U.S. patent application number 16/412986 was filed with the patent office on 2020-01-23 for crystalline salts of (4s,4as,5ar,12as)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me- thyl)amino)-methyl]-1,11-dioxo-1.
The applicant listed for this patent is Allergan Pharmaceuticals International Limited, Paratek Pharmaceuticals, Inc.. Invention is credited to Catherine COULTER, Sean M. JOHNSTON, Farzaneh SEYEDI.
Application Number | 20200024223 16/412986 |
Document ID | / |
Family ID | 47139728 |
Filed Date | 2020-01-23 |
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United States Patent
Application |
20200024223 |
Kind Code |
A1 |
COULTER; Catherine ; et
al. |
January 23, 2020 |
CRYSTALLINE SALTS OF
(4S,4AS,5AR,12AS)-4-DIMETHYLAMINO-3,10,12,12A-TETRAHYDROXY-7-[(METHOXY(ME-
THYL)AMINO)-METHYL]-1,11-DIOXO-1,4,4A,5,5A,6,11,12A-OCTAHYDRO-NAPHTHACENE--
2-CARBOXYLIC ACID AMIDE AND METHODS OF USING THE SAME
Abstract
A crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is disclosed having improved stability. In
addition, a crystalline mono mesylate salt and crystalline mono
sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(me-
thoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naph-
thacene-2-carboxylic acid amide are also disclosed having improved
stability. A pharmaceutical composition containing the crystalline
salts and methods of treating inflammatory skin disorders and
bacterial infections comprising administering the crystalline salts
are also disclosed.
Inventors: |
COULTER; Catherine;
(Ballymena, GB) ; JOHNSTON; Sean M.; (Doylestown,
PA) ; SEYEDI; Farzaneh; (Mansfield, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan Pharmaceuticals International Limited
Paratek Pharmaceuticals, Inc. |
Dublin
Boston |
MA |
IE
US |
|
|
Family ID: |
47139728 |
Appl. No.: |
16/412986 |
Filed: |
May 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15913504 |
Mar 6, 2018 |
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16412986 |
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14974813 |
Dec 18, 2015 |
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15913504 |
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13471275 |
May 14, 2012 |
9255068 |
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14974813 |
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61485179 |
May 12, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/10 20180101;
C07B 2200/07 20130101; A61P 31/04 20180101; C07C 239/20 20130101;
C07C 309/04 20130101; C07C 237/26 20130101; C07C 2603/46 20170501;
A61P 17/00 20180101; C07B 2200/13 20130101 |
International
Class: |
C07C 237/26 20060101
C07C237/26; C07C 239/20 20060101 C07C239/20; C07C 309/04 20060101
C07C309/04 |
Claims
1. A crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, wherein the salt is selected from a group
consisting of mono hydrochloride, mono mesylate and mono
sulfate.
2. The crystalline salt of claim 1, wherein the salt is
substantially pure.
3. The crystalline salt of claim 1, wherein the salt is mono
hydrochloride.
4. The crystalline salt of claim 3, having an XRPD pattern
substantially as illustrated in FIG. 1 after synthesis of the
crystalline salt.
5. The crystalline salt of claim 3, having characteristic peaks at
diffraction angle 2-theta degrees appearing at least at about 13.4,
about 20.5 and about 23.3, as measured by XRPD.
6. The crystalline salt of claim 3, having a DSC curve
substantially as illustrated in FIG. 2 after synthesis of the
crystalline salt.
7. The crystalline salt of claim 3, having a TGA curve
substantially as illustrated in FIG. 3 after synthesis of the
crystalline salt.
8. The crystalline salt of claim 3, wherein the salt has a J-isomer
content at 0 days of about 0.1% peak area to about 7.0% peak area,
as measured by HPLC.
9. The crystalline salt of claim 1, wherein the salt is mono
mesylate.
10. The crystalline salt of claim 9, having an XRPD pattern
substantially as illustrated in FIG. 4 after synthesis of the
crystalline salt.
11. The crystalline salt of claim 9, having characteristic peaks at
diffraction angle 2-theta degrees appearing at least at about 9,
about 15 and about 23.8, as measured by XRPD.
12. The crystalline salt of claim 9, having a DSC curve
substantially as illustrated in FIG. 5 after synthesis of the
crystalline salt.
13. The crystalline salt of claim 9, having a TGA curve
substantially as illustrated in FIG. 6 after synthesis of the
crystalline salt.
14. The crystalline salt of claim 9, wherein the salt has a
.beta.-isomer content at 0 days of about 2.0% peak area to about
10.0% peak area, as measured by HPLC.
15. The crystalline salt of claim 1, wherein the salt is mono
sulfate.
16. The crystalline salt of claim 15, having an XRPD pattern
substantially as illustrated in FIG. 7 after synthesis of the
crystalline salt.
17. The crystalline salt of claim 15, having characteristic peaks
at diffraction angle 2-theta degrees appearing at least at about
15, about 17.8 and about 23.5, as measured by XRPD.
18. The crystalline salt of claim 15, having a DSC curve
substantially as illustrated in FIG. 8 after synthesis of the
crystalline salt.
19. The crystalline salt of claim 15, having a TGA curve
substantially as illustrated in FIG. 9 after synthesis of the
crystalline salt.
20. The crystalline salt of claim 15, wherein the salt has a
.beta.-isomer content at 0 days of about 3.0% peak area to about
26.0% peak area, as measured by HPLC.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
Provisional Application No. 61/485,179, filed May 12, 2011, the
content of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] The instant disclosure relates to crystalline mono
hydrochloride, mono mesylate, and mono sulfate salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, and methods of using the same. More
specifically, the disclosure relates to crystalline mono
hydrochloride, mono mesylate, and mono sulfate salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide having improved stability over tetracycline
compounds known in the art. In addition, the instant disclosure
relates to pharmaceutical compositions comprising the crystalline
mono hydrochloride, mono mesylate, or mono sulfate salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, and methods of treating acne, rosacea or
gram positive bacterial infections using the crystalline mono
hydrochloride, mono mesylate, or mono sulfate salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
BACKGROUND OF THE INVENTION
[0003] Tetracyclines are known "broad spectrum" antibiotics and
have become widely used for therapeutic purposes. Tetracyclines
have been found to be highly effective pharmacologically against
rickettsiae; a number of gram-positive and gram-negative bacteria;
and the agents responsible for lymphogranuloma venereum, inclusion
conjunctivitis, and psittacosis. The first use of tetracycline
antibiotics dates as far back as 1948. Examples of pharmaceutically
active tetracycline and tetracycline analogue compositions may be
found in U.S. Pat. Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531;
3,454,697; 3,557,280; 3,674,859; 3,957,980; 4,018,889; 4,024,272;
and 4,126,680. Tetracyclines may also be used to treat inflammatory
skin disorders, including dermatitis, psoriasis, pyoderma
gangrenosum, acne and rosacea.
[0004] Acne vulgaris, also referred to as acne, is both an
inflammatory skin disorder and a bacterial infection. It is a
disorder resulting from hormones affecting the sebaceous glands,
which leads to plugged pores and outbreaks of lesions, or pimples.
Acne is the most common skin disease in the United States,
affecting nearly 17 million people. Severe acne can lead to
disfiguration, and permanent scarring.
[0005] Acne is described as a disorder of the pilosebaceous units
(PSUs). Found over most of the body, PSUs consist of sebaceous
glands, which make an oily substance that normally empties onto the
skin surface through the opening of the follicle, also called a
pore. When the pore is plugged, the mixture of oil and cells allows
bacteria that normally live on the skin to grow in the plugged
follicles, which produce chemicals and enzymes and attract white
blood cells that cause inflammation. The plugged follicle breaks
down, the sebum, shed skin cells and bacteria disseminate into the
nearby tissues, leading to lesions or pimples.
[0006] Acne is commonly treated with systemic antibiotics,
including tetracyclines, to reduce the growth of bacteria. Efficacy
is thought to be due to an effect on Propionibacterium acnes
(P.acnes) as well as the intrinsic anti-inflammatory properties of
these antibiotics. Propionibacterium acnes is a relatively slow
growing, typically aerotolerant anaerobic gram positive bacterium
(rod) that is linked to acne. Tetracyclines are known to be
effective in killing P.acnes and other bacteria and have been used
to treat acne because of their antibacterial and anti-inflammatory
properties.
[0007] Rosacea is a skin disorder characterized by facial redness,
mainly affecting individuals of north western European descent.
Early symptoms of rosacea include redness on the chin, nose, skin
or forehead; small visible blood vessels on the face; bumps or
pimples on the face; and watery and irritated eyes. Although the
causes of rosacea are poorly understood, systemic antibiotics, such
as tetracyclines, are commonly prescribed for the treatment of
rosacea, due to both their anti-inflammatory and antibacterial
properties.
[0008] After the widespread use of tetracyclines for both major and
minor illnesses and diseases led to resistance to these
antibiotics, substituted tetracycline compounds were developed to
treat bacterial infections, inflammation, neoplasms, and other
conditions. The term "tetracycline compound" includes many
compounds with a similar ring structure to tetracycline. Examples
of these tetracycline compounds include: chlortetracycline,
doxycycline, minocycline, oxytetracycline, demeclocycline,
methacycline, sancycline, chelocardin, rolitetracycline,
lymecycline, apicycline; clomocycline, guamecycline, meglucycline,
mepylcycline, penimepicycline, pipacycline, etamocycline,
penimocycline. For example, substituted tetracycline compounds have
been disclosed in WO 2008/079339 and WO 2008/079363.
[0009] One substituted tetracycline compound is
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, described in U.S. Patent Application
Publication Nos. 2008/0312193 and 2010/0305072. The free base of
this compound has proven unstable for use as an active
pharmaceutical ingredient. In addition, while those skilled in the
art have attempted to synthesize a salt of this compound
previously, only amorphous salts have been produced and these
amorphous salts have shown only minimal improved stability over the
free base. Accordingly, there exists a need in the art for improved
stability of this substituted tetracycline compound.
[0010] The present invention is directed to the novel crystalline
mono hydrochloride, mono mesylate, and mono sulfate salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, which exhibit superior stability over the
free base and previously known salts thereof. This is a significant
advancement in the state of the art.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to a crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, wherein the salt is selected from a group
consisting of mono hydrochloride, mono mesylate and mono sulfate.
In a certain embodiment, the crystalline salt is substantially
pure. One embodiment is directed to the crystalline mono
hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide. In a certain embodiment, the crystalline
mono hydrochloride salt has an X-ray powder diffraction (XRPD)
pattern substantially as illustrated in FIG. 1 after synthesis of
the crystalline salt, and, in a preferred embodiment, has
characteristic peaks in the XRPD pattern at diffraction angle
2-theta degrees appearing at least at about 13.4, about 20.5 and
about 23.3. In further embodiments, the crystalline mono
hydrochloride salt has a differential scanning calorimetry (DSC)
curve substantially as illustrated in FIG. 2 after synthesis, and a
thermo-gravimetric analysis (TGA) curve substantially as
illustrated in FIG. 3 after synthesis. In another embodiment, the
crystalline mono hydrochloride salt has a DJ-isomer content at 0
days of about 0.1 percent peak area (hereinafter referred to as "%
peak area") to about 7.0% peak area, as measured by High
Performance Liquid Chromatography (HPLC).
[0012] Other embodiments of the invention are directed to a
crystalline mono mesylate salt and a crystalline mono sulfate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide. In a certain embodiment, the crystalline
mono mesylate salt has an XRPD pattern substantially as illustrated
in FIG. 4 after synthesis of the crystalline salt, and, in a
preferred embodiment, has characteristic peaks in the XRPD pattern
at diffraction angle 2-theta degrees appearing at least at about 9,
about 15 and about 23.8. In further embodiments, the crystalline
mono mesylate salt has a DSC curve substantially as illustrated in
FIG. 5 after synthesis, and a TGA curve substantially as
illustrated in FIG. 6 after synthesis.
[0013] In a certain embodiment, the crystalline mono sulfate salt
has an XRPD pattern substantially as illustrated in FIG. 7 after
synthesis of the crystalline salt, and, in a preferred embodiment,
has characteristic peaks in the XRPD pattern at diffraction angle
2-theta degrees appearing at least at about 15, about 17.8 and
about 23.5. In further embodiments, the crystalline mono sulfate
salt has a DSC curve substantially as illustrated in FIG. 8 after
synthesis, and a TGA curve substantially as illustrated in FIG. 9
after synthesis.
[0014] In preferred embodiments, the crystalline mono mesylate salt
has a .beta.-isomer content at 0 days of about 2.0% peak area to
about 10.0% peak area, as measured by HPLC, and the crystalline
mono sulfate salt have a .beta.-isomer content at 0 days of about
3.0% peak area to about 26.0% peak area, as measured by HPLC.
