Reducing tetracycline resistance in living cells

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 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, C5 ester 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.

RESEARCH REPORT 
The research for the present invention was supported by funds obtained 
through Tufts University. 
1. 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, fungi, rickettsia, and the like. 
2. 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 CH.sub.3 
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. 
TABLE I 
______________________________________ 
TETRACYCLINE 
##STR1## 
At Carbon 
Congener Substituent(s) Position Nos. 
______________________________________ 
Chlortetracycline 
Cl (7) 
Oxytetracycline 
OH, H (5) 
Demeclocycline 
OH, H; Cl (6; 7) 
Methacycline 
OH, H; CH.sub.2 (5; 6) 
Doxycycline 
OH, H; CH.sub.3, H 
(5; 6) 
Minocycline 
H, H; N(CH.sub.3).sub.2 
(6; 7) 
______________________________________ 
Subsequent to these initial developments, much research effort was focused 
on developing new-tetracycline antibiotic compositions effective under 
varying therapeutic conditions and routes of administration; and for 
developing new tetracycline analogues which might prove to be equal or 
more effective than the originally introduced tetracycline families 
beginning in 1948. Representative of such developments are U.S. Pat. Nos. 
3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280; 
4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. It will be 
understood that these issued patents are merely representative of the 
range of diversity of investigations seeking tetracycline and tetracycline 
analogue compositions which are pharmacologically active. 
Historically, soon after their initial development and introduction, the 
tetracyclines regardless of specific formulation or chemical structure 
were 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. Hence, tetracyclines became known as "broad spectrum" 
antibiotics. With the subsequent establishment of their in-vitro 
antimicrobial activity, effectiveness in experimental infections, and 
pharmacological properties, the tetracyclines as a class rapidly became 
widely used for therapeutic purposes. However, this widespread use of 
tetracyclines for both major and minor illnesses and diseases led directly 
to the emergence of resistance to these antibiotics even among highly 
susceptible bacterial species both commensal and pathogenic--as for 
example pneumococci and Salmonella. The rise of tetracycline-resistant 
organisms has led not only to a general decline in use of tetracyclines 
and tetracycline analogue compositions as antibiotics of choice, but has 
also launched major efforts and investigations to uncover the mechanism 
for tetracycline resistance--in the hope that some effective means might 
be developed to overcome the problem of tetracycline-resistance and thus 
reestablish the pharmacological value and efficacy of tetracyclines as a 
whole. 
The following represents a current summary of the investigations and 
knowledge regarding the mechanism of action for tetracyclines in bacteria. 
The principal site of action for tetracyclines is the bacterial ribosome; 
at least two different processes appear to be required for tetracyclines 
to gain access to the cytoplasm and the ribosomes of bacteria. The first 
process is a passive diffusion of the tetracycline through hydrophilic 
pores located in the outer cell membrane. One of these structures is the 
major outer membrane protein, Omp F in E. coli. The second process 
involves an energy-dependent active transport system that pumps all 
tetracyclines through the inner cytoplasmic membrane into the cytoplasm of 
the cell. In the tetracycline-sensitive cell or organism, once the 
tetracycline gains access to the interior of the cell, it is able to bind 
to the ribosomes and inhibit protein synthesis. However, in many 
tetracycline resistant cells and organisms, an efflux pump system is 
present which appears to bind the tetracycline molecule and actively 
transports the tetracycline molecule out of the organism into the 
surrounding environment. This active efflux employs an inner membrane 
protein designated TET (or Tet) protein which is synthesized in the cell 
from a gene which is generally acquired by the organism. Often the gene is 
present on an extra-chromosomal, autonomously replicating plasmid or a 
transposon. 
Tetracycline resistance is often regulated--that is, inducible by 
tetracycline. Investigations of active tetracycline efflux systems and the 
details of the active efflux mechanism of action have been well documented 
and include the following publications, each of which is expressly 
incorporated by reference herein: Chopra et al., J. Antimicrobiol. 
Chemotherapy 8:5-21 (1981); Levy and McMurry, Biochem. Biophys. Res. Comm. 
56:1060-1068 (1974); Levy and McMurry, Nature 275:90-92 (1978); McMurry 
and Levy, Antimicrobial Agents And Chemotherapy 114:201-209 (1978); 
McMurry et al., Proc. Nat. Acad. Sci. U.S.A. 77:3974-3977 (1980); Ball et 
al., Biochem. Biophys. Res. Comm. 93:74-81 (1980); Curiale and Levy, J. 
Bact. 151:209-2115 (1982); Mendez et al., Plasmid 3:99-108 (1980); Curiale 
et al., J. Bact. 157:211-217 (1984); and Levy, S. B., Journal of 
Antimicrobial Chemotherapy 24:1-3 (1989). 
In addition, a second mechanism of tetracycline resistance for cells is 
known and in effect. This resistance mechanism involves a cytoplasmic 
protein which protects the intracellular ribosomes from the inhibitory 
action of tetracyclines. This form of tetracycline resistance is described 
within Burdett, V., J. Bact. 165:564-569 (1986); and Levy, S. B., J. 
Antimicrob. Chem. 24:1-3 (1989). 
With the increased understanding and knowledge regarding the origin and the 
mechanisms of tetracycline resistance in various cells and microorganisms, 
active investigations and developments seeking means for overcoming these 
mechanisms, notably the active efflux system have been attempted. One 
successful approach is described within U.S. Pat. No. 4,806,529 issued 
Feb. 21, 1989--an innovation which is a precursor of more recent 
developments, namely U.S. Pat. No. 5,064,821 issued Nov. 12, 1991. 
Clearly, additional methods and materials for overcoming 
tetracycline-resistance in bacteria and other organisms are most desirable 
and needed. Substantive advances which additionally overcome the active 
efflux system for tetracycline and/or the ribosomal protection mechanism 
in the resistant cell would be presently recognized by the ordinary 
practitioner in the art as a major asset and innovation. 
SUMMARY OF THE INVENTION 
The present invention provides methods and compositions for therapeutically 
treating a tetracycline-resistant cell and also provides a method for 
altering a cell from a tetracycline-resistant state into a 
tetracycline-sensitive state. In one preferred embodiment, this method 
comprises the steps of: administering to the cell a predetermined quantity 
of at least a first composition selected from the chemical group 
consisting of a blocking agent which is capable of interacting with, e.g. 
binding to, a product of at least one tetracycline resistance determinant 
capable of protecting ribosomes in the cell from tetracycline's inhibitory 
activity; and 
concomitantly administering to the cell a pre-determined quantity of at 
least a second composition selected from the chemical group consisting of 
tetracycline, tetracycline analogues, and tetracycline derivatives which 
are not said blocking agent. The cell is allowed to preferentially react 
with the blocking agent. 
The unique methodology is able to alter and to convert 
tetracycline-resistant cells or microorganisms into tetracycline-sensitive 
ones; and, accordingly, to provide a therapeutic treatment for those 
living subjects, human, animal, and plants, which have been previously 
refractory to a tetracycline therapeutic regimen. 
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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention represents a unique methodology by which to overcome 
the increasing resistance of many different varieties of cells and 
microorganisms to the antibiotic activity of tetracyclines, their 
analogues and derivatives. The present invention takes into account and 
acts upon the existence of specific DNA sequences, which are typically 
found on plasmids and transposons, and which specify proteins for 
tetracycline-resistance determinants. Some of these determinants act via 
an active efflux system which maintains an intracellular tetracycline 
concentration below those levels able to inhibit protein synthesis within 
the microorganism such as described in above-mentioned U.S. Pat. No. 
4,806,529. Other determinants act by protecting the ribosome from 
tetracycline's inhibitory activity, e.g. by binding with tetracycline. The 
present invention represents improvement in efficacious and reliable 
techniques for overcoming tetracycline resistance in living cells and thus 
for reestablishing tetracyclines as an antibiotic of choice in the 
treatment of infectious diseases caused by the ever-increasing variety and 
diversity of disease agents. The invention relies on the action of a 
blocking agent which is capable of interacting with a product of at least 
one tetracycline resistance determinant which acts by protecting the cell 
from tetracycline's inhibitory activity. The determinant is capable of 
making a product, such as a cytoplasmic protein, which interacts with the 
ribosomes to make them tetracycline resistant or a membrane protein which 
keeps tetracycline out of the cell. 
