U.S. patent application number 15/595248 was filed with the patent office on 2017-08-31 for phospholipid depot.
The applicant listed for this patent is Dr. Reddy's Laboratories SA. Invention is credited to Andrew Xian CHEN, Hailiang CHEN, Franklin OKUMU, Dushyanth SURAKANTI.
Application Number | 20170246109 15/595248 |
Document ID | / |
Family ID | 43928096 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170246109 |
Kind Code |
A1 |
CHEN; Hailiang ; et
al. |
August 31, 2017 |
PHOSPHOLIPID DEPOT
Abstract
The present invention provides a clear depot comprising at least
one hydrophilic water-soluble pharmaceutically active agent
selected from the group consisting of vancomycin, gentamicin, a
pharmaceutically acceptable salt thereof and a mixture thereof,
water, a phospholipid, an oil, optionally a pH adjusting agent, and
a viscosity modifying agent selected from the group consisting of
ethanol, isopropanol, and a mixture thereof, wherein the water
present in the depot is no more than about 4 wt % relative to the
total weight of the depot and the depot has a pH of between about 3
and about 6, method of making and administering same.
Inventors: |
CHEN; Hailiang; (San Diego,
CA) ; CHEN; Andrew Xian; (San Diego, CA) ;
SURAKANTI; Dushyanth; (Somerset, NJ) ; OKUMU;
Franklin; (Morristown, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dr. Reddy's Laboratories SA |
Basel |
|
CH |
|
|
Family ID: |
43928096 |
Appl. No.: |
15/595248 |
Filed: |
May 15, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15347949 |
Nov 10, 2016 |
9682040 |
|
|
15595248 |
|
|
|
|
14793552 |
Jul 7, 2015 |
9522169 |
|
|
15347949 |
|
|
|
|
13242778 |
Sep 23, 2011 |
9132144 |
|
|
14793552 |
|
|
|
|
PCT/US2010/061015 |
Dec 17, 2010 |
|
|
|
13242778 |
|
|
|
|
61375502 |
Aug 20, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/107 20130101;
A61K 9/1075 20130101; A61K 38/14 20130101; A61K 9/145 20130101;
A61K 47/10 20130101; A61K 9/16 20130101; A61K 47/44 20130101; A61P
31/04 20180101; A61K 9/0024 20130101; Y02A 50/30 20180101; A61P
31/00 20180101; A61K 9/1682 20130101; A61K 9/0019 20130101; A61K
31/7036 20130101; A61K 47/24 20130101; A61K 9/1617 20130101 |
International
Class: |
A61K 9/107 20060101
A61K009/107; A61K 47/44 20060101 A61K047/44; A61K 47/24 20060101
A61K047/24; A61K 38/14 20060101 A61K038/14; A61K 31/7036 20060101
A61K031/7036 |
Claims
1. A depot composition comprising: at least one hydrophilic
water-soluble pharmaceutically active agent selected from the group
consisting of vancomycin, gentamicin, and pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent; wherein said composition is
characterized by a major endothermic event up to about 100.degree.
C.
2. The depot of claim 1, wherein said phospholipid is present in
the depot from about 5 wt % to about 95 wt %.
3. The depot of claim 1, wherein said phospholipid is present in
the depot from about 25 wt % to about 75 wt %.
4. The depot of claim 1, wherein said phospholipid is present in
the depot from about 35 wt % to about 60 wt %.
5. The depot of claim 1, wherein said oil is selected from the
group consisting of: vegetable oil, animal oil, and a mixture
thereof.
6. The depot of claim 1, wherein said oil present in the depot is
from about 5 wt % to about 95 wt %.
7. The depot of claim 1, wherein said oil present in the depot is
from about 25 wt % to about 75 wt %.
8. The depot of claim 1, wherein said oil present in the depot is
from about 35 wt % to about 60 wt %.
9. The depot of claim 1, wherein said viscosity modifying agent is
selected from the group consisting of: ethanol, isopropanol and a
mixture thereof.
10. The depot of claim 1, wherein the amount of the viscosity
modifying agent in the depot is from about 1 wt % to about 20 wt %
relative to the total weight of the depot.
11. The depot of claim 1, wherein the amount of the viscosity
modifying agent in the depot is from about 2 wt % to about 18 wt %
relative to the total weight of the depot.
12. The depot of claim 1, wherein said pharmaceutically acceptable
salt of vancomycin and/or gentamicin is selected from the group
consisting of acetate, hydrochloride, hydrobromide, citrate,
formate, lactate, succinate, and sulfate.
13. The depot of claim 1, wherein the viscosity of the depot is
from about 100 centipoise to about 5000 centipoise.
14. The depot of claim 1, wherein the pH of the depot is from about
3 to about 6.
15. The depot of claim 1, wherein the amount of the water present
in the depot is no more than about 4 wt % relative to the total
weight of the depot.
16. The depot of claim 1, wherein the amount of the water present
in the depot is no more than about 2 wt % relative to the total
weight of the depot.
17. The depot of claim 1, wherein said endothermic event is
elucidated by Differentiating Scanning calorimetry (DSC).
18. A depot composition comprising: at least one hydrophilic
water-soluble pharmaceutically active agent selected from group
consisting of vancomycin, gentamicin, and pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent; wherein said composition releases no
more than about 80% of vancomycin and/or gentamicin at two hours
when measured in accordance with a USP method I using 500 ml of
deionized water as a medium.
19. The depot of claim 18, wherein said composition releases no
more than about 50% of vancomycin and/or gentamicin at two
hours.
20. The depot of claim 18, wherein said composition releases no
more than about 20% of vancomycin and/or gentamicin at two
hours.
21. A composition comprising: at least one hydrophilic
water-soluble pharmaceutically active agent selected from the group
consisting of vancomycin, gentamicin, and pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent; wherein said composition is selected
from an emulsion, a primary emulsion, a nanoemulsion, a solution,
dry paste, a gel, or a combination thereof.
22. The composition of claim 21, wherein said composition is an
emulsion and is prepared by a process comprising:
emulsification.
23. The composition of claim 21, wherein said composition is a
primary emulsion and is prepared by homogenization of an
emulsion.
24. The composition of claim 21, wherein said solution is
monophasic solution and is prepared by a process comprising
preparing an emulsion, performing homogenization to obtain a
primary emulsion, and performing microfludization to obtain a
monophasic solution.
25. The composition of claim 21, wherein said composition is a dry
paste and is prepared by a process comprising: emulsification,
homogenization of the emulsion, and microfluidization and
lyophilisation to obtain a dry paste.
26. The composition of claim 21, wherein said gel is prepared by a
process comprising steps of (1) emulsification, (2)
homogenization/microfluidization, (3) lyophilization, (4) dilution,
(5) pre-filtration, (6) viscosity modifying agent removal and (7)
filtration.
27. A composition comprising: vancomycin or pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent; wherein said composition is an
oil-in-water emulsion having an initial drug concentration of
vancomycin in water from about 1 mg/ml to about 50 mg/ml.
28. The composition of claim 27, wherein said vancomycin is
vancomycin hydrochloride.
29. The composition of claim 27, wherein said composition has an
initial drug concentration of vancomycin hydrochloride in water
from about 20 mg/ml to about 30 mg/ml.
30. A composition comprising: gentamicin or pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent; wherein said composition is oil-in-water
emulsion having an initial drug concentration of gentamicin in
water from about 1 mg/ml to about 75 mg/ml.
31. The composition of claim 30, wherein said gentamicin is
gentamicin sulfate.
32. The composition of claim 30, wherein said composition has an
initial drug concentration of gentamicin sulfate in water from
about 10 mg/ml to about 30 mg/ml.
33. A method of administering a depot comprising: administering
intradermally, intramuscularly, intraincisionally, subcutaneously,
by instillation or topically to a wound at least one hydrophilic
water-soluble pharmaceutically active agent selected from group
consisting of vancomycin, gentamicin, and pharmaceutically
acceptable salts thereof; water; a phospholipid; an oil; and a
viscosity modifying agent.
34. The method of claim 33, wherein said method comprises
administering a depot which provides local tissue concentrations
sufficient to treat and/or prevent infections at the wound.
35. The method of claim 33, wherein said depot is sufficient to
release the pharmaceutically active agent for a period of about at
least one day with a dosing volume from about 0.1 mL to about 100
mL.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/347,949, filed Nov. 10, 2016, which is a
continuation of U.S. patent application Ser. No. 14/793,552, filed
Jul. 7, 2015 and now issued as U.S. Pat. No. 9,522,169, which is a
continuation of U.S. patent application Ser. No. 13/242,778, filed
Sep. 23, 2011 and now issued as U.S. Pat. No. 9,132,144, which is a
continuation of International application No. PCT/US2010/061015,
filed Dec. 17, 2010, which claims the benefit of the filing date of
U.S. Provisional Patent Application No. 61/375,502 filed Aug. 20,
2010, each of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] A depot is a way of administering an active ingredient into
the body of a patient for systemic or local action. It is generally
administered by subcutaneous or intramuscular injection or
instillation into other body tissues, vessels or cavities. A depot
can also be applied to a wound before it is staunched, stitched,
bandaged or otherwise closed. Unlike removable depots,
biodegradable depots disintegrate or degrade within a pre-defined
time, typically after the entrapped active pharmaceutical
ingredient has been delivered. In other constructs, the
biodegradable injectable depot releases its active pharmaceutical
ingredient roughly simultaneously with, or as a function of, its
gradual degradation. A key advantage of certain biodegradable
delivery depots is their ability to deliver medication directly to
the intended site of action providing elevated local concentrations
of medication when compared to systemic levels.
[0003] Depots can also modulate delivery of medication to enable
various release profiles. The release profile could be immediate
release (burst) followed by a steady state, could be, among others,
"zero order" or constant rate of delivery, could provide a slow
rise to steady state, or could even provide for a delayed release.
In addition, depots have the advantage of allowing release over an
extended period of time, with a single administration. Blood levels
are not compromised by, for example, patient compliance issues.
[0004] Depots can be comprised of particulate systems such as
microsphere-based depots and nanosphere-based depots, or can also
be comprised of a biodegradable gel, typically made from soluble
matrix formers (polymers, lipids, carbohydrates) and either an
organic solvent or a mixture of water miscible and non-miscible
solvents.
[0005] Phospholipids have been used to prepare depots comprising a
lipophilic pharmacological active agent. Phospholipids are soluble
in oils or organic solvents but insoluble in water. To form a
depot, a high concentration of depot-forming phospholipids is often
required. This can impact the volume and viscosity of the resulting
depot and, accordingly, currently available phospholipid depots can
be very difficult to inject through a conventional needle or a
syringe. References describing phospholipids-based formulations
include WO 89/00077, WO 02/32395, EP 0282405 and U.S. Pat. Nos.
5,863,549, 4,252,793, 5,660,854, 5,693,337, and Wang et al.,
Lyophilization Of Water-In-Oil Emulsions To Prepare
Phospholipid-Based Anhydrous Reverse Micelles For Oral Peptide
Delivery, 39 European Journal of Pharmaceutical Sciences, at 373-79
(2010).