[0015] The present invention is further directed to a
pharmaceutical composition comprising a crystalline mono
hydrochloride salt, crystalline mono mesylate salt or crystalline
mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide and a pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition is
used for treating acne. In another embodiment, the pharmaceutical
composition is used for treating rosacea. In yet another
embodiment, the pharmaceutical composition is used for treating a
gram positive bacterial infection, wherein the gram positive
bacteria is selected from the group consisting of Propionibacterium
acnes, Staphylococcus aureus, Streptococcus pneumonia,
Streptococcus pyogenes, and Clostridium difficile.
[0016] The present invention is also directed to a method of
treating acne comprising administering to a subject a
therapeutically effective amount of a crystalline mono
hydrochloride salt, crystalline mono mesylate salt, or crystalline
mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
[0017] The present invention is also directed to a method of
treating rosacea comprising administering to a subject a
therapeutically effective amount of a crystalline mono
hydrochloride salt, crystalline mono mesylate salt, or crystalline
mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
[0018] The present invention is also directed to a method of
treating a gram positive bacterial infection, wherein the gram
positive bacteria is selected from the group consisting of
Propionibacterium acnes. Staphylococcus aureus. Streptococcus
pneumonia. Streptococcus pyogenes, and Clostridium difficile,
comprising administering to a subject a therapeutically effective
amount of a crystalline mono hydrochloride salt, crystalline mono
mesylate salt, or crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows X-ray powder diffraction (XRPD) analysis of
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis and after storage for 7
days at 40.degree. C. and 75% relative humidity (RH).
[0020] FIG. 2 is a differential scanning calorimetry (DSC) curve of
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0021] FIG. 3 is a thermo-gravimetric analysis (TGA) curve of
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0022] FIG. 4 shows XRPD analysis of crystalline mono mesylate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis and after storage for 7
days at 40.degree. C. and 75% RH.
[0023] FIG. 5 is a DSC curve of crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0024] FIG. 6 is a TGA of crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0025] FIG. 7 shows XRPD analysis of crystalline mono sulfate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis and after storage for 7
days at 40.degree. C. and 75% RH.
[0026] FIG. 8 is a DSC curve of crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0027] FIG. 9 is a TGA of crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after synthesis.
[0028] FIG. 10 shows XRPD analysis of amorphous bis hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
[0029] FIG. 11 is a TGA curve and DSC curve overlaid of amorphous
bis hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
DETAILED DESCRIPTION OF THE INVENTION
Crystalline Salts
[0030] Novel crystalline salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide are disclosed herein. After much
experimentation and discovery, the inventors determined the stable
and preferred salt forms of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(m-
ethoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-nap-
hthacene-2-carboxylic acid amide, which may be used as a
pharmaceutical active ingredient in a pharmaceutical composition.
The present disclosure teaches how to make these novel crystalline
salts and the superior benefits of them over the free base of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide and previously known amorphous salts
thereof.
[0031] Thus, one embodiment of the present invention is a
crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy-
(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthace-
ne-2-carboxylic acid amide, wherein the salt is selected from a
group consisting of mono hydrochloride, mono mesylate and mono
sulfate.
[0032] In a preferred embodiment, the crystalline salt is
substantially pure. A substantially pure crystalline salt contains
less than about 10% peak area and, preferably, less than about 4%
peak area, total impurity content, as measured by HPLC. In a more
preferred embodiment, the crystalline salt is substantially free of
an amorphous salt. Preferably, less than about 8% peak area of
amorphous salt is present, more preferably, less than about 5% peak
area of amorphous salt is present, and still more preferably, less
than about 3% peak area of amorphous salt is present.
[0033] As used herein in reference to the percent peak area of
impurity content, the term "about" generally means within 10
percent, e.g., within 5 percent of a given value or range.
[0034] The term "crystalline" as used herein refers to compounds in
a solid state having a periodic and repeating three-dimensional
internal arrangement of atoms, ions or molecules characteristic of
crystals. The term crystalline does not necessarily mean that the
compound exists as crystals, but that it has this crystal-like
internal structural arrangement. The term "amorphous" as used
herein refers to compounds lacking a crystalline structure: no
repeating pattern, only short range order, extensively
disordered.
[0035] The crystalline salts of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide may be used to treat, prevent, or otherwise
ameliorate bacterial, viral, parasitic, and fungal infections;
cancer (e.g., prostate, breast, colon, lung melanoma and lymph
cancers) and other disorders characterized by unwanted cellular
proliferation; arthritis; osteoporosis; diabetes; stroke; acute
myocardial infarction; aortic aneurysm; neurodegenerative diseases
and other conditions for which tetracycline compounds have been
found to be active (see, for example, U.S. Pat. Nos. 5,789,395;
5,834,450; 6,277,061; and 5,532,227, each of which is expressly
incorporated herein by reference). In addition, the salts of the
invention can be used to prevent or control important mammalian and
veterinary diseases such as rickettsial infections, sexually
transmitted infections, respiratory tract infections, bacterial
infections, ophthalmic infections, anthrax; may serve as therapy in
acute intestinal amebiasis, acne, and lyme disease; and may be used
for prophylaxis of malaria and the like. Preferably, the
crystalline salts of the present invention may be used to treat
bacterial infections and inflammatory skin disorders, which
include, without limitation, eczema, dermatitis, psoriasis,
pyoderma gangrenosum, acne and rosacea. In one embodiment, the
crystalline salts of the present invention may be used to treat
acne and/or rosacea. For example, the crystalline salts of the
present invention may be used to treat acne. Nonlimiting examples
of bacterial infections that can be treated by the salts of the
invention include infections with gram positive organisms
Propionibacterium acnes. Staphylococcus aureus. Streptococcus
pneumonia. Streptococcus pyogenes, or Clostridium difficile.
[0036] A certain embodiment is the crystalline mono hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,1-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-
-carboxylic acid amide.
[0037] The term "mono hydrochloride salt" as used herein refers to
an ionic compound that results from the neutralization reaction of
an acid and a base. The ionic compound (herein, HCl) is composed of
a cation and an anion so that the compound is neutral.
[0038] General methods for analyzing crystalline salts include
crystal analysis by X-ray powder diffraction (XRPD), differential
scanning calorimetry (DSC) and thermo-gravimetric analysis
(TGA).
[0039] XRPD analysis as disclosed herein was collected on a Bruker
AXS C2 GADDS diffractometer using Cu K.alpha. radiation (40 kV, 40
mA), automated XYZ stage, laser video microscope for auto-sample
positioning and a HiStar 2-dimensional area detector. X-ray optics
consisted of a single Gbbel multilayer mirror coupled with a
pinhole collimator of 0.3 mm. The software used for data collection
was GADDS for WNT 4.1.16 and the data was analyzed and presented
using Diffrac Plus EVA v 9.0.0.2 or v 13.0.0.2. Samples were
analyzed under ambient conditions as flat plate specimens using
powder as received. Approximately 1-2 mg of the sample was lightly
pressed on a glass slide to obtain a flat surface. Samples analyzed
under non-ambient conditions were mounted on a silicon wafer with a
heat conducting compound. The sample was then heated to the
appropriate temperature at approximately 20.degree. C.min.sup.-1
and subsequently held isothermally for approximately 1 minute
before data collection was initiated.
[0040] In certain embodiments, the crystalline mono hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide has an XRPD pattern substantially as
illustrated in FIG. 1 after synthesis of the crystalline salt.
[0041] The term "XRPD pattern" as used herein refers to the
graphical representation of the data collected by XRPD analysis.
XRPD analysis is a technique used to characterize the
crystallographic structure, size, and preferred orientation in
polycrystalline or powdered solid samples. This diffraction is also
used to characterize heterogeneous solid mixtures to determine the
percent of crystalline compounds present and can provide structural
information on unknown materials.
[0042] The terms "substantially" and "about" as used herein in
reference to an XPRD pattern refer to the XPRD pattern wherein a
listed peak(s) appears within 0.2 degrees 2-theta, including within
0.1 degrees 2-theta of a given 2-theta value.
[0043] In a preferred embodiment, the crystalline mono
hydrochloride salt has characteristic peaks at diffraction angle
2-theta degrees appearing at least at about 13.4, about 20.5 and
about 23.3, as measured by XRPD. In a more preferred embodiment,
the crystalline mono hydrochloride salt has characteristic peaks at
diffraction angle 2-theta degrees appearing at least at about 9.5,
about 13.4, about 15.5, about 20.5 and about 23.3, as measured by
XRPD, and still more preferable, the crystalline mono hydrochloride
salt has characteristic peaks at diffraction angle 2-theta degrees
appearing at least at about 9.5, about 13.4, about 15.5, about
16.6, about 19.2, about 20.5, about 22.2, and about 23.3.
[0044] The term "characteristic peak" as used herein refers to a
peak in the XRPD pattern having an intensity at least 20%, more
preferably 40% greater than the baseline noise.
[0045] TGA and DSC analysis are used to measure thermal behavior
and can be used to distinguish between polymorphs. One polymorphic
form may exhibit thermal behavior different from that of the
amorphous material or another polymorphic form.
[0046] DSC analysis as disclosed herein was collected on a TA
Instruments Q2000 equipped with a 50 position auto-sampler. The
instrument was calibrated for energy and temperature using
certified indium. The calibration for thermal capacity was carried
out using sapphire. Typically, 0.5-3.0 mg of each sample, in a
pin-holed aluminum pan, was heated at 10.degree. C.min.sup.-1 from
25.degree. C. to 250.degree. C. A nitrogen purge at 50
ml.min.sup.-1 was maintained over the sample. The instrument
control software used was Advantage for Q Series v2.8.0.392 and
Thermal Advantage v4.8.3 and the data was analyzed using Universal
Analysis v4.4A.
[0047] DSC is a thermoanalytical technique in which the difference
in the amount of heat required to increase the temperature of a
sample and reference is measured as a function of temperature. DSC
can be used to measure a number of characteristic properties of a
sample, allowing observation of crystallization events.
Specifically, with DSC, it is possible to observe small energy
changes that occur as matter transitions from a solid to a liquid
crystal and from a liquid crystal to an isotropic liquid. The
presence of events in the DSC curve can be used to assess the
compound's stability, as well as the presence of solvates or
hydrates.
[0048] TGA is used to determine changes in weight in relation to
change in temperature, which may reveal degradation of the compound
and the presence of solvates or hydrates. TGA analysis as disclosed
herein was collected on a TA Instruments Q500 TGA equipped with a
16 position auto-sampler. The instrument was temperature calibrated
using certified Alumel and Nickel. Typically, 5-30 mg of each
sample was loaded onto a pre-weighed platinum crucible and aluminum
DSC pan and was heated at 10.degree. C. min.sup.-1 from ambient
temperature to 300.degree. C. A nitrogen purge at 60 ml.min.sup.-1
was maintained over the sample. The instrument control and data
analysis software used was Advantage for Q Series v2.8.0.392 and
Thermal Advantage v4.8.3 and the data was analyzed using Universal
Analysis v4.4A.
[0049] In a certain embodiment, the crystalline mono hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a DSC curve substantially as
illustrated in FIG. 2. Preferably, the crystalline mono
hydrochloride salt analyzed by DSC exhibits no events up to
degradation of the crystalline salt.
[0050] The term "events" as used herein refers to a change in the
sample associated with absorption (endothermic) or evolution
(exothermic) of heat causing a change in differential heat flow
which is recorded as a peak in the thermogram. Such changes in the
sample include decomposition, degradation, and change of form or
morphology, solvate or hydrate. The absence of any events indicates
that the compound is stable and is in a low energy form.
[0051] The term "substantially," as used herein in reference to DSC
curve means the DSC curve demonstrating a peak(s) within 1.degree.
C., including within 0.5.degree. C. of a given temperature.
[0052] In a certain embodiment, the crystalline mono hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a TGA curve substantially as
illustrated in FIG. 3. Preferably, the crystalline mono
hydrochloride salt analyzed by TGA exhibits a weight loss of about
1% to about 5% from about 30.degree. C. to about 200.degree. C. and
a weight loss of about 12% to about 16% from about 200.degree. C.
to about 250.degree. C. and, more preferably, a weight loss of
about 3% from about 30.degree. C. to about 200.degree. C. and a
weight loss of about 14% to about 15% from about 200.degree. C. to
about 250.degree. C.
[0053] The term "substantially," as used herein in reference to the
TGA curve means the curve demonstrating a percent weight loss
within 1%, including within 0.5% of a given value in relation to
temperature change.
[0054] In certain embodiments, the crystalline mono hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is stable for at least 4 months, and more
preferably for at least 6 months.
