METHODS AND COMPOSITIONS FOR THE TREATMENT OF MAMMALIAN INFECTIONS EMPLOYING MEDICAMENTS COMPRISING HYMENOPTERA VENOM, PROTEINAGEOUS OR POLYPEPTIDE COMPONENTS THEREOF, OR ANALOGUES OF SUCH PROTEINACEOUS OR POLYPEPTIDE COMPONENTS

Abstract

Methods and compositions are described for the treatment of mammalian infections, including bacterial, viral and cancerous infections, in which hymenoptera venom, proteinaceous or polypeptide components thereof, and analogues of such proteinaceous or polypeptide components are employed to enhance the activity of primary anti-bacterial, anti-viral, anti-carcinogenic or carcinostatic agents.

Description

INTRODUCTION

[0002] This invention relates to the use of certain secondary agents derived from nature, as well as synthetic analogues thereof, in the enhancement of the activity of other primary chemotherapeutic agents useful against bacterial, viral and cancerous infections, and especially the activity of antibiotic agents. The identity of antibacterial, anti-viral and anti-carcinogenic agents, and in particular antibiotic agents, and the activities and therapeutic usage of these materials are well known. The secondary agents employed in the invention in the enhancement of the activity of these primary anti-infectious agents are also known per se and have, in some cases, been used in medicine, but their ability to enhance the activity of antibacterial, anti-viral and anti-carcinogenic agents and, particularly, antibiotic agents, has not been recognized previously. Some of the secondary agents employed in the invention are obtained primarily from the venom of species of the order hymenoptera, which includes, without limitation, and by way of example only, honeybees, bumblebees, yellow jackets, bald faced hornets, fire ants, and the like.

SUMMARY OF THE INVENTION

[0003] The invention resides in the discovery that hymenoptera venom, isolated active proteinaceous or polypeptide components of such venoms, and analogues of such proteinaceous or polypeptide components, enhance or potentiate the activity of antibacterial, anti-viral and anti-carcinogenic agents and, especially, antibiotic agents.

[0004] The present invention grew out of the work described in a thesis in veterinary science by Lorraine Smith Mulfinger, entitled “Synergistic Activity Of honeybee Venom With Antibiotics”, which is to be submitted to the Graduate School Department of Veterinary Science of the Pennsylvania State University. The entire contents of that thesis is hereby made a part of the disclosure herein by reference. References to earlier work by others below have been abbreviated here since the full references are set forth in the bibliography of the Mulfinger thesis and at the end of this application.

BACKGROUND AND PRIOR ART

[0005] The use of anti-bacterial, anti-viral carcinostatic and anti-carcinogenic substances, while widely known in the art, is still the subject of massive continuing research, much of which, in addition to the discovery of new agents, is directed to the discovery of means for the enhancement of the activity of known active agents.

[0006] Indeed, certain substances derived from bee venom have been studied and have been found useful in certain specific pharmacologic applications. For example, U.S. Pat. No. 4,444,753 issued Apr. 24, 1984, describes a composition comprising a component obtained by deproteinizing an extract from the poison pouch contents of bees. This product has an immuno-stimulating activity, a carcinostatic activity, an effect of enhancing the antibacterial activity of an anti-bacterial substance, and an effect of enhancing the carcinostatic activity of a carcinostatic substance. The invention disclosed in that patent is directed to cacinostatic, immuno-stimulating and antibacterial agents comprising the composition described. While that invention is similar in purpose to that of the present invention, it differs in that the bee extract is modified by deproteinizing it so that it is negative in biuret reaction and sulfosalicyclic acid reaction.

[0007] U.S. Pat. No. 4,370,316, issued Jan. 25, 1983 to the same inventors as the patent described above, also claims a method of treating a host animal having decreased immunity by administering an effective amount of the deproteinized extract from the poison pouch of the bee.

[0008] Therefore, while antibacterial, anti-viral and anti-carcinogenic substances are well known, and it is also known that a deproteinized extract from the poison pouch of a bee has certain useful activities, including antibacterial activity, activity in stimulating antibacterial activity and immuno-stimulating activity, it has not been recognized previously that proteinaceous hymenoptera venoms, proteinaceous or polypeptide extracts thereof, and analogues of such proteinaceous or polypeptide components, have an enhancing effect on virtually all antibacterial, anti-viral, carcinostatic and anti-carcinogenic agents. Such enhancement of the activity of such primary anti-infectious agents not only increases the effect of dosages of such agents which would be effective alone but can also render effective low dosages of such agents which would be ineffective if used alone.

[0009] As noted above, the present invention relates to the use of hymenoptera venom, proteinaceous or polypeptide components thereof, and analogues of such proteinaceous or polypeptide components to enhance the activity of anti-infectious therapeutic agents in general. To simplify the description of the invention, however, it will be discussed below for purposes of illustration, in the use of honeybee venom or its proteinaceous extract melittin, in the enhancement of the activity of antibiotics in the control of bacterial, viral, and cancerous infections. Honeybee venom (HBV) has been selected since it is readily available. It is to be understood, however, that the venom of other hymenoptera and proteinaceous or polypeptide components thereof, as well as analogues thereof, are also effective in the invention in varying degrees. Similarly, anti-infectious agents other than antibiotics may also be employed in the invention in the treatment of infections for which they have been used previously, but with enhanced effect when used in combination with the proteinaceous hymenoptera agents.

[0010] As further background, it is noted that honeybee venom is credited with a multitude of useful activities. Some of the activities are scientifically documented while others appear to be based on empirical data and folklore. The invitro antibacterial activity of honeybee venom is well documented (Schmidt-Lange, 1951; Ortel and Markwardt, 1955; Fennel et alia, 1968), however, few efforts have been made to put this activity to practical use. In the present invention, the data from several empirical experiments indicated that the antibacterial activity of honeybee venom may have a significant effect in vivo, in the presence of antibiotics. Based upon these observations, an investigation was designed to study the interactions of honeybee venom and antibiotics using an in vitro assay where the two compounds could be evaluated without the contributing effects of the natural immune responses of the host animal.

[0011] In this study, three strains of bacteria were tested initially against three different antibiotics using separate checkerboard titrations of honeybee venom with each antibiotic. Representatives of three major groups of antibiotics (penicillins, aminoglycosides, and polymyxins) were selected and assayed to determine if honeybee venom could improve the antibacterial efficacy of selected antibiotics. An antibiotic from a fourth major group was studied later as described below.

[0012] Once synergy was demonstrated in the checkerboard assay, a broader survey was attempted using a simplified procedure. Two automated minimal inhibitory concentration (MIC) assay plates, which titrate susceptibility to eleven antibiotics simultaneously, were inoculated in parallel with bacterial cultures with and without non-inhibitory doses of honeybee venom (HBV). Eight gram-positive and four gram-negative organisms were tested using this system in an effort to find classes of antibiotics that routinely produce synergy with HBV, and to determine the spectrum of synergistic action of these combinations among different groups of bacteria.

[0013] In addition to testing whole honeybee venom, the venom was fractionated by size exclusion chromatography. Each of four fractions were tested to determine if a specific component was responsible for antibacterial activity and could also act synergistically in antibacterial assays. It was shown that the fraction containing melittin, which had been previously identified as the antibacterial element of the honeybee venom (Fennel et alia, 1968), is active in its purified form and will act synergistically in a magnitude equal to that of whole honeybee venom.

[0014] Furthermore, the activity of various analogues of the active components of hymenoptera venoms was determined and compared with that of melittin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a diagram of the amino acid sequence of melittin;

[0016] FIG. 2 is a graph of optical density versus hours after inoculation which shows the antibacterial activity of honeybee venom (HBV) on S. aureus;

[0017] FIG. 3 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus S. aureus;

[0018] FIG. 4 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus S. aureus;

[0019] FIG. 5 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus S. aureus;

[0020] FIG. 6 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus E. coli;

[0021] FIG. 7 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus E. coli;

[0022] FIG. 8 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus E. coli;

[0023] FIG. 9 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus E. coli;

[0024] FIG. 10 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus kanamycin resistant S. aureus;

[0025] FIG. 11 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus kanamycin resistant S. aureus;

[0026] FIG. 12 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus kanamycin resistant S. aureus;

[0027] FIG. 13 shows the electrophoresis results of 100 ug of melittin protein;

[0028] FIG. 14 is a graph of optical density versus hours after inoculation showing antibacterial activities of melittin/HBV versus S. aureus;

[0029] FIG. 15 is a graph of optical density versus hours after inoculation for melittin/HBV and kanamycin versus S. aureus;

[0030] FIG. 16 is a graph of optical density versus hours after inoculation for rifampicin and HBV versus S. aureus;

[0031] FIG. 17 is a graph of optical density versus hours after inoculation for rifampicin and HBV versus Ps. inosa;

[0032] FIG. 18 is a graph of optical density versus hours after inoculation for polymyxin B and bumblebee venom versus E. coli;

[0033] FIG. 19 is a graph of optical density versus hours after inoculation for polymyxin B and yellow jacket venom versus E. coli;

[0034] FIG. 20 is a graph of optical density versus hours after inoculation for polymyxin B and bald faced hornet venom versus E. Coli;

[0035] FIG. 21 is a graph of the log 10 bacteria/ml blood versus treatment for a single treatment model of septicemia (polymyxin B and melittin interactions);

[0036] FIG. 22 is a graph of the log 10 bacteria/ml blood versus treatment for a repeated treatments model of septicemia (polymyxin B and melittin interactions); and

[0037] FIG. 23 is a graph of optical density versus hours after inoculation for melittin/analogue and polymyxin B versus E. coli.

Composition of Venoms

[0038] Venoms are heterogeneous mixtures of biochemical compounds. Most venoms are more than 90% protein. Toxins and enzymes make up this protein portion and are the cause of direct cell damage. While many enzymes such as phospholipase A2, acid phosphatase, and hyaluronidase are common to most venoms, toxins and other biologically active peptides contained in venoms are highly species specific.

[0039] Venom producing insects all belong to the insect order Hymenoptera. Like snake venoms, enzymatic activities such as phospholipase A2, hyaluronidase, and acid phosphatase are common to all insect venoms. The toxin and peptide components, however, vary from species to species. (Tu, 1977b)

[0040] The venom of the Italian honeybee (Apis mellifera) is the most extensively studied insect venom. The major component of honeybee venom is melittin. This peptide has a molecular weight of 2,847 daltons and accounts for approximately 50% of the venom's dry weight. A second peptide, apamine, is present as approximately five percent of the venom and several other peptides are present in trace amounts. (Haberman, 1972)

[0041] The venoms of other hymenoptera contain peptides having biological properties which are similar to those of melittin. Examples of such peptides are bombolitines I-V from the bumblebee, Megabombus pensylvanicus, mastoporan from wasps, hornets, and yellow jackets, and crabolin from European hornets. A common feature of these peptides is their amphiphilic nature. These peptides have been subjected to sequence analysis and their structures are well known. (A. Argiolas and J. J. Pisano, 1985)

Antibacterial Activity of Honeybee Venom

[0042] The bactericidal activity of honeybee venom was first documented in 1941 by W. Schmidt-Lange (1941). He tested E. coli and staphylococci and found both to be susceptible to the antibacterial activity of honeybee venom. Additionally, he noted that the minimal inhibitory dose of honeybee venom for E. coli was much higher than for staphylococci.

[0043] It wasn't until ten years later that Brangi and Pavan (1951) evaluated various extraction procedures to isolate the antibacterial activity of honeybee venom. They found the activity to be present in both water and acetone extracts of venom. They also showed that the activity was stable when heated to 100 degrees centigrade for up to 15 minutes.

[0044] In 1955, Ortel and Markwardt (1955) published the results of an investigation of the variability in sensitivity among different bacteria to honeybee venom's antibacterial activity. Two hundred ninety-six strains of bacteria were tested. The results showed that tolerance to honeybee venom is much greater in gram-negative organisms than in gram-positive organisms. Ranges for bactericidal concentrations were reported to be 12.5 to 25 ug/ml for gram-positive bacteria and 1 to 10 mg/ml for gram-negative bacteria. The bactericidal activity co-purified with the red blood cell “direct hemolytic fraction”. The name “melittin” had not yet been assigned to the active component of this fraction.

[0045] In 1963, Benton et alia published a bio-assay for honeybee venom. The bacteriostatic activity of venom was quantitated by a radial diffusion assay which measured zones of growth inhibition caused by serial venom dilutions in a lawn of bacterial growth. This assay was proposed to standardize the biological activity of honeybee venom intended for in vivo use. (Currently, allergy desensitization is the only in vivo honeybee venom treatment approved by the Food and Drug Administration of the United States.) The article also tested the heat sensitivity of the honeybee venom activity and found it could withstand sterilization procedures (121 degrees centigrade for 15 minutes) (Benton et al. 1963).

Melittin Isolation and Activities

[0046] Honeybee venom has several pharmacologically active compounds. The compound appearing in the greatest proportion in venom is melittin, a polypeptide with a molecular weight of 2,847 daltons, that acts as a direct hemolysin of red blood cells. Other active components include phospholipase A2, histamine, dopamine, noradrenaline, apaamin, and hyaluronidase (Haberman, 1972).

[0047] Antibacterial Activity of Melittin

[0048] Fennel, Shipman, and Cole (1968), purified melittin with Sephadex G-50 chromatography and showed that the melittin fraction had “potent antibacterial activity”. They tested 30 random strains of bacteria (including several streptococci, staphylococci, and enteric bacteria strains), comparing the activity of purified melittin to whole honeybee venom. They noted that one strain of S. aureus, a penicillin resistant isolate, showed no decrease in sensitivity to the melittin.

[0049] Although melittin had been reported to be the antibacterial factor of honeybee venom, no reports of its use in vivo have been found. It was noted by Mollay and Kreil (1974) that interactions between melittin and lecithin enhanced the activity of phospholipase A2 honeybee venom on lecithin. It has not previously been recognized, however, that melittin enhances the activity of antibiotics.

[0050] Haberman and Jentsch (1967) have purified melittin and published the amino acid sequence. They found that melittin exists in two natural forms, differing only by a formyl substitution at the N-terminces (FIG. 1).

[0051] Analogues of Proteinaceous and Polypeptide Components of Hymenoptera Venoms.

[0052] The following analogues of melittin have been prepared.

Analogue
No.	Composition
1.	Melittin	(1-20) - NH2
2.	Melittin	(1-20) - Orn-Orn-Orn-Orn-Gln-Gln-NH2
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-Gln-NH2
8.	Melittin	(1-20) - Asp-Asp-Asp-Asp-Asp-Asp-NH2
9.	Melittin	(1-20) - Lys-Lys-NH2
10.	Mastoporan	(1-14) - NH2 (native)
11.	Mastoporan	(1-14) - Orn-Orn-Orn-Orn-Gln-Gln-NH2
12.	Melittin	(1-20) - (CH2NH2)12
13.	Melittin	(1-20) - Orn-Orn-NH2

[0053] The analogues were prepared by conventional peptide synthesis as described by e.g. M. Bodanszky: “Principles of Peptide Synthesis”, Springer Verlag, 1984.

