BACKGROUND OF THE INVENTION

Despite the development of effective topical and systemic antibiotics, invasive wound sepsis and septicemia from

Pseudomonas aeruginosa remain problems in seriously burned patients. Emergence and development of drug resistant species of bacteria have defied the control obtained through the regimen of potent antibiotics. In recent years, numer- ous reports of gentamicin resistant gram negative organisms (Shulman, J.A., Terry, P.M., Hough, C.E.: Colonization with a gentamicin resistant Pseudomonas aeruginosa pyocine type 5 in a burn unit. J. of Inf. Diseases 124;S18, 1971), espe¬ cially Pseudomonas, have appeared in the literature. (Snelling, C.F.T., Ronald, A.R. , Cates, C.Y., et al.: Re¬ sistance of gram negative bacilli to gentamicin, J. of Inf. Diseases 124:S264, 1971; Chadwic , P.: Resistance of Pseudo¬ monas aeruginosa to gentamicin, Canadian Med. Assoc. J. 109:585, 1973; Bryan, L.E. , Shahrabadi, M.S., Van Denelzen, H.M. : Gentamicin resistance in Pseudomonas aeruginosa. R-factor mediated resistance. Antimicrobial Agents and Chemotherapy 6:191, 1974). -Although silver sulfadiazine (AgSD) , presently the most commonly used topical agent in the treatment of burn wound infections (Fox, Jr., C.L.: A new topical therapy for Pseudomonas in burns. Arch. Surg. 6_:184, 1968; Fox, Jr., CL. , Rappole, B. . , Stanford, J.W. : Control of Pseudomonas infection in burns by silver sulfa¬ diazine, Surg. Gyn. Obstr. 128:1021, 1969), appeared to sur¬ mount these problems, Pseudomonas infections resistant to silver sulfadiazine treatment have been reported recently in burned patients (Gayle, W.E., Mayhall, C.G., Lamb, A., et al.: Resistant enterobacter cloacal in a burn center. The effectiveness of silver sulfadiazine, J. of Trauma 18:327, 1978; Heggers, J.P., Robson, M.C.: The emergence of silver

OMPI

^

sulfadiazine resistant Pseudomonas aeruginosa. Burns 5_:184, 1978) .

Similar occurrences of AgSD-resistant Pseudomonas infections in patients have been observed in other parts of the world. Several such resistant strains have been obtained and the nature of their resistance studied in an experimental burn model. This investigation revealed an unusual phenomenon, namely, normal sensitivity of Pseudomonas to AgSD in vitro, but resistance to topical AgSD therapy in infected burn wounds in mice and rats. (Modak, S., Stanford, J.W., Bradshaw, W. , Fox, Jr., C.L.: Silver sulfadiazine resistant Pseudomonas infection in experimental burn wounds. 3rd

Intrl. Congr. of Pharma. Treatment of Burns, 1980 (in press) ed. Donati,L., Burke, J., Bertelli, A., Italy,)

Comparative studies of the virulence and drug sensitivity of in vivo AgSD sensitive and nonsensitive strains were carried out to investigate the possible mechanism of in vivo resis¬ tance. Since all the resistant strains obtained from burn patients appeared to be sensitive in vitro, the evaluation of a topical agent for its effectiveness was determined in experimental burn models. Several other antibacterial agents known to be effective in vitro were also ineffective against these strains.

The continued search for an effective topical agent led to the discovery that l-ethyl-6-fluoro-l,4-dihydro-4-oxo-7-

(l-piρerazinyl)-3-quinoline carboxylic acid and metal salts thereof (referred to hereinafter as QC and its metal salts as MeQC) possess high anti-Pseudomonas activity in vitro (Ito, A., Hira, K., Inoue, M. , et al.: In vitro antibacter- ial activity of AM-715, a new nalidixic acid analog.

