylsulfonyl)ethyl)-2-methyl-4-nitro-1H-imidazol-5-ol and
2-hydroxymethyl tinidazole.
[0097] In certain embodiments, the thermoreversible gelling agent
can be present in the formulations described herein at any suitable
concentration. In certain embodiments, the thermoreversible gelling
agent can be at a concentration of about 5% to about 50% (w/w) and
in certain embodiments, at a concentration of about 15% to about
35% (w/w). In particular embodiments, the thermoreversible gelling
agent can be present at a concentration of about 15% to about 25%
(w/w). In certain embodiments, the thermoreversible gelling agent
can be present in an amount ranging from about 15% to about 25%
(w/w); in an amount ranging from about 17% to about 22% (w/w); or
in an amount ranging from about 18% to about 20% (w/w). In certain
embodiments, the thermoreversible gelling agent can be present at a
concentration of about 18% (w/w), about 19% (w/w), or about 20%
(w/w). Exemplary thermoreversible gelling agents include, but are
not limited to, triblock copolymers having a central hydrophobic
block flanked on each side with a hydrophilic block. In certain
embodiments the hydrophobic block can be a block of polypropylene
oxide). In certain embodiments, the hydrophilic blocks can be
blocks of poly(ethylene oxide). In particular embodiments, the
thermoreversible gelling agent can be selected from the group
consisting of poloxamer 407 (CAS 9003-11-6), poloxamer 124,
poloxamer 188, poloxamer 237, poloxamer 338. In specific
embodiments, the thermoreversible gelling agent is poloxamer
407.
[0098] The one or more pharmaceutically acceptable C.sub.1-C.sub.7
alcohols can be present in the formulations at any suitable
concentration and can include any appropriate straight chain,
branched, cyclic, or aromatic alcohol having the designated number
of carbons. Exemplary pharmaceutically acceptable C.sub.1-C.sub.7
alcohols include, but are not limited to, ethanol, isopropanol,
propylene glycol, 2-(2-ethoxyethoxy)ethanol, and benzyl alcohol. In
certain embodiments, the one or more pharmaceutically acceptable
C.sub.1-C.sub.7 alcohols can be present at a concentration of about
2% to about 20% (w/w). In other embodiments, the one or more
pharmaceutically acceptable C.sub.1-C.sub.7 alcohols can be present
at a concentration of about 5% to about 15% (w/w). In particular
embodiments, the one or more pharmaceutically acceptable
C.sub.1-C.sub.7 alcohols can be present from about 8% to about 11%
(w/w).
[0099] In certain embodiments, the one or more pharmaceutically
acceptable C.sub.1-C.sub.7 alcohols can include one to ten, i.e. 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 pharmaceutically acceptable
C.sub.1-C.sub.7 alcohols. In certain embodiments, the formulations
can include a first C.sub.1-C.sub.7 alcohol and a second
C.sub.1-C.sub.7 alcohol. In certain embodiments the first
C.sub.1-C.sub.7 alcohol can be present at a concentration of about
2.25% to about 3.25% (w/w). In certain embodiments, the first
C.sub.1-C.sub.7 alcohol can be present in an amount of about 2.5%,
about 2.9%, or about 3.2% (w/w). In other embodiments, the second
C.sub.1-C.sub.7 alcohol can be present at a concentration of about
10% to about 12% (w/w), and in certain embodiments can be about 6%,
about 6.5%, or about 7.2% (w/w) of the formulation. In certain
embodiments, the first C.sub.1-C.sub.7 alcohol can be benzyl
alcohol. In certain embodiments, the second C.sub.1-C.sub.7 alcohol
can be isopropyl alcohol. In certain embodiments, the isopropyl
alcohol can be a 60% solution (w/w) in water.
[0100] The formulations can also include a solubility enhancer,
useful for enhancing the solubility of tinidazole in the
formulation. Exemplary solubility enhancers include, but are not
limited to, mono and di-alkyl ethers of isosorbide. In particular
embodiments, the solubility enhancer is dimethylisosorbide ("DMI").
The solubility enhancer can be present in the formulations at any
suitable concentration; however, in certain embodiments it can be
present at a concentration of about 10% to about 15% (w/w). In
other embodiments, the solubility enhancer can be present at a
concentration of about 11.5%, about 12.7%, or about 14% (w/w).
[0101] The formulations can further include an optional penetration
enhancer. The optional penetration enhancer can be used in the
formulations in any suitable concentration; however in certain
embodiments, it can be present in the formulations in a range of
about 0.01% to 15% (w/w). Exemplary penetration enhancers include,
but are not limited to terpenes and terpenoids (such as menthol and
the like); pyrrolidones (such as N-methyl-2-pyrrolidone,
1-dodecylazacycloheptan-2-one, and the like); sulfoxides (such as
DMSO and the like); phospholipids; cyclodextrins such as, but not
limited to, .beta.-cyclodextrin; dodecyl-N,N-dimethylamino acetate;
clofibric acid; and amino acid derivatives such as, but not limited
to, dodecyl N,N-dimethylamino isopropionate. While in certain
embodiments the optional penetration enhancer can be present, in
other embodiments, the penetration enhancer can be completely or
substantially absent.
[0102] The formulations can optionally include one or more
preservatives in a pharmaceutically acceptable amount. Suitable
exemplary preservatives include, but are not limited to,
methylparaben, propylparaben, BHA, BHT, and combinations of the
foregoing. In certain embodiments, the formulations can comprise
less than about 1% (w/w), less than about 0.5% (w/w), or less than
about 0.05% (w/w) of the formulation.