[0006] Vancomycin is a glycopeptide antibiotic used in the
prophylaxis and treatment of infections caused by Gram-positive
bacteria. It is generally the drug of choice for serious infection
and endocarditis caused by S. aureus, coagulase-negative
staphylococci, streptococcus pneumoniase, .beta.-hemolytic
streptococci, corynebacterium group JK, viridans streptococci, or
enterococci when .beta.-lactams cannot be used because of drug
allergy or resistance. Vancomycin can be combined with other
antimicrobials when treating, inter alia, methicillin-resistant
coagulase-negative staphylococcal prosthetic valve endocarditis,
and enterococcal endocarditis. It has also been used as an
alternative agent for pneumococcal meningitis caused by strains
with reduced penicillin sensitivity. Vancomycin has been used in
cardiac and vascular surgery to prevent post surgical infection.
See Rybak et al., Vancomycin Therapeutic Guidelines: A Summary of
Consensus Recommendations From The Infectious Diseases Society of
America, The American Society Of Health-System Pharmacists, and The
Society Of Infectious Disease Pharmacists, CID 2009:49 (1 August),
pg. 325.
[0007] Gentamicin is an aminoglycoside antibiotic used to treat
many types of bacterial infections particularly those caused by
susceptible Gram-negative bacteria. It has been used in a surgical
setting because it acts against pathogens such as pseudomonas
aeroginosa and escherichia coli. Gentamicin has been used in other
surgical applications (e.g. compounded with bone cement in
orthopedic settings). Gentamicin impregnated with biodegradable
collagen implant (sponge) is currently being used in several
markets outside of the US for the prevention of surgical site
infections (SSI). However, two large pivotal phase III studies
showed higher incidence of SSI in patients receiving the gentamicin
sponge (colorectal surgery) and no difference in the incidence of
SSI vs. standard of care (cardiothoracic surgeries). See generally,
E. Bennett-Guerrero, NEJM, 2010, 1-10; and E. Bennett-Guerrero,
JAMA, Aug. 18, 2010, 755-762.
[0008] Both vancomycin and gentamicin are very hydrophilic
antibiotics. They are also both difficult to formulate into
injectable depots based on phospholipids or other high oil phase
content formulations, as they are not freely soluble in
phospholipid or oil.
[0009] In addition, by conducting a series of stability tests, it
has now been found that vancomycin and gentamicin degrade by
different mechanisms. Vancomycin loses its stability through
hydrolysis while gentamicin degrades due to oxidation or adduct
formation. Thus, formulations containing either one of the actives
are generally sensitive to these conditions. Moreover, both
vancomycin and gentamicin are heat-sensitive and cannot be
sterilized by using heat, such as autoclaving or
gamma-radiation.
[0010] Accordingly, attempting to formulate a depot comprising
vancomycin, gentamicin or both along with a phospholipid and oil
provide many practical challenges. One such attribute includes the
formulation should not feature high viscosity since the formulation
has to be sterilized by filtering through a sterilizing membrane,
such as one having pores of about 0.2 micron or less. There also
remain certain dichotomous problems. For instance, these two
particular actives have compatibility problems with phospholipids
which, like viscosity, suggests a need to keep phospholipid content
low. However, the need for coherent and cohesive gel formation and
proper release characteristics suggest just the opposite.
[0011] Accordingly, there remains a long felt need for storage
stable phospholipid depots containing vancomycin, gentamicin, a
pharmaceutical salt thereof or a mixture thereof that can be
administered by subcutaneous or intramuscular injection, by
intraincisional injection or placement into surgical wound or other
body tissues, vessels or cavities.
BRIEF SUMMARY OF THE INVENTION
[0012] One aspect of the present invention provides a process for
making a depot comprising at least one hydrophilic water-soluble
pharmaceutically active agent comprising: (1) mixing at least one
hydrophilic water-soluble pharmaceutically active agent selected
from the group consisting of vancomycin, gentamicin, a
pharmaceutically acceptable salt thereof and a mixture thereof,
water, a phospholipid, and an oil to form an oil-in-water
"emulsion"; (2) homogenizing the emulsion to obtain a "primary
emulsion"; (3) microfluidizing the primary emulsion to obtain a
"monophasic solution," (4) ensuring that the pH of the primary
emulsion and/or the monophasic solution is between about 3 to about
6, and in one embodiment, from about 3 to about 5, and in another
embodiment, from about 3 to about 4 by adjusting the pH as
necessary, (5) lyophilizing the monophasic solution of desired pH
to obtain a dry paste, (6) adding a viscosity modifying agent to
the dry paste in an amount sufficient to obtain a clear solution,
(7) removing at least some of the viscosity modifying agent from
the clear solution to obtain a depot having from about 5.5 wt % to
about 7.5 wt % of the viscosity modifying agent relative to the
total weight of the depot, and (8) sterilizing the depot by
filtration.
[0013] In one embodiment, the steps of forming the emulsion and the
primary emulsion can be combined as one step as long as the
resulting product is the primary emulsion. In another embodiment,
the steps of forming a primary emulsion and the monophasic solution
can be combined as one step, as long as the resulting product is
the monophasic solution. In yet another embodiment, the steps of
forming the emulsion, the primary emulsion, and the monophasic
solution can be combined as one step thereby going directly to the
monophasic solution.
[0014] In an embodiment, the water present in the depot is no more
than about 4 wt % relative to the total weight of the depot. In
another embodiment, the water content of the depot is no more than
about 2 wt %, and in still another embodiment, no more than about 1
wt %. In still a further embodiment, there is no more than about
0.5 wt % of water relative to the total weight of the depot. In
other embodiments, the pharmaceutically active agents are
vancomycin hydrochloride and gentamicin sulfate. In other
embodiments, the depot is clear, and in yet other embodiments, the
depot is ultra clear.
[0015] Another aspect of the present invention provides a process
for making a clear depot comprising at least one hydrophilic
water-soluble pharmaceutically active agent comprising: (1)
dissolving at least one hydrophilic water-soluble pharmaceutically
active agent selected from the group consisting of vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof in water to form an aqueous solution; (2) forming
an oil-in-water emulsion comprising a phospholipid, an oil, and the
aqueous solution comprising; (3) homogenizing the emulsion to
obtain a primary emulsion; (4) microfluidizing the primary emulsion
to obtain a monophasic solution, (5) adjusting the pH of the
emulsion, primary emulsion and/or the monophasic solution to
between about 3 to about 6, in another embodiment, from about 3 to
about 5, and in yet another embodiment, from about 3 to about 4 as
necessary, (6) lyophilizing the monophasic solution of desired pH
to obtain a dry paste, (7) adding a viscosity modifying agent to
the dry paste in an amount sufficient to obtain a desired viscosity
and/or a desired clarity, (8) pre-filtering of the viscosity
modified solution to obtain a clear solution, (9) removing at least
some of the viscosity modifying agent from the clear solution to
obtain a depot having from about 5.5 wt % to about 7.5 wt % of the
viscosity modifying agent relative to the total weight of the
depot, and (10) sterilizing the depot without substantial heating.
Such sterilization procedures may be done by filtration among other
methods. In another embodiment, pre-filtering and removing the
viscosity modifying agent are optional steps. In one embodiment,
the at least one hydrophilic water-soluble pharmaceutically active
agent is vancomycin, gentamicin, a pharmaceutically acceptable salt
thereof and a mixture thereof.
[0016] Yet another aspect of the present invention provides a
method for making a depot comprising: (1) forming an oil-in-water
emulsion including a phospholipid, an oil, at least one hydrophilic
water-soluble pharmaceutically active agent selected from the group
consisting of vancomycin, gentamicin, a pharmaceutically acceptable
salt thereof or a mixture thereof and water; (2) converting the
emulsion to a monophasic solution having a pH of between about 3 to
about 6; (3) lyophilizing the monophasic solution to obtain a dry
paste, (4) adding a viscosity modifying agent to the dry paste in
an amount sufficient to obtain a viscosity modified solution, (5)
removing at least some of the viscosity modifying agent to obtain a
depot, and (6) sterilizing the depot, wherein the depot is
clear.
[0017] In an embodiment, the method further comprises a step of
aseptically filling the depot into a syringe, a vial or any other
appropriate device capable of storing and/or delivering the depot
to the treatment site or wound.
[0018] In accordance with another aspect of the invention, a
stabilizing agent is optionally dissolved in water along with the
pharmaceutically acceptable ingredient(s). In yet another aspect of
the invention, a stabilizing agent is optionally mixed along with
the pharmaceutically acceptable ingredient(s), water, a
phospholipid, and an oil. Examples of the stabilizing agent
includes, but are not limited to EDTA disodium, glycine,
L-histidine, citric acid, mithionine, ascorbic acid, L-cysteine,
alpha-tocopherol, and mixtures thereof. In yet another aspect of
the invention, the depot does not include a stabilizing agent.
[0019] In an embodiment, in the step of forming the oil-in-water
emulsion, the amount of water added is about 60 wt % to about 80 wt
% relative to the total weight of the resulting emulsion. In
another embodiment, the amount of water in the emulsion in the step
of forming the oil-in-water emulsion is about two times the weight
of the emulsion.
[0020] In yet another embodiment, after the step of microfluidizing
the primary emulsion, which results in a monophasic solution, also
referred to herein as "nanoemulsion", the nanoemulsion droplet size
has an average diameter of less than about 120 nm, less than about
100 nm, or less than about 80 nm.
[0021] The reduction of average diameter of the droplet size of the
nanoemulsion/monophasic solution is believed, without limitation,
to reduce the viscosity of the resulting monophasic solution,
allowing sterilization through a filter, rather than by using a
heat-based sterilization system, such as by autoclaving or
gamma-radiation sterilization, which can affect stability of
vancomycin and/or gentamicin.
[0022] Before the step of microfluidization, the primary emulsion
is generally a white, opaque, thick yogurt-like mass. After
microfluidization, the resulting monophasic solution is generally
clear, translucent, and water-like in viscosity and flow
properties.
[0023] Although the present invention is not limited by any
particular theory of operation, it is believed that very
hydrophilic vancomycin, gentamicin, a pharmaceutically acceptable
salt thereof or a mixture thereof, can be formulated with
phospholipids to form a monophasic solution as defined herein
resulting in storage stable depots with desirable properties. It is
believed that the extremely small nanoemulsion droplets provided
during microfluidization may be instrumental in the eventual
properties of the depots produced, among other factors that may be
involved.
[0024] In accordance with another embodiment of the present
invention, the pH of the emulsion, primary emulsion and/or the
monophasic solution is from about 3 to about 6, from about 3 to
about 5, or from about 3 to about 4. And if not, the pH could be
adjusted to that it fell in the desired range.
[0025] In accordance with yet another embodiment of the present
invention, the pH of the depot, the final product, is from about 3
to about 6, from about 3 to about 5, and in another embodiment,
from about 3 to about 4.
[0026] Another aspect of the present invention is a depot
comprising at least one hydrophilic water-soluble pharmaceutically
active agent selected from the group consisting of vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof, water, a phospholipid, and one or more of an oil,
optionally a pH adjusting agent, and a viscosity modifying agent,
wherein the water present in the depot is no more than about 4 wt
%, no more than about 2 wt %, no more than about 1 wt %, or no more
than about 0.5 wt % of water relative to the total weight of the
depot. In another embodiment, the depot is syringeable.
[0027] In one embodiment of the present invention, the depot
comprises both vancomycin and gentamicin. In another embodiment,
the depot comprises pharmaceutical salts of one or both vancomycin
and gentamicin. In another embodiment, the depot comprises either
vancomycin or gentamicin. In yet another embodiment, the depot
comprises a pharmaceutical salt of either vancomycin or
gentamicin.
[0028] The depots in accordance with the present invention are, in
one embodiment, "clear." This offers advantages in being able to
see entrapped air, foreign bodies, and the like to prevent the
unintended introduction of same into the body. Interestingly, it
has also been discovered that when both vancomycin and gentamicin
are present in the depot, the depot of the invention is clearer
than when the depot contains either vancomycin or gentamicin alone.
