CA2251704A1 - Topical administration of antimicrobial agents for the treatment of systemic bacterial diseases - Google Patents

Abstract

The present invention provides a method of topically administering antimicrobial agents such as premafloxacin, premafloxacin-like compound, premafloxacin ester, ciprofloxacin, enrofloxacin, cefquinome, cefpodoxime, gentamicin or erythromycin for the treatment of systemic bacterial diseases in mammals.

Description

CA 022~1704 1998-10-01 wo 97/42954PCT/US97/07124 TOPICAL ADMINISTRATION OF ANTIMICROBIAL AGENTS
FOR THE TREATMENT OF ~Y~ ;MIC BACTERIAL DISEASES

FIELD OF THE INVENTION
5The present invention relates to a method of topically ~rlminictering timicrobial agents for the treatment of systemic bacterial ~lice~cçs in m~mm~lc BACKGROUND OF THE INVENTION
It has been generally accepted that intravenous infusion, intramuscular lO injection, subcutaneous, buccal, oral, and rectal routes are the methods for ~tlmini.ctration of a wide variety of antimicrobial agents for the tre~tmQnt of systemic bacterial ~ice~ce~s~ Due to lack of systemic level effects with antimicrobial agents ~rlminictered topically, the topical ~minictration of ~ntimicrobial agents has been limited to the tre~tment of 10~1i7ed infect;onc of the skin or eyes.
15However, it is known that the aforementioned non-topical methods of ~rlminictration for the tre~tment of systemic bacterial tlice~Res have certain disadvantages. For ~Y~mple, buccal and rectal ~mini.ctration often produce ~licc~mfort and aggravation to the m~mm~l.c that are treated. The intravenous, subcutaneous and intramuscular routes are not only painful, but also must be performed by trained individuals. In addition, there is a risk of needle injury,inf~ctiol~, and other trauma in~ ]rlinE the emotion~l trauma inevitably Ac,soçi~t~d with injecti~nc. Oral ~rlmini.ctration, although generally acceptable, may have the disadvantages of poor absorption of the therapeutic agent from the gastroint~stin~l tract and/or degr~ t;on which may be caused by the acidic medium of the stQm~h, or causes digestive disflmrtion in rl~min~ntC Furthermore, in the case of treating :~nimAl.c, the aforementioned methods of ~minictration are labor and time consuming. Topical ~minictration of ~ntimi~robial agents would circumvent these problems by allowing a more convenient, non-invasive method for the treatment ofsystemic bacterial rli.ce~ces Thus, it is desirable to have antimicrobial agents that produce systemic effects when they are topically ~mini~trated for the tre~tm~nt of systemic ~lice~c~c It has been unexpectedly discovered, during the invPstig~tion of activities of ~ntimirrobial agents by topical ~rlmini.~ration, that certain antimicrobial q1lino10~es, such as pr~mAfloY~-~in, prem~floY~in-like compound, ciprofloY~- in, and enrofloY~in, and certain ceph~losporin derivatives, such as cefquinome and cefpotl~-Yim~, as well as gent~mitin and erythromycin, when a~lmini.ctrated topically CA 02251704 1998-lO-01 WO 97t42954 - PCT/US97/07124 using dimethyl sl-lfoYi-le and water as a carrier, are effective for the treatment of systemic bacterial infections. It has also been discovered that prçm~floY~rin, pr~m~floY~rin like compound and its esters, when ~lmini.qtrated topically using propylene glycol and oleyl alcohol or propylene glycol and A_one as a carrier, are effective for the treatment of systemic bacterial infectionR

