METHODS AND COMPOSITIONS FOR THE TREATMENT OF MAMMALIAN INFECTIONS EMPLOYING MEDICAMENTS COMPRISING HYMENOPTERA VENOM, PROTEINACEOUS 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: METHODS AND, COMPOSITIONS FOR THE TREATMENT OF MAMMALIAN INFECTIONS EMPLOYING MEDICAMENTS COMPRISING HYMENOPTERA VENOM, PROTEINACEOUS OR POLYPEPTIDE COMPONENTS THEREOF, OR ANALOGUES OF SUCH PROTEINACEOUS OR POLYPEPTIDE COMPONENTS Cross Reference This invention is a continuation-in-part of Application Serial No. 07/096,628 filed on September 14, 1987. Introduction 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 chemo herapeutic 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 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. 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 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. Indeed, certain substances derived from bee venom have been studied and have been found useful in certain specifi pharmacologic applications. For example, U.S. Patent 4,444,753 issued April 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-stimulati ng activity, a carcinostat activity, an effect of enhancing the antibacterial activi of an anti-bacterial substance, and an effect of enhancin the carcinostatic activity of a carcinostatic substance. The invention disclosed in that patent is directed to carcinostatic, immuno-stiinulating and antibacterial agent comprising the composition described. While that inventi is similar in purpose to that of the present invention, i differs in that the bee extract iε modified by deproteinizing it so that it is negative in biuret reacti and sulfosalicylic acid reaction. U.S. Patent 4,370,316, issued January 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. 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. 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. As further background, it is noted that honeybee venom is credited with a multitude of usef l 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 jln 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. 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 poly yxins) 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. 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 synergiεtic action of these combinations among different groups of bacteria. 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. 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 Figure 1 is a diagram of the amino acid sequence of melittin; Figure 2 is a graph of optical density versus hours after inoculation which shows the antibacterial activity of honeybee venom (HBV) on S^ aureus; Figure 3 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus S^ aureus; Figure 4 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus S^ aureus; Figure 5 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus S_ ; _ aureus; Figure 6 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus E_^ coli; Figure 7 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus E_ ; _ coli; Figure 8 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus E^_ coli; Figure 9 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus E^ coli; Figure 10 is a graph of optical density versus hours after inoculation for ampicillin and HBV versus kanamycin resistant S^ aureus; Figure 11 is a graph of optical density versus hours after inoculation for kanamycin and HBV versus kanamycin resistant §_;_ aureus; Figure 12 is a graph of optical density versus hours after inoculation for polymyxin B and HBV versus kanamycin resistant S^ aureus; Figure 13 shows the electrophoresis results of lOOug of melittin protein; Figure 14 is a graph of optical density versus hours after inoculation showing antibacterial activities of melittin/HBV versus S_ j _ aureus; Figure 15 is a graph of optical density versus hours after inoculation for melittin/HBV and kanamycin versus S. aureus; Figure 16 is a graph of optical density versus hours after inoculation for rifampicin and HBV versus S^ aureus; Figure 17 is a graph of optical density versus hours after inoculation for rifampicin and HBV versus Ps. aeruginosa; 8a Figure 18 is a graph of optical density versus hours after inoculation for polymyxin B and bumblebee venom versu E. coli; Figure 19 is a graph of optical density versus hours after inoculation for polymyxin B and yellow jacket venom versus E^ coli; Figure 20 is a graph of optical density versus hours after inoculation for polymyxin B and bald faced hornet ven versus Σ _ Coli; Figure 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); Figure 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); Figure 23 is a graph of optical density versus hours after inoculation for melittin/analogue and polymyxin B versus E^_ coli; Figure 24 is a graph of optical density versus hours after inoculation showing relative activities of melittin analogues; Figure 25 is a graph of optical density versus hours after inoculation showing relative activities of melittin analogues; and Figure 26 is a graph of optical density versus hours after inoculation showing relative activities of melittin analogues. 8b Composition of Venoms 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. 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) 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) 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 8c 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 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. It wasn't until ten years later that Brangi and Pavan (1951) evaluated various extraction procedures to isolate t antibacterial activity of honeybee venom. They found the activity to be present in both water and acetone extracts o venom. They also showed that the activity was stable when heated to 100 degrees centigrade for up to 15 minutes. 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. 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 vivo use. (Currently, allergy desensitization is the- only iii 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). 10 Melittin Isolation and Activities 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 ) . Antibacterial Activity of Melittin 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 puri fied 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. 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. 11 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 (Figure 1) . Analogues of proteinaceous and polypeptide components of Hymenoptera venoms. The following analogues of melittin have been prepared. Analogue Composition No. 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-NH 2 4. Melittin (1-20) - Lys-Arg-Lys-Arg-Gly-Gly-NH2 5. Melittin (1-20) - Arg-Arg-Arg-Arg-Gln-Gln-NH 2 6. Melittin (1-20) - Lys-Lys-Lys-Gln-Gln-NH 2 7. Melittin (1-20) - Gly-Gly-Gly-Gly-Gln-Gln-NH 2 8. Melittin ( 1-20) - Asp-Asp-Asp-Asp-Asp-Asp-NH2 9. Melittin (1-20) - Lys-Lys-NH 2 10. Mastoporan (1-14) - NH2 (native) 11. Mastoporan (1-14) - Orn-Orn-Orn-Orn-Gln-Gln-NH 2 12. Melittin (1-20) - (CH-NEu) Z Z 12 13 . Melittin ( 1-20 ) - Orn-Orn-Nlk 11a The analogues were prepared by conventional peptide synthesis as described by e.g. M. Bodanszky: "Principles of Peptide Synthesis", Springer Verlag, 1984. As an example the peptide synthesis of analogue No. 4, viz. melittin (l-20)-Lys-Arg-Lys-Arg-Gly-Gly-NH2 will now be described in further detail. A derivatized resin such as a polydimethylacrylamide gel which is commercially available under the trade name PEPSYN KA is reacted with (Fmoc-Gly^O wherein Fmoc is 9- fluorenylmethoxycarbonyl which serves as a temporary protecting group. 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. The ester is deprotected in the presence of 20% piperidine in DMF so as to form H-Gly-O-resin. The deprotected product is then reacted with an activated ester having the formula Fmoc-Gly-OPfp wherein Pfp is pentafluorophenyl so as to form Fmoc-Gly-Gly-O-resin lib The remaining 24 amino acids are coupled to the reaction product formed in 20 similar cycles of deprotection and coupling with active esters. 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 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. 