In such embodiment where both vancomycin and gentamicin are present
in the depot, the clarity of such depot is "ultra clear" as defined
herein. In an embodiment where the depot comprises either
vancomycin or gentamicin, the clarity of such depot is
"translucent" or "clear" as defined herein.
[0029] In one embodiment, the viscosity modifying agent is ethanol,
wherein the amount of ethanol present in the depot is from about 3
wt % to about 25.0 wt %, about 4 wt % to about 10 wt %. In still
another embodiment, the amount of ethanol present ranges from
between about 5 wt % to about 6.5 wt % relative to the total weight
of the depot. In yet another embodiment, the viscosity modifying
agent is absolute ethanol.
[0030] In an embodiment, the viscosity modifying agent may be added
to the dry paste until the amount of viscosity modifying agent is
about 75 wt % or more of the viscosity modified solution. In other
embodiments, the amount of viscosity modifying agent is about 50 wt
% or more, and in still another embodiment, about 30 wt % or more.
Finally, the amount of viscosity modifying agent is about 25 wt %
or more relative to total weight of the viscosity modified
solution.
[0031] In yet another embodiment, the amount of phospholipid
present in the depot is from about 5 wt % to about 95 wt %, and in
another embodiment, from about 25 wt % to about 75 wt % relative to
the total weight of the depot. In another embodiment, the amount of
phospholipids ranges from about 35 wt % to about 60 wt % relative
to the total weight of the depot.
[0032] In accordance with another embodiment of the present
invention, the amount of oil present in the depot is from about 5
wt % to about 95 wt %, and in another embodiment, from about 25 wt
% to about 75 wt % relative to the total weight of the depot. In
yet another embodiment, the amount of oil ranges from about 35 wt %
to about 60 wt % relative to the total weight of the depot.
[0033] In accordance with an embodiment of the present invention,
no more than about 80% of vancomycin and/or gentamicin are released
at two hours when measured in accordance with a USP method I using
500 ml of deionized water as a medium. In another embodiment, no
more than about 50%, and in yet another embodiment, no more than
about 20% of vancomycin and/or gentamicin are released at two hours
when measured in accordance with a USP method I using 500 ml of
deionized water as a medium.
[0034] In accordance with another aspect of the invention, the
depot optionally comprises a stabilizing agent to improve the
stability of vancomycin, gentamicin or both. Examples of the
stabilizing agent include, but not limited to EDTA (disodium
edentate), glycine, L-histidine, citric acid, mithionine, ascorbic
acid, L-cysteine, alpha-tocopherol, and mixtures thereof. In
accordance with yet another aspect of the invention, the depot does
not contain a stabilizing agent. In still another embodiment, the
amount of stabilizing agent used, if any, will not negatively
impact the stability of each active, vancomycin or gentamicin, in
the depot.
[0035] In another aspect of the invention, a depot as described
herein is provided in an applicator, syringe, vial or any other
device capable of storing and/or delivering the depot to the
treatment site, depot site or wound.
[0036] Another aspect of the present invention is a method of
administering, via intradermal, intramuscular, intraincisional,
subcutaneous, instillation or topically, the depot of the invention
comprising a hydrophilic water-soluble pharmaceutically active
agent selected from the group consisting of vancomycin, gentamicin,
a pharmaceutically acceptable salt thereof or a mixture thereof,
water, phospholipid, an oil, optionally a pH adjusting agent and a
viscosity modifying agent to a patient in need thereof.
[0037] Yet another aspect of the present invention is a method of
preventing and/or treating post surgical infection by introducing a
depot of the present invention.
[0038] Another aspect of the present invention is a method of
preventing and/or treating infection comprising administering a
depot of the present invention which achieves sufficiently high
local tissue concentrations sufficient to treat and/or prevent
infections at a local site, without toxicity to kidney and/or other
organs, and without contributing to the emergence of drug resistant
strains of bacteria.
[0039] In another aspect, there is a method of rendering localized
tissue unable to sustain pathogenic microorganisms by administering
a depot of the present invention to the wound.
[0040] Yet another aspect of the present invention is a method of
rendering localized tissue unable to sustain pathogenic
microorganisms by administering a depot of the present invention
without causing toxicity to kidney and other organs, and without
causing emergence of drug resistant strains of bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a process flow diagram of an embodiment of the
method of making an inventive composition in accordance with an
aspect of the invention.
[0042] FIG. 2 shows the assay recovery of vancomycin and gentamicin
of the formulation of EXAMPLE 1 after the autoclave treatment.
[0043] FIG. 3 is an in vitro release profile of gentamicin and
vancomycin of the formulation of EXAMPLE 6 using USP method I.
[0044] FIG. 4 illustrates plasma concentrations of vancomycin of
the formulation of EXAMPLE 1 in rabbits.
[0045] FIG. 5 illustrates tissue concentrations of vancomycin of
the formulation of EXAMPLE 1 in rabbits.
[0046] FIG. 6 illustrates plasma concentrations of gentamicin of
the formulation of EXAMPLE 1 in rabbits.
[0047] FIG. 7 illustrates tissue concentrations of gentamicin of
the formulation of EXAMPLE 1 in rabbits.
[0048] FIG. 8 illustrates mean vancomycin plasma concentrations in
rabbits after single SC wound instillation of the formulation of
EXAMPLE 6.
[0049] FIG. 9 illustrates mean total plasma concentration of
gentamicin of the formulation of EXAMPLE 6 in rabbits.
[0050] FIG. 10 illustrates tissue concentration in a pig when
administered intraincisionally with a depot of the present
invention vs. MIC 90 for top surgical site infection (SSI)
pathogens
[0051] FIG. 11 illustrates comparison of vancomycin plasma
concentration after therapeutic IV dose in humans vs.
intraincisional administration of a formulation in accordance with
the present invention in the pig.
[0052] FIG. 12 illustrates the small angle X-ray diffraction (SAXS)
patterns of Examples 10A to 10F.
[0053] FIG. 13 illustrates the thermal gravimetric analysis of
Examples 10A and 10D
[0054] FIG. 14 illustrates the differential scanning calorimetry
(DSC) analysis of Examples 10A and 10D
DETAILED DESCRIPTION
[0055] The present invention will be described in more detail
below.
[0056] While the specification concludes with the claims
particularly pointing and distinctly claiming the invention, it is
believed that the present invention will be better understood from
the following description. All percentages and ratios used herein
are by weight of the total composition and all measurements made
are at 25.degree. C. and normal pressure unless otherwise
designated. All temperatures are in Degrees Celsius unless
specified otherwise. The present invention can comprise (open
ended) or consist essentially of the components of the present
invention as well as other ingredients or elements described
herein. As used herein, "comprising" means the elements recited, or
their equivalent in structure or function, plus any other element
or elements which are not recited. The terms "having," "including,"
and "comprised of" are also to be construed as open ended unless
the context suggests otherwise. As used herein, "consisting
essentially of" means that the invention may include ingredients in
addition to those recited in the claim, but only if the additional
ingredients do not materially alter the basic and novel
characteristics of the claimed invention. Generally, such additives
may not be present at all or only in trace amounts. However, it may
be possible to include up to about 10% by weight of materials that
could materially alter the basic and novel characteristics of the
invention as long as the utility of the compounds (as opposed to
the degree of utility) is maintained. All ranges recited herein
include the endpoints, including those that recite a range
"between" two values. Terms such as "about," "generally,"
"substantially," and the like are to be construed as modifying a
term or value such that it is not an absolute. Such terms will be
defined by the circumstances and the terms that they modify as
those terms are understood by those of skill in the art. This
includes, at very least, the degree of expected experimental error,
technique error and instrument error for a given technique used to
measure a value.
[0057] Note that while the specification and claims may refer to a
final product such as, for example, a depot or other dosage form of
the invention as, for example, containing a pH at an intermediate
state, it may be difficult to tell from the final dosage form that
the recitation is satisfied. However, such a recitation may be
satisfied if the materials used prior to final production meet that
recitation. Similarly, the amount of ingredients introduced into,
for example, the emulsion, if described as being by weight may
change relative to the weight of the product at some other phase of
production such as, in the final depot, which may weight more or
less. It is sufficient that those weight percentages were correct
at any steps of production and/or in any intermediate. Indeed, as
to any property or characteristic of a final product which cannot
be ascertained from the dosage form directly, it is sufficient if
that property resides in the components recited just prior to final
production steps.
[0058] The term "emulsion" used herein is a system of two
immiscible liquid phases. One of the two phases (the internal
phase, discontinuous phase or discrete phase) is distributed as
droplets/globules through the second phase (the external or
continuous phase). As used herein, emulsions include oil-in-water
(O/W) emulsions, in which a less polar liquid commonly referred to
as an oil is in the internal phase; and water-in-oil (W/O)
emulsions, in which an aqueous or other relatively polar liquid is
in the internal phase.
[0059] The term "primary emulsion" used herein refers to a
resulting product of the homogenization step, which may employ, for
example, a high shear mixer.
[0060] The term "monophasic solution" and "nanoemulsion" are used
interchangeably herein. It is noted that the term "solution" in
"monophasic solution" does not mean that it is a homogeneous
mixture of two or more substances, but that it is a resulting
product of the microfluidization step, which may employ, for
example, a high-pressure microfluidizer.
[0061] The term "monophasic," "one phase" and "one phase-like" are
used to mean that the resulting product will remain as one phase
without separation of phases or precipitation even after 6000 g
centrifugation for 10 minutes at 25 deg C. in 1 g sample quantity,
using a centrifuge made by Heraeus, Model Biofuge Fresco or any
equivalent.
[0062] The term "viscous" as used here means that the viscosity of
the composition is from about 1 centipoise to about 5000
centipoise, from about 200 centipoise to about 2000 centipoise, or
from about 300 centipoise to about 1500 centipoise.
[0063] The term "syringeable" as used herein means that the
composition may be administered with a syringe or a catheter or
withdrawn from a vial into a syringe. It does not mean, however,
that the composition of the invention must actually be in a syringe
or administered using a syringe unless the specific recitation or
the context suggests that meaning.
[0064] The term "translucent" and "clear" are used interchangeably
herein to mean that the final depot or any of the intermediate step
composition, such as a solution, emulsion, primary emulsion,
nanoemulsion, and/or a gel, is not hazy or opaque, and that it is
free from visually suspended particles. It should also be free of
bubbles. Moreover, by translucent, it is meant that the depot
and/or any of the intermediate composition, such as a solution,
emulsion, primary emulsion, nanoemulsion, and/or a gel, is free
from visually suspended particles and should also be free of
bubbles. Moreover, by "translucent" or "clear," it is also meant
that the depot and/or any of the intermediate composition, such as
a solution, emulsion, primary emulsion, nanoemulsion, and/or a gel,
of the present invention has a light transmittance of greater than
about 90% measured at 800 nm (T800) in a 1 cm path quartz cuvette
using alcohol as blank when measured by a UV-visible
spectrophotometer, such as the one made by Pharmacia, Model
Ultrospec III.
[0065] By "hazy" or "opaque," it is meant that a T800 value of the
depot is less than about 90%.
[0066] By "ultra clear," it is meant that a T800 value of the depot
is greater than about 92%, or 95%.
[0067] The term "stable" as used herein means that (1) the
formulation remains clear at 25 deg C. for at least one year, or
(2) the formulation remains clear and does not separate out or
precipitate after centrifugation when the formulation is exposed to
40 deg C. for one week.