[0055] The term "stable" and "stability" as used herein refers to
both the physical form and the chemical purity of the salt. "The
salt" as used herein refers to the disclosed crystalline mono
hydrochloride, mono mesylate and mono sulfate salts of the present
invention.
[0056] One measure of the stability of the physical form of the
salt is hygroscopicity, which is the propensity of a substance to
absorb or adsorb water molecules from the surrounding environment.
Whenever moisture can promote degradation, the salt is stable if it
is non-hygroscopic or mildly hygroscopic above 70% relative
humidity (RH). In preferred embodiments, the salt is
non-hygroscopic or mildly hygroscopic above 80% RH, and in more
preferred embodiments, the salt is non-hygroscopic or mildly
hygroscopic to 90% RH. "Non-hygroscopic or mildly hygroscopic" as
used herein refers to a compound at about 40.degree. C. and at an
RH of about 75%, existing over about 80% w/w in solid crystalline
form, preferably over about 90% w/w in solid crystalline form, that
absorbs less than 10% w/w water, and preferably, less than 5% w/w
water in 8 hours or less. Hygroscopicity (hygroscopic degree) is
calculated based on increase in weight in a compound at comparative
points of measurement. Another measure of physical stability is the
crystal form of the salt, which may be measured by XPRD.
[0057] One measure of chemical purity is defined by the DJ-isomer
content of the salt. Many tetracyclines are optically active and
contain one or more asymmetric centers. The process by which the
asymmetry of such a center is altered to form the opposite
stereochemistry is referred to as epimerization. Tetracyclines
undergo reversible epimerization to the less active
epi-tetracycline. The rate at which epimerization occurs is
dependent on many factors, such as pH, temperature, counter ion,
and humidity. The naturally occurring epimer is typically referred
to as a, or the active epimer. The other epimer, known as 1, may or
may not possess biological activity.
[0058]
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(meth-
oxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphth-
acene-2-carboxylic acid amide has an epimeric center at C.sub.4.
The .alpha. and .beta. epimers of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide are separable and quantifiable by reversed
phase HPLC (High Performance Liquid Chromatography) with
ultraviolet detector (HPLC-UV) analysis, and measured as percent
area under the curve, also referred to as percent peak area.
Although the epimer of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is believed to be non-toxic, under certain
conditions it may lack the anti-bacterial efficacy of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide and, therefore, is considered an
undesirable degradation product.
[0059] The lower the .beta.-isomer content, the higher the chemical
purity of the salt. The .beta.-isomer content is measured after
synthesis of the salt and compared with the measured .beta.-isomer
content after storage for a designated period of time. Where the
.beta.-isomer content does not significantly increase after
storage, there has been no negative effect of storage on the
chemical purity of the salt and the salt is stable for that
designated period of time. Since moisture uptake by the
tetracycline may be a contributing factor to epimerization, a salt
form of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide that is not significantly hygroscopic may
provide an innate resistance to epimerization caused by
humidity.
[0060] Another measure of chemical purity is defined by the content
of other related impurities, by-products or degradation products of
the salt, which represents the morphology of the compound. The
lower the content of total impurities as measured by HPLC,
generally, by HPLC-UV, the higher the chemical purity of the salt.
The total impurity content is measured after synthesis of the salt
and compared with the measured total impurity content after storage
for a designated period of time. Where the total impurity content
does not significantly increase after storage, there has been no
negative effect of storage on the chemical purity of the salt and
the salt is stable for that designated period of time.
[0061] In certain embodiments, at 0 days, the crystalline mono
hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide has a total impurity content of less than
about 8% and, preferably, less than about 4%. In another
embodiment, the crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide has a total impurity content after storage
for about 6 months at about 40.degree. C. and about 75% RH of less
than about 10% and, preferably, less than about 6%. In a certain
embodiment, the salt has a total impurity content after storage for
about 6 months at about 40.degree. C. and about 75% RH not more
than about 80% peak area greater than the total impurity content at
about 0 days, and preferably, not more than about 50% peak area
greater than the total impurity content at about 0 days.
[0062] In a certain embodiment of the present invention, the
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is stable and has a .beta.-isomer content
after storage for about 75 days at about 40.degree. C. and about
75% RH not more than about 20% peak area greater than the 3-isomer
content at about 0 days. In a preferred embodiment, the salt has a
3-isomer content after storage for about 75 days at about
40.degree. C. and about 75% RH not more than about 10% peak area
greater than the .beta.-isomer content at about 0 days; in a more
preferred embodiment, the .beta.-isomer content after storage for
about 75 days at about 40.degree. C. and about 75% RH is not more
than about 1% peak area greater than the .beta.-isomer content at
about 0 days; and in a further preferred embodiment, the
.beta.-isomer content after storage for about 75 days at about
40.degree. C. and about 75% RH is about equal to the .beta.-isomer
content at about 0 days. "After synthesis" as used herein refers to
less than about one day from the time of confirmation of synthesis,
also referred to as "0 days."
[0063] In a certain embodiment, the .beta.-isomer content of the
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide at 0 days is about 0.1% to about 7.0% peak
area, preferably about 1.0% to about 6.0% peak area, more
preferably about 2.0% to about 4.0% peak area, and most preferably
about 3.0% to about 4.0% peak area. In a certain embodiment, the
.beta.-isomer content of the crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide after storage for at least 3 months is
about 2.0% to about 8.0% peak area, and more preferably about 3.0%
to about 4.0% peak area. In other embodiments, the .beta.-isomer
content after storage for at least 6 months is about 0.1% to about
10.0% peak area, and more preferably about 2.0% to about 8.0% peak
area. In a certain embodiment, the .beta.-isomer content of the
crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide at 0 days is less than about 6.0% peak area
and after storage for about 75 days at ambient conditions is less
than about 6.0% peak area. Preferably, the .beta.-isomer content at
0 days is less than about 4.5% peak area and after storage for
about 75 days at ambient conditions is less than about 4.5% peak
area, and in a still further embodiment, the .beta.-isomer content
at 0 days is about 3.8% peak area and after storage for about 75
days at ambient conditions is about 3.8% peak area.
[0064] Ambient conditions, as used herein, means a temperature of
about 20.degree. C. to about 25.degree. C. and an RH of about
40%.
[0065] Another embodiment of the present invention is the
crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide.
[0066] The term "mono mesylate salt" as used herein refers to an
ionic compound that results from the neutralization reaction of an
acid and a base. The compound is composed of a cation and an anion
(herein, CH.sub.3SO.sub.2) so that the compound is neutral.
[0067] In certain embodiments, the crystalline mono mesylate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide has an XRPD pattern substantially as
illustrated in FIG. 4 after synthesis of the crystalline salt.
[0068] In a preferred embodiment, the crystalline mono mesylate
salt has characteristic peaks at least appearing at diffraction
angle 2-theta degrees appearing at about 9, about 15 and about
23.8, as measured by XRPD. In a more preferred embodiment, the
crystalline mono mesylate salt has characteristic peaks at
diffraction angle 2-theta degrees appearing at least at about 9,
about 15, about 22.7 and about 23.8, as measured by XRPD, and still
more preferable, the crystalline mono mesylate salt has
characteristic peaks at diffraction angle 2-theta degrees appearing
at least at about 9, about 15, about 22, about 22.7 and about
23.8.
[0069] In a certain embodiment, the crystalline mono mesylate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a DSC curve substantially as
illustrated in FIG. 5. Preferably, the crystalline mono mesylate
salt analyzed by DSC exhibits no events up to degradation of the
crystalline salt.
[0070] In a certain embodiment, the crystalline mono mesylate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a TGA curve substantially as
illustrated in FIG. 6. Preferably, the crystalline mono mesylate
salt analyzed by TGA exhibits a weight loss of about 1% to about 4%
from about 30.degree. C. to about 200.degree. C. and a weight loss
of about 3% to about 10% from about 200.degree. C. to about
250.degree. C. and, more preferably, a weight loss of about 2% to
about 3% from about 30.degree. C. to about 200.degree. C. and a
weight loss of about 6% to about 7% from about 200.degree. C. to
about 250.degree. C.
[0071] In a certain embodiment of the present invention, the
crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-
-carboxylic acid amide is stable and has a .beta.-isomer content
after storage for about 75 days at about 40.degree. C. and about
75% RH not more than about 20% peak area greater than the
.beta.-isomer content at about 0 days. In a preferred embodiment,
the salt has a .beta.-isomer content after storage for about 75
days at about 40.degree. C. and about 75% RH not more than about
10% peak area greater than the .beta.-isomer content at about 0
days; in a more preferred embodiment, the 0-isomer content after
storage for about 75 days at about 40.degree. C. and about 75% RH
is not more than about 1% peak area greater than the .beta.-isomer
content at about 0 days; and in a further preferred embodiment, the
.beta.-isomer content after storage for about 75 days at about
40.degree. C. and about 75% RH is about equal to the 3-isomer
content at about 0 days.
[0072] In a certain embodiment, the .beta.-isomer content of the
crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-
-carboxylic acid amide at 0 days is about 2.0% to about 10.0% peak
area, preferably about 2.0% to about 6.0% peak area, and more
preferably about 2.0% to about 3.0% peak area.
[0073] Another embodiment of the present invention is a crystalline
mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-
-carboxylic acid amide.
[0074] The term "mono sulfate salt" as used herein refers to an
ionic compound that results from the neutralization reaction of an
acid and a base. The compound is composed of a cation and an anion
(herein, SO.sub.42) so that the compound is neutral.
[0075] In certain embodiments, the crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide has an XRPD pattern substantially as
illustrated in FIG. 7 after synthesis of the crystalline salt.
[0076] In a preferred embodiment, the crystalline mono sulfate salt
has characteristic peaks at diffraction angle 2-theta degrees
appearing at least at about 15, about 17.8 and about 23.5, as
measured by XRPD. In a more preferred embodiment, the crystalline
mono sulfate salt has characteristic peaks at diffraction angle
2-theta degrees appearing at least at about 15, about 17.8, about
22.5 and about 23.5, as measured by XRPD. In a still more preferred
embodiment, the crystalline mono sulfate salt has characteristic
peaks at diffraction angle 2-theta degrees appearing at least at
about 15, about 17.8, about 19.0, about 22.5 and about 23.5, as
measured by XRPD.
[0077] In a certain embodiment, the crystalline mono sulfate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a DSC curve substantially as
illustrated in FIG. 8. Preferably, the crystalline mono sulfate
salt analyzed by DSC exhibits no events up to degradation of the
crystalline salt.
[0078] In a certain embodiment, the crystalline mono sulfate salt
of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide exhibits a TGA curve substantially as
illustrated in FIG. 9. Preferably, the crystalline mono sulfate
salt analyzed by TGA exhibits a weight loss of about 1% to about 5%
from about 30.degree. C. to about 200.degree. C. and a weight loss
of about 12% to about 16% from about 200.degree. C. to about
250.degree. C. and, more preferably, a weight loss of about 3% to
about 4% from about 30.degree. C. to about 200.degree. C. and a
weight loss of about 13% to about 14% from about 200.degree. C. to
about 250.degree. C.
[0079] In a certain embodiment of the present invention, the
crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is stable and has a .beta.-isomer content
after storage for about 75 days at about 40.degree. C. and about
75% RH not more than about 20% peak area greater than the
.beta.-isomer content at about 0 days. In a preferred embodiment,
the salt has a .beta.-isomer content after storage for about 75
days at about 40.degree. C. and about 75% RH not more than about
10% peak area greater than the .beta.-isomer content at about 0
days; in a more preferred embodiment, the 0-isomer content after
storage for about 75 days at about 40.degree. C. and about 75% RH
is not more than about 1% peak area greater than the .beta.-isomer
content at about 0 days; and in a further preferred embodiment, the
.beta.-isomer content after storage for about 75 days at about
40.degree. C. and about 75% RH is about equal to the .beta.-isomer
content at about 0 days.
[0080] In a certain embodiment, the .beta.-isomer content of the
crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide at 0 days is about 3.0% to about 26.0% peak
area, preferably about 5.0% to about 20.0% peak area, and most
preferably about 6.0% to about 10.0% peak area.
Pharmaceutical Compositions
[0081] One embodiment of the invention is directed to a
pharmaceutical composition comprising a crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, wherein the salt is selected from a group
consisting of mono hydrochloride, mono mesylate, and mono sulfate,
and a pharmaceutically acceptable excipient.