[0103] In typical embodiments, the remainder of the formulations
comprises water. As used herein "water" refers to sterile deionized
water, buffered aqueous solutions comprising sterile deionized
water, or any combination of the foregoing. The amount of water in
the formulations can be at least about 10%, 20%, 30%, 40%, 45%,
47%, 50%, 55%, 58%, 60%, 61%, 65% or at least about 70% (w/w). In
certain embodiments, the amount of water in the formulations can
range from about 40% (w/w) to about 70% (w/w), from about 50% to
about 70% (w/w), or from about 50% to about 65% (w/w). In
particular embodiments, the amount of water in the formulations can
be about 54.4% (w/w), about 57.6% (w/w), or about 61% (w/w).
[0104] Buffered aqueous solutions can contain one or more
pharmaceutically acceptable buffering agents suitable for
maintaining the pH of the formulations in a range of about 3 to
about 5, and in certain embodiments, about 3.5 to about 4.5.
Suitable buffering agents and systems are well known to those of
ordinary skill in the art; however in certain embodiments, the
buffering agent is a citrate buffer. Other possible buffering
agents include, but are not limited to, veronal acetate buffer, and
acetate buffer. In particular embodiments, the pH of the
formulations is buffered to a pH of about 4 to about 4.5.
[0105] The viscosity of the formulations can range from about 1 cP
to about 100,000 cP, about 1 cP to about 50,000 cP, about 1 cP to
about 10,000 cP, or about 1 cP to about 1,000 cP in its ungelled
state when measured using a Brookfield DV-II+ Pro Extra LV.TM.
viscometer with spindle type 63 at 100 RPM at about 21.degree. C.
to about 22.degree. C. In particular embodiments, the ungelled
formulations can have a viscosity of about 1 to about 900 cP. In
certain embodiments, the ungelled formulations can have a viscosity
of about 200 cP to about 750 cP, and in other embodiments, the
ungelled formulations can have a viscosity of about 300 cP to about
550 cP. In particular embodiments, the composition can have a
viscosity of about 315 cP to about 345 cP. In other embodiments,
the ungelled formulations can have a viscosity of about 420 cP to
about 460 cP. In still other embodiments, the formulations can have
a viscosity in the ungelled state of about 480 cP to about 550
cP.
[0106] In certain embodiments, the formulations can have a
temperature of gelation (T.sub.gel) of about 18 to about 37.degree.
C. In other embodiments, the formulations can have a T.sub.gel of
about 22 to about 35.degree. C. In still further embodiments, the
formulations can have a T.sub.gel of about 30.degree. C. to about
35.degree. C. And in still other embodiments, the formulations can
have a T.sub.gel of about 32.degree. C.
[0107] In addition to providing the formulations discussed above,
the present disclosure also provides methods for treating BV,
trichomoniasis, or other disease or disorder suitable for treatment
via the intravaginal administration of a tinidazole. Suitable
methods for treating BV include intravaginally administering the
formulations to a subject in need thereof. Exemplary subjects
include mammals, preferably humans. In certain embodiments, the
formulations can be administered intravaginally once a day for a
period of up to about 30 days. However, in other embodiments, the
formulations can be administered chronically over a period of
months or years.
[0108] In certain embodiments, the formulations can be administered
intravaginally twice a day for a period of up to about 30 days. In
specific embodiments, the formulations can be administered
intravaginally once or twice a day for a period of 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the
formulations can be administered intravaginally once or twice a day
every other day, once or twice a day every third day, once or twice
a day every fourth day, once or twice a day every fifth day, once
or twice a day every sixth day, once or twice a day once a week,
once or twice a day one time every other week, once or twice a day
one time every third week, once or twice a day once a month. In
certain embodiments, the formulations can be administered
intravaginally in a single application, i.e., in the substantial
absence of further dosing until such time as symptoms recur or
until the subject is directed by a physician to resume
treatment.
[0109] The present formulations also have surprising stability at
low temperature. For example, in certain embodiments, the
formulations will show little or no tinidazole crystallization when
chilled to as low as about 6.degree. C. for 24 hours and then
warmed to 22 C for 24 hours. In other embodiments, the formulations
will show little or no tinidazole crystallization when chilled to
as low as about 14.degree. C. for 24 hours and then warmed to
22.degree. C. for 24 hours.
[0110] The present formulations similarly exhibit unexpected
long-term stability at 22.degree. C., showing little or no
tinidazole crystallization at 1, 2, and in certain embodiments 2.6
or 2.8 months. In other embodiments the formulations can be stable
for at least about 10 months, at least about 12 months, and in
other embodiments, at least about 18 months.
[0111] The present formulations have similarly shown a surprising
lack of irritation in a rabbit vaginal model, as described in
detail below.
[0112] In addition to the properties noted above, the formulations
described herein can exhibit a "Franz cell flux." As used herein, a
"Franz cell flux" refers to the rate at which tinidazole in the
formulations described herein crosses a polycarbonate membrane
having 0.40 .mu.m pores and an area of 1.77 cm.sup.2 in a Franz
cell diffusion study at 37.degree. C. wherein the receiver fluid is
0.05 M citrate buffer (pH 3.3). In some embodiments, Franz cell
flux can range from about 2 to about 15 (.mu.mol/cm.sup.2)/ t,
where t is measured in hours (" t(h)"). In other embodiments, the
Franz cell flux can range from about 4 to about 10
(.mu.mol/cm.sup.2)/ t. In still further embodiments, the Franz cell
flux can range from about 6 to about 9 (.mu.mol/cm.sup.2)/ t(h). In
particular embodiments, the Franz cell flux can be about 6, about
7.5, or about 8.7 (.mu.mol/cm.sup.2)/ t(h). In particular
embodiments, the formulations can be any of formulations F1, F2, or
F3 described elsewhere herein.