[0068] The term "gel" and "depot" are used interchangeably
herein.
Process Description
[0069] As shown in FIG. 1, one aspect of the present invention
provides a process for making a depot comprising a hydrophilic
water-soluble pharmaceutically active agent selected from the group
consisting of vancomycin, gentamicin, a pharmaceutically acceptable
salt thereof, and a mixture thereof, comprising: (1) mixing at
least one hydrophilic water-soluble pharmaceutically active agent
selected from the group consisting of vancomycin, gentamicin, a
pharmaceutically acceptable salt thereof and a mixture thereof,
water, a phospholipid, and an oil to form oil-in-water emulsion
(see FIG. 1, Step 1); (3) homogenizing the emulsion to obtain a
primary emulsion (see FIG. 1, Step 2); (4) microfluidizing the
primary emulsion to obtain a monophasic solution, also referred to
herein and in FIG. 1 as a nanoemulsion (see FIG. 1, Step 3), (4)
ensuring that the pH of the primary emulsion and/or the monophasic
solution is between about 3 to about 6, a range of from about 3 to
about 5, or a range of from about 3 to about 4 by adjusting the pH
as necessary (see FIG. 1, Step 4), (5) lyophilizing the monophasic
solution of desired pH to form a dry paste (see FIG. 1, Step 5),
(6) adding a viscosity modifying agent to the dry paste in an
amount sufficient to obtain a clear solution, (see FIG. 1, Step 6)
(7) removing at least some of the viscosity modifying agent from
the clear solution to obtain a depot having from about 5.5 wt % to
about 7.5 wt % of the viscosity modifying agent relative to the
total weight of the depot (see FIG. 1, Step 7), and (8) sterilizing
the depot without heating the depot (see FIG. 1, Step 8).
[0070] In an embodiment of the present invention, the step of
mixing at least one hydrophilic water-soluble pharmaceutically
active agent selected from the group consisting of vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof, water, a phospholipid, and an oil to form an
oil-in-water emulsion comprises (1) dissolving vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof in water to form an aqueous solution; and (2)
forming an emulsion comprising a phospholipid, an oil, and an
aqueous solution comprising the hydrophilic water-soluble
pharmaceutically acceptable ingredient(s) selected from the group
consisting of vancomycin, gentamicin, a pharmaceutically acceptable
salt thereof or a mixture thereof.
[0071] In an alternate embodiment, a viscosity modifying agent is
added to the dry paste in an amount sufficient to obtain a desired
viscosity, and then the viscosity modified solution is pre-filtered
to obtain a clear solution.
[0072] In one embodiment, the water present in the depot is no more
than about 4 wt %, no more than about 2 wt %, no more than about 1
wt %, or no more than about 0.5 wt % of water relative to the total
weight of the depot. In other embodiments, the pharmaceutically
active agents are vancomycin hydrochloride and gentamicin sulfate.
In other embodiment, the depot is clear, and in yet another
embodiment, the depot is ultra clear.
Forming Oil-in-Water Emulsion
[0073] At least one hydrophilic water-soluble pharmaceutically
active agent selected from the group consisting of vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof, water, a phospholipid, and an oil are mixed to
form an oil-in-water emulsion.
[0074] In another embodiment, first, vancomycin hydrochloride,
gentamicin sulfate or both are dissolved in water to form an
aqueous solution.
[0075] The initial drug concentration of vancomycin hydrochloride
in water is from about 1 mg/ml to about 50 mg/ml or from about 20
mg/ml to about 30 mg/ml, and initial drug concentration of
gentamicin sulfate in water is from about 1 mg/ml to about 75
mg/ml, or from about 10 mg/ml to about 30 mg/ml.
[0076] Then the aqueous solution of vancomycin and/or gentamicin,
phospholipid, oil, optionally a pH adjusting agent and optionally a
stabilizing agent is mixed to form an oil-in-water emulsion.
Homogenizing to Obtain a Primary Emulsion
[0077] Subsequently, the emulsion may be homogenized using a high
shear mixer (such as for example Silverson Model L5M mixer) to form
a primary emulsion.
Microfluidizing to Obtain a Monophasic Solution
[0078] The primary emulsion was then microfluidized by using, for
example, a high-pressure microfluidizer, to obtain a
nanoemulsion/monophasic solution. The resulting
nanoemulsion/monophasic solution has an average diameter of less
than 120 nm, less than 100 nm, and or less than 80 nm to form a
monophasic solution/nanoemulsion. It is found that the droplet size
greater than 180 nm may result in a cloudy solution.
[0079] The reduction of average diameter of the nanoemulsion
droplets is believed, without limitation, to reduce the viscosity
of the resulting monophasic solution, allowing sterilization
through a filter, rather than by using a heat-based sterilization
system, such as by autoclaving or gamma-radiation sterilization,
which can affect stability of vancomycin and/or gentamicin.
[0080] Before the step of microfluidization, the primary emulsion
is generally a white, opaque, thick yogurt-like mass. After
microfluidization, the resulting monophasic solution is generally
clear, translucent, and water-like in viscosity and flow
properties.
[0081] In order to produce a clear monophasic solution, the
oil-in-water emulsion advantageously contains about 10% to about
80% water, from about 30% to about 80% water, or from about 60% to
80% water relative to the total weight of the oil-in-water emulsion
in order to have the desired flow property to be processed in the
high-pressure homogenizer, such as a MICROFLUIDIZER.
Adjusting pH
[0082] The pH of the emulsion, primary emulsion or monophasic
solution may be adjusted by adding a pH adjusting agent so that the
pH of the emulsion, primary emulsion or monophasic solution is from
about 3 to about 6, a range of about 3 to about 5, or a range of
from about 3 to about 4.
[0083] In another embodiment, this step is performed by adding an
appropriate amount of a pH adjusting agent to the emulsion,
followed by high shear mixing homogenization step for about 1
minute. Then, after the homogenization step, the pH of the
composition is checked and may be adjusted again if necessary.
Lyophilization, Sublimation or Evaporation
[0084] By removing the water, gentamicin and/or vancomycin become
uniformly dispersed in the phospholipid/oil vehicle. Water is then
removed from the monophasic solution by lyophilization, sublimation
and/or evaporation so that the amount of residual water in the
resulting dry paste or the final syringeable clear depot is lower
than about 4 wt %, lower than about 2 wt %, or lower than about 0.5
wt % of water relative to the total weight of the dry paste or
viscous clear depot.
[0085] In another embodiment, the monophasic solution is
freeze-dried using a tray lyophilizer. In yet another embodiment,
the tray of the lyophilizer is stainless steel.
[0086] In yet another embodiment, the liquid filling height in the
stainless steel lyophilization tray is no more than about 3 cm. In
an embodiment, after the step of lyophilization, the resulting
product, which is the dry paste, has no more than 1 wt % of water
relative total weight of the dry paste.
Addition of Viscosity Modifying Agent
[0087] The viscosity modifying agent is added to the dry paste
until the dry paste is completely dissolved. The viscosity
modifying agent may be added to the dry paste until the amount of
viscosity modifying agent is about 75 wt % or more, about 50 wt %
or more, about 30 wt % or more or about 25 wt % or more relative to
total weight of the viscosity modified solution. In one embodiment,
the viscosity modifying agent and the dry paste may be mixed at a
temperature of about 10 deg C. to about 80 deg C., or about 25 deg
C. to about 60 deg C.
Pre-Filtration
[0088] This is an optional step and is not required for certain
embodiments of the invention. If the viscosity modified solution
obtained after adding the viscosity modifying agent is hazy, the
viscosity modified solution may be filtered using for example 0.65
micron filter to form a clear solution. The hazy component removed
by the pre-filtration steps consists of a small fraction of
vancomycin (about 2% target assay) and gentamicin (3-4% target
assay). This loss may be compensated by adjusting up the initial
load or dropping the assay targets. This is an optional step and is
not required for certain embodiments of the invention.
Removal of Viscosity Modifying Agent
[0089] Subsequently, the viscosity modifying agent which was added
to dissolve the dry paste is removed. Removal of the viscosity
modifying agent may be done until the amount of residual viscosity
modifying agent which may be present in the depot from about 1% to
about 50%, from about 2% to about 18%, or from about 5% to about
6.5% relative to the total weight of the depot.
[0090] If over-dried, the viscosity modifying agent may be added
back as needed. Removal of the viscosity modifying agent may be
done using a rotary evaporator or by blowing with nitrogen gas or
air. Thermal gravimetric Analysis (TGA) can be used to measure the
amount of viscosity modifying agent removed from the clear solution
to form a depot.
[0091] The viscosity of the resulting depot in accordance with the
present invention is from about 100 centipoise to about 5000
centipoise, from about 200 centipoise to about 2000 centipoise, or
from about 300 centipoise to about 1500 centipoise. Viscosity
measurement can be performed using any conventional method,
including using a Brookfield Digital Programmable Rheometer with
Model No. DV-III with Spindle No. SP-40. This is an optional step
and is not required for certain embodiments of the invention.
Sterile Filtration
[0092] The depot is then sterilized by filtering through a
sterilizing membrane, such as one having pores of about 0.2 micron
or less.
Depot
[0093] Another aspect of the present invention provides a depot
comprising at least one hydrophilic water-soluble pharmaceutically
active agent selected from the group consisting of vancomycin,
gentamicin, a pharmaceutically acceptable salt thereof and a
mixture thereof, water, a phospholipid, an oil, a pH adjusting
agent, and a viscosity modifying agent, wherein the water present
in the depot is no more than about 4 wt %, no more than about 2 wt
%, or no more than about 0.5 wt % of water relative to the total
weight of the depot.
[0094] In accordance with another aspect of the invention, the
depot optionally comprises a stabilizing agent to improve the
stability of vancomycin, gentamicin or both. In another aspect of
the invention, this depot is provided in a syringe, vial or any
other device capable of delivering the depot to the treatment site,
depot site or wound.
Pharmaceutical Active Ingredient
[0095] The pharmaceutical active ingredient in accordance with the
present invention is vancomycin, gentamicin, a pharmaceutically
acceptable salt thereof or a mixture thereof. In one embodiment,
the pharmaceutical active ingredient in accordance with the present
invention is vancomycin hydrochloride, gentamicin sulfate or a
mixture thereof. In another embodiment, the pharmaceutical active
ingredients in accordance with the present invention are vancomycin
hydrochloride and gentamicin sulfate. In yet another embodiment,
the pharmaceutical active ingredient in accordance with the present
invention is either vancomycin hydrochloride or gentamicin
sulfate.
[0096] Examples of the pharmaceutically acceptable salt include,
but not limited to, any acids that can form salts with either
vancomycin or gentamicin such as acetic acid, hydrochloric acid,
hydrobromic acid, citric acid, formic acid, lactic acid, succinic
acid, sulfuric acid, and the like.
[0097] The amount of the pharmaceutical active ingredients that may
be present in the depot can vary with a number of parameters
including the size of the total intended dose, the duration of
administration, the size of the depot and where and how it will be
administered, the type of active to be administered, the pattern of
administration (e.g., continuous, delayed, etc.) and the like.
However, generally, the total amount of the pharmaceutically
acceptable ingredient may be from about 0.001 wt % to about 20 wt
%, from about 0.01 wt % to about 10 wt %, or from about 0.1 wt % to
about 5 wt % relative to the total weight of the depot.