INFORMATION DISCLOSURE
Pr~m~floY~rin, ciprofloY~rin, enrofl~ Y~.in, c~nofloYA-~in, and their qllinolonefs~mili~s are series of new and potent ~ntimicrobial agents with a broad spectrum of antimicrobial activity. These quinolone f~miliPs are active against a variety ofhuman and veterinary pathogens, including both gram-positive and gram-negative bacteria. Prçm~floY~rin and its esters are ~ çlose-l in U.S. Patents 4,665,079 and 5,563,155, issued May 12, 1987 and October 8, 1996 to Warner-Lambert Company.
CiprofloY~rin and enrofll~Y~rin are tliRrl( se-~ in U.S. Patent 4,670,444, issued June 2, 1987 to Bayer Aktiengesçll.qrh~ft DAnofl-Y~rin is tliqçlose(l in European Patent215650, issued to Pfizer Inc.
Ceftiofur, cefquinome, and cefpo-l-Yime are derivatives of the family of ceph~losporins, which have been recogni7e(1 as highly active antimicrobial agents.
Ceftiofur i9 rliqrlose~l in U.S. Patent 4,464,367, issued August 7, 1984 to Sanofi.
CefpodoYime is disclosed in U.S. Patent 4,486,425, issued December 4, 1984 to Sankyo Company Limited. Cefquinome is a known compound which is rliqclosed in, among the others, U.S. Patent 4,754,031, European Patent 64,740, and European Patent 74,645.
Gçnt~mi~.in is a known ~mino~;lyco~ide antibiotic compl~lr derived from Micromonospora purpurea, or M. Echinospora. G~nt~mirin is effective against a wide range of aerobic gram-negative bacilli, especially the Enterobacteriaceae and Pseudomonas, and some gram-positive bacteria. Gent~mirin is diRrloserl in, interalia, U.S. Patents 3,091,572 and 3,136,704.
Erythrol,ly.;ill is an intermP~i~te spectrum macrolide antibiotic, produced by Streptomyces erythreus, effective against most gram-positive and certain gram-negative bacteria. Erythrollly~ is rli~rlnse~l in, among the others, U.S. Patents

2,653,899 and 2,823,203.

SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method of treating or preventing systemic bacterial tli~e~es in m~mm~l~ which comprises topically CA 0225l704 l998-lO-Ol ~rlminictering to such m~mm~l an effective amount of an antimicrobial agent selPcte~l from the group cnnCictinE of pr~mzfloY~in, prçm~floY~ in-like compound, ciprofloY~rin, enrofloY~-in, cefqtlinnme, cefporlnYime, gent~mi~in or erythlu",y~ in a DMSO/H20 carrier.
In another aspect, the present invention provides a m~thod of treating or preventing systemic bacterial tlice~c~.c in m~mm~lc which comprises topically ~mini.ctering to such ms-mmAl an effective amount of prem~flQY~rin~ prem~flnY~rin like compound, or its ester in a PG/OA or a PG/Azone carrier.

DETAILED DESCRIPTION OF THE INVENTION
The present invention resides in the unexpected result that certain antimicrobial qllinolorl~,s and ceph~losporin del.v~lives as well as gent~mi-~.in and eryth.c",lyci~l, when using DMSO/H20, as a carrier and when ~-imini~trated topically, are effective for the tre~t~n~nt of systemic bacterial infection.c. It is also unexpected that prem~flnY~-in and its esters, when using PG/OA or PG/Azone as a carrier and ~lmini.ctrated topically, are effective for the treatment of systemic bacterial infectionc For the purpose of the present invention, systemic bacterial flice~c~.s means systemic bacterial infect;onc caused by a variety of human and veterinary pathogens, including both gram-positive and gram-negative bacteria. Such rlice~ce.s and cor~r~itions are well known and readily ~ gnose-l by physicians and veterinarians of ordinary skill.
The term m~mm~l refers to man and Anim~lc of veterinary interest.
However, the invention may also be practiced with other vertebrates, and with the lower species comprising the non-vertebrates.
Prem~flnY~-~in refers to a compound of formula I or a pharmPl~elltic~lly acceptable acid ~cl-lition salt thereof C H 3 - N H~CF ~3,C O O H

I.
Prem~flnY~in ester refers to a compound of formula II or a pharm~ce~ltirz~lly acceptable acid addition salt thereof CA 02251704 1998-lO-01 CH3 NH~N ~COOR

OCH~

wherein R is Cl 10 straight or branched aL~cyl such as methyl, ethyl, propyl or butyl, Cl 10 straight or branched aLkenyl, Cl 6 cycloaLkyl, phenyl, benzyl, napthyl, or m~ntyl.
Prem~floY~rin-like compound refers to a compound of formula III or a 10 pharmaceutically acceptable acid addition salt thereof 16 R4Rs~F~'~CO2R2 III
wherein * denotes an asymmetric carbon atom; R1 is ethyl, cyclopropyl, or 2,4-difluorophenyl; R2 is hydrogen, Cl 4 aL~yl or a cation; R3 is hydrogen, amino, or methyl; R4 and R5 are each indep~n-lçntly hy-lro~en or methyl.
Ciprnfl- Y~rin refers to rh~mir-ql compound of formula IV or a pharmaceutically acceptable acid ~ lit;on salt thereof 26 f N~COOH
HN~J
IV.
EnrofloY~rin refers to r.hemicz~l compound of formula V or a pharmaceutically acceptable acid addition ~alt thereof o F ~ ,COOH

CH3CH2--N--J ~, V.