Antimicrobial Agents and Chemotherapy 17:103, 1980, and French patents 879,106 and 870,576), and are effective in controlling AgSD-resistant Pseudomonas infections in burned

OM

mice. See also our co-pending U.S. application. Serial No. 193,307, filed October 2, 1980, the disclosure of which is hereby incorporated by reference into this application.

Specifically, that application discloses QC and MeQC, e.g., AgQC, as topical antimicrobials useful in burn therapy.

SUMMARY OF THE INVENTION

It has been found that compositions containing l-ethyl-6- fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-guinoline car¬ boxylic acid (QC) and/or its metal salts (MeQC), such as its zinc salt (ZnQC) , cobalt salt (CoQC) , cerous salt (CeQC) ^' , silver salt (AgQC) and magnesium salt (MgQC) , together with silver sulfadiazine (AgSD), provide improved compositions use¬ ful in burn therapy and compositions generally useful for combatting topical or surface or skin infections, including microbial and/or fungal infections and the like.

Moreover, it has unexpectedly been found that compositions comprising the silver salt of l-ethyl-6-fluoro-l,4-dihydro- 4-OXO-7-(l-piperazinyl)-3-quinoline carboxylic acid and silver sufadiazine, in which the compounds are present in amounts which would be ineffective if only one of the co - pounds were present, are useful in the treatment of burns in animal and man. These compositions may be applied to the affected surface or burned surface of a burn victim, either directly, or preferably in the form of a composition which includes a physiologically acceptable carrier, such as a water-dispersible hydrophilic carrier, e.g., an oil-in-water dispersion.

DETAILED DESCRIPTION OF THE INVENTION

l-Ethyl-6-fluoro-l,4-dihydro-4-oxo-7- (l-piperazinyl)-3- quinoline carboxylic acid (QC) has the structure:

and is known to have antibacterial activity in vitro against standard bacterial strains such as B. subtilis, S. aureus, P. aeruginosa, and E. coli strains. Ito, A. et al.. Antimicrobial Agents and Chemotherapy 3/7:103, 1980, supra. The metal salts of QC, i.e., MeQC, viz.MgQC, CoQC, ZnQC, CeQC,and AgQC are also of interest and appear to be suitable topical antimicrobial agents. For example, AgQC, which would appear to have the structure wherein t e _^ ^COOH moiety of QC is changed to^-COO"" Ag ⁺ , is a potent antimicrobial. Unlike certain compounds which have high in vitro antibac¬ terial activity, but are ineffective in controlling silver sulfadiazine-resistant Pseudomonas infections in burned mice, QC and MeQC, are effective in con¬ trolling such infections when employed in amounts or concen¬ trations greater than about lOmM. At lesser amounts or con¬ centrations, the silver salt is ineffective.

The metal salts of l-eth l-6-fluoro-l,4-dihydro-4-oxo-7- (l-piperazinyl)-3-quinoline carboxylic acid, i.e., MeQC, are readily prepared. For example, AgQC may be prepared as fol¬ lows. QC is obtained directly or synthesized by known tech- niques. The sodium salt of l-ethyl-6-fluoro-l,4-dihydro-4- oxo-7-(l-piperazinyl)-3-quinoline carboxylic acid, i.e., NaQC, may then be prepared by adding an equimolar amount of sodium hydroxide to the QC. The silver salt, i.e., AgQC, may be prepared by reacting the NaQC in an aqueous solution

with a stoichiometric quantity of a suitable silver salt such as silver nitrate, silver chloride, or the like.

Silver sulfadiazine is well known as an effective agent in burn therapy. However, the silver sulfadiazine must be em¬ ployed in compositions in amounts or concentrations greater . than about 1.0 percent by weight. At lesser amounts, silver sulfadiazine (AgSD) is ineffective.

Not only have combinations of AgSD and QC and MeQC been found to provide potent antimicrobial compositions, but, also, it has unexpectedly been found that a synergistic re- suit is obtained when AgSD and AgQC are combined for use in burn treatment. Specifically, compositions useful in burn therapy have been discovered in which the amounts of AgSD and of AgQC are below the amounts required for antibacterial activity or effectiveness if only one of the compounds is present or included.