[0113] In other embodiments, Franz cell flux can range from 2 to 15
(.mu.mol/cm.sup.2)/ t(h). In other embodiments, the Franz cell flux
can range from 4 to 10 (.mu.mol/cm.sup.2)/ t(h). In still further
embodiments, the Franz cell flux can range from 6 to 9
(.mu.mol/cm.sup.2)/ t(h). In particular embodiments, the Franz cell
flux can be 6, 7.5, or 8.7 (.mu.mol/cm.sup.2)/ t(h). In particular
embodiments, the formulations can be any of formulations F1, F2, or
F3 described elsewhere herein.
[0114] The formulations described herein can further exhibit a mean
flux rate in EpiVaginal.TM. tissue culture. The "mean flux rate in
EpiVaginal.TM. tissue culture" refers to the rate at which
tinidazole in a 100 .mu.L aliquot of the formulations described
herein at 37.degree. C. crosses a full thickness tissue culture
produced from normal human-derived vaginal-ectocervical epithelial
cells (VEC-100-FT EpiVaginal.TM. tissue available from MatTek
Corporation, Ashland, Mass.), mounted on polycarbonate membranes
having 0.4 .mu.m pores, wherein the culture medium (alternatively
referred to as the receiver fluid) is 0.5 ml VEC-100-ASY available
from MatTek Corporation (Ashland, Mass.).
[0115] In particular embodiments, the mean flux rate
(nmol/cm.sup.2/min) can range from about 15 nmol/cm.sup.2/min to
about 30 nmol/cm.sup.2/min. In other embodiments, the mean flux
rate can range from about 15 nmol/cm.sup.2/min to about 25
nmol/cm.sup.2/min. In other embodiments, the mean flux rate can
range from about 17 nmol/cm.sup.2/min to about 25
nmol/cm.sup.2/min. In other embodiments, the mean flux rate can
range from about 18 nmol/cm.sup.2/min to about 22.5
nmol/cm.sup.2/min. In particular embodiments, the mean flux rate
can be about 18, about 19, or about 22.5 nmol/cm.sup.2/min.
[0116] In other embodiments, the mean flux rate (nmol/cm.sup.2/min)
can range from 15 nmol/cm.sup.2/min to 30 nmol/cm.sup.2/min; from
15 nmol/cm.sup.2/min to 25 nmol/cm.sup.2/min; from 17
nmol/cm.sup.2/min to 25 nmol/cm.sup.2/min; or from 18
nmol/cm.sup.2/min to 22.5 nmol/cm.sup.2/min. In particular
embodiments, the mean flux rate can be 18, 19, or 22.5
nmol/cm.sup.2/min.
[0117] The formulations described herein can also have a mean
tinidazole receiver fluid concentration. The "mean tinidazole
receiver fluid concentration" is the concentration of tinidazole in
receiver fluid at a given time point after application of a 100
.mu.L aliquot of the formulations described herein at 37.degree. C.
to full thickness tissue culture produced from normal human-derived
vaginal-ectocervical epithelial cells (VEC-100-FT EpiVaginal.TM.
tissue available from MatTek Corporation, Ashland, Mass.), mounted
on polycarbonate membranes having 0.4 .mu.m pores, wherein the
culture medium/receiver fluid is VEC-100-ASY available from MatTek
Corporation (Ashland, Mass.).
[0118] In particular embodiments, the mean tinidazole receiver
fluid concentration after 1 hour can be about 0.5 mM to about 2.5
mM. In other embodiments, the mean tinidazole receiver fluid
concentration after 1 hour can be about 1 mM to about 2 mM. In
still other embodiments, the mean tinidazole receiver fluid
concentration after 1 hour can be about 1.14 mM to about 1.74 mM.
In particular embodiments, the mean tinidazole receiver fluid
concentration after 1 hour can be about 1.14 mM, about 1.5 mM, or
about 1.74 mM.
[0119] In other embodiments, the mean tinidazole receiver fluid
concentration after 1 hour can range from 0.5 mM to 2.5 mM; from 1
mM to 2 mM; or from 1.14 mM to 1.74 mM. In particular embodiments,
the mean tinidazole receiver fluid concentration after 1 hour can
be 1.14 mM, 1.5 mM, or 1.74 mM.
[0120] In particular embodiments, the mean tinidazole receiver
fluid concentration after 2 hours can be about 1.5 mM to about 3.5
mM. In other embodiments, the mean tinidazole receiver fluid
concentration after 2 hours can be about 2 mM to about 3 mM. In
still other embodiments, the mean tinidazole receiver fluid
concentration after 2 hours can be about 2.3 mM to about 2.6 mM. In
particular embodiments, the mean tinidazole receiver fluid
concentration after 2 hours can be about 2.3 mM, about 2.9 mM, or
about 2.6 mM.
[0121] In other embodiments, the mean tinidazole receiver fluid
concentration after 2 hours can range from 1.5 mM to 3.5 mM; from 2
mM to 3 mM; or from 2.3 mM to 2.6 mM. In particular embodiments,
the mean tinidazole receiver fluid concentration after 2 hours can
be 2.3 mM, 2.9 mM, or 2.6 mM.