[0054] As an example the peptide synthesis of analogue No. 4, viz. melittin (1-20)-Lys-Arg-Iys-Arg-Gly-Gly-NH2 will now be described in further detail.

[0055] A derivatized resin such as a polydimethylacrylamide gel which is commercially available under the trade name PEPSYN KA is reacted with (Fmoc-Gly)2O wherein Fmoc is 9-fluorenylmethoxycarbonyl which serves as a temporary protecting group.

[0056] The reaction, which is carried out in the presence of 4-dimethylaminopyridine as a catalyst, results in the formation of the ester Fmoc-Gly-O-resin.

[0057] The ester is deprotected in the presence of 20% piperidine in DMF so as to form H-Gly-O-resin.

[0058] The deprotected product is then reacted with an activated ester having the formula

Fmoc-Gly-OPfp

[0059] wherein Pfp is pentafluorophenyl so as to form

Fmoc-Gly-Gly-O-resin

[0060] The remaining 24 amino acids are coupled to the reaction product formed in 20 similar cycles of deprotection and coupling with active esters.

[0061] The product thus formed is deprotected with 20% piperidine in DMF and the melittin analogue formed is cleaved from the resin in the presence of TFA (trifluoroacetic acid) and a scavenger, such as water.

Antibiotics

[0062] Antibiotics can be divided functionally into four groups based upon the active sites of the antibiotics (Volk, 1978a). Target structures of the four groups are the cell wall, the cell membrane, the protein synthesis machinery, and the nucleic acid replication machinery. Because of the complexity of the synergy assay, four antibiotics, one from each of the foregoing groups, were chosen for testing. The selected antibiotics were ampicillin, kanamycin, polymyxin B and rifampicin. Each has a different mode of action on procaryotic cells.

[0063] Ampicillin

[0064] Ampicillin belongs to the group of antibiotics affecting 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 enzyme which crosslinks the pentaglycine bridges of the peptidoglycan, therefore, only actively growing cells are affected by their presence.

[0065] Ampicillin is a semisynthetic derivative of penicillin. The synthetic step in ampicillin synthesis adds an amine group to the alpha carbon of penicillin G. This confers resistance to beta-lactamases (the predominant penicillin resistance factor of bacteria) giving ampicillin a much broader spectrum of efficacy among bacteria than penicillin (Volk, 1978b).

[0066] Kanamycin

[0067] Kanamycin is an aminoglycoside. This group of antibiotics blocks protein synthesis. Members of this group bind to the 30s ribosome of bacteria and sterically block the binding of aminoacyl-tRNA's or inhibit the translocation of the growing peptide chain at the ribosomal active site (Volk, 1979c). Since protein synthesis is required for many regulatory cell functions, aminoglycosides are effective on bacteria in either active or stationary growth phases.

[0068] Polymyxin B

[0069] Polymyxin B is a cyclic, amphiphatic peptide. Due to the combined hydrophilic and hydrophobic properties, polymyxin B has a detergent-like action that does not require cell growth to be effective. Like melittin, polymyxin B interacts with membranes to form small hydrophilic pores in the hydrophobic areas of membranes. In gram-negative organisms, which have a thick lipopolysaccharide layer acting as a selective permeability barrier, polymyxin B is effective in disturbing osmotic gradients. Therefore, polymyxin B is very effective on gram-negative organisms, while only minimally effective on gram-positive organisms. (Sebek, 1979). While melittin can form membrane pores simularly to polymyxin B, melittin is more active on gram-positive organisms, therefore the action of melittin cannot be totally analogous to that of polymyxin B.

[0070] Rifampicin

[0071] Rifampicin is an antibiotic from the group which acts at the level of nucleic acid synthesis, which completes examples of antibiotics from the four main categories referred to above.

Synergy Studies

[0072] A review of articles studying synergy between antibiotics and other compounds in bacterial systems showed that all investigators used the same basic approach. Bacterial growth was monitored in broth cultures with and without each compound separately, and then with both compounds together. In order to prove synergistic action as opposed to an additive effect, in each case, at least one of the compounds was used at a level where alone it would demonstrate minimal growth inhibition. Thus, with one compound relatively inactive, any increased activity of the second compound in its presence would be the result of synergistic interactions (Moellering at alia, 1971; Carrizosa and Levison, 1981; and Cynamon and Palmer, 1983). It is upon this type of design that experiments in this invention were based.

Materials and Methods

Materials

[0073] Honeybee (Apis melifera) venom was supplied by Vespa Laboratories, Spring Mills, Pa.

[0074] Bacteria strains were supplied by the Veterinary Science Department of the Pennsylvania State University. S. aureus #140A is a field isolate from a case of bovine mastitis. E. coli #G1880E was selected from the E. coli Reference Center systematic collection. A kanamycin resistant strain of S. aureus was isolated by a natural selection procedure described below.

[0075] Antibiotics were purchased from Sigma Chemical Company (St. Louis, Mo.) and activity units were based on their analyses.

[0076] Trypticase soy base (BBL Microbiology Systems, Cockeysville, Md.) was used to support all bacterial growth either as a broth or an agar.

[0077] Sephadex G-50 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden.

[0078] Minimal inhibitory concentration (MIC) assays of antibiotics with and without honeybee venom, were performed by the Microbiology Department of the Allegheny General Hospital, Pittsburgh, Pa., using the Sensititre™ assay system distributed by Gibco Laboratories, Lawrence, Mass.

Methods

[0079] Isolation of Kanamycin Resistant Mutant

[0080] S. aureus was grown in 5 ml of trypticase soy broth (TSB) overnight to an approximate density of 109 colony forming units/ml. 0.1 ml of the overnight culture was plated on a plate of trypticase soy agar (TSA) containing 39 ug/ml kanamycin and incubated for 48 hours at 37 degrees centigrade. Colonies appearing within 48 hours were subcultured onto a second TSA plate supplemented with 39 ug/ml kanamycin.

[0081] Checkerboard Titration Assay for Synergy

[0082] Bacteria cultures were prepared for this assay by freezing each strain while in logarithmic growth in TSB. For this purpose, a 5 nil overnight culture was used to inoculate 200 ml of TSB in a 500 ml erlenmeyer flask. The culture was incubated at 37 degrees centigrade with constant stirring and the optical density (OD) at 660 nm was read hourly. When the culture reached mid-log phase (approximately 0.500 OD units), 5 ml aliquots were transferred to 16×100 mm screw cap tubes. All cultures were frozen and stored at −20 degrees centigrade. E. coli required glycerol to be added to the medium to a final concentration of 20% to survive freezing. This was accomplished by mixing 1 ml of sterile glycerol with 4 ml of log-phase culture immediately before freezing.

[0083] To begin an assay, one tube of a 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 ml erlenmeyer flask, stirred, and the OD660 immediately measured and recorded as the “time zero” reading. The flask was then incubated at 37 degrees centigrade with constant stirring for two hours at which time the OD660 was again read and recorded, the culture was split into 16×100 mm screw-cap test tubes prefilled with the specified aliquots of honeybee venom (HBV) and antibiotic described

[0084] Stock solutions of HBV and antibiotics were made in distilled water, filter sterilized, and stored at −20 degrees centigrade in 5 ml aliquots at concentrations twice the concentration needed for the checkerboard titration system. The frozen stock concentrations required for each bacterial species are given in Table 1. The concentration used for each bacterium was based on preliminary experiments using the antibiotics alone to determine the minimal inhibitory ranges of each antibiotic for each microorganism.

[0085] For each assay, one vial of antibiotic and one vial of HBV were thawed at room temperature and diluted with an equal volume of 2×TSB and then serially diluted twofold into normal strength TSB to obtain four concentrations of venom and four concentrations of antibiotic. Seventy-five screw capped test tubes were numbered and arranged to correspond to the checkerboard pattern shown in Table 2. TSB, antibiotic, and HBV were then dispensed according to the design shown in Table 3. Tubes labeled as OO and O contained 2.5 ml of TSB and served as OD blanks and sterility control tubes. Tubes 1-75, each containing a total volume of 500 ul, was inoculated with 2 ml of the two hour culture described above. [Note: the final concentration of HBV and/or antibiotic in each tube was one tenth of the concentration added in the 250 ul aliquot (refer to Table 3).] Each tube was immediately sealed and inverted. After all tubes mere inoculated, they were placed in horizontal racks on a rocker platform at 37 degrees centigrade. The growth in each tube was individually monitored at four, six, eight, 12, and 24 hours by determining the optical density of each tube at 660 nm.

[0086] Minimal Inhibitory Concentration Assays with HBV

[0087] The microbiology laboratory of the Allegheny General Hospital, having the capacity to perform automated MIC assays, was contracted to perform a trial survey on 12 clinical bacterial isolates. The adaptation of the automated MIC assay had the following restrictions: (1) each assay could only test one dose level of HBV, and (2) the effect of the HBV alone could be evaluated only as completely inhibitory or non-inhibitory. Synergy of HBV with the 11 antibiotics in this system was evaluated by comparing two assays run simultaneously with and without HBV present. The dose of HBV used for each species was estimated to be a non-inhibitory dose, based on the checkerboard titration assays.

[0088] Melittin Purification

[0089] Sephadex® G-50 gel filtration bedding was swollen for 24 hours at room temperature in beta alanine-acetic acid buffer (BAAB), pH 4.3 (Guralnick et alia, 1986), and then equilibrated at five degrees centigrade overnight. A 2.5×60 cm column was poured and equilibrated at a flow rate of 1.0 ml/hour. One hundred mg of HBV was reconstituted in 5 ml of BAAB buf containing 200 sucrose. The HBV was layered on the column and eluted at a flow rate of 1 ml/hour. The effluent was monitored for absorbence at 280 nm. Fractions containing the main peak were pooled, an aliquot was assayed by the Lowry Protein Assay (Lowry, 1951), and the remainder was lyophilized.

[0090] Identification of the melittin fraction was based on the relative mobility and quantitation of bands appearing in polyacrylamide gel separations of each fraction (Benton, 1965). The melittin was also checked for purity by polyacrylamide gel electrophoresis. Electrophoresis was performed as described by Guralnick et alia, (1986). Lyophilized fractions were reconstituted to 2 mg/ml in the electrophoresis sample buffer and 50 ul samples were applied per sample well on the gels.

[0091] Whole Venom Equivalence of Melittin

[0092] The amount of the melittin fraction equivalent to its proportion in whole honeybee venom was determined by quantification of individual bands in electrophoresed samples of whole venom and the melittin fraction. Twenty, 40, 60, 80, and 100 ug samples of whole honeybee venom were separated by electrophoresis, stained with Coomassie® brilliant blue-perchloric acid stain, and scanned with a densitometer. A standard curve was established relating the peak area of the melittin band of the whole venom samples to the quantity of protein in the sample when it was applied. Six 40 ug samples of the purified melittin were assayed simultaneously and their equivalence in honeybee venom was determined from the standard curve. This procedure is described in detail by Mulfinger et alia. (1986).

[0093] Testing the Melittin Fraction for Synergistic Activity

[0094] To compare the antibacterial activity of whole honeybee venom and the melittin fraction, earlier checkerboard titration results were reviewed and the test system was selected where HBV dose effects could be easily seen alone and in combination with an antibiotic. Since staphylococci were susceptible to the HBV alone at concentrations used in the above checkerboard assays, and since kanamycin showed good synergistic action with the HBV, this system was chosen to compare the antibacterial activities of whole HBV and melittin. The doses of each component used in this analysis were 2 ug/ml HBV and 2.5 ug/ml kanamycin. These doses were in a range of bacterial reactivity where the effects of small dose changes were reproducible and easily measured. The equivalent dose of the melittin fraction for 2.0 ug/mI HBV was 1.6 ug/ml. Each experiment compared in parallel, triplicate samples of the melittin fraction and whole honeybee venom with and without kanamycin present to check for equivalent activity.

[0095] Statistical Analysis

[0096] Each checkerboard experiment was repeated five times. The averages of the five repetitions for each bacteria-antibiotic combination were tested at each time point for significant differences using a Waller-Duncan K-ratio T Test and families of curves were selected for synergy testing. A curve family consisted of an experiment control curve (bacterial growth with no antibiotic or HBV present), an antibiotic control curve (bacterial growth with antibiotic but no HBV present), a venom control curve (bacterial growth with HBV but no antibiotic present) and an interaction curve (growth with antibiotic and HBV present). Families in which the antibiotic control curve and the venom control curve showed small average OD decreases relative to the experiment control curve, and which also demonstrated large OD decreases in the interaction curve relative to the experiment control curve were tested for synergy.

[0097] A synergistic effect between compounds can he differentiated from an additive effect of the compounds since an additive effect is predictable. Additive effects can be predicted by summing the effects of the two compounds individually, thus, any greater effect would indicate synergistic interaction. An equation predicting OD readings for an additive interaction between HBV and an antibiotic was derived. See the Mulfinger thesis (1987) referred to above, pages 23-25.

RESULTS

Checkerboard Titration Assays

[0098] Three bacterial strains were tested against each of three antibiotics combined with honeybee venom. These nine combinations of bacteria, antibiotic, and HBV were analyzed using the checkerboard assay which provided for 25 treatnents (antibiotic and HBV combinations) for each bacterium-antibiotic combination. Each checkerboard experiment included triplicate samples for each treatment and was repeated five times. The data from triplicate samples repeated in five experiments were averaged and the mean and standard deviation for each time point of each treatment appear in the appendix. For each bacterium-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 of four curves, as described in “Statistical Analysis” above were compared for evidence of synergy. The family of curves showing the greatest OD difference between the interaction curve and the lowest of the experiment curve, antibiotic control curve and venom control curve, gas plotted and each time point was tested for synergy using the equation derived in the section “Statistical Analysis” above. For each family of curves, if the estimate of (−X+A+V−AV) for a time point is significantly greater than zero at 95% confidence level (i.e., synergy is indicated), the time point is noted on the interaction curve by a superscript “s” at the square representing that time point (FIGS. 2-11). S. aureus

[0099] S. aureus is sensitive to honeybee venom alone at low concentrations. It was important, therefore, to find the maximum dose of honeybee venom for which no effects were demonstrated. This concentration was approximately 2 ug/ml. Therefore, for all antibiotic/HBV combinations with S. aureus, the venom doses for the checkerboard titration system were 0, 2, 4, 8, and 16 ug/ml (Tables A-1 through A-3). FIG. 2 demonstrates the effects of these dosages of honeybee venom when used alone as an antibacterial compound.

[0100] S. aureus Versus Ampicillin/HBV

[0101] The final concentrations of ampicillin in tubes of the checkerboard system were 0, 0.05, 0.1, 0.2, and 0.4 ug/ml. FIG. 3 shows the results of the ampicillin/HBV combination using 2 ug/ml HBV and 0.05 ug/ml ampicillin. No synergy is seen at the 4 or 6 hour points; however, at both the 8 and 12 hour time points, it is evident that the interaction curve is much lower than would be predicted from the sum of the effects caused by ampicillin and HBV alone. Statistical analysis shows that at both time points, the summation (−X+A+V−AV) is significantly greater than zero.

