U.S. patent application number 16/627118 was filed with the patent office on 2020-06-04 for high potency stable formulations of vaginal lactobacillus.
The applicant listed for this patent is Osel, Inc.. Invention is credited to Angela Marcobal, Thomas P. Parks.
Application Number | 20200171107 16/627118 |
Document ID | / |
Family ID | 64950361 |
Filed Date | 2020-06-04 |
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United States Patent
Application |
20200171107 |
Kind Code |
A1 |
Parks; Thomas P. ; et
al. |
June 4, 2020 |
HIGH POTENCY STABLE FORMULATIONS OF VAGINAL LACTOBACILLUS
Abstract
This invention provides for a dry preserved formulation of
Lactobacillus suitable for administration to people as a pro-biotic
or live biotherapeutic treatment where the formulation is stable,
has high potency, and contains no animal-derived excipients.
Inventors: |
Parks; Thomas P.; (San
Mateo, CA) ; Marcobal; Angela; (Mountain View,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Osel, Inc. |
Mountain View |
CA |
US |
|
|
Family ID: |
64950361 |
Appl. No.: |
16/627118 |
Filed: |
July 5, 2018 |
PCT Filed: |
July 5, 2018 |
PCT NO: |
PCT/US2018/040884 |
371 Date: |
December 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62529756 |
Jul 7, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 47/02 20130101; A61K 9/10 20130101; A61K 47/22 20130101; C12N
1/20 20130101; A61K 9/19 20130101; A61P 15/02 20180101; C12N 1/04
20130101; A61K 35/747 20130101; A61K 2035/115 20130101; A61K 47/26
20130101; A61K 47/183 20130101; A61K 9/0034 20130101; A61K 47/10
20130101; A61P 31/04 20180101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; C12N 1/20 20060101 C12N001/20; A61K 47/26 20060101
A61K047/26; A61K 47/10 20060101 A61K047/10; A61K 47/22 20060101
A61K047/22; A61K 47/02 20060101 A61K047/02; A61K 47/18 20060101
A61K047/18; A61K 9/19 20060101 A61K009/19; A61K 45/06 20060101
A61K045/06; A61K 9/00 20060101 A61K009/00; C12N 1/04 20060101
C12N001/04; A61P 31/04 20060101 A61P031/04; A61P 15/02 20060101
A61P015/02 |
Claims
1. An aqueous bacterial suspension of vaginal Lactobacillus
species, having no animal-derived excipients, where the suspension
results from a combination of a cell pellet of vaginal
Lactobacillus species with an aqueous preservation medium
consisting essentially of: (i) trehalose at between 5-20%, w/v;
(ii) xylitol at between 2-9%, w/v; (iii) sodium ascorbate 0.5-1.5%,
w/v; and (iv) sodium phosphate at between 10-50 mM.
2. The bacterial suspension of claim 1, wherein the aqueous
preservation medium optionally comprises sodium glutamate at
between 0-5%.
3. The bacterial suspension of claim 1, wherein the vaginal
Lactobacillus species has the ability to produce greater than 0.5
ppm of hydrogen peroxide under effective culture conditions.
4. The bacterial suspension of claim 1, wherein the vaginal
Lactobacillus species is selected from the species consisting of
Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus
gasseri.
5. The bacterial suspension of claim 1, wherein the aqueous
preservation medium consists essentially of: (i) trehalose at
between 5-15%, w/v; (ii) xylitol at between 2-7%, w/v; (iii) sodium
ascorbate 0.5-1.0%, w/v; and (iv) sodium phosphate at between 10-30
mM.
6. The bacterial suspension of claim 5, wherein the aqueous
preservation medium optionally comprises sodium glutamate at
between 0-5%.
7. The bacterial suspension of claim 1, wherein the suspension is
lyophilized to yield a dry powder.
8. The dry powder of claim 7, wherein the powder has a water
activity value of less than 0.220.
9. The dry powder of claim 8, wherein the powder is combined with
an inactive excipient at a ratio of powder: excipient of between
1:1 and 1:10 w/w.
10. The dry powder of claim 9, wherein the excipient is
maltodextrin.
11. The dry powder of claim 9, wherein the ratio of powder:
excipient is between 1:1 and 1:5 w/w.
12. The dry powder of claim 7, wherein the powder is contained
within a plastic housing designed for vaginal administration.
13. A method of preserving Lactobacillus spp. under dry conditions
without animal-derived excipients, the method comprising: (i)
obtaining an aqueous suspension of vaginal Lactobacillus species
having a cell concentration between 10.sup.8 to 10.sup.13 per ml;
(ii) centrifuging the solution to form a bacterial cell pellet; and
(iii) resuspending the bacterial cell pellet in an aqueous
preservation medium consisting essentially of: (a) trehalose at
between 5-20%, w/v; (b) xylitol at between 2-9%, w/v; (c) sodium
ascorbate 0.5-1.5%, w/v; and (d) sodium phosphate at between 10-50
mM; where the weight ratio of cell pellet wet weight (grams) to
preservation media (mL) is between 1:1 and 1:5 grams of cell pellet
to milliliter of preservation media to yield a bacterial
suspension.
14. The method of claim 13, wherein the aqueous preservation medium
optionally comprises sodium glutamate at between 0-5%.
15. The method of claim 13, wherein the vaginal Lactobacillus
species has the ability to produce greater than 0.5 ppm of hydrogen
peroxide under effective culture conditions.
16. The method of claim 13, wherein the vaginal Lactobacillus
species is selected from the species consisting of Lactobacillus
crispatus, Lactobacillus jensenii and Lactobacillus gasseri.
17. The method of claim 13, wherein the aqueous preservation medium
consists essentially of: (a) trehalose at between 5-15%, w/v; (b)
xylitol at between 2-7%, w/v; (c) sodium ascorbate 0.5-1.0%, w/v;
and (d) sodium phosphate at between 10-30 mM.
18. The method of claim 17, wherein the aqueous preservation medium
optionally comprises sodium glutamate at between 0-5%.
19. The bacterial suspension of claim 13, wherein the bacterial
suspension is lyophilized to yield a dry powder.
20. The dry powder of claim 13 wherein the powder has a water
activity value of less than 0.220.
21. The dry powder of claim 20, where the dry powder is combined
with an inactive excipient at a ratio of powder: excipient of
between 1:1 and 1:10 w/w.
22. The dry powder of claim 21, wherein the excipient is
maltodextrin.
23. The dry powder of claim 21, wherein the ratio of
powder:excipient is between 1:1 and 1:5 w/w.
24. A method of treating abnormal vaginal microbiota in a woman
comprising the steps of: (i) selecting a woman having a diagnosis
of abnormal vaginal microbiota; (ii) administering an antibiotic in
an amount effective to reduce the level of abnormal vaginal
microbiota; (iii) following step ii, administering a dry powder
derived from an aqueous bacterial suspension of vaginal
Lactobacillus species with no animal-derived excipients where the
suspension results from a combination of a cell pellet of vaginal
Lactobacillus species with an aqueous preservation medium
consisting essentially of: (a) trehalose at between 5-20%, w/v; (b)
xylitol at between 2-9%, w/v; (c) sodium ascorbate 0.5-1.5%, w/v;
and (d) sodium phosphate at between 10-50 mM.
25. The method of claim 24, wherein the aqueous preservation medium
optionally comprises sodium glutamate at between 0-5%.
26. The method of claim 24, wherein step ii includes daily
administration of antibiotic for between 2 and 7 days and wherein
step iii begins at any time between two days before the completion
of antibiotic administration and two days after the administration
of antibiotic in step ii ends.
27. The method of claim 24, wherein the vaginal Lactobacillus
species has the ability to produce greater than 0.5 ppm of hydrogen
peroxide under effective culture conditions.
28. The method of claim 24, wherein the vaginal Lactobacillus
species is selected from the species consisting of Lactobacillus
crispatus, Lactobacillus jensenii and Lactobacillus gasseri.
29. The dry powder of claim 24, wherein the dry powder has a water
activity value of less than 0.220.
30. The dry powder of claim 29, wherein the dry powder is combined
with an inactive excipient at a ratio of powder: excipient of
between 1:1 and 1:10 w/w.
31. The dry powder of claim 30, wherein the excipient is
maltodextrin.
32. The dry powder of claim 30, wherein the ratio of
powder:excipient is between 1:1 and 1:5 w/w
33. The method of claim 24, wherein the cell pellet is combined
with an aqueous preservation medium consisting essentially of: (a)
trehalose at between 5-15%, w/v; (b) xylitol at between 2-7%, w/v;
(c) sodium ascorbate 0.5-1.0%, w/v; and (d) sodium phosphate at
between 10-30 mM.
34. The method of claim 33, wherein the aqueous preservation medium
optionally comprises sodium glutamate at between 0-5%.
35. The method of claim 24, wherein the antibiotic is clindamycin,
metronidazole, or tinidazole.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Pat. Appl. No. 62/529,756, filed on Jul. 7, 2017, the entire
content of which is incorporated in its entirety herein for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The mucosal membranes of all humans are naturally colonized
by bacterial microbiota. Recent studies have indicated that the
microbiota found in the human gut, mouth and vagina, interact
closely with cells and tissues of the body to regulate natural
biological processes such as non-specific host defense. See, e.g.,
Redondo-Lopez, et al. (1990) Rev. Infect. Dis. 12:856-872; Gilbert,
J. A., et al. Nature 2016 Jul. 7, 535(7610):94-103; McDermott, A.
J., et al. Immunology 2014 May, 142(1):24-31; Nelson, D. B., et al.
Anaerobe 2016 December, 42:67-73. Generally, healthy vaginal
microbiota is dominated by Lactobacillus species, which are gram
positive rods that play an important role in resisting infection
via production of lactic acid and acidification of the vagina, or
by production of other antimicrobial products, such as hydrogen
peroxide (H.sub.2O.sub.2). The species of Lactobacillus most
commonly isolated from the reproductive tracts of healthy women
worldwide include L. crispatus, L. jensenii, L. gasseri, and L.
iners. See, e.g., Antonio et al., (1999) J. Infect. Dis.
180:1950-1956; Vasquez et al., (2002) J. Clin. Microbiol.
40:2746-2749; Vallor, A. C., et al. J Infect Dis. 2001 Dec. 1,
184(11):1431-6; Ravel, J., et al. Proc Natl Acad Sci, USA. 2011
Mar. 15, 108 Suppl 1:4680-7. L. crispatus, L. jensenii, and L.
gasseri are capable of producing H.sub.2O.sub.2, whereas L. iners
strains generally do not produce H.sub.2O.sub.2. These species are
phylogenetically and functionally different from food and/or
environmental Lactobacillus species. These facultative anaerobes
metabolize glucose to lactic acid, contributing to the maintenance
of a low vaginal pH (4.0-4.5) that accounts for a major part of the
non-specific host defense of the vagina. An acidic pH has a
significant antagonistic effect on the growth of opportunistic
commensal and pathogenic organisms, and lactic acid has antiviral
activity against HIV and HSV-2.