[0082] The pharmaceutical composition of the present invention
comprises an effective amount of a crystalline salt, a
pharmaceutically acceptable excipient, and, in some embodiments, it
may also contain one or more additional active ingredients. The
content of crystalline salt in the pharmaceutical composition of
the present invention varies depending on the subject of
administration, route of administration and target disease, among
other variables. The pharmaceutical composition of the present
invention may be administered orally, topically (e.g., transdermal,
etc.), vaginally, rectally, or parenterally (e.g., intravenous,
etc.). Preferably, the pharmaceutical composition of the present
invention may be used for treating bacterial infections and
inflammatory skin disorders. For example, the pharmaceutical
composition of the present invention may be used for treating acne
and/or rosacea, e.g., for treating acne, or for treating infections
with gram positive bacteria, wherein the gram positive bacteria is
selected from the group consisting of Propionibacterium acnes.
Staphylococcus aureus. Streptococcus pneumonia. Streptococcus
pyogenes, and Clostridium difficile.
[0083] Examples of topical administration of the pharmaceutical
composition include transdermal, buccal or sublingual application.
For topical applications, the pharmaceutical composition can be
suitably admixed in a pharmacologically inert topical carrier, such
as a gel, an ointment, a lotion or a cream. Such pharmacologically
inert topical carriers include water, glycerol, alcohol, propylene
glycol, fatty alcohols, triglycerides, fatty acid esters, or
mineral oils. Other possible pharmacologically inert topical
carriers are liquid petrolatum, isopropylpalmitate, polyethylene
glycol, ethanol 95/%, polyoxyethylene monolauriate 5% in water,
sodium lauryl sulfate 5% in water, and the like. In addition,
materials such as anti-oxidants, humectants, viscosity stabilizers
and the like also may be added.
[0084] For oral administration, the crystalline salt of the present
invention may be administered as a capsule, tablet or granule.
Tablets may contain various excipients such as microcrystalline
cellulose, sodium citrate, calcium carbonate, dicalcium phosphate
and glycine, along with various disintegrants such as starch (and
preferably corn, potato or tapioca starch), alginic acid and
certain complex silicates, together with granulation binders like
polyvinylpyrrolidone, sucrose, gelatin and acacia. In a certain
embodiment, the tablet may be film coated. Additionally,
lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc are often very useful for tablets. Other solid
compositions may also be employed as fillers in gelatin capsules;
preferred materials in this connection also include lactose or milk
sugar as well as high molecular weight polyethylene glycols. When
aqueous suspensions and/or elixirs are desired for oral
administration, the crystalline salt may be combined with various
sweetening or flavoring agents, coloring matter or dyes, and, if so
desired, emulsifying and/or suspending agents, together with such
diluents as water, ethanol, propylene glycol, glycerin and various
like combinations thereof. The pharmaceutical compositions of the
invention may be formulated such that the crystalline salt is
released over a period of time after administration.
[0085] Preparing such pharmaceutical compositions of the
crystalline salt of the present invention along with a
pharmaceutically acceptable excipient and, optionally, an
additional active ingredient, may be done by any conventional
technique known in the art.
[0086] In an embodiment, the crystalline salt present in the
pharmaceutical composition is about 0.01% to about 90% by weight
relative to the whole composition. A suitable therapeutically
effective amount of the crystalline salt will typically range from
about 0.01 mg/kg to about 1 g/kg of body weight per day; in another
embodiment, from about 1 mg/kg to about 600 mg/kg body weight per
day; in another embodiment, from about 1 mg/kg to about 250 mg/kg
body weight per day; in another embodiment, from about 10 mg/kg to
about 400 mg/kg body weight per day; in another embodiment, from
about 10 mg/kg to about 200 mg/kg of body weight per day; in
another embodiment, from about 10 mg/kg to about 100 mg/kg of body
weight per day; in one embodiment, from about 10 mg/kg to about 25
mg/kg body weight per day; in another embodiment, from about 1
mg/kg to about 10 mg/kg body weight per day; in another embodiment,
from about 0.001 mg/kg to about 100 mg/kg of body weight per day;
in another embodiment, from about 0.001 mg/kg to about 10 mg/kg of
body weight per day; and in another embodiment, from about 0.001
mg/kg to about 1 mg/kg of body weight per day. In a certain
embodiment, when a pharmaceutical composition described herein is
administered orally, a suitable therapeutically effective amount of
the crystalline salt is about 0.01 to about 100 milligrams per
kilogram of body weight of recipient per day, preferably about 0.1
to about 50 milligrams per kilogram body weight of recipient per
day, more preferably from about 0.1 to about 20 milligrams per
kilogram body weight of recipient per day, and even more preferably
from about 0.1 to about 10 milligrams per kilogram body weight of
recipient per day. The desired dose may be administered once daily,
or by several sub-divided doses, e.g., 2 to 5 sub-divided doses, at
appropriate intervals through the day, or other appropriate
schedule.
[0087] The term "pharmaceutically acceptable excipient" as used
herein includes, but is not limited to, one of more of the
following: polymers, resins, plasticizers, fillers, lubricants,
diluents, binders, disintegrants, solvents, co-solvents,
surfactants, buffer systems, preservatives, sweetener agents,
flavoring agents, pharmaceutical-grade dyes or pigments, chelating
agents, viscosity agents, and combinations thereof.
Pharmaceutically acceptable excipients can be used in any component
in making the dosage form, i.e. core tablet or coating. Flavoring
agents and dyes and pigments among those useful herein include but
are not limited to those described in Handbook of Pharmaceutical
Excipients (4th Ed., Pharmaceutical Press 2003). Suitable
co-solvents include, but are not limited to, ethanol, isopropanol,
acetone, and combinations thereof. Suitable surfactants include,
but are not limited to, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene monoalkyl ethers, sucrose monoesters, simethicone
emulsion, sodium lauryl sulfate, Tween 80.RTM., and lanolin esters,
ethers, and combinations thereof. Suitable preservatives include,
but are not limited to, phenol, alkyl esters of parahydroxybenzoic
acid, benzoic acid and the salts thereof, boric acid and the salts
thereof, sorbic acid and the salts thereof, chlorbutanol, benzyl
alcohol, thimerosal, phenylmercuric acetate and nitrate,
nitromersol, benzalkonium chloride, cetylpyridinium chloride,
methyl paraben, propyl paraben, and combinations thereof. Suitable
fillers include, but are not limited to, starch, lactose, sucrose,
maltodextrin, and microcrystalline cellulose. Suitable plasticizers
include, but are not limited to, triethyl citrate, polyethylene
glycol, propylene glycol, dibutyl phthalate, castor oil, acetylated
monoglycerides, triacetin, and combinations thereof. Suitable
polymers include, but are not limited to, ethylcellulose, cellulose
acetate trimellitate, hydroxypropylmethylcellulose phthalate,
cellulose acetate phthalate, polyvinyl acetate phthalate, and
Eudragit.RTM. L 30-D, Eudragit.RTM. L 100-55, Eudragit.RTM. F530D
and Eudragit.RTM. S 100 (Rohm Pharma GmbH and Co. KG, Darmstadt,
Germany), Acryl-EZE.RTM. and Sureteric.RTM. (Colorcon, Inc., West
Point, Pa.), and combinations thereof. Suitable lubricants include,
but are not limited to, magnesium stearate, stearic acid, talc, and
combinations thereof.
[0088] The term "additional active ingredient" as used herein
includes any agent known in the art to treat, prevent or reduce the
symptoms of the condition being treated by the pharmaceutical
composition. Such agents, include but are not limited to agents
known to treat, prevent or reduce the symptoms of bacterial
infections and inflammatory skin disorders.
[0089] The improved stability of the crystalline salts of the
present invention means that the crystals are less hygroscopic,
i.e., less sensitive to humidity, so that a pharmaceutical
composition containing the crystalline salt can be stored for a
longer period of time than previously known pharmaceutical
compositions.
[0090] In a certain embodiment, the pharmaceutical composition
comprises the mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide and a pharmaceutically acceptable
excipient. In another embodiment, the pharmaceutical composition
comprises the mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide and a pharmaceutically acceptable
excipient. In a still further embodiment, the pharmaceutical
composition comprises the mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2--
carboxylic acid amide and a pharmaceutically acceptable
excipient.
[0091] In a certain embodiment, the invention is directed to a
pharmaceutical composition comprising
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable excipient for use in
treating a bacterial infection, e.g., a Streptococcus pyogenes and
Clostridium difficile bacterial infection. In a preferred
embodiment, the pharmaceutical composition comprises a crystalline
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide selected from the group consisting of mono
hydrochloride, mono mesylate and mono sulfate salt.
[0092] The phrase "pharmaceutically acceptable salt" of a compound
as used herein means a salt that is pharmaceutically acceptable and
that possesses the desired pharmacological activity of the parent
compound. Pharmaceutically acceptable salts include salts of acidic
or basic groups present in a compound of the invention.
Pharmaceutically acceptable acid addition salts include, but are
not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate,
mesylate, sulfate, bisulfate, phosphate, acid phosphate,
isonicotinate, acetate, lactate, salicylate, citrate, tartrate,
pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzensulfonate, p-toluenesulfonate and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Suitable base
salts include, but are not limited to, aluminum, calcium, lithium,
magnesium, potassium, sodium, zinc, and diethanolamine salts.
Preferably, the pharmaceutically acceptable salt is a crystalline
salt. Even more preferably, the pharmaceutically acceptable salt is
a crystalline salt selected from mono hydrochloride, mono mesylate,
and mono sulfate.
Methods of Use
[0093] One embodiment of the invention is directed to a method for
treating acne and/or rosacea comprising administering to a subject
a therapeutically effective amount of a crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, wherein the crystalline salt is selected
from a group consisting of mono hydrochloride, mono mesylate and
mono sulfate. In one embodiment, the invention is directed to a
method of treating acne. In another embodiment, the invention is
directed to a method of treating rosacea.
[0094] The term "treating" as used herein includes therapeutic
and/or prophylactic treatment of acne and/or rosacea or other
conditions described herein. The treatment includes the
diminishment or alleviation of at least one symptom associated with
acne and/or rosacea or at least one symptom associated with another
condition described herein.
[0095] The term "therapeutically effective amount" as used herein
means an amount of a compound or composition high enough to
significantly positively modify the symptoms and/or condition to be
treated, but low enough to avoid serious side effects (at a
reasonable risk/benefit ratio), within the scope of sound medical
judgment. The therapeutically effective amount of active ingredient
for use in the method of the invention herein will vary with the
particular condition being treated, the age and physical condition
of the patient to be treated, the severity of the condition, the
duration of the treatment, the nature of concurrent therapy, the
particular active ingredient being employed, the particular
pharmaceutically-acceptable excipients utilized, and like factors
within the knowledge and expertise of a skilled physician or
veterinarian. Various suitable therapeutically effective amounts
are described above.
[0096] The term "subject" as used herein is an animal. "Subject"
includes, without limitation, a human, mouse, rat, guinea pig, dog,
cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey.
In one embodiment, "subject" is a mammal. In another embodiment,
"subject" is a human.
[0097] A certain embodiment is directed to the method for treating
acne comprising administering to a subject a therapeutically
effective amount of the crystalline mono hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide. In an embodiment thereof, the amount of
the mono hydrochloride salt employed is between about 10 mg and
about 2000 mg, and preferably between about 25 mg and about 500 mg.
In a certain embodiment, the mono hydrochloride salt is
administered at least once monthly, preferably, weekly, more
preferably, bi-weekly, and most preferably, the mono hydrochloride
salt is administered daily.
[0098] Another embodiment is directed to the method for treating
acne comprising administering to a subject a therapeutically
effective amount of the crystalline mono mesylate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide. In certain embodiments thereof, the amount
of crystalline mono mesylate salt employed is between about 10 mg
and about 2000 mg, and preferably between about 25 mg and about 500
mg. In a certain embodiment, the mono mesylate salt is administered
at least once monthly, preferably, weekly, more preferably,
bi-weekly, and most preferably, the mono mesylate salt is
administered daily.
[0099] A further embodiment is directed to the method for treating
acne comprising administering to a subject a therapeutically
effective amount of the crystalline mono sulfate salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide. In certain embodiments, the amount of
crystalline mono sulfate salt employed is between about 10 mg and
about 2000 mg, and preferably between about 25 mg and about 500 mg.
In a certain embodiment, the mono sulfate salt is administered at
least once monthly, preferably, weekly, more preferably, bi-weekly,
and most preferably, the mono sulfate salt is administered
daily.
[0100] Yet another embodiment of the invention is directed to a
method of treating a gram positive bacterial infection comprising
administering to a subject a therapeutically effective amount of a
crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, wherein the crystalline salt is selected
from a group consisting of mono hydrochloride, mono mesylate and
mono sulfate. Gram positive bacterial infections include
Propionibacterium acnes. Staphylococcus aureus. Streptococcus
pneumonia. Streptococcus pyogenes, and Clostridium diflicile
infections.