[0102] S. aureus Versus Kanamycin/HBV

[0103] The final concentrations of kanamycin selected for testing S. aureus in the checkerboard system were 0, 1.25, 2.50. 5.0, and 10.0 ug/ml (Table A-2). FIG. 4 depicts the family of curves demonstrating the greatest contrast between control and interaction curves. In the experiment, synergy first becomes demonstrable near the 6 hour time point and is clearly seen by the 8 hours of incubation. At 12 hours, the cultures appear to have escaped the effects of the combined dose and the synergistic effect is lost since growth becomes limited by other (nutritional) factors in the medium. (This growth limitation is demonstrated by the control curve.) Despite the 12 hour growth restriction, statistical analysis of the data at 6, 8, and 12 hours suggest synergistic interaction between kanamycin and HBV in this assay.

[0104] S. aureus Versus Polymyxin B/HBV

[0105] The final concentrations of polymyxin B in these experiments were 0, 312, 624, 1250, and 2500 U/ml (Table A-3). Synergy was observed with 4 ug/ml HBV and 625 U/ml polymyxin B (FIG. 5). At both 8 and 12 hours of incubation, synergy is demonstrated by the interaction curve.

[0106] E. coli

[0107] Honeybee venom was not inhibitory alone to E. coli at the levels required to demonstrate synergy (Tables A-4 through A-6), thus, toxicity was not the limiting factor for HBV in the checkerboard assay with E. coli. However, experimental conditions limited the upper concentration of HBV at approximately 40 ug/ml; concentrations greater than this caused precipitation of medium components. Therefore, the final concentrations of HBV used in the checkerboard assays with E. coli were 0, 5, 10, 20, and 40 ug/ml.

[0108] E. coli Versus Ampicillin/HBV

[0109] The final concentrations of ampicillin selected for use in the E. coli checkerboard titration were 0.5, 1, 2, and 4 ug/ml (Table A-4). Synergy was less dramatic in all families of curves evaluated than for any of the above experiments. There was evidence of synergy only in the 40 ug/ml HBV-1 ug/ml ampicillin combination and only at the 6 hour time point (FIG. 6).

[0110] E. coli Versus Kanamycin/HBV

[0111] The final concentrations of kanamycin selected for the checkerboard assay were 0, 5, 10, 20, and 40 ug/ml (Table A-5). FIG. 7 shows the effects of honeybee venom with a minimally effective dose of kanamycin. In this situation, only the 8 hour time point shows synergy. Regardless of the HBV dose, no synergy was seen in any of the other combinations of HBV with low doses of kanamycin.

[0112] FIG. 8 shows a higher dose of kanamycin with HBV on E. coli. Here, synergism is statistically proven at all time points after 2 hours.

[0113] E. coli Versus Polymyxin B/HBV

[0114] The final concentrations of polymyxin B in the checkerboard titrations were 0, 1.5, 3, 6, and 12 U/ml (Table A-6). The combination of 3 U/ml polymyxin B and 5 ug/ml HBV gave the most dramatic illustration of synergism (FIG. 9). Synergy is evident at all time points during the treatment and the differences between the observed and the predicted values are large.

[0115] Kanamycin Resistant S. aureus

[0116] A kanamycin resistant S. aureus, obtained by the selection of spontaneous mutants, was assayed to evaluate the effect of HBV on drug resistant bacteria. A kanamycin resistant S. aureus was desirable because some synergy was seen for all antibiotics with this organism, and because synergistic effects were most easily seen with kanamycin.

[0117] No difference was found in the resistant strain's susceptibility to HBV, thus the venom concentrations in the checkerboard assays were the same as for the parent strain, 0, 2, 4, 8, and 16 ug/ml (Tables A-7 through A-9). It was noted that under identical conditions, the resistant strain had a slower growth rate than the parent strain, therefore, comparing optical density readings between experiments on the two different strains is not meaningful.

[0118] Kanamycin Resistant S. aureus Versus Ampicillan/HBV

[0119] The final concentrations of ampicillin used in this checkerboard assay were the same as for the parent S. aureus, 0, 0.05, 0.1, 0.2, and 0.4 ug/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 was seen at a higher ampicillin concentration than for the parent. Due to the slower growth rate, a longer growth period was considered. FIG. 10 shows the interaction of 2 ug/ml HBV and 0.4 ug/ml ampicillin. Statistical evaluation of the data shows synergy at the 8, 12, and 24 hour time points.

[0120] Kanamycin Resistant S. aureus Versus Kanamycin/HBV

[0121] The dosage of kanamycin required to reduce the growth rate of the kanamycin resistant strain of S. aureus was approximately four times higher than the dose required by the parent strain. The checkerboard assay range for the kanamycin resistant S. aureus was 0, 5, 10, 20, and 40 ug/ml of kanamycin (Table A-8). Again, the slow growth rate made it necessary to consider a longer growth period. The combination of 8 ug/ml honeybee venom and 10 ug/ml kanamycin is shown in FIG. 11. Although the dose of kanamycin used is twice as high as the dose needed for the parent S. aureus, it remains effective twice as long in the presence of honeybee venom. Synergy was observed only after 12 hours and was proven to be significant only at the 24 hour time point.

[0122] Kanamycin Resistant S. aureus Versus Polymyxin B/HBV

[0123] It was interesting to note that this mutant, selected for increased resistance to kanamycin, became more susceptible to polymyxin B than the parent strain. The polymyxin B doses used for the checkerboard assay was 0, 12.5, 25, 50, and 100 U/ml (Table A-9), whereas the polymyxin B dose range used for assaying the parent strain was between 312 and 2500 U/ml. FIG. 12 shows kanamycin resistant S. aureus versus 50 U/ml Polymyxin B and 4 ug/ml HBV. Synergy was shown at the 12 hour time point.

[0124] MIC Assays of Antibiotics With and Without HBV

[0125] The results of a preliminary survey of the effect of HBV on the MIC of antibiotics for eight gram-positive bacteria and four gram negative bacteria are shown in Table 4 and Table 5 respectively. Despite the apparent inadequacies of the assay system, definite trends where seen in the results of the survey. Synergy was strongly suggested where observations within a single MIC assay showed that identical doses of HBV affected some antibiotic MIC's while not affecting others. In Tables 4 and 5, a (+) was used to denote a decrease of more than one twofold dilution of the MIC of an antibiotic in the presence of HBV. A (−) indicates no difference or only a single dilution step variation (judged to be the variation of the assay) in the MIC of an antibiotic with HBV present.

[0126] Table 4 shows the results of several gram-positive organisms. The results indicate that trends exist within the species tested. For example, S. aureus appears to show synergy with all antibiotic/HBV combinations, while S. epidermidis shows consistent synergistic results only with the cephalothin/HBV combination and sporatic results with other antibiotic/HBV combinations. The one Streptococcus faecalis strain that was tested reflects none of the same synergistic trends shown by the two staphylocuccus organisms.

[0127] The data in Table 5 lists the results of four E. coli strains in the MIC assay system. Definite patterns of synergy 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 amikacin were lowered in every instance except one. The MIC of cefoxitin was also lowered by HBV in all E. coli assays.

Melittin Purification and Testing

[0128] Chromatography of Honeybee Venom

[0129] Purification of melittin on Sephadex G-50 gave well defined, base-line resolved peaks. The void volume was 100 ml and the melittin fraction eluted between 200 and 230 mls after tie void volume. Approximately 65 ug of the initial 100 ug sample were recovered in fractions 200 to 230. These fractions were pooled and were checked for purity by polyacrylamide gel electrophoresis. FIG. 13 shows the electrophoresis results of 100 ug of protein from the pooled fractions 200-230. Comparison of the relative mobility of this band to the relative mobilities of electrophoretically separated HBV components identified melittin as the only component of fractions 200-230 detectable in this separation.

[0130] Testing Melittin for Antibacterial Activity

[0131] Equivalent doses of melittin and whole honeybee venom were compared for antibacterial activity in combination with and without antibiotic (Table A-10). Since S. aureus was susceptible to HBV at levers used in the above assays, this organism was chosen to test the melittin fraction's activity. Kanamycin was chosen to evaluate the synergistic activity of the fraction, because the interaction curve seen in the above testing of S. aureus versus this antibiotic with HBV reflected synergy at all time points.

[0132] The Antibacterial Activity of Melittin

[0133] The results melittin versus whole HBV are shown in FIG. 14. No significant differences were observed in the antibacterial activity of whole HBV and the melittin fraction. For each time point represented in FIG. 14, the optical densities of the HBV curve and the melittin curve are statistically equal.

[0134] The Synergistic Activity of Melittin with Kanamycin

[0135] FIG. 15 compares the antibacterial activities of equivalent doses of the melittin fraction and whole HBV venom in combination with equal doses of kanamycin. None of the optical densities at any time point on the two curves are significantly different. Moreover, ignoring statistical evaluations, the interaction curve representing the melittin fraction is actually slightly lower at all time points than the interaction curve representing whole HBV. Thus, if the time points on both curves were accepted as the true means, the final conclusion would be that the melittin fraction is actually more active than whole HBV.

Interpretation of Checkerboard of Assay Results

[0136] The results of the checkerboard assays clearly demonstrate synergism between antibiotics and honeybee venom. FIG. 2 illustrated the effects of various doses of honeybee venom on S. aureus without antibiotics. It can be seen in this Figure that the addition of high doses of venom, such as 8 or 16 ug/ml, to the growing cultures actually lowered the optical density of the culture. This indication of cell lysis is evidence that honeybee venom is actually bactericidal. The mechanism of this bactericidal activity and its contribution to the synergy seen with antibiotics is not known. The varied results of the checkerboard titration assays suggest that several different synergistic mechanisms may be functioning in these experiments.

[0137] Questions may arise on the large standard deviations seen at some time points in the data tables. This variability in general is due to the sharp slope of the growth rate when the bacteria are in log phase. Time points taken in mid-log phase will have a much larger difference in optical density with time than will time points taken during a slower growth period. Thus, uncontrollable, small variations in sampling intervals could cause larger variations in optical density readings at time points during logarithmic growth. Since cultures are split during log phase into the various treatment groups, variations are even more noticeable between experiments. This type of error is taken into consideration, however, in the statistical evaluation procedure. By using a large sample number (15), estimation ranges for the means of the time points were made narrow enough to statistically evaluate the differences in these means.

[0138] Although melittin was only tested initially as the synergistic component of HBV in combination with one antibiotic with one bacterial strain, for the purpose of discussing possible mechanisms it has been assumed that melittin is the synergistic honeybee venom component in each of the bacterial-antibiotic-HBV combinations tested.

[0139] Apparent Increased Dosage

[0140] In most cases, honeybee venom seems to boost the initial effectiveness of the antibiotic, which is indicated by an increased ability to lower the bacterial growth rate immediately upon addition of the two compounds. This type of cooperativeness was most demonstrable with E. coli versus HBV and polymyxin B (FIG. 9). At the first time point after addition of the two compounds, synergism is apparent and it continues as the culture progresses through log phase. These results suggest that low, noneffective doses of antibiotics may be made effective with the addition of HBV.

[0141] The boosted dosage effect described above is the type of synergy seen in most of the experimental combinations that were tested. This type of effect could be explained by the action of melittin through several different mechanisms: (1) altering the solubility properties of the antibiotic molecules, (2) increasing the permeability of the bacterial membrane, and (3) increasing the effectiveness of the antibiotic molecules at their active sites.

[0142] Altered Solubility Properties of the Antibiotics

[0143] The melittin could increase antibiotic efficacy by allowing it to be more easily transported into the bacterial cell. The direct interaction of melittin with antibiotic molecules, making the molecules less polar or more hydrophobic might allow passive transport through the bacterial membranes. The amphiphatic nature and basicity of melittin makes it a likely candidate for such a function and adds to the plausibility of this mechanism. This type of mechanism would be simular to the facilitated diffusion of potassium ions with valinomycin.

[0144] Increased Membrane Permeability

[0145] The apparent dosage of an antibiotic could also be increased by reducing penetration barriers of the bacterium.

[0146] Although this role as a channel-forming peptide is easily supported, it cannot be the only function of melittin that is involved in the antibacterial synergy. Increased transport across membranes fails to explain why melittin alone is more effective on gram positive organisms which have less of a membrane barrier.

[0147] Increased Antibiotic Specific Activity

[0148] A third possible mechanism for synergistic interactions proposes the direct interaction of melittin and the antibiotic to make the antibiotic more effective once it reaches the active site. A more specific example is the possible interaction with kanamycin. Once kanamycin reaches the 30S ribosome, a melittin-kanamycin complex may have a greater affinity for the active site than unbound kanamycin (after all, melittin is a basic molecule, like nucleic acids), or the melittin-kanamycin complex may be more effective in sterically blocking transfer-RNA's from the ribosome due simply to the size of the complex.

[0149] Increased Active Life of Antibiotics

[0150] In several cases, it was difficult to detect an increase in effectiveness of the antibiotics with the addition of honeybee venom (melittin) until late in the growth period. In these cases it appeared that the melittin caused an increase in the duration of the antibiotic's effect. This effect was seen with the kanamycin resistant S. aureus treated with kanamycin/HBV. Shown in FIG. 9 is a relatively high dose of HBV, the reason being that no synergism was seen with lower doses. Thus, although it is difficult in FIG. 9 to rule out synergy at the early time points due to the effectiveness of the HBV alone, lower doses of HBV showed no synergy with kanamycin at these early time points. A synergistic effect is noted, however, at the 24 hour time point. Two explanations for this type of delayed effect are suggested: (1) elimination of resistant mutants or (2) extension of the antibiotic's half-life.

[0151] Decreased Probability for the Selection of Resistant Strains

[0152] If both the honeybee venom and antibiotic are present in a bacterial culture at bacteristatic doses, the probability that a resistant bacterium will survive the combined treatment is equal to the product of the probabilities that one would exist and survive either treatment. This would appear as a delayed synergistic effect, as it would take many generations for the mutants to multiply to a level detectable by increased OD readings. Mutant selection would be characterized as a sporadic occurrence of a drastically higher OD reading among replicate samples which would be reflected in the standard deviation of the treatment. For example, when the effects of HBV treatment alone on the kanamycin resistant S. aureus with kanamycin was evaluated, the mean OD of the 12 hour time point on the venom control curve was 0.65 with one standard deviation of 0.51 (Table A-8), indicating highly varied readings at this time point. Thus, it could be very possible that the synergistic effect seen here at the 24 hour time point is the result of suppression of HPV venom resistant mutants.