Cefpodoxime refers to a compound of formula VI or a pharmaceutically acceptable acid addition salt thereo~

rNI ~I c--CONH ~S
NOCH3 0 N'~CH20CH3 COOfHOCOOCCH(CH3)2 VI.
Cefquinome refers to a compound of formula VII or a pharmaceutically acceptable acid addition salt thereof Nr ~ C--CONH ~ O
NOCH3 0 o~ 2 N~9 VII.
Gent~milin refers to antibiotic complex produced from ferment~ti~ln of Micromonospora purpurea or M. echinospora and variants thereof.
Erythromycin refers to antibiotic substance produced by a strain of Streptomyces erythreus.
Prems~floY~-~in, ciproflnY7~in, enroflc-Y~r,in, cefqllinomP, cefpo-l(YimP, gent~mi~in and erythrol-ly~-ll are commercially available antibiotic agents.
PrPmAfloY~in esters and pr~m~fl~Y~in-like compounds can be prepared according 25 to the procedures described in U.S. Patents 4,665,079 and 5,563,156 incorporated herein by reference.
The term DMSO is the abbreviation of dimethyl slllf.~Yi.le The term PG is the abbreviation of propylene glycol.
The term OA is the abbreviation of oleyl ~ n~hol Azone refers to hexahydro-l-dodecyl-2H-azepin-2-one.
The term Ivermectin vehicle refers to the carrier and penetrant used to deliver ivermectin, a commo~ly used topical antiparasitic agent.
Topical ~rlmini.~tration or appli~tior means the direct contact of an ~ntimi-~robial formulation with skin such as, for example, by drops, spray, paint, or 35 pour on. The active antimicrobial form~ tion of the present invention is ?~lmini.qtrated 1 to 5 times daily until the bacterial infectirns is treated.