[0003] The H.sub.2O.sub.2-producing strains (e.g. L. crispatus and
L. jensenii) are more protective than those that do not produce
H.sub.2O.sub.2 (L. iners). Indeed, it has been demonstrated that
women with vaginal mucosa colonized with sufficient amounts of
protective Lactobacillus spp. have a 50% lower frequency of
gonorrhea, chlamydial infections, trichomoniasis and bacterial
vaginosis. The presence of H.sub.2O.sub.2-producing lactobacilli in
the vagina have been linked to a decreased frequency of bacterial
vaginosis, symptomatic yeast vaginitis and sexually transmitted
pathogens including Neisseria gonorrhea, Chlamydia trachomatis, and
Trichomonas vaginalis. In vitro studies have demonstrated that
H.sub.2O.sub.2-producing lactobacilli have potent bactericidal and
viricidal properties against vaginal pathogens, including human
immunodeficiency virus (HIV). Therefore, beneficial lactobacilli
associated with the vaginal mucosa can be considered to provide a
protective "biofilm". See e.g., Falagas et al., (2006) Drugs,
66:1253-1261.
[0004] Many vaginal and systemic medications may kill vaginal
Lactobacillus, and the depletion of the dominant vaginal
Lactobacillus species leads to a more diverse abnormal microbiota
populated with facultative and strict anaerobes, such as
Gardnerella vaginalis and Atopobium vaginae, higher vaginal pH, and
higher levels of proinflammatory cytokines, which can be associated
with the development of clinical syndromes, such as bacterial
vaginosis (BV), establishment of opportunistic infections, and an
increased risk of acquiring HIV-1 and Herpes simplex virus type 2
(HSV-2) in women. See, e.g., Sha et al. (2005) J. Infect. Dis.
191:25-32; Taha et al. (1998) AIDS 12:1699-1706; Bolton, M., et al.
Sex Trans Dis 2008 March 35(3):214-215 Hence, treatment of sexually
transmitted diseases with antibiotics may place women at increased
risk for repeated acquisition of the diseases. These findings,
along with the widespread belief that lactobacilli generally
promote vaginal health, have suggested to clinicians that women
should recolonize the vagina with Lactobacillus to prevent or treat
urogenital tract infections.
[0005] There has been considerable interest in the development of
non-antibiotic, ecologically appropriate approaches, such as
Lactobacillus Replacement Therapy (LRT) to replenish the healthy
vaginal microbiota and to prevent urogenital infections. The
success of LRT depends in part on selection of an ecologically
appropriate Lactobacillus strain, cell preservation, recovery of
the dry Lactobacillus formulation following rehydration, as well as
the extent and duration of vaginal colonization. Various methods
for administering beneficial bacteria and other substances to the
vaginal mucosa are known. In fact, Lactobacillus products for
intravaginal or oral use have been available for over 100 years in
the form of "acidophilus" preparations available in health food
stores, and acidophilus milk or yogurt bought in grocery stores
(e.g., these products typically advertise the inclusion of a strain
of Lactobacillus acidophilus). These products have included vaginal
tablets, capsules, and vaginal suppositories containing lyophilized
Lactobacillus acidophilus of human origin as well as various
nutritional supplements.
[0006] These products are largely non-efficacious due to the
failure of the products to colonize the vagina with the exogenous
lactobacilli. These failures are often due to the poor quality of
the commercially available products and that Lactobacillus species
contained in probiotics are not of vaginal origin, and thus are not
appropriate for the vagina. It has been documented that
Lactobacillus products sold as foods or as Lactobacillus
supplements are often contaminated with other potential pathogens.
In addition, Lactobacillus obtained from yogurt are unable to bind
to vaginal epithelial cells. The binding of lactobacilli to the
epithelial cells is a necessary step to establish colonization of
the host organism. Furthermore, the low percentage of
physiologically viable cells reflected by the low recovery in
simulated vaginal fluid significantly affects the actual bacterial
dosage.
[0007] Therefore, a product is needed for the treatment of vaginal
infections, which can be manufactured under exacting conditions and
which uses appropriate human strains of Lactobacillus having in
vivo microbicidal properties, adherence to vaginal epithelial
cells, and an effective potency of viable microbes. The present
invention addresses these and other needs.
BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides for an aqueous
bacterial suspension of vaginal Lactobacillus species, which has no
animal-derived excipients. The suspension can result from a
combination of a cell pellet of vaginal Lactobacillus species and
an aqueous preservation medium. The aqueous preservation medium can
be comprised of trehalose at between 5-20% (w/v), xylitol at
between 2-9% (w/v), sodium ascorbate at between 0.5-1.5% (w/v),
sodium phosphate at between 10-50 mM, and, optionally, sodium
glutamate at between 0-5% (w/v).
[0009] In another aspect, the present invention provides for a
method of preserving Lactobacillus spp. under dry conditions, in
the absence of animal-derived excipients. The method involves
obtaining an aqueous suspension of a vaginal Lactobacillus species
having a cell concentration between about 10.sup.8 to about
10.sup.10 per ml, centrifuging the solution to form a bacterial
cell pellet, and resuspending the bacterial cell pellet in an
aqueous preservation medium. The aqueous preservation medium can be
comprised of trehalose at between 5-20% (w/v), xylitol at between
2-9% (w/v), sodium ascorbate at between 0.5-1.5% (w/v), sodium
phosphate at between 10-50 mM, and, optionally, sodium glutamate at
between 0-5% (w/v). The resulting bacterial suspension can have a
weight ratio of the cell pellet wet weight (i.e., grams of cell
pellet following centrifugation and decanting the supernatant) to
preservation media (ml) of between 1:1 and 1:5 grams of pellet to
milliliter of preservation media.
[0010] Another aspect of the present invention provides a method of
treating abnormal vaginal microbiota in women. The method involves
selecting a woman having a diagnosis of abnormal vaginal
microbiota, administering an antibiotic in an amount effective to
reduce the level of abnormal vaginal microbiota, followed by the
administration of a dry powder derived from an aqueous bacterial
suspension of vaginal Lactobacillus species with no animal-derived
excipients. The aqueous suspension is the result of a combination
of a cell pellet of vaginal Lactobacillus species with an aqueous
preservation medium, which can be comprised of trehalose at between
5-20% (w/v), xylitol at between 2-9% (w/v), sodium ascorbate at
between 0.5-1.5% (w/v), sodium phosphate at between 10-50 mM, and,
optionally, sodium glutamate at between 0-5% (w/v).
[0011] In some embodiments, the method of treating abnormal vaginal
microbiota in a woman involves the daily administration of an
antibiotic for between 2 and 7 days and the administration of the
dry powder can begin at any time between two days before the
cessation of the administration of an antibiotic and two days after
the administration of an antibiotic. In some cases, the antibiotic
can be clindamycin, metronidazole or tinidazole.
[0012] The following embodiments can be combined with any of the
above aspects of the invention. For example, in some embodiments,
the vaginal Lactobacillus species of the aqueous bacterial
suspension can produce greater than 0.5 ppm of hydrogen peroxide
under effective culture conditions. In other embodiments, the
vaginal Lactobacillus species can be selected from the species
consisting of Lactobacillus crispatus, Lactobacillus jensenii and
Lactobacillus gasseri.
[0013] In some embodiments of the invention, the aqueous
preservation medium can be comprised of trehalose at between 5-15%
(w/v), xylitol at between 2-7% (w/v), sodium ascorbate at between
0.5-1.0% (w/v), sodium phosphate at between 10-30 mM, and,
optionally, sodium glutamate at between 0-5% (w/v).
[0014] In another embodiment, the bacterial suspension can be
lyophilized to yield a dry powder. The dry powder can have a water
activity value of less than 0.220. In some embodiments, the powder
can be combined with an inactive excipient at a ratio of powder:
excipient of between 1:1 and 1:10 w/w to adjust the concentration
of colony forming units. Alternatively, the dry bacterial powder
can be combined with an excipient blend to adjust the potency of
the final product. For convenience, the dry powder can be contained
within a plastic housing designed for vaginal administration.
[0015] In some embodiments of the invention, the aqueous
preservation medium of the bacterial suspension does not contain
skim milk. In another embodiment, the aqueous preservation medium
of the bacterial suspension does not contain .alpha.-tocopherol. In
some embodiments of the invention, the aqueous preservation medium
of the bacterial suspension does not contain skim milk or
.alpha.-tocopherol.
[0016] In some embodiments of the invention, the aqueous
preservation medium can be comprised of trehalose at between 5-20%
(w/v), xylitol at between 2-9% (w/v), sodium ascorbate at between
0.5-1.5% (w/v), sodium phosphate at between 10-50 mM, and,
optionally, sodium glutamate at between 0-5% (w/v), wherein the
aqueous preservation medium does not contain skim milk. In some
embodiments of the invention, the aqueous preservation medium can
be comprised of trehalose at between 5-20% (w/v), xylitol at
between 2-9% (w/v), sodium ascorbate at between 0.5-1.5% (w/v),
sodium phosphate at between 10-50 mM, and, optionally, sodium
glutamate at between 0-5% (w/v), wherein the aqueous preservation
medium does not contain .alpha.-tocopherol. In some embodiments of
the invention, the aqueous preservation medium can be comprised of
trehalose at between 5-20% (w/v), xylitol at between 2-9% (w/v),
sodium ascorbate at between 0.5-1.5% (w/v), sodium phosphate at
between 10-50 mM, and, optionally, sodium glutamate at between 0-5%
(w/v), wherein the aqueous preservation medium does not contain
skim milk or .alpha.-tocopherol.
[0017] In an aspect, the invention relates to a composition
comprising a dry powder derived from an aqueous bacterial
suspension of vaginal Lactobacillus species with no animal-derived
excipients for use in the treatment of a woman having a diagnosis
of abnormal vaginal microbiota, wherein said woman has previously
been treated with an antibiotic in an amount effective to reduce
the level of abnormal vaginal microbiota; and wherein said aqueous
bacterial suspension results from a combination of a cell pellet of
vaginal Lactobacillus species with an aqueous preservation medium
consisting essentially of: (a) trehalose at between 5-20%, w/v; (b)
xylitol at between 2-9%, w/v; (c) sodium ascorbate 0.5-1.5%, w/v;
and (d) sodium phosphate at between 10-50 mM. In some embodiments,
the aqueous preservation medium optionally comprises sodium
glutamate at between 0-5%.
[0018] Another aspect of the invention relates to a kit of parts
for use in the treatment of a woman having a diagnosis of abnormal
vaginal microbiota, said kit of parts comprising: a first container
comprising one or more antibiotics in an amount effective to reduce
the level of abnormal vaginal microbiota; a second container
comprising an amount of a composition comprising a dry powder
derived from an aqueous bacterial suspension of vaginal
Lactobacillus species with no animal-derived excipients where the
suspension results from a combination of a cell pellet of vaginal
Lactobacillus species with an aqueous preservation medium
consisting essentially of: (a) trehalose at between 5-20%, w/v; (b)
xylitol at between 2-9%, w/v; (c) sodium ascorbate 0.5-1.5%, w/v;
and (d) sodium phosphate at between 10-50 mM; and optionally,
instructions for use of said kit in the treatment of a woman having
a diagnosis of abnormal vaginal microbiota. In some embodiments,
the aqueous preservation medium optionally comprises sodium
glutamate at between 0-5%.