[0153] Increased Antibiotic Stability

[0154] Not to be excluded from possible mechanism is protection of the antibiotic from decomposition. A common technique in increasing antibiotic efficacy is to structurally alter the antibiotic to make it more stable in solution or resistant to enzymatic attack. These types of modifications account for many of the derivatives in the penicillin family of antibiotics. For example, penicillin V has a phenoxymethyl substitution which provides steric hinderance, protecting the antibiotic's beta-lactam ring from enzymatic attack (Volk, 1978c). Such substitutions may also prevent this end of the molecule from cyclization with the beta-lactam ring making the molecule more resistant to acid hydrolysis. These types of modifications would also produce a synergic effect demonstrable only at bacteristatic doses, since the antibiotic would not be any more effective initially and the prolonged life span of the antibiotic should be evident only if the bacterial culture had not reached a nutritionally limiting OD at that time. If, however, HBV could cause such a modification, more consistent results among replicate samples would be expected.

Evaluation of MIC Testing

[0155] The checkerboard titration assay which was developed for HBV/antibiotic synergy testing was too time-consuming for use in a broad survey of the effect of HBV on different antibiotics and on different bacteria. Such a survey was needed, however, in order to locate trends among antibiotic classes towards synergy with HBV, as well as to determine the spectrum of susceptibility among bacterial species to specific synergistic combinations of antibiotics and HBV. The modification of the automated MIC assays was designed to facilitate this type of a survey.

[0156] Due to the limitations of the automated MIC assays, the evaluation of the results are somewhat empirical. The results cannot be proven to be synergistic, as opposed to additive, interactions since the effect of HBV alone was recorded only as inhibitory or non-inhibitory. (Slightly inhibitory doses of HBV would have been recorded as non-inhibitory, thus some MIC decreases may actually be the result of an additive effect). In most assays, however, only certain antibiotics showed decreased MIC's, suggesting that the HBV dose was not additive. Therefore, when supported by the results of the checkerboard titration system, the use of these MIC assays should be reliable to point out the antibiotic/HBV combinations with the greatest potential for specific groups of bacteria. In this respect, the MIC's will be used to direct future research.

[0157] Identification of the Active Honeybee Venom Component

[0158] Although the results of these studies suggest that the synergistic activities of honeybee venom are entirely contained in the melittin fraction, careful interpretation should be made of these results. It is possible that small peptides or non-staining (Coomassie Blue) compounds comigrate with the melittin in the chromatography due to ionic or hydrophobic interactions with the melittin molecules. Melittin migrates as an aggregate of five times it's normal molecular weight both in native polyacrylamide gel electrophoresis and in Sephadex gel chromatography (Haberman, 1972). These small micelles could carry smaller hydrophobic compounds through the chromatography. Analyses to detect such types of contamination in the melittin fraction are involved and are discussed in Chapter 6.

[0159] As noted above, additional tests were conducted to demonstrate that HBV is also effective to enhance the activity of the fourth group of antibiotics referred to above, which is represented by rifampicin. The data are set forth below in Tables 6, 7 and FIGS. 16, 17.

[0160] The activity of hymenoptera venom other than HBV was also determined for bumblee venom, yellow jacket venom and bald faced hornet venom, as shown below in Tables 8, 9

[0161] Also some of then analogues mentioned above were tested to determine their relative activities with respect to native melittin. The relative activity is calculated as follows: 1$\frac{\mathrm{Dose}\mathrm{of}\mathrm{melittin}}{\begin{array}{c}\mathrm{Dose}\mathrm{of}\mathrm{analogue}\mathrm{needed}\mathrm{to}\mathrm{demonstrate}\\ \mathrm{equivalent}\mathrm{synergy}\mathrm{with}\mathrm{polymyxin}B\end{array}}\times 100$

[0162] The results obtained are set forth in Table 10.

[0163] It appears that analogues in which the NH2-terminal end mainly consists of basic amino acids are more active than analogues having an NH2-terminal end mainly consisting of neutral and/or acid amino acids.

IN VIVO EXPERIMENTS

[0164] Introduction

[0165] In vivo experiments demonstrate that melittin, the major peptide of honey bee venom, enhances the effectiveness of a proven 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, against an E. coli septicemia are compared in two basic sets of experiments. With both experimental protocols, a synergistic interaction between melittin and polymyxin B is evident and is verified statistically by a contrast of the treatment means in each analysis of variance. Thus, the ability of mellitin to enhance the effectiveness of polymyxin B and yield superior antibacterial activity in vivo is demonstrated clearly.

[0166] Numerous references cited above in the section entitled Background and Prior Art disclose the use of honey bee venom, or more specifically melittin, as an antimicrobial agent. However, these references demonstrate only in vitro effectiveness of honey bee venom or melittin.

[0167] Several other systems have used melittin as an artificial means of perturbing various immune responses in isolate in vitro systems. Goodman et al. (1984) reported B cell activation by melittin 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 phagocytes of both mice and guinea pigs. Lastly, one publication (Somerfield et al. 1986) relating the effect of honey bee venom on the immune system describes the inhibition of neutrophil O production by melittin. Somerfield et al. suggest a role for melittin as an anti-inflammatory agent. This activity would most likely weaken antibacterial defense in vivo.

[0168] Despite the substantial amount of research with melittin. It had not yet been demonstrated that melittin is effective in vivo against infectious organisms. More importantly, nowhere has the need for, or tire benefit of, the interaction of melittin with antibiotics been proposed. The results reported herein demonstrate beneficial interactions between melittin and polymyxin B when used in vivo to treat mice suffering a bacterial septicemia caused by E. coli.

[0169] Materials and Methods

[0170] Female Swiss CD-1 mice (Charles River) were obtained at weights of 18-20 grams. The mice were housed at 77 +/−1 degrees Fahrenheit and 30-45% relative humidity with a daily 12 hour photoperiod. Upon delivery, each shipment of mice was maintained for a two week acclimatization period before use in the experiment.

[0171] Polymyxin B (Sigma Chemical Company) was purchased in powder form with an activity of 7900 Units/mg. A stock solution was prepared at 0.1 mg/ml in 0.85% NaCl and frozen at −20 degrees Centigrade in 5 ml aliquots until use.

[0172] Honey bee venom (HBV) was provided by Vespa Laboratories, Inc., Spring Mills, Pa. The HBV was the source of melittin which was isolated using gel filtration as was described previously for in vitro experiments. Melittin was quantitated by the Lowry protein assay (Lowry et al. 1951) and then lyophilized. The lyophilized melittin was reconstituted in 0.85% NaCl to a concentration of 0.1 mg/ml and frozen at −20 degrees Centigrade in 1.5 ml aliquots until further use.

[0173] E. coli strain #G1108E was obtained from the Pennsylvania State University E. coli Reference Center, University Park, Pa. A 5.0 ml overnight trypticase soy broth culture was used to inoculate 800 ml of fresh trypticase sony broth. The culture was propagated overnight with mild shaking. Two hundred ml of sterile glycerol was added to the culture which was then aseptically dispensed, while stirring, into 5.0 ml aliquots. These aliquots were frozen and stored at −20 degrees Centigrade. Upon thawing, each aliquot yielded a (+/−3)×108 viable bacteria/ml. The culture was then diluted 1:400 with trypticase soy broth containing 2.5% gastric mucin (Sigma Chemical Company) prior to inoculation.

[0174] Mice were infected by intraperitoneal injection of 0.25 ml of the 1:400 dilution of bacteria (approximately 500,000 viable bacteria) suspended in trypticase soy broth with 2.5% mucin.

[0175] Prior to injection, Polymyxin B and melittin were thawed, filter sterilized, and diluted appropriately with sterile 0.85% NaCl such that the required dosage was contained in 0.2 ml of solution. Thirty minutes after infection, this volume was then delivered to the mice by subcutaneous injection into the skin fold at the base of the neck. The skin fold is formed between the thumb and forefinger in a basic restraining hold.

[0176] Bacterial levels in the blood were determined from blood samples obtained by aseptic heart puncture. After heart puncture, the needle was removed from the heparin coated syringe and 0.2 ml of blood was dispensed into a tube containing 0.2 ml of 0.85% NaCl and mixed well. All samples were kept on ice until plated. Duplicate spread plates of all samples, at appropriate dilutions, were prepared on trypticase soy agar and were incubated at 37 degrees Centigrade overnight. All plates containing less than 400 colonies were counted and recorded.

[0177] Results

[0178] In the first experimental design, 4 random groups of 4 mice each 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) 0.85% NaCl only (“no treatment”); 2) 2.0 ug polymyxin B; 3) 50 ng melittin; or 4) 2.0 ug polymyxin B +50 ng melittin. Twenty-one hours after the initial inoculation, blood samples were taken and the number of bacteria per milliliter of blood was calculated by averaging the results of duplicate plate counts of the appropriate blood dilution (Table A-11). This experiment was run in triplicate and the average number of bacteria per milliliter of blood for each of the four treatments was compared (FIG. 21). A p-value of 0.0015 for the treatment effect in a two-way analysis of variance (adjusted for unequal sample sizes) indicated a significant difference in at least one of the treatment means. 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 ug. polymyxin B and 50 ng melittin. By comparing the sum of the activities provided by melittin and polymyxin B used individually to their activity when used in combination, a contrast within the analysis of variance confirmed that the interaction was indeed synergistic (p-value=0.0493).

[0179] A second experimental design tested the effect of repeated treatments. Again four groups containing four mice each were inoculated with E. coli and treated thirty minutes later with the same four treatments: 1) 0.85% NaCl only (“no treatment”); 2) 2.0 ug polymyxin B; 3) 50 ng melittin; or 4) 2.0 ug polymyxin B+50 ng melittin. Eighteen hours after the initial infection, each mouse was challenged again with the same E. coli inoculum and thirty minutes later treated with the same antibiotic/melittin regime. Five hours later (23 hours after the initial infection), blood samples from each mouse were plated to quantitate the number of bacteria in the blood. This experiment was replicated 5 times and the results (Table A-12) were evaluated by analysis of variance. These analyses showed a significant difference (p-value=0.0001) in at least one treatment. Tukey's multiple mean comparison showed that repetitive polymyxin B treatments caused a significant decrease in the number of bacteria per milliliter blood. More importantly, Tukey's comparison showed that the bacterial counts in the blood of animals treated with polymyxin B plus melittin were significantly lower than the counts in the blood of animals treated only with polymyxin B or melittin (refer to FIG. 22). A contrast within the analysis of variance provided a high degree of confidence for the synergy of these two compounds (p-value=0.0007).

[0180] Conclusions

[0181] The above experiments clearly demonstrate synergistic interaction between the antibiotic polymyxin B and melittin. It is highly likely that melittin enhances the therapeutic effects of other pharmaceuticals due to its antimicrobial properties and ability to enhance membrane permeability.

[0182] The results of the first set of experiments (FIG. 21) lacked statistical significance for the effect of melittin alone, but comparisons of the absolute means suggested positive effects with the melittin treatment alone. The results of the second set of experiments (FIG. 22) again lacked a significant difference for the melittin only treatment. A comparison of the absolute treatment means suggested a negative effect of the melittin alone. Remembering that the mice in the second set of experiments received double doses of melittin, this suggests that higher doses of melittin, when used without antibiotics, may aggravate the infectious process. Previous experimentation with higher doses of melittin verifies this assumption. It is likely that detrimental activity occurs when melittin is used alone. Importantly, the effective use of melittin to treat infections was not found during a literature search. Antibiotics apparently counter the negative effects of melitin, thus making combined therapy a significant development.

Antibiotic Synergy

Demonstrated With A Synthetic Melittin Analogue

[0183] The synergy seen between antibiotics and melittin may also be achieved by replacing melittin with synthetic peptide analogues. Such an analogue was designed and synthesized for this purpose. When tested in parallel with natural melittin, it provided equivalent antibiotic enhancement.

[0184] Introduction

[0185] Analogue No. 6, the structure of which is shown below,

[0186] H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Lys-Lys-Lys-Gln-Gln-NH2

[0187] was tested in vitro as described previously for synergy with polymyxin B against E. coli. This peptide varies from melittin at amino acids 22 and 24 (underlined) where argenines have been replaced by lysines. When used in the aforementioned assay, it demonstrates activity equivalent to natural melittin.

[0188] Materials and Methods

[0189] Melittin was isolated from whole honeybee venom (Vespa Laboratories, Inc.) via gel filtration chromatography, quantitated by the Lowry protein assay, and stored lyophilized. For these assays, lyophilized melittin was reconstituted to 0.4 mg/ml with distilled water, filter sterilized, and stored in 4.0 ml aliquots at −20 degrees Centigrade until used.

[0190] Analogue No. 6 was synthesized by Dr. Torben Saermark (The Protein Laboratory, Copenhagen University, Sigurdsgade 34, DK-2200 Copenhagen N, Denmark). It was estimated to be better than 98% pure based on the high pressure liquid chromatograph elution profile from a C18 column using a 0-80% acetonitrile gradient in 0.1% trifluoroacetate. The peptide was received in lyophillized form and was reconstituted to approximately 0.2 mg/mil in 0.85% NaCl, filter sterilized, and stored in 0.5 ml aliquots at −20 degrees Centigrade until used.

[0191] 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 in 4.0 ml aliquots at −20 degrees Centigrade until used.

[0192] E. Coli strain No. G1108E was obtained from the Pennsylvania State University E. Coli Reference Center (105 Henning Building, University Park, Pa., 16802). Inoculums were prepared from a culture grown in trypticase soy broth to mid-log phase. Sterile glycerol was added to make a final concentration of 20% and the culture was dispensed and frozen in 5.0 ml aliquots at −20 degrees Centigrade until used.

[0193] A checkerboard titration synergy assay was performed, testing natural melittin and analogue #6 in parallel with polymyxin B against E. coli. Equivalent dosages of the natural melittin and analogue #6 were based on a Lowry protein assay performed simultaneously on aliquots of each after the final filtration. Both peptides were tested in the synergy assay at final concentrations in the medium of 5 ug/ml and 10 ug/ml. Polymyxin B was tested at final medium concentrations of 3 units/ml and 6 units/ml against both levels of both peptides.

[0194] Results

[0195] Synergy was best demonstrated with both the natural melittin and analogue #6 when tested at 10 ug/ml against 6 units/ml of polymyxin B ( Table 11). Under these conditions (refer to FIG. 23), the data for each peptide was statistically analyzed for synergistic activity at each time point. Using statistical contrasts, the mean activities of each peptide alone and polymyxin B alone were compared to the activity of the respective peptide-polymyxin B combination. Synergy was detected at the 4, 6, and 8 hour time points for both melittin and analogue #6 (p-values=0.0001).

[0196] Additionally, the synergy curves for melittin (10 ug melittin+6 units polymyxin B) and analogue #6 (10 ug analogue #6+6 units polymyxin B) were compared at each time point for different levels of activity. At no time could a significant difference be detected between these two curves.