CA 022~1704 1998-10-01 WO 97t42954 PCT/US97/07124 Topical form~ ffon.q of this invention may be prepared by employing conventional technique to dissolve an antimicrobial agent described above in DMSO
and water, in PG and OA, or in PG and Azone. Optionally, the compositions may cont~in conventional stabilizers and thi~k~nine agents.
For ~Y~mple, an ~ntimi~robial agent is transferred in a DMSO/H2O carrier, and the mixture is stirred until the solution is clear. When dissolution is complete, the r~m~ining solvents are added to prepare the final concentration of drug and ratio of solvents.
The term DMSO/H2O carrier refers to the mixture solution of dimethyl s-~lf~ and water. MSO and water are in a ratio (DMSO: water) of from 100: 0 to about 10: 90 by volume to volume (V/V). The use of 100% DMSO by volume achieves the best ~ntimicrobial effect in the present invention, while at least about 10% by volume of DMSO is required in the DMSO and water carrier to have systemic effect.
In ~not~r example, about 95% of PG by volume is mixed with about 5% of OA by volume, or about 95% of PG by volume is mixed with about 5% of Azone by volume. An appropriate amount of a pr~m~flr~Y~cin ester is weighed out and transferred to the a graduated mixing flask. The desired carrier is then added to the appropriate volume mark. The flask is sealed and the contents is mixed on a rotator until the solution is clear.
The term PG/OA carrier refers to the mixture solution of propylene glycol and oleyl ~l~ohol Propylene glycol and oleyl alcohol is in a ratio at least about 80% of propylene glycol by volume. The use of 95~o propylene glycol and 5% oleyl alcohol achieves the best antimicrobial effect in the present invention.
The term PG/Azone carrier refers to the mixture solution of propylene glycol and Azone. Propylene glycol and oleyl alcohol is in a ratio at least about 80% of propylene glycol by volume. The use of 95% propylene glycol and 5% Azone achieves the best antimicrobial effect in the present invention.
The amount of an antimicrobial agent in a formulation may be varied or adjusted widely depending upon the solubility of the particular antimicrobial agent, the potency of the particular antimicrobial agent being used, the severity of the bacterial infection, the particular form~ tion~ and the desired concentration.
C~enerally, the amount of an antimicrobial agent is present in the formulation in a range between about 0.5% to about 90% by weight of the fortnlll~tio~, preferablybetween about 1% to about 25% by weight of the formlll~tir~n~
In therapeutic use for treating systemic bacterial infections in m~mm~l~, an CA 022~1704 1998-10-01 ~ntimirrobial agent is ~lmini~tçred topically at a dosage to obtain and m~intqin a concentration, of which the blood-level of active agent in the m~mm~l being treated is antibacterial e~.;~ive. Generally, the effective amount of the active agent will be in the range of about 0.1 to about 100 mg/kg, more preferably about 1.0 to about 50 6 mg/kg of body weight/day. It is understood that the dosages may vary depending upon the requirements of the subject being treated, the severity of the bacterial infPction, and the particular antimicrobial agent being used. Also, it is to be understood that the initial dosage ~-lmini-ctered may be increased beyond the above upper level in order to rapidly achieve the desired blood-level or the initial dosage may be smaller than the optimum and the daily dosage may be pro~les~ively increased during the course of tre~tmçnt depending upon the particular sitn~tionThe present invention achieves the desired results as ~emonFtrated in the mouse systemic protection tests. Mice that had been infPcte~l with Pasteurella haemolytica are given doses of a formulation of the present invention. The nn~Yrected result of the present invention is realized by their ED50 values. Theunexpected results are also dem~ trated by the comparison with other routes of ~lmini.ctration, with other formtfl~tinn~ and with variety of antimicrobial agents.
In test 1, prem~fl-~Y~rin in an aqueous solution co~t~ininE 10% DMSO
~mini~tQred topically is compared with prem~fl~ Y~rin ~mini~tered s--hc--t~neously.
Hair is removed from ~nim~lR in the topically treated group. The ED50 for s--hc~ t~npous and topical iq~lmini.~trations are 0.12 mg/kg of body weight/day and 3.7 mg/l~g of body weightlday, respectively.
In test 2, oral ~-lminictration of prçm~fl~Y~r.in is compared with topical z~lmini.~tration of prem~fl~Y~rin The topical form~ tir)n of prem~floY~rin is prepared using 100% DMSO and is ~-lmini.~tered to two groups of mice. One group had hair removed while the hair coat is left nntli~tllrbed in the second group. The ED50s for the three groups were 0.6, 0.87, and 3.4 mg/kg for the oral, topical/hair removed, and topical/no hair removed groups, respectively. It appears that whileincreasing the DMSO from 10% to 100% increased the penetration of pr~m~fl(~Y~rin, the hair coat decreased the penetration of the ~ntimicrobial agent.
Test 3 illustrates the antimicrobial effect of various formlfl~ti~ on the activity of topically ~lminict~red pr~om~floY~cin The data ~lem~n~trate that prRm~flf~Y~rin in 100% DMSO formulation achieves the highest ~ntimirrobial activity.
Test 4 lists the antimicrobial effects of several qllinolr)n~s, ceph,qlosporin derivatives as well as gent~mirin and erythromycin in 100% DMSO when CA 0225l704 l998-lO-Ol miniQtrated topically.
Test 5 illustrates the antimicrobial effect of pr~m~floYP.. in in a formulation containing PG and OA (95:5 by volume). The result is compared with the ~ntim;rrobial effect of prçm~flt~Y~rin in 100% DMSO.
Test 6 lists ~ntimicrobial effects of prçm~floY~rin esters in a formulation cnnt~inine PG and Azone (95:5 by volume). The antimicrobial effects of prem~floY~rin esters are compared with the antimicrobial effect of pr~m~floY~rin in 100% DMSO and ~miniQtered orally.