[0019] In an embodiment, said one or more antibiotics are for
administration for e.g. between 2 and 7 days to said woman; and
wherein said dry powder is for administration at any time between
two days before the completion of antibiotic administration and two
days after the administration of antibiotic ends.
[0020] Thus, the kit of parts is preferably intended for sequential
administration of i) the one or more antibiotics followed by ii)
the composition comprising a dry powder derived from an aqueous
bacterial suspension of vaginal Lactobacillus species with no
animal-derived excipients, for use in the treatment of a woman
having a diagnosis of abnormal vaginal microbiota.
[0021] In an embodiment, said vaginal Lactobacillus species has the
ability to produce greater than 0.5 ppm of hydrogen peroxide under
effective culture conditions, wherein the Lactobacillus species is
e.g. selected from a group of species consisting of: Lactobacillus
crispatus, Lactobacillus jensenii and Lactobacillus gasseri.
[0022] It should be noted that embodiments and features described
in the context of one of the aspects of the present invention also
apply to the other aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. Stability of LACTIN-V formulated with skim milk
(lower line, circles) or without skim milk (upper line, inverted
triangles). Powder samples were stored at 25.degree. C. for one
year and activity determined at different time points by culturing
on MRS agar plates and counting colonies.
[0024] FIG. 2. Lactobacillus powder formulations with monosodium
glutamate or without monosodium glutamate. The accelerated
stability of four LACTIN-V formulations without monosodium
glutamate (circles, triangles) or with monosodium glutamate
(squares, inverted triangles) at 37.degree. C. was determined by
measuring viability over time. Powder samples were stored at
37.degree. C. for 90 days and viability determined at different
time points by culturing on MRS agar plates and counting
colonies.
DETAILED DESCRIPTION OF THE INVENTION
I. INTRODUCTION
[0025] This invention provides for a high potency, stable dry
preserved formulation of Lactobacillus suitable for administration
to people as a treatment where the formulation has high
colonization potency and no animal-derived excipients.
Specifically, as disclosed herein, the present invention provides
methods and compositions for Lactobacillus Replacement Therapy
(LRT) to repopulate the vaginal mucosa with protective
Lactobacillus microbiota as a means to correct dysbiosis and
maintain vaginal health. As described in more detail below, the
present invention teaches methods, compositions, and reagents for
the preparation and use of transiently buffered dried Lactobacillus
formulations.
II. DEFINITIONS
[0026] As used herein, the terms "vaginal microbiota" or "vaginal
microbiome" are used interchangeably and refer to the
microorganisms that colonize the vagina, although "microbiota" and
"microbiome" are the preferred terms. The terms "abnormal vaginal
microbiota" or "abnormal vaginal microbiome" or "vaginal dysbiosis"
refer to a condition in which the vaginal mucosa lacks protective
Lactobacillus spp. and is colonized by significant numbers of
diverse non-lactobacillus spp. The condition can be symptomatic or
asymptomatic.
[0027] As used herein, the terms "aqueous preservation medium" or
"preservation formulation" or "preservation medium" are used
interchangeably and refer to a composition capable of preserving
and maintaining a bacterial cell culture in a metabolically
inactive state while minimizing the damaging effects encountered
during the preservation process. Generally, a Lactobacillus strain
is converted from an actively growing metabolic state to a
metabolically inactive state upon addition to the preservation
medium, freezing and lyophilization. The preservation medium can
therefore be formulated for optimal cell resilience, such that the
cells can adhere to mucosal surfaces upon rehydration and return to
full metabolic activity. The aqueous preservation medium as used
herein is an aqueous solution which typically includes a
carbohydrate, a polyol (sugar alcohol), an anti-oxidant, a
buffering agent, and, optionally, an amino acid. The aqueous
preservation medium is used to resuspend a cell pellet of bacteria
to a concentration of about 10.sup.8 CFU/mL, where the suspension
can be dried, stored for at least 2 years at 2-8.degree. C., and
resuspended with a loss of CFU of less than 15%.
[0028] As used herein, the term "animal-derived excipients" refers
to inert substances derived from an animal, which may be included
in a composition comprised of substances that are considered active
ingredients. Non-limiting examples include milk, yogurt, butter
oil, chicken fat, lard, gelatin, and tallow.
[0029] As used herein, the term "excipient" and "inactive
excipient" are used interchangeably and refer to inert substances
formulated alongside the active ingredient of a medication,
included for the purpose of long-term stabilization, providing bulk
to the powder formulation (thus often referred to as "bulking
agents," "fillers," or "diluents"), or to confer a therapeutic
enhancement on the active ingredient in the final dosage form, such
as facilitating drug absorption, reducing viscosity, or enhancing
solubility. Examples of excipients include, without limitation,
maltodextrin, starch, pre-gelatinized starch, microcrystalline
cellulose, calcium carbonate, dicalcium phosphate, colloidal
SiO.sub.2, Pharmasperse.RTM., mannitol, xylitol, trehalose,
lactose, sucrose, polyvinyl pyrrolidone, crosspovidone, glycine,
magnesium stearate, sodium stearyl fumarate, cyclodextrins and
derivatives and mixtures thereof.
[0030] As used herein, the term "consisting essentially of" refers
to a composition or method that includes the disclosed components
or steps, and any other components or steps that do not materially
affect the basic and novel characteristics of the compositions or
methods. Compositions that consist essentially of listed
ingredients do not contain additional ingredients that would affect
the essential properties of those bacterial compositions. For
example, a Lactobacillus powder formulation of the present
invention can also be comprised of a pharmaceutically acceptable
excipient, such as a coloring agent and/or a filler, and an
antiviral or antibacterial agent, and/or an enzyme, without the
viability properties of the dry Lactobacillus powder being
affected.
[0031] As used herein, the term "Lactobacillus" refers to bacteria
that are Gram-positive facultative anaerobic bacteria,
characterized by the ability to produce lactate (lactic acid) from
carbohydrate sources such as glucose. These bacteria may be present
in food products or be commensal organisms that colonize the
vaginal or gastrointestinal mucosa.
[0032] As used herein, the terms "Lactobacillus crispatus" or "L.
crispatus" refer to a species of the Lactobacillus genus. The
species is generally distinguished from other lactobacilli based on
the polynucleotide sequence of the ribosomal 16S ribosomal RNA
gene. "Lactobacillus gasseri" or "L. gasseri" and "Lactobacillus
jensenii" or "L. jensenii" refer to other species of Lactobacillus.
L. crispatus, L. gasseri, L. jensenii are vaginal species capable
of producing hydrogen peroxide.
[0033] As used herein, the term "dry composition" refers to a
composition from which moisture has been removed. Drying or
desiccation techniques include, e.g., heating (e.g., sublimation),
application of low pressure or vacuum, lyophilization (i.e., freeze
drying), and combinations thereof. Compositions are commonly
desiccated for easy storage and transport.
[0034] As used herein, the term "effective culture conditions"
refers to the environment in which Lactobacillus cells are placed
in or are exposed to in order to promote growth of said cells.
Thus, the term refers to the medium, temperature, atmospheric
conditions, substrate, stirring conditions and the like which may
affect the growth of cells permitting a generation time (doubling
rate of cell population) of about 0.5 to 2.5 hours.
[0035] As used herein, the term "potency" refers to the number of
viable microbial cells delivered per medicant unit (i.e., medical
powder). According to the present invention, viable cells can grow
and reproduce. For a Lactobacillus dry powder to be efficacious in
vivo, both colonization of the vaginal epithelial cells by the
microbial cells at a potency of at least about 10.sup.8 CFU per
medicant unit and biological effect (e.g., as evidenced by absence
of an infected state such as bacterial vaginosis) are necessary.
"High potency" refers to the vaginal medicant containing at least
10.sup.8 viable microbial cells (CFUs) per medicant unit.
[0036] As used herein, the term "lyophilization" refers to the
process of freezing a substance and then reducing the concentration
of water, by sublimation and/or evaporation to levels which do not
support biological or chemical reactions.
[0037] As used herein, the term "water activity" and the notation
"a.sub.w" refer to and are defined to be equal to the Equilibrium
Relative Humidity ("ERH") divided by 100. ERH is the equilibrium
state at which the dry powder neither absorbs nor loses moisture to
the environment. The ERH is influenced by the composition of all
ingredients, particularly those with high water contents, which may
be present as free or bound water. The amount of free water can
influence the storage stability and purity of the dry powder which
could result in undesired degradation of activity or growth of
contaminating microorganisms during storage.
[0038] As used herein, the term "wet weight" refers to the weight
(grams) of the cell pellet following centrifugation and decantation
of the supernatant. In general, following the step of cell
harvesting, centrifuge bottles are pre-weighed, cells are spun
down, the supernatant is decanted, and the bottles are weighed
again. The difference in weight is the wet weight of the
pellet.
III. COMPOSITIONS AND METHODS
Obtaining Vaginal Bacteria
[0039] A Lactobacillus strain suitable for use in a medicant of the
present invention (i.e., medical powder) can be any Lactobacillus
strain that has the identifying characteristics described herein.
Lactobacillus strains can be detected and isolated from natural
sources using appropriate screening techniques that are known in
the art. Specifically, suitable strains of Lactobacillus for use in
a medicant of the present invention can be obtained through
publicly available resources, such as American Type Culture
Collection (ATCC) or Biodefense and Emerging Infections Research
Resources Repository (BEI, beiresources.org) or isolated from the
healthy vagina of a human. The identifying characteristics of
Lactobacillus strains suitable for use in the present invention and
methods to screen for these characteristics are discussed in detail
below.
[0040] One identifying characteristic of a Lactobacillus suitable
for use in the present invention is that the Lactobacillus strain
has a percent vaginal epithelial cell (VEC) cohesion value of at
least about 50%. A "percent VEC cohesion value" is defined as the
percentage of VECs to which at least one Lactobacillus cell is
adhered in the total number of VECs in an identified group.
According to the present invention, the terms "cohesion" and
"adherence" can be used interchangeably. Adherence of microbial
cells to vaginal epithelial cells is critical for colonization and
biological effect. Successful adherence of a Lactobacillus cell of
the medical powder to a vaginal epithelial cell results in
successful colonization of the vaginal epithelial cell. Long term
in vivo colonization is a goal of the products and methods of the
present invention, and "percent VEC cohesion value" is a good
predictor of whether a significant number of VECs will accept
microbial cells in vitro and in vivo. Methods used to determine
acceptable VEC cohesion values are well known in the art and can be
found in U.S. Pat. No. 6,468,526 and U.S. Pat. No. 6,093,394. See
also Kwok, et al., J. Urol. 2006, 176:2050-2054.
