DK167901B1 - Compositions for treating mammalian infections, which compositions comprise Hymenoptera venom, proteinaceous components or polypeptide components of such venom, or analogues of these proteinaceous components or polypeptide components, and a unit dose which comprises such a composition - Google Patents

Description

in DK 167901 B1

The invention relates to a composition for treating an infection in a mammal, comprising a first antibiotic-possessing activity against the infection and a second agent which is at least one Hymenoptera venom, or at least one active protein component of a Hymenoptera venom. , or at least one polypeptide component of a Hymenoptera venom, or at least one analog or chemically modified derivative of an active protein component of Hymenoptera venom, or at least one analog or chemically modified derivative of a polypeptide component of Hymenoptera venom or mixtures thereof, the other 10 agent is biuret positive and the proportions of the first antibiotic and second agent are such that the second agent enhances the activity of the first antibiotic.

The invention also relates to a unit dose for treating an infection in a mammal, which unit dose comprises an effective dose of a drug comprising an antibiotic with activity against the infection and another agent selected from the group consisting of at least one Hymenoptera poison, at least one active agent. protein component of a Hymenoptera venom, at least one polypeptide component of a Hymenoptera venom, at least one analog or chemically modified derivative of an active Hymenoptera venom protein component, at least one analog or chemically modified derivative of a Hymenoptera venom polypeptide component, and mixtures thereof, wherein the second agent is bioretpositive and the proportions of the antibiotic and the second agent are such that it enhances the activity of the antibiotic.

The identity of antibacterial, antiviral and anticarcinogenic agents, and in particular antibiotic agents, as well as the activities and therapeutic uses of these materials are well known. The secondary agents used in the invention in enhancing the activity of these primary anti-infectious agents are also known per se, and have been used in medicine in some cases, but their ability to enhance the activity of antibacterial agents. antiviral and anticarcinogenic agents and in particular antibiotic agents have not been previously known. Some of the secondary agents used in the invention are obtained primarily from the venom of species of the order hymenoptera, which include, without limitation, and by way of example only, honeybees, bumblebees, wasps, goats, bald faced hornets , ants and the like.

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It has been found that hymenopteran agents, isolated active proteinaceous components or polypeptide components from such poisons and analogues to these proteinaceous components or polypeptide components enhance or promote the activity of antibacterial, antiviral and anti-cancer agents, and in particular antibiotic agents.

The present invention is made on the basis of the work described in a dissertation in veterinary science by Lorraine Smith Mulfinger entitled "Synergistic activity of 10 honey bee antibiotics" to be submitted to the Graduate School Department of Veterinay Science, Pennsylvaina State University. The entire content of the thesis is hereby made part of the present description with this reference. References below to previous work by others have been abbreviated herein, with as many as 15 references cited in the literature record for Mulfinger's dissertation and at the end of this description.

BACKGROUND AND PRIOR ART 20 The use of anti-bacterial, antiviral, carcinostatic and anti-carcinogenic compounds is, although well known in the art, still subject to extensive, ongoing research, some of which, in addition to the discovery of new agents, is aimed at discovery of agents for improving the activity of known active agents.

In fact, certain compounds derived from bigift have been investigated and have been found to be useful for certain specific pharmacological applications. For example, U.S. Patent No. 4,444,753 issued April 24, 1984, discloses a composition comprising a component obtained by deproteinizing an extract from the contents of the bee's poison bag. This product has an immunostimulatory activity, a carcinostatic activity, an effect on enhancing the antibacterial activity of an antibacterial compound, and an effect on enhancing the carcinostatic activity of a carcinostatic compound. The invention disclosed in said patent is directed to carcinostatic, immunostimulatory and antibacterial agents comprising the composition described. Although the invention is similar in purpose to the present invention, it is different from DK 167901 B1 3 that the beech extract is modified by deproteinization so that it is negative in the biuret reaction and the sulfosalicylic acid reaction.

Also, the disclosure of U.S. Patent No. 4,370,316 issued January 25, 1983 to the same inventors as the above patent discloses a method of treating a host animal with diminished immunity by administering an effective amount of the deproteinized extract from the bee's poison bag.

Although antibacterial, antiviral and anticarcinogenic compounds are well known and it is also known that a deprotected extract from a bee's sachet has certain useful activities, including antibacterial activity, anti-bacterial activity and immunostimulatory activity, it has not previously It has been known that proteinaceous hymenopteran agents, proteinaceous extracts or polypeptide extracts thereof and analogues to such proteinaceous components or polypeptide components have an enhancing effect on virtually all antibacterial, antiviral, carcinostatic and anticarcinogenic agents. Such enhancement of the activity of such primary anti-infectious agents not only enhances the efficacy of doses of such agents which would be effective alone, but may also render low doses of such agents which would be ineffective if used alone.

As noted above, hymenopteran agents, protein components or polypeptide components and analogs to such proteinaceous components or polypeptide components can generally enhance the activity of anti-infectious therapeutics. However, to simplify the description of the invention, for purposes of illustration, the use of honey bee venom or its proteinaceous extract melittin in the composition will be described to enhance the activity of antibiotics to control bacterial, viral and cancer infections. Honey bee venom (HBV) has been selected as it is easily obtainable. However, it should be understood that venom from other hymenoptera species and the protein component or polypeptide components thereof, as well as analogues thereof, are also to varying degrees effective in the invention. Furthermore, anti-infectious agents other than antibiotics may be used in connection with the invention for the treatment of infections to which they have been previously used, but with improved efficacy when used in combination with the protein-aggravated hymenopteran agents.

As a further background it should be noted that honey bee has the honor 5 for several useful activities. Some of the activities are scientifically documented, while others appear to be based on empirical results and legends. The antibacterial in vitro activity of honey bee venom is well documented (Schmidt-Lange, 1951; Ortel and Markwardt, 1955; Fennel et alia, 1968), however, 10 few attempts have been made to utilize this activity in practice. According to the present invention, results from several empirical studies suggest that the antibacterial activity of honey bee venom may have a significant effect in vivo in the presence of antibiotics. Based on these findings, a study has been designed to investigate the interactions between honey bee venom and antibiotics using an in vitro assay, where the two compounds can be assessed without the contributing effect of the host's natural immune responses.

20 In this study, three bacterial strains were initially tested against three different antibiotics using separate "chessboard" titrations of honey bee venom with each antibiotic. Representatives from each of the three main groups of antibiotics (penicillins, aminoglycosides and polymyxins) were selected and analyzed to determine whether honey bee venom could enhance the antibacterial effect of selected antibiotics. As described below, an antibiotic from a fourth important group was examined later.

30 When synergy was demonstrated by the "chessboard" analysis, a broader overview was attempted using a simplified procedure. Two automated minimal inhibitory concentration (MlC) assay plates titrating the sensitivity to eleven antibiotics simultaneously were inoculated in parallel with bacterial cultures with and without 35 non-inhibitory doses of honey bee venom (HBV). Eight gram positive and four gram negative organisms were tested using this system to find classes of antibiotics that routinely produce synergy with HBV and to determine the spectrum of synergistic effect of these combinations among different groups of bacteria.

In addition to testing for whole honey bee venom, the venom was fractionated by size exclusion chromatography. Each of four fractions was tested to determine if a specific component was responsible for antibacterial activity and could also act synergistically in ant in-bacterial assays. It was found that the fraction containing melittin, previously identified as the antibacterial element of honey bee venom (Fenne! Et al., 1968), is active in its purified form and will act synergistically to an extent equivalent to whole honey bee venom. .

Furthermore, the activity of various analogs to the active components of hymenoptera poison was determined and compared to melitin tins.

Brief description of the drawing

Figure 1 shows a diagram of flour in tti n's amino acid sequence.

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Figure 2 shows a graph of optical density versus hours after inoculation showing the antibacterial activity of honey bee venom (HBV) on S. aureus.

Figure 3 shows a graph of optical density versus hours after inoculation for ampiciΠin and HBV versus S. aureus.

Figure 4 shows a graph of optical density versus hours after inoculation for kanamycin and HBV versus S. aureus.

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Figure 5 shows a graph of optical density versus hours after inoculation for polymyxin B and HBV versus S. aureus.

Figure 6 shows a graph of optical density versus hours after inoculation 35 for ampicillin and HBV versus E. coli.

Figure 7 shows a graph of optical density versus hours after inoculation for kanamycin and HBV versus E. coli.

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Figure 8 shows a graph of optical density versus hours after inoculation for kanamycin and HBV versus E. col i.

Figure 9 shows a graph of optical density versus hours after inoculation 5 for polymyxin B and HBV versus E. col i.

Figure 10 shows a graph of optical density versus hours after inoculation for ampici1 lin and HBV versus kanamycin resistant S. aureus.

Figure 11 shows a graph of optical density versus hours after inoculation for kanamycin and HBV versus kanamycin resistant S. aureus.

Figure 12 shows a graph of optical density versus hours after inoculation for polymyxin B and HBV versus kanamycin resistant S. aureus.

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Figure 13 shows the electrophoresis results of 100 µg melittin protein.

Figure 14 shows a graph of optical density versus hours after inoculation, showing antibacterial activities of melittin / HBV 20 versus S. aureus.

Figure 15 shows a graph of optical density versus hours after inoculation for meelittin / HBV and kanamycin versus S. aureus.

Figure 16 shows a graph of optical density versus hours after inoculation for rifampicin and HBV versus S. aureus.

Figure 17 shows a graph of optical density versus hours after inoculation for rifampicin and HBV versus Ps. aeruginosa.

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Figure 18 shows a graph of optical density versus hours after inoculation for polymyxin B and hop poison versus E. coli.

Figure 19 shows a graph of optical density versus hours after inoculation 35 for polymyxin B and wasp versus E. coli.

Figure 20 shows a graph of optical density versus hours after inoculation for polymyxin B and goat venom versus E. coli.

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Figure 21 shows a graph of 1 and 1Q bacteria / ml blood versus treatment for a single sepsis treatment model (polymyxin B and melittin interactions).

Figure 22 shows a graph of log1Q bacteria / ml of blood versus treatment for a repeated sepsis treatment model (polymyxin B and melittin interactions).

Figure 23 shows a graph of optical density versus hours after inoculation 10 for melittin / analogue and polymyxin B versus E. coli.

Figure 24 shows a graph of optical density versus hours after inoculation, showing relative activities of melittin and analogs thereof.

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Figure 25 shows a graph of optical density versus hours after inoculation, showing relative activities of melittin and analogs thereof.

Figure 26 shows a graph of optical density versus hours after inoculation, showing relative activities of melittin and analogs thereof.

The poison composition 25

Poisons are heterogeneous mixtures of biochemical compounds. Most poisons consist of more than 90% protein. Toxins and enzymes make up this protein component and are the cause of direct cell damage. Although many enzymes such as phospholipase A2, acid phosphatase and hyaluronidase are common to most poisons, toxins and other biologically active peptides contained in poisons are very species specific.

Poisonous insects all belong to the insect order Hymenoptera. Like snake venom, enzymatic activities such as phospholipase 35 A2, hyaluronidase and acid phosphatase are common to all insect poisons.

However, the toxin and peptide components vary widely from species to species (Tu, 1977b).

The poison from Italian honey bee (Apis mel li fera) is the most extensively investigated insect venom. The most important component of honey bee venom is melittin. This peptide has a molecular weight of 2,847 daltons and amounts to approx. 50% of the poison's dry weight. Another peptide, apamine, is present to an extent of approx. 5% of the venom and several other peptides 5 are present in trace amounts (Haberman, 1972).

Poisons from other hymenoptera contain peptides with biological properties similar to those of melittin. Examples of such peptides are bumblebees I-V from hops bee, Megabombus pensvilvani cus, mastoporan from wasp, goat ham and other wasp species, and krabolin from European goat ham. A common feature of these peptides is their amphiphilic nature. These peptides have been subjected to sequence analysis and their structure is well known (A. Argiolas and J. J. Pisano, 1985).

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Antibacterial activity of honey bee venom

The bactericidal activity of honey bees was first documented in 1941 by W. Schmidt-Lange (1941). He tested E. coli and staphylo-20 cocci and found that both were sensitive to the antibacterial activity of honey bee venom. He further noted that the minimal inhibitory dose of honey bee venom for E. coli was much higher than that for staphylococci.

25 It was not until ten years later that Brangi and Pavan (1951) evaluated various extraction procedures to isolate the antibacterial activity from honey bee venom. They found that the activity was present in both water and acetone extracts of the poison. They also showed that the activity was stable by heating up to 100 ° C for up to 15 30 min.

In 1955, Ortel and Markwardt (1955) published the results of a study on the variation in sensitivity among various bacteria to the antibacterial activity of honey bees. 296 bacterial strains were tested. The results showed that tolerance to honey bee venom is much greater in gram-negative organisms than in gram-positive organisms. Areas for bactericidal concentrations were described to be 12.5 - 25 µg / ml for gram-positive bacteria and 1 - 10 mg / ml for gram-negative bacteria. The bactericidal activity DK 167901 B1 9 was co-purified along with the "direct hemolytic fraction" of red blood cells. The name "melittin" had not yet been assigned to the active component of this fraction.

5 In 1963, Benton et al. a bioanalysis for honey bee venom. The bacteriostatic activity of the venom was quantified by a radial diffusion assay, which determined growth inhibition zones caused by serial poison dilutions in bacterial growth lawns. This analysis was proposed to standardize the biological activity of 10 honey bees for in vivo use. (Currently, allergy sensitization is the only in vivo honey supplement treatment accepted by the Food and Drug Administration, USA). The article also describes the testing of the heat sensitivity of the honey in the ngbacti, and it was found to withstand sterilization procedure (121 ° C for 15 minutes) (Benton et al., 1963).

Melittin insulation and activities

Honey bees poison includes several pharmacologically active compounds. The 20 compound present in the largest proportion of the venom is melittin, which is a polypeptide having a molecular weight of 2,847 daltons and which acts as a direct red blood cell hemolysin. Other active ingredients include phospholipase A2, histamine, dopamine, norepinephrine, apaamine, and hyaluronidase (Haberman, 1972).

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Antibacterial activity of melittin

Fennel, Shipman, and Cole (1968) purified melittin by Sephadex G-50 chromatography and showed that the meltin fraction possessed "potent antibacterial activity." They tested 30 random bacterial strains (including several streptococci, staphylococci and enteral bacterial strains) by comparing the activity of purified melittin with whole honey bee venom. They noted that a strain of S. aureus, a penicillin-resistant isolate, showed no decrease in sensitivity to 35 melittin.

Although melittin has been described as the antibacterial factor in honey bee venom, no description has been found of its use in vivo. It was noted by Mol 1ay and Kreil (1974) that DK 167901 B1 10 interactions between melittin and lecithin enhanced the activity of phospholipse A2 honey venom against lecithin. However, it has not been previously recognized that melittin boosts antibiotic activity.

5

Haberman and Jentsch (1967) have purified melittin and published the amino acid sequence. They found that melittin exists in two natural forms that differ only by a formyl substitution at the N-terminus (Figure 1).

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Analogues to proteinaceous components and polypeptide components of hematopoietic assays

The following analogues to melittin were prepared: Analog No. Composition 1 Melittin (1-20) - NH2 2 Melittin (1-20) - 0rn-0rn-0rn-0rn-Gln-Gln-NH2 20 3 Melittin (1-20) ) - D-Lys-D-Lys-D-Lys-D-Arg-D-Gln-D-

Gln-NH2 4 Melittin (1-20) - Lys-Arg-Lys-Arg-Gly-Gly-NH2 5 Melittin (1-20) - Arg-Arg-Arg-Arg-Gln-Gln-NH2 6 Melittin (1- 20) - Lys-Lys-Lys-Gln-Gln-NH2 7 Melittin (1-20) - Gly-Gly-Gly-Gly-Gln-GIn-NH2 8 Melittin (1-20) - Asp-Asp-Asp Asp-Asp-Asp-NH2 9 Melittin (1-20) - Lys-Lys-NH2 10 Mastoporan (1-14) - NH2 (natural) 11 Mastoporan (1-14) - 0rn-0rn-0rn-0rn-Gln Gln-NH2 12 Melittin (1-20) - (CH2 NH2) 13 Melittin (1-20) - Orn-Orn-NH2

The analogs were prepared by conventional peptide synthesis as described by e.g. M. Bodanszky: "Principles of Peptide Synthesis", 35 Springer Verlag, 1984.

An example of peptide synthesis of analog # 4, i.e. melittin (1-20) -Lys-Arg-Lys-Arg-Gly-Gly-NH2.

DK 167901 B1 11

A derivatized resin, such as a polydimethylacrylamide gel commercially available under the trademark PEPSYN KA, is reacted with (Fmoc-Gly 2 O, where Fmoc represents 9-fluorenylmethoxycarbonyl, which serves as a temporary protecting group).

5

The reaction carried out in the presence of 4-dimethylaminopyridine as a catalyst results in the formation of the ester Fmoc-Gly-O resin.

The ester is deprotected in the presence of 20% piperidine in DMF to form 10 of H-Gly-O resin.

The deprotected product is then reacted with an activated ester of the form a 15 Fmoc-Gly-OPfp, where Pfp represents pentafluorophenyl, to give

Fmoc-Gly-Gly-O-Resin.

20

The remaining 24 amino acids are coupled to the reaction product formed for 20 corresponding cycles of deprotection and coupling with active esters.

The product thus formed is deprotected with 20% piperidine in DMF and the melittin analog formed is cleaved from the resin in the presence of TFA (trifluoroacetic acid) and a scavenger such as water.

Antibiotics 30

Antibiotics can be functionally divided into four groups based on the active sites of the antibiotic (Volk, 1978a). Target structures for the four groups are the cell wall, the cell membrane, the protein synthesizer and the nucleic acid replicator. Due to the complexity of the synergy 55 complex in ten, four antibiotics were selected, one for each of the above groups for testing. The antibiotics selected were ampicillin, kanamycin, polymycin B and rifampicin. Each has a different mode of action on prokaryotic cells.