12 Ampicillin 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 transpeptidaεe enzyme which croεεlinkε the pentaglycine bridgeε of the peptidoglycan, therefore, only actively growing cells are affected by their presence. Ampicillin is a semisynthetic derivative of penicillin. The synthetic step in ampicillin syntheεis 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). Kanamycin Kanamycin is an aminoglycoside. This group of antibiotics block protein synthesis. Members of this group bind to the 30s ribosome of bacteria and εterically block the binding of aminoacyl-tRNA'ε or inhibit the tranεlocation of the growing peptide chain at the riboεomal active site (Volk, 1979c). Since protein εyntheεiε is required for many regulatory cell functions, aminoglycosides are effective on bacteria in either active or stationary growth phases. 13 Polymyxin B Polymyxin B is a cyclic, a phiphatic 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 εelective 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 organismε. (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. Rifampicin 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 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 Honeybee (Apis elifera) venom was supplied by Veεpa Laboratories, Spring Mills, Pennsylvania. Bacteria strains were supplied by the Veterinary Science Department of the Pennsylvania State University. S^ aureus #140A iε a field isolate from a case of bovine mastitis. E. coli #G1880E was selected from the E^_ coli Reference Center syεtematic collection. A kanamycin reεiεtant strain 15 of S_^ aureus was iεolated by a natural selection procedure described below. Antibiotics were purchased from Sigma Chemical Company (St. Louis, Missouri) and activity units were baεed on their analyses. Trypticase soy base (BBL Microbiology Systems, Cockeysville, Maryland) was uεed to εupport all bacterial growth either aε a broth or an agar. Sephadex G-50 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Minimal inhibitory concentration (MIC) assays of antibiotics with and without honeybee venom, were performed by the Microbiology Department of the Allegheny General Hospital, Pittsburgh, Pennsylvania, using the Sensititre™ aεεay εystem distributed by Gibco Laboratories, Lawrence, Massachusetts. Methods Isolation of Kanamycin Resistant Mutant S. aureus was grown in 5 ml of trypticase soy broth (TSB) overnight to an approximate density of 10° colony forming units/ml. 0.1 ml of the overnight culture was plated on a plate of trypticaεe 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 εecond TSA plate supplemented with 39 ug/ml kanamycin. 16 Checkerboard Titration Assay for Svnerqy Bacteria cultures were prepared for this assay by freezing each strain while in logarithmic growth in TSB. For this purpose, a 5 ml overnight culture was used to inoculate 200 ml of TSB in a 500 ml erlen eyer 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 unitε), 5 ml aliquots were transferred to 16 x 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. To begin an asεay, 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 OD550 immediately meaεured 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 005 0 was again read and recorded, the culture was split into 16 x 100 mm screw-cap test tubes prefilled with the specified aliquots of honeybee venom (HBV) and antibiotic described below. 17 Stock solutions of HBV and antibiotics were made in distilled water, filter sterilized, and stored at -20 degreeε centigrade in 5 ml aliquots at concentrationε 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 waε based on preliminary experiments using the antibiotics alone to determine the minimal inhibitory ranges of each antibiotic for each microorganism. For each assay, one vial of antibiotic and one vial of HBV were thawed at room temperature and diluted with an equal volume of 2X 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 00 and 0 contained 2.5 ml of TSB and served as OD blanks and sterility control tubes. Tubeε 1-75, each containing a total volume of 500 ul, waε 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 18 (refer to Table 3).] Each tube was immediately sealed and ' inverted. After all tubes v/ere inoculated, they were placed in horizontal racks on a rocker platform at 37 degreeε 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 n . Minimal Inhibitory Concentration Assays with HBV The microbiology laboratory of the Allegheny General 0 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) 5 the effect of the HBV alone could be evaluated only aε completely inhibitory or non-inhibitory. Synergy of HBV with the 11 antibiotics in this system was evaluated by comparing two assays run simultaneoulsy with and without HBV present. The dose of HBV used for each species was on estimated to be a non-inhibitory dose, based on the checkerboard titration assayε. Melittin Purification Sephadeχ G-50 gel filtration bedding waε εwollen for 24 hours at room temperature in beta alanine-acetic acid 5 buffer (BAAB), pH 4.3 (Guralnick et alia, 1986), and then 19 equilibrated at five degrees centigrade overnight. A 2.5 x 60 cm column waε 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 20% sucrose. The HBV was layered on the column and eluted at a flow rate of 1 ml/hour. The effluent was monitored for abεorbence 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. 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 waε alεo checked for purity by polyacrylamide gel electrophoreεis. Electrophoresiε 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 εampleε were applied per εample well on the gelε. Whole Venom Equivalence of Melittin The amount of the melittin fraction equivalent to itε proportion in whole honeybee venom waε determined by quantification of individual bands in electrophoresed samples of whole venom and the melittin fraction. Twenty, 40, 60, 80, and 100 ug sampleε of whole honeybee venom were εeparated by electrophoresis, stained tRJ with Coomasεie^ brilliant blue- 20 perchloric acid stain, and scanned with a denεitometer. 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 εimultaneouεly and their equivalence in honeybee venom was determined from the standard curve. This procedure is described in detail by Mulfinger et alia. (1986). Testing the Melittin Fraction for Synergistic Activity 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 εynergistic action with the HBV, this system waε choεen to compare the antibacterial activitieε 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/ml HBV was 1.6 ug/ml. Each experiment compared in parallel, triplicate samples of the melittin fraction and 21 whole honeybee venom with and without kanamycin preεent to check for equivalent activity. Statistical Analysis Each checkerboard experiment was repeated five times. 5 The averages of the five repetitions for each bacteria- antibiotic combination were tested at each time point for εignificant differenceε using a Waller-Duncan K-ratio T Teεt and families of curves were selected for synergy testing. A curve family conεiεted of an experiment control 0 curve (bacterial growth with no antibiotic or HBV preεent), an antibiotic control curve (bacterial growth with antibiotic but no HBV present), a venom control curve (bacterial growth with HBV but no antibiotic preεent) and an interaction curve (growth with antibiotic and HBV 5 preεent). Families in which the antibiotic control curve and the venom control curve εhowed εmall average OD decreases relative to the experiment control curve, and which also demonstrated large OD decreaseε in the interaction curve relative to the experiment control curve 0 were teεted for synergy. A εynergistic effect between compounds can be 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 5 compounds individually, thus, any greater effect would 22 indicate synergistic interaction. An equation predicting OD readings for an additive interaction betv/een HBV and an antibiotic waε derived. See the Mulfinger thesis (1987) referred to above, pages 23-25. RESULTS Checkerboard Titration Asεays 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 treatments (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 εampleε 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 valueε for each antibiotic/HBV treatment at each time point were arranged in deεcending order, and grouped according to εignificant differenceε 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 betv/een the interaction curve and 23 the lowest of the experiment curve, antibiotic control curve and venom control curve, was plotted and each time point was tested for synergy using the equation derived in the section "Statistical Analysiε" above. For each family of curveε, if the eεtimate 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 iε noted on the interaction curve by a superscript "s" at the square representing that time point (Figureε 2-11). S. aureus 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 doseε for the checkerboard titration system were 0, 2, 4, 8, and 16 ug/ml (Tables A-l through A- 3). Figure 2 demonstrates the effects of these dosageε of honeybee venom when uεed alone aε an antibacterial compound. S. aureus versus Ampicillin/HBV. The final concentrations of ampicillin in tubes of the checkerboard system were 0, 0.05, 0.1, 0.2, and 0.4 ug/ml. Figure 3 εhows the resultε of the ampicillin/KBV combination using 2 ug/ml HBV, and 0.05 ug/ml ampicillin. No synergy is εeen 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 εhows that at both time points, the summation (-X+A+V-AV) is significantly greater than zero. S. aureus versus Kanamycin/HBV. The final concentrations of kanamycin selected for testing j>. aureus in the checkerboard system were 0, 1.25, 2.50. 