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Patent US20090131696 - Reducing Tetracycline Resistance in Living Cells - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsThe present invention provides an improved methodology by which therapeutically to overcome resistance to tetracycline in living cells including bacteria, parasites, fungi, and rickettsiae. The methodology employs a blocking agent such as C5 ester derivatives, or 6-deoxy 13-(substituted mercapto) derivatives...http://www.google.com/patents/US20090131696?utm_source=gb-gplus-sharePatent US20090131696 - Reducing Tetracycline Resistance in Living CellsAdvanced Patent SearchPublication numberUS20090131696 A1Publication typeApplicationApplication numberUS 12/194,362Publication dateMay 21, 2009Filing dateAug 19, 2008Priority dateNov 6, 1991Also published asUS6756365, US7414041, US7732429, US20030153537, US20040157807Publication number12194362, 194362, US 2009/0131696 A1, US 2009/131696 A1, US 20090131696 A1, US 20090131696A1, US 2009131696 A1, US 2009131696A1, US-A1-20090131696, US-A1-2009131696, US2009/0131696A1, US2009/131696A1, US20090131696 A1, US20090131696A1, US2009131696 A1, US2009131696A1InventorsStuart B. LevyOriginal AssigneeTrustees Of Tufts CollegeExport CitationBiBTeX, EndNote, RefManReferenced by (6), Classifications (14), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetReducing Tetracycline Resistance in Living CellsUS 20090131696 A1Abstract The present invention provides an improved methodology by which therapeutically to overcome resistance to tetracycline in living cells including bacteria, parasites, fungi, and rickettsiae. The methodology employs a blocking agent such as C5 ester derivatives, or 6-deoxy 13-(substituted mercapto) derivatives of tetracycline, in combination with other tetracycline-type antibiotics as a synergistic combination of compositions to be administered simultaneously, sequentially or concurrently. In another embodiment, certain novel compositions are provided which may be administered alone against, for example, a sensitive or resistant strain of gram positive bacteria such as S. aureus and E. faecalis. The concomitantly administered compositions effectively overcome the tetracycline resistant mechanisms present such that the cell is effectively converted from a tetracycline-resistant state to a tetracycline-sensitive state.
28. A 6-deoxy-13-(substituted mercapto) tetracycline compound having the formula
wherein A is hydroxyl,
29. The tetracycline compound of claim 1, wherein R is ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, cyclopentyl, hexyl, decyl, cyclohexyl, benzyl, p-chloro-benzyl, p-methyl-benzyl, or dihydroxypropyl.
30. The tetracycline compound according to claim 1, wherein R is decyl.
31. The tetracycline compound according to claim 1, wherein R is hexyl.
32. The tetracycline compound according to claim 1, wherein R is benzyl.
33. The tetracycline compound according to claim 1, wherein R is p-chloro-benzyl.
34. The tetracycline compound according to claim 1, wherein R is p-methyl-benzyl.
35. The tetracycline compound according to claim 1, wherein R is cyclopentyl.
36. The tetracycline compound according to claim 1, wherein R is butyl.
37. The tetracycline compound according to claim 1, wherein R is t-butyl.
38. The tetracycline compound according to claim 1, wherein R is isobutyl.
39. The tetracycline compound according to claim 1, wherein R is propyl.
40. The tetracycline compound according to claim 1, wherein R is isopropyl.
41. The tetracycline compound according to claim 1, wherein R is dihydroxypropyl.
42. The tetracycline compound according to claim 1, wherein R is ethyl.
43. The tetracycline compound according to claim 1, wherein R is cyclohexyl.
RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 09/504,897, filed on Feb. 16, 2000; which is a continuation of U.S. patent application Ser. No. 09/158,175, filed on Sep. 21, 1998; which is a continuation of U.S. patent application Ser. No. 08/722,977, filed on Sep. 30, 1996, now U.S. Pat. No. 5,811,412; which is a continuation of U.S. patent application Ser. No. 08/232,247, filed on Jun. 14, 1994, now U.S. Pat. No. 5,589,470; which is a continuation of PCT/US92/0895, filed on Oct. 16, 1992; which is a continuation-in-part of U.S. patent application Ser. No. 07/788,693, filed Nov. 6, 1991 now abandoned.