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ampicillin

Ampicillin belongs to the group of antibiotics that affect cell wall structure. These antibiotics are all penicillin derivatives, each containing the functional beta-lactam ring. Collectively known as the beta-lactam group, these antibiotics block cell wall synthesis by inhibiting the transpeptidase system, which cross-links the peptidoglycan's pentaglycine bridges, therefore only actively growing cells are affected by their presence.

10

Ampicillin is a semi-synthetic derivative of penicillin. The synthesis steps of ampicillin synthesis add an amine group to the alpha carbon atom of pinicillin G. This contributes to resistance to beta-lactamases (the dominant penicillin resistance factor in bacteria), giving ampicillin a much broader spectrum of action among bacteria than penicillin (Volk, 1978b).

Kanamycin 20 Kanamycin is an aminoglycoside. This group of antibiotics blocks protein synthesis. Members of this group bind to the 30s ribosome in bacteria and block steric binding of aminoacyl tRNAs or inhibit translocation of the growing peptide chain at the ribosomal activation site (Volk, 1979c). As protein synthesis is required by many regulatory cell functions, aminoglycosides are effective on bacteria in either active or stationary growth phases.

Polyvinyl B

Polymyxin B is a cyclic amphiphatic peptide. Due to the combined hydrophilic and hydrophobic properties, polymyxin B has a detergent-like effect which does not require cell growth to be effective. Like melittin, polymyxin B interacts with membranes to form small hydrophilic pores in the hydrophobic region of the 35 membranes. In gram-negative organisms which have a thick lipopoly-saccharide layer that acts as a selective permeability barrier, polymyxin B is effective in disrupting osmotic gradients. Polymyxin B is therefore very effective on gram-negative organisms, even if it is minimally effective on gram-positive organisms (Sebek DK 167901 B1 13 1979). Although flour in ttin may form membrane pores similar to polymyxin B, melittin is more active on Gram-positive organisms, so the effect of mel ittin is not completely analogous to the action of polymyxin B.

5

rifampicin

Rifampicin is an antibiotic from the group that acts at nucleic acid synthesis levels, completing examples of antibiotics 10 from the four main categories discussed above.

Svnerqi examine! See

A review of articles describing synergy between antibiotics and 15 other compounds in bacterial systems showed that all the researchers used the same basic method. Bacterial growth was monitored in broth culture with and without each compound separately and then with both compounds together. To demonstrate synergistic action as opposed to an additive effect, at least one of the compounds 20 was used to an extent where it will exhibit only minimal fluid retention.

With a relatively inactive compound, any increased activity of the second compound in the presence of the first will be the result of synergistic interactions (Moellering et al., 1971; Carrizosa and Levison, 1981; and Cynamon and Palmer, 1983). It is on this basis that experiments in this specification are based.

Materials and Methods

Materials 30

Gift from honey bee (Api's flour in feral was provided by Vespa Laboratories, Spring Mills, Pennsylvania.

Bacterial strains were provided by the Veterinary Science Department 35 'at Pennsylvania State University. S. aureus No. 140A is a field iso-1 from a case of bovine mastitis. E. coli No. G188E was selected from the E. coli Reference Center systematic assembly. A kanamyci resistant strain of S. aureus was isolated by natural selection procedure, as described below.

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Antibiotics were purchased from Sigma Chemical Company (St. Louis, Missouri) and activity units were based on their analyzes.

Trypticase soy base (BBL Microbiology Systems, Cockeysville, Maryland) was used to study all bacterial growth either as a broth or an agar.

Sephadex G-50 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden.

10

Minimum inhibitory concentration (MIC) assays of antibiotics with and without honey bee venom were performed by the microbiology department at Allegheny General Hospital, Pittsburgh, Pennsylvania using the Sensititre® assay system distributed by Gibco Laboratories, 15 Lawrence, Massachusetts.

methods

Isolation of kanamycin resistant mutant 20 S. aureus was grown in 5 ml of trypticase soy broth (TSB) overnight g to a density of about 10 colony forming units / ml. 0.1 ml of this culture was plated on a plate of trypticase soy agar (TSA) containing 39 µg / ml kanamycin and incubated for 48 hours at 37 ° C. Colonies that appeared within 48 hours were sub-cultured on another TSA plate added 39 µg / ml kanamycin.

Chessboard Ten Serenity Analyzes 30 Bacterial culture was prepared for this analysis by freezing each strain in its logarithmic growth phase in TSB. For this purpose, a 5 ml 24-hour culture was used to inoculate 200 ml of TSB into a 500 ml erlenmeyer flask. The culture was incubated at 37 ° C with constant stirring and the optical density (OD) at 660 nm was read 35 every hour. When the culture reached the mid-logarithmic phase (about 0.500 OD units), 5 ml of sub-samples were transferred to 16 x 100 mm screw cap tubes. All the cultures were frozen and stored at -20 ° C. E. coli required glycerol to be added to the medium at a final concentration of 20% to survive freezing. This was done by mixing 1 ml of sterile glycerol with 4 ml of culture in the logarithm phase immediately before freezing.

To begin an analysis, a tube of frozen culture was thawed in a beaker of water at room temperature. The thawed culture was added to 175 ml of TSB in a 500 Erlenmeyer flask, stirred, and ODgG0 was determined immediately and recorded as reading at "time zero". The flask was then incubated at 37 ° C with constant stirring for 2 hours, after which the ODgg0 was read and recorded again, the culture was divided into 16 x 100 mm screw cap tubes pre-filled with specified honeycomb (HBV) and antibiotic specimens as described below. .

Stock solutions of HBV and antibiotics were prepared in distilled water, filter sterilized and stored at -20 ° C in 5 ml partial samples with concentrations twice the concentration needed for the chessboard titration system. The frozen stock solution concentrations required for each bacterial species are listed in Table 1 (page 43). The concentration used for each bacterium was based on prior experiments using antibiotics alone to determine the minimum inhibitory ranges for each antibiotic for each microorganism.

For each assay, a vial of antibiotics and a vial of HBV were thawed at room temperature and diluted with a corresponding volume of 2 x TSB and then serially diluted twice to normal strength TSB to obtain four concentrations of poison and four concentrations of antibiotics. 75 test tubes with screw cap were numbered and placed in a manner similar to the checkerboard pattern 30 shown in Table 2. TSB, antibiotics and HBV were then distributed according to the design shown in Table 3. Tubes marked as 00 and 0 contained 2.5 ml of TSB and served such as OD blind samples and sterile etch control tubes. Tubes 1 - 75, each containing a total volume of 500 µl, were inoculated with 2 ml of the above-described 2 hour culture. [Note: 35 final concentrations of HBV and / or antibiotics in each tube were 1/10 of the concentration added in 250μ1 sub-sample, (cf. Table 3)]. Each tube was immediately sealed and flipped over. After all the tubes were inoculated, they were placed in horizontal racks on an adjustable platform at 37 ° C. The growth of each tube was monitored individually for 4, 6, 8, 12 and 24 hours by determining the optical density of each tube at 660 nm.

Minimum inhibitory concentration analyzes with HBV 5

The microbiology laboratory at Allegheny Genral Hospital with the capacity to perform automated MIC assays was asked to perform an experimental review of 12 clinical bacterial isolates. The adaptation of each automated MIC analysis had the following limitations: (1) 10 Each assay could test only one dose of HBV, and (2) the effect of HBV alone could only be assessed as completely inhibitory or noninhibitory. HBV's synergy with the 11 antibiotics in this system was assessed by comparing two assays with and without the presence of HBV. The HBV dose used for each species was assessed to be a non-inhibitory dose based on the chessboard titration assays.

Melittin Purification 20 Sephadex® G-50 gel filtration bed material was swollen for 24 hours at room temperature in beta-alanine acetic acid buffer (BAAB), pH 4.3 (Guralnick et al., 1986) and then equilibrated at 5 ° C overnight.

A 2.5 x 60 cm column was poured and equilibrated at a flow rate of 1.0 ml / hr. 100 mg of HBV was reconstituted in 5 ml of 25 BAAB buffer containing 20¾ sucrose. HBV was put on the column and eluted at a flow rate of 1 ml / hr. The throughput was monitored for absorbance at 280 nm. Fractions containing the main peak were pooled, a sub-sample was analyzed by Lowry's protein analysis (Lowry, 1951) and the remainder was lyophilized.

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Identification of the melittin fraction was based on the relative mobility and quantization of bands resulting from polyacrylamide gel separations of each fraction (Benton, 1965). Melittin was also tested for purity by polyacrylamide gel electrophoresis. Eletrophoresis 35 was performed as described by Guralnick et al. (1986). Freeze-dried fractions were reconstituted to 2 mg / ml in the electrophoresis sample buffer and 50 µl samples were loaded per ml. sample well on the gels.

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Melittin equivalence to whole gi ft The amount of melittin fraction, which is equivalent to its proportion in whole honey bee venom, was determined by quantifying individual bands 5 in electrophorized samples of venom and melittin fraction. 20, 40, 60, 80 and 100 µg samples of whole honey bee venom were separated by electrophoresis, stained with Coomassie® brilliant blue perchloric acid color and scanned with a densitometer. A standard code was established linking the peak area of the meal band from whole venom samples to the amount of protein in the sample when applied. Six 40 µg samples of the purified melittin were analyzed simultaneously and their equivalence to honey bees was determined from the standard curve. This procudure is described in detail by Mulfinger et al. (1986).

15 Testing the Melittin Fraction for Snergistic Activity

To compare the antibacterial activity of whole honey bee venom and the melittin fraction, previous chessboard titration results were studied and the experimental system by which HBV dose effects could easily be seen alone and in combination with an antibiotic was selected. Since staphylococci were sensitive to HBV only at concentrations used in the above chessboard assays and kanamycin exhibited good synergistic effect with HBV, this system was selected to compare the antibacterial activities of whole HBV and melittin. Doses of each component used in this assay were 2 µg / ml HBV and 2.5 µg / ml kanamycin. These doses were in an area of bacterial reactivity where the effect of small dose changes was reproducible and easily determinable. The equivalent dose of melittin fraction to 2.0 µg / ml HBV was 1.6 µg / ml. Each trial compared 30 parallel triplicate samples of the melittin fraction and whole honey bee venom with and without the presence of kanamycin to test for equivalent activity.

Statistical Analysis 35

Each chessboard attempt was repeated five times. The average of the five repetitions for each bacterial-antibiotic kakombi night in on was tested for significant differences at each time using a Waller-Duncan K ratio T test and families of DK 167901 B1 18 curves were selected for synergy testing. A curve family consisted of a trial control curve (bacterial growth without the presence of antibiotics or HBV), an antibiotic control curve (bacterial growth with antibiotics but without HBV), a poison control curve (bacterial growth with HBV but no antibiotics present) and an interaction curve (growth with presence). of antibiotics and HBV). Families in which the antibiotic control curve and the poison control curve showed small mean OD decreases relative to the experimental control curve, and also showed greater OD decreases for the interaction curve compared to the experimental control curve, 10 were tested for synergy effect.

A synergistic effect can be differentiated between the compounds from the additive effect of the compounds, an additive effect being foreseeable. Additive effects can be predicted by summing up the effect of the two compounds individually, and thus any major effect will indicate a synergistic interaction.

An equation predicting OD readings for an additive interaction between HBV and an antibiotic was derived. Cf. Mulfinger's dissertation (1987), pages 23-25.

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results

Chessboarding nasal ointments 25 Three bacterial strains were tested against each of three antibiotics combined with honey bee venom. These nine combinations of bacteria, antibiotics and HBV were analyzed using the chessboard assay, which yielded 25 treatments (antibiotic and HBV combinations) for each bacterial antibiotic combination. Each chessboard trial 30 included triplicate samples for each treatment and was repeated five times. An average of the results from the triplicate samples was repeated in five experiments and the mean and standard deviation for each time point at each treatment are shown in the appendix. For each bacterial antibiotic combination, the mean OD values for each antibiotic / HBV treatment at each time point were arranged in descending order and grouped according to significant differences using the Waller-Duncan K ratio T test. From the Waller-Duncan profiles, families with four curves, as described in the "Statistical Analysis" section above, compared DK 167901 B1 19 for signs of synergy. The curve family, which showed the largest OD difference between the interaction curve and the bottom of the test curve, the antibiotic control curve and the poison control curve, was plotted and each time point tested for synergy using the equation derived in the above section "Statistical Analysis". If the estimate (-X + A + V-AV) for a time point is significantly greater than zero at 95% confidence level (i.e., suggestion of synergy), the time of each curve family will be noted on the interaction curve with an inscription "s" at the square, which represents this time 10 (Figures 2-11).

S. aureus S. aureus is sensitive to honey bees alone at low concentrations. Therefore, it was important to find the maximum dose of honey bee venom that did not produce any effects. This concentration was approx. 2 / ug / ml. Therefore, for all antibiotic / HBV combinations with S. aureus, the poison doses of the chessboard titration system were 0, 2, 4, 8 and 16 µg / ml (Table A-1 - A-3). Figure 2 shows the effects of these doses of 20 honey bees when used alone as an antibacterial compound.

S. aureus versus amoici11 in / HBV

The final concentration of ampicillin in tubes from the checkerboard system was 0, 0.05, 0.1, 0.2 and 0.4 µg / ml. Figure 3 shows the results of the ampicillin / HBV combination using 2 µg / ml HBV and 0.05 µg / ml ampicillin. No synergy is seen at the 4 or 6 hour point. However, at both the 8 and 12 hour points, it is clear that the interaction curve is much lower than would be expected from the sum of the effects caused by ampicillin and HBV alone. Statistical analysis shows that at both time points the addition (-X + A + V-AV) is significantly greater than 0.

35 S. aureus versus kanamvcin / HBV

The concentrations of kanamycin selected for testing S. aureus in the chessboard system were 0, 1.25, 2.50, 5.0 and 10.0 / tg / ml (Table A-2). Figure 4 depicts the curve family, which shows the greatest contrast between the control and interaction curve. In this experiment, the synergy first becomes detectable at the 6 hour point, and is evident at the 8 hour incubation. After 12 hours, the cultures appear to have avoided the effects of the combined dose and the synergistic effect 5 lost as growth is limited by other (nutritional) factors in the medium (this growth restriction is shown by the control curve). Despite the 12 h growth restriction, statistical analysis of the results after 6, 8 and 12 h suggests synergistic interaction between kanamycin and HBV in this analysis.

10

$. auresu versus polvmvxin B / HBV

The SI concentrations of polymyxin B in these experiments were 0, 312, 624, 1250 and 2500 U / ml (Table A-3). Synergy with 15 4 µg / ml HBV and 625 U / ml polymyxin B was observed (Figure 5). Synergy was shown after both 8 and 12 hours of incubation with the interaction curve.

E. coli 20 · Honey venom was not only inhibitory to E. coli at the levels required to show synergy (Table A-4 - A-6), and thus toxicity was not the limiting factor for HBV in the chessboard assay. E. coli. However, the experimental conditions limited the upper concentration of HBV to approx. 40 / ig / ml. Concentrations above 25 caused precipitation of constituents in the medium. . Therefore, the final concentration of HBV used in the chessboard analysis with E. col is at 0, 5, 10, 20 and 40µg / m 2.

E. coli versus ampicillin / HBV 30 The SI concentrations of ampicillin selected for use in the E. coli checkerboard titration were 0.5, 1, 2 and 4 µg / ml (Table A-4). The synergy was less drastic in all assessed curve families than for each of the above trials. There was only evidence of synergy with $ 0β $ / \ η \ 35 HBV-ljLtg / ml ampicillin combination and only at the 6 hour time point (Figure 6).

DK 167901 B1 21

E. coli versus kanamvcin / HBV

The final concentrations of kanamycin selected for the chessboard assay were 0, 5, 10, 20 and 40 μg / m 2 (Table A-5). Figure 7 shows the effects of 5 honey bees with a minimal effective dose of kanamycin. In this situation, only the 8 hour point exhibits synergy. Regardless of the HBV dose, there was no synergy in any of the other low dose HBV combinations kanamycin.

Figure 8 shows a higher dose of kanamycin with HBV on E. coli. In this case, the synergism is statistically displayed at all times after 2 hours.

E. coli versus polyvinyl B / HBV 15

The final concentrations of polymyxin B at the chessboard titrations were 0, 1.5, 3, 6 and 12 U / ml (Table A-6). The combination of 3 U / ml polymyxin B and 5 µg / ml HBV provided the most drastic illustration of the synergy blend (Figure 9). The synergy is evident at all times during the 20 treatment and the difference between the observed and predicted values is large.

Kanamycin-resistant S. aureus 25 A kanamycin-resistant S. aureus obtained by selection of spontaneous mutants was analyzed to assess the effect of HBV on drug-resistant bacteria. A kanamycin-resistant S. aureus was desirable, with some synergy being observed for all antibiotics with the organism and because the synergistic effects were most easily seen with kanamycin.

No difference was found with respect to the susceptibility of the resistant strains to HBV, and the poison concentrations in the chessboard assays were thus the same as for the parent strain, 0, 2, 35 4, 8 and 16 / tg / ml (Table A-7 - A 9). It was noted that under identical conditions, the resistant strain had a slower growth rate than the parent strain and therefore comparison of the optical density readings for the experiments on two different strains did not matter.

DK 167901 B1 22

Kanamvcin-resistant S. aureus versus ampicillIn / HBV

The final concentrations of ampicillin used in this chessboard analysis were the same as those of S. aureus of 0, 0.05, 0.1, 0.2 and 0.4 5 g / ml (Table A-7). Whether due to the slower growth rate or the resistance factor, the effects seen with this strain were not completely analogous to the parent strain. The best evidence of synergy could be seen at a higher ampicillin concentration than for the parent cell. Due to the slower growth rate, a longer growth period was investigated. Figure 10 shows the interaction between 2 µg / ml HBV and 0.4 µg / ml ampicill in. Statistical evaluation of the results shows synergy at 8, 12 and 24 hours time.