[0041] Another identifying characteristic of a Lactobacillus which
is suitable for use in a medicant of the present invention is the
ability to produce hydrogen peroxide (H.sub.2O.sub.2). The
H.sub.2O.sub.2 positive phenotype is also associated with sustained
vaginal colonization. See, e.g., Vallor, A. C., et al., J Infect
Dis. 2001 Dec. 1; 184(11):1431-6. As discussed above, hydrogen
peroxide has been shown to be responsible for the killing of other
microorganisms by the Lactobacillus. Preferably, the Lactobacillus
can produce greater than about 0.5 ppm of H.sub.2O.sub.2 under
normal growth conditions. More preferably, the Lactobacillus can
produce at least about 10 ppm of H.sub.2O.sub.2, and even more
preferably, the Lactobacillus can produce at least about 20 ppm of
H.sub.2O.sub.2 under effective growth conditions, herein defined as
any medium and conditions capable of promoting production of
H.sub.2O.sub.2. Effective growth conditions include both in vitro
growth conditions (e.g., an effective culture medium and
conditions) and in vivo growth conditions (e.g., successful
colonization of the vagina). Hydrogen peroxide producing vaginal
Lactobacillus include most L. crispatus and L. jensenii strains,
and approximately half of L. gasseri strains, as described in
Antonio et al. The Journal of Infectious Diseases 1999,
180:1950-6.
[0042] H.sub.2O.sub.2 production by a Lactobacillus of the present
invention can be quantitated by any means for measuring
H.sub.2O.sub.2 production. Methods used to measure H.sub.2O.sub.2
production are well known in the art, and can include the culture
method or the direct detection method. The culture method can
involve measuring H.sub.2O.sub.2 production by quantifying the
intensity of a blue pigment formed when Lactobacillus is inoculated
onto tetramethylbenzidine medium (TMB) and incubated under
anaerobic conditions. For example, Lactobacillus is incubated on a
TMB agar plate for about 48 hours under anaerobic conditions at
37.degree. C. The agar plate is then exposed to ambient air.
Exposure to the ambient air causes the H.sub.2O.sub.2 produced by
the Lactobacillus to react with horseradish peroxidase in the agar
to oxidize the TMB, causing the Lactobacillus colonies to turn
blue. See, e.g., Antonio et al. The Journal of Infectious Diseases
1999; 180:1950-6. The direct detection method can be used to
measure the quantity of H.sub.2O.sub.2 on a detection scale between
0 and 100 mg/L using commercially available H.sub.2O.sub.2
detection test trips (e.g., available from EM Sciences or Merck).
See, e.g., Strus, M. et al. The in vitro activity of vaginal
Lactobacillus with probiotic properties against Candida. Infect Dis
Obstet Gynecol. 2005 June; 13(2):69-75.
[0043] Another identifying characteristic of a Lactobacillus
suitable for use in a medicant of the present invention is the
genetic identity and stability of the Lactobacillus strain over
time both in vivo and in vitro. According to the present invention,
genetic stability refers to the ability of successive generations
of a Lactobacillus strain to substantially maintain the identical
genetic profile of the mother strain. In other words, successive
generations of a genetically stable strain will not acquire
substantial mutations in its genomic DNA over a period of time.
Repetitive Sequence Polymerase Chain Reaction (Rep PCR) can be used
to confirm genetic identity and stability of a strain of
Lactobacillus over time after either in vitro storage or in vivo
colonization of vaginal epithelial cells. Rep PCR methods used to
confirm genetic identity and stability Lactobacillus strains are
well known in the art and can be found in U.S. Pat. No. 6,093,394.
See also, Antonio & Hillier, J. Clin. Microbiol. 2003, 41:
1881-1887.
[0044] Another identifying characteristic of a Lactobacillus
suitable for use in a medicant of the present invention is the
ability to produce lactic acid. Lactic acid has been shown to
inhibit the growth of pathogens in vitro. Preferably, a
Lactobacillus produces at least about 0.75 mg/100 mL lactic acid,
and more preferably at least about 4 mg/100 mL lactic acid, and
even more preferably at least about 8.8 mg/100 mL lactic acid under
effective growth conditions.
[0045] A suitable Lactobacillus strain can have a relatively large
cell size. As provided in Bergey's Manual of Determinative
Bacteriology, typical Lactobacillus are 0.8-1.6 .mu.m in width and
2.3-11 .mu.m in length. A preferred Lactobacillus strain for use in
the present invention has a cell size of from about 1 to about 2
microns in width and from about 2 to about 4 microns in length.
Without being bound by theory, the present inventors believe that
the large dimensions exhibited by cells of a Lactobacillus strain
of the present invention may allow it to better serve as a
protective agent in biocompetitive exclusion. Biocompetitive
exclusion refers to the ability of the medical powder strain or
strains of the present invention to competitively inhibit the
growth of undesired bacterial strains. Such exclusion is attributed
to the occupation of available space on a vaginal epithelial cell
by the beneficial Lactobacillus cells (e.g., the medical powder
strain), thus preventing attachment of pathogenic, or undesirable,
microbial cells.
[0046] In addition to known species and strains of Lactobacillus,
newly identified species and strains from nature and mutant strains
derived from known or newly identified strains can be used in a
medicant of the present invention. Mutants of a parental strain of
Lactobacillus that have the identifying characteristics of a
Lactobacillus suitable for use in a medicant of the present
invention can be obtained by, for example, subjecting a parental
strain to at least one round of chemical and/or radiation
mutagenesis, to increase the rate of mutagenesis, thereby
increasing the probability of obtaining a microorganism having
improved desired characteristics. It will be obvious to one of
skill in the art that mutant microorganisms of the present
invention also include microorganisms that can be obtained by
genetically engineering microorganisms to, for example, have
increased percent VEC cohesion values. As used herein, a "mutated
microorganism" is a mutated parental microorganism in which the
nucleotide composition of such microorganism has been modified by
mutation(s) that occur naturally, that are the result of exposure
to a mutagen, or that are the result of genetic engineering.
[0047] Preferred species of Lactobacillus include Lactobacillus
crispatus, Lactobacillus gasseri and Lactobacillus jensenii, or a
species of Lactobacillus having 95% sequence homology to the 16S
rRNA gene sequence of any of the identified species. Particularly
preferred strains of lactobacilli are strains having all the
identifying characteristics of the Lactobacillus crispatus CTV-05
strain, Lactobacillus crispatus SJ-3C strain. Lactobacillus
crispatus CTV-05 is a preferred strain. Methods used to
differentiate between Lactobacillus strains include Rep-PCR, as
described in Antonio & Hillier, J. Clin. Microbiol. 2003, 41:
1881-1887, multilocus sequence typing (MLST), originally developed
to identify strains of pathogens (see, e.g., Maiden, M. C., et. al.
1998, Multilocus sequence typing: a portable approach to the
identification of clones within populations of pathogenic
microorganisms. Proc. Natl. Acad. Sci. USA., 95:3140-2145), and
whole genome sequencing.
Culturing Vaginal Bacteria
[0048] The Lactobacillus strains useful for the present invention
can be grown in liquid or on solid media (e.g., agar). Bacterial
media for growing Lactobacillus strains useful for the present
invention are known and commercially available (e.g., from BD
Difco) and include, e.g., de Man, Rogosa, and Sharpe (MRS) and
Rogosa media. The Lactobacillus are preferably cultured
anaerobically or microaerophilically and the temperature of the
culture medium can be any temperature suitable for growth of
Lactobacillus. Lactobacillus strains for the instant invention can
be cultured in anaerobic conditions and are generally grown at
about 37.degree. C. Effective culture conditions for vaginal
Lactobacillus strains useful for the instant invention are well
known in the art. Specific culture conditions, culture media and
methods of culturing Lactobacillus strains, particularly L.
crispatus and L. gasseri, can be found in, e.g., U.S. Pat. No.
8,329,447, U.S. Pat. No. 6,093,394, and Davis, C. Enumeration of
probiotic strains: Review of culture-dependent and alternative
techniques to quantify viable bacteria. J Microbiol Methods. 2014;
103:9-17.
[0049] The culture medium is inoculated with an actively growing
culture of the Lactobacillus strain in an amount sufficient to
produce, after a reasonable growth period, a suitable cell density
(or potency) for transfer to the preservation medium. A
non-limiting example of a reasonable growth period of the
Lactobacillus used herein is a generation time of between 1 to 2.5
hours. The cells are grown to a preferred cell density in the range
of from about 10.sup.8 CFU/mL to about 10.sup.10 CFU/mL. A
culture-based method is used to determine the cell density, in
which serial dilutions of Lactobacillus cultures are plated onto
MRS agar plates and incubated for 48 hr anaerobically at 37.degree.
C. Colonies on the plates are counted and the number of CFUs
(colony forming units) in the samples are calculated as CFU/mL or
CFU/gram. Methods of determining the CFUs are described in detail
below.
[0050] Once the cells are grown to preferred cell density, the
bacterial cells can be harvested using any suitable method to
remove the cells from the culture media. Non-limiting exemplary
methods for harvesting the cultured cells includes, filtration,
centrifugation, and sedimentation. In some examples, harvesting
cultured cells can involve hollow fiber filtration and washing via
diafiltration. Methods for harvesting cultured Lactobacillus cells
are well known in the art and are described in detail in the
Examples section. After separation of the cells from the culture
media and/or washing of the biomass, the cells are centrifuged to
form a cell pellet in preparation for suspension in a preservation
medium.
Preparation of the Aqueous Preservation Medium
[0051] The bacterial cell pellet formed from the methods described
above is resuspended in a suitable aqueous preservation medium,
where the weight ratio of cell pellet wet weight (grams) to
preservation media (mL) can be between 1:1 and 1:8 grams of cell
pellet to milliliter of preservation media. In some embodiments,
the bacterial cell pellet is resuspended in a suitable aqueous
preservation medium, where the weight ratio of cell pellet wet
weight (grams) to preservation media (mL) can be between 1:1 and
1:7 grams of cell pellet to milliliter of preservation media, or
between 1:1 and 1:6, or between 1:1 and 1:5, or between 1:1 and
1:4, or between 1:1 and 1:3, or between 1:1 and 1:2, or between 1:2
and 1:6, or between 1:3 and 1:5 grams of cell pellet to milliliter
of preservation media. In some embodiments, the bacterial cell
pellet is resuspended in a suitable aqueous preservation medium,
where the weight ratio of cell pellet wet weight (grams) to
preservation media (mL) can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
or 1:8 grams of cell pellet to milliliter of preservation media. In
some embodiments, the bacterial cell pellet is resuspended in a
suitable aqueous preservation medium, where the weight ratio of
cell pellet wet weight (grams) to preservation media (mL) can be
between 1:1 and 1:5 grams of cell pellet to milliliter of
preservation media. In some embodiments, the bacterial cell pellet
is resuspended in a suitable aqueous preservation medium, where the
weight ratio of cell pellet wet weight (grams) to preservation
media (mL) can be between 1:1 and 1:3 grams of cell pellet to
milliliter of preservation media.
[0052] The aqueous preservation medium is comprised of ingredients
that minimize the damaging effects encountered during the
preservation process. The preservation medium of this invention
includes a carbohydrate, a polyol, an anti-oxidant, a buffering
agent, and, optionally, an amino acid. The carbohydrate used in the
preservation medium functions as a lyoprotectant to protect and
stabilize the cells during freeze drying, and afterwards during
storage. Non-limiting exemplary carbohydrates suitable for use with
the invention include trehalose, dextrose, lactose, maltose,
sucrose and/or any other disaccharide or polysaccharide. In some
embodiments, the preservation medium comprises from about 0.5% to
about 30% carbohydrate by weight per volume (w/v) of the
preservation medium, or from about 1% to about 25%, or from about
5% to about 20%, or from about 10% to about 15% carbohydrate by w/v
of the preservation medium. In some embodiments, the preservation
medium comprises from about 0.5% carbohydrate by weight per volume
(w/v) of the preservation medium, or from about 1, 2, 5, 7, 10, 15,
20, 25, or 30% carbohydrate by w/v of the preservation medium. In
some embodiments, the preservation medium comprises from about 5%
to about 20% trehalose w/v of the preservation medium. In some
other embodiments of the invention, the preservation medium
comprises from about 5% to about 15% trehalose w/v of the
preservation medium.