RESEARCH REPORT The research for the present invention was supported by funds obtained through Tufis University.
FIELD OF THE INVENTION The present invention concerns therapeutic tetracycline treatment of living cells, and is particularly directed to methods and materials for altering and overcoming resistance to tetracycline within microorganisms such as bacteria, fingi, rickettsia, and the like.
BACKGROUND OF THE INVENTION The development of the tetracycline antibiotics was the direct result of a systematic screening of soil specimens collected from many parts of the world for evidence of microorganisms capable of producing bacteriocidal and/or bacteriostatic compositions. The first of these novel compounds was introduced in 1948 under the name chlortetracycline. Two years later oxytetracycline became available. The detailed elucidation of the chemical structure of these agents confirmed their similarity and furnished the analytical basis for the production of a third member of this group in 1952, tetracycline. By 1957, a new family of tetracycline compositions characterized chemically by the absence of the ring-attached CH3 group present in the earlier compositions was prepared and became publicly available in 1959 under the official name demeclocycline. Subsequently, methacycline, a derivative of oxytetracycline, was introduced in 1966; doxycycline became available by 1967; and minocycline was in use by 1972. For clarity, for general ease of understanding, and for comparison purposes, these individual tetracycline type agents are structurally compared within Table I below.
�OH, �H; ═CH2 (5;6)
�H, �H; �N(CH3)2 (6;7)
As noted above, in another embodiment, certain novel compositions are provided which may be administered alone against, for example, a sensitive or resistant strain of gram positive bacteria such as S. aureus and E. faecalis. Examples of products of a tetracycline resistance determinant are Tet M, Tet 0 and Tet Q proteins for cytoplasmic protein products and Tet A, Tet B, Tet K and Tet L for membrane products.
wherein R1 and R2 are selected from the group consisting of a methylene group, hydroxyl, hydrogen or a group consisting of organic entities comprising from 1-12 carbon atoms, with or without other heteroatoms including sulfur, oxygen, halogen, nitrogen, and the like, and takes form as linear, branched, or cyclic alkyl, aryl, or alkylaryl structures; and A is selected from the group consisting of a hydrogen atom, a methylene group, and any linear, branched, or ring structure comprising from 1-6 carbon atoms and optionally including heteroatoms such as oxygen and nitrogen atoms. Certain C5 esters have been described by Bernardi et al. (Il Farmaco, Ed. Sc. vol. 29�fasc. 12, pages 902-909 (1974)) as being useful against, for example. S. aureus. Methods of synthesizing these disclosed derivatives may be found, for example, in U.S. Pat. No. 3,579,564, the disclosure of which is incorporated by reference herein.
In general, the synthesis of these 13-thio-substituted-5-acyl-6-deoxy-tetracyclines (hereinafter �13,5 derivatives) may be accomplished by the anti-Markovnikov radical addition of alkyl or aryl thiols to the 6,13 exocyclic double bond of methacycline by the method of Blackwood et al., J. Am. Chem. Soc., 85:3943 (1963) the disclosure of which is incorporated by reference herein, followed by esterification with an appropriate carboxylic acid in anhydrous HF according to the method of Bernardi et al., Il Farmaco Ed. Sc., 29:9022 (1974) the disclosure of which is incorporated by reference herein, as depicted in Scheme I below.
The present invention requires that at least one other composition which is not chemically a blocking agent, such as the above-described 6-deoxy-13-(substituted mercapto)tetracycline or C5 ester, be administered concurrently or simultaneously with the blocking agent to the cell. This additional administered composition is any �tetracycline-type antibiotic� currently known which includes tetracycline itself; or any member of the tetracycline family including all analogues and derivatives which are NOT C5 ester derivatives nor 13-carbon substituted mercaptan compounds. Accordingly, the broadest definition for the additional tetracycline, analogue, or derivative to be administered concurrently is defined by Formula III below.