Kanamvcin resistant S. aureus versus kanamvcin / HBV 15

The kanamycin dose required to decrease the growth rate of the kanamycin resistant S. aureus strain was approx. four times higher than the dose required by the parent strain. The chessboard analysis range for the kanamycin resin stent S. aureus was 0, 5, 10, 20 and 20 40 µg / ml kanamycin (Table A-8). Again, the slow growth rate made it necessary to consider a longer growth period. The combination of 8 µg / ml honey bee venom and 10 µg / ml kanamycin is shown in Figure 11. Although the dose of kanamycin used is twice as high as the dose required for S. aureus originated, it remains effective. twice as long in the presence of honey bees. Only synergy was observed after 12 hours and it was found to be significant only after 24 hours.

Kanamvcin-resistant S. aureus versus polyvinoxin B / HBV 30

It was interesting to note that this mutant, selected for increased resistance to kanamycin, became more sensitive to polymyxin B than the parent strain. The polymyxin B doses used for the chessboard assay were 0, 12.5, 25, 50 and 100 U / ml (Table A-9), whereas the 35 polymyxin B dose range used to analyze the parent strain was between 312 and 2500 U / ml. Figure 12 shows kanamycin resistant S. aureus versus 50 U / ml polymyxin B and 4 µg / ml HBV. Synergy was shown at the 12 hour time point.

DK 167901 B1 23

MlC analyzes of antibiotics with and without HBV

The results of an initial study on the effect of HBV on the MIC of antibiotics for 8 gram-positive bacteria and 4 gram-negative 5 bacteria are shown in Table 4 and Table 5. Despite the apparent inadequacy of the assay system, clear trends emerged from the results of the study. Synergy was strongly suggested when the observations within a single MIC analysis showed that identical doses of HBV affected some antibiotic MICs without affecting others. In Tables 4 and 5, (+) was used to show a decrease of more than a 2-fold dilution of MIC by an antibiotic in the presence of HBV. One (-) indicates no difference or only a single dilution step variation (considered to be the variance of the assay) in the MIC of an antibiotic with HBV present.

15

Table 4 shows the results of several gram-positive organisms. The results suggest that trends exist within the species tested. For example, S. aureus is found to show synergy with all antibiotic / HBV combinations, whereas S. epidermidis only 20 exhibits concordant synergistic results with the cephalothin / HBV combination and single results with other antibiotic / HBV combinations. The one tested Streptococcus faecal in the s strain did not reflect any of the same synergistic tendencies exhibited by the two staphylococcus organisms.

25

Table 5 shows the results for four E. coli strains in the MIC assay system. Clear synergy patterns are seen with each of the beta-lactam antibiotics (ampicillin, carbenicillin and piperacillin) included in the MIC assay system. Also, the MIC of the aminoglycosides gentimicin and 30 amikacin was lowered in each case except one. MIC for cefoxitin was also lowered by HBV in all E. colj assays.

Melittinoprene Rino and Test 35 Chromatography of honeycomb

Purification of melittin on the Sephadex G-50 yielded well-defined dissolved baseline peaks. The void volume was 100 ml and the melittin fraction eluted between 200 and 230 ml after void volume. About 65 µg of the sample of the initial 100 µg was recovered in fractions 200 -230. These fractions were combined and then tested for purity by polyacrylamide gel electrophoresis. Figure 13 shows electrophoresis results for 100 µLg of protein from the pooled fractions 200 - 230.

5 Comparison of the relative mobility of this band with the relative mobility of electrophoretically separated HBV components identified mel ittin as the only component of fractions 200 - 230 which is detectable by this separation.

Testing of flour in ttin for antibacterial activity

Similar doses of melittin and whole honey bee venom were compared for antibacterial activity in combination with and without antibiotics (Table A-10). As S. aureus was sensitive to HBV at levels 15 used in the above assays, this organism was selected to test the activity of melittin fraction. Kanamycin was chosen to assess the synergistic activity of the fraction, as the interaction curve seen in the above test of S. aureus versus this antibiotic with HBV reflected synergy at all times.

20

Flour in ttin's antibacterial activity

The results for melittin versus whole HBV are shown in Figure 14. No significant differences in antibacterial activity were observed for whole HBV and melittin fraction. For each time point shown in Figure 14, the optical densities of the HBV curve and flour in the ttin curve are statistically similar.

Melittin's synergistic activity with kanamvcin 30

Figure 15 compares the antibacterial activities of equal doses with whole fraction of HBV venom in combination with equal doses of kanamycin. Neither optical density is significantly different at any point on the two curves. In fact, without taking into account 35 statistical assessments, the interaction curve representing the meal fraction is slightly smaller at all times than the interaction curve representing whole HBV. Thus, if the time on both curves is accepted as true averages, the final conclusion would be that the melittin fraction is actually more active than whole HBV.

Interpretation of chessboard analysis results 5 The results of the chessboard analyzes clearly show the synergy between antibiotics and honey bee venom. Figure 2 shows the effect of different doses of honey bee venom on S. aureus without antibiotics. It can be seen from this figure that adding high poison doses, such as 8 or 16 µg / ml, to growing cultures actually lowers the optical density of the culture. This indication of cell lysis is proof that honey bees are actually bactericidal. Mechanisms of this bactericidal activity and its contribution to the synergy with antibiotics are unknown. The varied results from the chessboard titration analyzes suggest that several different synergistic mechanisms may work in these experiments.

Questions may arise with regard to the large standard deviations seen at certain times in the result tables. This variation is due to the sharp slope of the growth curve when the bacteria are in the 20 logarithmic phase. Mid-logarithmic phase times will have a much greater difference in optical density as a function of time than times during a slower growth period. Thus, uncontrollable small variations in sampling intervals may cause greater variations in optical density readings at times below 25 logarithmic growth. As the culture is divided during the logarithmic phase into different treatment groups, the variations are even more visible between the experiments. However, this type of error is taken into account in the statistical assessment. Using a large sample number (15), the estimation ranges for the time averages were made 30 narrow enough to statistically estimate differences among these averages.

Although melittin was only initially tested as the synergistic component of HBV along with an antibiotic and bacterial strain to discuss possible mechanisms, it has been hypothesized that melittin is the synergistic honey bee component of each of the bacterial antibiotic HBV tested. combinations.

DK 167901 B1 26

Apparently increased dose In most cases, honey bee venom seems to increase the initial effectiveness of the antibiotic, which is suggested by an increased ability 5 to lower bacterial growth rate immediately after the addition of the two compounds. This type of interaction was most detectable with E. coli versus HBV and polymyxin B (Figure 9). At the first stage after the addition of the two compounds, the synergy is evident and it continues as the culture goes through the logarithmic phase. These 10 results suggest that low, ineffective doses of antibiotics can be made effective with the addition of HBV.

The increased dose effect described above is the synergy type seen in most of the tested combinations tested. This type of action can be explained by the action of melittin through several different mechanisms: (1) altering the solubility properties of antibiotic molecules, (2) increasing the permeability of the bacterial membrane, and (3) increasing the effectiveness of the antibiotic molecules at their active sites.

Altered solubility properties of antibiotics

Melittin could increase the effect of antibiotics by making it easier to transport into the bacterial cell. The direct interaction between melittin and antibiotic molecules, which makes the molecules less polar and more hydrophobic, may allow passive transport through bacterial membranes. Melittin's amphiphatic nature and basicity make it a likely candidate for such an effect and give increased probability of this mechanism. This type of mechanism will correspond to the easier diffusion of potassium ions with valinomycin.

Increased membrane permeability 35 The apparent antibiotic dose can also be increased by reducing the bacterial penetration barrier. Although this role as a channel-forming peptide is easily supported, it may not be the only effect of melittin involved in the antibacterial le synergy. Increased transport across membranes cannot explain why DK 167901 B1 27 melittin alone is more effective on Gram-positive organisms, which have less membrane barrier.

Increased antibiotic-specific activity 5

A third possible mechanism for synergistic interactions suggests the direct interaction between melittin and antibiotics, making antibiotics more effective when it reaches the active site. A more specific example is the possible interaction with kanamycin. When 10 kanamycin reaches the 30S ribosome, a melittin-kanamycin complex may have a greater affinity for the active site than unbound kanamycin (in fact, melittin is a basic molecule, like nucleic acid) or the mel ittin-kanamycin complex may be more effective in steric blocking of transfer. -RNAs from the ribosome simply due to the size of the complex.

Increased active life of antibiotics In several cases, it was difficult to detect an increase in the effectiveness of antibiotics by the addition of honey bees (melittin) until late in the growth period. In these cases, melittin was found to cause an increase in the duration of antibiotic action. This effect was seen with kanamycin resistant S. aureus treated with kanamycin / HBV. Figure 9 shows a relatively high dose of HBV, which is due to no synergy at the lower dose. Although Figure 9 is difficult to exclude synergy at the earlier times due to the efficacy of HBV alone, lower HBV doses showed no synergy with kanamycin at these early times. However, a synergistic effect is noted at the 24 hour time point. Two explanations for this type of delayed effect are proposed: (1) elimination of resistant mutants or (2) delay of half-life of antibiotics.

Decreasing probability of selection of resistant strains 35

If both honey bee venom and antibiotics are present in the bacterial culture at bacteriostatic doses, the probability that a resistant bacterium will survive the combined mixture is equal to the product of the probabilities that one will exist and survive each treatment. This would appear as a delayed synergistic effect as it will take many generations for the mutants to multiply to a level that can be detected by increased OD readings. Mutant selection would be characterized as a sporadic occurrence of a drastically higher OD reading among copy samples, which would be reflected in the standard deviation of the treatment. For example, when assessing the effects of HBV treatment on kanamycin-resistant S. aureus alone with kanamycin, the OD average of the 12-hour point on the poison control curve is 0.65 with a standard deviation of 0.51 10 (Table A-8), suggesting very varied readings at this time. Thus, it could be very likely that the synergistic effect seen here at the 24 hour time point is the result of suppression of HBV venom resistant mutants.

15 Increased antibiotic stability

One possible mechanism that cannot be ruled out is the protection of antibiotics from disintegration. A common method of increasing antibiotic efficacy is to change the antibiotic structurally to make it more stable in solution or resistant to enzymatic attack. These types of modification are responsible for many of the derivatives of the penicillin family in one of the antibiotics. For example, penicillin V has a phenoxymethyl substitution which is sterically hindered, protecting the beta-lactam ring of the antibiotic from enzymatic attack (Volk, 1978c). Such substitutions may also prevent this end of the molecule ring-ending with the beta-lactam ring, making the molecule more resistant to acid hydrolysis. These types of modification will also produce a synergistic effect that can only be detected in bacteriostatic doses, since the antibiotic will not initially be more effective and the longer life of the antibiotic would be evident only if the bacterial culture had not reached a nutrient-limiting OD at this time. However, if HBV could cause such a modification, more consistent results could be expected among copy samples.

35

Assessment of MIC testing

The chessboard titration assay developed for HBV / antibiotic synergy testing was for time consuming to be used in DK 167901 B1 29's study of the effect of HBV on different antibiotics and on different bacteria. However, such a study was needed to locate trends among antibiotic classes for synergy with HBV, as well as to determine the spectrum of sensitivity 5 among bacterial species to specific synergistic combinations of antibiotics and HBV. The modification of the automated MIC analysis was designed to facilitate this type of study.

Due to the limitations of the automated MIC analyzes, the 10 assessment of the results is somewhat empirical. The results cannot be shown to be synergistic interactions as opposed to additives, as the effect of HBV alone was only recorded as inhibitory or noninhibitory (slightly inhibitory HBV doses would have been recorded as noninhibitory, in this way some MIC decreases 15 actually be the result of an additive effect). However, in most cases, certain antibiotics exhibited decreasing MICs, suggesting that the HBV dose was not additive. Therefore, when supported by the results of the chessboard titration system, the use of these MIC assays should be reliable in identifying the antibiotic / -20 HBV combinations with the greatest potential for specific bacterial groups. In this regard, the MICs will be useful for direct investigation in the future.

Identification of the active honeycomb component 25

Although the results of these studies suggest that the honey bee synergistic activities are completely contained in the melite-tin fraction, careful examination of these results should be performed. It is possible that small peptides or non-staining (Coomassie blue) compounds migrate with the melittin by chromatography due to ionic or hydrophobic interactions with flour in the molecule. Melittin is migrated as an aggregate with five times its usual molecular weight both by natural polyacrylamide gel electrophoresis and by Sephadex gel chromatography (Haberman, 1972). These 35 small micelles can carry less hydrophobic compounds through the chromatography.

As stated above, further experiments were performed to show that DK 167901 B1 30 HBV is also effective in increasing the activity of the above mentioned fourth group of antibiotics represented by rifampicin. The results are listed below in Tables 6, 7 and in Figures 16, 17.

5

The activity of hymenopteran venom other than HBV, namely, for hop bee venom, wasp venom and goat venom, as shown below in Tables 8, 9 and Figures 18 - 20, was also determined. Some of the above-mentioned analogs were also tested to determine their relative activities in relation to natural flour. The relative activity is calculated as follows: _Melittine dose_ yqq

Analog dose required to show similar synergy

polymyxin B

The results obtained are listed in Table 10.

It turns out that the analogs in which the NHg terminal end consists mainly of basic amino acids are more active than analogs with an NHg terminal end consisting mainly of neutral and / or acidic amino acids.

25 In vivo experiments

Introduction

In vivo tests show that melittin, the major peptide in 30 honey bees, increases the efficacy of a tested antibiotic, polymyxin B. A disease model, bacterial sepsis, was developed in mice. For the experiments, the activities of polymyxin B and melittin, separately and in combination, are compared against E. coli septicaemia in two basic test kits. For both test protocols, a synergistic interaction between melittin and polymyxin B is seen and this is statistically confirmed by comparing the treatments in each analysis of variance. Thus, the ability of melittin to increase the effect of polymyxin B and to provide excellent antibacterial activity in vivo is shown at species.

DK 167901 B1 31

Several of the references cited above in the section entitled "Background and Prior Art" describe the use of honey bee venom and more specifically melittin as an antimicrobial agent. However, these references only describe in vitro efficacy of 5 honey bees in ft or melittin.

Several other systems have used melittin as an artificial means of eliciting various immune responses in solar in vitro systems. Goodman et al. (1984) reported on B cell activation with melittin jn.

In vitro. Two separate reports, one by Kondo and Kanai (1986) and one by Kondo (1986), describe the use of melittin in vitro to stimulate the bactericidal activity of membranes isolated from both mouse and guinea pig phagocytes. A publication (Somerfield et al., 1986) on the effect of honni ngbi gi ft on the immune system, 15. describes inhibition of neutrophil O production by melittin. Somerfield et al. suggests that melittin has a role as an anti-inflammatory agent. This activity is likely to impair antibacterial defense in vivo.

Despite the substantial scope of studies with melittin, melittin has not yet been shown to be effective in vivo against infectious organisms. More importantly, the need or benefit of mel ittin's interaction with antibiotics has never been proposed. The results described herein show beneficial interactions between melittin and polymyxin B when used in vivo to treat mice suffering from a bacterial septicemia caused by E. coli.

Materials and Methods 30

Swiss CD-I female mice (Charles River) were obtained with weights of 18-20 g. The mice were placed at 25 ± 0.6 ° C and 30 - 45% relative humidity with a daily light period of 12 hours. After delivery, each shipment of mice was maintained for a 2-week acclimation period prior to use in the experiment.

Polymyxin B (Sigma Chemical Company) was purchased as a powder form with an activity of 7900 units / mg. A stock solution of 0.1 mg / ml in 0.85% NaCl was prepared and frozen at -20 ° C in 5 ml DK 167901 B1 32 partial samples until use.

Honni ngbi gi ft (HBV) was provided by Vespa Laboratories, Inc. Spring Mills, PA. HBV was the source of ttin flour which was isolated using gel filtration as described previously in in vitro experiments. Flour in ttin was quantified by Lowry's protein analysis (Lowry et al., 1951) and then lyophilized. The freeze-dried flour in ttin was reconstituted in 0.85% NaCl to a concentration of 0.1 mg / ml and frozen at -20 ° C in 1.5 ml aliquots, until further use.

E. coli strain No. G1108E was obtained from Pennsylvania State University E. coli Reference Center, University Park. ON. 0.5 ml of a 24 hour trypticase soy broth was used to inoculate 800 ml of 15 fresh trypticase soy broth. The culture was propagated overnight under mild shaking. 200 ml of sterile glycerol was added to the culture, which was then aseptically distributed with stirring in 5.0 ml of sub-samples. These subsamples were frozen and stored at -20 ° C.

ISLAND

After thawing, each subsample gave (± 3) x 10 live bacteria / ml.

The culture was then diluted 1: 400 with trypticase soy broth containing 2.5% gastric mucin (Sigma Chemical Company) before inoculation.

Mice were infected by intraperitoneal injection of 0.25 ml of a 25 1: 400 dilution of bacteria (approximately 500,000 live bacteria) suspended in trypticase soy broth with 2.5% mucin.

Prior to injection, polymyxin B and melittin were thawed, filter sterilized and appropriately diluted with sterile 0.85% NaCl so that the required dose was contained in 0.2 ml of solution. Thirty minutes after infection, this volume was then administered to the mouse by subcutaneous injection into the skin fold at the base of the neck. The skin fold is formed between the thumb and forefinger with a basic grip.

35 Bacteria levels in the blood were determined from blood samples obtained by aseptic heart puncture. After cardiac puncture, the needle was removed from the hepatic nebuliser and 0.2 ml of blood was partitioned into a tube containing 0.2 ml of 0.85% NaCl and mixed well. All samples were kept on ice until plating. Double spread sheets of all samples at appropriate dilutions were prepared on trypticase soy agar and incubated at 37 ° C overnight. All plates containing less than 400 colonies were counted and recorded.

5 results

In the first experimental design, four random groups with each of four mice were inoculated with E. coli as described above. Thirty minutes later, the four mice in each group were each treated with 0.2 ml of 0.85% NaCl solution containing one of the following: 1) only 0.85% NaCl ("no treatment"), 2) 2, 0 µg polymyxin B, 3) 50 ng melittin or 4) 2.0 µg polymyxin B + 50 ng melittin. 21 hours after the initial inoculation, blood samples were taken and the number of bacteria per ml of blood was calculated by taking the mean of 1® results from double plate counts of appropriate blood dilutions (Table A-11). This experiment was performed in triplicate and the average number of bacteria per ml of blood for each of the four treatments was compared (Figure 21). A p-value of 0.0015 for the treatment effect of a two-way variance analysis (adjusted for 20 sample sizes) indicated a significant difference in at least one of the treatment methods. Tukey's multiple mean comparison showed that the only mean that was significantly different was the mean of the group receiving the combination of 2 µg polymyxin B and 50 ng melittin. By comparing the sum of activities obtained with melittin and polymyxin B used by the individual with their activity when used in combination, a contrast within the analysis of variance confirmed that the interaction was synergistic (p-value = 0.0493).