[0122] In other embodiments, the mean tinidazole receiver fluid
concentration after 4 hours can be about 3.5 mM to about 6 mM. In
other embodiments, the mean tinidazole receiver fluid concentration
after 4 hours can be about 4 mM to about 5.5 mM. In particular
embodiments, the mean tinidazole receiver fluid concentration after
4 hours can be about 4.4 mM, about 5.6 mM, or about 5.1 mM.
[0123] In other embodiments, the mean tinidazole receiver fluid
concentration after 4 hours can range from 3.5 mM to 6 mM or from 4
mM to 5.5 mM. In particular embodiments, the mean tinidazole
receiver fluid concentration after 4 hours can be 4.4 mM, 5.6 mM,
or 5.1 mM.
[0124] The formulations described herein can also have a
concentration dependent mean peak tinidazole concentration in
EpiVaginal.TM. tissue ("mean peak concentration"). The mean peak
concentration is the average maximum concentration of tinidazole in
full thickness tissue culture produced from normal human-derived
vaginal-ectocervical epithelial cells (VEC-100-FT EpiVaginal.TM.
tissue available from MatTek Corporation, Ashland, Mass.) after
application of a 100 .mu.L aliquot of the formulations described
herein at 37.degree. C. for a period of time sufficient to achieve
the maximum tinidazole concentration in the tissue, wherein the
tissue is mounted on polycarbonate membranes having 0.4 .mu.m
pores, wherein the culture medium/receiver fluid is VEC-100-ASY
available from MatTek Corporation (Ashland, Mass.).
[0125] In particular embodiments, the mean peak concentration can
range from about 1500 ng tinidazole/mg tissue to about 4500 ng
tinidazole/mg tissue. In other embodiments, the mean peak
concentration can range from about 1800 to about 4200 ng
tinidazole/mg tissue. In still further embodiments, the mean peak
concentration can range from about 2000 ng tinidazole/mg tissue to
about 4000 ng tinidazole/mg tissue. In certain embodiments, the
mean peak concentration can be about 2000 ng tinidazole/mg tissue,
about 2700 ng tinidazole/mg tissue, or about 3400 ng tinidazole/mg
tissue.
[0126] In other embodiments, the mean peak concentration can range
from 1500 ng tinidazole/mg tissue to 4500 ng tinidazole/mg tissue.
In other embodiments, the mean peak concentration can range from
1800 to 4200 ng tinidazole/mg tissue. In still further embodiments,
the mean peak concentration can range from 2000 ng tinidazole/mg
tissue to 4000 ng tinidazole/mg tissue. In certain embodiments, the
mean peak concentration can be 2000 ng tinidazole/mg tissue, 2700
ng tinidazole/mg tissue, or 3400 ng tinidazole/mg tissue.
[0127] In a further embodiment, the formulation can have a mean
peak tinidazole concentration that satisfies the formula:
y=2834x-804.83
over a range of tinidazole concentrations of from about 0.8% (w/w)
to about 1.8% (w/w), wherein y is the mean peak concentration of
tinidazole measured in the culture and x is the concentration of
tinidazole in the formulation.
Examples
[0128] The formulations and methods are now further detailed with
reference to the following examples. These examples are provided
for the purpose of illustration only and the formulations and
methods described herein should in no way be construed as being
limited to these examples. Rather, the formulations should be
construed to encompass any and all variations which become evident
as a result of the teaching provided herein.
[0129] The 60% IPA solution (w/w) described below was prepared by
adding 600 g of IPA USP and 400 g of water USP to a 1.0 L vessel
and mixing the combined solution well.
[0130] The 0.05 M citric acid buffer solution used and described in
the examples below was prepared as follows. 21.016 g of citric acid
monohydrate was added to a 2.0 L vessel with 2000 mL of water
("solution 1"). In a separate 2 L vessel, 25.806 g of sodium
citrate (anhydrous) was mixed with 2000 mL of water until the
material was dissolved ("solution 2"). Finally, 550 mL of solution
1 and 450 mL of solution 2 were combined and mixed in an
appropriately sized vessel. The pH of the resulting solution was
measured at 3.30 using a standard pH electrode.
Example 1: Tinidazole Gel Formulations
[0131] Formulations having 1%, 1.25%, and 1.5% tinidazole as shown
in Table 1, below, were prepared according to the following general
procedure.
[0132] An appropriate quantity of poloxamer 407 and citrate buffer
were combined in an appropriate vessel to give a suspension. The
vessel was then placed in an ice-water bath and the suspension was
mixed at 400 rpm for 6 hours to dissolve all of the poloxamer. Upon
complete dissolution, the clear solution was warmed to room
temperature (22.degree. C.) and this temperature was maintained
with a water bath.
[0133] Next, the appropriate quantities of 60% IPA, benzyl alcohol,
DMI and tinidazole were combined in an appropriately sized vessel.
The resulting mixture was then heated at 37.degree. C. for
approximately 30 minutes until all of the tinidazole was dissolved.
Once dissolved, the solution was then cooled to room temperature
and added slowly and with agitation to the poloxamer solution. The
resulting mixture was then stirred at low RPM until uniform. After
stirring, the mixing apparatus was removed and the resulting
material was allowed to settle for 24 hours to allow trapped air to
bubble to the surface.