Thus, compositions useful in burn therapy may be prepared in which the amount or concentration of AgQC is less than about lOmM, such as an amount from about ImM up to about lOmM, e.g., about 3mM; and the amount or concentration of AgSD is less than about 1.0 percent by weight, such as an amount from about lOmM up to about-1.0 percent, e.g., about 30mM.

The compositions of this invention may be applied directly to the surface of burn wounds or infections, or, preferably, may be employed in combination with a physiologically ac¬ ceptable carrier. When employed in a composition with a physiologically acceptable carrier, the carrier is desirably a"conventional water-dispersible, hydrophilic or oil-in-water carrier, particularly a conventional semi-soft or cream-like, water-dispersible or water-soluble, oil-in-water emulsion, which may be applied to an affected burn surface or infected surface with a minimum of discomfort.

Suitable compositions may be prepared by merely incorpora¬ ting or homogeneously admixing finely divided compounds with the hydrophilic carrier or base or ointment. One tech¬ nique in accordance with this invention for incorporating the metal salts, e.g., the silver salt, in a hydrophilic ointment, such as an oil-in-water emulsion, involves react¬ ing equimolar aqueous solutions of silver nitrate and NaQC to yield a white precipitate which is AgQC. The resulting precipitate, after washing and drying, is then mixed or blended with the candidate hydrophilic ointment, such as the oil-in-water emulsion, to yield a composition which in¬ cludes the silver salt dispersed in the ointment. The AgSD may be incorporated into the composition either together with the AgQC or independently.

Compositions in accordance with this invention containing AgSD and MeQC dispersed in a water-dispersible, hydrophilic carrier or ointment, e.g., a hydrophilic, oil-in-water emul¬ sion, are usually characterized by the following components and amounts by weight set forth in Table I:

TABLE I Component Amount Petrolatum 0-25

Water-insoluble C ₁₆ -C22 fatty alcohol 7-45 -Emollient 0-15

Emulsifying Agents, preferably non-ionic 4-16 Humectant 7-40 AgSD lOmM-1.0

QC or MeQC lmM-lOmM Preservative 0-0.3 Deionized or Distilled Water q.s. 0-80 100

Amounts are in percent by weight unless otherwise indicated.

The fatty alcohols, stearyl alcohol, cetyl alcohol, lauryl alcohol and yristyl alcohol are useful in the preparation of compositions in accordance with this invention. These preferential oil-soluble fatty alcohols act as stiffeners in the resulting compositions. As the emollient, isopropyl myristate, lanolin, lanolin derivatives, isopropyl palmi- tate, isopropyl stearate and the corresponding sebacates and other known emollients are suitable. As the emulsifying agent sorbitan monooleate, such as an amount in the range 0.5-4 percent by weight, and polyoxyl 40 stearate in an amount in the range 7-12 percent by weight, both non-ionic emulsifying agents, re satisfactory. A suitable humectant would be propylene glycol, sorbitol, or glycerin,or mixtures thereof, all being water-soluble compounds. A suitable pre¬ servative would be any of the useful conventional water- soluble preservatives which exhibit anti-microbial activity, such as sorbic acid, benzoic methylparaben, propylparaben, and mixtures thereof.

In the formulation of a composition having the make-up set forth in Table I hereinabove, as the amount of aqueous phase is increased, the solid content, i.e., the water-immiscible or water-insoluble components, e.g., fatty alcohol, such as stearyl alcohol, and/or petrolatum, must also be increased relatively to help stiffen the composition. The preserva¬ tive, e.g., methylparaben, is employed in the formulation only as a preservative for the overall composition and, as indicated, methylparaben was found to be a satisfactory preservative. Methylparaben, as indicated, however, may also be used in combination with propylparaben.