SUSCEPTIBILITY TESTING OF E. coli Class A Tcr Class B Tcr Tcs (MI 308-226)
150 � 0.25
45 � 20 150 � 50
30 � 0.25
45 � 6 60
14 � 4 45 � 5
�0.26 Indicates that the same MIC or MLC was determined in two or more experiments. Other values represent experimental error determined by averaging the values obtained in multiple experiments. If no value is given, the experiment has not be repeated. Larger numbers will consistantly have larger errors since all experiments were done by the standard 1 ml perial dilution liquid MIC procedure.
SUSCEPTIBILITY TESTING OF S. aureus Tcs(RN450)
0.75 � 0.25 >6 90 � 10 100
0.2 � 0.1 10 � 2
SUSCEPTIBILITY TESTING OF E. faecalis Tcr (L)
Tcs (ATCC9790r)
3.5 � 1 18 � 2 Cyclohexyl*
10 � 4 1 � 0.5
Susceptible Strains 1. E. coli (Table E1, Column 1)
None of these compounds was more active than tetracycline or minocycline against susceptible E. coli strains. The most active was the ethyl-5-derivative which showed an MIC of 5 μg/ml. 2. S. aureus (Table E2, Column 1)
Against susceptible S. aureus, all of the 13-S-derivatives were effective alone within therapeutic ranges. They were about as active as tetracycline and minocycline (except perhaps the cyclohexyl derivative). All 13-S-derivatives showed bacteriocidal activity better than tetracycline or minocycline of which 4 showed bacteriocidal activity at a level of about 5 μg/ml. 3. E. faecalis (Table E3, Column 1)
Against susceptible Enterococcus faecalis, all the tested compositions were effective well within a therapeutic range and all, but the isopropyl derivative, at 1 μg/ml or less. All showed greater bacteriocidal activity than did tetracycline or minocycline, especially the benzyl, cyclohexyl, and cyclopentyl S-derivatives. Resistant Strains 1. All the other compositions were more active than tetracycline against resistant E. coli strains (both Class A and Class B determinants). None individually was as active as minocycline. Most 13-S-derivatives showed bacteriocidal activity lower than tetracycline against resistant E. coli, but not within therapeutic ranges (Table E1, Columns 2 and 3).
Chart 1: MIC/MLC (μg/ml) dosages
Chart 2: MIC/MLC (μg/ml) dosages
Chart 3: MIC/MLC (μg/ml) dosages
Chart 4: MIC/MLC (μg/mL) dosages
1. Against the tetracycline resistant (Class A) E. coli (strain D1-299) synergy was observed. The most effective analogues were cyclopentyl, cyclohexyl, and ethyl. These all inhibited growth at concentrations of 5 μg/ml or less of analogue and tetracycline. Synergy was also demonstrated in bacteriocidal activity, although the amounts of the 13-S-derivatives needed were higher than 5 μg/ml in order to kill 99.9% of the cells with 4-5 μg/ml of tetracycline. 2. Against tetracycline resistant S. aureus, all the 13-S-derivatives tested showed synergistic activity at levels of both drugs below 4 μg/ml. In addition, cyclohexyl>cyclopentyl>benzyl showed bacteriocidal activity within therapeutic combinations with tetracycline where the combined dose of the two drugs was ≦6 μg/ml to achieve MLC. 3. Against E. faecalis (Tet L), all four 13-S-derivatives showed excellent synergy in inhibiting growth in combination: <1 μg/ml of analogue with 1 μg/ml tetracycline. While bacteriocidal effects were seen synergistically, the amounts of drugs needed to produce the MLC were higher than each at 4-5 μg/ml. 4. Against E. faecalis (Class M) cyclopentyl, cyclohexyl, and benzyl S-derivatives showed little, if any synergistic activity with tetracycline. However, the propyl-5-derivative, while not as active alone, did show meaningful synergy. Chart 5: MIC/MLC (μg/ml) dosages
SUMMARY 1. These studies show that a group of S-alkyl substitutions and the benzyl substitution at the 13th carbon position of methacycline can inhibit growth of both susceptible and tetracycline resistant gram-positive (and to a less extent gram-negative) organisms.