5.0, and 10.0 ug/ml (Table A-2). Figure 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 εeen by the 8 hourε of incubation. At 12 hours, the cultures appear to have escaped the effectε of the combined dose and the synergistic effect is lost since growth becomeε limited by other (nutritional) factors in the medium. (This growth limitation is demonεtrated 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 aεεay. 25 S. aureus versus Polymyxin B/HBV. 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 ug/ml HBV and 625 U/ml polymyxin B (figure 5). At both δ and 12 hours of incubation, synergy is demonεtrated by the interaction curve. E.coli Honeybee venom waε not inhibitory alone to E_ ; _ coli at the levels required to demonstrate synergy (Tables A-4 through A-6), thus, toxicity waε not the limiting factor for HBV i the checkerboard aεsay with E^_ coli. However, experimental conditions limited the upper concentration of HBV at approximately 40 ug/ml; concentrations greater than thiε caused precipitation of medium componentε. Therefore, the final concentrationε of HBV used in the checkerboard assayε with E. coli were 0, 5, 10, 20, and 40 ug/ml. E. coli versus A picillin/HBV. 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 waε evidence of synergy only in the 40 ug/ml HBV-1 ug/ml ampicillin combination and only at the 6 hour time point (figure 6). 26 E. coli versus Kanamycin/HBV. The final concentrations of kanamycin selected for the checkerboard assay were 0, 5, 10, 20, and 40 ug/ml (Table A-5) . Figure 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. Figure 8 shows a higher dose of kanamycin with HBV on E. coli. Here, synergism is statistically proven at all time points after 2 hours. E. coli versus Polymyxin B/HBV. The final concentrationε of polymyxin B in the checkerboard titrationε were 0, 1.5, 3, 6, and 12 U/ml (Table A-6). The combination of 3U/ml polymyxin B and 5 ug/ml HBV gave the most dramatic illustration of εynergism (Figure 9). Synergy is evident at all time points during the treatment and the differences between the observed and the predicted values are large. Kanamycin Reεistant S. aureus 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 . aureus was desirable because some synergy was seen for all antibioticε with this organism, and because εynergiεtic effects were moεt eaεily εeen with kanamycin. /11766 27 No difference v/as 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 v/as noted that under identical conditions, the resistant strain had a slower growth rate than the parent strain, therefore, comparing optical density reεdings between experiments on the two different strains is not meaningful. Kanamycin Resistant S. aureus versus Ampicillan/HBV. The final concentrations of airpicillin used in this checkerboard assay were the same as for the parent Σ3. aureus, 0, 0.05, 0.1, 0.2, and 0.4 ug/ml (Table A-7). Whether due to the slower grovth rate or the resiεtance factor, the effectε seen with this strain were not completely analogous to the parent strain. The best evidence of εynergy waε εeen at a higher ampicillin concentration than for the parent. Due to the εlower growth rate, a longer grov/th period was cσnεidered. Figure 10 εhows the interaction of 2 ug/ml HBV and 0.4 ug/ml ampicillin. Statistical evaluation of the data showε synergy at the 8, 12, and 24 hour time points. Kanamycin Reεiεtant S. aureus versus Kanamycin/HBV. The dosage of kanamycin required to reduce the growth rate of the kanamycin resistant εti≤in of S_^ aureus was approximately four timeε high≤r than the dose required by 28 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 Figure 11. Although the dose of kanamycin used iε twice as high as the doεe needed for the parent £>. aureuε, it remains effective twice as long in the preεence of honeybee venom. Synergy was obεerved only after 12 hours and was proven to be εignificant only at the 24 hour time point. Kanamycin Reεiεtant S. aureus versus Polymyxin B/HBV. It was interesting to note that this mutant, selected for increased resistance to kanamycin, became more suεceptible to polymyxin B than the parent strain. The polymyxin B doses used for the checkerboard asεay waε 0, 12.5, 25, 50, and 100 U/ml (Table A-9), whereaε the polymyxin B doεe range uεed for aεεaying the parent εtrain was between 312 and 2500 U/ml. Figure 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. MIC Assays of Antibiotics With and Without HBV The resultε of a preliminary εurvey 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 29 Table 5 respectively. Despite the apparent ina d equacies of the assay system, definite trends were seen in the results of the survey. Synergy was strongly suggested where observations within a single MIC assay showed that 5 identical doses of HBV affected some antibiotic MIC's while not affecting otherε. In Tableε 4 and 5, a ( + ) waε 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 10 variation ( judged to be the variation of the assay ) in the MIC of an antibiotic with HBV present. Table 4 showε the results of several gram-positive organismε. The results indicate that trends exist within the species tested. For example, . aureus appears to show 15 synergy with all antibiotic/HBV combinations, while S. epidermidis εhows consistent synergistic reεultε only with the cephalothin/HBV combination and εporatic reεultε with other antibiotic/HBV combinationε. The one S treptococcus faecalis strain that waε teεted reflectε none of the same 20 synergistic trends shown by the two staphylocuccus organisms. The data in Table 5 listε the reεultε of four E. coli εtrainε in the MIC aεεay εystem. Definite patternε of εynergy are εeen with each of the beta-lactam antibiotics - ■ - > ( ampicillin, carbenicillin, and piperacillin ) included in 30 the MIC assay εyεte . Also, the MIC of the aminoglycoεides 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 Chromatography of Honeybee Venom 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 is after the 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. Figure 13 showε the electrophoreεis resultε of 100 ug of protein from the pooled fractionε 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. Testing Melittin for Antibacterial Activity Equivalent doses of melittin and whole honeybee venom were compared for antibacterial activity in combination with and without antibiotic (Table A-10). Since J3. aureus was suεceptible to HBV at levels uεed in the above aεεays, this crganiεm waε chosen to test the melittin fraction's 31 activity. Kanamycin was chosen to evaluate the εynergistic 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. The Antibacterial Activity of Melittin. The reεultε melittin versus whole HBV are shown in Figure 14. No significant differences were observed in the antibacterial activity of whole HBV and the melittin fraction. For each time point represented in Figure 14, the optical densities of the HBV curve and the melittin curve are statistically equal. The Syner g istic Activity of Mellitin with Kanamycin. Figure 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 repreεenting the melittin fraction iε actually εlightly lower at all time points than the interaction curve representing whole HBV. Thus, if the time points on both curveε