BIOLOGICAL TESTING
1. Bacteria.
The rh~ n~e org~niQm, Pas~eurella haemolytica UC6531, was originally i.qol5~te~1 from a case of bovine pnellmoni~ The organism was kept frozen at -70 ~C
in trypticase soy broth cQnt~ininF 10% glycerol on 3 mm glas~ beads. Prior to use, 15 the org~niQm was snhcllltllred onto a trypticase soy agar plate containing 5% sheep blood and incnh~tetl for 18-24 hours at 35 ~C under aerobic contlition.q A single colony was removed from the blood agar plate, inoculated in 6 ml brain-heart infusion broth (BHIB) and incubated for 6 hours at 35 ~C in a ~% C02 ~trnosphere.
The org~ni~m was then mixed with an equal volume of BHIB containing 2%
20 brewer's yeast which served as the final inl cnlllm 2. Mouse Systemic Protection Tests The P. haemolytica mouse ~iy~ lC protection test was used to evaluate the activity of a new eYten~ l spectrum ~ntimirrobial agents by topical ~-lminiQtration.
25 For this model, 21-25 day old female CD-1 mice were used. Mice were injected with - apl,l~.. ;.. ~t~ly 100 LD50 doses ofthe rh~ n~e org~niQ~m intraperit~n~l ~ntimirrobial agents were ~rlminiFtered (0.1 m1) either by subcutaneous injection, orally (snhcllt~neous and oral ~tlmini~ration served as positive controls) or bytopical ~lminiFtration. For topical ~minictration~ the ~n*mirrobial agent was 30 dissolved in the ap~,p~;ate solution and placed in a 1 cm2 area on the back. All ~ntimicrobial agents were ~lmini~hred within one hour of Gh~llenEe and at 24 hours intervals for the two following days for a total tre~tm~t time of three days.
Ten mice were used in each tre~tm~nt group at each dosage level with five, two-fold serial ~lilntir)n.~ of the ~ntimirrobial agent in each determin~tior The post-rh~llenee

3~ effectiveness was c~lc~ t~-l based on post-rh~ nee day 6 and was reported as the ED50, the amount of antimicrobial agent (mg/kg body weight/day) required to

4 PCT/US97/07124 protect 50% of inf~cte~l mice. Antimicrobial agents with lower ED50 values are eYpecte-l to be more effective. It iB generally con~itlered that an agent with a ED50 value less than 10 will display effir~rinus therapeutic effects in treating ~y~ell~ic bacterial (ii~e~es in m~mm~lA.

Hair is removed from a 1 cm2 area of skin on the front shoulder of mice.
Pr~m~flnY~rin in 10% DMSO i9 ~r~mini~tered topically. The result is compared with a group of mice receiving subcutaneous injection of the same antimicrobial agent.
10~ntimir.robial~r1mini~tration Formulation ED50 Agent Route (mglkg/day) Prem~flnY~rin Subcutaneous Aqueous Solution 0.12 Prçm~flnY~rin Topical 10% DMSO 3.7 Topical ~miniFtration of prem~flnY~rin in 100% DMSO in both denuded and normal mice. The results are compared with oral ~timini~tration of pr~m~flnY~rin in aqueous solution.
20AntimicrobialA-lminiFtration Formulation ED50 Agent Route (mg/kg/day) Prem~flol~rin Oral Aqueous Solution 0.6 Prem~flny~rin Topical 100% DMSO 0.87 (hair removed) Prem~flnlr~rin Topical 100% DMSO 3.4 (no hair removed) CA 02251704 1998-lO-01 Topical ~lmini.cfration of the ~ntimicrobial agent in various form~ tinns.
Hair was not removed in this test. The results are compared to oral ~imini~tration of prem~floY~rin 5Antimicrobial ~rlminiAtration Formnl~tinn ED50 Agent Route (mg/kg/day) Pr~m~flcY~in Oral Aqueous Solution 0.6 Prem~flnY~-in Topical 1005~o DMSO 2.2 Pr.o.m~floY~-~in Topical Aqueous Solution >10 10Pr~m~flnY~in Topical Propylene Glycol/ >10 Glycerol Formal (60:40) Prem~flnY~in Topical PEG 400 >10 Prçm~floY~-~.in Topical Ivermectin Vehicle >10 Topical ~miniQtration of various ~ntimi~robial agents in 100% DMSO. Hair was not removed in this test. The results are compared to oral ~1mini~tration of pr.om~floY~in Antimicrobial ~r~mini~qtration Form-fl-qtion ED50 Agent Route (m~lkg/day) Prçm~flnY~in Oral Aqueous Solution 0.9 PrPm~fll Y~ in Topical 100% DMSO 2.6 CiproflnY~in Topical 100% DMSO 0.6 EnrnfloY~c;n Topical lOO~o DMSO 6.7 25DanoflnY~-~,in Topical 100% DMSO 18.6 Ceftiofur Topical lOO~oDMSO 15.6 Cefquinome Topical 100% DMSO 3.6 CefpodoYime Topical 100% DMSO 3.1 Gent~mirin Topical 100% DMSO 4.6 Erythlollly~ Topical 100% DMSO 3.3 Tilmicosin Topical 100% DMSO >50 Topical A~imini~tration of pr~m~fl~Y~rin and its esters in formlfl~tion~
cont~ining 95% propylene glycol and 5~o oleyl alcohol or 5% Azone. The result~ are 10 compared with topical ~-iminictration of prem~flnY~in in 100% DMSO and oral lmini~tration of pr~m~flnY~in in aqueous solution.
Antimicrobial ~flmini~tratiOn Formulation ED50 Agent Route (mglkg/day) Prçm~flnY~in Oral Aqueous Solution 1.6 15Prem~flnY~-in Topical 100% DMSO 1.7 Prem~flnY~in Topical PG/OA (95:5) 0.9 Prem~fl~Y~I~in Topical PG/OA (95:5) 0.6 butyl ester Pr~m~fl~.Y~in Topical PG/Azone (95:5) 0.3 20butyl ester Prçm~fl~Y~ in Topical PG/OA (95:5) 0.7 ethyl ester Prçm~fl~Y~in Topical PG/Azone (95:5) 0.4 ethyl ester