[0101] An additional embodiment of the invention is directed to a
method of treating a bacterial infection, e.g., a gram positive
bacterial infection selected from Streptococcus pyogenes and
Clostndium difficile infection, comprising administering to a
subject a therapeutically effective amount of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide or pharmaceutically acceptable salt
thereof. In a preferred embodiment, the method comprises
administering to a subject a therapeutically effective amount of a
crystalline salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, preferably the salt is mono hydrochloride,
mono mesylate or mono sulfate salt.
[0102] The following examples will illustrate the practice of the
present invention in some of the preferred embodiments. Other
embodiments within the scope of the claims will be apparent to one
skilled in the art.
EXAMPLES
[0103] The following examples illustrate the synthesis of the
compounds described herein.
Synthesis of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide ("the Free Base")
##STR00001##
[0105] A solution of 7-formylsancycline TFA salt (2.23 g) and
N,O-dimethylhydroxylamine hydrochloride (780 mg) in
N,N-dimethylacetamide (15 mL) was stirred for 10 minutes at room
temperature under argon atmosphere. To this solution was added
sodium cyanoborohydride (302 mg). The solution was stirred for 5
minutes and monitored by LC-MS. The reaction mixture was poured
into diethyl ether, and the resulting precipitates were collected
by filtration under vacuum. The crude product was purified by
prep-HPLC using a C18 column (linear gradient 10-40% acetonitrile
in 20 mM aqueous triethanolamine, pH 7.4). The prep-HPLC fractions
were collected, and the organic solvent (acetonitrile) was
evaporated under reduced pressure. The resulting aqueous solution
was loaded onto a clean PDVB SPE column, washed with distilled
water, then with a 0.1 M sodium acetate solution followed by
distilled water. The product was eluted with acetonitrile. The
eluent was concentrated under reduced pressure, 385 mg was obtained
as free base.
Synthesis of Crystalline Mono Hydrochloride Salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide (the "Crystalline Mono Hydrochloride
Salt")
##STR00002##
[0107] Crude
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide (100 g, app. 35% assay) was purified on
preparative column chromatography. The desired fractions (8-10
liters) were combined and the pH was adjusted to 7.0-7.5 using
ammonium hydroxide. This aqueous solution was extracted 3 times
with dichloromethane (4 liters each time). The dichloromethane
layers were combined and concentrated under reduced pressure. The
residue was suspended in ethanol (800 ml) and 20 ml water was
added. The pH was gradually adjusted to pH 1.6-1.3 using 1.25M
hydrochloric acid in methanol and the mixture was stirred for 20-60
minutes at which point the free base was completely dissolved. The
solution was concentrated under reduced pressure to 200-250 ml and
was seeded with
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide mono HCl crystals (100-200 mg). The
stirring was continued for 2-18 hours while the slurry was kept at
.ltoreq.5.degree. C. The resulting crystals were filtered, washed
with ethanol (50 mL) and dried under reduced pressure to a constant
weight. 20 g crystalline
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide mono hydrochloride was isolated in
.gtoreq.90% purity and .gtoreq.90% assay.
Synthesis of Crystalline Mono Mesylate Salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid (the "Crystalline Mesylate Salt")
[0108]
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(meth-
oxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphth-
acene-2-carboxylic acid amide free base (74 mg) was suspended in
ethanol (740 .mu.l) and heated with stirring to 60.degree. C. (bath
temperature). Methane sulfonic acid (1.1 eq, 167 .mu.l as 1M
solution in THF) was added and most of the solid dissolved. After
five minutes, the suspension was cooled to ambient temperature over
approximately 1.75 hours (uncontrolled in oil bath). By 53.degree.
C., solid had precipitated which was filtered at ambient
temperature under reduced pressure. A further portion of ethanol
(200 .mu.l) was added to aid filtration, as the suspension was
viscous. The cake was washed with n-hexane (400 .mu.l) and air
dried on filter for approximately 30 minutes to yield 59 mg (67%
yield) of yellow solid.
Synthesis of Crystalline Mono Sulfate Salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid (the "Crystalline Sulfate Salt")
[0109]
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(meth-
oxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphth-
acene-2-carboxylic acid amide free base (86 mg) was suspended in
ethanol (500 .mu.l) and heated with stirring to 63.degree. C. (bath
temperature) at which temperature most of the free base had
dissolved. Sulfuric acid (1.1 eq, 1941l as 1M solution in water)
was added and all of the solid dissolved. The solution was cooled
to ambient temperature over approximately 1.75 hours (uncontrolled
in oil bath) at which temperature no solid had precipitated. Methyl
t-butyl ether (MtBE) was added as an antisolvent (4.times.50
.mu.l). Each addition caused a cloud point, but the solid
re-dissolved on stirring. The solution was stirred with a stopper
for approximately 3 hours after which time solid precipitated. The
solid was filtered under reduced pressure and washed with MtBE
(3.times.200 .mu.l) and air dried on filter for approximately 45
minutes to yield 93 mg (90% yield) of yellow solid.
Comparative Example 1
Synthesis of Amorphous Bis Hydrochloride Salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide
[0110]
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(meth-
oxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphth-
acene-2-carboxylic acid amide free base (1 g) was suspended in
methanol (50 mL). The freebase was converted to the hydrochloride
salt by adding an excess of methanolic HCl followed by under
reduced pressure evaporation to give 1.1 g yellow solid: MS
(Mz+1=488). 1H NMR (300 MHz, CD3OD) .delta. 7.46 (d, 1H, J=8.6 Hz),
6.81 (d, 1H, J=8.6 Hz), 4.09 (d, 1H, J=1.0 Hz), 3.79 (d, 1H, J=13.1
Hz), 3.73 (d, 1H, J=13.1 Hz), 3.36 (m, 1H), 3.27 (s, 3H), 3.08-2.95
(8H), 2.61 (s, 3H), 2.38 (t, 1H, J=14.8), 2.22 (m, 1H), 1.64 (m,
1H). An XRPD pattern is shown in FIG. 10 and a TGA and DSC curve
overlaid are shown in FIG. 11.
Comparative Example 2
Synthesis of Amorphous Mono Hydrochloride Salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide
[0111] A sample of Crystalline Mono Hydrochloride Salt (2.09 g) was
dissolved in water (250 ml, 120 vols), filtered and frozen in a
-78.degree. C. bath. Water was removed from the solidified sample
using a lyophilizer for 110 hours to yield the amorphous mono
hydrochloride salt as a fluffy yellow solid, that was confirmed to
be amorphous by XRPD analysis
Testing
[0112] In order to determine the stability of Crystalline Mono
Hydrochloride Salt, the .beta.-isomer content of the salt was
determined by HPLC-UV analysis and compared to the .beta.-isomer
content calculated after the salt was stored in an amber glass vial
for approximately 75 days at ambient conditions. The results are
shown in Table 1 below. As evidenced by the data collected, the
.beta.-isomer content did not increase over time and therefore,
storage did not negatively affect the chemical purity of the
Crystalline Mono Hydrochloride Salt.
TABLE-US-00001 TABLE 1 Formation of .beta.-isomer in the
Crystalline Mono Hydrochloride Salt after Storage. Chemical Purity
Chemical Purity Sample at 0 days at 75 days Crystalline Mono 93.8%
(3.8% .beta.-isomer) 94.8% (3.8% .beta.-isomer) Hydrochloride
Salt
[0113] Other samples of the Crystalline Mono Hydrochloride Salt
were analyzed by HPLC-UV and due to minor variations in the
synthetic process, purity at 0 days was found to be 93.8% (3.5%
.beta.-isomer) and 95.8% (3.4% .beta.-isomer). The chemical purity
for these samples after storage was not tested.
[0114] Further evidence of stability was demonstrated by analysis
of the total impurity content of Crystalline Mono Hydrochloride
Salt by HPLC-UV at 0 days and again after storage for about 6
months at about 40.degree. C. and about 75% RH and was found to be
4% peak area and 6% peak area, respectively.
[0115] In tests conducted on the stability of Comparative Example
1, after just 48 hours of storage at about 40.degree. C. and about
75% RH, the .beta.-isomer content increased to 31.7% peak area from
3.6% peak area at 0 days. After storage for 36 days at about
25.degree. C. and about 60% RH, the .beta.-isomer content was
calculated as 8.7% peak area. In another test, the compound of
Comparative Example 1 had a total impurity content of 4.2% peak
area at 0 days and 34.6% peak area after storage for about 2 days
at about 40.degree. C. and about 75% RH. Accordingly, Comparative
Example 1 exhibited a much higher increase in total impurity
content and was significantly less stable than Crystalline Mono
Hydrochloride Salt.
[0116] The .beta.-isomer content of Comparative Example 2 was also
determined by HPLC-UV analysis at 0 days and compared to the
.beta.-isomer content calculated after the compound was stored in a
clear glass vial for approximately 75 days at ambient conditions.
The results are shown in Table 2 below. As evidenced by the data,
after storage for 75 days, the .beta.-isomer content increased by
over 80% and, therefore, the storage negatively affected the
chemical purity of Comparative Example 2.
TABLE-US-00002 TABLE 2 Formation of .beta.-isomer in Comparative
Example 2 after Storage. Chemical Purity Chemical Purity Sample at
0 days at 75 days Comparative 94.3% (4.4% .beta.-isomer) 90.4%
(7.8% .beta.-isomer) Example 2
[0117] The stability of Crystalline Mono Hydrochloride Salt was
compared to the amorphous mono hydrochloride salt of Comparative
Example 2. The results of various tests demonstrating advantages
and disadvantages of the Crystalline Mono Hydrochloride Salt and
Comparative Example 2 are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Advantages and Disadvantages of the
Crystalline Mono Hydrochloride Salt and Comparative Example 2.
Solid form Advantages Disadvantages Crystalline Non-hygroscopic to
90% RH Some loss of crystallinity upon Mono pressing and milling
Hydrochloride No change in form or .beta.-isomer -- Salt content
upon milling or pressing No increase in .beta.-isomer content --
upon storage under ambient conditions Comparative High glass
transition (166.degree. C.) Hygroscopic above 70% RH Example 2
Stable to crystallization upon Change in form (from amorphous
storage and heat/cool cycle to crystalline) to Crystalline Mono
Hydrochloride Salt observed above 75% RH -- Increase in
.beta.-isomer content upon storage at 63% RH -- Increase in
.beta.-isomer content upon pressing and milling -- Increase in
.beta.-isomer content upon storage under ambient conditions --
Faster rate of .beta.-isomer formation upon exposure to solvent as
compared to Crystalline Mono Hydrochloride Salt
[0118] HPLC-UV and XRPD analysis were conducted on samples of
Crystalline Mono Hydrochloride Salt, Crystalline Mesylate Salt and
Crystalline Sulfate Salt at 0 days and after storage for 7 days at
40.degree. C. and 75% RH. FIGS. 1, 4 and 7 show the XRPD analysis
of the Crystalline Mono Hydrochloride Salt, Crystalline Mesylate
Salt and Crystalline Sulfate Salt at 0 days and after storage at
40.degree. C. and 75% RH with the graphs overlaid for comparison.
As shown in FIG. 1, Crystalline Mono Hydrochloride Salt showed no
change in crystal form after storage. As shown in FIG. 4,
Crystalline Mesylate Salt also showed no change in crystal form
after storage. The changes shown in the figure are in intensity and
resolution rather than in peak position, which, if present, would
indicate change in crystal form. Accordingly, Crystalline Mono
Hydrochloride Salt and Crystalline Mesylate Salt are physically
stable, as shown by FIGS. 1 and 4. As shown in FIG. 7, Crystalline
Sulfate Salt showed increased crystalline content after
storage.
[0119] The results of HPLC-UV analysis for the same samples of
Crystalline Mesylate Salt and Crystalline Sulfate Salt are shown in
Table 4. As the .beta.-isomer content did not increase after
storage, these salts were not negatively affected by storage.
TABLE-US-00004 TABLE 4 Formation of .beta.-isomer in Crystalline
Mesylate Salt and Crystalline Sulfate Salt after Storage. Chemical
Purity Chemical Purity at 7 days, Sample at 0 days 40.degree. C.
and 75% RH Crystalline Mesylate Salt 92% (3% .beta.-isomer) 98% (2%
.beta.-isomer) Crystalline Sulfate Salt 88% (9% .beta.-isomer) 91%
(9% .beta.-isomer)
[0120] In addition, differential scanning calorimetry (DSC) and
thermo-gravimetric (TGA) analysis of Crystalline Mono Hydrochloride
Salt, Crystalline Mesylate Salt and Crystalline Sulfate Salt after
synthesis was conducted. The DSC curves are shown in FIGS. 2, 5 and
8 and the TGA curves are shown in FIGS. 3, 6 and 9. These figures
show that, by DSC analysis, there are no events up to degradation
of the salts, thereby, confirming stability of the salts at raised
temperatures. The TGA curves show that no hydrates or solvates were
present. The observed apparent weight loss is due to instability of
the machine.