were accepted aε the true meanε, the final conclusion would be that the melittin fraction is actually more active than whole HBV. interpretation of ch^ ^^.,, of ftq „„ „_^ The results of the checkerboard assays clearly demonstrate synergism between antibiotics and honeybee venom. Pigure 2 illustrated the effects of var . ous floses > f honeybee venom on s. aureus witho t antibiotics. It can be seen in this Figure that the addition of high d oses of venom, such as 8 or 16 ug/ml, to the growing cultures actually lowered the optical density of the culture. This i ndication of cell lysis is evidence that honey b ee venom is actually bactericidal. The mechanism of this bactericidal act i ity and its contribution to the synergy seen with ant i biotics is not known. The varied results of the checkerboard titration assays suggest that several different synergistic mechanisms may be functioning in these experiments. Q uestions 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 gro th rate when the bacteria are in log p hase . Time Po i nts taken in mid-log phase will have a much larger difference in optical density with time than will time Po i nts taken during a slower growth period. Thus uncontrollable, small variations in sampling intervals could cause larger variations in optical density rea d ings at trme points during logarithmic growth , since cultures 33 are split during log phase into the various treatment groups, variations are even more noticeable betv/een experiments. This type of error is taken into consideration, however, in the εtatistical evaluation procedure. By using a large sample number (15) , estimation ranges for the means of the time points were made narrow enough to εtatiεtically evaluate the differences in these means. Although melittin waε only teεted initially aε the εynergiεtic component of HBV in combination with one antibiotic with one bacterial strain, for the purpose of discussing posεible mechanisms it has been asεumed that melittin iε the synergistic honeybee venom component in each of the bacterial-antibiotic-HBV combinations teεted. Apparent Increaεed Doεage 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 waε moεt demonεtrable with E_ ; _ coli verεus HBV and polymyxin B (Figure 9). At the first time point after addition of the tv/o compounds, εynergism iε apparent and it continueε as the culture progreεεes through log phaεe. These reεults εuggeεt that low, noneffective doses of antibiotics may be made effective with the addition of HBV. 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. Altered SoJuhilitv Prnn. u.. of thf , an M ^^. τhe 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 ionε with valinomycin. Iϋ££eased_Membrane Permeability, τhe apparent dosage Qf an antibiotic could also be increased by reducing penetration barriers of the bacterium. 35 Although thiε role aε a channel-forming peptide iε easily supported, it cannot be the only function of melittin that iε involved in the antibacterial synergy. Increased transport across membranes fails to explain why melittin alone is more effective on gram positive organismε which have less of a membrane barrier. Increased Antibiotic Specific Activity. 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 posεible interaction with kanamycin. Once kanamycin reacheε the 30S ribosome, a melittin-kanamycin complex may have a greater affinity for the active site than unbound kanamycin (after all, melittin iε a basic molecule, like nucleic acids), or the melittin-kanamycin complex may be more effective in εterically blocking transfer-RNA's from the riboεome due εimply to the size of the complex. Increased Active Life of Antibiotics 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 Figure 9 is a 36 relatively high doεe of HBV, the reason being that no synergism was seen with lower doseε. Thus, although it iε difficult in Figure 9 to rule out synergy at the early time pointε 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. Decreased Probability for the Selection of Resistant Strains. If both the honeybee venom and antibiotic are present in a bacterial culture at bacteriεtatic doseε, the probability that a resistant bacterium will survive the combined treatment is equal to the product of the probabilities that one would exist and εurvive either treatment. This would appear aε a delayed εynergistic 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 aε 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 reεiεtant S^ aureuε with kanamycin was evaluated, the mean OD of the 12 hour 37 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 poεsible that the synergiεtic effect seen here at the 24 hour time point is the result of εuppression of HBV venom reεiεtant mutantε. Increased Antibiotic Stability. 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 modificationε account for many of the derivativeε in the penicillin family of antibioticε. For example, penicillin V haε a phenoxymethyl substitution which provides steric hinderance, protecting the antibiotic's beta-lactam ring from enzymatic attack (Volk, 1978c) . Such subεtitutionε may alεo prevent thiε end of the molecule from cyclization with the beta-lactam ring making the molecule more reεiεtant to acid hydrolysis. These types of modificationε would alεo produce a εynergic effect demonstrable only at bacteristatic doεeε, since the antibiotic would not be any more effective initially and the prolonged life εpan of the antibiotic would be evident only if the bacterial culture had not reached a nutritionally limiting OD at that time. If, however, HBV 38 could cause such a modification, more consistent results among replicate samples would be expected. Evaluation of MIC Testing 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. 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 39 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. Identification of the Active Honeybee Venom Component 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 resultε. It is posεible 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 aε 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 micelleε 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. 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. The activity of hymenoptera venom other than HBV was alεo determined for bumblee venom, yellow jacket venom and bald faced hornet venom, as shown below in Tables 8, 9 and Figs. 18-20. 39a Also some of the analogueε mentioned above were tested to determine their relative activities with reεpect to native melittin. The relative activity iε calculated aε followε: Dose of melittin x 100 Dose of analogue needed to demonstrate equivalent synergy with polymyxin B The results obtained are set forth in Table 10. It appears that analogues in which the NH, -terminal end mainly consiεtε of baεic amino acidε are more active than analogueε having an NH, -terminal end mainly consisting of neutral and/or acid amino acids. 40 IN VIVO EXPERIMENTS Introduction In vivo experimentε demonεtrate that melittin, the major peptide of honey bee venom, enhanceε the effectiveneεε of a proven antibiotic, polymyxin B. A diεeaεe model, bacterial εepsis, waε 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 setε of experiments. With both experimental protocols, a synergistic interaction between melittin and polymyxin B is evident and is verified statiεtically by a contraεt 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 jLn vivo is demonstrated clearly. 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 demonεtrate only in vitro effectiveness of honey bee venom or melittin. Several other systems have used melittin as an artificial means of perturbing various immune responses in 41 isolate i.n 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 iri vitro to stimulate the bactericidal activity of embraneε iεolated from phagocytes of both mice and guinea pigs. Lastly, one publication (Somerfield et a . 1986) relating the effect of honey bee venom on the immune system describes the inhibition of neutrophil 0 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. Despite the εubstantial amount of research with melittin. It had not yet been demonstrated that melittin is effective άji vivo against infectious organismε. More importantly, nowhere haε the need for, or the benefit of, the interaction of melittin with antibiotics been proposed. The reεultε reported herein demonεtrate beneficial interactions between melittin and polymyxin B when used jln vivo to treat mice suffering a bacterial septicemia caused by E. coli. 42 Materials and Methods Female Swiss CD-I 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. 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. 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 asεay (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. 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 43 trypticase soy broth. The culture was propagated overnight with mild shaking. Two hundred ml of sterile glycerol was added to the culture which was then aεeptically dispensed, while stirring, into 5.0 ml aliquots. These aliquots were frozen and stored at -20 degrees Centigrade. Upon thawing, o each aliquot yielded a (+/-3) x 10 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. Mice were infected by intraperitoneal injection of 0.25 ml of the 1:400 dilution of bacteria (approximately 500,000 viable bacteria) suεpended in trypticase soy broth with 2.5% mucin. 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. 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 44 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 lesε than 400 colonieε were counted and recorded. Results 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 εamples 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-ll). This experiment was run in triplicate and the average number of bacteria per milliliter of blood for each of the four treatments was compared (Figure 21). A p-value of .0015 for the treatment effect in a two-way analysis of variance (adjusted for unequal sample 45 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 contraεt within the analysis of variance confirmed that the interaction was indeed synergistic (p-value = .0493). 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 timeε and the results (Table A-12) were evaluated by analysis of variance. These analyses showed a significant difference (p-value = .0001) in at least one treatment. 46 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 εignificantly lower than the counts in the blood of animals treated only with polymyxin B or melittin (refer to Figure 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). Conclusions The above experiments clearly demonstrate εynergiεtic 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. The results of the first set of experimentε (Figure 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 (Figure 22) again lacked a significant difference for the melittin only treatment. A comparison of the absolute treatment means 47 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. 48 Antibiotic Synergy Demonstrated With A Synthetic Melittin Analogue 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. Introduction Analogue No. 6, the structure of which is shown below, H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro -Ala-Leu-Ile-Ser-Trp-Ile-Lyε-Lyε-Lys-L y s-Gln-Gln-NH2 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. 49 Materialε and Methods 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. Analogue No. 6 was synthesized by Dr. Torben Saermark (The Protein Laboratory, Copenhagen Univerεity, 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 lyophilized form and was reconstituted to approximately 0.2 mg/ml in 0.85% NaCl, filter sterilized, and εtored in 0.5 ml aliquotε at -20 degreeε Centigrade until used. Polymyxin B (Sigma Chemical Company) with a εpecific activity of 7900 unitε/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. E. Coli strain No. G1108E was obtained from the Pennsylvania State University E_ ; _ Coli Reference Center (105 Henning Building, University Park, Pennsylvania, 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 50 dispensed and frozen in 5.0 ml aliquots at -20 degrees Centigrade until uεed. 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 5ug/ml and 10 ug/ml. Polymyxin B was tested at final medium concentrations of 3 unitε/ml and 6 unitε/ml againεt both levelε of both peptides. Results Synergy was ^ beεt demonεtrated 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 Figure 23), the data for each peptide was statistically analyzed for synergiεtic 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-polymixin B combination. Synergy was detected at the 4 , 6, and 8 hour time points for both melittin and analogue #6 (p-values = .0001). 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 51 significant difference be detected between these two curves. Conclusions The results show that analogue #6, a synthetic melittin analogue, has activity very similar to that of melittin with regard to itε capacity to enhance the activity of polymyxin B. Although the twelve hour time point εuggests 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 lesε than 10%. Thiε inveεtigation shows that it is possible to synthesize melittin analogues with synergistic capabilities equivalent or superior to melittin. 51a Synergistic Antibacterial Activity of Melittin and Polymyxin B: Relative Activities of Melittin Analogues The synergy seen between antibiotics and melittin may also be achieved by replacing melittin with either synthetic analogues αr 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. colt. Their relative ac¬ tivities were compared to that of melittin from natural honey bee venom. Each peptide demonstrated synergistic Interaction with polymyxin Bj however, specific activities differed sig¬ nificantly. Several analogues provided synergistic activity superior to that of natural melittin. 51b Introduction Two types of melittin analogues, synthetic peptides and a chemical modification of natural melittin, were assayed In vi tro for βynergy with polymyxin B in an antibacterial activ- lty assay. The synthetic analogues include a group of syn¬ thetic peptides, all of which vary from the 26 amino acid se¬ quence of melittin by two or more residues. The chemical modification of melittin, NPS-rae 11 tt In, consists of the at¬ tachment of an _-ni trophenyl sulfenyl group tα 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 J_n yl tro, significant differences In peptide ac¬ tivities were evident. These differences define key at¬ tributes of the melittin molecule in Its role as a poten- tiator of polymyxin B activity. 51c Materials and Methods Natural melittin was Isolated from whole honey bee venom (Vespa Laborato ies, Inc.) via gel filtration chromatography, quantitated by the Lowry protein assay and stored lyophilized. For these assays, lyophilized melittin was re¬ constituted to 0.34 mg/ml with distilled water, filter ster¬ ilized, and stored in 4.0 ml aliquots at -20 degrees centi¬ grade until used. NPS-rae 11111 n was synthesized from natural melittin by reacting the peptide with o_-nl trophenyl sulfenyl chloride (NPS-Cl) as has been described for adr enocor t icot r op i n by Raraachandr an 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-Pharraaci a, Piscataway, N.J.) column to remove remaining NPS-Cl salts. A determina ion of the molar absorptivity of the derivative at 365 nm indicated that the melittin was better than 9596 modi fled. Synthetic melittin was purchased from Peninsula Labora¬ tories, Belmont, Ca. 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. Synthetic analogues were synthesized by Dr. Torben Seamark (The Protein Laboratory, Copenhagen University, Slgurdsgade 34, DK-2200 Copenhagen N, Denmark) . Each analogue was assayed for purity and was estimated to be bet- 51d ter than 98% pure based the chromatographic elution profile from a C-lβ column using a 0-100% acetonitrile gradient. Each peptide was received in lyophilized form and was recon¬ stituted to approximately 1.0 mg/ml In 0.85% NaCl , filter sterilized, and stored in 1.0 ml aliquots at -20 degrees cen¬ tigrade 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 we i ghts. Polymyxin B (Sigma Chemical Company, St. Louis, Mo. ) with a specific activity of 7900 unlts/mg was reconstituted to 240 units/mg in 0.85% sal ine, fi lter steri l ized, and stored in 4.0 ml aliquots at -20 degrees centigrade until used. E . co l t G1108E was obtained from the Pennsylvania State University E . co l t 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 concen- tratlon of 20% and 5.0 ml aliquots were dispensed and frozen at -20 degrees centigrade until used. A checkerboard titration synergy assay was per¬ formed, testing each peptide with polymyxin B against E. colt in parallel with melittin. Equivalent dosages of the natural melittin and each analogue were based on a Lowry protein as¬ say performed on aliquots of each peptide after the final filtration of the stock solution. All peptides were tested in 51e 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 8 units/ml. 51f Resul ts Table 126 hows the amino acid sequences of the synthetic melittin analogues. For each synthetic analogue, the first twenty N-ternlmal ami no acids are the same as natural melittin. Alterations occur in the six C-terminal amino ac¬ ids and are indicated by boldfaced print. 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 o E. colt when used alone at 10 ug/ml or less. Thus, th "control" is also a representation of the culture w e treated with each peptide alone. When the growth curve of bacterial cultures treated wit only polymyxin B (6 units/ml) is compared to the curve o cultures treated with polymyxin B plus melittin (5 ug/ml) increased antibacterial activity is demonstrated as an In crease in the time required for the culture to overcome th treatment and achieve log-phase growth (figure 24). As treat ment of the culture with melittin alone at 5 ug/ml would pro duce a growth curve which would essentially overlay the "con trol" curve, an increase in the time required for th culture to escape the polymyxin B Inhibition and reac 51g 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 treat- raent Is evidence of synergy. 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 flgure24 for both synthetic melittin and NPS-rae 11 tt in. All peptides were at equal concentr ions as determined by Lowry assay. Similar growth curves comparing the synergistic ac¬ tivities of the synthetic melittin analogues with polymyxin B are shown in figure 25. In order to more clearly visualize the differences in rael I ttin/analogue synergy with polymyxin B, figures 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 figure 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 designated by different bar markings In figure 26. 51h Conclus ions These results show that a variety of melittin analogue can be created by both amino acid substitutions and chemica modifications. These types of modifications can either en hance or diminish the peptide's relative synergistic activ ity. Based on Figure 26, the relative Jji vitro activities o melittin and Its analogues can be stated In ascending orde as follows: 1 Analogue #7 A 2 Natural Melittin B 3 NPS-Mellttln C 4 Analogue #6 C 5 Synthetic Melittin C 6 Analogue #2 C 7 Analogue #4 D 8 Analogue #5 D Peptides with signi icantly different (alpha = 0.05) syner gistic activities at the 5 ug/ml level are noted by lette groups . It should be noted that the parameter used to establis this order of efficacy, delay time until raid-log phase of t culture, Increases s to 1 ch lometr leal 1 y with the amount o peptide only over a narrow range of melittin concentration Since the boundaries of the linear range for each analog may not be equivalent, the data presented here can be us only to determine the relative order of efficacy of the peptides at the given concentration and can not be used estimate quantitative differences. The order of effica does suggest, however, that synergistic activity of t 51i peptide relies on the number and exposure of positively charged side chains on amino acids in the C-terminal region. Although the relative efficacy of these melittin analogues has been established J_n vitro, substantial dlffer- ences may occur Jji vivo. In vivo parameters such as absorp¬ tion into and clearance from the host may β igni leantly alter this order of efficacy in practical usage. Side effects must also be considered. While adrenoco t icotropic activity of melittin Is well documented the NPS-mellttln may have less adrenal act ivi ty than natur al me l i tt in as the NPS-der 1vat Ive of adrenocort Icotropin (ACTH) Induces 100 time less lipolytlc activity than unmodified ACTH. For these reasons, each of the analogues included In this study should be considered for in vivo evalutlon. 52 Table 1 The concentrations of stock solutions of antibiotics and honeybee venom tested against, three different bacteria. Organism Venom Ampicillin Kanamycin Poly ixin B E. coli 800 ug/ml 80 ug/ml 800 ug/ml 240 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 SOU ug/ml 2000 U/ml kana R 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 Control 0*\0 b 0\1:16 0\1:8 0\1:4 0\1: 1-3 C 4-6 7-9 10-12 13-1 o n D 1:16 1:16\0 l:16\.l:16 1:16\1:8 1:16\1:4 1:16\ e 1 16-18 19-21 Ϊ5-27 28-30 y 1 b u e t 1:9 1:8\0 1:8\1:16 l:S\l:8 1:8\1:4 1:8\1 e 1 31-33 34-36 37-39 40-42 43-45 o V n e s 1:4 1:4\0 1:4\1:16 1:4\1:8 1:4\1:4 1:4\1 n 46-48 49-51 52-54 55-57 58-60 o 61-63 64-66 67-69 70-72 73-75 a = numerator, the dilution level of the stock HBV solutio b = denomenator, the dilution level of the stock antibioti solution c = assay position in a sequential arrangement of 75 test tubes SUBSTITUTESHEET 53 Table 3 The volumes and distributions of each component of the checkerboard titration assay. Tube # TSB Antibiotic Venom Bacteria 00-0 2.5ml 1-3 500ul 2.0ml 4-6 250U1 250ul 1:16 2.0ml 7-9 250ul 250ul 1:8 2.0ml 10-12 250ul 250ul 1:4 2.0ml 13-15 250U1 250ul 1:2 2.0ml 16-18 250ul 250ul 1:16 2.0ml 19-21 250ul 1:16 250ul 1:16 2.0ml 22-24 250ul 1:16 250ul 1:8 2.0ml 25-27 250ul 1:16 250ul 1:4 2.0ml 28-30 250ul 1:16 250ul 1:2 2.0ml 31-33 250ul 250ul 1:8 2.0ml 34-36 250ul 1:8 250ul 1:16 2.0ml 37-39 250ul 1:8 250ul 1:8 2.0ml 40-42 250ul 1:8 250ul 1:4 2.0ml 43-45 250ul 1:8 250ul 1.-2 2.0ml 46-48 250ul 250ul 1:4 2.0ml 49-51 250ul 1:4 250ul 1:16 2.0ml 52-54 250ul 1:4 250ul 1:8 2.0ml 55-57 250ul 1:4 250ul 1:4 2.0ml 58-60 250ul 1:4 250ul 1:2 2.0ml 61-63 250ul 250U1 1:2 2.0ml 64-66 250ul 1 - 250ul 1:16 2.0ml 67-69 250U1 1 " 250ul 1:8 2.0ml 70-72 250U1 1:2 250U1 1:4 2.0ml 73-75 250U1 1:2 250ul 1:2 2.0ml SUBSTITUTESHEET 54 Table 4 The effect of 4 ug/ml HBV on the MIC's of eleven antibiotics on eight, gram-positive organisms. C« 1 9 9 9 9 9 f 0 8 0 0 " 0 0 . 7 1 5 7 5 8 2 7 Penicillin -5 +6 + — _ + + Methicillin + + + - - + + _ Ampicillin + + + - - + + - Cephalothin + + + + + + + - Gentamicin + + + - - — + — Kanamycin + + - - — + + - Erythromycin' + + - - — _ + — Chloramphenicol + + - - — — + + Clindamycin + + - - - - + — Tetracycline + + - - - — + - Vancomycin + + - — — — + — 1 Group "A" = two strains of S. aureus Group "B" = five strains of S. epider idis 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. SUBSTITUTESHEET 55 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 Q 1 4 1 C 1 1 3 9 7 0 0 3 2 3 3 Ampicillin +2 + + + Carbenicillin + + + + Piperacillin + + + + Cephalothin _3 Cefoxitin + + + + Cefamandole Moxalactam — + _ — Amikacin + + + + Genti icin + - + + 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. SUBSTITUTESHEET Table 6 56 Staphylococcus aureus Rif ampin= . Olug/ l or . OOlug/ml Honey Bee Venom=4ug/ιnl hours after innoculation 0 2 4 6 8 12 .046 .080 .850 1.17 > * ( * 1.34 Control .046 .073 .815 1.16 1.26 1 ~~io .046 .073 .815 1.16 1.26 1.35 AVERAGE .046 .075 .827 1.16 1.26 1.34 .046 .056 .140 .372 1.07 1.32 Rifampicin .046 .054 .068 .156 .625 1.34 .Olug/ml .046 .058 .112 .304 1.00 1.30 AVERAGE .046 .056 .107 .277 .898 1.32 .046 .081 .855 1.18 1.27 1.34 Rifampicin .046 .064 .765 1.16 1.26 1.34 .OOlug/ml .046 .072 .800 1.17 1.26 1.34 AVERAGE .046 .072 .807 1.17 1.26 1.34 .046 .062 .158 .705 1.20 1.29 Venom .046 .063 .284 .875 1.22 1.31 4ug/ml .046 .059 .068 .312 1.09 1.29 AVERAGE .046 .061 .170 .631 1.17 1.30 Rifampicin .046 .053 .078 .156 .665 1 1. -O y -Or .Olug/ml + .046 .055 .078 .162 .640 1.32 Venom 4ug/ml .046 .056 .062 .092 .332 1.32 AVERAGE .046 .055 .073 .137 .546 1.32 Rifampicin .046 .066 .068 .242 1.08 L . *c .OOlug/ml + .046 .063 .109 .485 1.19 1.34 Venom 4ug/ml .046 .067 .087 .381 1.16 1.33 AVERAGE .046 .065 .088 .369 1.14 1.33 SUBSTITUTESHEET Table 7 5 7 Pseudomonas aeruginosa Rifampicin=10ug/ml or 20ug/ml Honey Bee Venom=40ug/ml hours after innoculation 0 4 6 8 12 .033 .062 .735 1.00 1.02 1.00 Control .033 .069 .755 .955 1.00 .990 .033 .068 .775 .950 .990 .900 AVERAGE .033 .066 .755 .968 1.00 .963 .033 .078 .690 .890 .960 .980 Venom .033 .087 .687 .870 .960 .980 40ug/ml .033 .058 .685 .880 .953 .950 AVERAGE .033 .074 .685 .880 .953 .970 .033 .074 .630 .830 .885 .842 Rifampicin .033 .084 .672 .850 .895 .850 10 ug/ml .033 .082 .640 .830 .865 .832 AVERAGE .033 .080 .647 .837 .882 .841 .033 .053 .375 .660 .730 .730 Rifampicin .033 .056 .326 .645 .720 .730 20 ug/ml .033 .063 .380 .700 .760 .745 AVERAGE .033 .057 .351 .688 .737 .735 Rifampicin .033 .084 .452 .805 .860 .861 lOug/ml +n .033 .079 .475 .795 .820 .839 Venom 40ug/ml .033 .078 .490 .820 .860 .880 AVERAGE .033 .080 .466 .807 .847 .860 Rifampicin .033 .065 .180 .410 .580 .620 20ug/ml + .033 .082 .168 .375 .535 .620 Venom 40ug/nl .033 .058 .168 .373 .525 .612 AVERAGE .033 .068 .172 .386 .547 .617 SUBSTITUTESHEET Table 8 5 Escherichia coli Polymyxin B=6.25ϋnits/m.l and 3.125Units/ml . Bumblebee Venom=5ug/ml and 20ug/ml (Megabombus pennsyl vani cus) hours after innoculation 0 O 4 6 8 12 .030 .688 1.04 1.05 1.14 1.23 Control .030 .680 1.03 1.04 1.13 1.22 .030 .683 1.02 1.04 1.13 4 1. - *otC AVERAGE .030 .684 1.03 1.04 1.13 1.22 .030 .715 1.03 1.02 1.04 1.12 BB Venom .030 .712 1.03 1.04 1.04 1.14 5ug/ml .030 .730 1.03 1.03 1.04 1.13 AVERAGE .030 .719 1.03 1.03 1.04 1.13 .030 .672 1.03 1.03 1.04 1.13 BB Venom .030 .67 1.04 1.03 1.05 1.16 20ug/ml .030 .688 1.04 1.03 1.06 1.13 AVERAGE .030 .678 1.04 1.03 1.05 1.14 .030 .654 1.03 1.03 1.04 1.12 Pol B .030 .642 1.02 1.03 1.04 1.14 3.125Units/ml .030 .652 1.02 1.03 1.04 1.14 AVERAGE .030 .649 1.02 1.03 1.04 1.13 .030 .022 .102 .710 .960 1.03 Pol B .030 .024 .472 .940 .950 1.03 6.25U its/ml .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 3.125Units/ml .030 .008 .250 .910 1.02 1.06 BBV=5ug/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 6.25Units/ml .030 .009 .009 .008 .008 .012 BBV=20ug/ml .030 .011 .009 .008 .008 .013 AVERAGE .030 .009 .010 .008 .008 .013 SUBSTITUTESHEET 59 Table 9 Escherichia coli Polymyxi n B =3.125Units/ml Yel low acket Venoιn=5ug/ml (Vespula germanica) Baldfaced Hornet Veno =5ug/ml (Dol i chovespul a macul a ta ) hours after i nnocul atJ on 0 2 4 6 8 12 .038 .526 1.03 1.07 1.08 1.12 Control .038 .522 1.04 1.07 1.08 1.12 AVERAGE .038 .524 1.04 1.07 1.03 1.12 Pol B .038 .477 1.03 1.07 1.08 1.14 3.125U/ml .038 .482 1.03 1.07 1.08 1.14 AVERAGF .038 .480 1.03 1.07 1.08 1.14 YJ .038 .547 1.04 1.07 1-09 1.16 5ug/ml .038 .550 1.04 1.07 1.08 1.14 AVERAGF .038 .549 1.04 1.07 1.09 1.15 BF * » «038 .552 1.04 1.08 1.08 1.16 5ug/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 5U/ml .038 .029 .098 .850 1.06 1.12 AVERAGF .038 .029 .141 .893 1.07 1.13 BF 5ug/ml .038 .027 .118 .890 1.08 1.13 Pol B 5U/ml .038 .023 .096 .840 1.06 1.10 AVERAGE .038 .025 .107 .865 1.07 1.12 60 Table 10 Relative activity of analogues of proteinaceous or polypeptide components of Hymenoptera venoms. Analoque No. Relative Activity 1 20% 2 200% 5 300% 6 100% 7 20% 61 Table 11 Average optical densities (ODgβø) of bacterial cultures versus time and treatment 0 * hrs 2 hrs 4 hrs 6 hrs 8 hrs 12 hrs Contro 1 013 072 .831 1.08 1.10 1.17 Melittin - .013 .072 .827 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 uni ts 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 ϋ + .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 61a Table 12 Sequences of synthetic melittin analogues*. Natural Melittin Melittin (l-20)-Lys-Arg-Lys-Arg-Gln-Gln-NH2. Analogue #2 Melittin (l-20)-0rn-0rn-0rn-0rn-Gln-Gln-NH2. Analogue #4 Melittin (1-20) -Lys-Arg-Lys-Arg-Gly-Gly-NH 2 . Analogue #5 Mel ittln (l-20)-Arg-Arg-Arg-Arg-Gln-Gln-Nll2. Analogue #6 Mel ittln (l-20)-Lys-Lys-Lys-Lys-Gln-Gln-NH2. Analogue #7 Melittin (l-20)-Gly-Gly-Gly-Gly-Gln-Gln-NH 2 . * Amino acids shown in bold print represent alterations from the native melittin sequence. 61b Tab 1 e 1 Opt ical dens ities (ODgβø) of bacterial cultures for treatments versus time. Values represent the mean over the SEM In parenthesis (n=6). Hours after culture inoculation J 2 4 6 8 lø 12 Control ,007 .088 .692 1.01 1.05 1. 6 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 Pθl B ( .001) ( .003) ( .017) ( .118) ( .077) ( .025) ( .022) Synthetic + .007 .088 .018 .143 .662 .881 1 01 Pol B ( -001) ( .003) ( .004) ( .057) ( .181) ( .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 t'BBl) ( .003) (.009) ( .118) ( .146) ( .021) ( .022) Analog#4 + .007 .088 ,020 .023 .094 .533 860 P0l B (.flffiTl) (.003) (.004) (.001) ( .034) ( .157) (.125) Analog#5 + .007 .088 .036 .036 .038 .334 687 Pθl B t- β ai) (.003) (.001) (,øøl) (.004) ( .096) (.150) Analog#β + .007 .088 .020 .180 .793 1.00 i g 4 Pol B (.001) ( .003) (.007) ( .062) (.071) ( .025) (.023) Analog#7 + .007 .088 .148 .818 1.02 1.05 1.06 Pol B ( .001) (.003) (.028) (.041) (.021) ( .028) (.028) Table A-l The checkerboard assay results of ampicillin and H oneybee venom verus . aureus . -U E. MEAN A 66p S.D. TIME MEAN Aecn S .D. AMP-O.HBV-0 TO AMP-O.H:BV-2 0.013 0.002 T2 TO 0.013 0.002 0.085 0.018 T4 T2 0.085 0.018 0.573 0.178 T6 T4 0.213 0.135 1.102 0.159 T8 T6 0.844 0.311 1.223 0.101 T12 T8 1.119 0.193 1T213 0.307 T24 T12 1.198 0.306 1.329 0.069 T24 1.295 0.208 AHP-O.HBV-4 TO AMP-0,HBV-8 0 013 0.002 T2 TO 0. 0.013 0.002 086 0.018 T T2 0. 0.085 065 0.018 0.040 T6 T4 0. 0.026 217 0.019 0.181 T8 T6 0. 0,014 671 0.012 0.412 T12 T8 0.027 1. 0.036 147 0.317 T24 T12 1.028 1.278 0.273 0.165 T24 1.291 0.119 AMP-0.HBV-16 O AMP-0.05 .HBV-0 0 . 013 0.002 T2 TO 0.013 0.002 0 . 085 0.018 T4 T2 0.085 0.018 0 . 025 0.011 T6 T4 0.355 0.073 0 . 007 0.004 T8 T6 0.552 0.195 0 . 006 0.004 T12 T8 0.689 0.146 0 . 077 0.173 T24 T12 0.736 0.135 0 . 857 0.576 T24 0.760 0.114 AMP-0.05,HBV-8 TO AMP-0.05 , HBV-16 0.013 0.002 T2 O .0.013 0.085 0.002 0.018 T4 T2 0.085 0.018 0.026 0.021 T6 T4 0.025 0.012 0.009 0.012 T8 T6 0.006 0.008 0.004 0.007 T12 T8 0.007 0.004 0.009 0.004 T24 T12 0.008 0.331 0.005 0.395 T24 0.013 0.004 63 Table A-l (continued. Table A-l (continued.) TIME MEAN Λ 660 S.D, TIME H M A 660 S.D AMP-O. 4.HBV-0 TO AMP-O. 4.HBV-2 0.013 0 002 TO T2 0. 0.013 0 085 002 0 018 T4 T2 0, 0.085 191 0 018 0, 042 T6 T4 0 0.098 ,110 0 054 027 T8 T6 0 0.061 048 0 041 019 T8 T12 0, 0.034 0, 027 027 009 T24 T12 0.020 0, 0.027 Oil 005 T24 0.018 0.008 AMP-0.4.HBV-4 TO AMP-0.4.HBV-8 0.013 0.002 T2 TO 0.013 0.085 0 002 0.018 T4 T2 0.085 0.040 0 018 0.028 T6 T4 0.023 0.028 0 015 0.023 T8 T6 0.010 0.019 0 006 0.017 T12 T8 0.006 0.012 0 004 0.004 T24 T12 0.008 0.009 0 005 0.007 T24 0.010 0, 006 AMP-0.4.HBV-16 O 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 Table A- 2 The checkerboard as say results of kanamyc in and honeybee venom verus S.. aureus . TIME MEAN A«sn S.D. TIME MEAN Afifin S.D. KANA-0 ,HBV-0 KANA-0 ,HBV-2 TO 0.024 0.005 O 0.024 T2 0.005 0.094 0.012 T2 T4 0.095 0.011 0.854 0.157 T4 0.542 T6 0.183 1.219 0.052 T6 T8 1.132 0.146 1.275 0.032 T8 1.275 T12 0.042 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 TO 0.024 0.005 TO 0.024 T2 0.005 0.094 0.012 T2 0.094 T4 0.012 0.154 0.131 T4 T6 0.036 0.017 0.630 0.391 6 T8 0.062 0.048 1.100 0.233 T8 T12 0.571 0.403 1.322 0.048 T12 T24 1.275 0.062 1.405 0.040 T24 1.389 0.057 KANA-0, HBV-16 KANA-1. O 25.HBV-0 0.024 0.005 TO T2 0.024 0.005 0.094 0.012 T4 T2 0.094 0.012 0.029 0.014 T4 T6 0.747 0.125 0.020 0.008 T6 T8 1.199 0.060 0.066 0.078 T8 T12 1.269 0.043 0.666 0.556 T24 T12 1.315 0.046 1.336 0.195 T24 1.355 0.042 KANA-1.: 25.HBV-2 TO KANA-1. 25.HBV-4 0.024 0.005 TO T2 0.024 0.005 0.094 0.012 T4 T2 0.094 0.012 0.428 0.197 T4 T6 0.107 0.060 0.929 0.369 T8 T6 0.310 0.289 1.174 0.116 T8 T12 0.694 0.422 1.290 0.048 T24 T12 1.231 0.107 1.373 0.035 T24 1.350 0.077 £ANA-1.25,HBV-8 TO KANA-1. : 25.HBV-16 0.024 0.005 TO T2 0.024 0.005 0.094 0.012 T4 T2 0.094 0.012 0.039 0.014 T6 T4 0.030 0.031 0.014 0.011 T8 T6 0.017 0.009 0.095 0.129 T8 T12 0.018 0.012 0.712 0.487. T24 T12 0.179 0.344 1.343 0.096 T24 1.124 0.357 Table A-2 (continued.) 67 Tab le A- 2 ( continued. ) HUE. MEAN-A660 S.D. TIME MEAN Atx t- c. S.D. KANA-10.HBV-0 .HA-10 .HBV-2 TO 0 024 0.005 TO 0.024 0.005 T2 0, 094 0.012 T2 0.094 0.012 T4 0, 279 0.054 T4 0.167 T6 0.089 0, 359 0.063 T6 0.183 T8 0.112 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 HA-10, HBV-8 TO 0.024 0.005 TO 0.024 T2 0.005 0.094 0.012 T2 0.094 T4 0.012 0.064 0.023 T4 0.041 T6 0.023 0.054 0.021 T6 T8 0.027 0.019 0.052 0.024 T8 0.023 T12 0.019 0.314 0.299 T12 T24 0.022 0.018 1.193 0.080 T24 0.836 0.412 KANA-10.HBV-16 TO 0.024 0.005 T2 0.094 0.012 T4 0.031 0.014 T6 0.020 0.009 T8 0.014 0.010 T12 0.015 0.013 T24 0.614 0.567 Tab l e A - 3 Th e c he ckbo ard assay results of polymyxin B and honeybee venom verus E_. coli. TIME MEAN S.D TIME MEAN As6Q S .D, POLY B ■ =0,HBV-0 POLY B-0 TO HBV-2 0.006 0.002 TO T2 0.006 0 002 0.074 0.004 T2 T4 0 074 0 004 . 