Accordingly, compositions useful in the practices of this invention would include compositions comprising 0-25 per¬ cent by weight petrolatum, 7-45 percent by weight stearyl

alcohol, 0-15 percent by weight isopropyl myristate, 5-20 percent by weight of an emulsifying agent, 7-40 percent by weight propylene glycol, lOmM-1.0 percent by weight AgSD and ImM-lOmM of AgQC, the remainder being water, as re¬ quired, to bring the total percentage to 100 percent. Other useful compositions would include compositions con¬ sisting essentially of 3mM AgQC, 30mM AgSD, 7-8 percent by weight propylene glycol, 38-44 percent by weight water,

14-18 percent by weight petrolatum, 14-18 percent by weight stearyl alcohol, 5-8 percent by weight isopropyl myristate, 0.5-2 percent by weight sorbitan monooleate and 6-10 percent by weight polyoxyl 40 stearate. Another composition useful in the practice of this invention would include the composi¬ tion consisting essentially of 0-25 percent by weight petro¬ latum, 7-45 percent by weight of an aliphatic fatty alcohol having a carbon atom content in the range C15-C22' 0-15 per¬ cent by weight of an emollient, 7-16 percent by weight of an emulsifying agent, 7-14 percent by weight of a humectant, 3mM AgQC, and 30mM AgSD.

Although the preceding discussion has primarily concerned the use of AgSD in combination with QC or MeQC, especially AgQC, it is likely that other metal salts of sulfadiazine may be usefully employed in the practices of this invention. For example, zinc sulfadiazine, cerous sulfadiazine, and cobaltous sulfadiazine are known to be useful as topical antimicrobial agents in the treatmentof burn victims. Thus, it may be that combinations of one or more of these compounds with QC or MeQC will provide advantages when employed in burn therapy or the treatment of surface infections.

The results of various experiments illustrating the practices of this invention are now set forth.

EXPERIMENTAL DETAILS

METHODS AND MATERIALS

Bacterial Strains: Ps. Boston was the strain used in our previous investigations (Fox, Jr., C.L., Sampath, A.C, Stanford, J.W.: Virulence of pseudomonas infection in burned rats and mice. Arc. Surg. 101:508, 1970); Ps. Mangalore was isolated from a burn patient in Kasturba Medical College, Mangalore, India; Ps. 181 was obtained from Hospital de los Ninos, Lima, Peru; and AgSD resistant Ps. Boston was produced in our laboratory by repeatedly growing this organism in medium containing increasing amounts of AgSD.

In vitro assay of microbial inhibition: Inhibition in¬ dices are obtained by tube dilution tests using nutrient broth. Growth in the presence and absence of drugs was observed by turbidity measurement after incubation at 37°C for 24-48 hours (Fox, Jr., C.L., Modak, S.M., Stanford, J.W.: Cerium sulfadiazine as a topical agent for burn wound infections: A comparison with silver sulfadiazine and zinc sulfadiazine. Burns 4_:233, 1978) .

Animal experiments: Mice (female Swiss 18-22 grams) re¬ ceived scalds using methods reported previously (Fox, Jr. , C.L.: A new topical therapy for pseudomonas in burns. Arch. Surg. 96_:184, 1968; Fox, Jr., CL., Sampath, A.C, Stanford, J.W.: Virulence of pseudomonas infection in burned rats and mice. Arch. Surg. 101:508, 1970; Fox, Jr., CL. , Modak, S.M., Stanford, J.W.: Cerium sulfadiazine as a topical agent for burn wound infections: A comparison with silver sulfadiazine and zinc sulfadiazine. Burns : 233, 1978) . The wounds were contaminated one hour post burn with freshly prepared 18-20 hour broth culture of pseudomonas diluted to optical density 0.30. Infection was induced by immersing the tail in the culture.