30. Another experimental design tested the effect of repeated treatments. Again, four groups each containing four mice were inoculated with E. col i and treated 30 minutes later with the same four treatments: 1) only 0.85% NaCl ("no treatment"), 2) 2.0 µg polymyxin B, 3 ) 50 ng melittin or 4) 2.0 µg polymyxin B + 50 ng melittin. 18 Hours after the initial infection, each mouse was challenged with it

same E. coli inoculum and treated 30 minutes later with the same I

anti bi oti ka / meli tti treatment for 5 hours later (23 hours after the initial infection), blood samples from each mouse were plated to quantify the number of bacteria in the blood. This experiment was repeated 5 times and the results (Table A-12) were assessed by analysis of variance. These results showed a significant difference (p-value = 0.0001) in at least one treatment. Tukey's multiple average comparison showed that repeated polymoxyin B treatments 5 caused a significant decrease in the number of bacteria per ml of blood. More importantly, Tukey's comparison showed that the bacterial counts in the blood from animals treated with polymyxin B as well as melittin were significantly lower than the counts in blood from animals treated with polymyxin B or melittin only (see Figure 22). A contrast within 10 of the variance analysis yielded a high degree of confidence for the synergy of these two compounds (p-value = 0.0007).

Conclusion 15 The above test clearly shows the synergistic interaction between the antibiotic polymyxin B and melittin. Melittin is most likely to potentiate the therapeutic effects of other pharmaceuticals due to its antimicrobial properties and ability to increase membrane permeability in the theta.

20

The results from the first set of experiments (Figure 21) lacked statistical significance for the effect of melittin alone, but comparisons of the absolute averages indicated positive effects with melitin treatment alone. The results for the second set of 25 trials (Figure 22) again lacked a significant difference for treatment with melittin alone. A comparison of the absolute treatment averages suggested a negative effect of melittin alone. Recalling that mice in the second set of trials received double melitin doses, this suggests that higher melitin doses may aggravate the infectious process when used without antibiotics. Previous trials of higher melittin doses confirm this assumption. The harmful activity is likely to occur when melittin is used alone. It is important that the effective use of melittin for the treatment of infections was not found in litte-35 rats. Antibiotics apparently counteract the negative effects of melittin and thus make combined therapy a significant development.

DK 167901 B1 35

Antibiotikasynerqi

Shown with a synthetic flour ittin analogue and 5 The synergy seen between antibiotics and melittin can also be achieved by replacing melittin with a synthetic peptide analogue. Such an analog was designed and synthesized for this purpose. When tested in parallel with natural melittin, the corresponding antibiotic boost was given.

10

Introduction

Analog # 6, the structure of which is shown below, H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-ne-Ser TrD-ne-Lvs-Lvs-Lvs-Lvs-Gln-Gln-NHo was tested in vitro as described previously for polymxin B synergy against E. coli. This peptide varies from melittin at the 20 amino acids 22 and 24 (underlined), where argenic units have been replaced by lysine units. When used in the aforementioned analysis, it showed an activity similar to natural melittin.

Materials and Methods 25

Melittin was isolated from whole honni ngbi gi ft (Vespa laboratories, Inc.) by gel filtration chromatography, quantified by Lowry protein analysis, and stored freeze-dried. For these assays, lyophilized melittin was reconstituted to 0.4 mg / ml with 30 distilled water, filter sterilized and stored as 4.0 ml aliquots at -20 ° C until use.

Analog # 6 was synthesized by dr. Torben Saermark (at the Protein Laboratory, University of Copenhagen, Sigurdsgade 34, DK-2200 35 Copenhagen N, Denmark). It was estimated to be more than 98% pure based on the high pressure liquid chromatography elution profile of a C18 column using a 0-80% acetonitrile gradient in 0.1% trifluoroacetate. Pepti it was received in freeze-dried form and was reconstituted to ca. 0.2 mg / ml in 0.85% NaCl, filter sterilized and stored as 0.5 ml partial samples at -20 ° C until use.

Polymyxin B (Sigma Chemical Company) with a specific activity of 7900 units / mg was reconstituted to 240 units / ml in distilled water, filter sterilized and stored as 4.0 ml partial samples at -20 ° C until use.

E. coli strain No. G1108E was obtained from Pennsylvania State University E. coli Reference Center (105 Henning Building, University 10 Park, Pennsylvania, 16802). Inoculum was prepared from a culture grown in trypticase soy broth to the middle logarithmic phase. Sterile glycerol was added to give a final concentration of 20% and the culture was distributed and frozen in 5.0 ml aliquots at -20 ° C until use.

15

A chess boarding synergy analysis was performed for parallel testing of natural melittin and analog # 6 together with polymyxin B against E. coli. Equivalent doses of natural melittin and analog # 6 were based on a Lowry protein assay performed simultaneously on sub-samples 20 of each after the final filtration. Both peptides were tested in the synergy assay at final concentrations in the medium of 5 µg / ml and 10 µg / ml. Polymyxin B was tested at final media concentrations of 3 units / ml and 6 units / ml against both levels of both peptides.

25 results

The synergy was best demonstrated with both the natural melittin and analogue # 6 tested at 10 µg / ml versus 6 units / ml polymyxin B (Table 11). Under these conditions (cf. Figure 23), results for each 30 peptide were statistically analyzed for synergistic activity at each time point. Using statistical contrasts, activity averages for each peptide alone and polymyxin B alone were compared to the activity of the respective peptide polymyxin B

combination. Synergy was detected at 4, 6 and 8 hours at 35 for both melittin and analogue # 6 (p values = 0.0001).

Furthermore, synergy curves for melittin (10 µg melittin + 6 units polymyxin B) and analogue no. 6 (ΐθ / xg analogue no. 6 + 6 units polymyxin B) were compared at each time point for different activity levels. At no time could a significant difference be detected between these two curves.

Conclusions 5

The results show that analogue # 6, which is a synthetic melittin analog, possesses an activity very similar to the activity of melittin in terms of its ability to enhance polymyxin B's activity. Although the 12 hour point suggests that analog # 6 possesses 10 slightly better activity with polymyxin B than melittin does, this difference in activity is minimal in terms of the current quantitative difference in peptide that it will reflect. By comparing the difference in synergy produced with 10μg melittin versus 10μg analogue # 6 with the difference in synergy produced with 10μg melittin versus 5μg 15 melittin (Table 11), the difference between the specific activities of the melite versus analogue # 6 can be estimated to be smaller than 10%.

The study shows that it is possible to synthesize melittin analogs with synergistic capabilities equal to or better than melittins.

Synergistic antibacterial activity of melittin and polymyxin B:

Relative activities of melittin analogs_ 25

The synergy seen between antibiotics and melittin can also be achieved by replacing melittin with either a synthetic analogue or a chemically modified derivative of the natural peptide. Synthetic melittin, five synthetic peptide analogs, and a chemical modification of 30 natural melittin were tested for synergistic interaction with polymyxin B for growth inhibition of E. coli. Their relative activities were compared to the activity of melittin from natural honey bees. Each peptide exhibited synergistic interaction with polymyxin B. However, the specific activities were significantly different. Several analogs provided better synergistic activity than natural melittin.

DK 167901 B1 38

Introduction

Two types of melittin analogs were analyzed in vitro, namely synthetic peptides and a chemical modification of natural melittin, 5 for synergy with polymyxin B in an antibacterial activity assay.

The synthetic analogs included a group of synthetic peptides, all of which differed from melittin's 26 amino acid sequence by two or more units. The chemical modification of melittin, NPS-melittin, consisted of a fixed view of an o-nitrophenylsulphenyl group to No. 19 10 tryptophan unit in natural melittin. The activity of each of these analogs was compared to the activities of both natural and synthetic melittin. Although each of these analogs exhibited some synergistic interaction with polymyxin B In vitro, significant differences in peptide activities were evident. These differences define 15 key properties of flour in the molecule in its role as a potentiator of polymyxin B activity.

Materials and Methods Natural melittin was isolated from whole honey bee venom (Vespa Laboratories, Inc.) by gel filtration chromatography, quantified by Lowry protein analysis and stored lyophilized. For these assays, lyophilized melittin was reconstituted to 0.34 mg / ml with distilled water, filter sterilized and stored as 4.0 ml 25 samples at -20 ° C until use.

NPS-melittin was synthesized from natural melittin by reacting the peptide with o-nitrophenylsulfenyl chloride (NPS-C1), as has been described for adrenocorticortropin by Ramachandran et al.

The peptide was precipitated from solution with ethyl acetate, resuspended in 0.1N acetic acid and then passed through a Sephadex G-10 (LKB-Pharmacia, Piscataway, N.J.) column to remove residual NPS-C1 salts. A determination of the molar absorptivity of the derivative at 365 nm suggested that melittin was more than 95% modified.

35

Synthetic melittin was purchased from Penisula Laboratories, Belmont, CA. 0.5 mg sample was reconstituted to 0.3 mg / ml in 0.85 saline solution based on Lowry protein analysis. The sample was stored at -20 ° C until use.

DK 167901 B1 39

Synthetic analogues were synthesized by dr. Torben Seemark (Protein Laboratory, University of Copenhagen, Sigurdsgade 34, DK-2200 Copenhagen N, Denmark). Each analog was analyzed for purity and was estimated to be more than 98 purely based on the chromatographic elution profile of a C-18 column using a 0-100% acetonitrile gradient. Each peptide was received in lyophilized form and reconstituted to ca. 1.0 mg / ml in 0.85% NaCL, filter sterilized and stored as 1.0 ml partial samples at -20 ° C until use. Each peptide solution was quantified by Lowry protein assay before use. The Lowry results were in good agreement with the concentration estimates based on peptide dry weights.

Polymyxin B (Sigma Chemical Company, St. Louis, Mo.) with a specific activity of 7900 units / mg was reconstituted to 240 units / mg in 0.85% saline, filter sterilized and stored as 4.0 ml aliquots at -20 ° C until application.

E. coli G1108E was obtained from the Pennsylvania State University E. coli Reference Center (105 Henning Building, University Park, Pa.).

20 16802). Inoculum was prepared from a culture grown in trypticase column broth for midlogarithmic phase. Sterile glycerol was added to the culture to a final concentration of 20% and 5.0 ml aliquots were dispensed and frozen at -20 ° C until use.

A checkerboard titration synergy analysis was performed which tests each peptide with polymyxin B against E. coli in parallel with melittin.

Equivalent doses of the natural melittin and each analog were based on a Lowry protein assay performed on subsamples of each peptide after the last filtration of the stock solution. All peptides j 3 ° were tested by the synergy analysis at final concentrations in the medium of 5 µg / ml. The concentration of polymyxin B in the medium in all the assays was 6 units / ml.

Results 35

Table 12 shows the amino acid sequences of the synthetic melittin analogs. For each synthetic analogue, the first 20 N-terminal amino acids are the same as for natural melittin. Changes occur in the six C-terminal amino acids and are indicated by highlighted text.

DK 167901 B1 40

Table 13 contains the growth curve readings for each of the compounds tested for synergistic interaction with polymyxin B. The value for each time point represents the mean and standard error of the mean for six samples. The "control" curve represents growth of the culture without added polymyxin B or peptide. The effect of each peptide on the culture alone is not included in the table. However, like flour in ttin, these peptides had no effect on the growth of E. coli when used alone at 10 µg / ml or less. Thus, the "control" is also representative of the culture when treated with each peptide alone.

When the growth curve for bacterial cultures treated with polymyxin B (6 units / ml) alone is compared with the curve for a culture treated with polymyxin B as well as melittin (5 µg / ml), increased antibacterial activity is shown by increasing the time required for that the culture gets over the treatment and reaches the logarithmic growth phase (Figure 24}. As treatment of the culture with melittin alone at 5 µg / ml will produce a growth curve that would substantially overlap the "control" curve, an increase in the time required for the culture to escape the polymyxin B inhibition and reach the mid-logarithmic phase in the presence of the peptide as evidence of the synergistic activity of the peptide A script of the growth curve representing polymyxin B with peptide to the right of the curve representing with polymyxin B alone, will be evidence of synergy.

25

The relationship between polymyxin B, melittin and bacteria in these experiments was designed to produce minimal synergy so that increased activity of peptide anal ogers could be seen. Such an increase is shown in Figure 24 for both synthetic melittin and NPS melittin.

All peptides were present at equal concentrations determined by Lowry analysis.

Corresponding growth curves comparing the synergistic activities of the synthetic melittin analogs with polymyxin B are shown in Figure 25.

To more clearly visualize the difference in melittin / analog synergy with polymyxin B, Figures 24 and 25 were used to calculate the additional time delay until each growth curve reached the mid logarithmic phase due to addition of melittin or analogs in comparison. with the treatment with polymyxin B alone. These values are shown in a bar chart in Figure 26. A Tukey-studentized value set test was performed on the results in Table 13 5 to compare the readings obtained at each time point between the different treatments. The peptides were then grouped all depending on their ability to exhibit significantly different levels of growth inhibition (alpha = 0.05) for at least one of the growth curve times. These groupings have been denoted by various 10 column markings in Figure 26.

conclusions

These results show that a wide variety of melittin analogs can be created by both amino acid substitutions and chemical modifications. These types of modifications can either increase or decrease the relative synergistic activity of the peptide. On the basis of Figure 26, the relative in vitro activities of melittin and its analogs can be listed in descending order: 20

1 Analog No. 7 A

2 Natural melittin B

3 NPS melittin C

4 Analog No. 6 C

25 5 Synthetic melittin C

6 Analog No. 2 C

7 Analog No. 4 D

8 Analog No. 5 D

5 ° Peptides with significantly different (alpha = 0.05) synergistic activities at the 5 µg / ml level are listed in letter groups.

It should be noted that the parameter used to establish this order of action, namely delayed time until the mid-logarithmic phase of the culture, is increased stoichiometricly with the amount of peptide over a narrow range of melittin concentrations. Since the boundaries of the linear range of each analogue need not be equivalent, the present results can only be used to determine the relative order of activity of these peptides at the given concentration and cannot be used to estimate quantitative differences. However, the order of action suggests that the synergistic activity of the peptides depends on the number and exposure of positively charged side chains on the amino acids in the 5 C-terminal region.

Although the relative efficacy of these cells has been determined in vitro, significant differences may occur in vivo. In vivo parameters such as absorption in and clearance from the host can significantly alter this order of action when used in practice. Side effects must also be taken into account. Although adrenocortico-tropic activity of melittin is well documented, NPS-melittin may possess less adrenal activity than natural melittin, with the NPS derivative of adrenocorticotropin (ACTH) inducing 100 times less lipolytic activity than unmodified ACTH. Because of this, every analogue covered by this study should be considered in in vivo assessment.

20 25 · 30 35 5 43 DK 167901 B1

Table 1 Concentrations of antibiotic stock solutions and honey bee venom tested against three different bacteria.

Organism Gift Ampici11 in Kanamvein Polvmvxin B

E. col i 800 µg / ml 80 µg / ml 800 µg / ml 240 U / ml S. aureus 320 µg / ml 8 / ig / ml 200 / zg / ml 50/000 U / ml 10 S. aureuskana® 320µ9 / ιη1 8 µg / ml 800 µg / ml 2000 U / ml

Table 2 Design and distribution of honey bee venom and antibiotics by a chessboard titration analysis.

15 Antibiotic Disorders

Control 1:16 1: 8 1: 4 1: 2 H Control 0a \ 0b 0 \ 1: 16 0 \ 1: 8 0 \ 1: 4 0 \ 1: 2 20 0 f! _3c 4-6 7-9 10 -12 13-15 nonr 1:16 1: 16 \ 0 1: 16 \ 1: 16 1: 16 \ 1: 8 1: 16 \ 1: 4 1: 16 \ 1: 2 it 16-18 19-21 21 -24 25-27 28-30 • new 25 gn 1: 8 1: 8 \ 0 1: 8 \ 1: 16 1: 8 \ 1: 8 1: 8 \ 1: 4 1: 8 \ 1: 2 bd 31 -33 34-36 37-39 40-42 43-45 iign 1: 4 1: 4 \ 0 1: 4 \ 1: 16 1: 4 \ 1: 8 1: 4 \ 1: 4 1: 4 \ 1: 2 ig 46-48 49-51 52-54 55-57 58-60 30 9 etr 1: 2 1: 2 \ 0 1: 2 \ 1: 16 1: 2 \ 1: 8 1: 2 \ 1: 4 1 : 2 \ 1: 2 61-63 64-66 67-69 70-72 73-75 a = counter, dilution level of HBV stock solution 35 b = denominator, dilution level of antibiotic stock c = assay position in a sequential arrangement with 75 test tubes DK 167901 B1 44

Table 3 Volumes and distributions of each component from the chess board analysis.