[0197] Conclusions

[0198] The results show that analogue #6, a synthetic melittin analogue, has activity very similar to that of melittin with regard to its capacity to enhance the activity of polymyxin B. Although the twelve hour time point suggests that analogue #6 has slightly better activity with polymyxin B than does melittin, this difference in activity is minimal with respect to the actual quantitative difference in peptide which it would reflect. By comparing the difference in synergy produced by 10 ug melittin versus 10 ug analogue #6 to the difference in synergy produced by 10 ug melittin versus 5 ug melittin (Table 11), the difference between the specific activities of melittin versus analogue #6 can be estimated to be less than 10%.

[0199] This investigation shows that it is possible to synthesize melittin analogues with synergistic capabilities equivalent or superior to melittin.

Synergistic Antibacterial Activity of Melittin and Polymyxin B: Relative Activities of Melittin Analogues

[0200] The synergy seen between antibiotics and melittin may also be achieved (by replacing melittin with either synthetic analogues or chemically modified derivatives of the natural peptide. Synthetic melittin, five synthetic peptide analogues, and one chemical modification of natural melittin were tested for synergistic interaction with polymyxin B with respect to growth inhibition of E. coli. Their relative activities were compared to that of melittin from natural honey bee venom. Each peptide demonstrated synergistic interaction with polymyxin B; however, specific activities differed significantly. Several analogues provided synergistic activity superior to that of natural melittin.

Introduction

[0201] Two types of melittin analogues, synthetic peptides and a chemical modification of natural melittin, were assayed in vitro for synergy with polymyxin B In an antibacterial activity assay. The synthetic analogues include a group of synthetic peptides, all of which vary from the 26 amino acid sequence of melittin by two or more residues. The chemical modification of melittin, NPS-melittin, consists of the attachment of an o-nitrophenyl sulfenyl group to the number 19 tryptophan residue of natural melittin. The activity of each of these analogues was compared to the activities of both natural and synthetic melittin. While each of these analogues demonstrated some synergistic interaction with polymyxin B in vitro, significant differences in peptide activities were evident. These difference define key attributes of the melittin molecule in its role as potentiator of polymyxin B activity.

[0202] Materials and Methods

[0203] Natural melittin was isolated from whole honey bee venom (Vespa Laboratories, Inc.) via gel filtration chromatography,

[0204] quantitated by the Lowry protein assay and stored lyophilized. For these assays, lyophilized melittin was reconstituted to 0.34 mg/ml with distilled water, filter sterilized, and stored in 4.0 ml aliquots at −20 degrees centigrade until used.

[0205] NPS-melittin was synthesized from natural melittin by reacting the peptide with o-nitrophenyl sulfenyl chloride (NPS-Cl) as has been described for adrenocorticotropin by Ramachandran et al. The peptide was precipitated from solution with ethyl acetate, resuspended in 0.1 N acetic acid, and then passed through a Sephadex G-10 (LKB-Pharmacia, Piscataway, N.J. ) column to remove remaining NPS-Cl salts. A determination of the molar absorptivity of the derivative at 365 nm indicated that the melittin was better than 95% modified.

[0206] Synthetic melittin was purchased from Peninsula Laboratories, Belmont, Calif. A 0.5 mg sample was reconstituted to 0.3 mg/ml in 0.85% saline based on a Lowry protein assay. The sample was stored at −20 degrees centigrade until used.

[0207] Synthetic analogues were synthesized by Dr. Torben Seamark (The Protein Laboratory, Copenhagen University, Sigurdsgade 34, DK-2200 Copenhagen N, Denmark). Each analogue was assayed for purity and was estimated to be better than 98% pure based the chromatographic elution profile from a C-18 column using a 0-100% acetonitrile gradient. Each peptide was received in lyophilized form and was reconstituted to approximately 1.0 mg/ml in 0.85% NaCl, filter sterilized, and stored in 1.0 ml aliquots at −20 degrees centigrade until used. Each peptide solution was quantitated by the Lowry protein assay prior to use. Lowry results agreed well with concentration estimations based on peptide dry weights.

[0208] 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 sterliized, and stored in 4.0 in aliquots at −20 degrees centigrade until used.

[0209] E. coli G1108E was obtained from the Pennsylvania State University E. coli Reference Center (105 Henning Building, University Park, Pa. 16802). Inoculums were prepared from a culture grown in trypticase soy broth to mid-log 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 degrees centigrade until used.

[0210] A checkerboard titration synergy assay was performed, testing each peptide with polymyxin B against E. coli in parallel with melittin. Equivalent dosages of the natural melittin and each analogue were based on a Lowry protein assay performed on aliquots of each peptide after the final filtration of the stock solution. All peptides were tested in the synergy assay at final concentrations in the medium of 5 ug/ml. The concentration of polymyxin B in the media of all assays was 6 units/ml.

[0211] Results

[0212] Table 12 shows the amino acid sequences of the synthetic melittin analogues. For each synthetic analogue, the first twenty N-ternimal amino acids are the same as natural melittin. Alterations occur in the six C-terminal amino acids and are indicated by boldfaced print.

[0213] 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 the standard error of the mean from six samples. The “control” curve represents the growth of the culture with no polymyxin B or peptide added. The effect of each peptide alone on the culture is not included in the table; however, like melittin, these peptides have no effect on the growth of E. coli when used alone at 10 ug/ml or less. Thus, the “control” is also a representation of the culture when treated with each peptide alone.

[0214] When the growth curve of bacterial cultures treated with only polymyxin B (6 units/ml) is compared to the curve of cultures treated with polymyxin B plus melittin (5 ug/ml), increased antibacterial activity is demonstrated as an increase in the time required for the culture to overcome the treatment and achieve log-phase growth (FiG. 24). As treatment of the culture with melittin alone at 5 ug/ml would produce a growth curve which would essentially overlay the “control” curve, an increase in the time required for the culture to escape the polymyxin B inhibition and reach mid-log phase in the presence of the peptide is evidence of synergistic activity of the peptide. Thus, a shift of the growth curve representing polymyxin B with peptide to the right of the curve representing the polymyxin B only treatment is evidence of synergy.

[0215] The ratio of polymyxin B to melittin to bacteria in these experiments was designed to produce minimal synergy so that increased activity of peptide analogues would be evident. Such an increases are seen in FIG. 24 for both synthetic melittin and NPS-melittin. All peptides were at equal concentrations as determined by Lowry assay.

[0216] Similar growth curves comparing the synergistic activities of the synthetic melittin analogues with polymyxin B are shown in FIG. 25.

[0217] In order to more clearly visualize the differences in melittin/analogue synergy with polymyxin B, FIGS. 24 and 25 were used to calculate the additional time delay until each growth curve reached mid-log phase due to the addition of melittin or analogues when compared to the treatment with polymyxin B alone. These values are shown in a bar graph in FIG. 26. A Tukey's studentized range test was performed on the data in table 13 to compare the readings obtained at each time point between the various treatments. The peptides were then grouped depending on their ability to show significantly different levels of growth inhibition (alpha=0.05) for at least one of the growth curve time points. These groupings have been designted by different bar markings in FIG. 26.

[0218] Conclusions

[0219] These results show that a variety of melittin analogues can be created by both amino acid substitutions and chemical modifications. These types of modifications can either enhance or diminish the peptide's relative synergistic activity. Based on FIG. 26, the relative in vitro activities of melittin and its analogues can be stated in ascending order as follows:

[0220] 1 Analogue #7 A

[0221] 2 Natural Melittin B

[0222] 3 NPS-Melittin C

[0223] 4 Analogue #6 C

[0224] 5 Synthetic Melittin C

[0225] 6 Analogue #2 C

[0226] 7 Analogue #4 D

[0227] 8 Analogue #5 D

[0228] Peptides with significantly different (alpha=0.05) synergistic activities at the 5 ug/ml level are noted by letter groups.

[0229] It should be noted that the parameter used to establish this order of efficacy, delay time until mid-log phase of the culture, increases stoichiometrically with the amount of peptide only over a narrow range of melittin concentrations. Since the boundaries of the linear range for each analogue may not be equivalent, the data presented here can be used only to determine the relative order of efficacy of these peptides at the given concentration and can not be used to estimate quantitative differences. The order of efficacy does suggest, however, that synergistic activity of the peptide relies on the number and exposure of positively charged side chains on amino acids in the C-terminal region.

[0230] Although the relative efficacy of these melittin analogues has been established in vitro, substantial differences may occur in vivo. In vivo parameters such as absorption into and clearance from the host may significantly alter this order of efficacy in practical usage. Side effects must also be considered. While adrenocorticotropic activity of melittin is well documented the NPS-melittin may have less adrenal activity than natural melittin as the NPS-derivatlve of adrenocorticotropin (ACTH) induces 100 time less lipolytic activity than unmodified ACTH. For these reasons, each of the analogues included in this study should be considered for in vivo evalution.

TABLE 1
The concentations of stock solutions of
antibiotics and honeybee venom tested against
three different bacteria.
Organism	Venom	Ampicillin	Tobramycin	Polymixin B
E. coli	800 ug/ml	80 ug/ml	800 ug/ml	800 U/ml
S. aureus	320 ug/ml	8 ug/ml	200 ug/ml	50,000 U/ml
S. aureus-	320 ug/ml	8 ug/ml	800 ug/ml	2000 U/ml
kanaR

[0231]

TABLE 2
The design and distribution of honeybee venom and
antibiotics in a titration checkerboard titration
assay.
	Antibiotic Dilutions
	Control	1:16	1:8	1:4	1:2
Honeybee venom
Dilutions
Control	0a\0b	0\1:16	0\1:8	0\1:4	0\1:2
	1-3c	4-6	7-9	10-12	13-15
1:16	1:16\0	1:16\1:16	1:16\1:8	1:16\1:4	1:16\1:2
	16-18	19-21	22-24	25-27	28-30
1:8	1:6\0	1:8\1:16	1:8\1:8	1:8\1:4	1:8\1:2
	31-33	34-36	37-39	40-42	43-45
1:4	1:4\0	1:4\1:16	1:4\1:8	1:4\1:4	1:4\1:2
	46-48	49-51	52-54	55-57	58-60
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 = numerator, the dilution level of the stock HBV solution
b = denominator, the dilution level of the stock antibiotic solution
c = assay position in a sequential arrangement of 5 test tubes

[0232]

TABLE 3
The volumes and distributions of each component
of the checkerboard titration assay.
Tube #	TSB	Antibiotic	Venom	Bacteria
00-0	2.5 ml	—	—	—
1-3	500 ul	—	—	2.0 ml
4-6	250 ul	—	250 ul 1:16	2.0 ml
7-9	250 ul	—	250 ul 1:8	2.0 ml
10-12	250 ul	—	250 ul 1:4	2.0 ml
13-15	250 ul	—	250 ul 1:2	2.0 ml
16-18	250 ul	250 ul 1:16	—	2.0 ml
19-21	—	250 ul 1:16	250 ul 1:16	2.0 ml
22-24	—	250 ul 1:16	250 ul 1:8	2.0 ml
25-27	—	250 ul 1:16	250 ul 1:4	2.0 ml
28-30	—	250 ul 1:16	250 ul 1:2	2.0 ml
31-33	250 ul	250 ul 1:8	—	2.0 ml
34-36	—	250 ul 1:8	250 ul 1:16	2.0 ml
37-39	—	250 ul 1:8	250 ul 1:8	2.0 ml
40-42	—	250 ul 1:8	250 ul 1:4	2.0 ml
43-45	—	250 ul 1:8	250 ul 1:2	2.0 ml
46-48	250 ul	250 ul 1:4	—	2.0 ml
49-51	—	250 ul 1:4	250 ul 1:16	2.0 ml
52-54	—	250 ul 1:4	250 ul 1:8	2.0 ml
55-57	—	250 ul 1:4	250 ul 1:4	2.0 ml
58-60	—	250 ul 1:4	250 ul 1:2	2.0 ml
61-63	250 ul	250 ul 1:2	—	2.0 ml
64-66	—	250 ul 1:2	250 ul 1:16	2.0 ml
67-69	—	250 ul 1:2	250 ul 1:8	2.0 ml
70-72	—	250 ul 1:2	250 ul 1:4	2.0 ml
73-75	—	250 ul 1:2	250 ul 1:2	2.0 ml

[0233]

TABLE 4
The effect of 4 ug/ml HBV on the IOC's of eleven
antibiotic on eight gram-positive organisms.
	A1	B2
		5	3
		1	9	5	5	7	7	C3
		0	8	9	9	9	9	8
	0	7	1	0	0	0	0	1
	C4	2	7	5	7	5	8	-
Penicillin	−5	+6	+	−	−	+	+	−
Methicillin	+	+	+	−	−	+	+	−
Ampicillin	+	+	+	−	−	+	+	−
Cephalothin	+	+	+	+	+	+	+	−
Gentamicin	+	+	+	−	−	−	+	−
Kanamycin	+	+	−	−	−	+	+	−
Erythromycin	+	+	−	−	−	−	+	−
Chloramphenicol	+	+	−	−	−	−	+	+
Clindamycin	+	+	−	−	−	−	+	−
Tetracycline	+	+	−	−	−	−	+	−
Venomycin	+	+	−	−	−	−	+	−
1 Group “A” = two strains od S. aureus
2 Group “B” = five strains of S. epidermidis
3 “C” = a strain of Streptococcus faecalis
4 QC = a S. aureus strain used for routine quality control testing of this assay system.
5 A (−) indicates a MIC decrease of less than two dilution steps.
6 A (+) indicates a MIC decrease greater than or equal to two dilution steps.

[0234]

TABLE 5
The effect of 4 ug/ml HBV on the MIC's of eleven
antibiotics on four strains of E. coli
	E. coli strain
	QC1	1173	4302	19033
	Ampicillin	+2	+	+	+
	Carbenicillin	+	+	+	+
	Piperacillin	+	+	+	+
	Cephalothin	−3	−	−	−
	Cefoxitin	+	+	+	+
	Cefamandole	−	−	−	−
	Moxalactam	−	+	−	−
	Amikacin	+	+	+	+
	Gentimicin	+	−	+	+
	Chloramphenicol	−	−	−	+
	Tobramycin	−	−	−	−
	1 QC is a strain of E. coli used for routine quality control testing of this assay system.
	2 A (+) indicates a MIC decrease greater than or equal to two dilution steps.
	3 A (−) indicates a MIC decrease of less than two dilution steps.