The present invention is intended for use with tetracycline-resistant cells 
or organisms which are found to contain or carry a product of the genetic 
determinants responsible for tetracycline resistance, and in particular, 
those which are due to protection of the ribosome from the inhibitory 
activity of tetracycline. As described within the recent publication of 
Levy, S. B., Journal of Antimicrobial Chemotherapy 24:1-3 (1989), the text 
of which is expressly incorporated by reference herein, more than a dozen 
different distinguishable tetracycline resistance determinants have been 
uncovered [Levy, S. B., "Resistance to the Tetracyclines," in 
Antimicrobial Drug Resistance, (Bryan, L. E., editor), Academic Press, 
Orlando, Fla., 1984, pages 191-204; Levy, S. B., ASM News 54:418-421 
(1988)]. As these genetic determinants of these tetracycline-resistant 
cells have been elucidated, it has become generally accepted that the same 
or very similar genes are responsible for resistance in a large number of 
different aerobic and anaerobic microorganisms. 
The present invention is therefore believed suitable for use with at least, 
but not exclusively, the following genera: Gram-negative genera, in 
particular Enterobacteriaceae, which harbor Class A-E tetracycline 
resistance determinants; Gram-positive genera including streptococci, 
staphylococci, and bacillus species which bear the Class K and L 
tetracycline resistance determinants; aerobic and anaerobic microorganisms 
bearing the Class M, O or Q determinants represented by Streptococcus 
agalactiae, Bacteroides, Enterococcus, Gardnerella and Neisseria species, 
Mycoplasma and Ureaplasma, and Clostridium; Clostridium perfringens 
bearing the Class P tetracycline-resistant determinant. 
It will be recognized and appreciated that the above listed organisms are 
themselves only representative and illustrative of the range, variety, and 
diversity of cell types, bacterial species, fungi, parasites, and 
rickettsial disease agents which may be therapeutically treated using the 
present methodology. It will be expressly noted that no specific class, 
genus, species, or family of cell, microorganism, or parasite is excluded; 
to the contrary, it is expected that with future investigations into the 
determinants responsible for tetracycline resistance, ever greater numbers 
of different cells will be recognized as suitable for efficacious 
treatment using the present invention. In addition, in view of the recent 
use of tetracyclines for treatment of neoplasms, it is deemed that the 
present methodology would be useful in such therapies [van der Bozert et 
al., Cancer Res. 48:6686-6690 (1988)]. 
The present invention represents a major improvement over presently known 
methods for dealing with tetracycline resistance within disease-causing 
cells and organisms. In one preferred embodiment, the methodology requires 
only two essential steps: the administration to the tetracycline-resistant 
cell of a predetermined quantity of at least a first composition selected 
from the chemical group consisting of a blocking agent which is capable of 
interacting with a product of at least one tetracycline resistance 
determinant which is capable of protecting ribosomes in the cell from 
tetracycline's inhibitory activity; and 
concomitantly administering to the cell a pre-determined quantity of at 
least a second composition selected from the chemical group consisting of 
tetracycline, tetracycline analogues, and tetracycline derivatives which 
are not said blocking agent. 
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 O and Tet Q proteins for cytoplasmic protein products and Tet A, Tet 
B, Tet K and Tet L for membrane products. 
The resistance mechanism of the cell is allowed to preferentially react 
with the blocking agent so avoiding preferential reaction with the second 
administered composition which is the tetracycline, a tetracycline 
analogue or derivative composition. 
Clearly, therefore, it is recognized and understood that in this preferred 
embodiment two different compositions are to be administered concurrently, 
sequentially or simultaneously to the tetracycline-resistant cell. 
Moreover, it will be noted that the methodology requires and relies upon a 
preferential binding and reaction with the administered blocking agent 
in-situ; and consequently demonstrate a substantial lack of attraction or 
preference for the other administered tetracycline composition, analogue, 
or derivative present in-situ. The operation, utility, and efficacy of the 
present methodology is thus based upon an empirically demonstrable 
preference of the tetracycline-resistant cell for one class of composition 
over another when both classes of composition are introduced 
concomitantly--that is, concurrently, sequentially or simultaneously to 
the resistant cell. 
To date, there is no basis, system, or technique which can be employed to 
accurately predict which of two similar tetracycline formulations and 
chemical structures would be preferentially reactive with the resistance 
systems of cells. Earlier investigations as described within U.S. Pat. No. 
4,806,529 issued Feb. 21, 1989, have demonstrated that when tetracycline 
[i.e., 
4-(Dimethylamino)-1,4,4a,5,5a,6-11,12a-octahydro-3,6,10,12,12a-pentahydrox 
y-6-methyl-1-11-dioxo-2-naphth acenecarboxamide] is administered 
concurrently or simultaneously with other tetracycline analogues and 
derivatives such as minocycline or thiatetracycline, it is not actively 
effluxed from the cell and consequently enters tetracycline-resistant 
cells. Further studies have demonstrated that 13-thiol derivatives of 
methacycline are able to block the efflux protein and inhibit the 
resistance mechanism in both gram negative and gram positive cells, 
including different mechanisms of resistance, namely efflux and ribosome 
protection (See U.S. Pat. No. 5,064,821). The present invention expands 
upon these earlier investigations in substantial degree. It also provides 
the user with novel blocking agents which unexpectedly have been found to 
show very high inhibition of the mechanisms for ribosome protection as 
well as efflux. 
In one embodiment of the invention, the blocking agent is a tetracycline 
analogue which contains a sufficient part of tetracycline to interact with 
a product of at least one tetracycline resistance determinant capable of 
protecting cells from tetracycline's inhibitory activity. 
One specific class of blocking agents is the class of 13-(substituted 
mercapto) tetracyclines of the formula (Formula I): 
##STR2## 
wherein A is selected from the group consisting of hydrogen and a hydroxyl 
group; 
B 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; and 
R is selected from the 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. 
These 13-(substituted mercapto) tetracyclines are known in the art as 
tetracyclines possessing antimicrobial activity in and of themselves 
against a variety of gram-positive bacteria. This class of tetracyclines, 
its conventionally recognized pharmacological activity, and methods for 
its synthesis are described within U.S. Pat. No. 3,165,531, the text of 
which is expressly incorporated by reference herein. 
In one embodiment, the preferred compositions, as empirically demonstrated 
hereinafter, are S-substituted alkyl derivatives at the No. 13 carbon 
ranging from 1-10 carbon atoms in length. Nevertheless, it is expected 
that a wide variety of RCO, RCX where X is a halogen, RHC.sub.2, and 
NRHC.sub.2 analogue and derivative forms in linear, branched, or cyclic 
structural format would be useful and operative in the present methodology 
in varying degrees. Accordingly, all such embodiments are deemed to be 
within the scope of Formulation I above. 
As representative examples of the preferred embodiments of this class which 
were empirically evaluated, some preferred 6-deoxy-13-(substituted 
mercapto) tetracyclines (hereinafter "13-S-Derivatives") and their 
respective blocking activities are provided within Table II below. The 
K.sub.i represents the relative inhibitory activity of each compound. The 
lower the K.sub.i, the more activity against the efflux protein. The 
preferred compound will have a lower K.sub.i than tetracycline (i.e., 
lower than about 4-8 .mu.g/ml). 
TABLE II 
______________________________________ 
BLOCKING ACTIVITY OF 13-S-DERIVATIVES OF 
METHACYCLINE 
Number of 
13-S-Derivatives 
Carbon Atoms 
K.sub.i (.mu.g/ml).sup.1 
______________________________________ 
Decyl 10 8.0 
Hexyl 6 3.1 
Cyclohexyl 6 0.4 
Benzyl 7 0.9 
p-Cl-Benzyl 7 1.5 
p-Me-Benzyl 8 1.2 
Cyclopentyl, 5 0.5 
2-morpholinomethyl 
Cyclopentyl 5 0.2 
Butyl 4 0.5 
t-Butyl 4 0.3 
Isobutyl 4 0.1 
Propyl 3 0.4 
Isopropyl 3 0.4 
Dihydroxypropyl 
3 3.9 
Ethyl 2 0.4 
______________________________________ 
.sup.1 By everted membrane vesicle assay. 