[0121] Table 5 below compares the DSC and TGA analysis of the
crystalline salts of the present invention with the crystalline
free base and Comparative Example 1.
TABLE-US-00005 TABLE 5 DSC and TGA Analysis of Crystalline Mono
Hydrochloride Salt, Crystalline Mesylate Salt and Crystalline
Sulfate Salt, Crystalline Free Base and Comparative Example 1.
Differential Scanning Sample Calorimetry Thermo-Gravimetric
Analysis Crystalline Mono No events up to 3% weight loss from about
30.degree. C. to Hydrochloride Salt degradation about 200.degree.
C. 14% weight loss from about 200.degree. C. to about 250.degree.
C. Crystalline No events up to 3% weight loss from about 30.degree.
C. to Mesylate Salt degradation about 200.degree. C. 7% weight loss
from about 200.degree. C. to about 250.degree. C. Crystalline
Sulfate No events up to 3% weight loss from about 30.degree. C. to
Salt degradation about 200.degree. C. 14% weight loss from about
200.degree. C. to about 250.degree. C. Crystalline Free Endothermic
peak at 15% weight loss from about 30.degree. C. Base 175.degree.
C. (.DELTA.H 72 J g.sup.-1) to decomposition at less than
220.degree. C. Comparative Broad endothermic 7% weight loss from
about 30.degree. C. to Example 1 peak between about about
106.degree. C. 20.degree. C. and about 8% weight loss from about
106.degree. C. 200.degree. C. to about 200.degree. C. 23% weight
loss from about 200.degree. C. to about 300.degree. C.
[0122] Accordingly, Crystalline Mono Hydrochloride Salt,
Crystalline Mesylate Salt and Crystalline Sulfate Salt are more
stable than crystalline free base and Comparative Example 1.
Antimicrobial Activity
[0123] Antimicrobial activity of the Crystalline Mono Hydrochloride
Salt was assessed according to anti-anaerobic activity, mechanism
of action and in vivo efficacy studies as detailed herein. Whether
either amorphous bis hydrochloride salt or crystalline mono
hydrochloride salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide is used to prepare samples for these
studies, it is well understood in the art that efficacy data would
be the same for all salt forms, since the compound is placed into
solution prior to testing. Accordingly, whether starting with the
Crystalline Mono Hydrochloride Salt or amorphous bis hydrochloride
salt of
(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(me-
thyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene--
2-carboxylic acid amide, the free base (as defined above; herein
"the active") is what is being tested.
[0124] For the studies below, samples were prepared with bis
hydrochloride salt, and the data is expressed based on the free
base ("active"). The overall anti-anaerobic microbial activity of
the Crystalline Mono Hydrochloride Salt can be seen via in vitro
study of the active against 37 representative strains of anaerobic
bacteria and the results compiled in Table 6. The active
demonstrated relatively potent activity (i.e., minimum inhibitory
concentration (MIC) of 4 g/mL or less) against many species of
Gram-positive bacteria, including P. acnes. Overall, the activity
of the active was similar to that of tetracycline and doxycycline
but less than that of minocycline. Organisms with high MIC values
for the active (MIC.gtoreq.16 .mu.g/mL) included C. perfringens and
S. constellatus.
[0125] MIC values for the Gram-negative anaerobes are shown in
Table 6. The tetracycline-resistant strains were cross-resistant to
the active. The active and the other tetracyclines demonstrated
potent activity against E. corrodens and Fusobacterium spp.,
moderate activity against P. melaninogenica (1 of 2 strains) and V.
parvula, and poor activity against P. asaccharolytica.
TABLE-US-00006 TABLE 6 Summary of in vitro MIC testing of the
Active Against Anaerobic Gram-Positive and Gram-Negative Bacteria.
The Active TET DOX MIN Organism/ ATCC MIC MIC MIC MIC Micromyx No.
No. (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) Bifidobacterium
bifidum 15696 1 1 0.5 0.25 3965 Bifidobacterium brevi 15698 1 1 0.5
0.25 3967 Bifidobacterium infantis 15702 0.5 1 0.5 0.25 3966
Bifidobacterium longum 15707 4 2 1 1 3968 Clostridium perfringens
-- 16 >16 16 16 3414 Clostridium perfringens -- 16 >16 16 8
3518 Clostridium difficile -- 0.12 0.5 0.06 0.03 3579 Clostridium
difficile -- 0.12 0.5 0.06 0.03 3584 Lactobacillus -- 4 2 2 0.5
acidophilus 0681 Lactobacillus casei 1722 393 2 2 2 0.5
Lactobacillus plantarum 39268 2 2 2 0.5 2791 Peptostreptococcus --
2 8 2 1 anaerobius 3526 Peptostreptococcus -- 4 16 4 2 anaerobius
3531 Peptostreptococcus -- 0.25 0.25 0.12 0.06 micros 3432
Peptostreptococcus -- 1 1 0.5 0.25 micros 3545 Propionibacterium
acnes -- 0.25 0.25 0.12 0.06 1713 Propionibacterium acnes 11829 1 1
0.5 0.5 1286 Streptococcus 27823 32 >16 16 16 constellatus 1202
Streptococcus 27335 1 2 0.5 0.25 intermedius 1203 Bacteroides
fragilis 3374 -- 0.12 0.5 0.12 0.03 Bacteroides fragilis 3479 -- 16
>16 16 8 Bacteroides ovatus 3503 -- 8 >16 8 4 Bacteroides
ovatus 3508 -- 0.25 0.5 0.12 0.03 Bacteroides -- 0.25 1 0.25 0.03
thetaiotaomicron 3399 Bacteroides -- 16 >16 16 8
thetaiotaomicron 3496 Bacteroides vulgatus -- 16 >16 8 8 3389
Bacteroides vulgatus -- 16 >16 8 8 3494 Eikenella corrodens
43278 1 0.5 0.12 0.03 1206 Fusobacterium 25286 0.25 0.5 0.5 0.06
necrophorum 3963 Fusobacterium 25586 0.25 0.5 0.5 0.06 nucleatum
3962 Porphyromonas -- 16 >16 4 8 asaccharolytica 3552
Porphyromonas -- 8 16 2 4 asaccharolytica 3557 Prevotella -- 32
>16 16 16 melaninogenica 3437 Prevotella -- 4 8 1 1
melaninogenica 3443 Prevotella spp. 3564 -- 4 1 1 0.25 Prevotella
spp. 3568 -- 2 4 1 0.25 Veillonella parvula 1272 17745 4 1 1 0.5
"TET" is tetracycline; "DOX" is doxycycline; "MIN" is minocycline;
"ATCC" is American Type Culture Collection.
Antibacterial Spectrum of Activity
[0126] An assessment of the antibacterial spectrum of activity of
the active was determined in several studies by in vitro MIC
determination for a variety of Gram-positive and Gram-negative
aerobic and anaerobic organisms. The results of these assays
(summarized in Table 7) indicate that the active demonstrates
activity against propionibacteria and other Gram-positive organisms
with a narrower spectrum of activity than clinically-used
tetracyclines. Strains resistant to tetracycline are
cross-resistant to the active. The activity for each organism group
is discussed in the text that follows the table.
TABLE-US-00007 TABLE 7 Summary of In Vitro MIC testing against
propionibacteria and aerobic Gram-positive and Gram-negative
organisms MIC Organism [Type] (No. Range MIC.sub.50 MIC.sub.90
isolates) Compound (.mu.g/mL) (.mu.g/mL) (.mu.g/mL)
Propionibacteria P. acnes The Active 0.25-4 0.5 2 [tetS]
Tetracycline 0.5-4 0.5 4 (13) Doxycycline 0.25-1 0.25 1 Minocycline
.ltoreq.0.06->8 0.125 1 P. acnes The Active >8->8 [tetR]
Tetracycline >8->8 (2) Doxycycline 8-8 Minocycline 2-2 P.
acnes The Active 0.5-16 0.5 4 [clinical isolates] Tetracycline
0.5-32 1 2 (55) Doxycycline 0.25-16 0.5 2 Minocycline 0.125-8 0.25
1 Clindamycin .ltoreq.0.06-64 .ltoreq.0.06 4 Erythromycin
.ltoreq.0.06->128 .ltoreq.0.06 >128 P. acnes The Active
0.25-1 [tetS] Tetracycline 0.25-1 (2) Doxycycline 0.12-0.5
Minocycline 0.06-0.5 Clindamycin 0.06-0.25 Metronidazole
>32->32 Penicillin 0.03-0.5 Vancomycin 0.5-0.5 P. granulosum
The Active 1-1 [clinical isolates] Tetracycline 1-2 (3) Doxycycline
0.5-1 Minocycline 0.25-0.5 Clindamycin .ltoreq.0.06-.ltoreq.0.06
Erythromycin .ltoreq.0.06-.ltoreq.0.06 P. avidum The Active 1-4
[clinical isolates] Tetracycline 1-8 (4) Doxycycline 0.5-4
Minocycline 0.25-2 Clindamycin .ltoreq.0.06-0.5 Erythromycin
0.125-0.125 Gram-positive aerobic bacteria S. aureus The Active
.ltoreq.0.06-0.25 0.125 0.25 [tetS] Tetracycline .ltoreq.0.06-0.25
0.25 0.25 (20) Doxycycline .ltoreq.0.06-0.25 .ltoreq.0.06 0.25
Minocycline 0.125-0.5 0.25 0.5 S. aureus The Active 0.125-32 4 16
[tetR] Tetracycline 2-64 64 64 (10) Doxycycline 1-16 4 16
Minocycline 0.25-16 0.5 8 S. epidermidis The Active 0.12-2 0.25 2
[MSSE] Tetracycline 0.12-2 0.25 2 (31) Doxycycline 0.06-1 0.12 1
Minocycline 0.06-0.25 0.06 0.25 Erythromycin 0.12->32 0.25
>32 Clindamycin .ltoreq.0.03->32 0.12 >32 Oxacillin
0.06-0.25 0.12 0.25 Vancomycin 1-2 2 2 S. epidermidis The Active
0.25-2 0.5 2 [MRSE] Tetracycline 0.25->32 1 2 (32) Doxycycline
0.12-8 0.5 1 Minocycline 0.06-0.5 0.12 0.25 Erythromycin
0.12->32 >32 >32 Clindamycin 0.06->32 >32 >32
Oxacillin 0.5->32 32 >32 Vancomycin 1-2 2 2 S. pneumoniae The
Active .ltoreq.0.06-0.125 [tetS] Tetracycline .ltoreq.0.06-0.25 (5)
Doxycycline .ltoreq.0.06-0.125 Minocycline 0.25-0.25 S. pneumoniae
The Active 4-32 [tetR] Tetracycline 32-64 (5) Doxycycline 4-4
Minocycline 8-16 S. pneumoniae The Active .ltoreq.0.03-32 0.12 0.25
[PSSP] Tetracycline 0.06->32 0.12 0.25 (32) Doxycycline
0.03->16 0.06 0.12 Minocycline .ltoreq.0.015->16 0.06 0.12
Erythromycin .ltoreq.0.015->16 0.03 2 Clindamycin
.ltoreq.0.015->16 0.03 0.06 Penicillin .ltoreq.0.015-0.12
.ltoreq.0.015 0.06 Vancomycin 0.06-0.25 0.25 0.25 S. pyogenes The
Active 0.12-16 0.12 8 (32) Tetracycline 0.12-32 0.12 32 Doxycycline
0.06-8 0.12 4 Minocycline 0.03-8 0.06 8 Erythromycin 0.03->16
0.06 0.06 Clindamycin 0.03->16 0.03 0.06 Penicillin
.ltoreq.0.015-0.25 .ltoreq.0.015 .ltoreq.0.015 Vancomycin 0.25-0.5
0.25 0.25 S. pyogenes The Active .ltoreq.0.06-0.25 [tetS]
Tetracycline .ltoreq.0.06-0.125 (5) Doxycycline .ltoreq.0.06-0.125
Minocycline 0.25-0.5 S. pyogenes The Active 4-16 [tetR]
Tetracycline 32-64 (5) Doxycycline 4-8 Minocycline 4-8 S.