Stir No._TSB_Antibiotics_Gift_Bacteria 5 00-0 2.5 ml - - 1-3 500 / Ltl - - 2.0 ml 4-6 250 µl - 250 µl 1:16 2.0 ml 7-9 250µ1 - 250 / l 1: 8 2.0 ml 10-12 250 / l - 250 / l 1: 4 2.0 ml 13-15 250 / l - 250 / l 1: 2 2.0 ml 16-18 250 / l 250 il 1:16 - 2.0 ml 10 19-21 - 250 µl 1:16 250 µl 1:16 2.0 ml 22-24 - 250 µl 1:16 250 µl 1: 8 2.0 ml 25 -27 - 250 µl 1:16 250 µl 1: 4 2.0 ml 28-30 - 250 µl 1:16 250 µl 1: 2 2.0 ml 31-33 250 µl 250 µl 1: 8 - 2.0 ml 34-36 - 250 µl 1: 8 250 µl 1:16 2.0 ml 37-39 - 250 µl 1: 8 250 µl 1: 8 2.0 ml lt; 40-42 - 250 / l 1: 8 250 / l 1: 4 2.0 ml lb 43-45 - 250 / l 1: 8 250 / l 1: 2 2.0 ml 46-48 250 / l 250 / l 1: 4 - 2.0 ml 49-51 - 250 / l 1: 4 250 / l 1:16 2.0 ml 52-54 - 250 / l 1: 4 250 / l 1: 8 2.0 ml 55- 57 -. 250 / l 1: 4 250 / l 1: 4 2.0 ml 58-60 - 250 / l 1: 4 250 / l 1: 2 2.0 ml 61-63 250 / l 250 / l 1: 2 - 2 , 0 ml 20 64-66 - 250 / tl 1: 2 250 / l 1:16 2.0 ml 67-69 - 250µl 1: 2 250µl 1: 8 2.0 ml 70-72 - 250 / tl 1: 2 250µ1 1: 4 2.0 ml 73-75 - 250µ1 1: 2 250µ1 1: 2 2.0 ml

Table 4 The effect of 4μ9 / πι1 HBV on MICs of eleven antibiotics on eight Gram-positive organisms.

A1 _B2_ ΟΞ.

Q.535577S C4 1 9 9 9 9 9 f 0 8 0 0 0 0 7 1 5 7 5 8 2 7 30 - -

Penicillin -5 +6 + - - ++

Methicillin + + - * - + + -

Ampicillin + + + - + + “

Cephalothin + + + + + + + + -

Gentamicin + + + - - + + - 35 Kanamycin + + - - + + -

Ery thromyciri + + - - - + “

Chloramphenicol ++ ---- ++

Clindamycin + + - - + -

Tetracycline + + - - + “

Vancomycin + + - - + - 45 DK 167901 B1 1 - Group "A" = two S. aureus strains 2 - Group "B" = five S. aureus strains 3 - "C" = one Streptococcus faecal in S. strain 4 - QC = an S. aureus strain used for routine quality control testing of this assay system.

5 - A (-) indicates an MIC decrease of less than two dilution steps 6 - (+) indicates an MIC decrease of more than or equal to two dilution steps.

10 Table 5 The effect of 4 µg / ml HBV on MICs of eleven antibiotics on four E. coli strains.

_E, coli strain_ 0 14 1 15 C1 2 3 1 3 9 7 0 0 3 2 3 _3

Ampicillin + = +++ Carberiicillin ++ ++

Piperacillin ++++

Cephalothin _3 __-

Cefoxitin + + + +

Cefamandole -

Moxalactam - + - -

Amikacia +++++

Gentimicia + - + + ^ Chloramphenicol _ _ - +

Tobramycin. - 35 - QC is an E. coli strain used for routine quality control testing of this assay system 2 - (+) indicates a MIC decrease of more than or equal to two dilution steps 3 - (-) indicates a MIC decrease of less than two dilution steps DK 167901 Bl 46

Table 6 Staphylococcus aureus

Rifampin = OjOilgg / ml or 0.001 / g / ml

Honey supplement = 4 µg / ml

Hours after inoculation 0 2 4 6 Θ 12 0.046 0-030 0-850 1.17 1.26 1.34

Control 0-046 0-073 0.815 1.16 1.26 1.32

Gnm 0.046 0.073 o-815 1-16 1.26 1.35 _I_ ^ .046 0.075 0.827 1.16 1.26 1.34 0.046 0.056 0-140 0.372 1.07 1.32

Rifampicin 0.046 0.054 0.06S 0.156 0.625 1.34 0, Qlug / ml 0.046 0-058 0-112 0.304 1.00 1.30

Avg ._0.046 0.056 0.107 0.277 0.898 1.32 0-046 0-081 0-855 1.18 1.27 1.34

Rifampicin ° C) 46 q-064 0-765 1.16 1.26 1.34 0.001 µg / ml 0-046 0-072 0-800 1.17 1.26 1.34

Gnm · _0.046 0-072 0.807 1.17 1.26 1.34 0.046 0-062 0.158 0.705 1.20 1.29

Gift O- 046 0.063 0-284 0.g75 1.22 1.31 4ug / ml o-046 0-059 0.068 ° .312 1.09 1.29

Gnm · _0-046 0-061 0-170 ° .631 1.17 1.30

Rifampicin o.O46 0.053 ° .078 0.156 0-665 1.33 0.1g / ml + o- 046 ° .055 0-078 0-162 0.640 1.32

Poison 4M9 / ml 0-046 0.056 0.062 0-092 0.332 1.32

Gnm · _ <0.046 0.055 0.073 0.137 ° .546 1.32

Rifampicin 0.046 ° .066 0.068 0.242 1.08 1.32 0.00lug / ml + O-046 ° .063 0-109 0.485 1.19 1.34

Married 4ug / mlo.046 0-067 0-087 0.3S1 1.16 1.33

Gnm .__ 0.046 ° .065 0.088 0.369 1.14 1.33 DK 167901 B1 47

Table 7 Pseudomonas aeruginosa

Rifampin = 10 µg / ml or 20 µg / ml

Honey supplement = 40 / fg / ml

Hours after inoculation _ _ _ _ _ _ 0-033 0.062 0-735 1.00 1.02 1.00

Check £> .033 0-069 0-755 0-955 1.00 0-990 0-033 Q. 068 O- 775 0-950 0.990 0.900

Avg._0.053 0.066 0.755 0.968 1.00 0.965 0-033 O- 07 & Q.690 0-090 O- 960 0.980

Gift 0.033 0-087 O'-687 £ -870 0-960 0.980 40uq / ml 0-033 0.058 0.685 <£ 080 0.953 O 950

Avg._0.055 0.074 0.685 0.880 0.955 CJ.970 O-033 0-074. 0.630 0.830 0.885 0.842

Rifampicin 0-033 £ .084 0-672 £ '.350 £ .895 0-850 10 uq / ml 0-033 p.082 0-640 0-830 0-865 0.832

Avg._0.055 D.08Q 0.647 0.857 0.88? 0.841 0-033 0-053 0-375 O 660 0.730 0.730

Rifampicin £ .033 0.056 0-326 0.645 Q-720 0-730 20 µg / ml 0.033 Q. 063 0-380 0.700 0.760 0.745

Gnm._6.035 C. 057 0.551 0.688 0.737 0.735

Rifampicin Q- 033 C: 084 0.452 0-805 0-860 O- 861 10uq / ml + n O 033 Q. 079 O-475 0-795 £ -820 0-839

Married 40ug / mlO033 O-078 0-490 0-020 0 860 0.880

Prim, 6,053 O 080 0.466 0 807 0.847 0-860

Rifampicin 0033 0.065 0.180 0.410 o 580 CO620 20ug / ml + £ 033 O'.082 O'-168 O-375 Cl 5p5 O-620

Gift 40ug / nl 6-033 O-058 0-168 0.37a £ 525 0.612

Gnm._Q 053 O 068 0.172 0.386 C. 547 0.617 DK 167901 B1 48

Table 8 Escherichia col i

Polymyxin B = 6.25 units / ml and 3.125 units / ml

Humi ebigift (BBV) = 5 µg / ml and 20 µg / ml (Megabombus pennsulvanicus)

Hours after inoculation 5 2 4 6 8 12 0.030 0.688 1.04 1.05 1.14 1.23

Control Or 030 0-680 1.03 1.04 1.13 1.22 O-030 0-683 1.02 1.04 1.13 1.22 gnm-_.030 (9.684 1.03 1.04 1.13 1.22

Bumblebee gift 0.030 (9-715 1.03 1.02 1.04 1.12 0-030 0-712 1.03 lr04 1.04 1.14 5ug / ml 0-030 0-730 1.03 1.03 1.04 1.13

Avg._0.030 0.719 1.03 1.03_1.04 1.15

Bumblebee gift 0.030 0.672 1.03 1.03 1.04 1.13 0.030 0-673 1.04 1.03 1.05 1.16 20ug / ml 0.030 0-688 1.04 1.03 1.06 1.13

Avg._0,030 £ .678 1.04 1.03_1.05 1.14 0.030 0.654 1.03 1.03 1.04 1.12

Pol B 0.030 0.642 1.02 1.03 1.04 1.14 3.125./ml C'.030 0.652 1.02 1.03 1.04 1.14

Avg._9,030 0.649 1.02 1.03_1.04 1.13 0-030 0.022 0.102 0.710 0.960 1.03

Pol 8 C. 030 0-024 0.472 0-940 0.950 1.03 6.25 units / ml 9-030 9-022 0-180 0.830 0-970 1.04

Gnm- '0.030 £ .023 0.251 0.627 O.960 1.03

Pole B = 9.030 G-008 0-168 0.820 1.00 1.06 3.125 units / ml9.030 C - 008 0-250 0-910 1.02 1.06 BBV = 5ug / ml 9-030 C .009 0-333 0-950 1.02 1.07

Omn._9.030 0.008 0.250 0.893_1.01 1.06

Pol B = 9.030 9.008 0-012 0-008 0.009 0.013 6.25 units / ml 9-030 G.009 0-009 0-008 0.008 0-012 BBV = 20ug / ml 9-030 0-011 0-009 O- 008 0-008 0-013

Gnm._9.030 0.OQ9 Cl 010 G. 008 0.008 0.013 DK 167901 Bl 49

Table 9 Escherichia col i

Polymyxin B = 3,123 units (U) / ml Wasp poison (YJ) = 5 µg / ml (Vespula germanica)

Puffin poison (BF) = 5 µg / ml (Dolichovespula maculata)

Hours after inoculation 0 2 4 0 8 12 0.038 0.526 1.03 1.07 1-08 1-12

Control 0.038 0.522 1.04 1.07 1-08 1.12

Avg._0.038 0.524_1.04 1 07 1-08_1 = 12,

Pol B 0.038 0.477 1.03 1.07 1-08 1.14 3 125U / ml 0.038 0-482 1.03 1.07 1-08 1-14

GNM · _O '. 038 0.480 1.03 1.07 _LJA

YJ 0.038 O.547 1.04 1.07 1-09 1-16 5u9 / ml 0.038 O-550 1.04 1.07 1-08 1.14

Avg ._0.038 0-549_1.04, 1 07 1-09_1 = .15 BF 0.038 0.552 1.04 1.08 1-08 1-16

Suction / ml 0-038 0.565 1.04 1.07 1-09 1-15

Avg._0.038 0.559_1.04 1.08 1-09 1-16 YJ 5ug / ml 0.038 C-028 0-183 0.945 1-08 1-14

Pole B 5U / ml 0-038 0-029 0-098 O-350 1-06 1-12

Gnm ~ _G, 038 0-029 O. 141 0.893 KQZ_ BF 5ug / ml O.038 0-027 0.118 O.S90 1-08 1.13

Pol B 5U / ml Gi038 C-023 O.096 o-840 1-06 1-10

Gnm._G. 038 0.025 0.107 0.865 1-07_1 ^ 12

Table 10 Relative activity of analogues to proteinaceous or polypeptide components of Hymenoptera toxins.

Analog No. Relative Activity 1 20% 2 200% 5 300% 6 100% 7 20% DK 167901 B1 50

Table 11 Average optical densities (0DggQ) of bacterial cultures as a function of time and processing.

_Q hours 2 hours 4 hours 6 hours 8 hours 12 hours

Control 013 .072 .831 1.08 1.10 1.17

Mellttin - .013 .072 .827 1.09 1.10 l.ig 10 µg

Mellttin - .013 .072 .833 1.10 1.11 1.18 5 µg

Analog "No. · 6 - .013 .072 .824 1.09 1.11 1.17 10 µg

Analog # 6 -. 013 .072 .831 1.07 1.09 1.16 5 µg

Polymyxin B - .013 .072 .423 .840 .911 .930 6 units

Polymyxin B - .013 .072 .808 1.06 1.08 1.15 3 units

Poly B - 6 U + .013 .072 .015 .014 .036 .908

Flour - in 0 µg

Poly B - 6 U + .013 .072 .031 .229 .550 .977

Flour - 5 µg

Poly B - 6 U +, 013 .072 .018 .018 .019 .844

Ana # 6 - 10 µg

Poly B -'6 U + .013 .072 .018 .029 .224 .926

Ana # 6 - 5 µg

Poly B - 3 U + .013 .072 .359 1.03 1.07 1.14

Me 1 - 10 pm

Poly B - 3 U + .013 .072 .544 1.08 1.10 1.18

Me 1 - 5 pm

Poly B - 3 U + .013 .072 .206 .911 1.07 1.12

Ana # 6 - 10 µg

Poly B - 3 U + .013 .072 .460 1.06 1.09 1.16

Ana # 6 - 5 ug 51 DK 167901 B1

Table 12 Sequences of synthetic melaninal oger *.

Natural Melittin Melittin (1-20) -Lys-Arg-Lys-Arg-Gln-Gln-NH2 ·

Analog # 2

Melittin (1-20) -Orn-Orn-Orn-Orn-Gln-Gln-NH ^.

10

Analog # 4

Melittin (1-20) -Lys-Arg-Lys-Arg-Gly-Gly-NH 2.

Analog # 5

Melittin (1-20) -Arg-Arg-Arg-Arg-Gln-Gln-NH2 ·

Analog No. 6 20

Melittin (1-20) -Lys-Lys-Lys-Lys-Gln-Gln-NH2.

Analog No. 7 Melittin (1-20) -Gly-Gly-Gly-Gly-Gln-Gln-NH2.

* Bold amino acids indicate changes from the natural melittin sequence.

30 35 DK 167901 B1 52

Table 13 Optical densities (0D6g0) of bacterial cultures for treatments as a function of time. Values represent averages with SEM listed in parentheses (n = 6)

Hours after Cultural Inoculation —2-2-i_§_a Sat,

Control 0.007 .08 B .692 1.01 1. ø5 in increase in ffR

(.αβΐ) (. »03j (.ase, (, βιβ) (.ai«) (; „8)

Polymyxin Β .007 .088 .470 .326 1.02 l ga in σβ (.SOI) (.003) (.072) (.014) (.027) (^ 6)

Me 1 I tt 1η + .007 .088 .071, 514 .925 1 02 ι σς

Pol Β (.001) (.003) (.017) (.116) (.077) '('. 025) (BZZ)

Synthetic + .007 .088 .018 .143 .662 881 1 a ^

Pol B t * · «) (·« «, C-« 7, * (. I * a) NPS-Me1 + .007 .088 .042 .247 .766 1 04, op

Pol B (. <? *!) (- αοτ3) (.elg, (.ιο8) (.194) (! 069) {elZ)

AnalogS2 + .007 .088 .022 .208 .509 942 1

Pol B (-0 ^ 1) C-e * 3) (.age, (.ιιβ) (.i46) '(.azi) (. ”2J

Analog # 4 + .007 .088 .020 .023 094 533

Pole B (.OO 1) (.003) (.004) (.001) (.034) '(.157)' (125)

An a i o g # 5 +. 007 .088 .036. 036 .038 334

Pol B (-001) (.003) (.OOl) (.adl) (.034) (.096) [J5Ø)

Analog # 6 + .007 .088 .ø2ø .ieø> T93

Pol B (· »»!) (· "") (.007) «... i,, .0.1)

Analog #! + .007 .088 .146 .818 l.ø2 i σ5 Λ ac p „l B <·« «> (·« «) <·« «) (·« «!) (.« Li,}; ”β )

Table A-l Chessboard analysis results for ampi c i 11 i n and honey bee venom versus S. aureus.

53 DK 167901 B1 IM Gnm. Acco Std.afv. Time Avg. ACCQ Std.afv.

AHP-0, HBV-0 AMP-O.HBV-2 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 TA 0.573 0.178 Γ4 0.213 0.135 T 6 1.102 0.159 T6 0.844 0.311 T8 1.223 0.101 TB 1.119 0.193 T12 1.213 0.307 T12 1.198 0.306 T24 1.329 0.069 T24 1.295 0.208 AHP-0, HBV — 4 AHP-O, HBV-8 TO 0.013 0.002 TO 0.013 0.002 T2 0.086 0.018 T2 0.085 0.018 TA 0.065 O.OAO TA 0.026 0.019 T6 0.217 0.181 T6 0.014 0.012 T 8 0.671 0.412 T8 0.027 0.036 TI 2 1.147 0.317 T12 1.028 0.273 T2A 1.278 0.165 T24 1.291 0.119 AHP-0, HBV-16 AHP-0.05, HBV-0 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 TA 0.025 0.011 TA 0.355 0.073 T 6 0.007 0.004 T 6 0.552 0.195 T8 0.006 0.00AT 8 0.689 0.14 6 T12 0.077 0.173 T12 0.736 0.135 T2A 0.857 0.576 T24 0.760 0.114

AHP-0.05, HBV-2 AHP-0.05, HBV-A

TO 0.013 0.003 TO O. OT 3 0.002 T2 0.085 0.004 T2 '0.083 0.017 ΤΑ 0.142 0.039 ΤΑ 0.045 0.025 T 6 0.260 0.142 T6 0.041 0.033 T 8 0.296 0.196 T8 0.035 0.032 TI 2 1,372 0.093 Ti 2 0.131 0.307 T2A 1.647 0.063 T24 0.840 0.251 AHP-0.0 5, HBV -8 AHP-0.O 5, HBV-1 6 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 TA 0.026 0.021 TA 0.025 0.009 T6 0.012 0.012 TG 0.006 0.004 T8 0.008 0.007 TB 0.007 0.004 TI 2 0.009 0.004 TI 2 0.008 0.005 T2A 0.331 0.395 T24 0.013 0.004 j Table Al (continued) 54 DK 167901 B1

Time Avg. A, - ™ Std. Time Avg. A, - ™ Std.