From this representative listing, it will be noted that the shorter chain 
length substitutions or smaller adducts (cyclohexyl vs. hexyl; isobutyl 
vs. butyl; benzyl vs. parachlorobenzyl) are preferred inhibitors of the 
efflux system. Also, substitutions at the C2 position have only a small 
effect on the blocking activity. These results lead to a general 
conclusion that the activity of compositions having substitutions at the 
13th carbon relate more to the size of the molecule than to the charge 
despite the presence of the sulfur atom. The longer chain length 
substitutions at the 13th carbon atom (e.g., decyl and hexyl) are not as 
active as the shorter length substitutions (e.g., butyl, propyl, and 
ethyl). Furthermore, the dihydroxypropyl derivative behaves more poorly in 
the blocking assay than the propyl or isopropyl derivative forms. On this 
basis, therefore, it is expected that a most preferred composition would 
be one having mercapto-substitutions on the 13th carbon atom in which the 
elipsoidal volume of the substituent joined to the sulfur atom is in the 
approximate size range of that provided by the butyl, benzyl or 
cyclopentyl derivatized structures. 
Moreover, the data of Table II suggest that the administration to a 
resistant cell of a 13-substituted mercaptan derivative or a composition 
which appears structurally similar to a 13-substituted mercaptan 
derivative would effectively block the resistance mechanism of the cell; 
and allow the concomitant administration of another tetracycline, 
tetracycline analogue, or tetracycline derivative to effectively inhibit 
further cell growth. 
In another embodiment, the blocking agent which can be employed in 
practicing the present invention is the class of C5 esters of 
tetracyclines of the formula (Formula II): 
##STR3## 
wherein R.sub.1 and R.sub.2 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 yet another embodiment, hybrids of the above-described 
6-deoxy-13(substituted mercapto) and C5 ester may be employed as the 
blocking agent against resistant gram negative strains or alone against 
resistant gram positive strains. 
In general, the synthesis of these 
13-thio-substituted-5-acy-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. 
##STR4## 
FORMULATION, STRUCTURE, AND RANGE OF OTHER TETRACYCLINES, TETRACYCLINE 
ANALOGUES, AND TETRACYCLINE DERIVATIVE FORMS 
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. 
##STR5## 
wherein R.sub.1 -R.sub.5 may be a hydrogen atom, a halogen atom, a 
hydroxyl group, or any other organic composition comprising from 1-8 
carbon atoms and optionally include a heteroatom such as nitrogen, oxygen, 
in linear, branched, or cyclic structural formats. A very wide range and 
diversity of embodiments within the definition of Formula III as are 
described within Essentials of Medicinal Chemistry, John Wiley and Sons, 
Inc., 1976, pages 512-517, the text of which is expressly incorporated by 
reference herein. Preferably R.sub.1 and R.sub.2 are hydrogen or a 
hydroxyl group; R.sub.3 is a hydrogen or a methyl group; R.sub.4 is a 
hydrogen atom, a halogen, or a nitrogen containing entity; and R.sub.5 is 
a hydrogen atom, or a nitrogen containing ring structure. The commonly 
known tetracycline analogues and derivatives including the following: 
oxytetracycline; chlortetracycline; demeclocycline; doxycycline; 
chelocardin; minocycline; rolitetracycline; lymecycline; sancycline; 
methacycline; apicycline; clomocycline; guamecycline; meglucycline; 
mepyclycline; penimepicycline; pipacycline; etamocycline; and 
penimocycline. It will be recognized and appreciated that these specific 
tetracycline compositions (as well as many others conventionally known and 
available through the scientific literature or from commercial sources) 
may be employed as the alternative tetracycline-type composition which 
does not contain a C5 ester nor a 13-carbon substituted mercapto group as 
part of its formulation and chemical structure. 
The individual compositions embodying Formula I, 13-S-derivatives, or 
Formula II, C5 esters, or the 13,5 derivative, and Formula III, 
alternative tetracycline compounds, can be administered concurrently, 
sequentially or simultaneously in any appropriate carrier for oral, 
topical or parenteral administration. It is also possible that the two 
discrete compositions could be linked covalently or otherwise joined to 
each other and/or to other ligands. These compositions can be introduced 
by any means that affects an infectious or disease state caused by 
tetracycline-resistant microorganisms in humans and/or animals. The 
specific route of administration, the choice of carrying materials, and 
the particular means for introducing each composition concomitantly to the 
tetracycline-resistant cells are of no major importance or relevance. 
Accordingly, if the 13-S-derivative, the C5 derivative composition or the 
13,5 derivative and the other alternative tetracycline-type compound are 
to be applied topically, they can be individually or mutually admixed in a 
pharmacologically inert topical carrier such as a gel, an ointment, a 
lotion, or a cream. Such topical carriers include water, glycerol, 
alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid 
esters, or mineral oils. Other possible 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 may also be added if and 
when necessary. 
Similarly, if the 13-S-derivative, the C5 derivative composition or the 
13,5 derivative and the alternative tetracycline-type composition are to 
be introduced concurrently, sequentially or simultaneously in parenteral 
form, each composition will be prepared individually or in combination in 
sterile form; in multiple or single dose formats; and be dispersed in a 
fluid carrier such as sterile physiological saline or 5% dextrose 
solutions commonly used with injectables. 
Furthermore, if the present methodology is to be employed for oral 
administration, each of the two requisite compositions may be provided 
individually or in combination in the form of prepared capsules, cachets, 
or tablets each containing a predetermined quantity of the 
13-S-derivative, the C5 ester composition or the 13,5 derivative and the 
tetracycline-type antibiotic. Their preparation may also take form as a 
powder or granules; or dissolved or suspended in a solution or suspension 
within an oil-in-water emulsion or conversely within a water-in-oil liquid 
emulsion for ingestion or for oral cavity lavage treatments. These solid 
or liquid formulations may generally include one or more carrier materials 
such as flavoring agents, binders, buffers, diluents, surface active 
agents, thickeners, lubricants, preservatives, and the like. It is deemed 
that all of these methods for formulating, preparing, and administering 
the requisite compositions are conventionally known. 
The effective dosages to be employed in vivo are typically dictated by the 
intended application or use circumstances; and are generally decided by 
reconciling several different factors. First, it will be recognized and 
appreciated that each embodiment of the 13-S-derivative, the C5 derivative 
composition or the 13,5 derivative and each embodiment of the alternative 
tetracycline composition (analogue or derivative) will have individual 
specific pharmacological activity which can be represented and evaluated 
as the Minimal Inhibitory Concentration (hereinafter "MIC") and as the 
Minimal Lethal Concentration (hereinafter "MLC")--each of which varies 
with its specific formulation and chemical structure. Second, any given 
specific chemical formulation will also have varying MIC and MLC dosages 
which fluctuate with the cell type--as, for example, with the genus and 
species of microorganism; thus, the MIC and MLC of each individual 
composition will vary markedly--as, for example, when administered to 
gram-positive bacteria in comparison to gram-negative bacteria or to the 
various different genera of fungi, rickettsia, and parasites. Thirdly, the 
degree of tetracycline resistance is known to very substantially among the 
different cell types, their delineated genera, and among the different 
species comprising a single genus; this varying degree of tetracycline 
resistance is without regard to whether the mechanism of resistance is 
based upon an active efflux system or a ribosome protection system 
intracellularly. Lastly, each specific route of in vivo administration is 
conventionally recognized to require markedly different dose concentration 
of conventionally known tetracycline compounds; accordingly, in vivo 
therapeutic dosages will vary depending upon whether the tetracycline-type 
composition is given orally, parenterally, or topically. Each of these 
individual factors should be taken in consideration by the user when 
deciding the proper dosage or concentration for the 13-S-derivative, the 
C5 derivative composition or the 13,5 derivative and the other 
tetracycline antibiotic composition. 
In general, however, it is most desirable that the dosage and concentration 
of the 13-S-derivative, the C5 derivative composition (broadly defined by 
Formula I or Formula respectively) or the 13,5 derivative be administered 
in a subinhibitory quantity--that is, less than the minimum inhibitory 
quantity--that is, less than the minimum inhibitory concentration or the 
minimum lethal concentration for that specific composition when employed 
against a tetracycline-resistant cell. In comparison, it is essential that 
the chosen alternative tetracycline-type composition (tetracycline 
analogue or tetracycline derivative meeting the broad definitional 
requirements of Formula III above) be employed in at least a minimum 
inhibitory concentration; and preferably be administered at an effective 
dosage to provide a minimal lethal concentration in-situ. Accordingly, it 
is deemed that the concentrations for the two concomitantly administered 
compositions are conventionally known within the art; and can be 
optimalized with a minimum of difficulty. 