agalactiae The Active 0.12-32 16 16 (31) Tetracycline 0.12->32
32 >32 Doxycycline 0.06-16 8 16 Minocycline 0.03-16 16 16
Erythromycin 0.03->16 0.06 >16 Clindamycin 0.03->16 0.06
>16 Penicillin .ltoreq.0.015-2 0.03 1 Vancomycin 0.25-2 0.5 0.5
S. agalactiae The Active 0.125-0.25 [tetS] Tetracycline 0.25-0.25
(3) Doxycycline 0.25-0.25 Minocycline 0.5-0.5 S. agalactiae The
Active 16-32 [tetR] Tetracycline 16-64 (7) Doxycycline 8-16
Minocycline 8-16 S. haemolyticus The Active 0.12-2 0.12 2 (33)
Tetracycline 0.12->32 1 >32 Doxycycline 0.06-16 0.5 16
Minocycline .ltoreq.0.03-0.5 0.06 0.5 Erythromycin 0.12->32
>32 >32 Clindamycin 0.06->32 0.12 1 Oxacillin 0.06->32
0.25 >32 Vancomycin 0.5-2 1 1 Streptococcus The Active 0.12-16
0.25 16 spp. Tetracycline 0.12->32 0.25 32 [Group C] Doxycycline
0.06-16 0.12 8 (30) Minocycline 0.03-8 0.06 8 Erythromycin
.ltoreq.0.015->16 0.06 4 Clindamycin .ltoreq.0.015->16 0.06
0.12 Penicillin .ltoreq.0.015-0.03 .ltoreq.0.015 .ltoreq.0.015
Vancomycin 0.25-1 0.25 0.5 E. faecalis The Active
.ltoreq.0.06-.ltoreq.0.06 [tetS] Tetracycline 0.25-0.5 (4)
Doxycycline <0.06-0.125 Minocycline 0.25-0.5 E. faecalis The
Active 8-32 [tetR] Tetracycline 32-64 (6) Doxycycline 2-16
Minocycline 4-16 E. faecalis The Active 0.25-32 32 32 [VSE]
Tetracycline 0.25->64 32 64 (31) Doxycycline 0.12-16 8 8
Minocycline 0.06-16 8 16 Erythromycin 0.25->32 >32 >32
Clindamycin 4->32 >32 >32 Ampicillin 0.5-8 1 1 Vancomycin
0.5-4 1 2 E. faecium The Active .ltoreq.0.06-.ltoreq.0.06 [tetS]
Tetracycline 0.125-0.25 (4) Doxycycline .ltoreq.0.06-.ltoreq.0.06
Minocycline 0.25-0.5 E. faecium The Active 8-32 [tetR] Tetracycline
32-64 (6) Doxycycline 4-16 Minocycline 2-32 E. faecium The Active
0.12-32 0.5 32 [VSE] Tetracycline 0.12->64 1 >64 (32)
Doxycycline 0.06-32 0.5 16 Minocycline .ltoreq.0.03-16 0.12 16
Erythromycin 0.06->32 >32 >32 Clindamycin 0.12->32
>32 >32 Ampicillin 0.12->64 64 >64 Vancomycin 0.25-2 1
1 E. faecium The Active 0.12-32 2 32 [VRE] Tetracycline 0.12->64
2 >64 (30) Doxycycline 0.06-16 1 8 Minocycline .ltoreq.0.03-16
0.25 16 Erythromycin 0.12->32 >32 >32 Clindamycin
0.06->32 >32 >32 Ampicillin 8->64 >64 >64
Vancomycin >64 >64 >64 Gram-negative aerobic bacteria E.
coli The Active 4-32 [tetS] Tetracycline 1-4 (7) Doxycycline 0.5-4
Minocycline 0.5-4 E. coli The Active >64->64 [tetR]
Tetracycline >64->64 (3) Doxycycline 64-64 Minocycline 8-16
E. coli The Active 2->64 16 >64 (33) Tetracycline 1->64 2
>64 Doxycycline 0.5->32 2 32 Minocycline 0.25->32 1 8
Ampicillin 1->64 >64 >64 Ciprofloxacin 0.008->2 0.015
>2 Cephalothin 2->64 32 >64 Tmp/Sxt
.ltoreq.0.06/1.19->64/1216 0.25/4.75 >64/1216 K. pneumoniae
The Active 16-64 [tetS] Tetracycline 0.5-4 (7) Doxycycline 0.5-8
Minocycline 1-16 K. pneumoniae The Active >64->64 [tetR]
Tetracycline 8->64 (5) Doxycycline 16-64 Minocycline 16->64
K. pneumoniae The Active 16->64 >64 >64 (31) Tetracycline
1->64 8 >64 Doxycycline 1->32 8 >32 Minocycline
1->32 4 >32 Ampicillin >64 >64 >64 Ciprofloxacin
0.03->2 >2 >2 Cephalothin >64 >64 >64 Tmp/Sxt
0.12/2.38->64/1216 >64/1216 >64/1216 E. cloacae The Active
0.25->64 32 >64 (30) Tetracycline 0.5->64 2 >64
Doxycycline 0.06->32 2 32 Minocycline .ltoreq.0.03->32 1 16
Ampicillin 4->64 64 >64 Ciprofloxacin 0.008->2 0.25 >2
Cephalothin 2->64 >64 >64 Tmp/Sxt
.ltoreq.0.06/1.19->64/1216 0.25/4.75 >64/1216 P. mirabilis
The Active >64 >64 >64 (30) Tetracycline 16->64 32 64
Doxycycline 32->32 >32 >32 Minocycline 8->32 16 >32
Ampicillin 0.5->64 4 >64 Ciprofloxacin 0.015->2 >2
>2 Cephalothin 2->64 8 >64 Tmp/Sxt
.ltoreq.0.06/1.19->64/1216 2/38 >64/1216 P. aeruginosa The
Active 32->64 >64 >64 (11) Tetracycline 4->64 64 64
Doxycycline 4->32 >32 >32 Minocycline 8->32 >32
>32 Ampicillin >64 >64 >64 Ciprofloxacin 0.12->2
>2 >2 Cephalothin >64 >64 >64 Tmp/Sxt
2/38->64/1216 16/304 >64/1216 Salmonella The Active 8->64
16 >64 spp. Tetracycline 1->64 2 >64 (35) Doxycycline
2->32 2 32 Minocycline 1->32 2 8 Ampicillin 0.5->64 1
>64 Ciprofloxacin 0.015-0.25 0.015 0.03 Cephalothin 1->64 2
16 Tmp/Sxt .ltoreq.0.06/1.19->64/1216 .ltoreq.0.06/1.19->64/
0.12/2.38 1216 Abbreviations used in Table 7: tetS, tetracycline
sensitive; tetR, tetracycline resistant; VSE,
vancomycin-susceptible Enterococcus; VRE, vancomycin-resistant
Enterococcus; MSSE, methicillin-susceptible Staphylococcus
epidermidis; MRSE, methicillin-resistant Staphylococcus
epidermidis; PSSP, penicillin-susceptible Streptococcus
pneumoniae;
MIC, minimum inhibitory concentration; MIC.sub.50, MIC at which 50%
of isolates are inhibited; MIC.sub.90, MIC at which 90% of the
isolates are inhibited.
In Vitro Antibacterial Activity of the Active Against
Propionibacteria
[0127] The in vitro antimicrobial activity of the active against
the acne vulgaris pathogen P. acnes was assessed using the Clinical
and Laboratory Standards Institute (CLSI)-approved agar dilution
method for anaerobes. Susceptibility testing was performed by
measuring the MIC against a screening panel of P. acnes including
several macrolide-resistant strains. The values determined for the
active were compared with similar members of the tetracycline class
of antibiotics, including the clinically-used acne therapeutics,
doxycycline and minocycline (Table 7). Against a panel of 13
tetracycline-sensitive P. acnes, the active demonstrated MICs
comparable to doxycycline and minocycline.
[0128] In an expanded study, the active was tested against 62
recent clinical isolates of propionibacteria along with several
tetracycline comparators (Table 7). Against 55 strains of P. acnes
isolated within the past 7 years, including 26 isolates obtained
within the past 3 years, the active demonstrated activity similar
to that of doxycycline.
In Vitro Antimicrobial Activity of the Active Against
Staphylococci
[0129] Thirty strains of S. aureus were tested against the active
(Table 7) and conventional tetracyclines (tetracycline,
doxycycline, and minocycline). Typed strains with known resistance
mechanisms, ribosomal protection and active efflux, were included.
Activity was variable and compared to doxycycline and
minocycline.
In Vitro Antimicrobial Activity of the Active Against
Streptococci
[0130] Thirty strains of streptococci (10 each S. pyogenes, S.
agalactiae, and S. pneumoniae) were tested against the active and
conventional tetracyclines (tetracycline, doxycycline, and
minocycline). Typed strains with known resistance to tetracycline
and minocycline, characteristic of ribosomal protection mediated by
tetM or tetO, were included. Similar to staphylococci, MIC ranges
and MIC.sub.90 values (Table 7) were closest to minocycline and
doxycycline. The active showed good activity against susceptible
strains of S. pyogenes, with MICs comparable to doxycycline and
minocycline.
[0131] The active demonstrated a bimodal distribution of MIC values
for S. agalactiae with 7 strains inhibited at 0.25 .mu.g/mL or
less, and the remainder of the isolates requiring 16-32 .mu.g/mL
for inhibition. The elevated MIC values tracked with resistance to
tetracycline. The MIC.sub.50 and MIC.sub.90 values for the active
were similar to those of doxycycline and minocycline but less than
that of tetracycline.
[0132] The active demonstrated potent activity against PSSP with
all but 3 strains inhibited at 0.25 .mu.g/mL or less. The elevated
MIC values tracked with resistance to tetracycline. The activity
was similar to that of tetracycline, doxycycline and
minocycline.
[0133] The active demonstrated potent activity against S. pyogenes
with all but 6 strains inhibited at 0.25 .mu.g/mL or less. Elevated
MICs (.gtoreq.4 .mu.g/mL) were observed for 5 strains which tracked
with resistance to tetracycline. The MIC.sub.90 value for the
active was similar to those of doxycycline and minocycline but less
than that of tetracycline.
[0134] The active demonstrated a bimodal distribution of MIC values
for Group C streptococci with 19 strains inhibited at 0.25 .mu.g/mL
or less, and the remainder of the isolates requiring 4-16 .mu.g/mL
for inhibition. The elevated MIC values tracked with resistance to
tetracycline. The MIC.sub.50 and MIC.sub.90 values for the active
were similar to those of tetracycline but higher than those of
doxycycline and minocycline.
In Vitro Antimicrobial Activity of the Active Against
Enterococci
[0135] Twenty enterococcal strains (10 each E. faecalis and E.
faecium) were tested against the active and conventional
tetracyclines (tetracycline, doxycycline, and minocycline),
including 11 well characterized tetracycline-resistant strains
(Table 7). Unlike the staphylococci, the active was not active
against any tetracycline-resistant strains by efflux (mediated by
tetK or tetL) or by ribosomal protection (tetM or tetO). The active
did show activity against tetracycline-susceptible strains of E.
faecalis and E. faecium which was comparable to that of
doxycycline.
[0136] The active demonstrated a bimodal distribution of MIC values
for vancomycin-susceptible E. faecalis with 8 strains inhibited at
0.5 .mu.g/mL or less, and the remainder of the isolates requiring
16-32 .mu.g/mL for inhibition. The elevated MIC values tracked with
resistance to tetracycline. The MIC.sub.50 and MIC.sub.90 values
for the active were similar to those of tetracycline and greater
than those of doxycycline and minocycline.
[0137] The active also demonstrated bimodal distribution of MIC
values for vancomycin-susceptible E. faecium with 17 strains
inhibited at 0.5 .mu.g/mL or less, and the remainder of the
isolates requiring 16-32 .mu.g/mL for inhibition. The elevated MIC
values tracked with resistance to tetracycline. The MIC.sub.50 and
MIC.sub.90 values for the active were similar to those of
doxycycline and minocycline with an MIC.sub.90 value lower than
that of tetracycline.
[0138] The active demonstrated a broad range of MIC values for
vancomycin-resistant E. faecium. The elevated MIC values tracked
with resistance to tetracycline. The MIC.sub.50 value for the
active was similar to that of tetracycline and doxycycline and
8-fold higher than that of minocycline. The MIC.sub.90 value for
the active was lower than that of tetracycline and greater than
that of doxycycline and minocycline. A high percentage of strains
were resistant to the rest of the test agents.
In Vitro Antimicrobial Activity Against Gram-Negative Bacteria
[0139] Against 7 tetracycline-sensitive strains of E. coli, the
active was less active in vitro than doxycycline and minocycline
(Table 7). Even less activity for the active was observed against
tetracycline-sensitive strains of K. pneumoniae. In contrast,
doxycycline and minocycline demonstrated greater activity against
these organisms than the active. As expected, no activity for the
active was observed against 6 tetracycline resistant strains
demonstrating active efflux mediated by tetB or tetD.
[0140] The active had generally poor activity against E. cloacae
though a small number of strains (7 of 30) were inhibited at 1
.mu.g/mL or less. The MIC.sub.50 value was 16- to 32-fold higher
than those of the other tetracyclines. The MIC.sub.90 value for the
active was the same as tetracycline and higher than that of
doxycycline or minocycline.