[0235]

TABLE 6
Staphylococcus aureus
Rifampin = .01 ug/ml or .001 ug/ml
Honey Bee Venom = 4 ug/ml
	hours after innoculation
	0	2	4	6	8	12
Control	.046	.080	.850	1.17	1.26	1.34
	.046	.073	.815	1.16	1.26	1.32
	.046	.073	.815	1.16	1.26	1.35
AVERAGE	.046	.075	.827	1.16	1.26	1.34
Rifampicin	.046	.056	.140	.372	1.07	1.32
.01 ug/ml	.046	.054	.068	.156	.625	1.34
	.046	.058	.112	.304	1.00	1.30
AVERAGE	.046	.056	.107	.277	.898	1.32
Rifampicin	.046	.081	.855	1.18	1.27	1.34
.001 ug/ml	.046	.081	.765	1.16	1.26	1.34
	.046	.072	.800	1.17	1.26	1.34
AVERAGE	.046	.072	.807	1.17	1.26	1.34
Venom	.046	.062	.158	.705	1.20	1.29
4 ug/ml	.046	.063	.284	.075	1.22	1.31
	.046	.059	.068	.312	1.09	1.29
AVERAGE	.046	.061	.170	.631	1.17	1.30
Rifampicin	.046	.053	.078	.156	.665	1.33
.01 ug/ml +	.046	.055	.078	.162	.640	1.32
Venom 4 ug/ml	.046	.056	.062	.092	.332	1.32
AVERAGE	.046	.055	.073	.137	.546	1.32
Rifampicin	.046	.066	.068	.242	1.08	1.32
.001 ug/ml +	.046	.063	.109	.485	1.19	1.34
Venom 4 ug/ml	.046	.067	.087	.381	1.16	1.33
AVERAGE	.046	.065	.088	.369	1.14	1.33

[0236]

TABLE 7
Pseudomonas aeruginosa
Rifampicin = 10 ug/ml or 20 ug/ml
Honey Bee Venom = 40 ug/ml
	hours after innoculation
	0	2	4	6	8	12
Control	.033	.062	.735	1.00	1.02	1.00
	.033	.069	.755	.955	1.00	.990
	.033	.068	.775	.950	.990	.900
AVERAGE	.033	.066	.755	.968	1.00	.963
Venom	.033	.078	.690	.890	.960	.980
40 ug/ml	.033	.087	.687	.870	.960	.980
	.033	.058	.685	.880	.953	.950
AVERAGE	.033	.074	.685	.880	.953	.970
Rifampicin	.033	.074	.630	.830	.885	.842
10 ug/ml	.033	.084	.672	.850	.895	.850
	.033	.082	.640	.830	.865	.832
AVERAGE	.033	.080	.647	.837	.882	.841
Rifampicin	.033	.053	.375	.660	.730	.730
20 ug/ml	.033	.056	.326	.645	.720	.730
	.033	.063	.380	.700	.760	.745
AVERAGE	.033	.057	.351	.688	.737	.735
Rifampicin	.033	.084	.452	.805	.860	.861
10 ug/ml + n	.033	.079	.475	.795	.820	.839
Venom 40 ug/ml	.033	.078	.490	.820	.860	.880
AVERAGE	.033	.080	.466	.807	.847	.860
Rifampicin	.033	.065	.180	.410	.580	.620
20 ug/ml +	.033	.082	.168	.375	.535	.620
Venom 40 ug/nl	.033	.058	.168	.373	.525	.612
AVERAGE	.033	.068	.172	.386	.547	.617

[0237]

TABLE 8
Escherichia coli
Polymyxin B = 6.25 Units/ml and 3.125 Units/ml
Bumble Bee Venom = 5 ug/ml and 20 ug/ml
(Megabombus pennsylvanious)
	hours after innoculation
	0	2	4	6	8	12
Control	.030	.688	1.04	1.05	1.14	1.23
	.030	.680	1.03	1.04	1.13	1.22
	.030	.683	1.02	1.04	1.13	1.22
AVERAGE	.030	.684	1.03	1.04	1.13	1.22
BB Venom	.030	.715	1.03	1.02	1.04	1.12
5 ug/ml	.030	.712	1.03	1.04	1.04	1.14
	.030	.730	1.03	1.03	1.04	1.13
AVERAGE	.030	.719	1.03	1.03	1.04	1.13
BB Venom	.030	.672	1.03	1.03	1.04	1.13
20 ug/ml	.030	.673	1.04	1.03	1.05	1.16
	.030	.688	1.04	1.03	1.06	1.13
AVERAGE	.030	.678	1.04	1.03	1.05	1.14
Pol B	.030	.654	1.03	1.03	1.04	1.12
3.125 Units/ml	.030	.642	1.02	1.03	1.04	1.14
	.030	.652	1.02	1.03	1.04	1.14
AVERAGE	.030	.649	1.02	1.03	1.04	1.13
Pol B	.030	.022	.102	.710	.960	1.03
6.25 Units/ml	.030	.024	.472	.940	.950	1.03
	.030	.022	.180	.830	.970	1.04
AVERAGE	.030	.023	.251	.827	.960	1.03
Pol B =	.030	.008	.168	.820	1.00	1.06
3.125 Units/ml	.030	.008	.250	.910	1.02	1.06
BBV = 5 ug/ml	.030	.009	.333	.950	1.02	1.07
AVERAGE	.030	.008	.250	.893	1.01	1.06
Pol B =	.030	.008	.012	.008	.009	.013
6.25 Units/ml	.030	.009	.009	.008	.008	.012
BBV = 20 ug/ml	.030	.011	.009	.008	.008	.013
AVERAGE	.030	.009	.010	.008	.008	.013

[0238]

TABLE 9
Escherichia coli
Polymyxin B = 3.12 Units/ml
Yellowjacket Venom =5 ug/ml
(Vespula germanica)
Baldfaced Hornet Venom = 5 ug/ml
(Dolichovespula maculata)
	hours after innoculation
	0	2	4	6	8	12
Control	.038	.526	1.03	1.07	1.08	1.12
	.038	.522	1.04	1.07	1.08	1.12
AVERAGE	.038	.524	1.04	1.07	1.08	1.12
Pol B	.038	.477	1.03	1.07	1.08	1.14
3.125 U/ml	.038	.482	1.03	1.07	1.08	1.14
AVERAGE	.038	.480	1.03	1.07	1.08	1.14
YJ	.038	.547	1.04	1.07	1.09	1.16
5 ug/ml	.038	.550	1.04	1.07	1.08	1.14
AVERAGE	.038	.549	1.04	1.07	1.09	1.15
BF	.038	.552	1.04	1.08	1.08	1.16
5 ug/ml	.038	.565	1.04	1.07	1.09	1.15
AVERAGE	.038	.559	1.04	1.08	1.09	1.16
YJ 5ug/ml	.038	.028	.183	.945	1.08	1.14
Pol B 5 U/ml	.038	.029	.098	.850	1.06	1.12
AVERAGE	.038	.029	.141	.893	1.07	1.13
BF 5 ug/ml	.038	.027	.118	.890	1.08	1.13
Pol B 5 U/ml	.038	.023	.096	.840	1.06	1.10
AVERAGE	.038	.025	.107	.865	1.07	1.12

[0239]

TABLE 10
Relative activity of analogues of proteinaceous or
polypeptide components of Hymenoptera venoms.
Analogue No. Relative Activity
1            20%
2            200%
5            300%
6            100%
7            20%

[0240]

TABLE 11
Average optical densities (OD660) of bacterial
cultures versus time and treatment
	0 hrs	2 hrs	4 hrs	6 hrs	8 hrs	12 hrs
Control	.013	.072	.831	1.08	1.10	1.17
Melittin −	.013	.072	.0827	1.09	1.10	1.19
10 ug
Melittin −	.013	.072	.833	1.10	1.11	1.18
5 ug
Analogue #6 −	.013	.072	.824	1.09	1.11	1.17
10 ug
Analogue #6 −	.013	.072	.831	1.07	1.09	1.16
5 ug
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
Mel − 10 ug
Poly B − 6 U +	.013	.072	.031	.229	.550	.977
Mel − 5 ug
Poly B − 6 U +	.013	.072	.018	.018	.019	.844
Ana#6 − 10 ug
Poly B − 6 U +	.013	.072	.018	.029	.224	.926
Ana#6 − 5 ug
Poly B − 3 U +	.013	.072	.359	1.03	1.07	1.14
Mel − 10 ug
Poly B − 3 U +	.013	.072	.544	1.08	1.10	1.18
Mel − 5 ug
Poly B − 3 U +	.013	.072	.206	.911	1.07	1.12
Ana#6 − 10 ug
Poly B − 3 U +	.013	.072	.460	1.06	1.09	1.16
Ana#6 − 5 ug

[0241]

TABLE 12
Sequences of synthetic melittin analogues*.
Natural Melittin
Melittin (1-20)-Lys-Arg-Lys-Arg-Gln-Gln-NH2.
Anologue #2
Melittin (1-20)-Orn-Orn-Orn-Orn-Gln-Gln-NH2.
Analogue #4
Melittin (1-20)-Lys-Arg-Lys-Arg-Gly-Gly-NH2.
Analogue #5
Melittin (1-20)-Arg-Arg-Arg-Arg-Gln-Gln-NH2.
Analogue #6
Melittin (1∝20)-Lys-Lys-Lys-Lys-Gln-Gln-NH2.
Analogue #7
Melittin (1-20)-Gly-Gly-Gly-Gly-Gln-Gln-NH2.
*Amino acids shown In bold print represent alterations from the native melittin sequence.

[0242]

TABLE 13
Optical densities (OD660) of bacterial
cultures for treatments versus time. Values represent the
mean over the SEM in parenthesis (n = 6).
	Hours after culture inoculation
	0	2	4	6	8	10	12
Control	.007	.088	.692	1.01	1.05	1.06	1.08
	(.001)	(.003)	(.058)	(.019)	(.010)	(.008)	(.008)
Polymyxin B	.007	.088	.470	.926	1.02	1.04	1.06
	(.001)	(.003)	(.072)	(.014)	(.027)	(.036)	(.044)
Melittin +	.007	.088	.071	.514	.925	1.02	1.05
Pol B	(.001)	(.003)	(.017)	(.116)	(.077)	(.025)	(.022)
Synthetic +	.007	.088	.018	.143	.662	.881	1.01
Pol B	(.001)	(.003)	(.004)	(.057)	(.161)	(.128)	(.043)
NPS-Mel +	.007	.088	.042	.247	.766	1.04	1.08
Pol B	(.001)	(.003)	(.010)	(.108)	(.194)	(.069)	(.053)
Analog#2 +	.007	.088	.022	.208	.509	.942	1.03
Pol B	(.001)	(.003)	(.009)	(.116)	(.146)	(.021)	(.022)
Analog#4 +	.007	.088	.020	.023	.094	.533	.860
Pol B	(.001)	(.003)	(.004)	(.001)	(.034)	(.157)	(.125)
Analog#5 +	.007	.088	.036	.036	.038	.334	.687
Pol B	(.001)	(.003)	(.001)	(.001)	(.004)	(.096)	(.150)
Analog#6 +	.007	.088	.020	.160	.793	1.00	1.04
Pol B	(.001)	(.003)	(.007)	(.062)	(.071)	(.025)	(.023)
Analog#7 +	.007	.088	.146	.818	1.02	1.05	1.06
Pol B	(.001)	(.003)	(.028)	(.041)	(.021)	(.028)	(.028)

[0243]

TABLE A-1
The checkerboard assay results of ampicillin and
honeybee venom verus S. aureus.
TIME	MEAN A66O	S.D.	TIME	MEAN A660	S.D.
AMP = 0, HBV = 0	AMP = 0, HBV = 2
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	0.573	0.178	T4	0.213	0.135
T6	1.102	0.159	T6	0.844	0.311
T8	1.223	0.101	T8	1.119	0.193
T12	1.213	0.307	T12	1.198	0.306
T24	1.329	0.069	T24	1.295	0.208
AMP = 0, HBV = 4	AMP = 0, HBV = 8
T0	0.013	0.002	T0	0.013	0.002
T2	0.086	0.018	T2	0.085	0.018
T4	0.065	0.040	T4	0.026	0.019
T6	0.217	0.181	T6	0.014	0.012
T8	0.671	0.412	T8	0.027	0.036
T12	1.147	0.317	T12	1.028	0.273
T24	1.278	0.165	T24	1.291	0.119
AMP = 0, HBV = 16	AMP = 0.05, HBV = 0
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	0.025	0.011	T4	0.355	0.073
T6	0.007	0.004	T6	0.552	0.195
T8	0.006	0.004	TB	0.689	0.146
T12	0.077	0.173	T12	0.736	0.135
T24	0.857	0.576	T24	0.760	0.114
AMP = 0.05, HBV = 2	AMP = 0.05, HBV = 4
T0	0.013	0.003	T0	0.013	0.002
T2	0.085	0.004	T2	0.083	0.017
T4	0.142	0.039	T4	0.045	0.025
T6	0.260	0.142	T6	0.041	0.033
TB	0.296	0.196	TB	0.035	0.032
T12	1.372	0.093	T12	0.131	0.307
T24	1.647	0.063	T24	0.840	0.251
AMP = 0.05, HBV = 8	AMP = 0.05, HBV = 16
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	0.026	0.021	T4	0.025	0.009
T6	0.012	0.012	T6	0.006	0.004
T8	0.008	0.007	TB	0.007	0.004
T12	0.009	0.004	T12	0.008	0.005
T24	0.331	0.395	T24	0.013	0.004
AMP = 0.1, HBV = 0	AMP = 0.1, HBV = 2
T0	0.013	0.002	T0	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
AMP = 0.1, HBV = 4	AMP = 0.1, HBV = 8
T0	0.013	0.002	T0	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	T6	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
AMP = 0.1, HBV = 16	AMP = 0.2, HBV = 0
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	0.026	0.013	T4	0.202	0.038
T6	0.007	0.005	T6	0.112	0.026
T8	0.006	0.004	T8	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	AMP = 0.2, HBV = 4
T0	0.013	0.002	T0	0.013	0.002
T2	0.086	0.018	T2	0.085	0.018
T4	0.103	0.065	T4	0.045	0.024
T6	0.079	0.050	T6	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
T0	0.013	0.002	T0	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	T6	0.009	0.008
TB	0.007	0.007	T8	0.006	0.003
T12	0.008	0.002	T12	0.009	0.006
T24	0.009	0.005	724	0.011	0.003
AMP = 0.4, HBV = 0	AMP = 0.4, HBV = 2
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	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
T0	0.013	0.002	T0	0.013	0.002
T2	0.085	0.018	T2	0.085	0.018
T4	0.040	0.028	T4	0.023	0.015
T6	0.028	0.023	T6	0.010	0.006
T8	0.019	0.017	T8	0.006	0.004
T12	0.012	0.004	T12	0.008	0.005
T24	0.009	0.007	T24	0.010	0.006
AMP = 0.4, HBV = 16
T0	0.013	0.002
T2	0.085	0.018
T4	0.027	0.013
T6	0.008	0.004
T8	0.006	0.004
T12	0.008	0.005
T24	0.010	0.004

[0244]