TABLE-US-00001 TABLE 1 Citrate Benzyl Poloxamer 407 Buffer Alcohol
60% IPA DMI Tinidazole Formulation (g)/ (g)/ (g)/ (g)/ (g)/ (g)/ #
(% w/w) (% w/w) (% w/w) (% w/w) (% w/w) (% w/w) F1 10.00 24.79 1.56
6.00 6.90 0.750 (20.00) (49.58) (3.12) (12.00) (13.80) (1.50) F2
9.50 26.62 1.43 5.50 6.33 0.625 (19.00) (53.24) (2.86) (11.00)
(12.6) (1.25) F3 9.00 28.50 1.25 5.00 5.75 0.500 (18.00) (57.00)
(2.50) (10.00) (11.50) (1.00)
Example 2
[0134] Formulations 4 and 5, described in Table 2, below, were
prepared according to the following general procedure.
[0135] An appropriate quantity of poloxamer 407 and citrate buffer
were combined in an appropriate vessel to give a suspension. The
vessel was then sealed and placed on a shaker rotating at
.about.160 rpm in a cold room at 4.degree. C. and was left shaking
overnight until complete dissolution of the poloxamer was observed.
The resulting clear solution was allowed to stand at 4.degree. C.
for a few hours to remove air bubbles present in the solution. The
poloxamer solution was then placed in an incubator at 18.degree. C.
for approximately 30 min.
[0136] Next, the appropriate quantities of DMI, 70% IPA, benzyl
alcohol, and tinidazole were combined in an appropriately sized
vessel. The resulting mixture was heated at 37.degree. C. for
approximately 10 min until complete dissolution of the tinidazole
was observed. The clear solution was then cooled to room
temperature (22.degree. C.) and was added slowly with agitation
(shaking) to the poloxamer solution at room temperature (22.degree.
C.) until a uniform clear solution was obtained. The final
formulations were allowed to stand at room temperature (22.degree.
C.) to remove air bubbles.
TABLE-US-00002 TABLE 2 26% Formu- Poloxamer 70% Benzyl Citrate
Tini- Tini- lation Gel Soln DMI IPA Alcohol Buffer dazole dazole #
(g) (ml) (ml) (ml) (ml) (mg) % F4 14.23 2.2 2.4 0.55 0.37 250 1.25
F5 14.23 2.0 2.2 0.50 0.87 200 1.00
[0137] The cold temperature stability of formulations 4 and 5 was
tested by cooling the formulations to a specified temperature for
24 hours and then warming the formulations to room temperature
(22.degree. C.) where they were allowed to stand for a further 24
hours. During the temperature challenge, the formulations were
visually evaluated for the formation of crystals either at low
temperature or upon subsequent warming. The results are shown in
Table 3.
TABLE-US-00003 TABLE 3 Temp (.degree. C.) Formulation # 18 14 10 6
F4 No crystals No crystals Crystals Crystals F5 No crystals No
crystals No crystals No crystals
[0138] This data demonstrates that the formulations can be
subjected to relatively cool temperatures and maintain tinidazole
in solution, even at high tinidazole concentrations.
Example 3: In Vitro Drug Release
[0139] To determine whether tinidazole would be released upon
intravaginal administration, an in vitro test (membrane-free model)
simulating formulation erosion in the vaginal cavity was conducted.
Two different tinidazole 1.25% gel formulations were prepared
(F6=18.6% gel, 1.75% hydroxypropyl methylcellulose (HPMC),
Tmelt=31.degree. C.; F7=19.01% gel, no HPMC, Tm=29.degree. C.).
Specifically, 1 g of each formulation was added to a pre-weighed
vial equilibrated at 37.degree. C. Next, 0.5 ml of deionized water
was slowly added to the top of the vial and the vial was allowed to
stand for 2 hours, 6 hours, 24 hours, or 4 days. After the
appropriate amount of time passed, the liquid supernatant sitting
on top of the formulation was carefully removed, weighed, and
analyzed for its tinidazole concentration using standard HPLC
methodology. All studies were conducted in triplicate to ensure
quality data. This membrane-free system is a suitable choice to
mimic natural gel erosion in the vaginal cavity.
[0140] These studies showed that the formulations described herein
are susceptible to gradual erosion/dissolution with minimal or no
agitation.
Example 4: Vaginal Irritation in Rabbits
[0141] Formulations F1, F2, and F3 (1 mL) (as described in Example
1) and Vehicle Formulations V1, V2, and V3 (as described below in
Table 4) were each administered into the vaginal vaults of 3 female
test rabbits (New Zealand White strain) for five consecutive days.
The formulations were administered with a blunt tipped
animal-feeding needle attached to a 3 cc syringe. Control animals
were dosed with 1 mL of a 0.9% saline solution under identical
conditions.
TABLE-US-00004 TABLE 4 Vehicle Formulations Citrate Benzyl 60%
Tini- Poloxamer Buffer Alcohol IPA DMI dazole (g)/ (g)/ (g)/ (g)/
(g)/ (g)/ (% w/w) (% w/w) (% w/w) (% w/w) (% w/w) (% w/w) V1 10.00
40.00 N/A N/A N/A N/A (20.00) (80.00) V2 10.00 25.54 1.56 6.00 6.90
N/A (20.00) (51.10) (3.12) (12.00) (13.80) V3 8.75 30.45 1.13 4.50
5.18 N/A (17.50) (60.90) (2.26) (9.00) (10.36)
[0142] Prior to the first dose of the control, vehicle, or active
formulations, and before each daily dose thereafter, the external
vaginal tissues of each animal was observed for erythema, exudate,
and edema according to the scoring system shown in FIG. 1. The
results of the daily monitoring of the external vaginal tissue are
shown in FIGS. 2 (V1), 3 (V2), 4 (V3), 5 (Formulation 1), 6
(Formulation 2), and 7 (Formulation 3). As can be seen in the
Figures, each of the formulations caused minimal or no visual
changes in the external vaginal tissue of the subject animals.