- --- 660 - - · -660 - AHP-0.1,1IBV-0 AHP-0.1, HDV-2 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.083 0.017 T4 0.257 0.043 T4 0.124 0.068 T6 0.248 0.061 T6 0.109 0.057 T8 0.155 0.059 T8 0.056 0.025 T12 0.095 0.033 T12 0.034 0.015 T24 0.347 0.178 T24 0.259 0.229 AHP-0.1, HBV-4 AHP-0.1, HBV-8 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 T4 0.042 0.026 T4 0.022 0.016 T6 0.031 0.030 T 6 0.007 0.006 T8 0.026 0.021 T8 0.005 0.004 T12 0.272 0.534 T12 0.011 0.013 T24 0.511 0.552 T24 0.246 0.497 AHP-0. 1, IIBV —16 AHP-0.2, HBV-0 T ° 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 T4 0.026 0.013 T4 0.202 0.038 16 0.007 0.005 T 6 0.112 0.026 T8 0.006 0.004 T 8 0.052 0.016 T12 0.007 0.004 T12 0.037 0.009 T24 0.011 0.004 T24 0.042 0.008 AMP-0.2, HBV-2 AKP-0.2, HBV-4 TO 0.013 0.002 TO 0.013 0.002 T2 0.086 0.018 T2 0.085 0.018 T4 0.103 0.065 T 4 0.045 0.024 T6 0.079 0.050 T 6 0.029 0.022 T8 0.036 0.027 T8 0.021 0.015 T12 0.026 0.021 T12 0.013 0.006 T24 0.069 0.179 T24 0.011 0.008 AMP-0.2.HBV-8 AMP-0.2.HBV-16 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 T4 0.023 0.019 T4 0.024 0.012 T6 0.011 0.010 T 6 0.009 0.008 T8 0.007 0.007 T 8 0.006 0.003 T12 0.008 0.002 T12 0.009 0.006 T24 0.009 0.005 T24 0.011 0.003 55

Table A-1 (continued) DK 167901 B1

Time Gnnh_Agg0 Std.vv. Time Avg. AggQ Std.afv.

AMP-0. 4, HBV-0 AMP-0. 4, HBV-2 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 TA 0.191 0.042 T4 0.098 0.054 T6 0.110 0.027 T6 0.061 0.041 T8 0.048 0.019 T8 0.034 0.027 T12 0.027 0.009 T12 0.020 0.011 T24 0.027 0.005 T24 0.018 0.008 AMP-0.4 , HBV-4 AMP-0.4, HBV-8 TO 0.013 0.002 TO 0.013 0.002 T2 0.085 0.018 T2 0.085 0.018 T4 0.040 0.028 T4 0.023 0.015 T 6 0.028 0.023 T6 0.010 0.006 T8 0.019 0.017 T8 0.006 0.004 TI 2 0.012 0.004 T12 0.008 0.005 T24 0.009 0.007 T24 0.010 0.006 AMP-0.4, HBV-16 TO 0.013 0.002 T2 0.085 0.018 T4 0.027 0.013 T 6 0.008 0.004 T8 0.006 0.004 T12 0.008 0.005 T24 0.010 0.004

Table A-2 Chessboard analysis results for kanamycin 'and honey bee venom versus S. aureus.

56

Time Gnnu_A660 Std.vv. Time Gm, _A660 Std.afv,.

KANA-0, HBV — 0 KANA-0, HBV-2 TO 0.024 0.005 TO 0.024 0.005 T2 -0.094 0.012 T2 0.095 0.011 T4 0.854 0.157 T4 0.542 0.183 T6 1.219 0.052 T6 1.132 0.146 T8 1.275 0.032 T8 1.275 0.042 T12 1.320 0.044 T12 1.333 0.041 T24 1.358 0.031 T24 1.402 0.040 CANA-0, HBV-4 CANA-0, HBV-8 TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.154 0.131 T4 0.036 0.017 T6 0.630 0.391 T6 0.062 0.048 T8 1.100 0.233 T8 0.571 0.403 T12 1.322 0.048 T12 1.275 0.062 T24 1.405 0.040 T24 1.389 0.057

KANA-0, HBV-16 KANA-1.25, HBV-O

TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.029 0.014 T4 0.747 0.125 T6 0.020 0.008 T6 1.199 0.060 T8 0.066 0.078 T8 1.269 0.043 T12 0.666 0.556 T12 1.315 0.046 T24 1.336 0.195 T24 1.355 0.042 KANA-1.25, HBV-2A -1.25, HBV-4 TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.428 0.197 T4 0.107 0.060 T6 0.929 0.369 T6 0.31C 0.289 T8 1.174 0.116 T8 0.694 0.422 T12 1.290 0.048 T12 1.231 0.107 T24 1.350 0.077 T24 1.350 0.077 -1.25.HBV-8 CANA-1.25.HBV-16 TO 0.024 0.005 TO 0.-024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.039 0.014 T4 0.030 0.014 T6 0.031 0.011 T6 0.017 0.009 T8 0.095 0.129 T8 0.018 0.012 T12 0.712 0.487. T12 0.179 0.344 T24 1.343 0.096 T24 1.124 0.357

Table A-2 (continued) 57 DK 167901 B1

Time £ ηηκ_Α660 Std.afv, Time Grim ...... A66Q Std ^ afv ^ CAN A-2. 5, HBV-0 KANA-2.5, HBV-2 TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.630 0.081 T4 0.358 0.203 T6 1.090 0.093 T6 0.747 0.438 T8 1.227 0.042 T8 0.925 0.462 T12 1.248 0.046 T12 1.229 0.079 T24 1.315 0.056 T24 1.320 0.073 KANA-2.5, HBV — 4 KANA-2.5, HBV-8 TO 0.024 0.005 TO 0.025 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.089 0.070 T4 0.037 0.015 T6 0.124 0.191 T6 0.026 0.010 T8 0.186 0.279 T8 0.021 0.010 T12 0.842 0.381 T12 0.187 0.224 T24 1.284 0.062 T24 1.287 0.100 KANA-2.5.HBV-16 KANA-5, HBV-0 TO 0..024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.028 0.014 T4 0.448 0.076 T6 0.017 0.009 T 6 0.696 0.159 T8 0.026 0.041 T8 0.888 0.193 T12 0.246 0.481 T12 1.008 0.195 T24 0.950 0.589 T24 1.085 0.093 KANA-5, HBV-2 KANA-5.HBV-4 TO 0.024 0.005 .TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.265 0.152 T4 0.065 0.026 T 6 0.371 0.260 T6 0.057 0.029 T8 0.483 0.329 T8 0.065 0.047 T12 0.915 0.189 T12 0.653 0.380 T24 1.119 0.098 T24 1.242 0.068 KANA-5, HBV-8 KANA-5, HBV-16 TO 0.024 0.005 'TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.035 0.015 T4 0.030 0.015 T6 0.023 0.011 T 6 0.019 0.009 T8 0.018 0.012 T8 0.015 0.010 T12 0.054 0.048 T12 0.012 0.015 T24 1.245 0.096 T24 0.484 0.544 DK 167901 Pg 58

Table A-2 (continued)

Time Avg. Acco Std.afv. Out Gnm, ._ A660 Std.vv.

KANA-10, HBV-0 KARA-10, HBV-2 TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.279 0.054 T4 0.167 0.089 T6 0.359 0.063 T6 0.183 0.112 T8 0.416 0.082 T8 0.205 0.128 T12 0.667 0.175 T12 0.666 0.168 T24 0.995 0.074 T24 1.153 0.070 KANA-10.HBV-4 KANA-10, HBV-8 TO 0.024 0.005 TO 0.024 0.005 T2 0.094 0.012 T2 0.094 0.012 T4 0.064 0.023 T4 0.041 0.023 T 6 0.054 0.021 T6 0.027 0.019 T8 0.052 0.024 T8 0.023 0.019 T12 0.314 0.299 T12 0.022 0.018 T24 1.193 0.080 T24 0.836 0.412 CANA-10.HBV-16 TO 0.024 0.005 T2 0.094 0.012 T4 0.031 0.014 T6 0.020 0.009 T8 0.014 0.010 T12 0.015 0.013 T24 0.614 0.567

Table A-3 Chessboard analysis results for polymyxin B and honey bee venom versus E. coli.

59 DK 167901 B1

Time Gnnk_A660 Std.vv. Time Gnnk_å660 hrs · - POLY B- = 0, HBV-0 POLY B = 0, HBV-2 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.785 0.061 T4 0.195 0.116 T6 1.243 0.011 T6 0.886 0.304 T8 1.295 0.024 T8 1.264 0.027 T12 1.343 0.018 T12 1.316 0.026 T24 1.396 0.023 T24 1.405 0.020 POLY BO.HBV-4 POLY B-0.HBV-8 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 -0.004 T4 0.038 0.013 T4 0.018 0.008 T6 0.070 0.046 T6 0.012 0.014 T8 0.589 0.235 T8 0.022 0.012 T12 1.315 0.081 T12 0.769 0.503 T24 1.415 0.024 T24 1.405 0.028 POLY B-0, HBVOM-16 POLY B- = 312, HBV-0 TO 0.006 0.002 TO 0.006 0.022 T2 0.074 0.004 T2 0.074 0.004 T 4 0.015 0.007 T4 0.526 0.138 T 6 0.006 0.003 T6 1.046 0.269 T8 0.007 0.004 T8 1.244 0.057 T12 0.012 0.005 T12 1.305 0.051 T24 0.457 0.566 T24 1.429 0.053 POLY B-312.HBV-2 POLY B-312.HBV-4 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 • T4 0.167 0.071 T4 0.023 0.013 T 6 0.795 0.231 T6 0.064 0.132 T8 1.195 0.117 T8 0.216 0.357 T12 1.303 0.041 T12 0.812 0.513 T24 1.422 0 .066 T24 1.415 0.040 POLY B-312, HBV-8 FpLY B-312.HBV-16 TO 0.006 0.002 TO 0.006 0.002. T2 0.074 0.004 T2 0.074 0.004 T4 0.014 0.005 T4 0.023 0.008 T 6 0.007 0.005 T6 0.013 0.004 TB 0.011 0.005 T 8 0.013 0.004 TI 2 0.384 0.383 T12 0.031 0.048 T24 1.294 0.393 T24 0.334 0.579

Table A-3 (continued) 60 DK 167901 B1

Time Avg. Acgq Std.afv. Time Avg. A66Q Std, _afv.

POLY B-625, HBV-0 POLY B-625, HBV-2 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.330 0.117 T4 0.165 0.076 T6 0.766 0.386 T6 0.553 0.267 T8 1.048 0.314 T8 '1.037 0.260 T12 1.238 0.125 T12 1.261 0.067 T24 1.401 0.123 T24 1.405 0.075 POLY B-625, HBV-4 POLY B-625, HBV-8 TO 0.006 0.002 TO 0.007 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.025 0.011 T4 0.015 0.005 T6 0.030 0.034 T6 0.009 0.004 18 0.073 0.128 TB 0.011 0.005 T12 0.627 0.428 T12 0.051 0.062 T24 1.405 0.050 T24 1.323 0.307 POLY B-625.HBV-16 · PQLY B-1250, HBV-0 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.039 0.013 T4 0.159 0.032.

T6 0.023 0.008 T6 0.172 -0.093 T8 0.022 0.007 T8 0.259 0.261 T12 0.022 0.007 T12 0.778 0.437 T24 0.294 0.538 T24 1.362 0.094 POLY B-1250, HBV-2 POLY B-1250, HBV-4 • TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0, .110 0.043 T4 0.038 0.012 16 0.115 0.085 T6 0.020 0.009 T8 0.203 0.237 T8 0.018 0.006 T12 0.552 0.557 T12 0.033 0.042 T24 1,207 0.487 T24 1.150 0.449 POLY B-1250, HBV-8 POLY B-1250, HBV-16 TO 0.006 0.002 TO 0.006 0.002 12 0.074 0.004 T2 0.074 0.004 TA 0.028 0.010 T4 0.071 0.014 T6 0.019 0.007 T6 0.054 0.012 18 0.019 0.006 T8 0.046 0.009 T12 0.021 0.010 T12 0.036 0.006 T24 1.013 0.556 T24 0.223 0.440 61 DK 167901 B1

Table A-3 (continued)

Time Gnnk_A66Q Std.vv. Time Avg. ACCQ Std.afv.

POLY B-2500.HBV-0 POLY B-2500, HBV-2 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.123 0.013 T4 0.107 0.022 T6 0.109 0.019 T6 0.085 0.021 T8 0.167 0.276 T8 0.072 0.020 T12 0.075 0.010 T12 0.056 0.013 T24 1.037 0.423 T24 0.879 0.530 POLY B — 2500, HBV — 4 POLY B-2500.HBV-8 TO 0.006 0.002 TO 0.006 0.002 T2 0.074 0.004 T2 0.074 0.004 T4 0.080 0.013 T4 0.070 0.020 T6 0.065 0.013 T6 0.067 0.010 T8 0.057 0.008 T8 0.058 0.015 T12 0.049 0.011 T12 0.052 0.007 T24 0.416 0.491 T24 0.301 0.524 POLY B-2500, HBV-16 TO 0.006 0.002 T2 0.074 0.004 T4 0.110 0.009 T6 0.091 0.008 T8 0.078 0.009 T12 0.061 0.006 T24 0.210 0.425

Table A-4 Chessboard analysis results for ampici11 in and honey bee venom versus E. cgl_L.

62 DK 167901 B1

Time Gnnk_A660 Std.vv. Fire Gnm. A66Q std-afv ·· - AMP-O.HBV-O AMP-O.HBV-5 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 T4 0.644 0.098 T4 0.624 0.102 T6 1.053 0.067 T6 1.049 0.081 T8 1.071 0.071 T8 1.070 0.078 T12 1.144 0.075 T12 1.146 0.098 T24 1.244 0.101 T24 1.258 0.128 AMP-0, HBV-10 AMP-0, HBV-20 TO 0.015 0.015 TO 0.015 -0.015 T2 0.084 0.032 T2 0.084 0.032 T4 0.646 0.103 T4 0.643 0.132 T6 1.056 0.085 T6 1.031 0.088 TB 1.066 0.091 T8 1.052 0.097 T12 1.154 0.110 T12 1.127 0.113 T24 1.260 0.139 T24 1.244 0.155 AMP-0, HBV-40 AMP-0.5, HBV-0 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 T4 0.587 0.204 T4 0.600 0.099 T6 1.026 0.092 T6 1.001 0.078 TB 1.050 0.094 T8 0.999 0.101 T12 1.119 0.111 T12 1.085 0.111 T24 1.210 0.167 T24 1.156 0.222 AMP-0.5, HBV-5 AMP-0.5, HBV-10 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 TA 0.603 0.099 T4 0.624 0.111 16 0.998 0.095 'T6 1.001 0.097 T8 1.011 0.097 T8 1.013 0.100 T12 1.099 0.120 T12 1.100 0.136 T24 1.215 0.159 T24 1.219 0.176

AMP-0.5.HBV-20 AMP-0.5, HBV-4O

TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 TA 0.614 0.148 T4 0.508 0.205 T6 0.980 0.094 T6 0.961 0.097 TB 0.993 0.093 T8 0.991 0.098 T12 1.073 0.123 T12 1.063 0.138 T24 1.182 0.155 T24 1.162 0.172 63 DK 167901 B1

Table A-4 (continued) ll Gnm. ftcc0 SUUflu Ud Sfl! lJ660 SLdjf », AMP-1, HBV-0 AMP -1, 11BV-5 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 T 4 0.538 0.094 T4 0.545 0.095 T 6 0.628 0.1 75 T6 0.621 0.126 T8 0.493 0.157 T8 0.470 0.147 TI 2 0.475 0.230 T12 0.407 0.125 T24 0.504 0.228 T24 0.447 0.028 AMP-1, HBV-10 AMP-1, HBV-2 0 TO 0.015 0.016 TO 0.015 0.015 T2 0.083 0.033 T2 0.084 0.032 T4 0.561 0.116 T4 0.543 0.122 T 6 0.506 0.077 T6 0.513 0.080 T 8 0.4 53 0.120 T8 0.43 2 0.132 T12 0.396 0.106 T12 0.367 0.1 04 T24 0.414 0.028 T24 0.395 0.047 ΛΜΡ-1, 11BV-40 AMP-2, HBV-0 TO 0.015 0.016 TO 0.015 0.015 T2 0.084 0.031 T2 0.084 0.032 T4 0.439 0.183 T4 0.428 0.112 T6 0.456 0.125 T6 0.125 0.042 T 8 0.4 35 0.191 T8 0.133 0.055 T12 0.385 0.163 T12 0.136 0.090 T 2 4 0.4 84 0.082 T 2 4 0.64 7 0.1 94 AMP-2, 1IBV-5 ΛΜΡ-2, HBV-10 TO 0.015 0.015 TO 0.015 0.015 T 2 0.08 4 0.0 32 T2 0.084 0.032 T4 0.440 0.130 T4 0.432 0.122 T 6 0.134 0.052 T6 0.1 2 7 0.052 T 8 0.14 8 0.073 T8 0.133 0.070 TI 2 0.192 0.14 7 TI 2 0.18 2 0.137 T24 0.685 0.175 T24 0.654 0.253

AMP-2, HBV-20 AMP-2, HBV-4O

TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 T4 0.406 0.151 T4 0.300 0.173 T 6 0.114 0.054 T 6 0.08 6 0.058 T 8 0.123 0.073 T8 0.096 0.07 1 T1 2 0.209 0.1 9 3 T12 0.098 0.055 T 2 4 0.687 0.205 ' T24 0.618 0.241

Table A-4 (continued) 64 DK 167901 B1

Ud Gnm. Acco Std.afv. Ud Gnm. A66Q Std ^ åiv.

AMP-4, HBV-0 AHP-A.HBV-5 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 TA 0.158 0.118 TA 0.154 0.108 T 6 0.063 0.019 T6 0.076 0.037 T8 0.126 0.230 T8 0.084 0.044 T12 0.055 0.023 T12 0.057 0.022 T24 .0.056 0.015 T24 0.076 0.071 AKP-4.HBV-10 AMP-4, HBV-20 TO 0.015 0.015 TO 0.015 0.015 T2 0.084 0.032 T2 0.084 0.032 TA 0.128 0.092 T4 0.090 0.070 T6 0.075 0.039 T6 0.066 0.043 T8 0.074 0.045 TB 0.066 0.045 TT 2 0.066 0.034 T12 0.050 0.031 T2A 0.063 0.032 T24 0.052 0.026

AMP-4, HBV-4O

TO 0.015 0.015 T2 0.084 0.032 TA 0.062 0.040 T6 0.055 0.040 TB O.054 0.039 TI 2 0.051 0.028 T24 0.042 0.022 DK 167901 B1

Table A-5 Chessboard analysis results for kanamycin and honey bee venom versus E. coli,.

