CA1192491A - Silver salts of phosphanilic acid - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Monosilver phosphanilate and disilver phos-phanilate were prepared and found to be potent anti-bacterial agents, especially against strains of Pseudomonas, suitable for use in the topical therapy of burns. A method is also disclosed for inhibiting the growth of bacteria on an inanimate object, comprising contacting the object with a bacterial growth inhibiting amount of a silver salt of phosphanilic acid.

Description

S~-1600 ~~

S~LVE~ S~LTS OF PHOSP~ANILIC ACID

BACXG~OUND o~ T~E INVENTION

1~ Field of th~ Invention~
Th~ present invention provi~es the ~ilver salts oE phosphanilic acid which are potent anti-bacterial agents ~p~clally aga~n~t strain~ o Pseudomonas and thus useful for topic21 application in the therapy of burns.

2, Description of the Prior Art.
Silver sulfadiazine is an an~ibacterial agent used in the topical treatment of burns. It has dis-advantagPs of sulfonamide sensitivity and the n~ed to monitor sulfonamid~ concentrations ~n the bloodstream to preve~t renal dys~unctio~ and crystalluria when thz burned area covers more ~han twenty percent of the total body surface. It has also been reported to be effectiYe in the topical trea~ment o~ skin ulcers and herpes virus.
It is o~ particular value in burn therapy because of its po~ent antibacterial activi~y agai.~st infections by strains of Pseudomonas which occur often in such patients.
Silver Sulfadiazine wa~ apparently first dis-closed in ~.S. ~,422,~8 and se~ al~o UOS~ 3f761,590 and U.S. 3,79~,161. Various ~omplexes are described in U~S~ 2,536,09~ and U~S. 2,629,682. Sllver derivative~ of pyrimidines and purines are disGlo~ed in Farmdo~
69657R~ Other preparation.~ and-uses are disclosed ln Chemiral Abstracts, 81, 158672c, Farmdoc 04862X, U,S.
.__ 4,0~0,150, Farmdoc 70776Y and ~armdoc 39S20A. For com~inations with other, novel metal salts of sulfa-diazine, ~.g, zinc and cerium, see U,S. 4,088,754, U~S, 4,078,058 and Fanmdoc abstracts 63047Y, 73999X and 04160A.
Silver zi~c allantoinate is disclosed in U.S. 3,856,805 as a new compound with bactericidal and wound~healing properties~ Silver allan~oin~te has been described for the treatment of burns in U.K, 1,346,544. See also U.S~ 3,830~824, ~,830,825 and 3,830,908 which broaden ~he concept to include other acids and metals such as zinc.
Silver sulfadiazine is marketed as "SIrVADENEI' Cream by ~arion La~oratories Inc., Xansas City, ~issouri;
see pages 1073 - 1074 of the 32nd edition of Phvsicians' Desk Reference published bv Medical Economics Company, OradPll, N. J. (19781. Its preparation is described in Farmdoc abstract 39 520A. The in vitro antibacterial activity of silver sulfadia2ine has been renorted, for exam~le, by C~rr et al., ~ntimicrobial ~ents and Chemothera~y, 4 (5~, 585-587 (1973) .
Current Science (India), 16 , 223 225 (1947) repc)rts the ln vitro antibacterial activity o~ phos-~hanilic acid and some of its derivatives asai~st E. coli, S. aureus, T~hl muriu~, C. xerosis and Boyd II.
Current Science (India), 17, 125-6 (1948) reports blood level and toxicity studies with ~hos-phanilic acid in laboratorY animals. Their results indicated poor absorption and low toxicitvO
}~ Bauer, J. l~ner. Chem. Soc,, 63, 2137 2138 (19411 reported a synthesls of phosphanilic acld as have others such as G. O. 3Oak et al., J. Amer.
Chem. Soc., 74, 753-754 (19~2). A few salts are also described in these publicationsO

J. Pharmacol. EXpl Therap., 74, 163~173 (1942) re~orts th~ testing of various sulfonamides, sulfones and relate~ phcsphorus compound~ against experl~ental tuberculosis~ They foun~ that phosphanilic acid had good tuberculars~atic activity in vi~ro but no effec~
in -~ivo, pres~mably due to low bloo~ concentrations ~: =
of phosphanilic acida Chemical Abstracts, 55, 2883 g ~1961) ~from Texas Repts. Biol. and Med., 18, 379-3g4 ~1960)] reports that phosphanilic acid had some protective erfect when tested in white mice aaainst a standardized infection produced by intravenous injection of the yeast phase of Histoplasma capsulatum.
Antibiotics and Chemotherapy, 3, ~56-~64 (1953) report the in vitro activity of some aromatic phosphonic and phosphinic acids against a variety of bactPrial species. Phosphanilic acid was reported as being generally the mos~ activa of th~ compounds tested.
Its activity approached that or sulfa~hiazole when com-pared on a molar basi3 and its antibacterial s~ectrum was similar to tha~ of sulfanilamide. It was also st~ted that a later paper would report in detail the findings that phosphanilic acld was effective ln vivo in mice infected with S, tvphosa, Ps fluorescens and Plasmodium ber~hei.
U. S. Patent 3~159,537 discloses the poten-tiation of various tetracycline antibiotics by admixture with ~or salt formation with) vaxiou~ organic oxvphos-phorous compounds, including phosphanilic acid.
Ciencia (Mexico), 17, 71-73 (1957) reports studies of the ln vitro synergism of mi~tures o~ phos-phanilic acid with neomycin or strep~omycin on various clinicallv isola~ed strains or Salmonella, Shi~ella and Pro~eus as well as colifo~m bacteria capable o inducing fermentation of lactose. The combination of ~hosphanilic acid and neomvcln showed 5vnerglsm a~ainst Salmonella, Proteus and the colif orm bacteria . With Shi~ella, however, there was onlv ~light svnergism for low con-centrations and onl~ an additive effect, or no effec~, at medium and hi~h concentrations~
It was the o~ject o~ the present inven~ion to provide improved ag~nts for burn therapy which, in additions lacked the disadvantage of silver ~ulfa-diazine.

SU~RY OF THE INVE~TION

The objec~i~re of the pre~ent ir~vention wer~
achieved by the provi~ion, acc:ording tca . he present i.nv~tion, of silver ~iill~t5 of phosph~nilic acid and particularly of monosilv~r phosphanilate and di~i~v~x pho~phanilate .
Thexe i3 ~urther provided by the ~resent ir~vention the process for prepar~g a ~ ter salt o phosphanilic acid which comprises mixing, pre~era~ly at roolr. t~p~rature, an aqueous ~uspen~ion of phosphanili ::
acid with an aqu~ous solution containing approximately ~ither c~ne or two moles of ~ilver nitrate per mole o phosphanilic acid to or~ a mixture which i5 then mixed with a water miscible organic s01vent, preferably acetc)ne and prefera~ly by slow addition to the organic solvent with the optional addition of a small amount of ammonium hydroxide, ~o precipitate the deslred proc~uct.
In a typlcal for;nula~ion a salt o the present invention is incorporated, preferably in micronized form in a water-miscible cream which is preferably a hypo-al~ergenic cream at a concen~ation, for ex~npla, in the ranse of 1 to 100 mgm, ~nd pre erably about 10 mgm, of salt per gram of cream vehicle. P~ ~uitable cream vehicle consists of white petrolatum USP, stearyl alcohol USP ~ isoproPyl myristate ~ sorbitan monooleate, poly-oxyl 4a stearate tJSP, propylene gl~Jcol USP and water, with METHYLPARABEN* USP 0.3~6 as a pre~3ervative~ The propor~ions are those cus~omary in the art.
In use the burn wounds are cleansed and debrided and the silver phosphanilate cream is appïied with stexile, gloved hand~ The b~lrn areas should be ~overed with this cream at all timi2!~;o The cream is best applied once to twice dailv to a thickness of * Trade Mark appro~imately 1~16 inch~ Whenever necessary, this cream should be reapplied ~o any areas from which it has been removed by patient activity. Treatment with this cream should be contin~led until satisfactory healing has occurred or until the burn site is ready for grafting.
In this divisional specification there is provided a method for inhibiting the growth of bacteria on an inanimate object, comprising contacting the object with a bacterial growth inhibiting amoun-t of a silver salt of phosphanilic acid.
In a preferred embodiment the silver salt is monosilver phosphanilate or disilver phosphanilate. Also in one embodiment the method as set out above applies to inhibiting the growth of pseudomonas aeruginosa.

DESCRIP~ION OF TH~ PREFERR'r~D Er~oDI~ Ts EXAMPI.E

C6H7~03PAg : H2N / /_1_OAg Synthesis of Monosilver Phos~hanilate To 1.50 g. (0.0087 mole~) of Phosphanilic Acid in 525 ml. of Deioni~ed ~1ater at 25C wa~ added 1.47 g. (0.0087 moles) of Silver Nitrate ~re~iouqlv dissolved in 75 ml. of Deioni~ed ~'ater. The ab~ve mixture wa~ added slowly to 600 ml. of Acetone and 1.3 ml. Concentrated Ammonium Hydroxide added.
Aft~r stirring at 0C for one hour~ the rPaction mix~ure was filtered and the precipitate was dried in a vacuum oven for 16 hours at 4~C. After dr~in~
the material is an off-white to pink, crvstalline powder with a yield of 1.90 g., 78% vield; I.R. and elemental analvsis were con~istent. ~he product contained 2 . 4 8 % water ( KF ) .

C6~7~1O3PAg P~equire~: C - 25~74 E~ - 2.53 _ 5~00 ~ g_ 3~.53 Found: C - ~3 . 50 Ei - 2,36 N - 4.72 Ag 37.40 Crvstal Form: Diamond shaped and crystal fra~ments.

f~

The crystalline Silver ~hosphanilate so formed has been shown to hava considerable anti pseudomonal activity.

EXAMP~E 2 /~=\
C6H6N03PAg2 ~2N ~ ~ ~ \ ~ 0 S~nthesis of Di~ilver Phosphanilate To 100 mg. (0.0058 moles) of Phosphanilic Acid in 35 ml. of Deionized llater at 25~C was added 200 ~g. ~0.00118 moles) of Silver .Titrate previouslv dissolved in 5 ml. of Deionized ~later. The above mixture was added 510wly to 100 ml. of Acetone and two drops of Concentrated Ammonium Hvdro~ide added.
After stirring at O~C for one hour~ the reaetion mixture was filtered and the precipi~ate was ~ried in a vacuum oven for 16 hours at 40C. After dr~ing the material i5 an off-white to pink, cr~stalline powder with a ~ield of 150 mg., 67~ vield.

Crvstal Form: Crvs~al fraqments.

The produc~ containe~ 0O4~ ~Jater (~

C6H6`~O3PAg2 Requires C - 18.63 H - 1.57 N - 3.62 Ag- 55.77 _ Found: C 18.~3 H ~ 1~50 _ 3.74 ~ 54.00 The crys~allln~ Di~ilv~r Phosphanilake so formed has been shown to have con~iderable anti-pseudomonal activity.

I~ VIT~0 ACTIVI-Y

Bacteria. ~he orsanisms, preponderantlv of r~c~nt clinical o.rigin, were obtain~d from numerous sources of broad geographical distribu~ion. O~ligate anaerobes were maintained in Egg ~eat r~edium tDifco);
Mycobact~rium wa~ stoxed on ~owenstein ~edium [J~nsen ?~odification; Difco). The techniques of storin~ all other organisms have been describ2d pre~iously ~Leit-ner et al., BL-S640~ A Ce~halosporiA with a Broad Spec~rum of Antibact~rial Activitv: Properties in vitro, Antimicrob. h~nt~ Ch~mother. 70298 305 ~1975)].
An~ibio~ic spectrum. ~he growth-inhibitor~
activit~ of the compounds was d~e~mined by the anti-biotic dilution technique. Procedures wer~ as follo-~s:
Aerobic organisms (excluding MycobactPrlum)0 Except for Haemophilu~ and ,lelsseria~ the assay was performed in ~uellPr~inton ~1e~ium (Difco). ~or astldious organisms, i.e., Streptococcus, Lis~eria, Pasteur211a, Bordetella and Vibrio~ the medlum was suppl2mented with 4~ defibri.nated sheep hlood. ~he anti~iotic susceptibility of Ha~mophilus and Meisseria was d~tPrmined in GC Medium Base ~BB~) suppl~nented ith 1~ Hemoglobin (BBL~ and 1% Isovltalex (BBL)~

'10--Overnight broth cultures of an exponenti allv growing culture (Neisseria) served as the source of inoculum. P, volume o a~proxir~atel~ O . 003 ml . of ~he undiluted or dllu~ed culture was applled to the surface of ~he antibiotic~containin~ agar plate~ wi~h the in~
oculator of Steers et al., AII Inocula Replicating Apparatus for Routine mesting of Bacterial Susce~ti-bilit~ to Antibiotics, Antibiot. Chemot;herO 9:307-311 (195~. Cultures of Meisseria, Streptococcus pneumoniae, S. viridan~ and S. pyogenes were used without dilution;
those of all other organisms were diluted lOO~fold.
The inoculum contained about 103 viable cells or ~leisseria, 105 for S. pneumoniae and SO Dvoqenes, 106 for S. viridans, and 104 for all other s~ecies.
The culture plates were incubated at 37C. either overnight or for 24 hours (Ha~ophilus) and ~he minimum inhibitorv concentration~ ~MIC), i . e., the lowest concentration of antibiotic which prevents visible grow~h, was recorded.

MIC ~cg/~l.) organismMono~ilver Silver Phosphanilate Suladiazine of E~ample 1 Str. pneumoniae 32 32

3~ 32 Str. pyogen~s 16 3 Staph. aureus 8 Staph. a~reus ~ 50~ serum 63 ~63 63 >63 Staph. aureus (Pen-Res) ~125 ~125 >125 ~1~5 Staph. aureus (Meth-Res) 16 8 Str. faecalis 8 8 ~ 8 E. cali 8 16 E. coli 16 8 K. pneumoniae 16 63 16 ~3 K, pneumoniae 16 16 Pr. mirabilis 8 8 Pr. mirabilis 4 8

4 8 Pr, morganii 4 8 MIC (mcg/ml. ) Organism Mono5ilver Silver Phosphanilate Sulfadiazine of Example Pr. rettgeri 8 Ser. Marcescen~ 4 16 ~, 16 Ent. cloacae 16 16 En t . c loacae 6 3 3 2 Ps. aeruginosa 4 1 Ps. aeruglnosa 8 8 P~. aeruginosa Ps. aeruginosa Ps . aerug inosa P~. aeruginosa 13 ~

The antibact~rial activity of mono~ and di-silver phosphanilate was also compared with that of silver sulfadiazine agains~: a select group of gram~
po~itive organisms, En~erobacteriacea~ and Pseudomonas aeruyinosa. These compounds were tested in a medi-lm essPntially fr~e of the phosphanilic acid antagonist thymid1ne ~Mueller-~inton Broth + 0 . 04 IU/ml ~hymidina phosphorylase) ~ whereas the da~a reported above on the activi~y of mono~ilver phosphanilate were obtained in nutrient broth wi~hou~ added ensyme.
The table below shows that disilver phos-phanilate was rnarginally more active than monosilver phosphanilate against gram-positive strains and most Enterobacteriaceae. In addition, its antipseudomonal activity was twice that of mono~ilver phosphanilat2.
M. X.C. (mcg/ml) Or~anism Mono- Disilv~r Silver silver Phosph- Sulfa-Phosph- anilate diazin~
anilate S. pneumoniae 8 8 16 S. pyogenes 8 8 16 S~ aureus 8 ~ 16 S~ aureus (Pen-res. ) 16 8 16 S. aureus ~Meth-res. ) 16 ~ 16 S. faeoalis 8 4 4 E. coli 4 4 E. coli 4 4 3 K. pneumoniae 16 a 16 K~ pneun~oniae 8 4 16 P . mirabil is 8 4 16 P. vulgaris 4 4 4 M . I . C . (mcg~ml ) Organism Mono-Disilver Silver ~ilverPhosph- Sulfa-Phosph-anilate diazine P. mor~anii 4 4 P. rettgeri 4 4 S. marce~cens 4 ~ 4 E. cloacae 4 4 8 E. cloacae 32 32 8 P~ aeruginosa 4 2 4 P. aerugino~a 4 2 4 P. aeruginosa 4 2 8 P. aeruginosa 4 2 4 P. aeruginosa 4 2 8 P. aeruginosa 4 2 8 MIC ' s w~re de~enTIined in .~ueller-Hinton Broth + thymidine phosphorylase at 0.04 I,U./ml in every case, except with S. pneumoniae and S. pyogenes wh2re 2~ laked horse blood wa~ added to Mueller Elinton Broth.

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. The method for inhibiting the growth of bacteria on an inanimate object, comprising contacting the object with a bacterial growth inhibiting amount of a silver salt of phosphanilic acid.

2. The method for inhibiting the growth of pseudomonas aeruginosa bacteria on an inanimate object, comprising contacting the object with a bacterial growth inhibiting amount of a silver salt of phosphanilic acid.

3. The method of claim 1 or 2 wherein the silver salt is monosilver phosphanilate.

4. The method of claim 1 or 2 wherein the silver salt is disilver phosphanilate.