Oil
[0098] An oil in accordance with the present invention may be, for
instance, natural oils such as vegetable oils, animal oil, vitamin
E, vitamin E ester, and the like and/or synthetic or semisynthetic
oils, or mixtures thereof.
[0099] A vegetable oil refers to oil derived from plant seeds or
nuts. Examples of vegetable oils include, but are not limited to,
almond oil, borage oil, black currant seed oil, castor oil,
safflower oil, soybean oil, sesame oil, cottonseed oil, grapeseed
oil, sunflower oil, canola oil, coconut oil, palm oil, orange oil,
corn oil, olive oil and the like.
[0100] An animal oil refers to triglyceride oil derived from an
animal source. Examples of animal oil can be fish oil, or from
other sources such as tallow, lard and the like.
[0101] Examples of synthetic or semisynthetic oils are mono-, di-
or triglycerides, whose acid components are C6 to C20 saturated
and/or unsaturated fatty acids, CAPTEX.RTM. (various grades of
propylene glycol esters such as propylene glycol didecanoate, and
glycerol esters such as glyceryl tricaprylate/caprate);
MIGLYOL.RTM. (caprylic/capric acid triglycerides; or
caprylic/capric/linoleic acid triglycerides; or
caprylic/capric/succinic acid triglycerides; or propylene glycol
diester of caprylic/capric acid and admixtures with other agents;
CAPMUL.RTM. (available in different grades, e.g. Capmul MCM. It is
mainly mono- and di-esters of glycerol and of propylene glycol,
such as glyceryl mono-oleate and propylene glycol monocaprylate.
Another grade consists of polyethylene glycol glyceryl
monostearate. In one embodiment, the oil used in accordance with
the present invention is sesame oil.
[0102] The amount of the oil that may be present in the depot may
be from about 5 wt % to about 95 wt %, from about 25 wt % to about
75 wt %, or from about 35% to about 60% relative to the total
weight of the depot.
[0103] In certain embodiments, the oil to phospholipid ratio in the
depot may be within a range of from about 20:1 to about 1:20, from
about 3:1 to about 1:3, or from about 1:2 to about 1:1, by
weight.
Phospholipid
[0104] Phospholipid in accordance with the present invention refers
to a lipid molecule containing one or more phosphate groups,
including those derived from either glycerol (phosphoglycerides,
glycerophospholipids) or sphingosine (sphingolipids).
[0105] In some embodiments, phospholipids are triglyceride
derivatives in which one fatty acid has been replaced by a
phosphate group and one of several nitrogen-containing molecules.
The fatty acid chains are hydrophobic and the charges on the
phosphate and amino groups make that portion of the molecule
hydrophilic. The result is an amphiphilic molecule.
[0106] According to the United States Pharmacopoeia (USP), lecithin
is a non-proprietary name describing a complex mixture of
acetone-insoluble phospholipids, which comprise mainly of
phosphotidylcholine, phosphotidylethanolamine, phosphotidylserine
and phosphotidylinositol, combined with various amounts of other
substances such as triglycerides, fatty acids and carbohydrates.
The composition of lecithin and hence its physical properties vary
depending upon the source of the lecithin and phospholipid
composition, e.g., phosphotidylcholine content, etc.
[0107] In accordance with an embodiment of the present invention,
lecithin used herein are pharmaceutical grade lecithins derived
from egg or soybean, which have been used in parenteral products
and are substantially free from irritating, allergenic,
inflammatory agents or agents that cause other adverse biological
reactions.
[0108] In accordance with the practice of the present invention,
the selection of phospholipid for preparing the depot is determined
based on the ability of the phospholipid to (1) be chemically
compatible with the at least one hydrophilic water-soluble
pharmaceutically active agent selected from the group consisting of
vancomycin, gentamicin and a mixture thereof, (2) form a monophasic
solution and maintain the small droplet size through the
manufacturing process and during storage, and (3) provide the
desired depot and provide the desired release of the
pharmaceutically active agent.
[0109] Examples of the phospholipid include, but not limited to,
sphingolipids in the form of sphingosine and derivatives (obtained
from soybean, egg, brain and milk), gangliosides, and
phytosphingosine and derivatives (obtained from yeast).
[0110] Phospholipids can also be synthesized and examples of common
synthetic phospholipids include, but not limited to, diglycerols,
such as 1,2-dilauroyl-sn-glycerol (DLG),
1,2-dimyristoyl-sn-glycerol (DMG), 1,2-dipalmitoyl-sn-glycerol
(DPG), 1,2-distearoyl-sn-glycerol (DSG); phosphatidic acids, such
as 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid, sodium salt
(DMPA,Na), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, sodium
salt (DPPA,Na), 1,2-distearoyl-sn-glycero-3-phosphatidic acid,
sodium salt (DSPA, Na); phosphocholines, such as
1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),
1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),
1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine (EPA-PC),
1,2-didocosahexaenyl-sn-glycero-3-phosphocholine (DHA-PC),
1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC),
1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),
1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),
1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),
1-stearoyl-2-palmitoy-sn-glycero-3-phosphocholine (SPPC),
1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC),
1-palmitoyl-2-oleoy-sn-glycero-3-phosphocholine (POPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
phosphoethanolamines, such as hydrogenated soybean
phosphoethanolamine (HSPE), non-hydrogenated egg
phosphoethanolamine (EPE),
1,2-dilauroyl-sn-glycero-3-phosphoethanolamin (DLPE);
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamin (DMPE);
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamin (DPPE);
1,2-distearoyl-sn-glycero-3-phosphoethanolamin (DSPE);
1,2-dioleoyl-sn-glycero-3-phosphoethanolamin (DOPE);
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamin (DLoPE);
1,2-dierucyl-sn-glycero-3-phosphoethanolamin (DEPE),
1,2-palmitoyl-sn-glycero-3-phosphoethanolamin (POPE);
phosphoglycerols such as hydrogenated soy bean
phosphatidylglycerol, sodium salt (HSPG, Na), non-hydrogenated egg
phosphatidylglycerol, sodium salt (EPG, Na),
1,2-dilauroyl-sn-glycero-3-phosphoglycerol, sodium salt (DLPG, Na),
1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG,
Na), 1,2-dimyristoyl-sn-glycero-3-phospho-sn-1-glycerol, ammonium
salt (DMP-sn-1-G, NH.sub.4),
1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG,
Na), 1,2-distearoyl-sn-glycero-3-phosphoglycerol, sodium salt
(DSPG, Na), 1,2-distearoyl-sn-glycero-3-phospho-sn-1-glycerol,
sodium salt (DSP-sn-1G, Na),
1,2-dioleoyl-sn-glycero-3-phosphoglycerol, sodium salt (DOPG, Na),
1,2-dierucyl-sn-glycero-3-phosphoglycerol, sodium salt (DEPG, Na),
1,2-palmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (POPG, Na);
phosphotidylserines such as
1,2-dimyristoyl-sn-glycero-3-phospho-L-sine, sodium salt (DMPS,
Na), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-sine, sodium salt
(DPPS, Na), 1,2-distearyl-sn-glycero-3-phospho-L-sine, sodium salt
(DSPS, Na), 1,2-dioleoyl-sn-glycero-3-phospho-L-sine, sodium salt
(DOPS, Na), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-sine,
sodium salt (POPS, Na); mixed chain phospholipids, such as
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phospoglycerol, sodium salt
(POPG, Na), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol,
ammonium salt (POPG, NH.sub.4); lysophospholipids, such as
1-myristoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC),
1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-lyso-PC),
1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC); and
pegylated phospholipids, such as
N-(carbonyl-methoxypolyethyleneglycol 2000)-MPEG-2000-DPPE,
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt,
N-(carbonyl-methoxypolyethyleneglycol 5000)-MPEG-5000-DSPE,
1-2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt,
N-(Carbonyl-methoxypolyethyleneglycol 5000)-MPEG-5000-DPPE,
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt,
N-(carbonyl-methoxypolyethyleneglycol 750)-MPEG-750-DSPE,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt,
N-(carbonyl-methoxypolyethyleneglycol 2000)-MPEG-2000-DSPE,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt.
[0111] The amount of the phospholipids that may be present in the
depot can vary with a number of parameters including the viscosity
of final formulation, the duration of administration, the size of
the depot and where and how it will be administered, the type of
active to be administered, the pattern of administration (e.g.,
continuous, delayed, etc.) and the like. However, generally, the
amount of the phospholipid that may be present in the depot may be
from about 5% to about 95% relative to the total weight of the
composition, or about 35% to about 60% relative to the total weight
of the composition.
[0112] Water
[0113] Water which can be used in accordance with the present
invention includes, but not limited to distilled and deionized
water, or any other aqueous liquid which is capable of dissolving
the hydrophilic water-soluble vancomycin and/or gentamicin and
capable of subliming/evaporating during the lyophilization
step.
[0114] In order to obtain a monophasic solution, for example, by
using a high pressure microfluidizer, the oil-in-water emulsion may
contain from about 50% to about 90% water, from about 60% to about
80% water, or from about 70% to about 80% water relative to the
total weight of the oil-in-water emulsion in order to have the
desired flow property to be processed in the homogenizer, such as a
MICROFLUIDIZER.
[0115] However, once the monophasic solution is obtained, most of
water may be removed by for example, lyophilization, sublimation
and/or evaporation.
[0116] Vancomycin degrades due to hydrolysis, and the amount of
residual water in the final depot affects the long term stability
of vancomycin. When vancomycin precipitates, the depot turns from
translucent to hazy or separates into two phases as shown in
EXAMPLE 3 herein.
[0117] Accordingly, in accordance with the present invention, the
amount of residual water must be maintained lower than about 4 wt
%, lower than about 2 wt % or lower than about 0.5 wt % of water
relative to the total weight of the viscous clear depot in order to
maintain the vancomycin stable during storage.
pH Adjusting Agent
[0118] The pH adjusting agent in accordance with the present
invention is any non-toxic acid, base or salt. Examples of pH
adjusting agents include, but not limited to, hydrochloric acid,
acetic acid, sulfuric acid, sodium hydroxide, potassium hydroxide,
ammonium hydroxide, lysine, arginine, and the like.
[0119] As mentioned above, gentamicin degrades due to oxidation or
adduct formation. As shown in EXAMPLE 4 hereinbelow, pH affects the
long term stability of gentamicin, and when gentamicin
precipitates, the depot turns from translucent to hazy.
[0120] Accordingly, pH of the depot may be from about 3 to about 6,
a range of from about 3 to about 5, or a range of from about 3 to
about 4.
Stabilizing Agent
[0121] A stabilizing agent in accordance with the present invention
is a material which reduces catalytic effect of metal ion on the
oxidation, hydrolysis or other degradation reactions and or
increases stability of the hydrophilic water-soluble
pharmaceutically active agent. Examples of such stabilizing agent
include, but not limited to, EDTA (disodium edentate), glycine,
L-histidine, citric acid, methionine, ascorbic acid, L-cysteine,
alpha-tocopherol, and mixtures thereof. In certain embodiments, the
amount of the stabilizing agent present in the depot is from about
0.001% to about 5.0% relative to the total weight of the
composition, or about 0.01% to about 1.0% relative to the total
weight of the composition. In another embodiment, the depot does
not contain a stabilizing agent.
Viscosity Modifying Agent
[0122] A viscosity modifying agent in accordance with the present
invention is an aqueous or non-aqueous (other than having a
contaminant level of water) liquid which is capable of dissolving
the dry paste formed after lyophilization, sublimation and/or
evaporation.
