CA2191627A1

CA2191627A1 - Synergistic antibiotic compositions containing a perphyrin and an antibiotic

Info

Publication number: CA2191627A1
Application number: CA002191627A
Authority: CA
Inventors: Zvi Malik; Yeshayahu Nitzan
Original assignee: Individual
Current assignee: Bar Ilan University
Priority date: 1994-06-02
Filing date: 1995-05-30
Publication date: 1995-12-14
Also published as: WO1995033463A2; EP0765161A2; AU2816195A; NO965109D0; WO1995033463A3

Abstract

Porphyrins are used as antimicrobial agents in synergistic antibiotic composition, comprising as an antibiotic active ingredient a mixture of one or more porphyrins and at least one antibiotic compound. An antimicrobial composition, comprises as an active ingredient a porphyrin or a mixture of one or more porphyrins, alone or in admixture with conventional antibiotic materials and/or pharmaceutically acceptable or beneficial agents or additives, in a water-based cream composition. Also described is a sterilizing liquid composition, comprising as an active ingredient a porphyrin or a mixture of one or more porphyrins, in an aqueous solution, together with a surface-active agent.

Description

~! W0~5/33463 2 1 9 1 ~127 r~ J. el s~

rlERG~llC ANTlBlOllC COMPOSmONS CONTAINING
A PERPHYRIN AND AN ANTlBlOTlC

Fi~ld of ThP InvPnt;nn The present invention relates to dl "i~ ul,ial and to ~1,,. ", .~
c More particularly, the invention relates to the use of pul~h~lilLs to increase antibiotic efficacy, to ~uLil i~lubial liquid ~Ulllpc,siliul s useful for hygienic and medical uses, and tû ~ILulu~lubio cream ~u~ osiLiu.~s.

BA('~ ROUND OF T~F INVE~TION
It is accepted in modern medicine that the ever increasing need for large amounts of antibiotic ~u~ uuul~ds is undesirable, since it adversely affects thepatients and gives rise to antibiotic-resistant strains, which in turn need new and more potent antibiotics. It is therefore a 5nh5t~nti~l goal of todayls research to be able to increase the efficac,v of antibiotic compounds, ~ithout g them in large dosages.

FulLII~lllulc:, antibiotic materials are widelv used in today's medicine, both for the treatment and for the ~ Y~lR;ul~ of in,ections. Antibiûtics are ~.1",i"i..~ d mostly systemically, orally or illLl~ lwu~ly, but for a number of uses, such as for superficial cuts and skin uounds, antibiotic ointments are also available.

However, in a number of cases antibiotic ointments are ineffective or cannot be used. One such case is the treatment of severe injuries, which are prone to become seriously infected by gram-positive bacteria.

Wo gs/33463 2 1 9 1 6 2 7 - 2 - PCT/lTSgS/0699X

Addihonally, str-ril;7~tion of surfaces is also an important task, particularly for medical applications. One of the problems r ~l~uulrL~ d is that of sterili_ing the hands of surgeons and of medical staff involved in surgery.
This problem is particularlv severe, because gram-positive bacterial nest in the various irreguiarities of the skin, whence they are very difticultly removed.

Th~? Prinr Art Pu~,ully~ have been known for some time to be efficient in the treatment of infections by photodvnamic therapy (PDT) [Malik et al., in "Photodynamic Therapy, Basic Principles and Clinical Applications", B. l~'. Henderson and T.J. Dougherty, Eds., Rosewell Park center Ir~stitute, Buffalo, ~ew Yorkl. The Anrih:~rtPri~l properties of hemin have also been described [Ladan et al., FEMS /\VIiLIU~IIUII~8!;I Zetters, 112(1993),1~3-178].

However, nowhere in the art an attempt has been made to exploit porphyrins to increase the efficacy of antibiotics, and to reduce the dosages of antibiotic materials needed to combat infections.

Another problem which has remained so far unsolved, is that of irfection resulting from exposed bones, either from injuries or from surgery. It is not u~r_u~ wll to discover that severe infection results after an exposed bone inJury, recluiring heavy systemic antibiohc treatment, which is not alwavs capable of uvt~ulllirrg the problem. In chest surgen~, the problem is complicated by the advent of staphylococcus infection, w hich may lead to the death of an othervvise successfully treated pahent. To overcome this problem, the chest bones are today covered with honey. However, this treatment is ~wossl33463 2 1 9 1 627 r~~ S(~!

only partially effective because, although it has some ,UI~Ve:~lLiVt! effect, honey does not possess anv antibiotic activity.

The problem of sterilization of surfaces is dealt with in the art b,v providing soaps of various types, ~hich are effective to remove superficial bacteria.
However, such soaps are practically ineffective against deep-nested bacteria which are not easily removed by a simple washing operation.

Another problem which has remained so far unsolved, is that of sterilizing exposed bones, either from injuries or from surgery, to avoid infection. It is not ~ lul l to discover that severe infechon results after an exposed bone injury requiring heavy systemic antibiotic treatment, which is not always capableofuvt:l.ullLll,gtheproblem.

Porphyrins are known to be effective ~LL~LiLLLL~lubi~ll agents. However, the arthas so far not addressed the use of pull~hyLilLs and LULl~,UUl~ILL~:I of sterilizing solutions for sterilizing hands or other surfaces which come into contact with exposed wounds.

S--mm~ f thP lnvPntinn It is an object of the present invention to provide a ayll~ Lic method and ~olllpobiLiulL~ for increasing the efficacy of antibiotic compounds.

It is another object of the invention to provide means to reduce the amount of antibiotic material needed to be ~. l l "; "; ~ d to a patient in need thereof .

It is still another object of the invention to provide LylLel~,ialL~ oaiLiolLs useful for the treatment of infections caused by gram-positive bacteria.

,, ... . , , ,, ~ . _ . , WO~/33463 2 1 9 1 627 PCIIUS9~106998 It is a further object of the invention to provide ~L-~r,i~Lic .ul.L~o~iLiul,s useful for the treatment of infections caused by gram-negative bacteria.

It has further been found, and this is another object ûf the present invention, that it is possible to provide antibiotic creams which can be used effectively to treat and to prevent gram-positive intection in severe wounds.

It is another object of the invention to provide a cream which can be used to treat burns and to prevent infections resulting from skin burns.

It is a further object of the invenhon to provide a method an compositions for sterilizing exposed bones in the course of surgery or the treatment of exposed bone injuries.

It is yet another object of the present invention to provide solutions which can be used effectively to sterilize hands and other surfaces which mav house bacteria in recesses from which a m!~rh:~nir:ll action is ineffective to remove them.

It is still another object of the invention to provide a solution which can be used to sterilize bones which have become exposed in the course of surgery, or as the result of an injurv.

Other objects of the invention will become apparent as the description proceeds.

~Wo~S/33463 21 ~1621 r~l"~ (tcs~

ln one aspect, therefore, the invention is directed to a ~ylle~ ,Li~ antibiotic ~v~lL~osiLiv~ ULI.IJ.i~il~g as an antibiotic active ingredient a mixture of one or more pul~uhy~ s and at least one antibiotic compound. In another aspect, ~ the invention is directed to a method of treating bacterial infections using reduced amounts of antibiotic materials.

In a further aspect, the invention is directed to an al~Li~ uLial ~olllpv~iLiu.., " " "I " ;~; "g as an active i~ di~l~L a porphyrin or a mixture of one or more pVl~Ul-ylills, alone or rn admixture with conventional antibiotic materials and/or phalll~ ~LILi~ly acceptable or beneficial agents or additives, rn a vrater-based cream ~U~ O~iliUl~, as well as to the use of pu~l~hylills for the treatment and the ,ul~:vellliùn of bacterial infections, and to the method of treatment which utilizes such ~ulll~o~iLiuns Preferred ~ulluhylil~ include d~uL~.u,uu.,ul.y.i.. and hemin.

The invention has as another objective a sterilizing liquid ~u~luv~iLivl~, " " "1'' ;~; ~ ~g as an active ingredient a porphyrin or a mixture of one or more ,uv~hy~ s, in an aqueous soiution, together with a surface-active agent. The combined actiûn of the porphy-rin and the surface-active agent is effective to reduce the ~ al;l)l~ of bacteria on a surface, even when deeply nested, and to maintain it at a low vaiue for a long period of time.

pri~f D.~ iull of th.~ Draw-n~
In the drawings:
Fig. 1 shows the effect of the combined treatment of lt:lla~ y ~lille and hemin on the growth of 5. aureus;

W09~133463 2 1 9 1 627 ~ c~

Fig. 2 illustrates the effect of the combined treatment of mPthirillin and hemin on cell growth of S. RureUS, Fig. 3 illustrates the effect of mr-thirillin, ampicillin, hemin and their ~ ,1,; "~ , on the viabilit~ of S. Rureus;

Fig. 4 shows the effect of the combined treatment of tetracycline and phuLua~liv~L~d d~ui~lutJu-luhy~ on cell growth of 5. aureus;

Fig.; shows the effect of the combined treatment of tetracycline ancd photoactivated d~uL~Iu,Jul~uhy~ on the viabilit~ of 5. aureus;

Fig. 6 shows the ~ylL~ liC effect of a .~ t;.." of an irradiated ~ "~ ;",, rr~mpri~ing dl:uL~lu,uulluh~ and polymyxin B
n~ ulide~ v~ith tetracycline, on the gram negative bacteria E. coii;

Fig. 7 illustrates the effect of d~uLe~ uullJllylill, alûne and with hernin, in a specific base cream (Merck);

Fig. ~ illustrates the effect of tetracycline, and of deLIl~uy~ llylill, alone and with hernin, in another specific base cream aohnson~s);

Fig. 9 iliustrates the effect of Lcll~y~lille, and of d~ uLt:~ u~uul~llylill, alone and with hemin, in yet another specific base cream(Carbûpol);

~I w0 9~33463 2 1 9 1 6 2 7 r~

Fig. 10 illustrates the effect of LL h~v~lilLe and of hemin, aione and in I . ,1 . ,1,; . ,;~ t;' ~- 1, in the Merck base cream; and Fig. 11 illustrates the activity of different sterilizing liquid ,n.~ on S. ~urEus.

Det~ile~ Dec.,;.,1;.... nfThe Inven~inn The ~ylLL L~ Li~ antibiotic ~ulLI~Ju~iLLul~ of the invention comprises as an antibiotic active ingredient a mixture of one or more yul~l,ylh,~ and at least one antibiotic compound.

The invention can be ~ullv~LLL-lLlly exploited with a variety of antibiotics.
Examples of suitable antibiotic materials are those which owe their activity to their ability to inhibit the synthesis of bacterial cell walls by blocking the terminal cross-linking of linear ~,ly~u~,uLides into the complex peptidoglycan, and which activate autolytic enzymes in the cell w alls.
Exarnples of such antibiotic materials are the penicillins and the ceph~lnspnrinc Other antibiotic materials which can be used according to the imrention are those which are active because they inhibit protein synthesis. Illustrative and non-limitative examples of such materials are, e.g.,the lr~L~a~y~ which act by inhibiting the binding of aminoacyl-tRNA to the 30S unit of bacterial ribisomes, chlulud~l",JI"-l".ul, which blocks the .. hlllrlll of amino acids to the nascent peptide chain on the 50S unit of "".-c by Illlrlrr~ g with the action of peptidyl ~ rl~l~r and the macrolide antibiotics, which attach to a receptor on the ~05 subunit of the bacteriai ribosome.

w09~33463 ~ 1 9 1 62 7 P~ 5~ ~

As can be seen, the antibiotic materials useful in ~ulljull Liull with the invention are many and of manv different types. A-~uldi~ly, while the following description will be limited to specific materials, for the sake of brevity, it is not to be construed as intending to limit the invention to any particular antibiotic material.

Illustrative, but non-limitative, examples of specific antibiotic materials which can be ~ul~v~lLL l~Lly used in the context of the invention are penicillin(G and V), arnpicillin, m~thirillin, oYacillin, ~mnYiri71in, cephalexin, cephradine, LtLL~y~lhle, ~lyiluullly-iul and chlu,u~ul,l.h~lu ul, and mixtures of two or more of such ~uulyuuulLlb with or without additional antibiotic and/or antibiotic byllt~ Li- materials.

According to a preferred ~mho~lim~nt of the invenhon, the porphyrin isselected from the group consisting essentially of d~uL~.uluulluhyrin, hemin, herm~,Lu,uu.~,l,y.iul, I luLu~ul~hyLilL, and meta-, para- or orto-hvdroxy ylpul~lly~iul.

The .~ .,l;.". of the invention can further comprise a peptide of the formula:

L-DAB-D-Leu-L-Leu L-Thr-L-DAB-L-DAB~
L-Thr-L-DAB-L-DAB

~I wos~/33463 21916 2 7 r~llL~

wherein DAB* is a 2,4-diaminobutyric acid residue, and DAB is a 2,~
diarninobutyric acid residue font~ining a free 4-amino group. This compound is a known material, which is useful to di~L~l~jdlLi~ the bacterial ~ membrane of gram-negative bacteria. A specific peptide is polSmyxin Bnonapeptide. Therefore, it can be ~ullv~ Lly used when the invention is exploited to combat infections generated by gram-negative bacteria.
However, alterr.ative additives can of course be used for this purpose, and the invention is not limited to the use of any particular additive. It should beunderstood that the L ulll~u~iLiJlls of the invention can be used together with any useful and ph~"~ "~ llv-acceptable additive, carrier and adjuvant, which are not listed nor discussed in detail herein, for the sake of brevity, since they are readily apparent to the skilled person.

Thus, in one aspect, the invention is drrected to the use of a culll~osiLiu~l comprising one or more Luu~,uhylilLs and one or more antibiotic materials, for the ,1.,.l~"r.,. I.11e of a ph~ LL~-tiUll for the treatment of infections caused by gram-positive bacteria.

In another aspect, the invention is directed to the use of a .UllLpU~iLiull .u~ hlg one or more luul~hylilLs and one or more antibiotic materials, for the nn~n-1f~~h1re of a p1,~",-... ~"1.. ,.1 ~JIelJdLdLiOll for the treatment of infections caused by gram-negative bacteria.

The synergistic composition of the invention is useful, in~er alia, in a method of treating an infection in an animal, including humans, which infection is susceptible of treatment by a given antibiotic material or mixture of antibiotic materials, ~ol, ~ ; l ,g ~l l ", ~ , ,g to the anirnal in need thereof an amount lower than the effective amount of the said antibiotic material or mixture of ... . . ... . . ..... .. .. . .. . . .. . , . .... .. .. . .. _ _ .. . . _ . _ .. . .. .

WO 9Sr33463 2 l 9 l 6 2 7 PCT/US95JU6998 antibiohc materials, together with one or more porphyrins. The ~UIlL~U~iliLlLs of the invention can be used effectively to treat infections which, for practical purposes, cannot be treated by the antibiotic material or by the ~
of antibiotic materials cor,tained therein alone, because the dosage required to affect the bacteria exceeds acceptable boundaries. Thus, the ~ylL~ L~,iaLic aiLiLLJlLS of the invention not only sl~hct~nti~lly reduce the amount of antibiotic material needed for a given treatment, which is an extremely important result by ibelf~ but also permits to use antibiotic materials which are essentially ineftective as such, for the treatment of infections so far untreatable or only difficultv treatable by the same antibiotics.

Illustrative examples of bacteria which can be treated according to theinvention are Gram-Positive bacteria, Anaerobic Gram-Negative bacteria, Streptococci sp., Diplococci sp., 5~ ,; sp., P. aemginosa, E. coZi Klebsiella sp., and Proteus vulgaris.

Aâ stated, in another aspect the invention is directed to an dl~LiuLLLLL~
composition, LUIlllJlLaiLLg as an active ingredient a porphyrin or a mixture of one or more pul~lLy-iLLa~ alone or in admixture with Lullvl:lLLiulLdl antibioticmaterials and~or pl,..""~. ~"1;l .~liy acceptable or beneficial agents or additives, in a water-based cream Lull.pusiLiull.

According to a preferred embodiment of the invention, the porphyrin is d~uL~L~ hyliuL or hemin. One preferred LuLll~cl~iLiul, of the invention comprises a mixture of d~uLL-lu~u~ uilL and hemin. Another preferred embodiment of the invention comprises a mixture of hemin and tetracycline.

~ wo gsl33463 2 ~ 9 1 6 2 7 P~

Without wishing to be bound bv any specific theory, the inventors believe that the high efficiencv of the cream .U.ll~ositiul~ of the inven.*on derives from the fact that the cream. is water based. Creams which are based on organic solvents, such as oils, tend to dissolve the porphyrins which thus loose their efficacy.

By ~Icream~ is meant to indicate all water-based ~ulLI~ù:~iLiul~ which are creamy in ~ lc,~ and which can be easily applied in thin layer to a surface. Water-based LOllLI~O~iLiulls may, of course, contain small amounts of materials which may be considered water-l1nmi~rihlr- solvents, such as for the purpose of creating emulsions, or as jellvfying agenb, or for any other purpose, as long as such materials do not s--hst~n*~llv dissolve the f~u~hy dll therein.

An illustrative and non-limitative dl,Li,lli.,uLidl cream ~U-I-~osiLiull according to the invention may comprise, along with other components, the following major ~u---~u-~r"L~. paraffin, propvlenglycol, polysorbat 4Q, cetylstearyl alcohol, ~'aseline and water Another illustrative and non-limitative a..Ll.. i..ubial cream ~ulllpusiLiu~l according to the invention may comprise, along with other .ULIl~Vll~llL~, the following major Lu~ LInellL~. synthetic beeswax, glvceryl stearate, stearic acid, propvlene glycol, al~ d-al,~l.(s), ~ , vailv~s, and water.

The invention is also directed to the use of a cream ~ulll~o~iLiol~ according tothe invention, for the treatment of infections caused by gram-posi.*ve bacteria. Such infections may comprise, inter alia, ir fec*ons caused by flesh wounds, surgical cuts, or exposed bone injuries.

wos~r33463 21915;~7 r.~

-Iq -The cream ~ulllpo~iLiun according to the invention can be Cullv~ llLly employed for the st~rili7.~hqn of bones. A particularly important use, which addresses a need so far unsolved, is the sterilization of chest bones exposed during chest surgery, to prevent the occurrence of post-surgical infechon.

In all the uses described above, when a photoporphyrin is employed, the treatrnent may further be rendered more effective by irradiating the treated area with light. Such irradiation procedure is ~~ithin the scope ûf the rouhneer, and is therefore not described herein in detail, for the sake of brevitv.

The invention therefore provides a method of treating or preventing the occurrence of an infection caused by Gram-positive bacteria, ~Ulll,Uli~ g applying to the area to be treated a cream composition as described above.
Of course, the amount of active material used in a given ~ulll,uusiLiull depends on the type or porphyrin, or porphyrin combination or antibiotic used, as well as on additional additives that may be emploved for a specific purpose. The skilled person wiil easily deterrnine the amount of each active material which it is desirable to employ in a given ~ulll,uo~iLion, by simple e~ .,L~ of the kind described in the examples given below. As an indicative value, however, an average ~ulll~o:,iLiull may comprise each of the pul~hylill(s) auld of the antibiotic material(s) in an amount comprised behveen 5 and 100 llglml.

The synergistic antibiotic ~ulllpo~iliuns described above, which comprise, as an antibiotic active ingredient, a mixture of one or more pul~l-ylills and at least one antibiotic compound, can of course be emploted also in ~ulljul~Liu ~ w0ss/33463 2 1 9 1 627 r ~ o with the antibiotic creams of the invention. However, the creams of the invention are not limited to those which comprise ~y~ Lic ~u~ o~iLiu.ls, and are intended to ~n~mp~cc all the antibiotic ~ulllbiildLiul~s described or referred to herein, which can be ill~ul,uuldL~d in water-based creams, whether they exhibit ~v~ clly improved activity or not. Of course, all the antibiotic materials described above, as well as many others, can be used in ~ulLjull-liu-- with the creams of the invention.

The ~eam ~ull~luo~ihul~ of the invention can of course be used together with any useful and ph~rm:~r~llti~lly-acceptable additive, carrier and adJuvant, which are not listed nor discussed in detail herein, for the sake of brevity, since they are readily apparent to the skilled person.

As stated, the invention has as a further ob~ective a sterilizing liquid ~UIII~JO~iliUII, """1~ ;-''"~ as an active ingredient a porphvrin or a mixture of one or more pu~ ylilc~, in an aqueous solution, together with a surface-active agent.

It has been found that in order to obtain a quick and prolonged sterilizing effect, and to reduce the amount of bacteria present on a surface, it is necessary to utilize both a porphyrin and a surface-active agent. The combined action of these two ...",l,.,l,.-"l~, is effective quickly to reduce the . of bacteria on a surface, even when deeply nested, and to maintain it at a low v alue for a long period of time By "~ lili~,g ~ulllpo~il;u-l" is meant to indicate a composihon that, when applied to a surface infected with bacteria, is capable of reducing the l-~ll~l.l,,.li,.,~ of the bacteria to a non-dangerous level, preferably to a non-Wo gS/33463 2 ~ 2 7 r~ ,s3~ ~

detectable level. However, in some instances a reduction of several orders of m~cgnitl-flP uLil be considered a ~ , when dlJ~UU~id~C~ according to the case, even if the bacteria are stiil detectable on the sterilized surface.

Whiie a variety of pùl~vhy~ can be used in the solutions of the invention, accordmg to a preferred Pmho~iimPnt of the invention the porphyrin is dt:ul~u~ul~hyliul or hemin, or a mixture thereof. These pu~hyliu~ are preferred because they are widely used and mostly do not present regulatory problems, but as stated, other pul~vllyliu-~ are also useful. Other pulyhy~
include, e.g" hematoporphyrin, protoporph-rin, and meta-, para- or orto-hydroxy ph~lyl~ullvllylill.

The surface-active agent appears to be ilc~L u~ lidl in allowing a better contact between the bacteria and the porphyrin, although the actual m~h~ni~m through v.~hich it acts has not been fulk~ eiucidated. The art is crowded with many surface-active agents, some of which may be unsuitable for the purposes of the invention because the may be hazardous to health.
According to a preferred ~mho~iim~nt of the invention, therefore, the surface-active agent is a detergent, preferably a detergent which is in use and is l~co~,.u,ed as being non-hazardous to health. Illustrative and non-~limitative examples of suitable detergents which are ~u~ul~ ially availableinclude Triton X-100 (which is iso-o.Lyl,,h~.lu;.yTvol~t:Lilv~yelll~ulol~
.."~I,.i~;llg d~l~lUXil~ L~l~ 5 moles/lit of ethvlene oxide, 1~ bv BDH Chemicais Ltd., England), sodium dodecyl sulphate and sodium lauryl suiphate, or a mixture of two or more of such agents.

The .~ ,.l".l;..,l of the porphyrin in the sterilizing solution may vary, according to the porphyrin or mixture of ~UI~JBy~ills empioyed, the surface-9sl33~63 ~ P~r/usss106998 active agent used, and the purpose for which it is desired to employ it. Theskilled person will be able to tailor specific sterilizing solutions for specific uses, which is within the scope of the routineer. By way of example, ~ however, in sterilizing ~ulllyOaiLiul1S of the invention the porphyrin may be present in a . . ~ l of l to lOO Ilg/ml of sterilizing solution.

Likewise, the ~u~ Ll.lLiuL~ of the surface-active agent may v ary over a wide range, depending on the surface-active agent employed, the porphyrin used and the purpose for which the solution is intended. Illustrative ~UI1~ .ti~ of the surface-active agent in the solution are O. i - a i~t%.

The invention also Pn~nmp~cc-lc the use of a liquid ~u~ uaiLiOl~ according to the invention, for the 5~ ;nll of surfaces. The surfaces to be sterilized can be of many types, e.g. the human hand or a bone. A particularly iul~uulL~ L case of bone ~ ,1, is when the bones are chest bones exposed during chest surgery.

While in most cases this rnay be ~ lly, it is possible to enhance the sterilizing activity of the solution of the invention, or to speed-up the ~1-. ;li,..lli. .~ 1 process, by irr~ tingr the area to be sterilized with light. Such a use is of course also within the scope of the invention.

The invention therefore provides a method of preventing the O~U11~ 1 of an infection caused by Gram-positive bacteria, .ullL~Iiaillg applying to the area to be treated a liquid sterilizing ~ulll~OaiLiul~ as described above.

Of course, the sylu:L~iaLic antibiotic ~Ulll~UsiLiul1s described above, which comprise, as an antibiotic active ingredient, a rnixture of one or more woss/33~3 2 ] 9 1 627 P~

pu~ ylilL~ and at least one antibiotic compound, can be employed also in with the sterillzing ~ullll~usiLiv~s of the invention. However, ~ullvL-I~Liullal antibiotics are not required in the liquid ~u~ uo~iLiu~ s of the invention. Nevertheless, the addition of amounts of ~UII~:IILiU11al antibiotics to the sterilizing liquid ~u~ osiliuos does not exceed the scope of the invention.

The ~ulll~usiLiulLs of the invention can of course be used together with any useful and IUlIAI ",~ iv-acceptable additive, carrier and adjuvant, which are not listed nor discussed in detail herein, for the sake of brevity, since they are readily apparent to the skilled person. FU-Lh~:...W.-, when the human hand is to be sterilized, ~U~V~-LiU~ d~ aLulo~ dlly-acceptable additives, e.g., perfumes, can of course be employed.

All the above and other ~ . and advantages of the invention will be better Imr1~arch-u-l through the following illustrative and non-limitative examples.

B~~tl~ria1 Strainq This e~ .Ls detaiied below were carried out using both Gram-positive and ~ram-negative bactena. Illustrative Gram-positive bacteria are the coagulase and DNAse positive Staphylococcus aureus which belong to phage h~pe 0[88J89/95] and resistant to the majorih~ of the common antibiotics such as M~thiriliin, Ampicillin, ChlulalllluhLIli~ul, Tetrac,vcline, Ofloxacin, lmipinen, Ciprofloxacin, (:~nt~mirin, and E~yLl~lullLy~ill. Illustrative Grarn-~j Wo 9~/33463 2 1 9 1 G 2 7 P.~ "~

negative bacteria are Fscnerichia coli of serotype 0111jB4, resistant to Ampicillin, Chlu~ uh~lliLul, Tetrac~cline and Sulf,~
LLi~ lho~ . All of these strains were recovered from clinical material submitted to the Bacteriological Laboratory of the Ivleir Hospital, Kfar-Saba, Israel.

~ o- t~ori;l I (~rowth Overnight cultures of S. eureus grown in Nutrient .4.gar (Difco, U.S.A.~ were L~ fc~d into Nutrient Broth (Difco, U.S.A.) at a pH of 6.5, to a final volume of 2O ml, at an initial densit,v of 0.1 at 660 mn and allowed to grow at 37~C with aeration. Pu~hy-iL s (10~g/ml) were added at the beginning of the log phase. When pllulua~liv~ (excluding hemin) PUL~h!~illS were used, the cultures were then irradiated, using two tungsten larnps which were placed 30 cm above both sides of the fiasks. Bacterial growth was deterrnined by the increase of the optical density as a hnchon of time at 66û
nm with a Novaspec Biochrom LKB ~ L u~l oLu.~lc~ . Viable bacteria were ~u~uiul~d by counting the number of colony-forming units on nutrient agar plates.

~nHhi~-*l~c The indicated antibiotics were added, as cdescribed specifically in each example, at the indicated ~u~L~llLL~lLiulls, to the cultures at the time of porphyrin addition, not prior to that, and the e~ i..lenL v~as continued as described above.

~r.-~rn r.. ~ 5 The relevant base cream were mixed with pre-dissolved d~ut~lu~ull~hylill or hemin, from a stock soluhon. The stock solution was prepared by dissolving WO 95133463 2 1 ~ 1 6 2 7 PCT/US95/06998 ~;

~ mg of porphyrin in 2 drops of 0.1 N NaOH, and then diluting in ~hu~haL~
buffer to pH 6.8, to a co~rPntrAhrm of 1 mg/ml. The diluted solution was mixed with a base crearn, to a final c~ ~"~ i. "~ of porph,vrin of lOO ,ug/rnl and, when used, 100 ~Lg/ml ~un~ ~"1,, linn of antibiotic was added.

Prep~rATinn of StPriili7ir~ So1~-~innc The sterili_ing solutions were prepared by adding pre-dissolved d~uLt:~u~ulphylill or hemin, from a stock solution. The stock solution was preyared by dissolving a mg of porphyrin in 2 drops of 0.1 N NaOH, and then diluting in phosphate buffer to pH 6.&, to a ~ull~enL~Lion of 1 mg/ml.
The diluted solution was mixed with phosphate buffer saline (PBS) 0.1i M, pH 7, to a final ~ul~ lLldLiuu of porphyrin of 10 llglml.

The procedures detailed above were used throughout the following examples, unless otherwise specified, and therefore are not reproduced again in each example, for the sake of brevity. Throughout the following examples, I IS given in ug/ml refer to ml of nutrient broth, or culture broth, or buffer, as a~.u~ Lt.

FY~TmrlL~ 1 The eftfect of the combined treatment of tetracycline and herr~in was tested on the cell growth of 5. aL~reLLs The following ~ulll~o~iLiu~ls were tested:

a. 10 ~lg~ml of hemin;
b. 30 llg~rnl of terracycline;
c. 10 llg/ml of hemin + 10 ~Lg/ml of tetracycline;
d. control cells.

~ wo ~33463 2 1 9 ~ ~ 2 7 r~.,u ~ t~s~

The optical density of the nutrient broth containing the calls was measured at 660 nrn, as detailed above, as a function of time. The results are shown in Fig.~ 1, from which it can be seen that a ~ulllbiL~Liull of 10 llg/ml hemin + 10 llg/ml tetracycline was the most effective, and was sTlhc~ntiAlly more effective than 30 llgjml LeLL~lLv~liLle alone.

EY ITrt!tle 2 The effect of the combined treatment of mrthirillin and hemin was tested on the cell growth of S. mlreus. The following ~u~ JOsiLiolLa were tested:

a. 10 ,ug/ml of hemin;
b. lQ ug/ml of Ampirillin;
c. 5 ~Lg/ml of mr-thirillin;
d. 10 )lg/ml of hemin + 10 ~g/ml of ampicillin;
e. 10 ,ug/ml of hemin + 5 llg/ml of mPthifillin;

The optical density of the nutrient hroth r~" ,IA;";"g the calls was measured at660 nm, as detailed above, as a function of time, and the results are plotted inFig. 2. From the results of this e~ LIL it is apparent that the control cells ~- line), to which no active material was added, behaved ven~ similarly to those to which there were added 5 llg/ml mrthi~illin (O line), or 10 ~Lg/ml ampicillin (o line). The optical densitv remained low were 10 ~g/ml of hemin (0 line) were added, but increased rapid}y after about 5 hours. In contrast, theoptical density of ~U~ ,o~iLiul- "d" (n line) and of ~oll~o~iLiu-l ~e" (X line),remained 5lthc~Anti-AIly lower than that of the ~U~ U~ g ~u~ v~iLiolls woss/33~63 2191627 r~".~ 9;~ ~

~o without hernin (~ "b" and "c"~. It should be noted that hemin alone inhibits the growth of S. aureus for onl- a hours, while the combined effect of hemin and methycillin (in amounts in which mPthirillin alone does not sho-v any effect) inhibit the grot~rth for 7-8 hours, which represents a striking ~Lc~ LiC effect.

EY~mplP 3 The effect of the :~yllt:L~i ~Li~ 'l of the invention was tested using S.
m~reus, and mPthirillin, ampicillm and hemin as the active matenals. Viable counts were taken at the begirming of the ~ ~ , and after one and two hours. The results are shown in Fig. 3.

The tested L ulL~Ju~iLiulL~ are identified as follows:
control - no active m-~terials added;
AMP - ampicillin, 10 ~g/ml;
MET - methicillin, 10 ~g/rnl;
HM - hernin, 10 llg/ml;

As it can be seen from the figure, there is virtually no difference in the ~ iable count after two hours, beh,veen the conbrol, .~.MP and MET samples. The HM
sample shows an rmproved antibiotic effect, and the HM+AMP and ~I+~vlET show a further iL-L~luv~llLclLL of one order of m~gnihl~lP over the HM.

~WO9S133463 ~1 21 91 627 r~ s!o~

EY~mple 4 Penicillase activit,v in S. aureus was measured by the phenol red test. In the test there were used l'u4 cells induced by methicillin, 50 llg/ml penicillin (inthe mother liquor~, and 0.5~iO phenol red. The e~ liu~ L was carried out according to the method described by Escamilla J., [ri~ci:~uLibiliiy of Hael.w~llilu~ influenzae to ampicillin as dr-t~Arm-n~-- by use of a modified, one-rninute beta-lactamase test", Antimiero~. Agents Chemoter., 9, 196-198 (1976~.

The results are shown in Table I below. 11+ " indicates that color change trom recl to yellow takes place in less than 1 minute, and 11 11 indicates that no color change from red to yeLow took piace in the course of the i'i~ lL.

Table T
PPnirillin~ce a~tivity in S. nvrrllC tr~atp~ tPria Penirillin~-A TreatmPnt TimP (min ) Penicillin ~in pPnirillin + }~Pmin after treatment lO~Lgjml 1011g/ml 10ilgjrnl + 1O~Lgjm 1 ++ I j 60 1 ++

woss/33463 21 9 1 f~27 P~ Cigs~ ~

FY~mple 5 The combined effect of L~LL~y~liL~e and photoachvated dru~lu~uul,ullyLiLL
waq tested on cell growth of S. aur2~s. The pho~ua~LivdLiul~ was effected as detailed above under I~Lx~lLLl~ k-l Procedures". Optical densit,v of the various samples was measured at 660 nm, as a function of time, and the results are plotted in Fig. 4. The ~u~ osiLiu~s used are identified as follows:

C - control cells;
Dp 0.1+L - d~uL~luLuuL~oilyLill~ 0.1 ~Lg/ml, irradiated with light at 5 J!cm2;
Tc 30 ~Lgjml - tetracycline, 30 llgiml;
Tc+DpO.1+L - Le:hG~yLliLle 2 ~Lg/ml + dt:uit:lol)u,luol~yli~, 0.1 llgjml, irradiated with light at 5 J/cm2;
Tc 2 ~Lg/ml - L~LL~y~ e~ 2 ~g/ml.

As can be seen with the figure, the best results were obtained using tetracycline 2 ~lgjml + d~uL~lu~ul~ohvrin, 0.1 ~g/rnl, irradiated with light at 5 J/cm2, and they were substantially better than those obtained with 30 gjml of tetracycline.

EY Impie 6 The effect of the ay~ ,lalic combination of the tetracycline and photoactivated dt:uL~u~uL~ was tested using S. m~reus. Viable counts were taken at the beginning of the ~ elilll~llL, and after one and two hours.
The results are shown in Fig. 5. The tested ~uul~uSiLiulla are identitied as in Example 5.

~ WO 9s/334fi3 L2 1 9 1 6 2 7 r~ 99 FY~mrle 7 The effect of the syllt:L~i~LiC ~nmhin~tinn of L~LL~y~LL,e with a ~ n~.;l;ll,l comprising photoactivated de,LL~lvpu.lul.y.iL. together with the nonapeptide if Formula ~I), was tested on the Gram negative bacteria E. coli. The e. coli cells were grûwn to the log phase under the conditions shoun in Fig. 6. Fig.
6A shows the optical density that was measured to determine total biomass, and Fig. 6B represents the same results, given in terms of colony forming units. The following materials were usecd:

Tc = 10~1g/ml of LL-LL~y~li,Le~
DP = 5 ~Ig/rnl of dellLt:lul~uL,ulLyliLL;
NP = 650 llg/ml of polymyxin B nonapeptide.

In all e~L~liLL~ LL~ the cells uere irradiated with 10 J/cm~ of light.

As can be clearly seer. from Fig. 6, the E. CL7li cells were essentially LLLs~ iLiv~
to the pulyL~Iy~CiLl B nu~L~Llv~ e alone, to its ..,.,.I,;"..ti".. with tetracycline, and to Lt hd.y.li,le alone. The cells treated with these ~ ;l .l ,.c behaved essentially as the untreated control cells (Cont). The LullliJiLIdLiull of the invention, on the other hand, comprising polymyxin B nonapeptide, de~lLt:l u~o.~l,y-i,- and L~LL ~I~y~liL ,e shows a surprising activity against E. coli.

wo g~l33463 ~ 1 9 1 6 2 7 ~ U~ ~C

EY~mple 8 The PYrPrimPntc described above were repeated with a nurnber of porphyrins and antibiotics, and an illustrative spectrum of activity on different bacteria is illustrated in Table Il below. Results ~nmr~rAhlP to thosedetailed in Examples 1-7 where obtained with the bacteria, pulyhy~ and antibiotics listed in Table II, and these e~ are therefore not described herein in detail, for the sake of brevity T~hle IT
Spectrum of ~u~yLy~ a and ~u-LI iOli~a activity on bacteria.

IT~ - r~ y deLILC~lUyUlyllyli hem~lL.,yu,yl.yLi yluLu~ulylly~
hernin m. ~. or o. hydroxy phenyl ~Vl,Ull~lills An~ih;n~irc ampicill n mPthi~il in L~:Ll~v~ e ~ Ll u ~ll l y ~
chlv.
polym~xin B
l~uwu~-u~,de ~wogs/33463 21~627 P~/f~-'~lj-Tahle IT (Corftinued) TTC - BAfrtPri~

General to Gram (+) Streptococci sp.
Diplococci sp.
Staphylococci sp.
P. aeruginosc E. coli Klebsiella sp.
Proteus vulgans Anaerobic Gram ~-) EY:~mrlP g The following base cream f~DECODERM, ~ r.~ d by Ivlerck & CCf., U.S.A.) was used Icontents in mg/1 gr base crearn):

2 mg sorbic acid 1 mg silicum dioxide 20 mg medi~m chain triglyceride 30 mg glycerol " 1. ./.~ f.
30 mg paraffin 50 mg ~ ylt:LLy,ly~ol 80 mg polvsorbat 40 90 mg .elybLe~yl alcohol 320 mg vaseline

3 21 91 ~)27 r~ sc~
-~6-double distilled water, q.s.

~he following ~u~ uuaiLiulls were tested:
control (base cream only);
d~uL~luluu~ yli~l~ 100 llg/ml - in the dark (Dp);
d~uLulu~u~,ullyliJl, 100 llg/ml - with a light irr~ tinn of 10 J/cm2;
d~ul.lu~w~hylil~, 100 llg/rnl + hemin, 100 ~Lg/ml - in the dark.

StaphyZococcus ~lureus bacteria grown in nutrient broth media to their log phase, were spinned and bacterial .. ,. ,. ~ cnnt~ining lO~ - 109 bacteria were added to the various cream ~ul~l~usiLiuL~s. Creams tested in the dark were kept at 37~C, while irradiated ~eams were illllminzlt~l as described above. At the end of the ;,.. .,I,,,li.". intervals, 100 ~Lg samples of the cream were diluted in PBS and plated on nutrient agar plates to determine the colony forming units / ml sample. Inrnh~n-)n intervals of 30 minutes and 180 minutes were employed.

The results are shown in Fig. 7. It is clearly seen that d~uLI:~upul~ullylil~ in the dark is entirely ineffective, and the CFU found are of the same order as the control cream, i~ ;ul~ of the d~uL~:lupu~ulLylill was only partially effective, while creams containing a ~uulbi~laLiul~ of dt:uLt:lulJu~lJhv~ill andhemin were highly effective even in the dark.

~ WO 9~33463 2 1 9 1 6 2 7 r~ f ~

EY lmrlP 10 Example 9 was repeated, with the following changes:

The base cream was a uJ~ .iallv available crearn, the so-called Johnson's baby I~ r cream (",.",--t,~ d by Johnson & Johnson Ltd., U.K.), I l~lll."ll;ll"

isopropyl palmitate;
pol~sorbate til j sorbitan stearate;
myristyl myristate;
cetyl alcohol;
stearyl alcohol;
synthetic beeswax;
glyceryl stearate;
stearic acid;
oleic acid;
propylene glycol;
carbomer 941;
Lhy l~ ben;
propylparaben;
buty-lparaben;
BHT;
~L~Iu~yL~ Lhylsilane;
stearyl alcohol;
benzyl alcoholj sodium hydroxide;
essence;

. "

-28- 2~ 91627 water. ~ ~X~

Instead of an irradiated deuteroporphyrin composition, a 100 ug/rnl tetracycline composition was tested.

The results are shown in Fig. 8. As before, deuteroporphyrin in the dark is entirely ineffective. The tetracycline-containing cream was also ineffective.
Creams containing a combination of deuteroporphyrin and hernin were ~ ect~ ~Je highly~as was the case with the previous base cream.

E.Y;~mple 11 Example 10 was repeated, but instead of the Johnson & Johnson base cream, Carbopol 940 was used. Carbopol 940 is a commercially available acrylic copolymer (produced by B. F. Goodrich Company, U.S.A.).

The results are sho~n in Fig. 9, from which it can be seen that the behavior is essentially identical to that obtained in Exarnple 10.

FY:~mple ~?

Using the base cream of Example 9, the following compositions were tested:

hemin, 100 ~gf ml;
tetracycline (Tc), 100 ~Lgjml;
hemin, 100 ~Ig/rrLl + tetracycline, 100 ~Ig/ml.

AMENDED SHEET

~l W0 9S/33463 2 1 9 1 6 2 7 P. ~

The results are shown in Fig. lO. The results shovw that hemin alone in the base cream was effective, but the ( ~ ~ of hemin and L~L~c~y~ e was substantially more effective.

E~amplf~c 1~ -16 Examples 9 - 12 were repeated, but instead of adding the bacterial ~u.L.~lLLIc~l~ directly to the cream, it was smeared on a bovine bone, and left to incubate at 37~C on the bone before a thin laver of crea~n was applied thereto. At the end of the i". ,~ ." periods, the bone was washed uith equal volumes of PBS, and the washings were plated on nutrient agar plates to determine the CFU.

EYImple 17 Example 16 was repeated, using S. c~,idi,,,.i~i~ instead of S. ~ureus, and z~lcwing i","l".l;(.l. times of up to 6 hours. The results obtained were rrlm~r:lhlr to those obtained with S. ~ureus.

When an; ~ " 1 li 1.1~ 1) time of up to 6 hours U as allowed, complete 11 i ~ I r, . 1 ;, ll, of the bone was observed, using compositions containing d~LLLe:lu~L~lLyLLLL
and hemin (100 llg~'ml of each) and hemin with tetracycline (also 100 ~lg/ml of each). This was checked by immr-rcing the bone in PBS buffer for 30 minutes, and using the PBS buffer in a growth experiment on agar plates. No CFU were observed in these t~ LLll~llL:~.

w0~3s/33463 2 1 9 1 6 2 7 ~ , li(3~

FY~mrle 18 The creams of Examples 9 -12 were tested in rnice, to treat surgical cuts infected with S. aurells . "". ~"1, ~ The cuts were of identical lengths, and the bacterial ~ w as taken from the sarne stock solution. The cuts were tested periodically for bacterial presence. The results were essentially the same as those obtained in Examples 9 -12.

FY~rle 19 The following sterilizing Lul~lposiLiul~ were tested, the liquid base solution being in all cases a PBS buffer 0.15 M at pH 7:
Con - Control: PBS without any addition;
Dp- Deu~elu,uu,luliy.il~, 10 ~Lg/ml;
Hm- Hemin, 10 llg/ml;
Dp+Det D~:ULI:IU~U~IJhY~ 0 ugjml, with 1 ~/c of the relevant detergent;
Hm+Det Hemin, 10 llg/ml, with l~o of the relevant detergent;

Three detergents were tested, all of which gave ~r~r~r~ results. Triton X-100, sodium dodecyl sulphate, and sodium laur~ l sulphate.

5S ~ 10~" ~ r~c aure~ls bacteria grown in nutrient broth media to their log phase, were spinned and bacterial cu~ llL~L~ containing 10~ -109 bacteria were added to the various sterilizing compositions. The solutions were kept at 37~C. At the end of the i~ Lull intervals, 10 ml samples of the solution ~woss/33~63 ~ 1 91 627 r~ 6s~i were plated on nutrient agar plates to determine the colony forming units /
mi sample. Inrnh~*nn intervals of 1, 2 and 3 hours were employed.

The results are shown in Fig. 11. Tbe results obtained with the rnmhinrtinn of porphyrin and detergent were excellent, and u ere better by one order of magnitude than those obtained with the luul~hyliLLs alone, and eight orders of magnitude better than the control.

FY~n~ple 20 Example 19 was repeated, with the exception that the bacterial ~ ~.". ~
was smeared on a human hand, uhich was allowed to incubate for 10 rninutes and was then washed with one of the solutions of Example 19. The washing liquid was then plated on an agar plate and tested for bacteriai growth. The control solution showed substantial bacterial ..." ~,,I,.,Ii,lll, the deut~u~u,,Jl.yli,L and the hemin solution showed traces of bacteria, which the bacteria were non-detectable in the washings rnnt~ining both a porphyrin and a detergent.

F.Y~Tnr~l' 21 Example 20 w as repeated, but this time the hands which had previouslJ been washed with one of the solutions were immersed in PBS for 2 minutes, and the PBS was then analyzed for bacterial growth. The results obtained were consistent with those of Example 20.

WO 95/33463 2 l 9 1 6 2 7 32 - PCT/US95/116998 FY~mrle 2'~

Example 21 was repeated, but instead of smearing the bacterial . ~ L
onto a human hand, it was smeared on a bovine bone, and left to incubate at 37~C on the bone before the bone was washed with one of the solutions of Example 19. At the end of the Lu~ubatiLJlL periods, the bone was washed with equal volumes of PBS, and the washings were plated on nutrient agar plates to df~h~rmin~o the CFIJ.

E~ Imrle 2~
Exampie 22 was repeated, using S. epiderm2dis instead of S. aîLreus The results obtained were cnmrAr~hl~ to those obtained with S. mlreus.

EY~mple 24 The solutions of Example 19 were tested in mice, to treat sur~,ical cuts infected with S. aureus . I~ u ..1, The cuts were of identical lengths, and the bacterial . . .n. ~ was taken from the same stock solution. The cuts were tested periodically for bacterial presence. The results were essentially the sarne as those obtained in Example 19 - 23.

All the above exarnples have been given for the purpose of illustration, and are not intended to limit the invention in any way. The im~ention extends to the aylleL~iaLc acti~,ity obtainable using pull~hylilLa together with antibioticmaterialsr in general, and is not intended to be limited to any specific pu~.hyLil,/antibiotic combination, or to any particular bacteria. As will be apparent to the skilled person, many different cream and liquid ~ullL~uaiLc~ns ~wog~/33463 2~916 2 7 r ~ c ;s3~

can be used, and many different combinations of IJu~ y~ and other antibiotic materials, synergists and additives can be employed, to combat a variet,v of infections, all u ithout exceeding the scope of the invention.

' ~ ~

Claims

1. A synergistic antibiotic composition, comprising as an antibiotic active ingredient a mixture of hemin, alone or together with one or more additional porphyrin(s), and at least one antibiotic compound.

2. A composition according to claim 1, wherein the antibiotic compound is a compound which inhibits protein synthesis.

3. A composition according to claim 1, wherein the antibiotic compound is a compound which inhibits the synthesis of bacterial cell walls.

4. A composition according to claim 1, wherein the antibiotic compound is selected from among the group consisting essentially of penicillins, cephalosporins tetracyclines, chloroamphenicol and macrolide antibiotics.

5. A composition according to claim 4, wherein the antibiotic is selected from the group consisting essentially of ampicillin, methicillin, tetracycline, erythromycin and chloroamphenicaol, and mixtures of two or more of such compounds with or withoutadditional antibiotic and/or antibiotic synergistic materials.

6. A composition according to any one of claims 1 to 5, wherein the one or more additional porphyrin is selected from the group consisting essentially of deuteroporphyrin, hematoporphyrin, protoporphyrin, and meta-, para- or ortho-hydroxy phenylporphyrin.

7. A composition according to any one of claims 1 to 6, further comprising a peptide of the formula:

wherein DAB* is a 2,4-diaminobutyric acid residue, and DAB is a 2,4-diaminobutyric acid residue containing a free 4-amino group.

8. A composition according to claim 7, wherein the peptide of Formula (I) is polymyxin B
nonapeptide.

9. Use of a composition according to any one of claims 1 to 6, for the manufacturing of a pharmaceutical preparation for the treatment of infections caused by gram-positive bacteria.

10. Use of a composition according to claim 7 or 8, for the manufacturing of a pharmaceutical preparation for the treatment of infections caused by gram-negative bacteria.

11. A method of treating an infection in an animal, including humans, which infection is susceptible of treatment by a given antibiotic material or mixture of antibiotic materials, comprising administering to the animal in need thereof an amount lower than the effective amount of the said antibiotic material or mixture of antibiotic materials, together with hemin, alone or in admixture with one or more additional porphyrin.

12. A method according to claim 11, wherein the antibiotic compound is a compound which inhibits protein synthesis.

36

14. A method according to claim 11, wherein the antibiotic compound is selected from among the group consisting essentially of penicillins, cephalosporins, tetracyclines, chloroamphenicol and macrolide antibiotics.

15. A method according to claim 14, wherein the antibiotic is selected from the group consisting essentially of ampicillin, methicillin, tetracycline, erythromycin and chloroamphenicol, and mixtures of two or more of such compounds with or without additional antibiotic and/or antibiotic synergistic materials.

16. A method according to any one of claims 11 to 15, wherein the additional porphyrin is selected from the group consisting essentially of deuteroporphyrin, hematoporphyrin, protoporphyrin, and meta-, para- or ortho-hydroxy phenylporphyrin.

17. A method according to any one of claims 11 to 16, further comprising administering to the animal in need thereof, together with the mixture of hemin and antibioticmaterial(s) and in the absence of photoactivation, also a peptide of the formula:

wherein DAB* is a 2,4-diaminobutyric acid residue, and DAB is a 2,4-diaminobutyric acid residue containing a free 4-amino group.

18. A method according to claim 17, wherein the peptide of Formula (I) is polymyxin B
nonapeptide.

18. A method according to claim 17, wherein the peptide of Formula (I) is polymyxin B
nonapeptide.

19. A method according to any one of claims 11 to 16, for the treatment of infections caused by gram-positive bacteria.

20. A method according to claim 18 or 19, for the treatment of infections caused by gram-negative bacteria.

21. A method according to claim 11, wherein the infection is caused by bacteria selected from among the group consisting essentially of Gram-Positive bacteria, Anaerobic Gram-Negative bacteria, Streptococci sp., Diplococci sp., Staphylococci sp., P.
aeruginosa, E. coli, Klebsiella sp., and Proteus vulgaris.

22. An antimicrobial composition, comprising as an active ingredient hemin or a mixture of hemin with one or more additional porphyrins, alone or in admixture with conventional antibiotic materials and/or pharmaceutically acceptable or beneficial agents or additives, in a water-based cream composition.

23. A composition according to claim 22, wherein the additional porphyrin is deuteroporphyrin.

24. A composition according to claim 22 or 23, comprising a mixture of deuteroporphyrin and hemin.

25. A composition according to claim 22 or 23, comprising a mixture of hemin and tetracycline.

26. An antimicrobial cream composition according to claim 22, wherein the base cream comprises: paraffin, propylene glycol, polysorbate 40, cetylstearyl alcohol, Vaseline and water.

27. An antimicrobial cream composition according to claim 22, wherein the base cream comprises: synthetic beeswax, glyceryl stearate, stearic acid, propylene glycol,alkylparaben(s), preservatives, and water.

28. Use of a cream composition according to any one of claims 22 to 27, for the treatment of infections caused by gram-positive bacteria.

29. Use of a cream composition according to any one of claims 22 to 27, for the treatment of flesh wounds.

30. Use according to claim 29, wherein the wound is a surgical cut.

31. Use of a cream composition according to any one of claims 22 to 27, for the sterilization of bones.

32. Use according to claim 31, wherein the bones are chest bones exposed during chest surgery.

33. A method of treating or preventing the occurrence of an infection caused by Gram-positive bacteria, comprising applying to the area to be treated a cream composition according to any one of claims 22 to 27.

34. A method according to claim 33, wherein each of the porphyrin(s) and of the antibiotic material(s) is present in an amount comprised between 5 and 100 µg/ml

35. A cream composition for the treatment or the prevention of bacterial infections, essentially as described.

36. Use of a porphyrin for the manufacture of an antibacterial cream composition, essentially as described.

37. A sterilizing liquid composition, comprising as an active ingredient a porphyrin or a mixture of one or more porphyrins, in an aqueous solution, together with a surface-active agent.

38. A composition according to claim 37, wherein the porphyrin is deuteroporphyrin or hemin, or a mixture thereof.

39. A composition according to claim 37 or 38, wherein the surface-active agent is a detergent.

40. A composition according to claim 39, wherein the surface-active agent is selected from among Triton X-100, sodium dodecyl sulfate and sodium lauryl sulfate, or a mixture of two or more of such agents.

41. A composition according to any one of claims 37 to 40, wherein the porphyrin is present in a concentration of 1 to 100 µg/ml of sterilizing solution.

42. A composition according to any one of claims 37 to 41, wherein the surface-active agent is present in an amount of 0.5 - 5 wt%.

43. Use of a liquid composition according to any one of claims 37 to 42, for thesterilization of surfaces.

44. Use according to claim 43, wherein the surface to be sterilized is the human hand.

45. Use according to claim 43, wherein the surface to be sterilized is a bone.

46. Use according to claim 45, wherein the bones are chest bones exposed during chest surgery.

47. Use according to any one of claims 43 to 46, for sterilizing areas infected with Gram-positive bacteria.

48. A sterilizing liquid composition, substantially as described.

49. Use of a sterilizing liquid composition, substantially as described.