TABLE A-2
The checkerboard assay results of kanamycin and
honeybee venom verus S. aureus.
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
KANA = 0, HBV = 0	KANA = 0, HBV = 2
T0	0.024	0.005	T0	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
KANA = 0, HBV = 4	KANA = 0, HBV = 8
T0	0.024	0.005	T0	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 = 0
T0	0.024	0.005	T0	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 = 2	KANA = 0, HBV = 2
T0	0.024	0.005	T0	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	0.310	0.289
T8	1.174	0.116	T8	0.694	0.422
T12	1.290	0.048	T12	1.231	0.107
T24	1.373	0.035	T24	1.350	0.077
KANA = 1.25, HBV = 8	KANA = 1.25, HBV = 16
T0	0.024	0.005	T0	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
KANA = 2.5, HBV = 0	KANA = 2.5, HBV = 2
T0	0.024	0.005	T0	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 = 0, HBV = 8
T0	0.024	0.005	T0	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
T0	0.024	0.005	T0	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	T6	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
T0	0.024	0.005	T0	0.024	0.005
T2	0.094	0.012	T2	0.094	0.012
T4	0.265	0.152	T4	0.065	0.026
T6	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
T0	0.024	0.005	T0	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	T6	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
KANA = 10, HBV = 0	KANA = 10, HBV = 2
T0	0.024	0.005	T0	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
T0	0.024	0.005	T0	0.024	0.005
T2	0.094	0.012	T2	0.094	0.012
T4	0.064	0.023	T4	0.041	0.023
T6	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
KANA = 10, HBV = 16
T0	0.024	0.005
T0	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

[0245]

TABLE A-3
The checkerboard assay results of polymyxin B and
honeybee venom verus E. coli
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
POLY B = 0, HBV = 0	POLY B = 0, HBV = 2
T0	0.006	0.002	T0	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 B = 0, HBV = 4	POLY B = 0, HBV = 8
T0	0.006	0.002	T0	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 = 2
T0	0.006	0.002	T0	0.006	0.022
T2	0.074	0.004	T2	0.074	0.004
T4	0.015	0.007	T4	0.526	0.138
T6	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
T0	0.006	0.002	T0	0.006	0.002
T2	0.074	0.004	T2	0.074	0.004
T4	0.167	0.071	T4	0.023	0.013
T6	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	POLY B = 312, HBV = 16
T0	0.006	0.002	T0	0.006	0.002
T2	0.074	0.004	T2	0.074	0.004
T4	0.014	0.005	T4	0.023	0.008
T6	0.007	0.005	T6	0.013	0.004
T8	0.011	0.005	T8	0.013	0.004
T12	0.384	0.383	T12	0.031	0.048
T24	1.294	0.393	T24	0.334	0.579
POLY B = 625, HBV = 0	POLY B = 625, HBV = 2
T0	0.006	0.002	T0	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
T0	0.006	0.002	T0	0.006	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
T8	0.073	0.128	T8	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	POLY B = 1250, HBV = 0
T0	0.006	0.002	T0	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
T0	0.006	0.002	T0	0.006	0.002
T2	0.074	0.004	T2	0.074	0.004
T4	0.110	0.043	T4	0.038	0.012
T6	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
T0	0.006	0.002	T0	0.006	0.002
T2	0.074	0.004	T2	0.074	0.004
T4	0.028	0.010	T4	0.071	0.014
T6	0.019	0.007	T6	0.054	0.012
T8	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
POLY B = 2500, HBV = 0	POLY B = 2500, HBV = 2
T0	0.006	0.002	T0	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 = 0, HBV = 8
T0	0.006	0.002	T0	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
T0	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

[0246]

TABLE A-1
The checkerboard assay results of ampicillin and
honeybee venom virus E. coli
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
AMP = 0, HBV = 0	AMP = 0, HBV = 5
T0	0.015	0.015	T0	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 = 10
T0	0.015	0.015	T0	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
T8	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
T0	0.015	0.015	T0	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
T8	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
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.603	0.099	T4	0.624	0.111
T6	0.998	0.095	T6	1.001	0.097
T8	1.011	0.097	T8	1.013	0.100
T12	K099	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 = 40
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.614	0.148	T4	0.508	0.205
T6	0.980	0.094	T6	0.961	0.097
T8	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
AMP = 1, HBV = 0	AMP = 1, HBV = 5
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.538	0.094	T4	0.545	0.095
T6	0.628	0.175	T6	0.621	0.126
T8	0.493	0.157	T8	0.470	0.147
T12	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 = 20
T0	0.015	0.016	T0	0.015	0.015
T2	0.083	0.033	T2	0.084	0.032
T4	0.561	0.116	T4	0.543	0.122
T6	0.506	0.077	T6	0.513	0.080
T8	0.453	0.120	T8	0.432	0.132
T12	0.396	0.106	T12	0.367	0.104
T24	0.414	0.028	T24	0.395	0.047
AMP = 1, HBV = 40	AMP = 2, HBV = 0
T0	0.016	0.015	T0	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
T8	0.435	0.191	T8	0.133	0.055
T12	0.385	0.163	T12	0.136	0.090
T24	0.484	0.082	T24	0.647	0.194
AMP = 2, HBV = 5	AMP = 2, HBV = 10
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.440	0.130	T4	0.432	0.122
T6	0.134	0.052	T6	0.127	0.052
T8	0.148	0.073	T8	0.133	0.070
T12	0.192	0.147	T12	0.182	0.137
T24	0.685	0.175	T24	0.654	0.253
AMP = 2, HBV = 20	AMP = 2, HBV = 40
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.406	0.151	T4	0.300	0.173
T6	0.114	0.054	T6	0.086	0.058
T8	0.123	0.073	T8	0.096	0.071
T12	0.209	0.193	T12	0.098	0.055
T24	0.687	0.205	T24	0.618	0.241
AMP = 4, HBV = 0	AMP = 4, HBV = 5
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.158	0.118	T4	0.154	0.108
T6	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
AMP = 4, HBV = 10	AMP = 4, HBV = 20
T0	0.015	0.015	T0	0.015	0.015
T2	0.084	0.032	T2	0.084	0.032
T4	0.128	0.092	T4	0.090	0.070
T6	0.075	0.039	T6	0.066	0.043
T8	0.074	0.045	T8	0.066	0.045
T12	0.066	0.034	T12	o.oso	0.031
T24	0.063	0.032	T24	0.052	0.026
AMP = 4, HBV = 40
T0	0.015	0.015
T2	0.084	0.032
T4	0.062	0.040
T6	0.055	0.040
T8	0.054	0.039
T12	0.051	0.028
T24	0.042	0.022

[0247]

TABLE A-5
The checkerboard assay results of kanamycin and
honeybee venom verus E. coli.
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
KANA = 0, HBV = 0	KANA = 0, HBV = 5
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.118	0.028
T4	0.701	0.136	T4	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	1.104	0.084
T24	1.144	0.119	T24	1.191	0.101
KANA = 0, HBV = 10	KANA = 0, HBV = 20
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.747	0.108	T4	0.764	0.087
T6	1.005	0.073	T6	1.001	0.060
T8	1.028	0.065	T8	1.026	0.063
T12	1.099	0.094	T12	1.094	0.090
T24	1.188	0.114	T24	1.198	0.102
KANA = 0, HBV = 40	KANA = 5, HBV = 0
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.124	0.033
T4	0.736	0.075	T4	0.473	0.120
T6	0.984	0.064	T6	0.800	0.132
T8	1.005	0.062	T8	0.889	0.081
T12	1.080	0.080	T12	0.930	0.091
T24	1.163	0.103	T24	1.019	0.119
KANA = 5, HBV = 5	KANA = 5, HBV = 10
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.484	0.128	T4	0.480	0.146
T6	0.827	0.129	T6	0.805	0.141
T8	0.908	0.080	T8	0.893	0.093
T12	0.955	0.101	T12	0.939	0.108
T24	1.050	0.122	T24	1.044	0.127
KANA = 5, HBV = 20	KANA = 5, HBV = 40
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.493	0.169	T4	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	0.942	0.116	T12	0.950	0.107
T24	1.046	0.126	T24	1.041	0.118
KANA = 10, HBV = 0	KANA = 10, HBV = 5
T0	0.025	0.009	T0	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
KANA = 10, HBV = 10	KANA = 10, HBV = 20
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.258	0.142	T4	0.257	0.153
T6	0.364	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
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.258	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
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.156	0.072	T4	0.144	0.075
T6	0.133	0.083	T6	0.095	0.069
T8	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
KANA = 20, HBV = 20	KANA = 20, HBV = 40
T0	0.025	0.009	T0	0.025	0.009
T2	0.119	0.028	T2	0.119	0.028
T4	0.128	0.081	T4	0.103	0.074
T6	0.078	0.065	T6	0.063	0.051
TB	0.151	0.128	T8	0.063	0.048
T12	0.174	0.061	T12	0.179	0.083
T24	0.692	0.113	T24	0.716	0.087
KANA = 40, HBV = 0	KANA = 40, HBV = 5
T0	0.025	0.009	T0	0.024	0.009
T2	0.119	0.028	T2	0.117	0.029
T4	0.136	0.049	T4	0.128	0.062
T6	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
KANA = 40, HBV = 10	KANA = 40, HBV = 20
T0	0.025	0.009	T0	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
TB	0.045	0.026	TB	0.038	0.016
T12	0.042	0.026	T12	0.039	0.017
T24	0.416	0.310	T24	0.404	0.318
KANA = 40, HBV = 40
T0	0.025	0.009
T2	0.119	0.028
T4	0.080	0.054
T6	0.041	0.020
T8	0.036	0.013
T12	0.040	0.019
T24	0.342	0.344

[0248]

TABLE A-6
The checkerboard assay results of polymyxin B
and honeybee venom verus E. coli.
TIME	MEAN A660	S.D.	TIME	MEAN A660	S. D.
POLY B = 0, HBV = 0	POLY B = 0, HBV = 5
T0	0.012	0.005	T0	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
T0	0.012	0.005	T0	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 = 40	POLY B = 1.5, HBV = 0
T0	0.012	0.005	T0	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 = 1.5, HBV = 10
T0	0.012	0.003	T0	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 = 40
T0	0.012	0.005	T0	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
POLY B = 3, HBV = 0	POLY B = 3, HBV = 5
T0	0.012	0.005	T0	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
T0	0.012	0.005	T0	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
T0	0.012	0.005	T0	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
T0	0.012	0.005	T0	0.012	0.005
T2	0.040	0.004	T2	0.040	0.004
T4	0.024	0.006	T4	0.029	0.006
T6	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 = 0, HBV = 40
T0	0.012	0.005	T0	0.012	0.005
T2	0.040	0.004	T2	0.040	0.009
T4	0.030	0.007	T4	0.041	0.008
T6	0.016	0.004	T6	0.019	0.006
T8	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
POLY B = 12, HBV = 0	POLY B = 12, HBV = 5
T0	0.012	0.005	T0	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
T0	0.012	0.005	T0	0.012	0.005
T2	0.040	0.004	T2	0.040	0.004
T4	0.029	0.004	T4	0.030	0.006
T6	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
T0	0.012	0.005
T2	0.040	0.004
T4	0.048	0.007
T6	0.022	0.006
T8	0.016	0.005
T12	0.015	0.006
T24	0.047	0.085

[0249]

TABLE A-7
The checkerboard assay results of ampicillin
and honeybee venom verus kanamycin resistant
S. aureus.
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
AMP = 0, HBV = 0	AMP = 0, HBV = 2
T0	0.020	0.016	T0	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 = 0, HBV = 4	AMP = 0, HBV = 8
T0	0.020	0.016	T0	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 = 0, HBV = 16	AMP = 0.05, HBV = 0
T0	0.020	0.016	T0	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 = 0.05, HBV = 2	AMP = 0.05, HBV = 4
T0	0.020	0.016	T0	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 = 0.05, HBV = 8	AMP = 0.05, HBV = 16
T0	0.020	0.016	T0	0.020	0.016
T2	0.064	0.020	T2	0.064	0.020
T4	0.025	0.013	T4	0.035	0.016
T6	0.013	0.009	T6	0.013	0.004
T8	0.008	0.005	T8	0.008	0.004
T12	0.010	0.007	T12	0.008	0.004
T24	0.150	0.295	T24	0.011	0.002
AMP = 0.1, HBV = 0	AMP = 0.1, HBV = 2
T0	0.020	0.016	T0	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 = 0.1, HBV = 8
T0	0.020	0.016	T0	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	T12	0.008	0.004
T24	0.044	0.102	T24	0.011	0.004
AMP = 0.1, HBV = 16	AMP = 0.2, HBV = 0
T0	0.020	0.016	T0	0.020	0.016
T2	0.064	0.020	T2	0.064	0.020
T4	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 = 8
T0	0.020	0.016	T0	0.020	0.016
T2	0.064	0.020	T2	0.064	0.020
T4	0.071	0.052	T4	0.047	0.029
T6	0.062	0.047	T6	0.033	0.026
T8	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	T0	0.020	0.016
T2	0.064	0.020	T2	0.064	0.020
T4	0.031	0.019	T4	0.036	0.015
T6	0.016	0.010	T6	0.015	0.005
T8	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
AMP = 0.4, HBV = 0	AMP = 0.4, HBV = 2
T0	0.020	0.016	T0	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
T0	0.020	0.016	T0	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
T12	0.014	0.006	T12	0.008	0.005
T24	0.011	0.003	T24	0.011	0.003
AMP = 0.4, HBV = 16
T0	0.020	0.016
T2	0.064	0.020
T4	0.033	0.014
T6	0.015	0.004
T8	0.008	0.005
T12	0.009	0.006
T24	0.012	0.003

[0250]

TABLE A-8
The checkerboard assay results of kanamycin
and honeybee venom verus kanarnycin resistant
S. aurreus.
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
KANA = 0, HBV = 0	KANA = 0, HBV = 2
T0	0.016	0.005	T0	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	T24	1.418	0.020
KANA = 0, HBV = 4	KANA = 0, HBV = 8
T0	0.015	0.004	T0	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
T8	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
KANA = 0, HBV = 16	KANA = 5, HBV = 0
T0	0.015	0.004	T0	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	0.351	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	T0	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
KANA = 5, HBV = 8	KANA = 5, HBV = 16
T0	0.015	0.004	T0	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	T6	0.016	0.008
T8	0.012	0.007	T8	0.014	0.008
T12	0.015	0.008	T12	0.016	0.005
T24	0.876	0.403	T24	0.237	0.378
KANA = 10, HBV = 0	KANA = 10, HBV = 2
T0	0.016	0.005	T0	0.015	0.004
T2	0.047	0.009	T2	0.047	0.009
T4	0.135	0.065	T4	0.045	0.026
T6	0.172	0.080	T6	0.044	0.029
T8	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
KANA = 10, HBV = 4	KANA = 10, HBV = 8
T0	0.015	0.004	T0	0.015	0.004
T2	0.047	0.009	T2	0.047	0.009
T4	0.030	0.016	T4	0.022	0.012
T6	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
KANA = 10, HBV = 16	KANA = 20, HBV = 0
T0	0.015	0.004	T0	0.016	0.005
T2	0.047	0.009	T2	0.047	0.009
T4	0.022	0.011	T4	0.123	0.061
T6	0.016	0.007	T6	0.145	0.073
T8	0.014	0.009	T8	0.166	0.079
T12	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
T0	0.015	0.004	T0	0.015	0.004
T2	0.047	0.009	T2	0.047	0.009
T4	0.044	0.020	T4	0.036	0.035
T6	0.041	0.020	T6	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
KANA = 20, HBV = 8	KANA = 0, HBV = 16
T0	0.015	0.004	T0	0.015	0.004
T2	0.047	0.009	T2	0.047	0.009
T4	0.023	0.011	T4	0.022	0.011
T6	0.017	0.007	T6	0.016	0.011
T8	0.014	0.006	T8	0.015	0.009
T12	0.017	0.007	T12	0.016	0.008
T24	0.240	0.340	T24	0.081	0.151
KANA = 40, HBV = 0	KANA = 40, HBV = 2
T0	0.016	0.005	T0	0.015	0.004
T2	0.047	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 = 0, HBV = 8
T0	0.015	0.004	T0	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
KANA = 40, HBV = 16
T0	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