[0123] Examples of a viscosity modifying agent include, without
limitation, ethanol, isopropanol, and a mixture thereof. In one
embodiment, the viscosity modifying agent is substantially
non-aqueous. In another embodiment, the viscosity modifying agent
is ethanol.
[0124] The viscosity modifying agent is added to the dry paste
until the dry paste is completely dissolved in the agent. The
resulting viscosity modified solution may also become "hazy." In
one embodiment, the viscosity modifying agent and the dry paste are
mixed at a temperature of about 10 deg C. to about 80 deg C., or in
a range of about 50 deg C. to about 70 deg C., or in a range of
about 25 deg C. to about 60 deg C.
[0125] The viscosity modifying agent is added to the dry paste
until the amount of viscosity modifying agent is about 10 wt %, 20
wt %, 25 wt % or 30 wt % relative to total weight of the resulting
solution. The resulting viscosity of the solution can be from about
10 to about 200 centipoise, from about 15 to about 100 centipoise,
or about 20 centipoise to about 50 centipoise.
[0126] Viscosity can be determined using a Brookfield digital
programmable rheometer with the SP-40 spindle or any other
equivalent rheometer. More specifically, the starting RPM of the
rheometer can be from 0.1 to 1.0, then reducing the RPM to 0.1 in
0.1 RMP increment every 30 seconds. The viscosity measurement can
be recorded at 0.8 RMP at an ambient temperature of about 30 deg
C.
[0127] Subsequently, some amount of the viscosity modifying agent
used to dissolve the dry paste may be removed. The removal of the
viscosity modifying agent may be done until the residual amount of
viscosity modifying agent which may be present in the depot is from
about 1 wt % to about 20 wt %, from about 2 wt % to about 18 wt %,
or from about 5 wt % to about 6.5 wt % relative to the total weight
of the depot. If over-dried, the viscosity modifying agent may be
added back as needed.
[0128] The viscosity of the resulting depot in accordance with the
present invention is from about 100 centipoise to about 5000
centipoise, from about 200 centipoise to about 2000 centipoise, or
from about 300 centipoise to about 1500 centipoise.
Method Of Treatment
[0129] Another aspect of the present invention is a method of
administering via intradermal, intramuscular, intraincisional,
subcutaneous, instillation or topically a depot of the present
invention comprising vancomycin, gentamicin, a pharmaceutically
acceptable salt thereof or a mixture thereof, water, phospholipid,
an oil, optionally a pH adjusting agent and a viscosity modifying
agent. The depot can be dosed at the desirable site using various
dosages and at various dosing intervals depending upon the need.
That is the depot should be sufficient to release the
pharmaceutically active agent for a period of about at least one
day with a dosing volume from about 0.1 mL to about 100 mL. For
example, dosing intervals of once-a-day, once-every-other-day,
once-every-3-days, once-a-week or once-a-month with a dosing volume
from about 0.1 mL to about 100 mL can be used. Typically, the depot
may be used in a single application and is generally instilled at
the wound site before suturing the wound site.
[0130] Another aspect of the present invention is a method of
preventing and/or treating infection including, without limitation,
surgical site infection, comprising administering a depot of the
present invention which achieves sufficiently high tissue
concentration to treat and/or prevent infections at a local site,
yet does not cause toxicity to kidney and/or other organs, and also
does not cause and/or contribute to the emergence of drug resistant
strains of bacteria.
[0131] In another aspect, there is provided a method of rendering
localized tissue unable to sustain pathogenic microorganisms by
administering a depot of the present invention to the wound.
[0132] In another embodiment, this is accomplished without causing
toxicity to kidney and/or other organs, and without causing and/or
contributing to the emergence of drug resistant strains of
bacteria.
[0133] In each of the foregoing methods, the dose of vancomycin,
gentamicin or both should be such that, when released from the
depot, the localized tissue is unable to sustain pathogenic
bacteria for at least 24 hours and, in another embodiment, at least
48 hours. In still another embodiment, the localized tissue is
unable to sustain pathogenic bacteria for at least 3 days, for at
least one week or for a period of one month.
[0134] As shown in FIG. 10, and according to published data, the
minimum inhibitory concentration required to inhibit the growth of
90% of the organisms (MIC.sub.90 (mcg/ml)) for well known surgical
site infection (SSI) pathogens, such as staphylococcus aureus,
coagulase-negative staphylococci, enterococci, pseudomonas
aeruginosa, and Escherichia coli, are in the range of 1-4 mcg/ml.
See generally, M. J. Rybak, et al., Vancomycin Therapeutic
Guidelines, CID 2009:49 (1 August), 325-327; and A. I. Hidron, et
al., Infection and Hospital Epidemiology, November 2008, vol. 29,
No. 11, 996-1011. These are based on use of vancomycin and
gentamicin individually as illustrated. When the depot of the
present invention comprising vancomycin (4.37 mg/kg) and gentamicin
(3.89 mg/kg) was administered to a pig, the localized pig tissue
concentration of vancomycin achieved by the depot of the present
invention was over 19 mcg/ml at 48 hrs, which is scientifically
higher than the MIC 90 for the above-identified SSI pathogens.
Similarly, the pig tissue concentration of gentamicin achieved by
administering the depot of the present invention was about 12
mcg/ml at 48 hrs.
[0135] To this end, it is noted that the inventors did not
inoculate pigs with the above-identified SSI pathogens and then
administer the depot to the local site to determine the efficacy of
the present invention. Nevertheless, the published MIC 90 data for
the above-identified SSI pathogens, and the achievable tissue
concentration of the formulation of the present invention
comprising vancomycin and gentamicin demonstrate that the use of
the depot of the present invention would be highly effective in
providing localized drug levels effective for treating and/or
preventing infection by rendering the localized tissue unable to
sustain pathogenic microorganisms.
[0136] FIG. 11 illustrates that the depots of the present invention
when administered intraincisional to pigs provides high local
tissue concentrations of both actives, vancomycin and gentamicin,
and low systemic concentrations (plasma). The low systemic
concentrations of gentamicin observed using the depot of the
present invention provides a significant safety margin since
gentamicin toxicity (renal and ototoxicity) are known to be related
to plasma concentrations greater than 10 mg/L. See generally, D. S.
Reeves, Infection 8 (1980) Suppl. 3, S 313-S320.
[0137] Vancomycin renal toxicity is also related to excessive drug
exposure but cannot as easily be correlated to a specific peak
concentration. However, the low systemic concentrations observed
using the depot administration of the present invention have shown
no systemic toxicity in the pig model for either drug and clearly
are well below published values for gentamicin.
[0138] A second concern with vancomycin is the development of
bacterial resistance. Vancomycin resistance can develop when the
target tissues or ancillary tissues that colonize bacteria are
exposed to sub-effective concentrations of vancomycin for
significant periods of time. After systemic administration of
vancomycin alone, the desired minimum plasma concentrations at
steady-state are at least 10 mg/L or maybe in the range of about
15-20 mg/L. Given the low uptake of vancomycin by tissues from
blood, these concentrations are sufficient to push therapeutically
effective concentrations into the tissues and achieve a therapeutic
effect. Blood concentrations serve as a surrogate marker for the
target tissues wherein the goal is to achieve a plasma
AUC.sub.0-t/MIC.sub.90 ratio of from about 400 to about 300,000 or
AUC.sub.0-t/MIC.sub.90 ratio of >400 or AUC.sub.0-t/MIC.sub.90
ratio of >1600. This ratio is achieved when trough levels are
maintained at the levels noted above.
[0139] With the depot of the present invention, very low
concentrations of vancomycin are observed in circulating plasma
while supra-therapeutic concentrations are present in the incision
site where the gel was administered as shown in FIG. 10. Given the
low level of uptake of vancomycin from plasma to tissue, the low
systemic concentrations of vancomycin lead to negligible levels of
vancomycin in tissues distal from the incision site. The only
mechanism for transport is via the plasma and the amount of uptake
by the tissues from the plasma is low. Therefore, the probability
of vancomycin resistance developing at sites distal from the
incision should be very low.
[0140] Thus in another aspect of this invention there is provided a
method of treating a patient comprising administering to said
patient a therapeutically effective dose of vancomycin alone or in
combination with gentamicin or pharmaceutically acceptable salts
thereof such that a plasma AUC.sub.0-t/MIC.sub.90 ratio of >400
is achieved for vancomycin so as to prevent emergence of resistance
in S. aureus. In yet another aspect of this invention a patient
receiving the above noted administration exhibits 1/10.sup.th the
steady-state trough serum concentration so as to avoid any
nephrotoxicity exhibited by high dose administration of vancomycin
by conventional methods. See generally, M. J. Rybak, Vancomycin
Therapeutic Guidelines, CID 2009:49 (1 August), 325-327.
EXAMPLES
Example 1: Depot in Accordance with the Present Invention
TABLE-US-00001 [0141] TABLE 1 List Of Ingredients Of The Depot In
Accordance With The Present Invention Component w/w % Gentamicin
sulfate Equivalent to 0.36% in the "USP Gentamicin Assay" value
Vancomycin hydrochloride Equivalent to 0.24% in the "USP Vancomycin
Assay" value Soy lecithin (Phospholipon 53.3 90G or PL90G)
L-Histidine 0.1 Ethanol 6.0 Sesame oil 40.0 TOTAL 100%
[0142] First, a 500 mL beaker was charged with 0.36 g gentamicin
sulfate, 0.24 g vancomycin hydrochloride, 53.3 g PL90G, 40 g sesame
oil and 0.1 g L-histidine. To this was then added Water for
Injection (WFI) and the mixture was homogenized by a high shear
mixer at 5000 RPM for 15 min. The resulting monophasic solution was
lyophilized to remove water to obtain a dry paste with less than
0.2% residual moisture.
Example 2: Effect of Water Content on Appearance of Example 1
[0143] This dry paste was mixed with water and/or ethanol, to form
a viscosity modified solution and used in several of the studies,
including EXAMPLE 2 to EXAMPLE 5 as set forth hereinbelow.
[0144] Various amounts of water (from 1.1 wt % to 4.1 wt %) and
ethanol (at 6 wt %) were added into the dry paste of EXAMPLE 1 to
produce several samples. Samples were mixed well by a BeadBeater
mixer, centrifuged to remove air bubble, and then observed for
initial appearance ("Initial sample"). Also, samples were passed
thru 0.45 .mu.m filter and the filtrates were stored at 2-8.degree.
C. for further appearance observation ("Filtered sample"). Table 2
shows the effect of water content on the appearance of the
formulations. It was found that water content significantly
affected the appearance of the formulations:
TABLE-US-00002 TABLE 2 Effect Of Water Content On The Appearance Of
Example 1 Formulations Sample ID S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8
Water (%) 1.14 1.46 1.83 2.05 2.61 3.06 3.70 4.07 Initial Hazy
Clear 2 sample phases Filtered All clear after filtration. However,
Not sample with more water, a delayed tested precipitation occurred
at 2-8.degree. C. after about 3 to 7 days.
Example 3: Effect of Water Content on Gentamicin and Vancomycin
Stability
[0145] The effect of residual water content on gentamicin and
vancomycin stability of the formulations of EXAMPLE 1 was evaluated
by a 60 min autoclave treatment. As summarized in Table 3 below, it
was found that vancomycin had reduced stability in terms of
recovery or purity at the higher residual water level. No
significant effect of water on gentamicin stability was observed in
the same range.