IC50 Cmpd
R1 (μM)a 1
CHART 6 A, B
Analog 5-proprionate
Strain E. coli DI-299
(Tet A)
25 12.5
37.5 43.8
48.4 49.2
49.6 49.3 49.9 50.0 C
18.3 21.9
24.2 24.5
24.3 24.9 25.0 25.0 D
12.1 12.3
12.4 12.5 12.5 12.5 +
6.1 6.2
25.0 12.5
0.0 Control
Strain S. aureus 4250
(Tet K)
46.7 48.4 49.2 49.6 49.3 49.9 50.0 C
23.4 24.2 24.5 24.3 24.9 25.0 25.0 D
11.7 12.1 12.3 12.4 12.5 12.5 12.5 ↓
6.1 6.2 6.23
3.1 3.12
CHART 6 C, D
Strain E. faecalis 158
(Tet L)
49.2 49.6 49.3 49.9 50.0 C
24.5 24.3 24.9 25.0 25.0 D
12.3 12.4 12.5 12.5 12.5 E
6.2 6.23
Strain E. faecalis 211
(Tet M)
46.7 48.4 49.2 49.6 49.3 49.9 50.0 C
23.4 24.2 24.5 24.3 24.9 25.0 25.0 ↓
11.7 12.1 12.3 12.4 12.5 12.5 12.5 +
5.9 6.1 6.2 6.23
3.0 3.05
1.5 1.53
CHART 7 A, B
50 25 12.5
25.0 37.5
48.4 49.2 49.6 49.3 49.9 50.0 C
12.5 18.3
24.2 24.5 24.3 24.9 25.0 25.0 +
12.1 12.3 12.4 12.5 12.5 12.5 +
50.00 25.0
12.4 12.5 12.5 12.5 E
CHART 7 C, D
46.7 48.4 49.2 49.6 49.3 49.9 50.0
23.4 24.2 24.5 24.3 24.9 25.0 25.0
11.7 12.1 12.3 12.4 12.5 12.5 12.5
CHART 8 A, B
Analog 13-cyclopentyl-thio-5-
50 25 12.5
25.0 37.5 43.8
12.5 18.3 21.9
23.4 24.2 24.5 24.3 24.9 25.0 25.0 ↓
11.7 12.1 12.3 12.4 12.5 12.5 12.5 +
50.00 25.0 12.5
49.3 49.9 50.0 C
24.9 25.0 25.0 D
12.5 12.5 12.5 E
CHART 8 C, D
49.3 49.9 50.0
24.9 25.0 25.0
12.5 12.5 12.5
12.5 12.5 12.5 +
CHART 9 A, B
Analog 13-propyl-thio
5-proprionate
46.7 48.4 49.2
23.4 24.2 24.5
11.7 12.1 12.3
Analog 13-propyl-thio-
49.3 49.9
50.03 C
24.9 25.0
25.0 D
12.5 12.5
12.5 +
CHART 9 C, D
12.4 12.5 12.5 12.5 ↓
49.6 49.3 49.9
24.3 24.9 25.0
12.4 12.5 12.5
Example 1 Synthesis of 13-propylthio-5-hydroxy-6-α-deoxy-tetracycline Methacycline hydrochloride (5.0 g, 10.4 mmol) was placed in a round-bottom flask and suspended in 100 mL of EtOH. Twenty mL of propanethiol (16.8 g, 0.270 mol) and AIBN 250 mg, were added and the reaction mixture refluxed with stirring for 12 h while under N2. The mixture was reduced to ⅕ volume by distillation and filtered. The filtrate was dripped slowly into cold Et2O while stirring resulting in the formation of a yellow precipitate. The precipitate was filtered, dissolved in H2O and brought to pH 4.5 with 1.0 M NaOH. This solution was filtered, and extracted with CH2Cl2 yielding a dark yellow solid (620 mg). The solid was dissolved in MeOH and treated with charcoal yielding a yellow solid in low yield (25%, 256 mg) mp=130-140� C. (dec.). TLC rf=0.70 (I); HPLC Rt=20.18 min. 1HNMR (DMSO-d6) δ 7.50 (t, 1H), 7.05 (d, 1H), 6.85 (d, 1H), 4.32 (d, 2H), 3.15 (s, 1H), 2.65 (s, 6H), 2.32-2.52 (m, 2H), 1.51-1.80 (m, 2H), 0.9-1.22 (m, 3H); HRMS (FAB); calc for C25H30N2O8S 519.1801 (M+1). found 519.1815 (M+1).