Again, as noted above, certain novel compounds, in particular, the 13,5 
derivatives, have been found to be particularly useful against certain 
gram positive bacteria when administered alone. 
In this context, and as empirically demonstrated by the data of Charts 1-5 
for the 13-S-derivatives, Charts 6-7 for the C-5 derivatives and Charts 
8-9 for the 13,5 derivatives which follow hereinafter, the concomitant 
administration of the 13-S-derivative, the C5 derivative composition or 
the 13,5 derivative and the other tetracycline-type composition together 
provides not only means for overcoming tetracycline resistance but also 
offers the capability to enhance the pharmacological activity of the known 
tetracycline-type composition to exert cidal activity and cidal effects 
upon the cell. Contrary to the universally accepted conventional view that 
tetracyclines, regardless of formulation, are only bacteriostatic 
agents--i.e., agents that do not kill but only inhibit future growth, the 
present method provides a synergistic combination of compositions which 
enhances the antibiotic activity of the tetracycline-type composition; 
and, for the first time, allows the enhanced tetracycline-type composition 
to exert bacteriocidal powers, "cidal" capability, i.e., the ability to 
kill the cell rather than merely inhibit its growth, against a broad 
spectrum of bacteria. 
In addition, the general molar ratio of 13-S-derivative, the C5 derivative 
composition, or the 13,5 derivative to alternative tetracycline-type 
composition is expected generally to be from 0.01:100.0, and is preferably 
in the range from 0.05:2.0. It is most desirable, however, that in no 
instance should the dosage of the 13-S-derivative, the C5 derivative 
composition or the 13,5 derivative be employed in a concentration which is 
within the MIC or MLC values. In comparison, the alternative, 
tetracycline-type composition (tetracycline or tetracycline analogue or 
tetracycline derivative) should be administered in accordance with 
conventional practice for the efficacious therapeutic treatment of 
infection or disease in humans and/or animals. Accordingly, for 
therapeutic purposes, the daily dosage of 13-S-derivative, the C5 
derivative composition or the 13,5 derivative for treatment of disease in 
living mammals is expected to lie in the range from 0.01-100 mg/kg 
(preferably from 15 to 30 mg/kg) of normal body weight while the dosage of 
the other tetracycline, analogue or derivative should continue to be given 
in the range from 500 milligrams to 2.0 grams per day depending upon the 
age, weight, and route of administration. When the 13,5 derivative is 
administered alone, for example, for treating infections caused by gram 
positive bacteria such as S. aureus or E. faecalis, the dosage employed is 
preferably that used in conventional tetracycline therapy. 
It will also be understood that the normal, conventionally known, 
precautions will be taken regarding the administration of tetracyclines 
generally in order to ensure their efficacy under normal use 
circumstances. Especially when employed for therapeutic treatment of 
humans and animals in vivo, the practitioner should take all sensible 
precautions to avoid conventionally known contradictions and toxic 
effects. Thus, the conventionally recognized adverse reactions of 
gastrointestinal distress and inflammations, the renal toxicity, 
hypersensitivity reactions, changes in blood, and impairment of absorption 
through aluminum, calcium, and magnesium ions should be duly considered in 
the conventional manner. 
MODE AND MANNER OF PHARMACOLOGICAL ACTIVITY 
It must be emphasized again that the present methodology is useful with all 
cells, regardless of type, source, family, genus, or species which have 
genetic determinants for tetracycline resistance. The methodology of the 
present invention is suitable for use with both tetracycline resistance 
attributable to an active efflux transport system utilizing one or more 
TET proteins which actively bind with tetracycline-type antibiotics and 
transport the tetracycline composition out of the cytoplasm of the cell; 
and also with tetracycline resistance which is a nonefflux system and 
typically involves a ribosome protection mechanism which causes a 
tetracycline antibiotic to fail to inhibit protein synthesis 
intracellularly. Regardless of which tetracycline resistance mechanism is 
present within the resistant cell, the present methodology is effective in 
overcoming tetracycline resistance and in rendering the cell 
tetracycline-sensitive. Although the sequence of molecular reactions 
remains far from understood at the present time, the concurrent or 
simultaneous administration of at least one 13-S-derivative or the C5 
derivative composition prepared in accordance with Formula I or Formula II 
or the 13,5 derivative and at least one other tetracycline antibiotic 
composition in accordance with Formula III causes an in situ conversion of 
the cell from a resistant state into a tetracycline-sensitive state. 
The efficacy and utility of the present methodology is based upon the 
cell's unexpected preferential reaction with the 13-S-derivative, the C5 
derivative composition or the 13,5 derivative which is desirably present 
in a subinhibitory concentration; and the comparable absence of avidity by 
the cell for the other tetracycline-type composition concomitantly 
administered. The resistance mechanism of the cell--be it the active 
efflux system or the ribosome protection system--focuses upon and 
interacts with the 13-S-derivative, the C5 derivative composition or the 
13,5 derivative primarily and predominantly; the concurrent or 
simultaneous presence of the other tetracycline-type antibiotic 
composition is relatively ignored and effectively overlooked by the 
tetracycline-resistance mechanism of the cell. 
Consequently, the other tetracycline-type antibiotic composition is allowed 
to accumulate intracellularly in at least a minimum inhibitory 
concentration (and preferably in a minimum lethal concentration); and this 
other tetracycline-type antibiotic is able to bind to the ribosomes and to 
exert its recognized pharmacological activity intracellularly to prevent 
further protein synthesis within that cell. In this respect, the 
administered 13-S-derivative of Formula I or C5 derivative composition of 
Formula II or 13,5 derivative is clearly the preferred composition for 
reaction with the tetracycline-resistance mechanism present; and by this 
preferred reactivity, acts as a blocking agent to engage and to divert the 
tetracycline resistance mechanism of that cell to the extent that the 
concurrently or simultaneously administered other tetracycline-type 
antibiotic composition of Formula III is able to exert its characteristic 
pharmacological activity efficaciously against the cell and to prevent 
further protein synthesis intracellularly. The present methodology is thus 
effective and useful by the cell's own preference for engagement and 
reaction with the 13-S-derivative, the C5 derivative composition or the 
13,5 derivative, rather than with the concomitantly administered other 
tetracycline-type antibiotic. In this manner, the cumulative effect is to 
render the cell tetracycline-sensitive for therapeutic purposes. 
EXPERIMENTAL STUDIES 
A series of experiments and resulting empirical data demonstrate and 
evidence the efficacy and utility of the present invention. These 
experiments will illustrate the essential components of the present 
methodology, and will demonstrate the value of the preferred embodiments 
comprising 13-S- or C5 derivative compositions prepared in accordance with 
Formula I, or Formula II and document the range and diversity of some 
tetracycline-resistant microorganisms which can be rendered sensitive to 
tetracycline therapy by employing the present methodology. 
For purposes of conducting the experimental model, tetracycline for the 
13-S-derivative or doxycycline for the C5 derivative was employed 
uniformly in combination with a variety of different C5 or 13 substituted 
mercapto-tetracyclines. Nevertheless, it will be understood that 
doxycycline and tetracycline are employed merely as a representative of 
all the different compositions and embodiments of tetracyclines, 
tetracycline analogues, and tetracycline derivatives conforming to the 
definition of Formula III given previously; and that the present invention 
is not limited to the use of tetracycline alone as a specific chemical 
formulation and structure. Moreover, it will be understood that the 
experiments and empirical data presented hereinafter are merely 
illustrative examples of the present invention without regard to specific 
applications or particular uses; and that the described experiments are 
merely representative of the totality of embodiments encompassed within 
the scope of the present invention. 