65

Time Avg. A ^ q Std.afv. Time Avg. AggQ Std.afv, KANA-0, HBV-0 KANA-0, HBV-5 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.118 0.028 TA 0.701 0.136 TA 0.726 0.108 T6 0.980 0.075 T6 1.002 0.065 T8 0.988 0.068 T8 1.028 0.063

T12 1.062 0.090 T12 1.10A 0.08A

T2A 1.1AA 0.119 T2A 1.191 0.101 KANA-O, HBV-10 KANA-0, HBV-20 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 ΤΑ 0.7A7 0.108 ΤΑ 0.76A 0.087 T 6 1.005 0.073 T6 1.001 0.060 T8 1.028 0.065 T8 1.026 0.063 T12 1.099 0.09A T12 1.09A 0.090 T2A 1.188 0.11A T2A 1.198 0.102 CANA-0, HBV-AO 5-5, HBV-0 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.12A 0.033 TA 0.736 0.075 ΤΑ 0.A73 0.120 T 6 0.9 8A 0.06A T6 0.800 0.132 T 8 1.005 0.062 T8 0.889 0.081 T12 1.080 0.080 T12 0.930 0.091 T2A 1.163 0.103 T2A 1.019 0.119 KANA-5, HBV-5 KANA-5.HBV-10 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 ΤΑ 0.A8A 0.128 ΤΑ 0.A80 0.1A6 T6 0.827 0.129 T6 0.805 0.1A1 T8 0.908 0.080 T8 0.893 0.093 T12 0.955 0.101 T12 0.939 0.108 T2A 1.050 0.122 T2A l.OAA 0.127

KANA-5.HBV-20 KANA-5, HBV-AO

TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 TA O. A 9 3 0.169 TA 0.503 0.177 T6 0.765 0.192 T6 0.783 0.181 T8 0.862 0.108 T8 0.873 0.096 T12 0.950 0.106 T2 0.950 0.107 T2A 1.0A6 0.126 T2A 1.0A1 0.116 DK 167901 B1

Table A-5 (continued)

Time Gnm- Acco Std.afv,. Tjd 6nni - ^ 660 Std.afv, KANA-10, HBV-0 KANA-10, HBV-5 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 T4 0.263 0.114 T4 0.267 0.135 T6 0.417 0.209 T6 0.414 0.242 T8 0.576 0.222 T8 0.563 0.248 T12 0.814 0.084 T12 0.807 0.098 T24 0.878 0.095 T24 0.894 0.095 CANA-10, HBV-10 CANA-10, HBV-20 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 T4 0.258 0.142 T4 0.257 0.153 0.243 T6 0.361 0.262 T8 0.511 0.242 T8 0.520 0.259 T12 0.738 0.180 T12 0.754 0.171 T24 0.873 0.078 T24 0.881 0.071 KANA-10.HBV-40 KANA-20, HBV-0 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 TA 0.25 0.176 T4 0.161 0.054 T6 0.356 0.303 T6 0.161 0.065 T8 0.494 0.292 T8 0.170 0.079 T12 0.784 0.147 T12 0.268 0.108 T24 0.906 0.103 T24 0.631 0.103 KANA-20.HBV-5 KANA-20, HBV-10 TO 0.09 0.009 TO 0.09 TO 0.028 T2 0.119 0.028 TA 0.156 0.072 T4 0.144 0.075 16 0.133 0.083 T6 0.095 0.069 18 0.119 0.086 T8 0.085 0.063 T12 0.233 0.122 T12 0.209 0.081 T24 0.678 0.112 T24 0.667 0.100 CANA -20, HBV-20 CANANA-20, HBV-40 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 TA 0.128 0.081 T4 0.103 0.074 T6 0.078 0.065 T 6 0.063 0.051 T8 0.151 0.128 T 8 0.063 0.048 T12 0.174 0.061 T12 0.179 0.083 T24 0.692 0.113 T24 0.716 0.087 DK 167901 B1 67

Table A-5 (continued)

Time Gnnk_A660 Std.vv. Tjd Gnm, ..... A66Q Std.afv, CANA * »4 O, HBV-0 CANA-4 O, HBV-5 TO 0.025 0.009 TO 0.024 0.009 T2 0.119 0.028 T2 0.117 0.029 T4 0.136 0.049 T4 0.128 0.062 T 6 0.126 0.052 T6 0.098 0.071 T8 0.120 0.057 T8 0.074 0.055 T12 0.100 0.055 T12 0.043 0.024 T24 0.617 0.108 T24 0.432 0.301 ΚΑΝΑ-4O, HBV — 10 ΚΑΝΑ-4 O, HBV-20 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 T4 0.117 0.068 T4 0.096 0.059 T6 0.066 0.047 T6 0.046 0.025 • T8 0.045 0.026 T8 0.038 0.016 T12 0.042 0.026 T12 0.039 0.017 T24 0.416 0.310 T24 0.404 0.318 CANA-40, HBV-40 TO 0.025 0.009 T2 0.119 0.028 T4 0.080 0.054 T6 0.041 T8 0.036 0.013 TI 2 0.040 0.019 T24 0.342 0.344 DK 167901 Bl

Table A-6 Chessboard analysis results for polymyxin B and honey bee venom versus E. col i.

68

Time Gnm ^ -AggQ Std.afv. Time Avg. A ^ q Std.afv.

POLY BO.HBV-O POLY B-0.HBV-5 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.506 0.076 T4 0.529 0.080 T6 1.011 0.110 T6 1.018 0.116 T8 1.043 0.096 T8 1.049 0.095 T12 1.103 0.116 T12 1.113 0.119 T24 1.201 0.137 T24 1.227 0.150 POLY B-0, HBV-10 POLY B-0, HBV-20 TO. 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.557 0.087 T4 0.544 0.061 T6 1.010 0.130 T6 1.005 0.117 • T8 1.049 0.100 T8 1.040 0.102 T12 1.104 0.142 T12 1.092 0.139 T24 1.228 0.162 T24 1.217 0.157 POLY B-0, HBV-4 POLY B-1.5.HBV-0 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.439 0.058 T4 0.411 0.078. T6 0.992 0.129 T6 0.984 0.105 T8 1.036 0.116 T8 1.020 0.091 T12 1.082 0.139 T12 1.075 0.107 T24 1.188 0.157 T24 1.200 0.141 POLY B-1.5, HBV- = 5 POLY B- = l. 5, HBV-10 TO 0.012 0.003 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.176 0.039 T4 0.160 0.078 T6 0.851 0.142 T6 0.837 0.133 T8 1.012 0.196 T8 1.015 0.091 T12 1.068 0.093 T12 1.073 0.134 T24 1.200 0.063 T24 1.203 0.145

POLY B-1.5.HBV-20 POLY B = 1.5.HBV-4O

TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 -T4 0.058 0.026 T4 0.024 0.010 T6 0.507 0.196 T6 0.147 0.262 T8 0.948 0.128 T8 0.438 0.390 T12 1.046 0.120 T12 1.016 0.102 T24 1.201 0.129 T24 1.153 0.143 69 DK 167901 B1

Table A-6 (continued)

Time 6pm Accq Std.afv ^ Time GnnL_A660 Stdjifx.

POLY B-3.HBV-0 POLY B-3.HBV-5 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.138 0.094 T4 0.029 0.018 T6 0.642 0.139 T6 0.105 0.188 T8 0.943 0.117 T8 0.174 0.339 T12 0.985 0.147 T12 0.471 0.390 T24 1.116 0.184 T24 1.117 0.132 POLY B-3, HBV-10 POLY B-3.HBV-20 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.030 0.019 T4 0.023 0.007 T6 0.092 0.169 T6 0.013 0.004 T8 0.200 0.339 T8 0.016 0.013 T12 0.442 0.414 T12 0.445 0.351 T24 1.105 0.111 T24 1.126 0.111 POLY B-3, HBV-40 POLY B-6.HBV-0 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.033 0.014 T4 0.022 0.007 T6 0.018 0.006 T6 0.014 0.006 T8 0.054 0.101 T8 0.011 0.004 T12 0.444 0.357 T12 0.109 0.188 T24 1.123 0.123 T24 0.975 0.140 POLY B-6.HBV-5 POLY B-6, HBV-10 TO 0.012 0.005 TO 0.012 0.005 'T2 0.040 0.004 T2 0.040 0.004 T4 0.024 0.006 T4 0.029 0.006 T 6 0.016 0.007 T6 0.017 0.005 T8 0.011 0.004 T8 0.012 0.004 T12 0.056 0.115 T12 0.065 0.111 T24 0.733 0.398 T24 0.701 0.441 POLY B-6, HBV-20 POLY B-6, HBV-40 • TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.042 0.009 T4 0.030 0.007 T 4 0.041 0.008 T 6 0.016 0.004 T 6 0.019 0.006 T 8 0.012 0.004 T8 0.014 0.006 T12 0.066 0.116 T12 0.016 0.006 T24 0.486 0.448 T24 0.270 0.374

Table A-6 (continued) 70 DK 167901 B1

Time Gnnk_A660 Std.vv. Time Avg. AG {-0 Std.

POLY B-12.HBV-0 POLY B-12.HBV-5 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.018 0.005 T4 0.025 0.005 T6 0.011 0.005 T6 0.016 0.006 T8 0.009 0.003 T8 0.011 0.005 T12 0.075 0.150 T12 0.010 0.005 T24 0.472 0.498 T24 0.196 0.361 POLY B-12, HBV-10 POLY B-12.HBV-20 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.029 0.004 T4 0.030 0.006 T 6 0.017 0.005 T6 0.017 0.006 T8 0.012 0.003 T8 0.013 0.003 T12 0.024 0.051 T12 0.012 0.004 T24 0.201 0.352 T24 0.073 0.184 POLY B-12, HBV-40 TO 0.012 0.005 T2 0.040 0.004 T4 0.048 0.007 T 6 0.022 0.006 T8 0.016 0.005 T12 0.015 0.006 T24 0.047 0.085 HH ~ - T —__ _ 7l DK 167901 B1

Table A-7 Chessboard analysis results for ampiciTI in and honey bee venom versus kanamycin resistance stent S. aureus.

Time GnnL-A660 Std.vv. Ud Gnm. A66Q Std, .afy._ AMP-O, HBV-0 AMP-O.HBV-2 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.382 0.155 T4 0.150 0.134 T6 0.885 0.173 T6 0.533 0.286 T8 1.108 0.041 T8 0.937 0.207 T12 1.191 0.035 T12 1.167 0.038 T24 1.233 0.049 T24 1.217 0.041

AMP-O, HBV-4 AMP-O, HBV-B

TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.038 0.021 T4 0.032 0.019 T6 0.040 0.029 T6 0.015 0.010 T8 0.155 0.184 T8 0.011 0.007 T12 0.903 0.263 T12 0.234 0.326 T24 1.181 0.050 T24 0.894 0.441 AMP-O, HBV-16 AMP -O.05, HBV-0 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.033 0.016 T4 0.230 0.054 T6 0.013 0.005 T6 0.338 0.076 T8 0.007 0.004 T8 0.372 0.144 T12 0.008 0.004 T12 0.352 0.220 T24 0.126 0.305 T24 0.461 0.139 AMP-O.05, HBV-2 AMP-OO 5, HBV-4 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.112 0.099 T4 0.044 0.025 T6 0.175 0.144 T6 0.031 0.021 T8 0.190 0.153 T8 0.025 0.018 T12 0.130 0.131 T12 0.018 0.012 T24 0.440 0.260 T24 0.581 0.239 AMP-O.05, HBV-8 AMP-O.05, HBV-16 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.025 0.013 T4 0.035 0.016 T 6 0.013 0.009 T6 0.013 0.004 T8 0.008 0.005 T8 0.008 0.004 T12 0.010 0.007 TI 2 0.008 0.004 T24 0.150 0.295 T24 0.011 0.002 72 DK 167901 B1

Table A-7 (continued)

Time GjTflL_Aggo Std.vv. Time 6pm A ^ q Std.afv.

AMP-0.l.HBV-0 AMP-0.l.HBV-2 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.379 0.430 T4 0.109 0.079 T6 0.156 0.045 T6 0.108 0.063 T8 0.112 0.038 T8 0.075 0.037 T12 0.050 0.011 T12 0.037 0.023 T24 0.053 0.009 T24 0.052 0.034 AMP-0.1, HBV-4 AMP-0.l.HBV-8 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.045 0.029 T4 0.030 0.018 T6 0.030 0.022 -T6 0.015 0.008 T8 0.023 0.016 T8 0.010 0.004 T12 0.018 0.012 ΤΓ2 0.008 0.004 T24 0.044 0.102 T24 0.011 0.004 AMP-0.1, HBV-16 AMP-O.2, HBV-0 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 0.031 0.017 T4 0.131 0.026 T6 0.014 0.004 T6 0.110 0.024 T8 0.007 0.005 T8 0.073 0.018 T12 0.009 0.005 T12 0.030 0.009 T24 0.012 0.004 T24 0.037 0.051 AMP-0.2, HBV-2 AMP-0.2, HBV-4 T0 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 0.071 0.052 T4 0.047 0.029 T6 0.062 0.047 T6 0.033 0.026 Τβ 0.039 0.026 T8 0.024 0.018 T12 0.018 0.011 T12 0.017 0.010 T24 0.017 0.010 T24 0.068 0.212 AMP-0.2, HBV-8 AMP-0.2, HBV-16 T0 0.020 0.016 'TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T * 0.031 0.019 T4 0.036 0.015 T6 0.016 0.010 T 6 0.015 0.005 TB 0.010 0.007 T8 0.008 0.005 T12 0.007 0.006 T12 0.008 0.005 T24 0.010 0.004 T24 0.012 0.003 73

Table A-7 (continued) DK 167901 B1 Time Gnnu_A650 Std.afv ^. Fire Gnm. _A66Q Std ^ L, AMP-0.4, HBV-0 AMP-0.4, HBV-2 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.202 0.184 T4 0.080 0.055 T6 0.290 0.415 T6 0.073 0.044 T8 0.285 0.472 T8 0.043 0.023 T12 0.271 0.514 T12 0.021 0.012 T24 0.277 0.530 T24 0.020 0.012 AMP-0.4, HBV-4 AMP-0.4, HBV-8 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.044 0.026 T4 0.030 0.019 T6 0.028 0.016 T6 0.015 0.008 T8 0.021 0.011 T8 0.008 0.005 TI 2 0.014 0.006 T12 0.008 0.005 T24 0.011 0.003 T24 0.011 0.003 AMP-0.4, HBV-16 TO 0.020 0.016 T2 0.064 0.020 T 4 0.033 0.014 T 6 0.015 0.004 T 8 0.008 0.005 TI 2 0.009 0.006 T24 0.012 0.003 DK 167901 B1 74

Table A-8 Chessboard analysis results for kanamycin and honey bee venom versus kanamycin resistance stent S. aureus.

Time yesOnL — Aggø Std.vv. Time Gnm .. Aqqq $ td..afv.

CANA-0, HBV-0 CANA-0, HBV-2 T0 0.016 0.005 TO 0.015 0.005 T2 0.047 0.009 T2 0.047 0.009 T4 0.636 0.151 T4 0.187 0.116 T6 1.246 0.026 T6 0.980 0.205 T8 1-331 0.015 T8 1.056 0.481 T12 1.356 0.025 T12 1.100 0.498 T24 1.417 0.039 124 1.418 0.020 KANA-0, HBV — 4 KANA-O, HBV-8 TO 0.015 0.004 TO 0.015 0.004, T2 0.047 0.009 T2 0.047 0.009 T4 0.030 0.016 T4 0.021 0.013 T6 0.065 0.075 T6 0.016 0.009 18 0.373 0.354 T8 0.043 0.056 T12 1.306 0.062 T12 0.655 0.507 T24 1.437 0.016 T24 1.402 0.034 CANA-0.HBV-16 ΚΑΝΑ-5, HBV-0 TO 0.015 0.004 TO 0.016 0.005 T2 0.047 0.009 T2 0.047 0.009 T4 0.025 0.012 T4 0.204 0.103 T6 0.014 0.007 T6 0.282 0.140 T8 0.013 0.008 T8 Q.351 0.176 T12 0.117 0.263 T12 0.751 0.288 T24 0.454 0.582 T24 1.152 0.121 KANA-5, HBV-2 KANA-5, HBV-4 T0 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T4 0.057 0.034 T4 0.031 0.017 T6 0.059 0.038 T6 0.024 0.012 T8 0.068 0.044 T8 0.022 0.011 T12 0.660 0.271 T12 0.147 0.223 T24 1.299 0.046 T24 1.279 0.063 CANA-5, HBV-8 ΚΑΝΑ-5, HBV-16 T0 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009. T2 0.047 0.009 T4 0.022 0.013 T4 0.024 0.010 T6 0.016 0.008 T 6 0.016 0.008 T8 0.012 0.007 T 8 0.014 0.008 T12 0.015 0.008 'T12 0.016 0.005 T24 0.876 0.403 T24 0.237 0.378

Table A-8 (continued) 75 DK 167901 B1

Time GjTm _ ^ _ AggQ Std.vv., Fire Gnm. AggQ Std.afv.