[0053] The polyol (i.e., polyhydric alcohol) of the preservation
medium is a lyoprotectant that helps protect cells from the
stresses of dehydration during freeze drying. Non-limiting
exemplary polyols suitable for use with the present invention
include xylitol, adonitol, glycerol, dulcitol, inositol, mannitol,
maltitol, isomalt, lactitol, erythritol, sorbitol and/or arabitol.
In some embodiments, the preservation medium comprises from about
0.1% to about 12% polyol by weight per volume (w/v) of the
preservation medium, or from about 1% to about 10%, or from about
2% to about 9%, or from about 3% to about 7% polyol by w/v of the
preservation medium. In some embodiments, the preservation medium
comprises from about 0.1% polyol by weight per volume (w/v) of the
preservation medium, or from about 0.5, 1, 2, 3, 5, 6, 7, 8. 9, 10,
11, or 12% polyol by w/v of the preservation medium. In some
embodiments, the preservation medium comprises from about 2% to
about 9% xylitol w/v of the preservation medium. In some other
embodiments of the invention, the preservation medium comprises
from about 2% to about 7% xylitol w/v of the preservation
medium.
[0054] The antioxidant of the preservation medium retards oxidative
damage to the microbial cells during the preservation and storage
process. Non-limiting exemplary antioxidants suitable for use with
the instant invention include sodium ascorbate, ascorbic acid,
palmityl ascorbate, propyl gallate and vitamin E
(.alpha.-tocopherol). In some embodiments, the preservation medium
comprises from about 0.1% to about 5% antioxidant by weight per
volume (w/v) of the preservation medium, or from about 0.5% to
about 3.0%, or from about 1.0% to about 2.0% antioxidant by w/v of
the preservation medium. In some embodiments, the preservation
medium comprises from about 0.1% antioxidant by weight per volume
(w/v) of the preservation medium, or from about 0.3, 0.5, 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0% antioxidant by w/v of the
preservation medium. In some embodiments, the preservation medium
comprises from about 0.5% to about 1.5% sodium ascorbate w/v of the
preservation medium. In some other embodiments of the invention,
the preservation medium comprises from about 0.5% to about 1.5%
sodium ascorbate w/v of the preservation medium.
[0055] Buffering agents suitable for use in the preservation medium
enhance the stability and recovery of the bacteria cells. A
buffering agent suitable for use in the preservation medium is a
physiological agent that does not exert any toxic effects on the
bacteria, vaginal epithelial cells or a female patient using a
pharmaceutical composition. Non-limiting exemplary buffering agents
suitable for use with the instant invention include sodium
phosphate, disodium phosphate, potassium phosphate, sodium
bicarbonate, histidine, arginine and sodium citrate. In some
embodiments, the buffering agent can have a pKa of from about 4.3
to about 8.0, or from about 4.6 to about 7.7, or from about 5.0 to
about 7.3, or from about 5.4 to about 7.0, or from about 6.0 to
about 6.7. In some other embodiments, the preservation medium
comprises a buffering solution having a pKa of at least 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or
higher. In some embodiments, the preservation medium comprises a
buffering solution having a pKa in the physiological range. In
other embodiments, the preservation medium comprises a buffering
solution having a pKa of from about 6.7 to about 7.8.
[0056] In still further embodiments, the preservation medium
comprises from about 5 mM to about 70 mM buffering agent, or from
about 10 mM to about 65 mM, or from about 15 mM to about 60 mM, or
from about 20 mM to about 55 mM, or from about 25 mM to about 50
mM, or from about 30 mM to about 45 mM, or from about 35 mM to
about 40 mM buffering agent. In some embodiments, the preservation
medium comprises from about 5 mM buffering agent, or from about 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 mM. In some
embodiments, the preservation medium comprises from about 10 mM to
about 50 mM sodium phosphate. In some other embodiments of the
invention, the preservation medium comprises from about 10 mM to
about 30 mM sodium phosphate.
[0057] In some embodiments, the preservation medium can optionally
include an amino acid that helps enhance stability of the
Lactobacillus cells at elevated temperatures without significantly
affecting cryopreservation during the lyophilization process. In
some embodiments, the optional amino acid can be in the salt form
of a suitable amino acid. Non-limiting exemplary amino acids and/or
their salts suitable for use with the instant invention include
sodium glutamate, glutamine, glycine, arginine, histidine, and
lysine. In some embodiments, the preservation medium optionally
comprises from about 0% to about 5% amino acid by weight per volume
(w/v) of the preservation medium, or from about 0.5% to about 3.0%,
or from about 1.0% to about 2.0% amino acid by w/v of the
preservation medium. In some embodiments, the preservation medium
optionally comprises from about 0.1% amino acid by weight per
volume (w/v) of the preservation medium, or from about 0.3, 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0. 4.5, or 5.0% amino acid by w/v
of the preservation medium. In some embodiments, the amino acid
optionally included in the preservation medium is amino acid salt
sodium glutamate, preferably monosodium glutamate. In some
embodiments, the preservation medium optionally comprises from
about 0% to about 5% sodium glutamate w/v of the preservation
medium. In some other embodiments of the invention, the
preservation medium optionally comprises from about 0% to about 5%
monosodium glutamate w/v of the preservation medium. In some
embodiments, the preservation medium optionally comprises from
about 1% to about 4% sodium glutamate w/v of the preservation
medium. In some other embodiments of the invention, the
preservation medium optionally comprises from about 1% to about 4%
monosodium glutamate w/v of the preservation medium.
[0058] The preservation medium of the present invention includes a
carbohydrate that is between about 5% and 20% of the preservation
medium by weight per volume, a polyol that is between about 2% and
9% of the preservation medium by weight per volume, an antioxidant
that is between about 0.5% and 1.5% of the preservation medium by
weight per volume and a buffering agent that is between 10 mM and
50 mM. In other embodiments, a preservation medium suitable for use
with the present invention can include a carbohydrate that is
between about 5% and 15% of the preservation medium by weight per
volume, a polyol that is between about 2% and 7% of the
preservation medium by weight per volume, an antioxidant that is
between about 0.5% and 1.0% of the preservation medium by weight
per volume and a buffering agent that is between 10 mM and 30
mM.
[0059] In some embodiments, the preservation medium of the present
invention includes a carbohydrate that is between about 5% and 20%
of the preservation medium by weight per volume, a polyol that is
between about 2% and 9% of the preservation medium by weight per
volume, an antioxidant that is between about 0.5% and 1.5% of the
preservation medium by weight per volume, a buffering agent that is
between 10 mM and 50 mM, and, optionally, an amino acid that is
between about 0% and 5% of the preservation medium by weight per
volume. In other embodiments, a preservation medium suitable for
use with the present invention can include a carbohydrate that is
between about 5% and 15% of the preservation medium by weight per
volume, a polyol that is between about 2% and 7% of the
preservation medium by weight per volume, an antioxidant that is
between about 0.5% and 1.0% of the preservation medium by weight
per volume, a buffering agent that is between 10 mM and 30 mM, and,
optionally, an amino acid that is between about 0% and 5% of the
preservation medium by weight per volume.
[0060] An example of a particularly useful preservation medium of
the present invention includes trehalose as the carbohydrate that
is between about 5% and 20% of the preservation medium by weight
per volume, xylitol as the polyol that is between about 2% and 9%
of the preservation medium by weight per volume, sodium ascorbate
as the antioxidant that is between about 0.5% and 1.5% of the
preservation medium by weight per volume and sodium phosphate as
the buffering agent that is between 10 mM and 50 mM. In some
embodiments, a particularly useful preservation medium of the
present invention includes trehalose as the carbohydrate that is
between about 5% and 20% of the preservation medium by weight per
volume, xylitol as the polyol that is between about 2% and 9% of
the preservation medium by weight per volume, sodium ascorbate as
the antioxidant that is between about 0.5% and 1.5% of the
preservation medium by weight per volume, sodium phosphate as the
buffering agent that is between 10 mM and 50 mM, and, optionally,
sodium glutamate as the amino acid that is between about 0% and 5%
of the preservation medium by weight per volume. In other
embodiments, a preservation medium suitable for use with the
present invention includes trehalose that is between about 5% and
15% of the preservation medium by weight per volume, xylitol that
is between about 2% and 7% of the preservation medium by weight per
volume, sodium ascorbate that is between about 0.5% and 1.0% of the
preservation medium by weight per volume and sodium phosphate that
is between 10 mM and 30 mM. In some other embodiments, a
preservation medium suitable for use with the present invention
includes trehalose that is between about 5% and 15% of the
preservation medium by weight per volume, xylitol that is between
about 2% and 7% of the preservation medium by weight per volume,
sodium ascorbate that is between about 0.5% and 1.0% of the
preservation medium by weight per volume, sodium phosphate that is
between 10 mM and 30 mM, and, optionally, sodium glutamate that is
between about 0% and 5% of the preservation medium by weight per
volume. Representative preservation media compositions, which are
in no way meant to be limiting, are included in Table 1 below.
TABLE-US-00001 TABLE 1 Exemplary preservation media compositions
and ingredient ratios. Ingredient (%, w/w) Sodium Sodium No.
Trehalose Xylitol ascorbate NaPO.sub.4 * glutamate i 5-15 2-9
0.5-1.5 10-30 0-5 ii 5, 10, 2 0.5 10 0-5 or 15 iii 7.5 2, 3, 0.75
15 0-5 or 9 iv 7.5 3 0.5, 1.0, 15 0-5 or 1.5 v 7.5 3 0.75 10, 12,
0-5 or 15 * Amount of sodium phosphate is measured in mM
[0061] Prior to addition of the above described harvested
Lactobacillus cells to the medium, the cells may be washed in a
saline buffer. Upon introduction of the harvested Lactobacillus
cells to the preservation medium described herein, the resulting
mixture is referred to as the cell-preservation medium slurry. In
some embodiments, a cell-preservation medium slurry can have an
activity of between 10.sup.8 CFU/mL and 10.sup.11 CFU/mL. A more
preferred cell-preservation medium slurry can have an activity of
at least about 10.sup.10 CFU/mL. It is to be understood that one of
ordinary skill in the art will appreciate variations to the basic
culturing, harvesting and suspending steps disclosed herein and as
such, the present invention incorporates such variations.
Drying the Cell-Preservation Medium Slurry
[0062] The cell-preservation medium slurry can be dried to produce
the resulting bulk drug powder using any suitable drying method
known in the art. Typically the effect of drying is to place the
bacteria in a state of dormancy to protect the bacteria from
environmental elements that negatively impact the viability of the
bacteria. The standard way to bring about dormancy is through the
removal of water. Generally, sufficient water is removed so that
the normal cellular processes (e.g. enzymatic activity) come to a
halt or are at least greatly diminished.