EXPERIMENTAL SERIES 1 
Initially, the inhibitory effects of a variety of different 
13-S-derivatives in comparison to tetracycline and minocycline were 
examined using a variety of different bacteria. These included 
tetracycline sensitive (hereinafter "Tc.sup.s ") and tetracycline 
resistant (hereinafter "Tc.sup.r ") strains of E. coli, S. aureus, and E. 
faecalis. The general protocol for performing these experiments is as 
follows: Cultures were grown up fresh in L broth in the morning from an 
overnight culture. After 4-6 hours of growth, each bacterial culture was 
diluted to an A.sub.530 of 0.2-0.5 depending on the strain (E. coli, 0.5; 
S. aureus, 0.4; E. faecalis, 0.2). Individual tubes, containing 1 ml of L 
broth and different concentrations of 13-S-derivatives, were inoculated 
with the different bacterial cultures and then incubated at 37.degree. C. 
After 17-18 h of incubation, the concentration of each 13-S-derivative at 
which no observed cloudiness was seen was called the minimal inhibitory 
concentration (MIC). The minimal lethal concentration (MLC), i.e., that 
concentration which kills 99.9%, was based on the number of bacteria 
initially inoculated into the assay tubes. Those culture tubes showing no 
bacterial growth after incubation at 37.degree. C. were evaluated for the 
number of bacteria remaining. 
The results obtained in this experimental series are provided by Tables 
E1-E3 below. 
TABLE E1 
__________________________________________________________________________ 
SUSCEPTIBILITY TESTING OF E. coli 
Class A Tc.sup.r 
Class B Tc.sup.r 
Tc.sup.s (MI308-225) 
(DI-299) (DI-209) 
Drug MIC (.mu.g) 
MLC (.mu.g) 
MIC (.mu.g) 
MLC (.mu.g) 
MIC (.mu.g) 
MLC (.mu.g) 
__________________________________________________________________________ 
Tetracycline 
0.6 .+-. 0.25 
40 .+-. 0.25 
160 .+-. 0.25 
200 &gt;200 &gt;200 
Minocycline 
&lt;4 &lt;4 &lt;4 20 6 .+-. 2 
30 .+-. 10 
Benzyl* 
16 .+-. 6 
25 .+-. 5 
46 .+-. 20 
160 .+-. 50 
30 .+-. 0.25 
90 .+-. 10 
Cyclohexyl* 
60 .+-. 10 
120 100 .+-. 0.25 
&gt;200 ND ND 
Cyclopentyl* 
20 60 40 .+-. 0.25 
80 80 .+-. 40 
100 .+-. 50 
Propyl* 
30 .+-. 5 
60 .+-. 10 
40 .+-. 0.25 
60 60 80 
Isopropyl* 
22 .+-. 2 
60 46 .+-. 6 
60 35 .+-. 5 
46 .+-. 16 
Ethyl* 5 .+-. 0.25 
60 14 .+-. 4 
46 .+-. 6 
35 .+-. 15 
60 .+-. 10 
__________________________________________________________________________ 
*Note: .+-.0.25 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 
consistently have larger errors since all experiments were done by the 
standard 1 ml serial dilution liquid MIC procedure. 
ND = not done. 
Tc.sup.s = tetracycline sesnsitive strain. 
Tc.sup.r = tetracycline resistant strain. 
.sup.a Smercapto derivative of methacycline. 
TABLE E2 
______________________________________ 
SUSCEPTIBILITY TESTING OF S. aureus 
Tc.sup.s (RN450) 
Tc.sup.r (RN4250) 
Drug MIC (.mu.g) 
MLC (.mu.g) 
MIC (.mu.g) 
MLC (.mu.g) 
______________________________________ 
Tetracycline 
0.75 .+-. 0.25 
&gt;6 90 .+-. 10 
100 
Minocycline 
&lt;0.25 8 &lt;0.25 &gt;80 
Benzyl 0.2 .+-. 0.1 
10 .+-. 2 1 .+-. 0.25 
10 .+-. 4 
Cyclohexyl 
2.5 .+-. 1.25 
10 .+-. 5 1.5 .+-. 0.5 
10 .+-. 4 
Cyclopentyl 
1 5 2 .+-. 0.25 
6 .+-. 2 
Propyl 0.5 .+-. 0.25 
5 4 .+-. 0.25 
16 .+-. 4 
Isopropyl 
0.5 .+-. 0.25 
6 .+-. 2 4.5 .+-. 0.5 
8 
Ethyl 0.5 .+-. 0.25 
4 5 .+-. 2 
30 .+-. 10 
______________________________________ 
Tc.sup.s = tetracycline sensitive strain 
Tc.sup.r = tetracycline resistant strain 
TABLE E3 
__________________________________________________________________________ 
SUSCEPTIBILITY TESTING OF E. faecalis 
Tc.sup.r (L) 
Tc.sup.r (M) 
Tc.sup.s (ATCC9790r) 
ATCC9790r/TelL) 
(ATCC09790r/TelM) 
Drug MIC (.mu.g) 
MLC (.mu.g) 
MIC (.mu.g) 
MLC (.mu.g) 
MIC (.mu.g) 
MLC (.mu.g) 
__________________________________________________________________________ 
Tetracycline 
0.25 &gt;200 90 .+-. 10 
&gt;300 100 300 
Minocycline 
&lt;0.25 &gt;40 &lt;0.25 &gt;80 10 &gt;80 
Benzyl* 
0.5 .+-. 0.25 
8 .+-. 0.25 
0.75 .+-. 0.25 
8 .+-. 2 
3.5 .+-. 1 
18 .+-. 2 
Cyclohexyl* 
1.25 .+-. 0.25 
8 1.5 .+-. 0.5 
8 .+-. 2 
2.5 .+-. 0.5 
10 .+-. 0.25 
Cyclopentyl* 
1 .+-. 0.25 
10 .+-. 4 
1 .+-. 0.5 
18 .+-. 2 
3 .+-. 1 
&gt;16 
Propyl* 
1.6 .+-. 0.25 
40 .+-. 0.25 
2.5 .+-. 0.5 
60 .+-. 10 
16 .+-. 2 
30 .+-. 10 
Isopropyl* 
3 .+-. 1 
20 .+-. 0.25 
3 .+-. 1 
100 .+-. 0.25 
22 .+-. 2 
&gt;200 
Ethyl* 1 .+-. 0.5 
40 .+-. 0.25 
4 .+-. 1 
100 .+-. 0.25 
25 .+-. 5 
&gt;200 
__________________________________________________________________________ 
Tc.sup.s = tetracycline sensitive strain. 
Tc.sup.r = tetracycline resistant strain. 
A close inspection and reading of Tables E1-E3 will reveal the following 
points regarding the tetracycline susceptible strains and the tetracycline 
resistant strains tested. These are: 
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-S-derivative which showed an MIC of 5 .mu.g/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 
.mu.g/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 .mu.g/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). 
2. Against resistant S. aureus, all the tested compounds showed an MIC 
within a therapeutic range, at least 20-100 fold more active than 
tetracycline. None individually was as active as minocycline. All 
13-S-derivatives were more bacteriocidal than tetracycline or minocycline 
alone with cyclopentyl showing an MLC of 6.+-.2 .mu.g/ml. Benzyl, 
cyclohexyl, and cyclopentyl S-derivatives each showed similar MIC values 
and MLC values against susceptible and resistant S. aureus. The most 
active 13-S-derivative was the cyclopentyl form (Table E2, Column 2). 
3. All the tested compositions had an MIC within a therapeutic range 
against E. faecalis bearing the Tet L determinant: 
benzyl&gt;cyclopentyl&gt;cyclohexyl, followed by the others. The 
13-S-derivatives were equally effective by MIC against susceptible and Tet 
L containing Enterococcus. All were more bacteriocidal than tetracycline 
and minocycline individually; the MLC for benzyl and cyclohexyl was 8.+-.2 
.mu.g/ml (Table E3, Column 2). 
4. Against Tet M containing E. faecalis, all the other tested compounds 
were considerably more antibacterial than tetracycline. Three of them, the 
benzyl, cyclohexyl, and cyclopentyl derivatives also had MIC values below 
minocycline and within therapeutic levels (Table E3, Column 3). 
Bacteriocidal activity was observed, but above therapeutic levels. 
5. While the MLC against resistant S. aureus and E. faecalis was 8-10 
.mu.g/ml for the most active drugs, a killing effect (seen as a 10-99% 
drop in cell viability) by the analogues occurred at considerably lower 
drug concentrations (see charts). 