TABLE-US-00003 TABLE 3 Effect Of Water Content On Gentamicin And
Vancomycin Stability Vancomycin Gentamicin Recovery Recovery (%
over Pre- Purity (% over Pre- ID autoclave) (%) autoclave) EXAMPLE
1* Pre- 69.4 89.2 67.7 (0.76% H.sub.2O) autoclave Autoclave 68.8
EXAMPLE 1* Pre- 65.7 89.9 80.2 (1.26% H.sub.2O) autoclave Autoclave
64.5 Example 1* Pre- 62.0 89.5 / (1.76% H.sub.2O) autoclave
Autoclave 63.2 Example 1* Pre- 60.5 88.8 77.5 (2.26% H.sub.2O)
autoclave Autoclave 58.8 Autoclave 69.4 *pH 5.7
Example 4: pH-Stability and pH-Solubility Profiles of Gentamicin
and Vancomycin in Example 1 Formulation
[0146] The pH adjusted formulations of EXAMPLE 1 were placed at 2-8
deg C. for appearance examination. (See Table 4 below.)
TABLE-US-00004 TABLE 4 Effect of pH on Appearance of EXAMPLE 1
Formulations Appearance pH Water (%) Before filtration Filtrate at
2-8.degree. C. 3.21 0.17 Clear Clear 5.54 0.15 Hazy Clear for 5-7
days, 5.63 0.13 then hazy 6.02 0.04 6.01 0.11 6.99 0.11 7.67
0.09
[0147] A pH-stability profile was generated by heating the samples
from EXAMPLE 1 with a 60 minute autoclave treatment. (See Table 5
below.)
TABLE-US-00005 TABLE 5 Effect of pH on Stability of EXAMPLE 1
formulations Assay Recovery Vancomycin Purity (%) (% over the
pre-treatment) Pre- Post- pH Water (%) Vancomycin Gentamicin
treatment treatment 3.21 0.17 82.9 94.1 91.6 79.4 5.54 0.15 82.3
79.6 91.5 81.6 5.63 0.13 78.0 81.6 90.4 73.6 6.02 0.04 77.0 79.6
90.5 74.3 6.01 0.11 80.1 82.7 89.6 75.0 6.99 0.11 80.5 73.1 90.7
76.2 7.67 0.09 77.4 75.3 91.2 77.1 5.99 0.201 80.8 84.3 87.8 71.5
The results indicated that: (1) pH affected EXAMPLE 1 formulations'
appearance. The formulation was clear at pH 3.2; (2) pH affected
gentamicin's stability in the formulation. A low pH (e.g., from pH
of 3 to 4) is preferred for gentamicin stability; and (3) pH did
not affect vancomycin stability significantly.
Example 5: pH Stability Profile of Gentamicin in Example 1
Formulation Between pH of 3.0 to 5.5
[0148] Samples of EXAMPLE 1 formulation at three different pH
levels between 3.0 to 5.5 were prepared. In addition, the effect of
L-histidine on the stability of the formulation of EXAMPLE 1 was
also tested comparing the formulation containing L-histidine with
those that do not contain L-histidine. The stability of gentamicin
and vancomycin was evaluated in the same way as set forth in
EXAMPLE 3. It was found that [0149] (1) Stability of gentamicin in
the formulation is pH-dependant (gentamicin preferred a low pH
(e.g., from pH of 3 to 4)); [0150] (2) Stability of vancomycin in
the formulation is less pH-sensitive in the pH range studied;
[0151] (3) L-histidine increased gentamicin stability in the pH
range studied; and [0152] (4) L-histidine decreased vancomycin
stability in the pH range studied.
[0153] FIG. 2 shows the assay recovery after the autoclave
treatment.
Example 6: Another Depot in Accordance with the Present Invention
and the Process of Making the Formulation
TABLE-US-00006 [0154] TABLE 6 List Of Ingredients Of Another Depot
In Accordance With The Present Invention Component w/w % Gentamicin
sulfate Equivalent to 1.675% in the "USP Gentamicin Assay" value
Vancomycin hydrochloride Equivalent to 1.876% in the "USP
Vancomycin Assay" value Soy lecithin (PL90G) 50.0 Ethanol 6.0
Sesame oil Qs to 100 HCl Enough to adjust to pH of 3.3 +/- 0.2
[0155] A clear yellow sterile depot (batch size: 1500 g), which
contained less than 0.5 wt % of residual water having a pH of 3.3
was prepared by a multi-step process following the steps of: (1)
emulsification, (2) homogenization/microfluidization, (3)
lyophilization, (4) ethanol dilution, (5) pre-filtration, (6)
ethanol removal and (7) filtration. Simple mixing of all of the
ingredients listed above does not form a clear depot.
[0156] Detailed procedures for each of the above noted steps are as
follows: First, water was added to gentamicin sulfate, vancomycin
hydrochloride, to allow complete dissolution of gentamicin sulfate
and vancomycin hydrochloride. Then, PHOSPHOLIPON.RTM. 90G (from
Phospholipid GmbH) and sesame oil was added, followed by high shear
mixing at 5000 rpm for 60 minutes to obtain a uniform primary
emulsion. Then the pH of the primary emulsion was adjusted to
3.3.+-.0.2 by adding 1N of HCl. This was done by adding an
appropriate amount of 1N HCl to the emulsion, followed by high
shear mixing for 1 minute. Then, the measurement of pH was taken to
ensure that the primary emulsion had a pH of 3.3.+-.0.2.
[0157] Subsequently, the primary emulsion was placed in a
microfluidizer to produce a monophasic solution. The average
diameter of the droplets of the monophasic solution was measured
using a laser light scattering device.
[0158] Then, the monophasic solution was lyophilized to remove
water to obtain a dry paste with less than 0.5% residual water.
Then the dry paste was mixed with dehydrated alcohol. The mixture
was then sonicated in a 60-deg C. water bath until a clear solution
(viscosity modified) was obtained. Then the solution was cooled to
room temperature, and was pre-filtered through a 0.65 micron
sterile filter.
[0159] Then the alcohol from the solution was removed by blowing
nitrogen gas until the residual amount of dehydrated alcohol was
6.5 wt %-7 wt % to obtain a viscous and clear gel. Dehydrated
alcohol was added back as needed, if it was over dried.
[0160] In a biosafety hood, argon gas at 40 psi was applied to
filter the depot through a 0.2 micron filter to sterilize the
formulation. Then, in a biosafety hood, filtered depot was filled
into a glass vial.
Example 7: In Vitro Release Profile
[0161] In vitro release profile of the formulation of EXAMPLE 6
containing gentamicin and vancomycin was measured using the USP
method I using basket apparatus (100 rpm at 37 deg C.). 1.36 g of
EXAMPLE 6's formulation was filled in a 000 size capsule and the
filled capsule was placed in 40 mesh basket with baffles. FIG. 3
shows an in vitro release profile of gentamicin and vancomycin of
the formulation of EXAMPLE 6 using USP method I.
Example 8: Pharmacokinetic Studies in Rabbits
[0162] New Zealand white rabbits were used to conduct
pharmacokinetic ("PK") studies to evaluate the delivery of the
formulations made in accordance with this invention. Two
formulations were made in accordance with the procedures as set
forth in EXAMPLE 1 and EXAMPLE 6, respectively, and administered
into a surgical wound or subcutaneous pocket. Table 7 below shows
the Rabbit PK study design in more detail:
TABLE-US-00007 TABLE 7 Genta Gel Vanco Genta Body Inj. Vanco Conc.
Formu- Dose Dose Wt Vol. Con. (mg/ Study lation (mg/kg) (mg/kg)
(kg) (ml) (mg/g) g) 1.sup.st EXAM- 2.06 3.08 2.5 2.0 2.57 3.85
experiment PLE 1 2.sup.nd EXAM- 12.6 or 11.5 or 3.0 2 or 4 18.76
16.75 experiment PLE 6 25.2 22.9
[0163] In a first experiment, two New Zealand white rabbits were
tested. After wound instillation of the formulation of EXAMPLE 1,
vancomycin (Vanco) and gentamicin (Genta) were rapidly absorbed,
with a plasma Tmax of 1-2 hours. Plasma Cmax concentrations were
similar to those observed in the mouse. Plasma concentrations
decreased to near the limit to quantification by 36 hours. Tissue
concentrations of vancomycin peaked at 72 hours and were above the
Minimum Inhibitory Concentration (MIC) through 168 hours, as shown
in FIGS. 4 and 5.
[0164] Tissue concentrations of gentamicin peaked at 72 hours and
were at or below the MIC through 168 hours, as shown in FIGS. 6 and
7.
[0165] Plasma and tissue analysis was performed by Liquid
Chromatography/Mass spectrometry (LC-MS/MS) analysis and the
Pharmacokinetic (PK) results of the formulation of EXAMPLE 1 are
summarized in Tables 8 and 9, respectively below:
TABLE-US-00008 TABLE 8 Rabbit Plasma PK Parameters Of EXAMPLE 1
Formulation PK Vanco Genta Param- Genta Genta Genta Genta AUC/
C.sub.max/ eters Vanco C1a C1 C2/C2a Total MIC MIC C.sub.max 0.702
0.278 1.063 0.746 2.085 0.3 (.mu.g/ml) T.sub.max 2 1 1 1 1 (hr) AUC
11.60 3.44 14.13 9.85 27.42 15.5 (hr * .mu.g/ ml) T.sub.1/2 (hr)
39.16 10.50 23.24 23.83 22.91
TABLE-US-00009 TABLE 9 Rabbit Tissue PK Parameters Of EXAMPLE 1
Formulation Vanco Genta PK Genta Genta Genta Genta AUC/ C.sub.max/
Parameters Vanco C1a C1 C2/C2a Total MIC MIC C.sub.max 3.73 1.0525
5.865 3.23 10.1475 1.3 (.mu.g/ml) T.sub.max (hr) 72 72 72 72 72 AUC
(hr * .mu.g/ml) 354.88 104.28 573.10 324.86 1002.25 473.2 T.sub.1/2
(hr) 35.32 50.32 49.68 51.60 50.35
[0166] In a second experiment, six New Zealand rabbits (Group I)
were tested by wound instillation of the formulation of EXAMPLE 6
containing the dose of 12.6 mg/kg of vancomycin and 11.46 mg/kg of
gentamicin; and six additional New Zealand rabbits (Group II) were
tested by wound instillation of the formulation of EXAMPLE 6
containing the dose of 25.2 mg/kg of vancomycin and 22.9 mg/kg of
gentamicin.
[0167] The lower concentration (Group I) gel averaged 4 .mu.g/g for
both vancomycin and gentamicin total in the wound site, while the
higher concentration gel (Group II) averaged and 19.4 .mu.g/g for
vancomycin and gentamicin, respectively, which are greater than
four times MIC (minimum inhibitory concentration) values. Plasma
concentrations of vancomycin and gentamicin from the MPI study of
the formulation of EXAMPLE 6 exhibited vancomycin AUC/MIC (area
under the concentration curve/minimum inhibitory concentration)
ratios greater than 400 at both doses and gentamicin Cmax/MIC
(maximum concentration/minimum inhibitory concentration) ratios
greater than 800 at both doses.
[0168] FIG. 8 illustrates mean vancomycin plasma concentrations in
rabbits after single subcutaneous (SC) wound instillation and FIG.
9 illustrates mean total gentamicin plasma concentration in
rabbits.