Claims (25)

We claim

1. A use of an antimicrobial agent selected from the group consisting of premafloxacin, premafloxacin-like compound, ciprofloxacin, enrofloxacin, cefquinome, cefpodoxime, gentamicin and erythromycin for the preparation of a medicament to treat or prevent systemic bacterial diseases in mammals which comprises topically administering to such mammal an effective amount of said antimicrobial agent in a DMSO/H2O carrier wherein said DMSO/H2O carrier comprises at least 10% of DMSO by volume.

2. The use according to claim 1 wherein said DMSO/H2O carrier comprises 60%
DMSO by volume.

3. The use according to claim 1 wherein said DMSO/H2O carrier comprises 100% DMSO by volume.

4. The use of claim 1 wherein the antimicrobial agent is premafloxacin.

5. The use of claim 1 wherein the antimicrobial agent is a premafloxacin-like compound.

6 The use of claim 1 wherein the antimicrobial agent is ciprofloxacin.

7. The use of claim 1 wherein the antimicrobial agent is enrofloxacin.

8. The use of claim 1 wherein the antimicrobial agent is cefpodoxime.

9. The use of claim 1 wherein the antimicrobial agent is cefquinome.

10. The use of claim 1 wherein the antimicrobial agent is gentamicin.

11. The use of claim 1 wherein the antimicrobial agent is erythromycin.

12. A use of an antimicrobial agent selected from the group consisting of premafloxacin, premafloxacin-like compound and premafloxacin ester for the preparation of a medicament to treat or prevent systemic bacterial diseases in mammals which comprises topically administering to such mammal an effective amount of said antimicrobial agent in a PG/OA carrier.

13. The use of claim 12 wherein said PG/OA carrier comprises about 95% of PG
and about 5% OA by volume.

14. The use of claim 12 wherein said antimicrobial agent is premafloxacin or premafloxacin ester.

16. The use of claim 12 wherein said premafloxacin ester is a compound of formula II or a pharmaceutically acceptable acid addition salt thereof wherein R is C1-10 alkyl, C1-10 alkenyl, C1-6 cycloalkyl, phenyl, benzyl, or napthyl.

16. The use of claim 15 wherein R is C1-4 alkyl.

17. The use of claim 15 wherein R is ethyl or butyl.

18. A use of an antimicrobial agent selected from the group consisting of premafloxacin, premafloxacin-like compound and premafloxacin ester for the preparation of a medicament to treat or prevent systemic bacterial diseases in mammals which comprises topically administering to such mammal an effective amount of said antimicrobial agent in a PG/Azone carrier.

19. The use of claim 18 wherein said PG/Azone carrier comprises about 95% of PG and about 5% Azone by volume.

20. The use of claim 18 wherein said antimicrobial agent is premafloxacin or premafloxacin ester.

21. The use of claim 18 wherein said premafloxacin ester is a compound of formula II or a pharmaceutically acceptable acid addition salt thereof wherein R is C1-10 alkyl, C1-10 alkenyl, C1-6 cycloalkyl, phenyl, benzyl, or napthyl.

22. The use of claim 21 wherein R is C1-4 alkyl.

23. The use of claim 21 wherein R is ethyl or butyl.

24. The use of claim 1, 12 or 18 wherein said antimicrobial agent is topicallyadministered in an amount of from about 0.1 to about 100 mg/kg body weight/per day.

25. The use of claim 1, 12 or 18 wherein said antimicrobial agent is topicallyadministered in an amount of from about 1.0 to about 50 mg/kg body weight/per day.