[0141] The active was the least active of the tetracyclines against
E. coli with MIC.sub.50 and MIC.sub.90 values of 16 and >64
.mu.g/mL, respectively. The active was the least active of the
tetracyclines against K. pneumoniae. The active had generally poor
activity against Salmonella spp. and was the least active of the
tetracyclines.
Mechanism of Action
[0142] For the studies below, samples were prepared with bis
hydrochloride salt, and the data is expressed based on the free
base ("the active"). The mechanism of action of the Crystalline
Mono Hydrochloride Salt was determined by two different approaches
via study of the active, as described below.
[0143] In the first approach, Antibacterial Mechanism of Action: In
Vitro Inhibition of Bacterial Transcription and Translation, the
ability of the active to inhibit bacterial protein synthesis was
assessed using an in vitro cell-free bacterial transcription and
translation assay (commercially-available from Promega Corporation,
Madison, Wis.) (Beckler, G., Promega Notes 31 (1991) pp. 3-6). The
active inhibited the synthesis of reporter protein with an
IC.sub.50 of 8.3.+-.0.18 .mu.M. This value was comparable to the
IC.sub.50 values determined for the comparator tetracyclines,
doxycycline and minocycline (IC.sub.50 values of 4.7.+-.0.48 and
2.4 f 0.22 .mu.M, respectively). These results provide evidence
that the active functions as a classical tetracycline by inhibiting
bacterial protein synthesis.
[0144] In the second approach, Antibacterial Mechanism of Action:
Inhibition of Macromolecular Synthesis in Staphylococcus aureus,
the ability of the active to target bacterial protein synthesis was
further confirmed in a whole-cell assay of macromolecular synthesis
in the Gram-positive organism, S. aureus. The active inhibited, in
a dose-dependent manner, the incorporation of [.sup.3H]-leucine
into proteins of the growing organism within the concentration
range of 0.25-8 fold the MIC (0.063-2 .mu.g/mL). A maximum
inhibition of 80% was observed at 8-fold the MIC which was
comparable to the values obtained for the tetracycline comparators
doxycycline and minocycline. In contrast, the active at 8-fold the
MIC demonstrated less than 20% inhibition for the synthesis of cell
wall, DNA, RNA and lipid components of the test bacteria. The
results of this study indicate that the active acts as a selective
inhibitor of bacterial protein synthesis at concentrations
comparable to known tetracyclines.
[0145] The in vitro susceptibility studies described above included
tetracycline-resistant strains with characterized tetracycline
resistance genes. Strains were selected that harbored the most
common tetracycline resistance genes: efflux (tetK, tet38, tetL,
tetS, tetB, and tetD), ribosomal protection (tetM and tetO), as
well as P. acnes resistant by rRNA point mutation. The MIC values
for these selected strains demonstrated a degree of
cross-resistance between the active and other tetracyclines, as
shown in Table 8. The presence of a tetracycline resistance gene
increased the MIC of the active relative to susceptible strains
(with the exception of tetK in S. aureus), with MIC values similar
to those of doxycycline and/or minocycline, but generally lower
than those of tetracycline.
TABLE-US-00008 TABLE 8 Activity of the Active Against Bacterial
Strains with Characterized Tetracycline Resistance Mechanisms. The
Strain Active Doxycycline Minocycline PBS Mech./ MIC MIC MIC
Organism # Genotype (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) P. acnes 1073
16S rRNA >8 8 2 point mutation S. aureus 1739 tet38 4 2 0.5 E.
coli 669 tetB >64 64 16 K. pneumoniae 266 tetD >64 64 64 S.
aureus 1309 tetK 0.5 2 0.5 E. faecium 1323 tetK 8 4 2 E. faecalis
274 tetL 32 16 16 S. aureus 1310 tetM 8 16 4 S. pyogenes 792 tetM 4
4 4 S. agalactiae 897 tetM 16 8 16 S. pneumoniae 511 tetM 4 4 8 E.
faecalis 276 tetM 16 8 16 E. faecium 965 tetM 8 4 8 S. pyogenes 330
tetO 16 8 8 S. agalactiae 316 tetO 32 8 16 E. faecium 1324 tetO 16
4 2 E. faecalis 949 tetS 8 2 4
Animal Models of Infection
[0146] For the studies below, samples were prepared with bis
hydrochloride salt, and the data is expressed based on the free
base ("the active"). In vivo efficacy studies were conducted with
the active in three distinct animal infection models and one
inflammation model. By possessing comparable activity to the
active, the studies show: 1) anti-infective efficacy of the
Crystalline Mono Hydrochloride Salt compared to other commercially
available tetracycline acne medications (doxycycline and
minocycline) against representative Gram-positive pathogens with
similar in vitro susceptibility as P. acnes; and 2) the
anti-inflammatory activity of Crystalline Mono Hydrochloride
Salt.
[0147] The comparators were dosed at a concentration of 10 mg/mL in
a vehicle of sterile water and adjusted for percent of their active
moiety. All studies, except for the thigh wound and rat footpad
edema studies, were conducted in CD-1 male mice. The thigh wound
model was conducted using CD-1 female mice and the rat footpad
edema studies were conducted with Sprague-Dawley male rats.
[0148] Table 9 presents the data collected from the following three
studies, which evaluated the efficacy of the active.
[0149] The first study evaluated the active in an S. aureus
Systemic Intraperitoneal Challenge (IPC) Model. The objective was
to assess the in vivo activity of the active against a
Gram-positive organism in an acute infectious model compared to
commercially-available tetracycline treatments for acne vulgaris.
Bacterial cultures were prepared by growing tetracycline-sensitive
S. aureus RN450-1 overnight, then diluting with sterile
phosphate-buffered saline (PBS). For each experiment, a total of 30
mice were infected by injection into the intraperitoneal cavity
with 500 .mu.l of 7.5.times.10.sup.7 CFU in 5% sterile
bacteriological mucin. Four to five treatment groups of 5 mice each
were treated with a single injection of the active, doxycycline,
and/or minocycline at doses ranging from 0.01-0.5 mg/kg in sterile
water. The doses were administered subcutaneously (SC) at 1 hour
post infection. An additional infected group of 5 mice was included
as a negative control (untreated) group. For all studies but one, a
positive control group(s) was included (e.g., doxycycline,
minocycline, or ciprofloxacin). Mice were monitored for survival
for up to 7 days. Efficacy was determined by calculating the
PD.sub.50 at 48 hours post infection. PD.sub.50 (protective dose,
50%) is the dose required to achieve 50% survival. The PD.sub.50
values reported are a mean of 2-3 independent experiments for each
drug tested.
[0150] The active displayed potent activity against S. aureus
RN450-1 resulting in a PD.sub.50 of 0.25 mg/kg and demonstrated
activity similar to doxycycline against a representative
Gram-positive pathogen.
[0151] The second study, Efficacy of the Active in a S. aureus
Thigh Wound Infection Model, looked at the activity of the active
against a representative Gram-positive infection in a tissue-based
infection model. The efficacy of the active was studied against S.
aureus RN450-1 in a thigh wound model of immunocompromised mice. A
total of 40 mice (n=4-8 mice per group) were rendered neutropenic
by cyclophosphamide treatment four days before (150 mg/kg) and one
day before (100 mg/kg) infection. Bacterial cultures were prepared
by growing S. aureus RN450-1 overnight and diluting with sterile
PBS. An injection of 100 .mu.L of .about.1.times.10.sup.6 CFU/mL in
sterile PBS was injected into the thigh. Four groups of mice for
each drug received doses of 0.33, 1, 3, or 9 mg/kg intravenously at
2 and 6 hours post infection. An additional group of untreated mice
served as a negative control group. At 24 hours post infection, the
thighs were collected aseptically, homogenized, and plated to
enumerate bacterial load per thigh. Data are reported as ED.sub.50
values. ED.sub.50 (effective dose, 50%) is the dose required to
achieve a 2 log.sub.10 reduction in bacterial burden (colony
forming units [CFU]/whole organ) in the target organ compared to
untreated controls.
[0152] As shown in Table 9, the active demonstrated efficacy
equivalent to doxycycline, a commonly used treatments for acne
vulgaris, in the S. aureus thigh wound model.
[0153] The third study, Efficacy of the Active in an Acute S.
pneumoniae Respiratory Tract Infection (RTI) Model, demonstrated
activity of the active in an additional tissue-based infection
model compared to doxycycline. The active and doxycycline were
tested independently against an S. pneumoniae-induced acute
pneumonia in immunocompromised mice. In each experiment, 35 mice
were rendered neutropenic by an intraperitoneal (IP) injection of
cyclophosphamide four days before (150 mg/kg) and one day before
(100 mg/kg) infection. Tetracycline-sensitive S. pneumoniae PBS
1339 was grown on plates overnight, colonies collected, and
resuspended in sterile PBS. Mice were infected intra-nasally with
50 .mu.L of this bacterial suspension containing
.about.6.times.10.sup.6 CFU/mouse. At 2 hours post infection, 4 to
5 groups of 5 mice each were treated with a single intravenous (IV)
dose of the active or doxycycline at 5 (the active only), 10, 20,
40, or 80 mg/kg dissolved in sterile water. Each study also had an
untreated control group and a group that received a positive
control compound (e.g., vancomycin at 20 mg/kg IV). Efficacy was
determined by calculating the PD.sub.50 at 72 hours post
infection.
[0154] A single IV dose of the active exhibited activity in a
neutropenic, lethal S. pneumoniae RTI model at a PD.sub.50 of 4.66
mg/kg (as shown in Table 9). This dose was slightly lower than the
7.18 mg/kg dose required to achieve the PD.sub.50 for doxycycline.
The active demonstrated activity in this additional tissue-based
infection model that was comparable to, or slightly better than,
that of doxycycline.
TABLE-US-00009 TABLE 9 Efficacy Summary of the Active and
Comparators in Murine Infection Models. S. aureus S. aureus S.
pneumoniae RN450-1 IPC RN450-1 Thigh PBS1339 RTI Compound PD.sub.50
(mg/kg) ED.sub.50 (mg/kg) PD.sub.50 (mg/kg) The Active 0.25 8.23
4.66 Doxycycline 0.30 8.31 7.18 Minocycline 0.03 -- --
[0155] The study, In Vivo Efficacy of the Active in an Inflammation
Model of Rat Carrageenan-Induced Footpad Edema, was conducted to
evaluate the anti-inflammatory properties of the active compared to
minocycline and doxycycline. Groups of 3-8 rats were injected IP
with the active, doxycycline, minocycline, and/or saline control at
5 minutes preceding an injection of the inflammatory carrageenan
solution (1 mg/100 .mu.L) in the hind paw. Each study also had a
saline treated control group (3-8 rats/group). The active was
tested at 5, 10, 25, 50, 75, 100, or 150 mg/kg. Minocycline was
tested at 25, 50, 75, or 100 mg/kg and doxycycline was tested at 75
and 100 mg/kg. Immediately following the carrageenan injection and
3 hours post-injection, the hind paw volume was measured with a
digital water plethysmometer. Results were calculated as a percent
change in paw volume over the 3 hours, divided by the baseline paw
volume, and then adjusted for the mean percent inflammation in the
untreated controls, and are presented in Table 10. The active,
doxycycline, and minocycline all demonstrated anti-inflammatory
activity at all doses tested. The active exhibited
anti-inflammatory activity in a standard animal model of
inflammation comparable to other commercially-available
tetracyclines commonly used for the treatment of acne vulgaris.
TABLE-US-00010 TABLE 10 Mean inhibition of Inflammation by the
Active in a Carrageenan- Induced Rat Footpad Edema Model. Mean
Percent Inflammation Compared to Untreated Controls Compound 150
mg/kg 100 mg/kg 75 mg/kg 50 mg/kg 25 mg/kg 10 mg/kg 5 mg/kg 1 mg/kg
The Active 26 53 56 52 59 65 78 103 Doxycycline -- 36 68 -- -- --
-- -- Minocycline -- 21 54 33 47 -- -- --
[0156] Numerous alterations, modifications, and variations of the
preferred embodiments disclosed herein will be apparent to those
skilled in the art, and they are all anticipated and contemplated
to be within the spirit and scope of the claimed invention. For
example, although specific embodiments have been described in
detail, those with skill in the art will understand that the
preceding embodiments and variations can be modified to incorporate
various types of substitute, additional or alternative materials.
Accordingly, even though only few variations of the present
invention are described herein, it is to be understood that the
practice of such additional modifications and variations and the
equivalents thereof, are within the spirit and scope of the
invention as defined in the following claims. All patent
applications, patents, and other publications cited herein are
incorporated by reference in their entirety.
* * * * *