[0251]

TABLE A-9
The checkerboard assay results of polymyxin B
and honeybee venom verus kanamycin resistant
S. aureus .
TIME	MEAN A660	S.D.	TIME	MEAN A660	S.D.
POLY B = 0, HBV = 0	POLY B = 0, HBV = 2
T0	0.009	0.003	T0	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	0.122
T8	0.887	0.107	T8	0.851	0.112
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 B = 0, HBV = 8
T0	0.009	0.003	T0	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
T0	0.009	0.003	T0	0.009	0.003
T2	0.068	0.004	T2	0.068	0.004
T4	0.034	0.010	T4	0.266	0.051
T6	0.013	0.004	T6	0.640	0.120
T8	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
T0	0.009	0.003	T0	0.009	0.003
T2	0.068	0.004	T2	0.068	0.004
T4	0.132	0.039	T4	0.035	0.012
T6	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	POLY B = 12.5, HBV = 16
T0	0.009	0.003	T0	0.009	0.003
T2	0.068	0.004	T2	0.068	0.004
T4	0.023	0.012	T4	0.036	0.012
T6	0.010	0.005	T6	0.013	0.004
T8	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
POLY B = 25, HBV = 0	POLY B = 25, HBV = 2
T0	0.009	0.003	T0	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
T0	0.009	0.003	T0	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
T0	0.009	0.003	T0	0.009	0.003
T2	0.068	0.004	T2	0.068	0.004
T4	0.035	0.013	T4	0.208	0.034
T6	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
T0	0.009	0.003	T0	0.009	0.003
T2	0.068	0.004	T2	0.067	0.004
T4	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 = 50, HBV = 8	POLY B = 50, HBV = 16
T0	0.009	0.003	T0	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	T8	0.011	0.005
T12	0.011	0.004	T12	0.012	0.004
T24	0.807	0.222	T24	0.023	0.028
POLY B = 100, HBV = 0	POLY B = 100, HBV = 2
T0	0.009	0.003	T0	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
T0	0.009	0.003	T0	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
T0	0.009	0.003
T2	0.068	0.004
T4	0.042	0.012
T6	0.020	0.006
T8	0.015	0.005
T12	0.014	0.004
T24	0.106	0.232

[0252]

TABLE A-10
The results of equivalent doses of melittin and
whole honeybee venom with and without kanamycin
on S. aureus.
	TIME	MEAN A660	S.D.
	KANA = 0,	MEL = 0,	HBV = 0
	T0	0.021	0.002
	T2	0.080	0.007
	T4	0.899	0.025
	T6	1.262	0.015
	T8	1.327	0.013
	T12	1.355	0.018
	T24	1.398	0.037
	KANA = 0,	MEL = 1.6,	HBV = 0
	T0	0.021	0.002
	T2	0.080	0.007
	T4	0.374	0.189
	T6	1.099	0.108
	T8	1.288	0.029
	T12	1.339	0.025
	T24	1.415	0.022
	KANA = 2.5,	MEL = 0,	HBV = 2
	T0	0.021	0.002
	T2	0.080	0.007
	T4	0.167	0.129
	T6	0.259	0.250
	T8	0.428	0.370
	T12	1.100	0.080
	T24	1.290	0.053
	KANA = 2.5,	MEL = 1.6,	HBV = 0
	T0	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	0.124
	T24	0.286	0.064
	KANA = 0,	MEL = 0,	HBV = 2
	T0	0.021	0.002
	T2	0.080	0.007
	T4	0.381	0.201
	T6	1.089	0.139
	T8	1.289	0.041
	T12	1.347	0.033
	T24	1.417	0.024
	KANA = 2.5,	MEL = 0,	HBV = 0
	T0	0.021	0.002
	T2	0.080	0.007
	T4	0.692	0.106
	T6	1.114	0.182
	T8	1.217	0.180
	T12	1.265	0.115
	T24	1.330	0.105
	KANA = 2.5,	MEL = 0,	HBV = 2
	T0	0.021	0.003
	T2	0.068	0.004
	T4	0.266	0.051
	T6	0.640	0.120
	T8	0.826	0.110
	T12	0.976	0.095
	T24	0.962	0.074

[0253]

TABLE A-11
Raw data for single treatment model.
	Log1Ø Bacteria/ml Blood
Exp.	Treatment	mouse 1	mouse 2	mouse 3	mouse 4
1	no treatment	2.62	3.49	2.91	3.18
I	melittin - 5Ø ng	3.27	2.88	2.76	—*
1	polymyxin B - 2 ug	3.2Ø	3.61	2.3Ø	3.42
I	mel 5Ø ng + pol 2 ug	1.9Ø	2.38	2.58	2.94
2	no treatment	2.96	4.16	3.77	3.89
2	melittin - 5Ø ng	3.39	2.79	2.58	2.88
2	polymyxin B - 2 ug	3.88	3Ø0	3.34	3.27
2	mel 5Ø ng + pol 2 ug	2.38	Ø.ØØ	2.62	2.15
3	no treatment	2.34	2.62	2.51	1.9Ø
3	melittlin - 5Ø ng	3.52	2.34	1.61	3.Ø8
3	polymyxln B - 2 ug	3.Ø8	3.11	2.91	Ø.ØØ
3	mel 5Ø ng + pol 2 ug	2.41	1.32	1.32	Ø.ØØ
*missing observation due to inadequate blood sample

[0254]

TABLE A-12
Raw data for repeated treatments model.
	LoglØ Bacteria/ml Blood
Exp.	Treatment	mouse 1	mouse 2	mouse 3	mouse 4
1	no treatment	4.8Ø	4.38	4.57	4.4Ø
1	melittin - 5Ø ng	4.Ø4	4.74	5.Ø7	4.62
1	polymyxin B - 2 ug	5.Ø4	4.56	3.48	Ø.ØØ
1	mel 5Ø ng + pal 2 ug	Ø.ØØ	Ø.ØØ	3.3Ø	Ø.ØØ
2	no treatment	4.67	4.36	4.45	4.41
2	melittin - 5Ø ng	4.67	4.51	4.89	4.9Ø
2	polymyxin B - 2 ug	1.78	3.Ø9	2.57	4.Ø6
2	mel 5Ø ng + pal 2 ug	1.3Ø	1.85	Ø.ØØ	3.21
3	no treatment	4.51	4.46	3.78	1.95
3	melittin - 5Ø ng	4.99	4.43	4.61	4.41
3	polymyxin B - 2 ug	3.2Ø	3.26	3.95	3.16
3	mel 5Ø ng + pal 2 ug	3.8Ø	3.24	3.22	3.46
4	no treatment	4.92	3.18	3.78	4.93
4	melittin - 5Ø ng	5.14	4.18	4.28	4.76
4	polymyxin B - 2 ug	3.35	3.51	3.51	3.89
4	mel 5Ø ng + pal 2 ug	2.6Ø	3.68	3.51	2.23
5	no treatment	3.53	4.3Ø	4.46	4.Ø8
5	melittin - 5Øng	4.Ø8	4.76	4.32	4.45
5	polymyxin - 2 ug	4.43	2.94	3.34	3.72
5	mel 5Ø ug + pal 2 ug	2.34	3.41	3.Ø5	2.93

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[0273] Mollay, C. and G. Kreil. 1973. Fluorometric measurements on the interaction of melittin with lecithin. Biochim. Biophys. Acta 316:196-203.

[0274] Mulfinger, L. M., A. W. Benton, H. W. Guralnick and R. A. Wilson. 1986. A qualitative and quantitative analysis of proteins found in vespid venoms. J. Allergy Clin. Immunol. 77:681-686.

[0275] Ortel, S. and P. Markwardt. 1955. Investigations on the bactericidal properties of bee venom (translated title, in German). Pharmazie 10:743-746. Abstracted in Chemical Abstracts. 1956. 50:1279c.

[0276] Schmidt-Lange, W. 1941. The bactericidal action of bee venom (Translated title, in German). Munchemer Medizinische Wochenschrift 88:935-936.

[0277] Sebek, O. K. 1980. Antibiotics: volume 1; mechanism of action. D. Gottlieb and P. D. Shaw (eds.). Springer-Verlag, New York. pp. 142-149.

[0278] Tu, A. T. 1977a. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc., New York, London, Sydney, and Toronto. pp. 1-16.

[0279] Tu, A. T. 1977b. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc., New York, London, Sydney, and Toronto. pp. 501-512.

[0280] Tu, A. T. 1977c. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc., New York, London, Sydney, and Toronto. pp. 505-509.

[0281] Volk, W. A. 1978a. Essentials of medical microbiology. C. May and J. Frazier (eds.). J. P. Lippincott Company, Phila., New York, San Jose and Toronto. pp. 121-122.

[0282] Volk, W. A. 1978b. Essentials of medical microbiology. C. May and J. Frazier (eds.). J. P. Lippincott Company, Phila., New York, San Jose and Toronto. pp. 122-126.

[0283] Volk, W. A. 1978c. Essentials of medical microbiology. C. May and J. Frazier (eds.). J. P. Lippincott Company., Phila., New York, San Jose and Toronto. pp. 130-133.

[0284] Volk, W. A. 1978d. Essential of medical microbiology. C. May and J. Frazier (eds.). J. P. Lippincott Company, Phila., New York, San Jose and Toronto. pp. 133-135.

[0285] Yunes, R. A. 1982. A circular dichroism study of the structure of Apis melifera melittin. Arch. Biochem. Biophys. 216(2):559-565.

ADDITIONAL BIBLIOGRAPHY

[0286] Goodman, M. G. and W. O. Weigle. Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on membrane-directed versus intracellular activators. J. Allergy Clin. Immunol. 74:418-425, 1984.

[0287] Kondo, E. and K. Kanal. Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin. Japan. J. Med. Sci. Biol. 39:9-20, 1986.

[0288] Kondo, E. Melittin-stimulated antimycobacterial activity of the membrane fraction isolated from phagocytes of guinea pigs. Japan. J. Med. Sci. Biol. 30:21-24, 1986.

[0289] Somerfield, S. D., J. Stach, C. Mraz, F. Gervals, and E. Skamene. Bee venom melittin blocks neutrophil O2 production. Inflammation 10:175-182, 1986.

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[0291] Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. 1951.

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Claims

1. A method for the treatment of an infection in mammal which comprises: administering an effective dosage of a medicament comprising: an antibiotic agent having activity against said infection; and a second agent selected from the group consisting of at least one analogue of an active protein component of a Hymenoptera venom, at least one analogue of a polypeptide component of a Hymenoptera venom, and mixtures thereof: the proportions of said antibiotic agent and said second agent being such that said second agent enhances the activity of said antibiotic agent.

2. The method of claim 1 wherein the antibiotic agent comprises an antibiotic selected from a family of antibiotics represented by a member of the group consisting of ampicillin, kanamycin, polymixin B, and rifampicin.

3. The method of claim 2 wherein the second agent is an analogue of an active protein component or an analogue of an active polypeptide component of a venom selected from the group consisting of honeybee venom, bumblebee venom, yellow jacket venom, bald faced hornet venom, and mixtures thereof.

4. The method of claim 3 wherein the antibiotic agent comprises ampicillin and the venom is honeybee venom.

5. The method of claim 3 wherein the antibiotic agent comprises ampicillin and the second agent is an analogue of melittin.

6. The method of claim 3 wherein the antibiotic agent comprises kanamycin and the venom is honeybee venom.

7. The method of claim 3 wherein the antibiotic agent comprises kanamycin and the second agent is an analogue of melittin.

8. The method of claim 3 wherein the antibiotic agent comprises polymixin B and the venom is honeybee venom.

9. The method of claim 3 wherein the antibiotic agent comprises polymixin B and the second agent is an analogue of melittin.

10. The method of claim 3 wherein the antibiotic agent comprises rifampicin and the venom is honeybee venom.

11. The method of claim 3 wherein the antibiotic agent comprises rifampicin and the second agent is an analogue of melittin.

12. A dosage unit for the treatment of an infection in a mammal which comprises: an effective dosage of a medicament comprising: an antibiotic agent having activity against said infection; and a second agent selected from the group consisting of at least one analogue of an active protein component of a Hymenoptera venom, at least one analogue of a polypeptide component of a Hymenoptera venom, and mixtures thereof: the proportions of said antibiotic agent and said second agent being such that said second agent enhances the activity of said antibiotic agent.

13. The dosage unit of claim 12 wherein the antibiotic agent comprises an antibiotic selected from a family of antibiotics represented by a member of the group consisting of ampicillin, kanamycin, polymixin B, and rifampicin.

14. The dosage unit of claim 13 wherein the second agent is an analogue of an active protein component or an analogue of an active polypeptide component of a venom selected from the group consisting of honeybee venom, bumblebee venom, yellow jacket venom, bald faced hornet venom, and mixtures thereof.

15. The dosage unit of claim 13 wherein the antibiotic agent comprises ampicillin and the venom is honey bee venom.

16. The dosage unit of claim 13 wherein the antibiotic agent comprises ampicillin and the second agent is an analogue of melittin.

17. The dosage unit of claim 13 wherein the antibiotic agent comprises kanamycin and the venom is honeybee venom.

18. The dosage unit of claim 13 wherein the antibiotic agent comprises kanamycin and the second agent is an analogue of melittin.

19. The dosage unit of claim 13 wherein the antibiotic agent comprises polymixin B and the venom is honeybee venom.

20. The dosage unit of claim 13 wherein the antibiotic agent comprises polymixin B and the second agent is an analogue of melittin.

21. The dosage unit of claim 13 wherein the antibiotic agent comprises rifampicin and the venom is honey bee venom.

22. The dosage unit of claim 13 wherein the antibiotic agent comprises rifampicin and the second agent is an analogue of melittin.

23. The method of claim 2 wherein the second agent is selected from the group consisting of analogues of melittin, bombilitin I-V, mastoporan and crabolin, and mixtures thereof.

24. The dosage unit of claim 13 wherein the second agent is selected from the group consisting of analogues of melittin, bombilitin I-V, mastoporan and crabolin.