EXPERIMENTAL SERIES 2 
Subsequently, another series of experiments was conducted which employed 
concurrent administrations of tetracycline and at least one other 
13-S-derivative composition in accordance with Formula I above. The 
general experimental protocol for synergy studies followed substantially 
that procedure employed for the standard MIC and MLC assays. The organisms 
were grown in fresh L broth and inoculated in culture tubes containing 
different concentrations of 13-S-derivative compositions and tetracycline 
together. The previously described methods for determining MIC and MLC 
were otherwise followed. 
Accordingly, the results are provided by Charts 1-5 in which: Chart 1 
represents the concurrent administration of 13-cyclopentyl sulfide 
derivative of methacycline in varying proportional ratios to tetracycline; 
Chart 2 represents 13-propyl-sulfide derivatives of methacycline varying 
proportional ratios with tetracycline; Chart 3 represents varying 
proportional ratios with tetracycline; Chart 3 represents varying 
proportional ratios of 13-cyclohexyl-sulfide derivatives of methacycline 
and tetracycline administered concurrently; Chart 4 represents varying 
proportional ratios of 13-benzyl-sulfide derivatives of methacycline 
delivered concurrently with tetracycline; and Chart 5 illustrates the 
concurrent administration of varying proportions of 13-ethyl-sulfide 
derivatives of methacycline and tetracycline. 
CHART 1 
__________________________________________________________________________ 
MIC/MLC (.mu.g/ml) dosages for tetracycline-resistant strains 
using cyclopentyl-sulfide derivatives of methacycline with and without 
tetracycline 
##STR6## 
##STR7## 
__________________________________________________________________________ 
Note: 
* Growth; 
0 No Growth (MIC); 
.dwnarw. Killing; 
##STR8## 
Tc = tetracycline concentration (.mu.g/ml). 
A = 6deoxy-13-(cyclopentyl mercapto) tetracycline concentration (.mu.g/ml 
CHART 2 
__________________________________________________________________________ 
MIC/MLC (.mu.g/ml) dosages for tetracycline-resistant strains 
using propyl-sulfide derivatives of methacycline with and without 
tetracycline 
##STR9## 
##STR10## 
__________________________________________________________________________ 
Note: 
* Growth; 
0 No Growth (MIC); 
.dwnarw. Killing; 
##STR11## 
Tc = tetracycline concentration (.mu.g/ml). 
B = 6deoxy-13-(propyl mercapto) tetracycline concentration (.mu.g/ml) 
CHART 3 
__________________________________________________________________________ 
MIC/MLC (.mu.g/ml) dosages for tetracycline-resistant strains 
using cyclohexyl-sulfide derivatives of methacycline with and without 
tetracycline 
##STR12## 
##STR13## 
__________________________________________________________________________ 
Note: 
* Growth; 
0 No Growth (MIC); 
.dwnarw. Killing; 
##STR14## 
Tc = tetracycline concentration (.mu.g/ml). 
C = 6deoxy-13-(cyclohexyl mercapto) tetracycline concentration (.mu.g/ml) 
CHART 4 
__________________________________________________________________________ 
MIC/MLC (.mu.g/ml) dosages for tetracycline-resistant strains 
using benzyl-sulfide derivatives of methacycline with and without 
tetracycline 
##STR15## 
##STR16## 
__________________________________________________________________________ 
Note: 
* Growth; 
0 No Growth (MIC); 
.dwnarw. Killing; 
##STR17## 
Tc = tetracycline concentration (.mu.g/ml). 
D = 6deoxy-13-(benzyl mercapto) tetracycline concentration (.mu.g/ml) 
CHART 5 
__________________________________________________________________________ 
MIC/MLC (.mu.g/ml) dosages for tetracycline-resistant strains 
using ethyl-sulfide derivatives of methacycline with and without 
tetracycline 
##STR18## 
##STR19## 
__________________________________________________________________________ 
Note: 
* Growth; 
0 No Growth (MIC); 
.dwnarw. Killing; 
##STR20## 
Tc = tetracycline concentration (.mu.g/ml). 
E = 6deoxy-13-(ethyl mercapto) tetracycline concentration (.mu.g/ml) 
As evidenced by the data of Charts 1-5, the results of administering 
13-S-derivative tetracycline compositions concurrent with varying 
proportional ratios of tetracycline clearly support the following 
conclusions: 
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 
.mu.g/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 .mu.g/ml in order to kill 99.9% 
of the cells with 4-5 .mu.g/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 
.mu.g/ml. In addition, cyclohexyl&gt;cyclopentyl&gt;benzyl showed bacteriocidal 
activity within therapeutic combinations with tetracycline where the 
combined dose of the two drugs was .ltoreq.6 .mu.g/ml to achieve MLC. 
3. Against E. faecalis (Tet L), all four 13-S-derivatives showed excellent 
synergy in inhibiting growth in combination: &lt;1 .mu.g/ml of analogue with 
1 .mu.g/ml tetracycline. While bacteriocidal effects were seen 
synergistically, the amounts of drugs needed to produce the MLC were 
higher than each at 4-5 .mu.g/ml. 
4. Against E. faecalis (Class M) cyclopentyl, cyclohexyl, and benzyl 
S-derivatives showed little, if any synergistic activity with 
tetracycline. However, the propyl-S-derivative, while not as active alone, 
did show meaningful synergy. 
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. 
2. In combination with tetracycline, all of these 13-S-derivatives show 
synergy, both in growth inhibition and in bacteriocidal activity for 
gram-positive as well as gram-negative susceptible and resistant strains. 
3. All of the 13-S-derivatives tested show bacteriocidal activity, although 
this is most evident against the gram-positive bacteria tested alone and 
in synergy with tetracycline, and against E. coli in synergy with 
tetracycline. 
4. All the 13-S-derivatives tested alone show greater bacteriocidal 
activity than minocycline against S. aureus and E. faecalis, chiefly the 
benzyl, cyclohexyl, and cyclopentyl derivatives. 
EXPERIMENTAL SERIES 3 
Everted vesicles, to which the TET proteins responsible for tetracycline 
efflux are attached, provide a reliable method for measuring efflux from 
tetracycline-resistant bacteria. By exposing the vesicles to different 
concentrations of tetracycline and measuring the amount of tetracycline 
taken up by the vesicle, the affinity of tetracycline for the efflux 
system may be determined. Similarly, exposure of the vesicles to solutions 
having both tetracycline and a potential efflux protein blocking agent 
produces a competition between tetracycline and the blocking agent for the 
limited number of binding states on the TET proteins. Subsequent 
measurement of the tetracycline concentration within the everted vesicles 
thus provides a sound measurement of the function of the bacterial efflux 
system for tetracycline in the presence of the potential blocking agent. 
This assay has also identified agents which affect resistance specified by 
other mechanisms, namely by a cytoplasmic protein which protects ribosomes 
from the inhibition of tetracycline (see U.S. Pat. No. 5,064,821). 
In the experiments, 0.5 mg/ml of everted membrane vesicles of E. coli 
strain D1-209 bearing the Class B tetracycline resistance determinant were 
incubated with about 4 .mu.M of .sup.3 H-tetracycline in a volume of 300 
.mu.l. Different potential blocking agents with substitutions at the C5 
position were separately tested at concentrations of 0.2, 0.5 and 2 
.mu.g/ml. A control experiment, wherein no blocking agent was used, was 
also performed. After incubation for 2.5 minutes, the vesicles were 
collected on membrane filters and the effect of the blocking agent on 
uptake of .sup.3 H-tetracycline was assessed by liquid scintillation 
counting of the radioactivity on the filters. 
The assay showed the relative inhibition of tetracycline by the different 
drugs vis a vis drug amounts (Table E4 below). Using the uptake at 2.5 
minutes (when the system reaches equilibrium) the IC.sub.50 of the analogs 
was determined. Using this method, the IC.sub.50 of different C5 esters 
ranged from 0.2 .mu.M (5 proprionate methacycline) to 9.4 .mu.M (5 
cyclopropanoate methacycline). Some showed no effect in this assay, 
suggesting they have poor, if any, blocking activity. These studies 
suggested that the smaller substitution at the 5 position, e.g., the 
proprionates and phenyl acyl, were more effective blockers of the efflux 
system than were those with larger substitutions. Derivatives bearing a 
substitution at C13 and C5 were also effective (e.g., 
13-cyclopentyl-thio-5-proprionate tetracycline, IC.sub.50 =3.3 .mu.M). 