KANA-10, HBV-0 KAKA-10, HBV-2 TO 0.016 0.005 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T4 0.135 0.065 T4 0.045 0.026 T 6 0.172 0.080 T6 0.044 0.029 T 8 0.200 0.086 T8 0.043 0.031 T12 0.397 0.186 T12 0.185 0.182 T24 1.164 0.145 T24 1.056 0.412 CANA-10, HBV-4 CANA-10, HBV-8 TO 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T4 0.030 .016 T4 0.022 0.012 T 6 0.023 0.010 T6 0.016 0.008 T8 0.020 0.008 T8 0.014 0.011 T12 0.061 0.070 T12 0.015 0.006 T24 1.135 0.305 T24 0.264 0.385 ΚΑΝΑ-10, HBV-16 K.ANA-20, HBV-0 TO 0.015 0.004 TO 0.016 0.005 T2 0.047 0.009 T2 0.047 0.009 T4 0.022 0.011 T4 0.123 0.061 T 6 0.016 0.007 T6 0.145 0.073 T 8 0.014 0.009 T 8 0.166 0.079 TI 2 0.017 0.006 T12 0.220 0.081 T24 0.028 0.024 T24 0.975 0.266 KANA-20.HBV-2 KANA = 20, HBV-4 TO 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T 4 0.044 0.020 T4 0.036 0.035 T6 0.041 0.020 T 6 0.022 0.013 T8 0.038 0.019 T8 0.019 0.011 T12 0.096 0.067 T12 0.025 0.018 T24 1.155 0.074 T24 0.666 0.488 CANA-20.HBV- 8 KANA-20, HEV «16 TO 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T4 0.023 0.011 T4 0.022 0.011 T 6 0.017 0.007 T 6 0.016 0.011 T8 0.014 0.006 T8 0.015 0.009 TI 2 0.017 0.007. T12 0.016 0.008 T24 0.240 0.340 T24 0.081 0.151 76 DK 167901 B1

Table A-8 (continued)

Time GnnL_AggQ Std.vv. Time Avg. Aggg Std.afv.

KANA-4 O, HBV-0 KANA-40.HBV-2 TO 0.016 0.005 TO 0.015 O.004 T2 O.OA 7 0.009 T2 0.047 0.009 T4 0.116 0.057 T4 0.048 0.021 T6 0.146 0.069 T6 0.047 0.021 T8 0.161 0.075 T8 0.043 0.020 T12 0.184 0.084 T12 0.049 0.022 T24 0.697 0.396 T24 0.692 0.463 KANA-40.HBV-4 KANA-4 O, HBV-8 TO 0.015 0.004 TO 0.015 0.004 T2 0.047 0.009 T2 0.047 0.009 T4 0.033 0.023 T4 0.023 0.011 T6 0.029 0.016 T6 0.017 0.007 T8 0.026 0.015 T8 0.015 0.008 T12 0.043 0.068 T12 0.016 0.007 T24 0.433 0.401 T24 0.113 0.176 KANA-40, HBV-16 TO 0.015 0.004 T2 0.047 0.009 T4 0.023 0.011 T6 0.017 0.008 T8 0.016 0.008 T12 0.019 0.007. T24 0.023 0.009 77 DK 167901 B1

Table A-9 Chessboard analysis results for polymyxin B and honey bee venom versus kanamycin resistance stent S. aureus.

Time GmTL_A660 Std.afv ^. Out Gnnk_A660 std-afv - ^ - POLY BO.HBV-O POLY BO.HBV-2 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.329 0.079 T4 0.178 0.042 T6 0.726 0.149 T6 0.621 0.127 T8 0.851 0.112 T8 0.851 T12 1.020 0.078 T12 1.065 0.072 T24 1.027 0.093 T24 1.106 0.083 POLY B-0, HBV-4 POLY BO.HBV-8 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.046 0.019 T4 0.023 0.013 T6 0.050 0.020 T6 0.012 0.011 T8 0.162 0.087 T8 0.007 0.003 T12 0.921 0.053 T12 0.138 0.142 T24 1.029 0.090 T24 1.038 0.068 POLY B-0, HBV-16 POLY B ~ 12.5, HBV-0 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.- 034 0.010 T4 0.266 0.051 T 6 0.013 0.004 T6 0.640 0.120 T 8 0.010 0.002 T8 0.826 0.110 T12 0.011 0.003 T12 0.976 0.095 T24 0.142 0.268 T24 0.962 0.074 POLY B-12.5, HBV-2 POLY B-12.5.HBV-4 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068. 0.004 T4 0.132 0.039. T4 0.035 0.012 T 6 0.490 0.142 T6 0.024 0.007 T8 0.742 0.196 T8 0.039 0.018 T12 1.027 0.093 T12 0.684 0.171 T24 1.083 0.063 T24 0.996 0.077 POLY B-12.5, HBV-8 PQLY B-12.5, HBV-16 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.023 0.012 T4 0.036 0.012 T 6 0.010 0.005 T6 0.013 0.004 T 8 0.007 0.003 T8 0.009 0.004 T12 0.050 0.056 T12 0.011 0.004 T24 0.993 0.073 T24 0.161 0.202

Table A-9 (continued) 78 DK 167901 B1

Time Gnj! L_Ag5o Std.vv. Time Avg. AC (-0 Std.vv.

POLY B-25.HBV-0 POLY B-25.HBV-2 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.243 0.033 T4 0.123 0.037 T6 0.629 0.073 T6 0.375 0.130 T8 0.835 0.114 T8 0.619 0.228 T12 1.008 0.096 T12 1.994 0.088 T24 1.048 0.091 T24 1.075 0.046 POLY B-25.HBV-4 POLY B-25.HBV-8 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.034 0.013 T4 0.022 0.012 T6 0.018 0.007 T6 0.009 0.003 T8 0.024 0.012 T8 0.007 0.003 T12 0.489 0.198 T12 0.016 0.014 T24 0.973 0.093 T24 0.906 0.171 POLY B-25.HBV-16 POLY B-50, HBV-0 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.035 0.013 T4 0.208 0.034 16 0.015 0.008 T6 0.376 0.123 T8 0.009 0.003 T8 0.567 0.192 T12 0.011 0.004 T12 0.841 0.115 T24 0.178 0.291 T24 0.968 0.048 POLY B-50, HBV-2 POLY B-50.HBV-4 TO 0.009 0.003 TO 0.009 0.003 12 0.068 0.004 T2 0.067 - 0.004 TZi 0.083 0.046 T4 0.027 0.013 T6 0.158 0.122 T6 0.012 0.006 T8 0.253 0.229 T8 0.011 0.006 T12 0.674 0.275 T12 0.263 0.177 T24 0.971 0.105 T24 0.951 0.105 POLY B-5O, HB V-8 POLY B-50.HBV-16 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.021 0.010 T4 0.038 0.012 T6 0.009 0.002 T6 0.015 0.005 T8 0.006 0.003 T 8 0.011 0.005 T12 0.011 0.004 T12 0.012 0.004 T24 0.807 0.222 T24 0.023 0.028

Table A-9 (continued) 79 DK 167901 B1

Time 6ηπκ_Α660 Std.afv. Fire Gnm. jA66q Std.afv ,, POLY B-100, HBV-0 POLY B-100, HBV-2 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.243 0.033 T4 0.123 0.037 T6 0.629 0.073 T6 0.375 0.130 T8 0.835 0.114 T8 0.619 0.228 T12 1.008 0.096 T12 1.994 0.088 T24 1.048 0.091 T24 1.075 0.046 POLY B-100, HBV-4 POLY B-100, HBV-8 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 • T4 0.034 0.013 T4 0.022 0.012 T6 0.018 0.007 T6 0.009 0.003 T8 0.024 0.012 T8 0.007 0.003 T12 0.489 0.198 T12 0.016 0.014 T24 0.973 0.093 T24 0.906 0.171 POLY B-100, HBV-16 TO 0.009 0.003 T2 0.068 0.004 T4 0.042 0.012 T 6 0.020 0.006 T8 0.015 0.005 T12 0.014 0.004 T24 0.106 0.232 DK 167901 B1 80

Table A-10 Results from equivalent doses of melittin and whole honey bee ft with and without kanamycin on S. aureus.

Time Avg. Accq Std.afv. Out Gnfn._A660 Std.afy_ ..

KANA-0, MEL-O, HBV-0 ΚΑΝΑΔΟ, MEL-O, HBV-2 TO 0.021 0.002 TO 0.021 0.002 T2 0.080 0.007 T2 0.080 0.007 TA 0.899 0.025 TA 0.381 0.201 T6 1.262 0.015 T6 1.089 0.139 T8 1.327 0.013 T8 1.289 0.041 T12 1.355 0.018 T12 1.347 0.033 T2A 1.398 0.037 T2A 1.417 0.024 KANA-0, MEL-1.6, HBV-0 KANA-2.5, KEL-O, HBV-0 TO 0.021 0.002 TO 0.021 0.002 T2 0.080 0.007 T2 0.080 0.007 TA 0.374 0.189 T4 0.692 0.106 T6 1.099 0.108 T6 1.114 0.182 T8 1.288 0.029 T8 1.217 0.180 T12 1.339 0.025 T12 1.265 0.115 T24 1.415 0.022 T24 1.330 0.105 KANA-2.5, MEL-0, HBV-2 KANA-2.5, MEL-O, HBV-2 TO 0.021 0.002 TO 0.021 0.003 T2 0.080 0.007 T2 0.068 0.004 TA 0.167 0.129 TA 0.266 0.051 T6 0.259 0.250 T6 0.640 0.120 T8 0.428 0.370 T8 0.826 0.110 T12 1.100 0.080 T12 0.976. 0.095 T24 1.290 0.053 T24 0.962 0.074 KANA-2.5.MEL-1.6, HBV-0 TO 0.021 0.002 T2 0.080 0.007 T4 0.152 0.121 T6 · 0.219 0.218 T8 0.366 0.363

T12 0.030 0.12A

T24 0.286 0.064 3i DK 167901 B1

Table A-11 "Raw results" from single treatment model

Log ^ p bacteria / ml blood

Test Treatment_mice 1 mouse 2 mouse 3 mouse 4 1 no treatment 2.62 3.49 2.91 3.18 1 medium tin - 50ng 3.27 2.88 2.76 * 1 polymyxin B - 2 µg 3.2 0 3.61 2.30 3742 * 1 flour 50 µg + pol 2ug 1.90 2.38 2.58 2.94 2 no treatment 2.96 4.16 3.77 389 2 medium tin - 50 ng 3.39 2.79 2.58 2.88 2 polymyxin B-2ug 3.88 3.00 3.34 3.27 2 flour 50ng + pol 2ug 2.38 0.00 2.62 2.15 3 no treatment 2.34 2. 62 2.51 1.90 3 medium tin - 50 ng 3.52 2.34 1.61 3.00 3 polymyxin B - 2ug 3.08 3.11 2.91 q -00 3 flour 50 ng + pole 2ug 2.41 1.32 1.32 0 -00 * lacks observation due to deficient blood test

Table A ~ 12 "Raw Results" from Repeated Treatment Model

Log10 bacteria / ml blood

Experiment Treatment mouse 1 mouse 2 mouse 3 mouse 4 1 No treatment 4.8 0 4.38 4.57 4.4Ο 1 luel in ttin - 50ng 4.ø4 4.74 5.07 4.62 1 polymyxin B - 2ug 5.04 4.56 3.48 q.00 1 me in 50 ng + pol 2ug 0. 00 0-00 3.30 0.00 2 No treatment 4.67 4.36 4.45 4.41 2 medium tin - 50ng 4.67 4.51 4.89 4.9q 2 polymyxin B - 2ug 1.78 3 .0 9 2.57 4 .0 6 2 flour 50ng + pole 2ug 1.30 1.85 0-00 3.21 3 No treatment 4.51 4.46 3.78 1.95 3 melittln-50ng 4.99 4.43 4.61 4.41 3 polymyxin B - 2ug 3.20 3.26 3.95 3.16 3 flour 50ng + pol2ug 3.80 3.24 3.22 3.46 4 No treatment 4.92 3.18 3.78 4.93 4 medium - 50ng 5.14 4.18 4.28 4.76 4 polymyxin B - 2ug 3.35 3.51 3.51 3.89 4 flour 50 ng + pol 2ug 2.60 3.6 8 3.5 1 2. 23 5 No treatment 3.53 4.30 4.46 4 .o 8 5 medium tin - SOng 4.0 8 4.7G 4.32 4.45 5 polymyxin - 2ug 4.43 2.94 3.34 3.72 5 flour 50ng + pol 2ug 2.34 3.41 3.05 2.93 DK 167901 B1 82

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5

Benton, A.W., R.A. Morse and F.V. Kosikowski, 1963. Bioassay and standardization of venom of the honeybee. Nature 198: 295-296.

Brangi, G.P. and M. Pavan. 1954. Bactericidal properties of bee venom 10 (Translated title, in Italian). Isoctex sociaux 1: 209-217.

Brown, L. R., J. Lauterwein, and K. Wuthnich. 1980. High-resolution * H-NMR studies of self-aggrogation of melittin in aqueous solution. Biochim. Biophys. Acta 622: 231-244.

15

Carrizosa, J. and M.E. Levison, 1981. Minimal concentration of aminoglycoside that can synergize with penicillin in entrococcal endocarditis. Antimicrob. Agents Chemother. 20: 204-409.

Coulson, C.C. and R.L. Kincaid. 1985. Gram preparative purification of calmodulin and S-100 protein using melittin-sepharose X L.

chromatography. 69ΐΠ Annual Meeting of the Federation of American Society for Experimental Biology. Federation Procedings 44: 1777.

25 Cynamon, Μ. H. and G.S. Palmer. 1983. In vitro activity of amoxicillin in combination with clavulanic acid against Mycobacterium turberculosis. Antimicrob. Agents Chemother. 24: 429-431.

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Franklin, T.J. and G.A. Snow. 1981a. Biochemistry of antimicrobial action. Chapman and Hall, New York, New York, pages 67-72.

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5 Haberman, E. 1972. Bee and wasp venoms: The biochemistry and pharmacology of their peptides and enzymes are reviewed. Science 177: 314-322.

Haberman, E. and J. Jentsch. 1967. Sequence analysis of melittins from the 10 tryptic and peptic clefts. Hoppe-Seyler's Z. Physiol. Chem. 348-37-5.

Hanke, W., C. Methfessel. H.U. Wiltnsen, E. Katz, G. Jung, and G. Boheim. 1983. Melittin and a chemically modified trichotoxin from 15 alamethiein-type multi-state pores. Biochim. Biophys. Acta 727: 100-114.

Lauterwein, J., C. Bosch, L.R. Brown and K. Wuthrich. 1979. Physiochemical studies of the protein-lipid interactions in 20 melittin-containing micelles. Biochim. Biophys. Acta 556: 244-264.

Lauterwein, J., L.R. Brown and K. Wuthrich. 1980. High-resolution 3 H-MNR studies of monomeric melittin in aqueous solution. Biochim. Biophys. Acta 622: 219-230.

25

Lowry, O.H., N.J. Rosenbrough, A.L. Farr and R.O. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193-265-275.

30 Moellering, R.C., C. Wennersten, and A.N. Weinberg. 1971. Studies of antibiotic synergism against enterococci. J. Lab. Clin. With. 77: 821-827.

Mollay, C. and G. Kreil. 1973. Fluorometric measurements on the interaction of melittin with lecithin. Biochim. Biophys. Acta 316: 196-203.

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Yunes, R.A. 1982. A circular dichroism study of the structure of Apis melifera melittin. Arch. Biochem. Biophys. 216 (2): 559-565.

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Goodman, M.G. and W.O. Weigle. Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on 5 membrane-directed versus intracellular activators. J. Allergy Clin. Immunol. Z4: 418-425, 1984.

Kondo, E. and K. Kanai. Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by 10 melittin. Japan. J. Med. Sci. Biol. 39: 9-20, 1986.

Kondo, E. Melittin-stimulated anti mycobacterial activity of the membrane fraction isolated from phagocytes of guinea pigs. Japan. J. Med. Sci. Biol. 39: 21-24, 1986.

15

Somerfield, S.D., J. Stach, C. Mraz, F. Gervais, and E. Skarnene. Bee venom melittin blocks neutrophil Oz production. Inflammation 10: 175-182, 1986.

Mulfinger, L.M. The synergistic activities of honey bee venom with antibiotics. Unpublished M.S. thesis, Pennsylvania State University, 1986 (covered by U.S. Patent Application Serial No. 096,628).

Lowry, O.H., N.J. Rosenbrough, A.L. Farr, and R.J. Randall. Protein 25 measurement with the folin phenol reagent. J. Biol. Chem.

193: 265-275. 1,951th

Ramachandran, J. and Virginia Lee. Preparation and properties of the o-nitrophenyl sulfenyl derivative of ACTH: an inhibitor of the lipolytic action of the hormone. Biochem. Biophys. Res. Com.

38 (3): 507-512. 1970th

Scoffone, E., A. Fontana, and R. Rocchi. Sulfenyl halides as modifying reagents for polypeptides and proteins. I. Modification of 35 tryptophan residues. Biochemistry 7 (3): 971-979. 1968th

Couch, T. and A. Benton. The effect of the venom of the honey bee, Apis mellifera L., on the adrenocortical response of the adult male rat. Toxicon 10: 55-62. 1,972th

Claims (5)

A composition for treating an infection in a mammal, characterized in that it comprises: a first antibiotic possessing activity against the infection, and a second agent which is at least one Hymenoptera venom, or at least one active protein component of a Hymenoptera venom, or at least one polypeptide component of a Hymenoptera venom, or at least one analog or chemically modified derivative of an active Hymenoptera venom protein component, or at least one analog or chemically modified derivative of a Hymenoptera venom or mixed polypeptide component. thereof, wherein the second agent is biuret positive and the proportions of the first antibiotic and second agent 20 are such that the second agent enhances the activity of the first antibiotic. Composition according to claim 2, characterized in that the first antibiotic comprises an antibiotic selected from ampicillin, kanamycin, polymyxin B or rifampicin. Composition according to claim 1 or 2, characterized in that the other agent is honey poison, hop poison, wasp poison, goat's venom, active protein components of at least one of these poisons, active polypeptide components of at least one of these poisons or analogs or chemically modified derivatives of flour in ttin, bombilittin, mastoporane and crabolin or mixtures thereof. Composition according to claim 1, 2 or 3, characterized in that the first antibiotic comprises ampicillin, kanamycin, polymyxin B or rifampicin and the second agent is honey bee poison or melittin. Unit dose for treating an infection in a mammal, 5 88 UK iD / aui D1, characterized in that it comprises an effective dose of a medicament comprising a composition according to any of the preceding claims. 10 o 15 20 25 30 35