Example 2 Synthesis of 13-cyclopentylthio-5-hydroxy-6-α-deoxy-tetracycline This compound was prepared substantially as described in Example 1. Purification was either by column chromatography on EDTA silica, extraction pH 4.5 into CH2Cl2, or by HPLC chromatography. An analytical sample was produced by HPLC as a yellow solid of mp=132-139� C. (dec.) in moderate yield (28.3%). Higher yields were obtained by the extraction method and treatment with activated charcoal in MeOH (32.1%); TLC rf=0.80 (I); HPLC Rt=21.19 min. 1HNMR (MeOH-d4) δ 7.38 (t, 1H), 7.02 (d, 1H), 6.72 (d, 1H), 4.10 (s, 2H), 2.70 (br s, 6H), 1.81-2.01 (br m, 2H), 1.28-1.75 (br m, 6H), (br m, 2H); HRMS (FAB); calc for C27H32N2O8S 545.1957 (M+1). found 545.1960 (M+1).
Referenced byCiting PatentFiling datePublication dateApplicantTitleUS7732429Aug 19, 2008Jun 8, 2010Trustees Of Tufts CollegeReducing tetracycline resistance in living cellsUS7935687Apr 14, 2008May 3, 2011Paratek Pharmaceuticals, Inc.Methods for treating spinal muscular atrophy using tetracycline compoundsUS8012951Jan 21, 2009Sep 6, 2011Trustees Of Tufts College7-N-substituted phenyl tetracycline compoundsUS8088820Oct 24, 2003Jan 3, 2012Paratek Pharmaceuticals, Inc.Substituted tetracycline compounds for the treatment of malariaUS8293931Jun 30, 2011Oct 23, 2012Trustees Of Tufts College7-N-substituted phenyl tetracycline compoundsUS8399437May 3, 2011Mar 19, 2013Paratek Pharmaceuticals, Inc.Methods for treating spinal muscular atrophy using tetracycline compoundsClassifications U.S. Classification552/203International ClassificationC07C237/26, A61K31/65, C07C323/29, C07D295/13Cooperative ClassificationA61K31/65, C07C2101/08, C07C323/29, C07C237/26, C07D295/13European ClassificationC07C323/29, A61K31/65, C07D295/13, C07C237/26Legal EventsDateCodeEventDescriptionJun 8, 2014LAPSLapse for failure to pay maintenance feesMar 14, 2014ASAssignmentOwner name: HBM HEALTHCARE INVESTMENTS (CAYMAN) LTD., AS COLLAFree format text: SECURITY INTEREST;ASSIGNOR:PARATEK PHARMACEUTICALS, INC.;REEL/FRAME:032448/0001Effective date: 20140307Jan 17, 2014REMIMaintenance fee reminder mailedMar 8, 2013ASAssignmentOwner name: MINTZ LEVIN COHN FERRIS GLOVSKY AND POPEO PC, MASSFree format text: NOTICE;ASSIGNOR:PARATEK PHARMACEUTICALS, INC.;REEL/FRAME:029940/0106Effective date: 20130308Apr 27, 2010ASAssignmentOwner name: TRUSTEES OF TUFTS COLLEGE,MASSACHUSETTSFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LEVY, STUART B.;US-ASSIGNMENT DATABASE UPDATED:20100427;REEL/FRAME:24293/936Effective date: 20100326Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LEVY, STUART B.;REEL/FRAME:24293/936Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LEVY, STUART B.;REEL/FRAME:024293/0936RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google