[0143] Following completion of the protocol, the animals were
sacrificed with sodium pentobarbital and the vaginal tissue of each
animal was carefully removed. The tissue was then observed
macroscopically and scored for irritation and injury to the
epithelial layer. Following macroscopic observation, the tissue was
sectioned to provide the cervical, central, and caudal portions.
The samples were then individually placed in 10% neutral buffered
formalin and submitted for histopathological evaluation. Samples
were evaluated based on the criteria shown in FIG. 8.
[0144] Specific results, corresponding to formulations V1, V2, V3,
F1, F2, and F3 are shown in FIGS. 9A and 9B through FIGS. 14A and
14B, respectively. Overall, each of the vehicle and drug-containing
formulations showed minimal observable effects, suggesting that the
formulations are suitable for intravaginal administration despite
the propensity for the individual components of the formulations to
cause irritation.
Example 5: Isolation and Identification of Bacterial Strains
Associated with Bacterial Vaginosis
[0145] To obtain clinical isolates from women suspected of having
bacterial vaginosis, 192 microscope slides from 192 vaginal swabs
(obtained from women consulting in the Quebec City area) submitted
for Nugent scoring were analyzed. Based on microscopic observations
of bacterial cell morphology and Nugent score, 46 swabs were
selected for bacterial isolation.
[0146] Bacterial isolates were identified by polymerase chain
reaction (PCR) and DNA sequencing of the 16S rRNA gene. A total of
30 swabs were positive for at least one isolate of Bacteroides
spp., Gardnerella vaginalis, Peptostreptococcus spp., Prevotella
spp., Lactobacillus spp., or Mobiluncus spp. Isolates from all
bacterial strains of interest were identified, resulting in 39
strains in total. Isolates were frozen and stored for later
use.
Example 6: Antimicrobial Susceptibility Testing
[0147] Reference type strains (8 strains total) were thawed and
tested against the formulations described herein. After initial
culture of the selected frozen preserved bacteria from Example 5,
and 2 additional passages (sub-cultures)--(total=3 passages) on
blood agar and colony suspension in saline, the saline suspension
was used to inoculate the surface of Brucella Agar with 5% Sheep
Blood. Small (6 mm) discs with 10 .mu.L of formulations F1, F2, or
F3 and control gel formulation were centered on the agar plates and
incubated at 35.degree. C. under anaerobic conditions. The diameter
of the inhibition zone in mm was measured after 48 to 96 hours of
incubation, depending on bacterial strains. Testing was conducted
in triplicate.
[0148] Testing showed that a specific Lactobacillus jensenii strain
and Mobiluncus curtisii subsp. Holmesii strain were not affected by
the presence of formulations F1, F2, and F3. But B. fragilis, G.
vaginalis, L. crispatus, M. mulieris, P. anaerobius, and P. bivia
were each inhibited by the formulations described herein, with
inhibition zones varying from about 20 to about 60 mm. No
inhibition was observed using control formulations F1, F2, and F3
prepared without tinidazole.
Example 7: MIC Determination
[0149] Minimum inhibitory concentrations were determined for 39
clinical isolates and 8 reference strains as described below.
[0150] A hemin stock solution (5 mg/mL) was prepared by dissolving
0.1 g of hemin in 2 mL of 1.0 N NaOH. Next, the volume was adjusted
to 20 mL with distilled water and the mixture was sterilized at
121.degree. C. for 15 minutes.
[0151] A stock vitamin K solution (10 mg/mL) was prepared by
combining 0.2 mL of vitamin K.sub.1 with 20 ml of 95% ethanol. A
working solution (1 mg/ml) was prepared by combining 1 ml of the
stock vitamin K solution with 9 ml sterile deionized water.
[0152] Laked sheep blood was prepared by freezing defibrinated
sheep blood at -80.degree. C. The blood was then thawed slowly at 2
to 8.degree. C. overnight. The thawed blood was mixed thoroughly
before use and warmed in a 48 to 50.degree. C. water bath
immediately before adding to a molten brucella agar base.
[0153] Serial dilutions of formulations F1 (1.5% tinidazole), F2
(1.25% tinidazole), and F3 (1.0% tinidazole) were prepared at the
following concentrations--F3: 1,000 .mu.g/ml; 500 .mu.g/ml; 250
.mu.g/ml; 125 .mu.g/ml; 62.5 .mu.g/ml; 31.25 .mu.g/ml; and 15.63
.mu.g/ml; F2: 1,250 .mu.g/ml; 625 .mu.g/ml; 312.5 .mu.g/ml; 156.25
.mu.g/ml; 78.125 .mu.g/ml; 39.06 .mu.g/ml; and 19.53 .mu.g/ml; F1:
1,500 .mu.g/ml; 750 .mu.g/ml; 375 .mu.g/ml; 187.5 .mu.g/ml; 93.75
.mu.g/ml; 46.875 .mu.g/ml; 23.44 .mu.g/ml; and 11.72 .mu.g/ml.
[0154] Brucella laked blood agar with vitamin K.sub.1 and hemin was
prepared as follows. 28 g of brucella broth powder was combined
with 15 g of agar and 1 ml each of the hemin stock solution and
vitamin K working solution in 1 L distilled water. The solution was
boiled to dissolve the agar and subsequently autoclaved at
121.degree. C. for 15 minutes. The resulting agar solution was then
cooled to about 50.degree. C. and 50 ml of sterile laked sheep
blood was added. Next, 2 ml of each of a given serial dilution of
formulation F1, F2, or F3 (as described above) was combined with 18
ml of the laked sheep blood containing agar solution. The resulting
solution was then added to a 15.times.100 mm Petri dish and allowed
to solidify and prereduce in an anaerobic chamber for at least 24
hours. This gave tinidazole infused agar plates having tinidazole
concentrations of 100 .mu.g/ml, 50 .mu.g/ml, 25 .mu.g/ml, 12.5
.mu.g/ml, 6.25 .mu.g/ml, 3.125 .mu.g/ml, and 1.563 .mu.g/ml (F3);
125 .mu.g/ml, 62.5 .mu.g/ml, 31.25 .mu.g/ml, 15.63 .mu.g/ml, 7.81
.mu.g/ml, 3.91 .mu.g/ml, and 1.95 .mu.g/ml (F2); and 150 .mu.g/ml,
75 .mu.g/ml, 37.5 .mu.g/ml, 18.75 .mu.g/ml, 9.37 .mu.g/ml, 4.69
.mu.g/ml, and 2.34 .mu.g/ml (F1).
[0155] As in Example 6, cultures of the frozen preserved bacteria
from Example 5, were grown on blood agar for 24 to 96 hours.
Colonies of similar morphology were selected and suspended in
prereduced brucella broth to achieve a turbidity equivalent to 0.5
McFarland.
[0156] The resulting suspension was then used to inoculate the
tinidazole infused and control agar plates in triplicate. MIC was
determined by comparing control plates (not infused with
tinidazole) against each of the inoculated tinidazole-infused
plates. MIC was read as the minimum concentration at which a marked
reduction in bacterial growth was observed. The data is reported in
Table 5.
TABLE-US-00005 TABLE 5 Bacteria MIC (.mu.g/ml) Bacteroides fragilis
Less than about 5, and in certain instances, less than about 1.6
Gardnerella vaginalis From less than about 2 to about 150; about 2
to about 125; or about 2 to about 100 Lactobacillus crispatus Less
than about 50, and in certain instances, less than about 30, or
less than about 25 (reference strain) Mobiluncus mulieris From
about 15 to about 25 Peptostreptococcus anaerobius, Prevotella Less
than about 2 bivia, Prevotella disiens, and Prevotella
timonensis
[0157] The MICs for each identified strain of Lactobacillus
gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus
vaginalis, and Mobiluncus curtisii were each greater than 150
.mu.g/ml. The non-reference strain of Lactobacillus crispatus was
similarly unaffected by tinidazole at the highest concentration
tested. This data demonstrates that these strains, and in
particular the lactobacillus strains which are important for
vaginal health, are not inhibited by tinidazole at concentrations
sufficient to inhibit bacterial strains associated with bacterial
vaginosis.
[0158] In addition to testing the formulations including tinidazole
as noted above, formulations equivalent to formulations F1, F2, and
F3 were prepared without tinidazole to determine the anti-microbial
activity attributable to the components of the F1, F2, and F3
formulations. These tinidazole-free formulations were appropriately
diluted and tested against the various identified bacterial
strains. No inhibition of growth for any bacterial isolate was
observed using the tinidazole free formulations.
Example 8: Franz Cell Diffusion Testing
[0159] Formulations F1, F2, and F3 were subjected to Franz cell
diffusion testing. In the Franz cell (PermeGear, Inc. Hellertown,
Pa.), a 0.40 .mu.m pore polycarbonate membrane (Fisher Scientific,
Pittsburgh, Pa.) was used. The membrane area was 1.77 cm.sup.2. A
480 .mu.L sample of each test formulation was loaded in the donor
cell. The receptor cell contained 8 mL of 0.05 M citrate buffer, pH
3.3 ("receiver fluid"). At 0.5, 1, 2, 3, 4, and 6 hours (n=6
replicates), a 500 .mu.l sample was removed from the receptor cell
and replaced with 500 .mu.L of fresh buffer. The study was
conducted at 37.degree. C. Concentration of tinidazole in the
receiver fluid removed from the Franz cell was determined by
LC-MS/MS.
[0160] Drug concentration per unit membrane area was plotted
against the square root of time in hours. FIG. 15 shows the
mean.+-.standard deviation of tinidazole released
(.mu.mol)/cm.sup.2 versus square root of time in hours.
[0161] There was no gel left in any of the chambers at the end of 6
hours. Due to tinidazole saturation in the receiver chambers
occurring around 180 minutes, all in vitro release rates (slopes)
were calculated using only the linear portion of the curve
(R.sup.2.gtoreq.0.9, i.e., samples up to and including 180
minutes). Rate and recovery data are shown in Table 6, below.
TABLE-US-00006 TABLE 6 Rate.sup.a % Recovery.sup.a (at Formulation
([.mu.mol/cm.sup.2/ t(h)]) 6 hours) F3 6.04 .+-. 0.893 98.5 .+-.