[0063] The cell-preservation medium slurry can be dried using any
of the numerous methods known in the art for drying a bacterial
preparation to increase their stability for long term storage.
Drying methodologies and protective agents are disclosed in the
review by Morgan et al. (2006) J. Microbiol. Meth. 66:183-193.
Suitable drying methods include air drying, vacuum drying, oven
drying, spray drying, flash drying, fluid bed drying, controlled
atmosphere drying, and lyophilization (i.e., freeze drying). In
some embodiments, a desiccant is used to aid in the drying process,
and/or to prevent reabsorption of moisture into the dried
formulation. In some embodiments, the drying is carried out using a
lyophilizer (i.e., Virtis, SP Scientific). Detailed freeze-drying
methods known to persons of skill in the art and are disclosed in
U.S. Pat. Nos. 6,093,394; 8,329,447; and 8,642,029. The resulting
dry formulation referred to as the bulk powder is tested for
potency using the methods described below. The potency of the dry
bulk drug powder can be between 10.sup.9 CFU/g and 10.sup.12 CFU/g.
A more preferred bulk powder can have an activity of at least about
10.sup.10 CFU/g.
Measuring Residual Water
[0064] A dried formulation can be tested for the presence of
residual water using any suitable method known in the art. In some
cases, residual water in the dried formulation can be measured
gravimetrically, as described in U.S. Pat. Nos. 8,329,447 and
8,642,029. Alternatively, an instrument for measuring water content
in powders could be used to monitor the moisture content of the
formulation during drying, e.g., the IR-120 Moisture Analyzer
(Denver Instruments, Denver, Colo.). Residual water moisture can
also be determined by performing well known coulometric or
volumetric titration techniques, such as the Karl Fischer
titration.
[0065] Water content in a Lactobacillus powder can also be measured
as the free water or water activity (a.sub.w) using a water
activity meter, e.g., AquaLab CX-2 Model series (Decagon
Instruments, Pullman, Wash.), or a Rotronic Model series (Rotronic
Instrument Corp., Huntington, N.Y.). The water activity meter
(AquaLab CX-2, Decagon Instruments) uses a chilled-mirror dew point
technique to measure the a.sub.w of a product. When a sample is
placed in the AquaLab, a stainless-steel mirror within the chamber
is repeatedly cooled and heated while dew forms and is driven off.
The instrument's fan circulates the air in the sensing chamber,
speeding up the equilibration process. Each time dew forms on the
mirror, AquaLab measures the temperature and calculates the a.sub.w
of the sample, saving these values to compare to previous values.
When the a.sub.w values of consecutive readings are less than 0.001
apart, the measurement process is complete.
[0066] The water energy level or water activity (a.sub.w)
determines the overall stability of the resulting dry bulk
Lactobacillus drug powder. One of ordinary skill in the art will
appreciate the importance of the water activity of pharmaceuticals,
such as the a.sub.w of the drug powder of the invention. By
maintaining a low water activity of a pharmaceutical product,
degradation of the active pharmaceutical ingredient (i.e., the
Lactobacillus drug powder) can be avoided. Furthermore, a
pharmaceutical product, such as the Lactobacillus drug powder of
the present invention, having a low water activity can be less
susceptible to crystallization, caking and clumping, which
contributes to the drug's degradation and ineffectiveness. These
are time-dependent reactions with rates influenced by water
activity. Details on the influence of a.sub.w on a product
formulation can be found in United States Pharmacopeial Method
<1112> Microbiological Attributes of Non-sterile
Pharmaceutical Products--Application of Water Activity
Determination.
[0067] In some embodiments, the dry bulk Lactobacillus drug
substance can have a measured a.sub.w of from about 0.001 to about
0.220, or from about 0.005 to about 0.200, or from about 0.010 to
about 0.150, or from about 0.025 to about 0.100, or from about
0.050 to about 0.075. In other embodiments, the dry bulk
Lactobacillus drug substance can have a measured a.sub.w of from
about 0.001, 0.003, 0.005, 0.007, 0.010, 0.030, 0.050, 0.070,
0.100, 0.150, 0.170, 0.200, 0.220. In particular embodiments, the
dry bulk Lactobacillus drug substance can have a measured a.sub.w
of less than 0.220.
Measuring Potency
[0068] The Lactobacillus formulations (wet and/or dry) of the
present invention are tested for potency at different times
throughout the preparation process using any suitable method known
in the art. Such methods used to determine the potency that of the
Lactobacillus formulations include, but are not limited to, the
culture-based method. The light scattering method for determining
cell density of Lactobacillus is used to monitor the fermentation
process and involves measuring the optical density at 600 nm of a
sample of bacteria.
[0069] The preferred method used to measure the potency of the
Lactobacillus formulations is the culture-based method involving
serial dilutions. A sample of the Lactobacillus formulation to be
tested is obtained and serial dilutions are made. A small aliquot
(i.e., 100 .mu.L) of serial dilutions are plated onto MRS agar
plates. The samples are allowed to incubate anaerobically at
37.degree. C. for 48 hours. After a suitable amount of time has
passed, the plates are illuminated by placing the Petri dishes in
transmitted light. The separate colonies are counted manually or
with a camera and computer using commercially available bacterial
counting software, and the number of CFUs in the samples are
calculated as CFU/ml or CFU/gram. More details involving the
culture-based methods are disclosed in Brugger, S. D., et al.
Automated Counting of Bacterial Colony Forming Units on Agar
Plates. PLOS ONE 2012; 7(3): e33695.
Purity and Identity
[0070] In addition to measuring the potency and the water activity,
the bulk drug powder produced from the above described drying
methods can be tested for purity and identity. The purity of the
bulk drug powder is determined using methods well known in the art
and as described in United States Pharmacopeial Method <61>
Microbial Enumeration Tests and United States Pharmacopeial Method
<62> Tests for Specified Microorganisms. Genetic
identification of the Lactobacillus species in the bulk powder and
final drug product is carried out by isolating genomic DNA using a
commercially available kit (e.g. Power Soil DNA Isolation Kit, Mo
Bio), amplifying the 16S rRNA gene using specific primers by PCR,
sequencing the gene using a commercial DNA sequencing service
(MCLAB), and comparing the sequence to a reference standard.
Identification of the Lactobacillus strain in the bulk drug powder
is determined using methods well known in the art, such as
Repetitive Sequence Polymerase Chain Reaction (Rep PCR) and as
described in U.S. Pat. Nos. 6,093,3941; 8,329,447; and
8,642,029.
Diluting Bacterial Powder with Inactive Excipients
[0071] In order to adhere to the potency and dosage guidelines
agreed upon and developed by the U.S. Food and Drug Administration
(FDA), the activity of the bulk Lactobacillus drug powder is
diluted using a pharmaceutically acceptable excipient. Any suitable
inactive pharmaceutically acceptable excipient (i.e., diluent)
known in the art can be used to dilute the potency of the
Lactobacillus drug powder. In some embodiments, a diluent can be
maltodextrin, pre-gelatinized starch, lactose, Pharmasperse.RTM.,
mannitol, xylitol, microcrystalline cellulose, sugar or a
combination thereof. In other embodiments, an inactive bulking
agent can be used in combination with another diluent. In other
embodiments, a maltodextrin or pre-gelatinized starch can be used
to dilute the bulk lactobacilli drug powder. In some embodiments,
maltodextrin is used to dilute the bulk lactobacilli drug
powder.
[0072] In some embodiments, the bulk Lactobacillus drug powder is
diluted with an inactive excipient by between 3-fold and 10-fold.
In other embodiments, the bulk Lactobacillus drug powder can be
combined with an inactive excipient at a ratio of powder to
inactive excipient of between 1:1 and 1:12 w/w. In some
embodiments, the bulk Lactobacillus drug powder can be combined
with an inactive excipient at a ratio of powder to an inactive
excipient of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,
1:11, or 1:12 w/w. In particular embodiments, the bulk
Lactobacillus drug powder can be combined with an inactive
excipient at a ratio of powder to an inactive excipient of between
1:1 and 1:10 w/w. In particular embodiments, the bulk Lactobacillus
drug powder can be combined with an inactive excipient at a ratio
of powder to an inactive excipient of between 1:1 and 1:5 w/w. In
some embodiments, the bulk Lactobacillus drug powder can be
combined with an inactive excipient at a ratio of powder to an
inactive excipient of between 1:1 and 1:3 w/w.
[0073] In particular embodiments, the bulk Lactobacillus drug
powder can be combined with maltodextrin at a ratio of powder to
maltodextrin of between 1:1 and 1:10 w/w. In particular
embodiments, the bulk Lactobacillus drug powder can be combined
with maltodextrin at a ratio of powder to maltodextrin of between
1:1 and 1:5 w/w. In some embodiments, the bulk Lactobacillus drug
powder can be combined with maltodextrin at a ratio of powder to
maltodextrin of between 1:1 and 1:3 w/w. The potency of the diluted
dry bulk Lactobacillus drug powder, referred to as the drug
product, can be between 10.sup.8 CFU/g and 10.sup.11 CFU/g, or
between 10.sup.8 CFU/g and 10.sup.10 CFU/g. A more preferred drug
product can have an activity of greater than 10.sup.9 CFU/g.
[0074] The drug product can be packaged in dosages of between about
100 mg and 600 mg. In some embodiments, the drug product dosage can
be packaged in a dosage of about 100 mg, or of about 150, 200, 250,
300, 350, 400, 450, 500, 550, or 600 mg. In other embodiments, the
drug product can be packaged in dosages of between about 150 mg and
450 mg, or about 150 mg and about 400 mg, or about 150 mg and about
350 mg. In some embodiments, the drug product can be packaged in
dosages of between about 150 mg and 250 mg. In a particular
embodiment, the drug product can be packaged in a dosage of about
200 mg.
[0075] The drug product can be placed in a medical powder
applicator, referred to as the final drug product, and packaged to
protect against moisture and oxygen during transport and storage.
The package can be comprised of any suitable material for such
protection such as Mylar or metallic film pouches. In some
embodiments, the final drug product is sealed into individual
packages, e.g., for individual dosages.
Using the Dry Preserved Lactobacillus Final Drug Product
[0076] The final drug product (i.e., dry powder) of any suitable
Lactobacillus species and strain, as described herein, can be used
to prevent and/or treat a vaginal infection (i.e., abnormal vaginal
microbiota). Such vaginal infections include, but are not limited
to, bacterial vaginosis, yeast vaginitis, gonorrhea, chlamydia,
trichomoniasis, human immunodeficiency virus infection, herpes
simplex virus type 2 (HSV-2), urinary tract infection, and pelvic
inflammatory disease. In some embodiments, the final drug product
can be used to prevent and/or treat bacterial vaginosis, yeast
vaginitis, or urinary tract infection. In a particular embodiment,
the dry powder can be used to prevent and/or treat bacterial
vaginosis (BV).
[0077] Abnormal vaginal microbiota can be detected and diagnosed
using any suitable means known in the art. A vaginal infection can
be symptomatic or asymptomatic. Symptoms generally include abnormal
odor and/or discharge, and discomfort from itching and/or pain.
Depending on the vaginal infection, it can be detected by a woman
without medical consultation or diagnostic apparatuses or kits. For
example, a few inexpensive, non-prescription kits for detecting
yeast vaginitis are available (e.g., Vagisil.TM.).