[0169] Plasma and tissue analysis was performed by LC-MS/MS
analysis, and the PK results of the formulation of EXAMPLE 6 are
summarized in Table 10 below:
TABLE-US-00010 TABLE 10 Genta Genta Total Total Vanco Genta C1 C1a
C2 + C2a Genta Vanco Genta 1 2 1 2 1 2 1 2 1 2 AUC/MIC
C.sub.max/MIC Grp Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean
1 2 1 2 C.sub.max 3125 3150 2737 3586 769 1023 3188 4318 6679 8927
835 1116 T.sub.max 3 2 1 1 1 1 1 1 1 1 AUC 47392 60114 19216 25447
5463 7351 23286 31652 47966 64451 63190 80151 last
[0170] The key difference between the formulation of EXAMPLE 6
(high strength) and the formulation of EXAMPLE 1 (lower strength)
is that although the PK profiles of these two formulations in small
animals, such as mice, were similar, there was a greater difference
in performance when tested on larger animals, such as rabbits,
since the tissue concentrations for the formulation of EXAMPLE 1
fell below the 4 times MIC value sooner, therefore describing a
lower area under the concentration curve (AUC) with respect to the
time/area spent 4 times over the MIC.
TABLE-US-00011 Comparative Example 1 Ingredients Weight %
Gentamicin sulfate 3 Vancomycin hydrochloride 2 Phospholipon 90G 63
Sesame oil 27 Ethanol 5 TOTAL 100.00
[0171] Comparative Example 1 was produced using the same methods as
Examples 1 or 6, except that the homogenization, ethanol removal
and/or pre-filtration steps were not performed.
[0172] Comparative Example 1 formed an opaque hard paste after
lyophilization and was not clear and not filterable after adding
viscosity modifying agent (ethanol).
Example 9: This Example Further Illustrates a Process for Making a
Depot Formulation of this Invention
TABLE-US-00012 [0173] TABLE 11 List Of Ingredients Of A Formulation
In Accordance With The Present Invention Component w/w % Gentamicin
sulfate 2.67* Vancomycin hydrochloride 1.83** Soy lecithin (PL90G)
50.0 Ethanol 6.0 Sesame oil 39.50 1N HCl Enough to adjust to pH of
3.3 +/- 0.2 *Equivalent to 16.75 mg/g gentamicin **Equivalent to
18.76 mg/g vancomycin
[0174] Gentamicin sulfate, vancomycin hydrochloride, PL90G and
sesame oil and water was added to a beaker, mixed and homogenized
by a high shear mixer at 500 RPM for 30 minutes to obtain a primary
emulsion. The pH of the primary emulsion was then adjusted to 3.3
by 1N HCl.
[0175] A microfluidizer (M-110EH, Microfluidics Corp) was applied
to reduce the droplet size of the primary emulsion. The operating
pressure was set up at 25000 psi. After 6 passes, the droplet size
(Z-Ave) of monophasic solution was less than 80 nm by laser light
scattering scatter (Nano-ZS, Malvern). The pH of the monophasic
solution was checked and adjusted to 3.3 as needed.
[0176] The monophasic solution was transferred on a stainless steel
container with a filling height less than 3 cm and then lyophilized
to remove water to less than 1% residual water (by Karl Fisher
titration) to obtain a dry paste. After lyophilization, the dry
paste was collected into a 2 L beaker. Dehydrated alcohol was added
into the paste to final 25% (w/w). The mixture was dissolving by
stirring at room temperature to form a clear yellow solution.
[0177] The clear solution was evaporated to reduce the alcohol
content by nitrogen gas blowing to obtain a viscous and clear depot
with 6% alcohol (w/w). Then the depot was sterilized by passing
through two 0.2 .mu.m SARTOPORE.RTM. 2 filters.
Comparative Example 2: Formulation Made without the Step of
Microfluidization
[0178] A primary emulsion was prepared in the same way as described
in Example 9. This primary emulsion was further shaken overnight or
homogenized with additional high shear mixing at 5000 RPM for 2
hours, and then lyophilized in the same way as described in Example
9. For this example, a step of microfluidization was not employed.
After lyophilization, dehydrated alcohol was added into the paste
so that the amount of the dehydrated alcohol was about 25% (w/w).
The resulting mixture was not clear even after stirring or heating
for an extended period of time. The process could not be continued
because a clear or filterable solution was not obtained.
Comparative Example 3: Formulation Made Using a Non-Stainless Steel
Container for Lyophilization
[0179] A primary emulsion and monophasic solution were prepared in
the same way as described in Example 9. The nanoemulsion was
lyophilized in the same way as described in Example 9 except a
glass container instead of a stainless steel container was used.
After lyophilization, dehydrated alcohol was added into the dry
paste so that the amount of the dehydrated alcohol is about 25%
(w/w) relative to the total weight of the resulting viscosity
modified solution. The resulting mixture was not clear even after
stirring or heating for an extended period of time. The process
could not be continued because a clear or filterable solution was
not obtained.
Comparative Example 4: Formulation Made without Adding Dehydrated
Alcohol to about 25% (w/w)
[0180] A primary emulsion, monophasic solution and dry paste were
prepared in the same way as described in Example 9. After
lyophilization, dehydrated alcohol was added into the paste so that
the amount of the dehydrated alcohol is about 6% (w/w) relative to
the total weight of the resulting viscosity modified solution, and
not 25% w/w. The resulting mixture was hazy and not clear even
after stirring or heating for an extended period of time.
[0181] As mentioned above, the clearness of the solution is
measured by appearance, e.g., that it is free from visually
suspended particle, and the intermediate solution has a light
transmittance of greater than about 90% measured at 800 nm (T800)
in a 1 cm path quartz cuvette and alcohol as blank when measured by
a UV-visible spectrophotometer, such as the one made by Pharmacia,
Model Ultrospec III
TABLE-US-00013 Examples 10A-10F Components Example 10A Example 10B
Example 10F Gentamicin In an amount In an amount 0 sulfate
equivalent to equivalent to 1.68% (w/w) 1.68% (w/w) gentamicin in
gentamicin in USP assay USP assay Vancomycin In an amount 0 0
hydrochloride equivalent to 1.88% (w/w) gentamicin in USP assay Soy
lecithin 50.00 51.00 50.00 Dehydrated 6.00 6.00 6.00 alcohol Sesame
oil Add to 100 Add to 100 Add to 100
[0182] Example 10A identified above was prepared according to the
method of the present invention.
[0183] Example 10B identified above was also prepared according to
the method of the present invention.
[0184] Example 10C identified above was prepared according to the
method of present invention without containing any hydrophilic
water-soluble pharmaceutically active agent.
[0185] Example 10D was prepared by mixing the formulation of
Example 10C with gentamicin sulfate and vancomycin hydrochloride.
Example 10D therefore was not prepared according to the method of
the present invention.
[0186] Example 10E was prepared by mixing the formulation of
Example 10C with gentamicin sulfate only. Example 10E therefore was
not prepared in accordance with the present invention.
[0187] Example 10F was prepared without the step of
microfluidization step. Accordingly, Example 10F also was not
prepared in accordance with the present invention. Omission of the
fluidization step resulted in precipitation in the depot. The
resulting depot, therefore, was not clear.
Example 11: Structural Characterization by Small Angle X-Ray
Diffraction (SAXS) of Examples 10A-10F
[0188] Procedure: Small angle X-ray scattering (SAXS) data were
collected in a helium chamber using a Bruker M18XHF22 rotating
anode generator operating at 50 kV and 50 mA supplying a CuK.alpha.
(.lamda.=1.541838 .ANG.) radiation beam that was collimated using a
pinhole collimator. K.beta. radiation was filtered out with a Ni
filter. A Highstar multiwire detector was used to collect the data.
The samples were loaded without modification into 0.9 mm
borosilicate glass capillaries and sealed with epoxy. The samples
were mounted in the He chamber on an automated goniometer at sample
to detector distance of 64.55 cm. To prevent scatter from air He
gas was purged into the chamber for 1 hour and then each sample was
collected for 7200 seconds. The data were smoothed and integrated
over the 360.degree. .chi. circle from 0.8 to 4.7.degree. 2.theta.
in 0.1 and 0.02 degree widths. The patterns were compared and the
0.1 degree width integrations were used for the refinements of peak
positions.
[0189] Results: FIG. 12 illustrates the small angle X-ray
diffraction (SAXS) patterns of Examples 10A-10F. Two distinct
diffraction peaks were observed. Examples 10A, 10B and 10F
exhibited at low angles diffraction peak at about 2 Theta (degree)
and the two physical mixtures (Examples 10D and 10E) and the depot
vehicle without any active agent (Example 10C) showed a much
broader diffraction peak at about 2.5 Theta (degree).
[0190] The formulations produced using the method of the present
invention (Examples 10A and 10B) had a unique SAXS diffraction peak
formed at about 2 Theta (degree), which was not found in Example
10C or physical mixtures of the depot vehicle with the same drugs
(Examples 10D and 10E). Example 10C has smaller lattice spacing
than Examples 10A, 10B and 10F.
[0191] There is approximately 8-9 .ANG. increase calculated in the
lattice spacing when gentamicin and vancomycin are incorporated
into the depot in accordance with the present invention, forming
such unique structure (herein referred to as the "2-Theta
Structure").
[0192] The two physical mixtures (Examples 10D and 10E) showed
lattice spacing of the primary diffraction peak that was consistent
with depot vehicle (Example 10C), indicating the physical mixing of
the depot vehicle with gentamicin and vancomycin does not change
the structure of the vehicle.
[0193] It is only after the vancomycin and/or gentamicin are
incorporated into the depot vehicle using the process of the
present invention that the 2-Theta Structure is formed.
[0194] This clearly indicates that the compositions of the present
invention have a unique 2-Theta Structure and such structure can
only be obtained by using the method of preparation of the present
invention.
[0195] The reduced diffraction intensity at 2 Theta (degree)
observed for Example 10F suggests that there exists partially the
"2-Theta Structure" in the composition prepared without the
microfluidization step.
[0196] Conclusion: The compositions of the present invention,
Examples 10A and 10 B contain uniquely different 2-Theta
Structure.
Example 12: Structural Characterization by Thermal Gravimetric
Analysis (TGA) of Examples 10A and 10D
[0197] Procedure: TGA experiments were carried on a Seiko
Instruments TG/DTA 220 nit. Temperature and enthalpy were
calibrated using Indium and Tin standards. Scans were completed
using a rate of 10.degree. C./min from 25-300.degree. C. with a
nitrogen purge rate of 80 ml/min in open pans and a sample size
between 5 and 10 mg.
[0198] Results: As shown in FIG. 13, the TGA results showed small
difference in weight loss profile between Example 10A which was
prepared according to the method of the present invention and
Example 10D, which was not prepared according to the method of the
present invention.
Example 13: Structural Characterization by Differentiating Scanning
Calorimetry (DSC) of Examples 10A and 10D
[0199] Procedure: DSC experiments were carried using a Seiko
Instruments DSC 120 single cell Modulated DSC with RSC
(refrigerated cooling) unit. The DSC was calibrated for temperature
and cell constant by using an Indium standard. Scans were run in
normal DSC mode at a rate of 10.degree. C./min in sealed pans with
a Nitrogen purge rate of 40 ml/min with weights between 5 and 10 mg
used for each sample. Scans were run from 25-300.degree. C.
[0200] Results: As shown in FIG. 14, the DSC profiles for both
samples (Example 10A and 10D) are characterized by a major
endothermic event up to about 100.degree. C., which is likely
related to desolvation of the samples. Example 10D, however,
exhibited an additional endothermic peak at about .degree. C.,
possibly due to melting of a solid drug, i.e., gentamicin sulfate
and/or vancomycin hydrochloride.
[0201] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *