WO1996029071A1

WO1996029071A1 - Uses of antibacterial compounds

Info

Publication number: WO1996029071A1
Application number: PCT/US1996/003778
Authority: WO
Inventors: Tomer Sivron
Original assignee: Ramot University Authority For Applied Research & Industrial Development Ltd.; Dippert, William, H.
Priority date: 1995-03-21
Filing date: 1996-03-21
Publication date: 1996-09-26
Also published as: AU5426096A

Abstract

Uses of inherently adherent antibacterial compounds such as TA having formula (I), and its derivatives such as Focusin having formula (II) are claimed. As these compounds inherently adhere to solid or semi-solid substrates, this property can be used to advantage in various therapeutic situations. One use of the described compounds is in a method of medical treatment in which an effective amount of these compounds are applied to the surface of a tissue at or in proximity to the site of a diseased tissue treatable by the compounds. The local application of these compounds can also be used as a means for preventing infection of the tissue. Another use is in a method for preventing infections in which the compounds are coated on the surfaces of a medical device which comes into contact with the body. A medical device coated with these compounds is also claimed.

Description

USES OF ANTIBACTERIAL COMPOUNDS

FIELD OF THE INVENTION

The present invention concerns novel uses of adherent antibacteri¬ al compounds in medical treatments. The invention is particularly drawn to the compounds "TA" and its derivatives, such as "Focusin". The present invention also concerns medical devices coated by these compounds.

BACKGROUND OF THE INVENTION AND PRIOR ART

In the following, reference will be at times made to prior art documents, the full particulars of which are to be found at the end of the description before the claims.

Myxobacteria are Gram-negative, rod-shaped bacteria commonly found in soil and decay vegetation (Kaiser et al., 1979). Myxobacteria are generally a rich source for antibacterial agents that exhibit a wide range of chemical structures, mode of action and anti-microbial spectrum (Rosenberg and Varon, 1984). One strain, M. xanthus TA, produces a broad spectrum antibacterial agent when grown under nutritionally limited conditions, during the end of its exponential growth phase. This antibacterial agent, disclosed in Rosenberg, U.S. Patent No. 3,973,005, was termed "TA", and is capable of inhibiting growth of a variety of Gram-positive and Gram-negative bacteria (Dworkin & Kaiser, 1985; Rosenberg et al , 1973). The growth inhibition is caused by lysis of growing bacteria as a result of blocking cell wall synthesis at the stage of polymerization of the lipid disaccharide- pentapeptide (Rosenberg et al , 1973; Zafriri et al. , 1981). This mechanism of growth inhibition differs from the mechanism of the β-lactam antibiotics, which are the major type of antibiotics in use today. As a result, strains which have developed resistance to β-lactams are not resistant to the TA antibacterial agent.

TA is a molecule with a molecular weight of 623.4 dalton, and having the molecular formula C_3SH_6]0₈N (see below for its structure). TA binds tightly to a variety of tissues and hard surfaces and retains its activity while in the bound form (Manor et al., 1985; Rosenberg et al. , 1984). For example, TA binds to dental hard and soft tissues, which allowed the use of this antibiotic to treat severe gingivitis and to reduce plaque in humans (Manor et al., 1989). One of the disadvantages of the TA antibiotic is that it loses its activity following simultaneous exposure to air and light (Rosenberg et al. , 1982). Recently, an improved derivative of TA, named "Focusin ", has been prepared and found to be more stable and adherent than the parent compound. This derivative is described in Applicant's co-pending Patent Application No. USSN 08/332,964. Focusin was found to have a similar activity spectrum to that of TA although with a somewhat lower specific activity. However, Focusin was found to be much more stable to light and air exposure than the TA antibiotic. Focusin further has a higher aqueous solubility than TA and it also adheres more strongly than TA to a variety of surfaces and is released therefrom more slowly than TA. Like TA, Focusin adheres to soft tissues and hard surfaces, and it retains its bactericidal activity in the bound form. These properties of Focusin and TA allow their use for a wide range of applications where other antibiotics would be unsuitable. Helicobacter pylori infection of the stomach lining is now regarded as one of the major etiological factors in the pathogenesis of inflammation of the stomach lining (gastritis) and of duodenal and gastric ulcers. H. pylori may also be implicated in the development of gastric malignancy such as gastric lymphoma. A panel of experts convened by the U.S. National Institutes of Health has recently noted that aggressive antibiotic regimens can "markedly decrease the recurrence rate of ulcers..." (ASM News, 1994). Although systemic treatments using antimicrobial agents such as bismuth subsalicylate, tetracycline and metronidazole have been somewhat effective, localized treatment at the site of infection would be preferable to systemic treatment which requires maintenance of therapeu¬ tic blood levels of the antibiotic. However, most antibacterial compounds do not adhere by themselves to the gastric mucosa, and necessitate various carriers, which can cause secondary complications. Infections caused by the insertion of medical devices into body cavities are widespread. The contact of the inserted device with the surrounding tissues for prolonged periods of time often results in bacterial infections. For example, over 900,000 episodes of catheter-associated urinary tract infections occur annually in acute care hospitals in the U.S. (Warren, 1987). Various types of bactericidal coated catheters have been described in the scientific literature, (e.g. Johnson, et al, 1993), in which either the bactericidal agent or the catheter or both undergo pre-treatment in order to stably bind one to the other. None of these coated catheters, however, have found acceptance in the commercial market except for silver oxide coated catheters, which are very expensive and have not as yet received approval for use in the U.S.

Recurrent tonsillitis is a major cause of morbidity as well as worker and student absenteeism. Approximately 40% of tonsillectomies performed are as a result of recurrent tonsillitis. This disease is usually treated using long term prophylactic systemic antibiotics. Such treatments are not always advisable since they often result in undesirable side effects. Although it would be preferable to prevent bacterial inoculation on the tonsil surface by local application of antibiotics, no treatment has been described as vet which involves coatinq the tonsil surface with an adherent antibiotic substance.

SUMMARY OF THE INVENTION

In accordance with the invention there are provided novel uses of an antibacterial compound or a derivative thereof having antibacterial properties and having an inherent ability to adhere to solid or semi-solid substrates.

A preferred compound is the antibacterial compound TA having the following formula:

Me

A preferred derivative compound is Focusin having the following formula:

II

In both of the above formulae, Me represents a methyl group.

Such adherent antibacterial compounds, and particularly TA and its derivatives, especially Focusin, will at times be referred to herein collectively as "said antibacterial compound".

In accordance with one aspect of the invention there is provided a method of medical treatment involving the use of said antibacterial compound, wherein the method comprises applying an effective amount of said compound to the surface of a tissue. The term "effective amount" should be understood as meaning an amount of said antibacterial compound sufficient to bring about a bactericidal activity.

In a first embodiment of this aspect of the invention, the compound is used to treat diseased tissue. In one preferred embodiment, the compound is applied to the gastric mucosa in order to treat gastric inflammations such as gastritis and gastric ulcer, as will be more particularly described below. In further preferred embodiments, the compound is applied to the eye for the treatment of eye infections, or to the ear for the treatment of Otitis media. However, it will be understood by the skilled man of the art that the above antibacterial compound can be applied through other means to other tissues or to the same tissue affected by other diseases which can be advantageously treated by an adhered antibacterial agent which is slowly released over a period of time.

In a second embodiment of this aspect of the invention, the compound is used to prevent a tissue at risk from becoming diseased. In a preferred embodiment, the compound is sprayed on the tonsils of a patient suffering from recurring tonsillitis. In a further embodiment, the said antibacterial compound may be used to prevent bodily odors, e.g. in the armpit, caused by bacteria. In accordance with a second aspect of the invention there is provided a method of preventing infections involving the use of said antibacterial compound, wherein the method comprises coating the surfaces of a medical device which comes into contact with the body with said com- pounds, the surfaces coming into contact with surrounding body tissues or fluids on employment of the device.

In one preferred embodiment of this second aspect of the invention, the medical device is a urinary catheter intended for insertion into the urethra. The external and internal surfaces of the catheter are coated by said antibacterial compound which adheres to the catheter's surface and is slowly released over a period of time, thus inhibiting growth of bacteria at the contact area of the catheter with the surrounding tissue. This slow- release property is especially important in the use of indwelling catheters. In a further preferred embodiment, the medical device is a stent. In a still further preferred embodiment, contact lenses are coated with said bacterial compound to prevent eye infections frequently caused by the use of such lenses or to treat an existing infection. It will be understood, however, by the skilled man of the art that other medical devices which are wholly or partially inserted into bodily cavities and whose inserted parts come into contact with the surrounding body tissue can also be coated by said antibacterial compounds in accordance with the invention.

The present invention also provides a medical device which comes into contact with the body, wherein the surfaces of the device which come into contact with surrounding body tissues or fluids on employment of said device are coated with said antibacterial compound.

The present invention further provides an antibacterial conjugate comprising a first said antibacterial compound, and a second non-adherent antimicrobial compound, wherein the conjugate retains the adherent properties of the first antibacterial compounds. In a preferred embodiment, the second antimicrobial compound has an activity selected from the group comprising antifungal, antiviral and antibacterial activities.

By conjugating the second antimicrobial compound to the first compound, the antimicrobial activity spectrum of the conjugate includes the combined spectrums of each of the component compounds, and the adherent properties of the first antibacterial compound are imparted to the second non-adherent compound.

The above conjugate may be used in all of the methods described above for use with said antibacterial compounds. The present invention takes advantage of the inherent adhesive properties of said antibacterial compound which obviate the necessity for using auxiliary means to bind the antibacterial substance to a substrate. Although the said compound strongly adheres to solid or semi solid substrates, it is slowly released from the substrate over a prolonged period of time thereby lengthening the effective period of action. This mode of action is unique in that until now, sustained released properties were, as a rule, achieved by virtue of the choice of an appropriate carrier. Since said antibacterial compound has the unique characteristic of being capable of binding to soft tissue as well as to hard surfaces, this compound can find use both in in situ application to body tissues, as well as with various medical instruments. The wide spectrum of activity exhibited by said compound allows it to be used in the treatment of a large number of bacterial infections.

Some examples of diseases potentially and advantageously treatable by said antibacterial compound include otitis media infection of the ears, acute and chronic sinusitis, chronic gingivitis, recurrent tonsillitis, eye infections, burns and other local skin infections. The compound can be applied to the tissue by spraying, or by creams, drops or pills. Since the compound exerts its activity locally, low concentrations are sufficient, lessening the possibility of side effects and lowering the cost of the treatments. The adhesiveness and slow release properties of said compound means that only a small number of treatments at a low frequency will be required. Some examples of solid substrates capable of being coated by said antibacterial compound include plastic and silicone tubing used for catheters and IV lines, as well as stents. Due to the unique adhesive properties of said compound and its ability to inhibit bacterial growth at very low concentrations, the amount of antibiotic required per device (e.g. catheter or stent) would be very low, making the coating process relatively inexpensive. The direct contact of the coated surfaces with the surrounding tissue, such as the urethra, can result in some of the compound adhering to these tissues, resulting in improved antibacterial protection.

The present invention will now be illustrated in the following description of examples carried out within the framework of the present invention. As will be appreciated, the invention is not limited to the specifically disclosed embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings:

Fig. 1 shows the amount of TA (■), Focusin (+) and Ampicillin (*) absorbed to a polystyrene surface, release to a medium within several time intervals;

Fig. 2 shows the amount of TA (■) and Focusin (+) absorbed onto polystyrene surfaces, released to a medium within several time intervals,

(similarly as in the experiment of Fig. 4). At the end of the experiment

(after 22 hours) the amount of the antibacterial agent remaining absorbed to the polystyrene surface was determined by extraction with ethanol; and Fig. 3 shows the effect of the antibacterial agents' concentration on the ratio of antibiotic bound to polystyrene versus that which was released ("bound/released"): Fig. 3a - TA antibiotic; Fig. 3b - Focusin.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The experiments performed within the framework of the present invention will now be described below. Although the invention is described with respect to TA and especially with respect to its preferred derivative Focusin, it is to be understood that other inherently adherent, antibacterial compounds, and other inherently adherent, antibacterial derivatives of TA are within the scope of the invention.

MATERIALS AND METHODS 1. Bacterial strains The following bacterial strains were used in the experiments

Escherichia coli ESS Kan^r, is a kanamycin resistant strain of

E.coli ESS, derived by transducing E.coli ESS with Pl :TnV, KanM This strain was used for the standard antibiotic assays. Other strains which were used were the following:

Salmonella thyphimorium, Erv.>inia herbicolla, Serratia marcescens, Providencia strautii and Proteus vulgaris, Enterobacter,

Kleb iella Gr47, Klebsiella pneumoniae, Klebsiella sp., Citrobacter diversus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli K-12, Bacillus suhtilis and Staphylococcus alhus and Mycobacterium smegmatis, Helicohacter pylori. 2. Media and growth conditions

E.coli ESS Kan^r was maintained by periodic transfer of single colonies for overnight incubation on LB Kan agar at 37°C, followed by further storage at 10°C for 4-6 weeks. LB Kan medium is 1% Tryptone (Difco Laboratories, Detroit Michigan U.S.A.) containing 0.5% Yeast Extract (Difco), 0.5% NaCl (Merck & Co. Inc. Rahway, New Jersey U.S.A.) and 50 μg/ml Kanamycin monosulfate (Sigma Chemical Company, St. Louis, U.S.A.).

LB Kan medium was solidified by 1.5% Bacto agar (Difco). For the antibiotic assay, Nutrient Broth agar plates were used. NB medium is 0.8% Nutrient Broth (Difco) containing 0.5% NaCl (Merck). NB was solidified by 1.8% Bacto agar (Difco). The NB plates were overlaid by 3.5 ml NB soft agar (0.9% Bacto agar) mixed with 0.1 ml E.coli ESS Kan^r overnight culture.

3. Paper disc filter antibiotic assay

Antibiotic activity against E.coli ESS Kan^r was determined by the paper disc filter assay (Loo et al, 1945). Unknown quantities of antibiotic TA or Focusin, dissolved in ethanol, saline or NB were applied to paper disc filters (6 mm diameter; Schleicher and Schull). After allowing the ethanol to evaporate in the hood for about 15 mins, the dry filters were placed on nutrient broth plates which had been overlaid with an overnight culture of E.coli ESS Kan^r as described above.

The diameter of the zone of inhibition was recorded after 18 hours incubation at 37°C. The concentration of the antibacterial agents were determined from a standard inhibition curve prepared with known quantities of TA and Focusin. 4. Testing for antimicrobial spectrum of TA and Focusin

Saturated solutions of TA and Focusin were prepared by adding 1.14 μg TA and 7 μg Focusin to 1 ml LB (final cone, of 1% ethanol). About 10^s bacteria of various strains were then added. The control was bacteria in the same medium with no antibacterial agents. The bacteria were introduced at 37°C with shaking at 100 φm. When the control became turbid, viable cell count was performed on LB agar plates. Growth inhibition is presented as percentage of the no antibiotic control.

5. Adherence to polystyrene

5.1 Adherence to polystyrene

The antibacterial agents (Focusin and TA) dissolved in ethanol were applied to small polystyrene petri dishes (3 cm diameter) and the ethanol was allowed to evaporate over a period of 15 minutes in a hood. For control, ethanol without the agents was applied to such dishes. To each petri dish, 1 ml of a nutrient broth was added. The petri dishes were then incubated at 37°C at 50 rpm in a shaking device

(New Brunswick Scientific, USA). Following each time interval, the

NB was removed and a fresh NB was added. At the end of the experiments, a solution of 100% ethanol was added to the petri dishes in order to extract the residual antibacterial agents that remained stuck to the polystyrene.

In order to check the antibacterial agents that were released from the polystyrene to the NB, 30 μ\ and 60 μ\ samples of the NB were applied to the paper disc filters and assayed as described under 3 above.

5.2 Adherence to polystyrene in the presence of E.coli ESS In some experiments, the release of antibiotics was tested in the presence of E.coli ESS. In these experiments, the nutrient broth (NB) which was added to the petri dishes contain 10⁵ E.coli ESS (from a log culture). Each fresh NB which was added to the dishes contained the same amount of E.coli cells.

Prior to the performance of the disc assay, the E.coli cells were killed by the application of 20 μ\ of ethanol onto the disc, so as not to disrupt the bioassay.

6. Adherence to a biological tissue and activity of bound antibiotic

6.1 Adherence of TA and Focusin to mouse tissues

Two mice were sacrificed by exposure to ether. The bladder, eyes, intestines, lungs, heart, teeth, liver, spleen and kidneys were removed by dissection. Each experimental set consisted of three test tubes. A piece of each tissue type was placed in each of the three test tubes. The amount of tissue per tube were as follows: bladder Vfe, eye \_, intestine 1 cm, lung 2, heart Va, fang tooth ^lΔ, liver V3, spleen Vfe and kidney Vi. One test tube contained a control tissue which was treated only with the solvent used for dissolving the antibacterial agent, usually 50% ethanol. The second and third test tubes contained tissues were treated with 4 μg TA or 20 μg Focusin, respectively.

Following application of the antibacterial agent or the control solution, the tissues were allowed to dry in a hood for 30 mins., and then placed in the test tubes. One milliliter of sterile saline was then added to each test tube and the tubes were then incubated with shaking (50 rpm) at 37°C for one hour. The amount of antibacterial agent that was released to the saline was measured following chloroform extraction by the disc filter assay. The tissues were then transferred to exponentially growing cultures of E.coli ESS in NB at a concentration of 10⁵ cells/ml. Incubation was at 37°C and continued for 5 hours, following which the number of viable cells was checked.

6.2 Release of Focusin from mouse tissue

Focusin was tested in the same manner as that described above (under 6.1) with the exception that the tissues in the test tube were washed four times with a solution of 1 ml sterile saline. Each saline wash was for 1 hour at 37°C. Following incubation, the washing saline solution was added to an exponentially growing cultures of E.coli ESS in a concentration of 10⁵ cells/ml.

The cultures with the saline wash were incubated al 37°C for 6 hours and then viable cell count was performed in order to estimate the amount of killing by the antibacterial agent that were released from the tissues. After the four washes the tissues were transferred to 10⁵ E.coli ESS log cultures and were then incubated for 5 hours. The tissues were transferred in this way for three times, each time the percentage of inhibition by the treated tissue was measured by viable count.

EXAMPLES

A. TISSUE SUBSTRATES

I. Antimicrobial spectrum of TA and Focusin In order to determine which diseases can be treated by the present invention, the sensitivities of various bacterial strains to TA and Focusin were tested in liquid media. The results are shown in the following Table 1. Table 1 - Sensitivity of different bacteria to saturating concentrations of TA and Focusin

Strain TA Focusin killing (%) killing (%)

E.coli K-12 100 100

Ervi'inia herbicola 100 100

Staphylococcus aureas 100 100

E.coli ESS 100 100

Salmonella thyphimurium 60 40

Enterobacter 100 100

Bacillus suhtilis 100 100

Citrobacter diversus 100 100

Klebsiella Gr47⁺ 100 100

Klebsiella sp* 100 60

Klebsiella pneumoniae " 100 96

Proteus vulgaris 100 90

Serratia marcescens 1 0 67

Staphylococcus alhus 83 73^*

Pseudomonas aeruginosa 0 0

Providencia strautii 100 100

Mycobacteria smegmatis 0 0

Helicobacter pylori 100 100

Streptococcus salivarius 100 100

On the 46th hour Hospital strains

The above results show that overall, TA and Focusin had a similar antibacterial activity spectrum in the tested bacterial strains. All the tested strains, except two, Pseudomonas aeruginosa and Mycobacterium smegmatis, were sensitive to both TA and Focusin. However, six of the strains {Salmonella thyphimurium, Klebsiella sp., Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens and Staphylococcus alhus) were only partially killed by Focusin, and two strains {Salmonella thyphimurium and Staphylococcus albus) were only partially killed by TA. The strain Staphylococcus albus began to show sensitivity to the antibacterial agents only after 46 hours of incubation.

It can be further seen that H. pylori, which is implicated in gastritis, and Streptococcus, which is implicated in tonsillitis, are equally sensitive to TA and Focusin. Bacteria such as E. coli and S. albus, which are often implicated in urinary tract infections (UTI), are also sensitive. II. Adherence of TA and Focusin to mouse tissue and their bacteri¬ cidal effect when bound to the tissue

In order to determine the binding properties of TA and Focusin to soft tissues, the antibacterial compounds were applied to mouse tissue as described under paragraph 6.1 above. Following incubation, the amount of inhibition was assayed by viable cell counts (VCC) and the results are shown in the following Table 2:

Table 2 - Adherence of TA and Focusin to mouse tissues and their bactericidal effect when bound to the tissues

Tissue Released TA alter Inhibition %' bv Released Focusin after Inhibition %' by

1 h saline wash (%) the TA-tissue 1 h saline wash (%) the Focusin-tisstie

Liver <3.7 96 <2.9 94

Lungs 4.1 100 4.9 100

Intestine <3.7 99 <3.1 100

Heart <3.0 100 <3.2 90

Teeth 19.7 1 0 <4.1 100

Spleen 6.0 100 <4.1 100

Kidney 3.9 " 100 <3.8 100

Bladder 15.8 100 <4.1 100

% Inhibition 100 - ^{vcc eχ}P^eriment _x 100 VCC control As can be seen in the above table, the teeth and bladder released relatively large quantities of TA; however, the bound TA still caused 100% inhibition of E.coli. Against this, the liver and intestine lost only less than 3.7% of the initial amount of TA, and the remaining bound TA showed a 96% and 99% inhibition, respectively. Focusin was bound tightly to all tissues tested, more than 95% remained bound after the initial wash. After 5 hours with E.coli ESS more than 90% of the E.coli was inhibited (compared to control without antibacterial agent). The liver and heart tissues treated with Focusin did not completely inhibit the E.coli.

III. Release of Focusin from mouse tissue and the killing effect of the treated tissue

Two sets of mouse tissue were treated, the first with 10 μg Focusin dissolved in 50% ethanol and the second only with ethanol, serving as a control (this solution did not contain an antibacterial agent). The tissues were treated as described in paragraph 6.2 above.

After the fourth wash, the treated tissues were transferred to E.coli ESS cultures (lO^"* cells) in nutrient broth. The culture tubes were then incubated at 37°C for 6 hours and the extent of growth inhibition was measured by viable cell count. The treated tissues were transferred to E.coli cultures two more times (a total of three times), and each time the percentage of growth inhibition by the treated tissues was checked. The results are shown in the following Table 3: Table 3 - Release of Focusin from mouse tissues and the antimicrobial effect of antibiotic-bound tissues

Tissue Inhibition %' by the Inhibition %' by the Inhibition %' by the

4th wash liquid treated tissue" treated tissue""

Liver 67 99 100

Lungs 100 100 17

Intestine 100 100 38

Heart 100 100 98

Teeth 100 100 91

Spleen 100 100 98

Kidncv loo 100 90

Bladder 100 >99 0

Eye 100 >99 0

VCC experiment

% Inhibition = 100 - A- 100 VCC control

After a single wash After two more transfers

The above results show that the bactericidal activity of the fourth liquid wash except for the liver tissue, contained enough Focusin to completely inhibit the E.coli growth. The washed tissues containing bound focusin showed a 99% inhibition or more After the third transfer to the NB containing E.coli (a total of seven washes from the beginning), the liver was the only tissue that completely inhibited the E.coli. All the other tissues caused some inhibition, in decreasing order: heart and spleen (98%), teeth, kidney, intestine and lungs However in bladder and eye insufficient Focusin was left and no inhibition was observed. IV. Protocol for treating gastric H. pylori infection with adherent antibiotic

As TA and Focusin are unstable at acidic pΗs, the patient having known gastric disease associated with H. pylori will need to be made achlohydric prior to the treatment. This can be done, for example, by giving the patient 20mg of Omeprazole twice a day for 3 days. On the third day, an upper gastrointestinal endoscopy (gastroscopy) is performed. The presence of H. pylori is confirmed by a biopsy CLO test, and the presence of a pΗ greater than 6 in the stomach is confirmed by testing a gastric aspirate taken at gastroscopy.

The pylorus is blocked off using a Gruntzig balloon. The stomach is then emptied and slightly expanded with air, after which TA or

Focusin is sprayed through the endoscope canal in 20 separate squirts, each squirt comprising 0.05ml of TA in a water/alcohol (50:50 V/V) solution of 3mg/ml, as follows:

4 squirts on the walls of the upper antrum; 4 squirts on the walls of the lower antrum; 4 squirts on the wall of the lower body of the stomach; 4 squirts on the wall of the upper body of the stomach; and 4 squirts on the wall of the fundus.

After 20 minutes, the Gruntzig balloon is deflated and the gastroscope is removed. One month after therapy, the residual presence of H. pylori is determined using a C¹⁴ urea breath test.

An alternate method of application is by orally administered pills or tablets in which the said adherent antibiotic is coated with a pharmaceuti¬ cally acceptable excipient which dissolves in the gastric area. In such applications, the gastric pΗ is adjusted as necessary prior to administration of the medicament. V. Ear and Eye infections

Otis media of the car is one of the most common causes of morbidity in children. Approximately 30% of children with fever seen in regular pediatric practice are diagnosed as suffering from this disease. Eye infections are less frequent, but are nevertheless quite prevalent in both children and adults.

Most of the above infections are caused by bacteria, and are therefore currently treated by local or systemic administration of antibiotics. When locally administered, the antibiotics are generally applied by ear/eye drops 2 to 3 times daily for a period of 1-2 weeks.

In contrast, treatment of the above infections with said antibacte¬ rial compound could involve only a single application, due to its inherent adhesiveness and slow release properties. One example of a formulation would be 1-5 mg/ml of said antibacterial compound in 50% DMSO + 1% ethyl alcohol.

VI. Prevention of recurring tonsillitis

One embodiment of a therapeutic procedure for the prevention of recurring tonsillitis comprises spraying said antibacterial compound onto the tonsils once every month, for a period of four months. The compound can be sprayed directly onto the tonsils after the patient performs a few deep expirations in order to dry the tonsils of saliva. Each treatment may consist, for example, of spraying a 0.4 ml aliquot of a 2.5 mg/ml solution of antibacterial compound in 50% ethanol. In an ongoing clinical trial performed in Israel for the prophylaxis of recurrent tonsillitis with TA, a 100% success rate has been demonstrated so far with 7 patients, having 2 to 4 months follow up. VII. Prevention of underarm odors

Underarm odor is known to result from, inter alia, axillary odorless precursor molecules such as testosterone being metabolized by axillary bacteria into odorous compounds. Axillary microflora include micrococci, staphyloaocci, anaerobic propionibacteria, and aerobic and anaerobic gram-positive coryneform bacteria. The latter are believed to play an important role in the production of axillary odorous compounds.

Thus, by destroying the axillary microflora, underarm odor is diminished.

Due to the adhesive properties of said antibacterial compound, the compound can be used for the purpose of killing off sensitive axillary bacteria. For example, a Focusin or TA alcoholic solution at a concentration of 5-10 mg/ml is periodically (once every 2-4 weeks) sprayed under the armpits at a dosage of 1-5 mg per spray. Once sprayed, the alcohol evaporates leaving the active compound adhered to the axillary skin.

B. SOLID SUBSTRATES

I. Adherence of TA, Ampicillin and Focusin to polystyrene, in the presence of E.coli ESS

4 μg of TA, 20 μg of Focusin and 4 μg of Ampicillin, were each dissolved in 100% ethanol and were dried on polystyrene petri dishes, for 15 minutes in a hood. One ml of an exponentially growing culture of E.coli ESS (1(P cell/ml) in NB was then added to the plate. The plates were incubated at 37°C under shaking at 50 φm for various time intervals. Following each time interval, the NB was removed and fresh NB was applied with 10⁵ E.coli ESS. Release of antibacterial agents to the NB was measured by the paper disc filter assay and the results are shown in Fig. 1. As can be seen in Fig. 1, all the Ampicillin was released to the NB at the first wash while the release of TA and Focusin was much slower. TA was released continuously for four washes (40 hours), whereas Focusin, even after 88 hours, was still bound to the polystyrene and was slowly being released.

II. Adherence of TA and Focusin to polystyrene in the absence of E.coli ESS

This experiment was performed in a similar manner to that described above with the exception that no E.coli ESS was added to the saline solution. Samples of 0.1 ml were withdrawn at varying time intervals and were assayed in the paper filter bioassay. At the end of the experiment the remaining antibacterial agent which was bound to the polystyrene was extracted by ethanol, and also measured by the paper filter bioassay.

The results are shown in Fig. 2 and as can be seen, Focusin was released much slower than TA. At the beginning, TA was released quickly and following four hours, a small constant amount remained through the 22 hours of incubation. Against this, Focusin showed a slower initial and a muc more prolonged release curve than TA. After 22 hours, 8 μg of Focusin and only 1 μg of TA were still bound to the polystyrene.

III. The effect of concentration on the adherence of TA and Focusin to polystyrene

Several concentrations of TA (2 μg, 4 μg, 10 μg and 20 μg) and Focusin (1 μg, 20 μg, 50 μg and 100 μg), and 0.1 ml 100% ethanol (control) were dried on 3 sets of polystyrene petri dishes for 20 mins. in a hood. Sterile saline (1 ml) was added to each plate, after one hour incubation in 37°C with shaking at 50 φm, the amount of antibacterial agent released to the saline was checked by the paper disc assay. The remaining agent bound to the polystyrene, was dissolved by ethanol and measured in the same manner. The results are shown in Fig. 3 depicting the effect of the antibacterial agent concentration on the bound/released ratio of the antibiotic to polystyrene. Fig. 3a shows that as the TA concentration increases, the amount of bound TA decreases; against this, as can be seen in Fig. 3b, the Focusin binding showed an increase bound/released ratio with an increase in concentration.

IV. Coating of urinary catheter with TA

One embodiment of the present invention will now be described with respect to a Standard Foley catheter. The catheter is dipped at room temperature in an alcoholic solution of 2-10 mg/ml of Focusin or TA, so that the entire catheter is coated by 1-5 mg of compound. The active antibacterial compound forms a uniform coating on the inner and outer surfaces of the catheter. The Focusin or TA antibiotic is expected to adhere to the catheter and urethral epithelium for a period of 4-21 days.

Standard urethral catheterization is performed, including lubrication with Ezracain gel 2%, or other hydrophilic lubricants, and the catheter's balloon is inflated to fix the catheter tip in the bladder. As a routine management, attachment and replacement of the urine collection bag is carried out while maintaining full sterility of the area between the catheter and the urine bag by use of an antiseptic solution.

V. Coating of contact lens

Approximately 20% of eye infections are associated with contact lens wear. A majority of these infections are caused by gram-negative bacteria, and a minority by gram-positive bacteria. The most frequently implicated bacteria are Pseudomonas sp., Staphylococcus epidermidis and Corynebacterium sp. Only about 3% of contact lens associated eye infections are caused by fungi. Contact lenses can be periodically coated with said antibacterial compound so as to avoid contact lens associated eye infections. For example, the lenses may be coated once every 2-4 weeks, preferably by dipping them in a solution containing 1-5 mg of compounds such as Focusin or TA.

In addition, therapeutic lenses can be coated with said compounds for the puφose of treating an existing eye infection. In this case, the lenses will preferably be precoated by dipping (0.1-0.5 mg compound per lens) and distributed as such under medical prescription.

C. ANTIMICROBIAL CONJUGATE

An additional novel use of said antibacterial compound is by the chemical attachment of an additional compound. For example, a second, different antibiotic could be linked to Focusin or TA. This second antibiotic, which is normally non-adherent and diffusible, will become adherent due to its being conjugated to said antibacterial compound, and will be slowly released together with said compound.

The antimicrobial activity spectrum of said antibacterial compound can be greatly expanded by the expedient choice of the second antibiotic. Furthermore, the chance that a bacterial strain will appear that is resistant to the conjugated antimicrobial substance will be very small.

The added antibiotic may have antibacterial, antifungal, and/or antiviral properties.

An example of a chemical reaction which can be used to conjugate a second antimicrobial compound to said antibacterial compound is a simple procedure in which a ketone group of Focusin or TA is transformed into an enol ether which is bound to the second compound. REFERENCES

I . ASM News, May 1994, 60:240-241.

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6. Manor, A., Varon M., and Rosenberg E., 1985. Absorption of antibiotic TA to dental hard tissues, J. Dental Res. , 64: 1371 -1373.

7. Manor, A., Eli, I., Varon, M., Judes, H. and Rosenberg, E, 1989. Effect of adhesive antibiotic TA on plaque and gingivitis in man, J. Clin. Periodontol, 16:281-284.

8. Rosenberg, E., 1976, U.S. Patent No. 3.973,005.

9. Rosenberg, E., Vaks, B., and Zuckerberg, Z., 1973. Bactericidal action of an antibiotic produced by Myxococcus xanthus, Antimicrob.

Agents and Chemother. , 4:507-513.

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Claims

CLAIMS:

1. A method of medical treatment involving the use of an antibacte¬ rial compound or a derivative antibacterial compound thereof, wherein said antibacterial compounds are inherently adherent to solid or semi-solid substrates, said method comprising applying an effective amount of one or more of said compounds to the surface of a tissue.

2. A method according to claim 1 wherein said antibacterial compound has the following formula:

Me

3. A method according to claim 1 wherein said derivative antibacte¬ rial compound has the following formula:

4. A method according to claim 1 wherein said tissue is diseased, and wherein said disease is treatable by said compounds.

5. A method according to claim 4 wherein said tissue is a mucosal tissue.

6. A method according to claim 5 for the therapeutic treatment of bacterial-induced gastric inflammations comprising applying said com- pounds to the mucosal surface of the stomach.

7. A method according to claim 6 wherein said compounds are applied by spraying.

8. A method according to claim 6 wherein said compounds are administered by pills.

9. A method according to any of claims 6-8 wherein said bacterial- induced gastric inflammation is caused by Helicohacter pylori.

10. A method according to claim 4 wherein said tissue is eye tissue.

11. A method according to claim 4 wherein said tissue is car tissue.

12. A method according to claim 1 wherein said tissue is at risk of being diseased, and said disease can be prevented by said compounds.

13. A method according to claim 12 wherein said tissue is a tonsil.

14. A method according to claim 13 for the prevention of recurrent tonsillitis comprising applying said compounds to the tonsils.

15. A method according to claim 1 for the prevention of bodily odors caused by bacteria.

16. A method involving the use of an antibacterial compound or a derivative antibacterial compound thereof for preventing infections, wherein said antibacterial compounds are inherently adherent to solid or semi-solid substrates, said method comprising coating the surfaces of a medical device which comes into contact with the body with said compounds, said surfaces coming into contact with surrounding body tissues or fluids on employment of said device.

17. A method according to claim 16 wherein said antibacterial compound has the following formula: M e

18. A method according to claim 16 wherein said derivative antibacterial compound has the following formula:

19. A method according to claim 16 wherein said device is a catheter.

20. A method according to claim 16 wherein said device is a stent.

21. A method according to claim 16 wherein said device is a contact lens.

22. A medical device which comes into contact with the body, wherein the surfaces of said device which come into contact with surround¬ ing body tissues or fluids on employment of said device are coated with an antibacterial compound or a derivative antibacterial compound thereof, wherein said antibacterial compounds are inherently adherent to solid or semi-solid substrates.

23. A medical device according to claim 22 wherein said antibacterial compound has the following formula:

^ A device according to claim 22 wherein said derivative antibacte¬ rial compound has the following formula:

0

25. An antimicrobial conjugate comprising:

(i) a first antibacterial compound or a derivative antibacterial compound thereof, wherein said first antibacterial compounds are inherently adherent to solid or semi-solid substrates; and D (ii) a second non-adherent antimicrobial compound; wherein said conjugate retains the adherent properties of said first antibacterial compounds.

26. A conjugate according to claim 25 wherein said second antimi¬ crobial compound has an activity selected from the group comprising 0 antifungal, antiviral and antibacterial.

27. A method of medical treatment involving the use of the conjugate of claim 25, said method comprising applying an effective amount of said conjugate to the surface of a tissue.

28. A method for the prevention of infection involving the use of the conjugate of claim 25, said method comprising coating the surfaces of a medical device which comes into contact with the body with said conjugate, said surfaces coming into contact with surrounding body tissues or fluids on employment of said device.

29. A medical device which comes into contact with the body, wherein the surfaces of said device which come into contact with surround¬ ing body tissues or fluids on employment of said device are coated with the conjugate of claim 25.