[0078] In some cases, medical practitioners will detect and
diagnose the vaginal infection. Clinical criteria require the
presence of at least three symptoms, including those mentioned
above, a vaginal fluid pH of >4.5, and the presence of clue
cells (e.g., vaginal epithelial cells studded with adherent
coccobacilli) on microscopic examination. For example, bacterial
vaginosis can be detected, e.g., by Amsel clinical criteria or Gram
stained vaginal smears (Nugent scoring system). The Gram stained
vaginal smear is used to determine the relative concentration of
lactobacilli (Gram-positive bacteria), Gram-negative and
Gram-variable rods and cocci (i.e., G. vaginalis, Prevotella,
Porphyromonas, and peptostreptococci), and curved Gram-negative
rods (i.e., Mobiluncus) characteristic of BV. Detection and
diagnostic methods for various vaginal infections are well known in
the art and are described in U.S. Pat. No. 8,329,447. See also
https://www.cdc.gov/std/tg2015/bv.htm.
[0079] The dry powder of the present invention can be administered
alone or in combination with (e.g., simultaneously with, before,
and/or after) any other therapy for the prevention and/or treatment
of vaginal infections. Administration of any other therapy for the
prevention and/or treatment of vaginal infections can be
administered in an amount effective to reduce the level of abnormal
vaginal microbiota. Other therapies for the prevention and/or
treatment of vaginal infections can include antibiotics or
antiviral agents, which are well known in the art. In some
embodiments, the other therapy for the prevention and/or treatment
of vaginal infections can be an antibiotic. Suitable antibiotics
for the prevention and/or treatment of abnormal vaginal microbiota
are well known in the art. Such antibiotics include, but are not
limited to, clindamycin, metronidazole, and tinidazole. An
antibiotic for use in conjunction with the dry powder of the
invention can be in any suitable form for administration. For
example, an antibiotic can be delivered topically (as a gel or
cream), or as an oral or vaginal tablet, capsule or suppository. In
a particular embodiment, the antibiotic is administered as a
topical gel.
[0080] The antibiotic treatment can be administered between 1 and 2
times per day. In a particular embodiment, the antibiotic treatment
can be administered 1 time per day.
[0081] In some embodiments of the invention, the antibiotic can be
administered for between 2 and 7 days. In other embodiments, the
antibiotic can be administered for 2, 3, 4, 5, 6, and 7 days. In
some other embodiments, the antibiotic can be administered for
between 2 and 7 days, or 3 and 6 days, or 4 and 5 days, or 4 and 7
days. In particular embodiments, the antibiotic can be administered
for between 2 and 7 days. In another embodiment, the antibiotic can
be administered for 7 days. In another embodiment, the antibiotic
can be administered for 5 days.
[0082] In some embodiments, the administration of the dry powder is
during the final few days of the administration regimen of an
antibiotic (i.e., 2 to 4 days before the completion of the
administration regimen of an antibiotic). In other embodiments, the
dry powder is administered after the completion of the
administration regimen of an antibiotic. The dry powder can be
administered 1 or 2 times per day after the completion of the
administration regimen of an antibiotic. In some embodiments, the
dry powder can be administered 2 times per day after the completion
of the administration regimen of an antibiotic. In a particular
embodiment, the dry powder can be administered 1 time per day after
the completion of the administration regimen of an antibiotic.
[0083] In some embodiments of the invention, the dry powder can be
administered for between 1 and 14 days after the completion of the
administration regimen of an antibiotic. In other embodiments, the
dry powder can be administered for 1, 2, 3, 4, 5, 6, 7, 10, 12, or
14 days after the completion of the administration regimen of an
antibiotic. In some other embodiments, the dry powder can be
administered for between 2 and 12 days, or 3 and 10 days, or 4 and
7 days, or 5 and 6 days after the completion of the administration
regimen of an antibiotic. In particular embodiments, the dry powder
can be administered for between 5 and 7 days after the completion
of the administration regimen of an antibiotic. In another
embodiment, the dry powder can be administered for 5 days after the
completion of the administration regimen of an antibiotic.
[0084] After completion of the antibiotic administration regimen
and the initial dry powder administration regimen, the dry powder
of the present invention can be administered for an additional
period of time. For example, following the completion of the
initial treatment using the dry powder (i.e., 1 dose per day for 7
days), the dry powder can be administered between 1 and 5 times per
week. In some embodiments, the dry powder can be administered 1, 2,
3, 4, or 5 times per week after the completion of the initial dry
powder treatment regimen. In other embodiments, the dry powder can
be administered between 1 and 4 times per week, or between 2 and 5
times per week, or between 1 and 3 times per week, or between 1 and
2 times per week after the completion of the initial dry powder
treatment regimen. In a particular embodiment, the dry powder can
be administered 1 time per week after the completion of the initial
dry powder treatment regimen.
[0085] In some embodiments of the invention, the dry powder can be
administered for between 1 and 26 weeks after the completion of the
initial dry powder treatment regimen. In other embodiments, the dry
powder can be administered for 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14,
16, 18, 20, 22, 24, or 26 weeks after the completion of the initial
dry powder treatment regimen. In some other embodiments, the dry
powder can be administered for between 2 and 26 weeks, or 3 and 24
weeks, or 4 and 22 weeks, or 5 and 20 weeks, or 6 and 18 weeks, or
7 and 16 weeks, or 8 and 14 weeks, or 10 and 12 weeks after the
completion of the initial dry powder treatment regimen. In
particular embodiments, the dry powder can be administered for
between 5 and 10 weeks after the completion of the initial dry
powder treatment regimen. In another embodiment, the dry powder can
be administered for 10 weeks after the completion of the initial
dry powder treatment regimen.
[0086] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0087] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
IV. EXAMPLES
[0088] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which could be
changed or modified to yield essentially similar results.
Example 1. Preparing The Dry Composition of Lactobacillus
[0089] This example details the general strategy for preparing the
dry composition of Lactobacillus in powder form as a medical
product, involving bacterial cultivation, suspension in
preservation medium, drying, dilution, and packaging. The procedure
described here, for the culture and processing of Lactobacillus
crispatus SJ-3C, is applicable for any microorganism suitable for
use with the present invention.
[0090] The initial Lactobacillus crispatus SJ-3C (SJ-3C) cells can
be obtained from the deposit American Type Culture Collection
(ATCC) under ATCC number PTA-10138. A Master Cell Bank and Working
Cell Bank of these cells are prepared and can be subsequently used
in the preparation of the dry Lactobacillus compositions.
[0091] The SJ-3C cells are initially plated onto modified de Man,
Rogosa, and Sharpe (MRS) agar plates and grown under anaerobic
conditions for 72 hours at 37.degree. C. Cells from the plates are
inoculated into 10 mL of modified MRS and incubated anaerobically
for 24 hours at 37.degree. C. This culture is then transferred to
490 mL of growth medium and incubated for 24 hours at 37.degree.
C., followed by transfer to 4.5 L of medium in a 5 L Bellco
Bioreactor. The 5-liter culture is incubated anaerobically at
37.degree. C. for an additional 24 hours to serve as the fermentor
inoculum.
[0092] Fermentation is performed in a fermentor (100 L fermentor)
at pH 6.0 in the presence of modified MRS medium sparged with
nitrogen gas. Fermentation is initiated by addition of the inoculum
and completed after approximately 15 hours when the cells reach
early stationary phase and growth stops. At this point, glucose is
depleted, lactic acid production stops, the optical density of the
culture at 600 nm (OD600) remains constant and the cells are
harvested.
[0093] Cells are harvested, concentrated, and washed by buffer
exchange into phosphate-buffered saline (diafiltration) in a
sterile closed hollow fiber system using a tangential flow
membrane. When the residual lactate concentration reaches 10% of
the starting value at harvest and pH of the permeate remains
constant, the cells are aseptically removed from the harvest system
and collected by centrifugation at 1500.times.g for 20 minutes,
2-8.degree. C.).
[0094] Cell pellets are resuspended in a preservation medium
solution, using 2.5 mL of preservation solution per gram of cell
paste. The preservation medium solution contains 15% trehalose, 6%
xylitol, and 1% sodium ascorbate in a 10 mM sodium phosphate buffer
(pH 7.4), which is used to prepare batches of the harvested SJ-3C
slurry. The resulting batches of the preservation medium cell
slurry are to have calculated activities of between
1.times.10.sup.10 CFU/mL and 5.times.10.sup.10 CFU/mL. The slurry
is transferred to sterile Lyoguard.TM. trays and lyophilized in a
Virtis Genesis Lyophilizer. Viability of the cell slurry is
determined prior to lyophilization by plate counting. The
Lyoguard.TM. trays containing the cell cakes are placed in
heat-sealed bags with desiccant and purged with nitrogen gas, and
held at 2-8.degree. C. until milling.
[0095] The SJ-3C bulk drug substance is produced by milling the
lyophilized cell cakes with 0.5% colloidal silicon dioxide as an
anti-caking agent using a cone mill. The bulk powder is purged with
nitrogen (N.sub.2) gas and stored with desiccant in a heat-sealed
bag at 2-8.degree. C. until used for manufacture of the drug
product. The SJ-3C bulk drug substance is tested for purity,
potency (CFU), identity, and residual moisture using the methods as
described previously and those known to one of skill in the art.
The ideal activity of the resultant batches of the dry SJ-3C bulk
drug substance should be between 5.times.10.sup.10 CFU/g and
1.0.times.10.sup.11 CFU/g. The ideal water activity of the dry
SJ-3C bulk drug substance should be <0.220. When tested for
purity, the resulting SJ-3C bulk drug substance will contain
<200 CFU/g of total aerobic counts, <20 CFU/g of total yeasts
and molds, and an absence of objectionable organisms. The identity
of the resulting SJ-3C bulk drug substance is confirmed by the 16S
rRNA gene sequence.
[0096] The bulk drug substance is diluted by 3 to 10-fold with
maltodextrin to give a final dose of 2.times.10.sup.9 CFU/dose to
5.times.10.sup.9 CFU/dose. The dose is 200 mg. One dose of the
diluted drug substance is placed in a medical powder applicator and
packaged as the final drug product.
Example 2. Formulation Studies of Preservation Media with and
without Animal-Derived Excipients
[0097] The following example demonstrates the development and
identification of a preservation medium formulation without skim
milk that exhibits with good room temperature stability for a dry
powder Lactobacillus crispatus. The example illustrates the
increased stability of a dry Lactobacillus powder when
animal-derived excipients are eliminated from the preservation
medium. The following procedure describing the formulation and
development of the preservation medium culture using Lactobacillus
crispatus CTV-05 is applicable for any microorganism suitable for
use with the present invention.
[0098] For the preservation formulation development studies,
Lactobacillus crispatus CTV-05 was grown in a modified MRS medium
at pH 6.0 on a 1 L scale in a stirred bioreactor BioFlo 110
Fermentor and Bioreactor (New Brunswick Scientific, Edison, N.J.).