Some of the drugs from this assay were then tested for their activity 
against the whole bacterial cells (See Experimental Series 4). 
TABLE E4 
______________________________________ 
5-Esters 
##STR21## 
IC.sub.50 
Cmpd R R.sub.1 (.mu.M).sup.a 
______________________________________ 
1 CH.sub.3, H COCH.sub.2 CH.sub.3 
1.0 
2 CH.sub.2 COCH.sub.2 CH.sub.3 
0.2 
3 CH.sub.3, H COCH.sub.2 C.sub.6 H.sub.5 
1.6 
4 CH.sub.3, H CO(CH.sub.2).sub.6 CH.sub.3 
9.3 
5 CH.sub.2 S-cyclopentyl, H 
COCH.sub.2 CH.sub.3 
3.3 
6 CH.sub.3, H COCH.sub.2 CH.sub.2 CO.sub.2 H 
2.0 
7 CH.sub.3, H CO(CH.sub.2).sub.3 NH.sub.2 
6.4 
8 CH.sub.2 S-propyl 
COCH.sub.2 CH.sub.3 
1.4 
9 CH.sub.2 S-cyclopentyl, H 
cyclohexanoate 
NE* 
10 CH.sub.2 S-cyclopentyl, H 
cyclopentanoate 
NE* 
11 CH.sub.2 cyclopropanoate 
9.4 
______________________________________ 
.sup.a by everted vessicle assay 
*NE = no effect (&gt;30 .mu.M) 
EXPERIMENTAL SERIES 4 
The growth inhibitory effect of different C5 derivatives of tetracyclines, 
with and without doxycycline, were determined using sensitive and 
resistant E. coli, Staphylococcus aureus, and Enterococcus faecalis. The 
general protocol for these experiments was as follows: 
Cultures were grown up fresh in L broth in the morning from an overnight 
culture. After 4-6 hours of growth, each bacterial culture was diluted to 
approximately 5.times.10.sup.5 cells/mi. The drugs were diluted in 
two-fold dilutions from 50 .mu.g/ml to &lt;1 .mu.g/ml and tested alone and in 
mixtures by incubation for 18 h at 37.degree. C. The MIC was that 
concentration of drug alone or combination of drugs in which no growth (no 
cloudiness) was observed. The minimal lethal concentration (MLC) was that 
concentration which killed 99.9% of the cells and was based on the number 
of bacteria initially inoculated into the assays. Those cultures showing 
no bacterial growth after incubation at 37.degree. C. were evaluated for 
the number of viable bacteria remaining by plating onto nutrient agar 
plates; these data determined the MLC. The results of four prototype 
drugs, the C5 propyl ester of methacycline, the C5 propyl ester of 
doxycycline and the combination of C5 propyl, C13 derivatives ( 
13-cyclopentylthio-5-proprionate tetracycline and 
13-propyl-thio-5-proprionate tetracycline) are presented in Table E5 and 
Charts 6-9. 
TABLE E5 
______________________________________ 
Susceptiblity of Tetracycline Susceptible Strains (MIC, .mu.g/ml) 
E. coli S. aureus 
E. faecalis 
C5 Ester ML308 450 ATCC 9790 
______________________________________ 
5 proprionate methacycline 
10 1.3 .8 
5 proprionate doxycycline 
10 .6 .4 
13-cyclopentyl-thio-5- 
20 .4 .4 
proprionate tetracycline 
13-propyl-thio-5- 
20 .3 2.5 
proprionate tetracycline 
______________________________________ 
Summary of Charts 6-9 
Chart 6 MIC/MLC (.mu.g/ml) dosages for tetracycline resistant strains using 
5 proprionate methacycline with and without doxycycline. 
Chart 7 MIC/MLC (.mu.g/ml) dosages for tetracycline resistant strains using 
5 proprionate doxycycline with and without doxycycline. 
Chart 8 MIC/MLC (.mu.g/ml) dosages for tetracycline resistant strains using 
13-cyclopentyl-thio-5-proprionate tetracycline with and without 
doxycycline. 
Chart 9 MIC/MLC (.mu.g/ml) dosages for tetracycline resistant strains using 
13-propyl-thio-5-proprionate tetracycline with and without doxycycline. 
+=growth 
0=no growth (MIC) 
.dwnarw.=killing 
.box-solid.=99.9% killing (MLC) 
.quadrature.=no growth, but no microbiologic testing 
The analog concentration is given in columns A and H. It is this 
concentration which is within the squares 2-10. The deoxycycline 
concentration in the control is in Column 11 and its concentration in each 
of the boxes is given as small numbers within each of the squares. 
__________________________________________________________________________ 
CHART 6 A, B 
Analog 5-proprionate methacycline Strain E. coli D1-299 (Tet A) 
A 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.largecircle. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.largecircle. 
.largecircle. 
+ + + + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.largecircle. 
+ + + + + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
+ + + + + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
+ + + + + + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control 
.box-solid. 
+ + + + + + + + + 
__________________________________________________________________________ 
Analog 5-proprionate methacycline Strain S. aureus 4250 (Tet K) 
B 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.dwnarw. 
.dwnarw. 
+ 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.largecircle. 
+ + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
.dwnarw. 
+ + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
.largecircle. 
+ + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ + + + + 
__________________________________________________________________________ 
CHART 6 C, D 
Analog 5-proprionate methacycline Strain E. faecalis 158 (Tet L) 
C 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.5 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.largecircle. 
.largecircle. 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.largecircle. 
.largecircle. 
+ + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.dwnarw. 
.largecircle. 
+ + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.largecircle. 
+ + + + 
__________________________________________________________________________ 
Analog 5-proprionate methacycline Strain E. faecalis 211 (Tet M) 
D 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.dwnarw. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
.largecircle. 
+ + + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.box-solid. 
+ + + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.dwnarw. 
+ + + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
+ + + + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ + + + + + + 
__________________________________________________________________________ 
CHART 7 A, B 
Analog 5-proprionate doxycycline Strain E. coli D1-299 (Tet A) 
A 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.largecircle. 
.largecircle. 
.largecircle. 
+ + 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
.largecircle. 
+ + + + + + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
+ + + + + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
+ + + + + + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.largecircle. 
+ + + + + + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control 
.dwnarw. 
+ + + + + + + + + 
__________________________________________________________________________ 
Analog 5-proprionate doxycycline Strain S. aureus 4250 (Tet K) 
B 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.largecircle. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.dwnarw. 
.largecircle. 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
.largecircle. 
+ + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
.box-solid. 
+ + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
.box-solid. 
.dwnarw. 
+ + + 
__________________________________________________________________________ 
CHART 7 C, D 
Analog 5-proprionate doxycycline Strain E. faecalis 158 (Tet L) 
C 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.box-solid. 
.box-solid. 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
.box-solid. 
+ + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
.box-solid. 
+ + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.largecircle. 
+ + 
__________________________________________________________________________ 
Analog 5-proprionate doxycycline Strain E. faecalis 211 (Tet M) 
D 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
.box-solid. 
.largecircle. 
+ + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.dwnarw. 
.largecircle. 
+ + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
.largecircle. 
+ + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
.largecircle. 
+ + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.largecircle. 
+ + + + + 
__________________________________________________________________________ 
CHART 8 A, B 
Analog 13-cyclopentyl-thio-5-proprionate tetracycline Strain E. coli 
D1-299 
(Tet A) 
A 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
.box-solid. 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
.dwnarw. 
+ + 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
.largecircle. 
+ + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.23 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
+ + + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
+ + + + + + + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
+ + + + + + + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control 
+ + + + + + + + + + 
__________________________________________________________________________ 
Analog 13-cyclopentyl-thio-5-proprionate tetracycline Strain S. aureus 
4250 
(Tet K) 
B 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.1 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.largecircle. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.box-solid. 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
+ 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
+ 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ 
__________________________________________________________________________ 
CHART 8 C, D 
Analog 13-cyclopentyl-thio-5-proprionate tetracycline Strain E. faecalis 
158 
(Tet L) 
C 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.box-solid. 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.box-solid. 
+ 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.box-solid. 
+ 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ 
__________________________________________________________________________ 
Analog 13-cyclopentyl-thio-5-proprionate tetracycline Strain E. faecalis 
211 
(Tet M) 
D 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.box-solid. 
.box-solid. 
+ 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
.box-solid. 