O

The first treatment was administered 4 hours post in- fection by rubbing the medicated creams over all burned surfaces. All drugs used were mixed in a cream base such as described hereinabove. Thereafter, all animals were observed and treated once daily. The primary criteron was survival. Animals that succumbed were autoposied and the cardiac blood cultured to verify the presence of pseudomonas.

RESTJLTS

In vivo efficacy of silver salt of l-eth l-6-fluoro-l,

4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid in combination with silver sulfadiazine

In vivo efficacy of the silver salt of the carboxylic acid and of silver sulfadiazine against Ps. Mangalore, Ps. 181, and AgSD-resistant Ps. Boston were tested and compared with that of a combination of both compounds in burned mice. The results are summarized in Table II. After infection with these resistant strains, the mortality with 30mM silver sulfadiazine therapy was 80 percent by the eighth day post burn. In the groups of mice receiving topical therapy with the silver salt of the carboxylic acid, the mortality was 0 percent for both Ps. Mangalore and Ps. 181 infection when the concentration of the drug in the cream was lOmM/kg. When lower amounts were used, there was 45- 80 percent mortality. The synergistic results obtained when both compounds are present are clearly shown.

TABLE II

Topical Therapy < 3f Burned ] Mice

Infected with AgSD Resistant Pseudomonas

No. of -% Mortality (Days Post Burn)

Groups Mice 2 3 4 5 6 7 8

Control 10 40 100 100 100 100 100 100

30mM AgSD 10 10 10 30 50 80 80 80 lOmM Ag salt 10 0 0 0 0 0 0 0

6mM Ag salt 9 0 10 10 10 10 30 45

3mM Ag salt 5 20 40 40 40 40 80 80

3mM Ag salt

+ 30mM AgSD 5 0 0 0 0 0 0 0

-13 -

In Vivo Efficacy

Mice anesthetized with ether were given a 30 percent scald by dipping the lower third of their body into a water bath at 68°C for 7 seconds. One hour post burn, mice were given 1 ml of Normosol by I.P. injection and then infected by dipping the tail in an overnight culture of pseudomonas diluted to 0.30 O.D. at 600nm.

The animals were divided at random into groups of 5, and topical therapy was initiated 4 hours post infection. Thereafter they were treated once daily. The primary cri¬ terion was survival. Animals that succumbed were autopsied and their cardiac blood was cultured to verify the presence of pseudomonas sepsis.

The results were as follows:

Ps. Boston and Ps. Mangalore Infection:

Cream used in Silvadene; see footnote 2.

TErade aik for silver sulfadiazine with a cream carrier manufactured and sold by Marion Laboratories, Inc., Kansas City, Missouri 64137.

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TABLE III

Weights of Rats Showing Results of Tbpical Therapy with Various Coπpositions

Topical Agents Days Post Burn 1 3 5 7 9 11 13 15 17 18 21

Control #1 195163 - ¹ - - - - - - - -

Control #2 193159 - - - - - - - - -

Control #3 201172 - - - - - - - - -

Ag Sulfadiazine #1 202181150 - - - - - - - -

Ag Sulfadiazine #2 194178148 - - - - - - - -

Ag Sulfediazine #3 194166 - - - - - - - - -

Silver Salt #1 202191189203205212203202210206207 Silver Salt #2 194187190198200210212215217210216

Silver Salt #3 195190183191197200 218219223225225

Ag Salt + AgSD #1 207200203192188206210214214215226

Ag Salt + AgSD #2 195190193196200209213218223216217 AG Salt + AgSD #3 193193199 207213220219219223218224

i3ashι ed lines indicate rats died.

TABLE IV

Topical Therapy of Burned Mice Infected with Ps. 181

% Mortality

Topical Agents Concentration (Days P.B.) 2 7

None 80 100

Silver Salt 3mM 100 100 Silver Salt 6mM 20 100

Silver Sulfadiazine 1.0% and Silver Salt 3mM 0 0

Silver Sulfadiazine 1.0% and Silver Salt 6mM 0 0

SILVADENE 1.0% 40 100