5.34 F2 7.56 .+-. 1.32 96.5 .+-. 4.24 F1 8.70 .+-. 0.964 89.6 .+-.
4.51 .sup.a= Mean .+-. Standard Deviation
[0162] As can be seen in Table 6, release rate increased as
tinidazole concentration increased. Based on 90% confidence
intervals computed for the ratios of the median in vitro release
rate for pairs of formulations F1, F2, and F3 (e.g. F1 and F2; F1
and F3; F2 and F3), the F1 and F2 comparison showed these
formulations can be considered to have equivalent release rates
(default "no difference" 90% confidence intervals limits were 75%
to 133.33%). The statistical equivalence of release rates of the F2
and F3 formulations was not determined by the present comparison,
while the difference in release rate between F1 and F3 was
statistically significant.
Example 9: Flux and Tissue Association Using EpiVaginal.TM.
Tissue
[0163] EpiVaginal.TM. tissue (produced from normal human-derived
vaginal-ectocervical epithelial cells) full thickness tissues
(VEC-100-FT) on polycarbonate membranes (NUNC.RTM. single well
tissue culture plate, pore size 0.4 .mu.m) and culture medium
(VEC-100-ASY) were purchased from MatTek Corporation (Ashland,
Mass.).
[0164] The culture medium (VEC-100-ASY) was pre-warmed at
37.degree. C. Under sterile conditions and using sterile forceps,
the EpiVaginal.TM. tissues were transferred into 12-well plates
containing the pre-warmed culture medium. Then, the 12-well plates
containing the EpiVaginal.TM. tissue samples were placed in a
humidified incubator (37.+-.2.degree. C., 5.+-.1% CO.sub.2) for
overnight recovery. The next morning, the culture medium in the
plates was aspirated, and the EpiVaginal.TM. tissues were
transferred into new 24-well plates.
[0165] An aliquot (100 .mu.L) of one of formulation F1, F2, or F3
was added to the apical (AP) side of EpiVaginal.TM. tissue in the
culture insert (see FIG. 16), and 500 .mu.L of the culture medium
was added to the basolateral (BL) side of the insert. For a control
group, an aliquot (100 .mu.L) of culture medium was added to the AP
side of the insert, and 500 .mu.L, of culture medium was added to
the BL side (the "Mediuim" in FIG. 16). All dosing was performed
under sterile conditions in a laminar flow hood.
[0166] The plates containing the tissue were incubated in a
humidified incubator (37.+-.1.degree. C., 5.+-.1% CO.sub.2) during
the study and removed only for assays at 1, 2, 4, 6, 8, 24, or 48
hours. Four replicates were studied at each time point, for a total
of 28 samples per formulation. The noted studies were run in
parallel such that plates assayed at a given time point were not
assayed at any other time point.
[0167] At the completion of each incubation time point, the 500
.mu.L of culture medium on the BL side was analyzed for tinidazole
content using LC-MS/MS. The tinidazole concentrations recorded at
the 1, 2, and 4 hour time points were used to calculate the mean
flux rate in EpiVaginal.TM. tissue culture.
[0168] Simultaneously, the EpiVaginal.TM. tissue culture was
removed from the reactor and the contents in both the AP and BL
sides were carefully aspirated and the tissues were washed twice
with assay media (300 .mu.L) to remove residual formulations. The
rinses were discarded and the tissue was gently blotted on a Kim
wipe and weighed. The tissues were stored at -80.degree. C. until
further analysis, which comprised homogenization with the resulting
tissue lysates analyzed by LC-MS/MS to determine the tissue
tinidazole concentration at each time point.
TABLE-US-00007 TABLE 7 Mean Peak Tinidazole Con- centration in Epi-
Mean Flux Vaginal .TM. Mean.sup.a Tinidazole Rate Tissue at 1
Receiver Fluid Concentration Formu- (nmol/cm.sup.2/ Hour (nM)
lation min) (ng/mg).sup.b 1-Hour 2-Hour 4-Hour F3 18 .+-. 0.993
2,017 .+-. 161 1,144,500 2,290,000 4,400,000 .+-. .+-. .+-. 149,089
303,425 199,499 F2 22.5 .+-. 2.31 2,762 .+-. 533 1,517,500
2,855,000 5,567,500 .+-. .+-. .+-. 207,746 245,289 279,687 F1 19.0
.+-. 3.52 3,434 .+-. 498 1,735,000 2,627,500 5,105,000 .+-. .+-.
.+-. 270,000 257,472 713,559 .sup.an = 4 replicates per treatment
at 1-, 2-, and 4-hour time point. .sup.bMean peak EpiVaginal .TM.
tissue concentrations occurred at the 1-hour time point for all
treatments (4 replicates).
[0169] As can be seen in Table 7, the mean tinidazole tissue flux
rates differed by less than 20% between formulations F1, F2, and
F3, implying that flux rates were not concentration dependent. Even
though flux rates were not concentration dependent,
concentration-dependent increases in the mean maximal
EpiVaginal.TM. tissue-associated tinidazole concentrations were
observed at the 1 hour incubation time point. All of the maximal
tissue-associated tinidazole concentrations exceeded 2,000 ng/mg of
tissue across the 3 formulations evaluated. Additionally, for each
of formulations F1, F2, and F3, the receiver fluid mean tinidazole
concentrations increased approximately proportionally with
incubation times of up to 4 hours duration.
[0170] It is to be understood that the phraseology or terminology
herein is for the purpose of description and not of limitation,
such that the terminology or phraseology of the present
specification is to be interpreted by the skilled artisan in light
of the teachings and guidance.
[0171] The breadth and scope of the present invention should not be
limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
* * * * *