.box-solid. 
+ + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.largecircle. 
+ + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.largecircle. 
+ + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .largecircle. 
+ + 
__________________________________________________________________________ 
CHART 9 A, B 
Analog 13-propyl-thio-5-proprionate Strain E. coli D1-299 (Tet A) 
A 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.largecircle. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.largecircle. 
+ + + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
+ + + + + + + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
+ + + + + + + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
+ + + + + + + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control 
+ + + + + + + + + + 
__________________________________________________________________________ 
Analog 13-propyl-thio-5-proprionate Strain S. aureus 4250 (Tet K) 
B 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.box-solid. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
+ 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.largecircle. 
+ 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
+ + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ + 
__________________________________________________________________________ 
CHART 9 C, D 
Analog 13-propyl-thio-5-proprionate Strain E. faecalis 158 (Tet L) 
C 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
.dwnarw. 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
+ 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.box-solid. 
.largecircle. 
+ + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.box-solid. 
.largecircle. 
+ + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.box-solid. 
+ + + + 
__________________________________________________________________________ 
Analog 13-propyl-thio-5-proprionate Strain E. faecalis 211 (Tet M) 
D 1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Analog 
A Doxy 50 25 12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
Doxy 
Control 
B 50 25.0 
37.5 
43.8 
46.7 
48.4 
49.2 
49.6 
49.8 
49.9 
50.0 
C 25 12.5 
18.8 
21.9 
23.4 
24.2 
24.6 
24.8 
24.9 
25.0 
25.0 
.dwnarw. 
D 12.5 6.25 
9.4 
11.0 
11.7 
12.1 
12.3 
12.4 
12.5 
12.5 
12.5 
+ + + 
E 6.25 3.12 
4.7 
5.5 
5.9 
6.1 
6.2 
6.23 
6.25 
6.25 
6.25 
+ + + + + 
F 3.12 1.56 
2.4 
2.8 
3.0 
3.05 
3.1 
3.12 
3.12 
3.12 
3.12 
.largecircle. 
+ + + + + 
G 1.56 .78 
1.2 
1.4 
1.5 
1.53 
1.55 
1.56 
1.56 
1.56 
1.56 
.largecircle. 
+ + + + + 
H Analog 
50.00 
25.0 
12.5 
6.25 
3.12 
1.56 
.78 
.39 
.195 
0.0 
Control .box-solid. 
.largecircle. 
+ + + + + 
__________________________________________________________________________ 
All four tetracycline derivatives effectively inhibit growth of sensitive 
Staph aureus and E. faecalis when used alone. None was effective alone 
against susceptible E. coli (Table E5). Against the tetracycline resistant 
S. aureus and E. faecalis strains, the analogs were also very effective 
alone (Charts 6-9). While they were not effective alone against resistant 
E. coli D1-299, they did show synergistic effects achieving both decreased 
growth and cell killing. For instance, at a combination of 5.9 .mu.g/ml of 
doxycyline and 6.25 .mu.g/ml of 13-cyclopentyl-thio-5-proprionate Tc, 
there was an MLC of E. coli D1-299 (Chart 8A). The 
13-cyclopentyl-thio-5-proprionate tetracycline was equally effective alone 
against Tet K in S. aureus and Tet L and Tet M determinants in E. 
faecalis. In fact, the values of the MLC/MLC were close to those of the 
sensitive strains (Table E5). 
The C5 proprionate esters were also effective alone against resistant S. 
aureus and E. faecalis, and showed further efficacy in additiveness and 
synergy when used with doxycycline in MIC and MLC. For instance, in Chart 
6B, the combination of 1.55 .mu.g/ml doxycycline and 1.56 .mu.g/ml 
5-proprionate methacycline or 6.23 .mu.g/ml doxycycline and 0.78 .mu.g/ml 
analog achieved MIC. 
The growth inhibition included cells bearing different tetracycline efflux 
systems (Class A, B, K & L) and a ribosomal protection system (Class M). 
These unexpected results confirm that the substitution at the C5 position 
produces an effective efflux blocking agent which demonstrates synergistic 
antibacterial activity against tetracycline resistant bacteria bearing 
different resistance determinants. 
EXAMPLE 1 
Synthesis of 13-propylthio-5-hydroxy-6-.alpha.-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 N.sub.2. The mixture was reduced to 
1/5 volume by distillation and filtered. The filtrate was dripped slowly 
into cold Et.sub.2 O while stirring resulting in the formation of a yellow 
precipitate. The precipitate was filtered, dissolved in H.sub.2 O and 
brought to pH 4.5 with 1.0M NaOH. This solution was filtered, and 
extracted with CH.sub.2 Cl.sub.2 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.degree.-140.degree. C. 
(dec.). TLC r.sub.f =0.70 (I); HPLC R.sub.t =20.18 min. HNMR 
(DMSO-d.sub.6).delta. 7.50 (t, 1 H), 7.05 (d, 1 H), 6.85 (d, 1 H), 4.32 
(d, 2 H), 3.15 (s, 1 H), 2.65 (s, 6 H), 2.32-2.52 (m, 2 H), 1.51-1.80 (m, 
2 H), 0.9-1.22 (m, 3 H); HRMS (FAB); calc for C.sub.25 H.sub.30 N.sub.2 
O.sub.8 S 519.1801 (M+1), found 519.1815 (M+1). 
EXAMPLE 2 
Synthesis of 13-cyclopentylthio-5-hydroxy-6-.alpha.-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 CH.sub.2 Cl.sub.2, or by HPLC chromatography. An 
analytical sample was produced by HPLC as a yellow solid of 
mp=132.degree.-139.degree. 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 r.sub.f =0.80 (I); HPLC R.sub.t =21.19 min. 
HNMR (MeOH-d.sub.4).delta. 7.38 (t, 1H), 7.02 (d, 1H), 6.72 (d, 1H), 4.10 
(s, 2 H), 2.70 (br s, 6 H), 1.81-2.01 (br m, 2 H), 1.28-1.75 (br m, 6 H), 
(br m, 2 H); HRMS (FAB); calc for C.sub.27 H.sub.32 N.sub.2 O.sub.8 S 
545.1957 (M+1), found 545.1960 (M+1). 
EXAMPLE 3 
Synthesis of 13-propylthio-5-proprionate-6-deoxy-tetracycline 
100 mg of 13-propylthio-5-hydroxy-6-.alpha.-deoxy-tetracycline of Example 1 
and 2.0 g of propionic acid were dissolved in 20 mL of anhydrous 
hydrofluoric acid and the resultant solution sealed in a polypropylene 
tube for 3 days at room temperature. The hydrofluoric acid was removed by 
a slow steady stream of nitrogen and the residue taken up in diethyl 
ether. The precipitate was dissolved in MeOH (4 mL) and injected into a 
preparative HPLC utilizing a C18 reverse-phase column and mobile phases of 
phosphate buffer (pH 4.5) and MeOH over a linear gradient (30%-100% over 
30 minutes) at 30 mL/minute. The compound was collected at 26.7-29.3 
minutes, extracted into 40 mL n-butanol, and the solvent removed in vacuo 
to yield 34 mg of pure product. MS data=M+1 (FAB) 575, 558, 541, 484. 
EXAMPLE 4 
Synthesis of 13-cyclopentylthio-5-proprionate-6-deoxy tetracycline 
100 mg of 13-cyclopentylthio-5-hydroxy-6-.alpha.-deoxytetracycline of 
Example 2 and 5.0 g of propionic acid were dissolved in 35 mL of anhydrous 
hydrofluoric acid and the resultant solution sealed in a polypropylene 
tube for 3 days at room temperature. The hydrofluoric acid was removed by 
a slow steady stream of nitrogen and the residue taken up in diethyl 
ether. The precipitate was dissolved in MeOH (4 mL) and injected into a 
preparative HPLC utilizing a C18 reverse-phase column and mobile phases of 
phosphate buffer (pH 4.5) and MeOH over a linear gradient (30%-100% over 
30 minutes) at 30 mL/minute. The compound was collected at 26.7-29.3 
minutes, extracted into 40 mL n-butanol, and the solvent removed in vacuo 
to yield 34 mg of pure product. MS data=M+1 (FAB) 601, 492, 391. 
The present invention is not to be restricted in form nor limited in scope 
except by the claims appended hereto.