A batch fermentation process was used and cells were harvested at
early stationary phase when glucose consumption and lactic acid
production were completed. The fermentation process generally
yielded 1.0.times.10.sup.9-1.5.times.10.sup.9 CFU/mL with >90%
cell viability. The cells were recovered by centrifugation, washed
with phosphate buffered saline, and mixed with one or more
preservation matrices. The mixture was then freeze-dried in a
Virtis Advantage lyophilizer.
[0099] Initially, the cryopreservation and accelerated stability of
CTV-05 was evaluated in 16 preservation matrices containing
different concentrations of excipients for preserving Lactobacillus
including skim milk, trehalose, xylitol, ascorbic acid,
.alpha.-tocopherol, and phosphate buffer (Table 2).
TABLE-US-00002 TABLE 2 Composition of preservation matrices
containing skim milk Ingredient (%, w/w) Sodium .alpha.- Skim No.
ascorbate Tocopherol Xylitol milk Trehalose NaPO.sub.4 * 1 0.1 0.2
2 5.0 5.0 10 2 1.0 0.2 2 15 5.0 10 3 0.1 1.2 2 15 15 10 4 1.0 1.2 2
5.0 15 10 5 0.1 0.2 6 15 15 10 6 1.0 0.2 6 5.0 15 10 7 0.1 1.2 6
5.0 5.0 10 8 1.0 1.2 6 15 5.0 10 9 0.1 0.2 2 5.0 15 20 10 1.0 0.2 2
15 15 20 11 0.1 1.2 2 15 5.0 20 12 1.0 1.2 2 5.0 5.0 20 13 0.1 0.2
6 15 5.0 20 14 1.0 0.2 6 5.0 5.0 20 15 0.1 1.2 6 5.0 15 20 16 1.0
1.2 6 15 15 20 * Amount of sodium phosphate is measured in mM
[0100] The samples were placed in serum vials and frozen at
-40.degree. C. for 5 hr, then subjected to primary drying at
-40.degree. C. under vacuum for 30 hr and secondary drying at
25.degree. C. for 20 hr. The samples were packaged in foil pouches
with desiccant and stored at 37.degree. C. Powder samples were
removed at regular intervals and viability (i.e., activity)
measured by plating on MRS agar and colony counting at 0, 10, and
30 days. As shown in Table 3, formulations #6 and #16 exhibited the
best storage stability at 37.degree. C. (70%-75% retention of
initial viability).
TABLE-US-00003 TABLE 3 Stability of CTV-05 in preservation matrices
containing skim milk at 37.degree. C. T.sub.30 stability No. CFU/g
at T.sub.0 CFU/g at T.sub.10 CFU/g at T.sub.30 (% of T.sub.0
activity) 1 4.62 .times. 10.sup.10 3.17 .times. 10.sup.9 2.05
.times. 10.sup.9 4.4 2 2.34 .times. 10.sup.10 8.24 .times. 10.sup.9
4.20 .times. 10.sup.9 17.9 3 1.04 .times. 10.sup.10 1.11 .times.
10.sup.9 5.49 .times. 10.sup.8 5.3 4 1.69 .times. 10.sup.10 3.78
.times. 10.sup.9 2.15 .times. 10.sup.9 12.7 5 8.99 .times. 10.sup.9
6.80 .times. 10.sup.9 4.05 .times. 10.sup.9 45.1 6 1.60 .times.
10.sup.10 .sup. 1.51 .times. 10.sup.10 .sup. 1.13 .times. 10.sup.10
70.6 7 2.43 .times. 10.sup.10 2.43 .times. 10.sup.8 7.41 .times.
10.sup.7 0.3 8 2.10 .times. 10.sup.10 9.41 .times. 10.sup.9 6.27
.times. 10.sup.9 29.9 9 2.18 .times. 10.sup.10 2.06 .times.
10.sup.9 3.62 .times. 10.sup.8 1.7 10 1.21 .times. 10.sup.10 6.56
.times. 10.sup.9 3.05 .times. 10.sup.9 25.2 11 1.54 .times.
10.sup.10 1.82 .times. 10.sup.9 6.04 .times. 10.sup.8 3.9 12 2.07
.times. 10.sup.10 4.52 .times. 10.sup.9 1.86 .times. 10.sup.9 9 13
2.14 .times. 10.sup.10 .sup. 1.14 .times. 10.sup.10 5.32 .times.
10.sup.9 24.9 14 3.80 .times. 10.sup.10 .sup. 1.59 .times.
10.sup.10 4.68 .times. 10.sup.9 12.3 15 9.79 .times. 10.sup.9 5.65
.times. 10.sup.9 3.70 .times. 10.sup.9 37.8 16 7.34 .times.
10.sup.9 7.32 .times. 10.sup.9 5.55 .times. 10.sup.9 75.6
[0101] A similar experiment was performed using the same 16
formulations described above, with the skim milk component removed
(Table 4).
TABLE-US-00004 TABLE 4 Composition of preservation matrices without
skim milk Ingredient (%, w/w) Sodium No. ascorbate
.alpha.-Tocopherol Xylitol Trehalose NaPO.sub.4 * 1 0.1 0.2 2 5.0
10 2 1.0 0.2 2 5.0 10 3 0.1 1.2 2 15 10 4 1.0 1.2 2 15 10 5 0.1 0.2
6 15 10 6 1.0 0.2 6 15 10 7 0.1 1.2 6 5.0 10 8 1.0 1.2 6 5.0 10 9
0.1 0.2 2 15 20 10 1.0 0.2 2 15 20 11 0.1 1.2 2 5.0 20 12 1.0 1.2 2
5.0 20 13 0.1 0.2 6 5.0 20 14 1.0 0.2 6 5.0 20 15 0.1 1.2 6 15 20
16 1.0 1.2 6 15 20 * Amount of sodium phosphate is measured in
mM
[0102] As shown below in Table 5, formulations #6 and #16 without
skim milk exhibited similar initial potencies and the highest
storage stability at 37.degree. C. (.about.25% of initial
viability).
TABLE-US-00005 TABLE 5 Stability of CTV-05 in preservation matrices
without skim milk at 37.degree. C. T.sub.31 stability No. CFU/g at
T.sub.0 CFU/g at T.sub.10 CFU/g at T.sub.31 (% of T.sub.0 activity)
1 6.70 .times. 10.sup.10 1.50 .times. 10.sup.9 2.03 .times.
10.sup.8 0.3 2 8.10 .times. 10.sup.10 .sup. 1.09 .times. 10.sup.10
2.90 .times. 10.sup.9 3.6 3 3.00 .times. 10.sup.10 1.89 .times.
10.sup.9 8.61 .times. 10.sup.8 2.9 4 3.77 .times. 10.sup.10 9.09
.times. 10.sup.9 2.80 .times. 10.sup.9 7.4 5 3.16 .times. 10.sup.10
4.41 .times. 10.sup.9 7.79 .times. 10.sup.8 2.5 6 3.07 .times.
10.sup.10 .sup. 1.32 .times. 10.sup.10 7.36 .times. 10.sup.9 24 7
7.53 .times. 10.sup.10 1.36 .times. 10.sup.8 7.90 .times. 10.sup.6
0.01 8 6.45 .times. 10.sup.10 1.66 .times. 10.sup.9 5.47 .times.
10.sup.7 0.08 9 2.77 .times. 10.sup.10 1.91 .times. 10.sup.9 2.82
.times. 10.sup.8 1.0 10 3.00 .times. 10.sup.10 5.52 .times.
10.sup.9 1.68 .times. 10.sup.9 5.6 11 4.87 .times. 10.sup.10 6.91
.times. 10.sup.8 1.92 .times. 10.sup.8 0.39 12 4.80 .times.
10.sup.10 5.42 .times. 10.sup.9 5.19 .times. 10.sup.9 10.8 13 6.07
.times. 10.sup.10 2.69 .times. 10.sup.8 4.09 .times. 10.sup.7 0.067
14 5.69 .times. 10.sup.10 2.02 .times. 10.sup.9 2.30 .times.
10.sup.8 0.4 15 2.49 .times. 10.sup.10 2.84 .times. 10.sup.9 6.56
.times. 10.sup.8 2.6 16 2.60 .times. 10.sup.10 .sup. 1.39 .times.
10.sup.10 6.66 .times. 10.sup.9 25.6
[0103] While skim milk in Formulations #6 and #16 appeared to
improve the storage stability, higher concentrations of sodium
ascorbate and xylitol also appeared to improve the storage
stability. However, higher concentrations of .alpha.-tocopherol did
not seem to improve the storage stability of CTV-05, and subsequent
experiments demonstrated that removal of .alpha.-tocopherol (and
its vehicle, Tween 20) led to improved stability of CTV-05.
[0104] Following the removal of .alpha.-tocopherol (and Tween 20)
from the preservation matrix of Formulation 6 in Table 2 above, it
was determined that skim milk could also be removed without
adversely affecting cryoprotection during freeze-drying or long
term storage stability at 25.degree. C. (FIG. 1). Thus, the best
combination of cryoprotection and room temperature stability was
achieved using a preservation solution containing 15% trehalose, 6%
xylitol, 1% sodium ascorbate, and 10 mM sodium phosphate at pH 7.4
(FIG. 1, upper line). This formulation provided better stability
than the same formulation containing 5% skim milk (FIG. 1, lower
line).
Example 3. Use of Monosodium Glutamate to Improve Stability
[0105] Following the procedure of Example 2, cultured L. crispatus
CTV-05 (LACTIN-V) cells were formulated in four different
preservation media: 1) 15% trehalose, 6% xylitol, 1% sodium
ascorbate and 10 mM sodium phosphate at pH 7.4 (triangles); 2) the
same preservation medium as 1), and additionally 5% monosodium
glutamate (inverted triangles); 3) 12% trehalose, 8% xylitol, 1%
sodium ascorbate and 10 mM sodium phosphate at pH 7.4 (circles);
and 4) the same preservation medium as 3), and additionally 5%
monosodium glutamate (squares). As shown in FIG. 2, the monosodium
glutamate improved the stability of both powder formulations at
elevated temperatures (37.degree. C.), while having no effect on
the initial cryopreservation.
Example 4. Use of a Dry Composition to Treat Abnormal Vaginal
Microbiota
[0106] The example details an in vivo study which demonstrates that
the Lactobacillus of the present invention can be used in vivo,
delivered to the patient as a powder, to recolonize the vagina of
women having recurrent bacterial vaginosis.
[0107] A 25-year old female patient having recurrent bacterial
vaginosis (BV) is suffering from the symptoms of a BV infection,
such as abnormal vaginal odor and discharge, and discomfort from
itching and pain. BV is detected in the female patient by the Amsel
clinical criteria (.gtoreq.3 criteria satisfied), and confirmed
microbiologically by the Nugent scoring system (Nugent Score of
7-10).
[0108] The patient receives initial standardized antibiotic
treatment with 0.75% topical metronidazole (MetroGel.RTM.) once a
day for 5 days. After completion of the metronidazole treatment,
the female patient begins treatment using the Lactobacillus SJ-3C
drug product of the invention. A 200-mg dose of the SJ-3C drug
product having an activity of 2.times.10.sup.9 CFU per dose is
administered to the patient vaginally using the packaged medical
powder applicator. The patient receives a dose of the SJ-3C drug
product once per day before sleeping for 5 consecutive days,
followed by twice weekly treatments for 10 weeks.
* * * * *
References