0.785 0.061 T4 T6 0, 195 0 116 1.243 0.011 T6 T8 0.886 0.304 1.295 0.024 T8 T12 1.264 0, 027 1.343 0.018 T24 T12 1.316 0, 026 1.396 0.023 T24 1,405 0.020 POLY B-O.HBV-4 TO POLY B-O.HBV-8 0.006 0.002 TO T2 0.006 0 002 . 0.074 0.004 T4 T2 0.074 0 004 0.038 0.013 T6 T4 0.018 0 008 0.070 0.046 T8 T6 0.012 0, 014 0.589 0.235 T8 T12 0.022 0, 012 1.315 0.081 T24 T12 0.769 0, 503 1.415 0.024 T24 1.405 0.028 POLY B- 0.HBVOM-16 TO POLY B- 312.HBV-0 0.006 0.002 T2 TO 0.006 0 022 0.074 0.004 T4 T2 0, 074 0 004 0.015 0.007 T6 T4 0, 526 0 138 0.006 0.003 T8 T6 1.046 0, 269 0.007 0.004 T8 T12 1.244 0, 057 0.012 0.005 T24 T12 1.305 0.051 0.457 0.566 T24 1.429 0.053 POLY B=: 312.HBV-2 TO POLY B- 312 ,HBV-4 0.006 0.002 T2 TO 0.006 0 0.074 002 0.004 T4 T2 0 074 0 004 0.167 0.071 T6 T4 0 023 0 013 0.795 0.231 T8 T6 0.064 0 132 1.195 0.117 T12 T8 0, 216 0, 1.303 357 0.041 T24 T12 0, 812 0 1.422 513 0.066 T24 1.415 0, 040 POLY B-312.HBV-8 TO POLY B-312.HBV-16 0.006 0.002 T2 TO 0 006 0.074 0.002 0.004 T4 T2 0 074 0.014 0.004 0.005 T6 T4 0 023 0.007 0.008 0.005 T8 T6 0 0.011 013 0.004 0.005 T12 T8 0, 0.384 013- 0.004 0.383 T24 T12 0, 031 1.294 0.048 0.393 T24 0. 334 0.579 Table A-3 (continued.) IIME AN_A660 S.D. TIME MEAN S.D. POLY B-625.HBV-0 POLY B- -625.HBV-2 TO 0.006 0 ,002 TO 0.006 T2 0.002 0.074 0,,004 T2 0.074 T4 0.004 0.330 0.117 T4 0.165 0.076 T6 0.766 0.386 T6 0.553 T8 0.267 1.048 0.314 T8 ' 1.037 0.260 T12 1.238 0.125 T12 T24 1.261 0.067 1.401 0.123 T24 1.405 0.075 POLY B-625.HBV-4 TO POLY B-625.HBV-8 0 006 0.002 TO 0 T2 007 0 0. .002 074 0.004 T2 T4 0, 074 0 0. .004 025 0.011 T4 T6 0, 015 0 .005 0.030 0.034 T6 T8 0.009 0 .004 0.073 0.128 T8 T12 0. Oil 0 .005 0.627 0.428 T12 T24 0.051 0 .062 1.405 0.050 T24 1.323 0 .307 POLY B-625.HBV-16 TO POLY B«1250,HBV-0 0.006 0.002 T2 O 0 006 0 .002 0.074 0.004 T4 T2 0 074 0 .004 0.039 013 T6 T4 0 159 0 .032 0.023 008 T8 T6 0, 172 0 .093 0.022 007 T8 T12 0.259 0 .261 0.Q22 007 T24 T12 0.778 0 .437 0.294 538 T24 1.362 0, .094 POLY B-1250.HBV-2 TO POLY B-1250.HBV-4 0.006 0.002 T2 TO 0.006 0. 0.074 .002 0.004 T4 T2 0.074 0. .004 0.110 0.043 T6 T4 0.038 0. ,012 0.115 0.085 T8 T6 0.020 0. .009 0.203 0.237 T8 T12 0.018 0. 006 0.552 0.557 T24 T12 0.033 0. 042 1.207 0.487 T24 1.150 0. 449 POLY B-1250.HBV-8 TO 0 POLY B-1250.HBV-16 006 0.002 T2 O 0 0 006 074 0. 002 0.004 T4 T2 0, 0, 074 028 0. 004 0.010 T6 T4 0. 0, 071 019 0. 014 0.007 T8 T6 0. 0. 054 019 0. 012 0.006 T12 T8 0. 0. 046 021 0. 009 0.010 T24 T12 0.036 1.013 0. 006 0.556 T24 0.223 0. 440 Table A-3 (continued.) IIME. MEAN_£660 S_ IME MEAN_A 660 S.D. POLY B-2500.HBV-0 TO POLY B-2500 f HBV-2 0 006 0.002 T2 TO 0 0, 006 074 0.002 0.004 T4 T2 0, 074 0.123 0.004 0.013 T6 T4 0.107 0.109 0.022 0.019 T8 T6 0.085 167 0.021 0.276 T12 T8 0.072 075 0.020 0.010 T24 T12 0.056 037 0.013 0.423 T24 0.879 0.530 POLY B-2500.HBV-4 TO POLY B-2500.HBV-8 0.006 0.002 T2 TO 0.006 0.074 0.002 0.004 T4 T2 0.074 0.080 0.004 0.013 T6 T4 0.070 0.065 0.020 0.013 T8 T6 0.067 0.057 0.010 0.008 T12 T8 0.058 0.049 0.015 0.011 T24 T12 0.052 0.416 0.007 0.491 T24 0.301 0.524 POLY B-2500.HBV-16 TO 0.006 0.002 T2 0.074 0.004 T4 110 0.009 T6 091 0.008 T8 078 0.009 T12 061 0.006 T24 210 0.425 71 Table A-4 The checkerboard assay results of ampicillin- and honeybee venom verus E_. coli. HME. HEAN_A660 S_ TIME MEAN S.D, AMP-O,HBV-0 TO AHP-0.HBV-5 0.015 0.015 T2 TO 0.015 0.084 0.015 0.032 T4 T2 0.084 0.644 0.032 0.098 T6 T4 0.624 1.053 0.102 0.067 T6 T8 1.049 0.081 1.071 0.071 T8 1.070 T12 0.078 1.144 0.075 T12 1.146 T24 0.098 1.244 0.101 T24 1.258 0.128 AMP-0,HBV-10 TO A P-O,HBV-20 0.015 0.015 T2 TO 0.015 0.084 0.015 0.032 T4 T2 0.084 0.646 0.032 0.103 T6 T4 0.643 1.056 0.132 0.085 T8 T6 1.031 1.066 0.088 0.091 T12 T8 1.052 1.154 0.097 0.110 T24 T12 1.127 1.260 0.113 0.139 T24 1.244 0.155 AMP-O .HBV-40 TO AMP-0.5 ,HBV-0 0.015 0, 015 T2 TO 0.015 0.084 0. 0.015 032 T4 T2 0.084 0.587 0. 0.032 204 T6 T4 0.600 1.026 0. 0.099 092 T8 T6 1.001 1.050 0. 0.078 094 T12 T8 0.999 1.119 0. 0.101 111 T24 T12 1.085 1.210 0.111 0.167 T24 1.156 0.222 AMP-0.5 HBV-5 TO AMP-0.5 .HBV-10 0.015 0 015 T2 O 0.015 0.084 0, 0.015 032 T4 T2 0.084 0.603 0. 0.032 099 T6 T4 0.624 0.998 0.111 0.095 T8 T6 1.001 1.011 0. 0.097 097 T12 T8 1.013 1.099 0. 0.100 120 T24 T12 1.100 1.215 0. 0.136 159 T24 1.219 0.176 AMP-0.5 HBV-20 TO ft.MP-0.5, HBV-40 0.015 0 015 T2 O 0.015 0.084 0, 0.015 032 T4 T2 0.084 0.614 0.148 0.032 T6 T4 0.980 0.508 0.094 0.205 T8 T6 0.961 0.993 0.093 0.097 T12 T8 1.073 0.991 0. 0.098 123 T24 T12 1.182 1.063 0.155 0.138 T24 1.162 0.172 Table A-4 (continu ed.) Table A-4 (continued.) TIME MEAN A660 S.D, TIME — MEAN ϋO o u -> .-, . AMP-4,HBV-0 AMP-4.HBV -5 TO 0 .015 0.015 TO T2 0.015 0, 0.015 .084 0.032 T2 T4 0.084 0. 0.032 ,158 0.118 T4 T6 0.154 0. 0.108 063 0.019 T6 T8 0.076 0. 126 0.037 0.230 T8 T12 0.084 0.044 0.055 0.023 T12 T24 0.057 0.022 .0.056 0.015 T24 0.076 0.071 AMP-4.HBV-10 TO AHP-4.HBV. -20 0.015 0.015 TO T2 0.015 0.015 0.084 0.032 T4 T2 0.084 0.032 0.128 0.092 T4 T6 0.090 0.070 '' 0ι £ 075 0.039 T6 T8 0.066 0.043 '0:074 0.045 T8 T12 0.066 0.045 0.066 0.034 T24 T12 0.050 0.031 0.063 0.032 T24 0.052 0.026 AMP-4.HBV-40 TO 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 Table A-5 The checkerboard assay results of kanamycin and h oneybee venom verus E.. coli. HUE MEAN Ag 60 s_ TIME S.D. KANA-0,HBV-0 TO KANA-0 .HBV-5 0.025 0.009 T2 TO 0.119 0.025 0.009 0.028 T4 T2 0.701 0.118 0.028 0.136 T6 T4 0.980 0.726 0.108 0.075 T8 T6 0.988 1.002 0.065 0.068 T12 T8 1.062 1.028 0.063 0.090 T24 T12 1.144 1.104 0.084 0.119 T24 1.191 0.101 KANA-0,HBV-10 TO KANA-0 , ,HBV-20 0.025 0, 009 T2 TO 0.025 0.119 0, 0.009 028 T4 T2 0.119 0.747 0. 0.028 108 T6 T4 0.764 1.005 0. 0.087 073 T8 T6 1.001 1.028 0. 0.060 065 T12 T8 1.099 1.026 0. 0.063 094 T24 T12 1.188 1.094 0. 0.090 114 T24 1.198 0.102 KANA-0.HBV-40 TO KAN A- 5 , 0.025 HBV-0 0.009 T2 TO 0.119 0.025 0.009 0.028 T4 T2 736 0.124 0.033 T6 0.075 T4 984 0.473 0.120 T8 0.064 T6 005 0.800 0.132 0.062 T12 T8 080 0.889 0.081 0.080 T24 T12 163 0.930 0.091 0.103 T24 1.019 0.119 75 Table A-5 (continued.) HUE. £EAN_A660 S.D. TIME MEAN A* en S.D. KANA-10,HBV-0 KANA-10 .HBV-5 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 T4 0.263 0.114 T4 0.267 0.135 T6 0.417 0.209 T6 0.414 0.242 T8 0.576 0.222 T8 0.563 0.248 T12 0.814 0.084 T12 0.807 0.098 T24 0.878 0.095 T24 0.894 0.095 KANA-10.HBV-10 KANA-10 .HBV-20 TO 0.025 0.009 TO 0.025 0.009 T2 0.119 0.028 T2 0.119 0.028 T4 0.258 0.142 T4 0.257 0.153 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 TO 0 025 0. 009 TO 0.025 T2 0.009 0, 119 0.028 T2 T4 0.119 0.028 0.258 0.176 T4 0.161 T6 0.054 0.356 0.303 T6 0.161 T8 0.065 0.494 0.292 T8 0.170 T12 0.079 0.784 0.147 T12 0.268 0.108 T24 0.9Q6 0.103 T24 0.631 0.103 KANA-20.HBV-5 KANA-20, TO HBV-10 0 .025 0.009 TO T2 0.025 0.009 0.119 0.028 T2 T4 0.119 0.028 0,.156 0.072 T4 T6 0.144 0.075 0..133 0.083 T6 T8 0.095 0.069 0.,119 0.086 T8 T12 0.085 0.063 0.233 0.122 T12 T24 0.209 0.081 0.678 0.112 T24 0.667 0.100 KANA-20.HBV- ■ 20 KANA-20, TO HBV-40 0 025 0.009 TO T2 0.025 0.009 0 119 0.028 T2 T4 0.119 0.028 0 128 0.081 T4 T6 0.103 0.074 0, 078 065 T6 T8 0.063 0.051 0.151 128 T8 0.063 T12 0.048 0.174 061 T12 T24 0.179 0.083 0.692 113 T24 0.716 0.087 Table A-5 (continued.) 77 Table A-6 The checkerboard assay results of polymyxin E and honeybee venom verus E.. coli . TIME MEAN A t- c- t, S.D. TIME MEAN Afi ς, Q S ,D POLY B-( ) ,HBV-0 POLY B-0 : .HBV-5 TO 0.012 0.005 TO 0.012 0.005 T2 0.040 0.004 T2 0.040 0.004 T4 0.506 0.076 T4 0.529 0.080 T6 1.011 0.110 T6 1.018 0.116 T8 1.043 0.096 T8 1.049 0.095 T12 1.103 0.116 T12 1.113 0.119 T24 1.201 0.137 T24 1.227 0.150 POLY B-0 .HBV-10 POLY B-O.HBV-20 O . 0.012 0.005 TO 0.012 0 005 T2 0.040 0.004 T2 0.040 0 004 T4 0.557 0.087 T4 0.544 0 T6 061 1.010 0.130 T6 1.005 0 T8 117 1.049 0.100 T8 1.040 0 102 T12 1.104 0.142 T12 1.092 0 T24 139 1.228 0.162 T24 1.217 0 157 POLY B-O .HBV-40 POLY B-l.5,HBV-0 TO 0.012 0.005 TO 0.012 T2 0 . 005 0.040 0.004 T2 0 040 T4 0 . 004 0.439 0.058 T4 0 411 T6 0 . 078 0.992 0.129 T6 0 984 T8 0 . 105 1.036 0.116 T8 1, 020 T12 0 . 091 1.082 0.139 T12 1, 075 T24 0 . 107 1.188 0.157 T24 1.200 0 . 141 POLY B-l. 5.HBV-5 • TO POLY B-1.5.HBV-10 0.012 0.003 TO T2 0.012 0.005 0.040 0.004 T2 0 T4 040 0 004 0.176 0.039 T4 0 T6 160 0 078 0.851 0.142 T6 0 837 T8 0 133 1.012 0.196 T8 1, 015 0 T12 091 1.068 0.093 T12 1.073 T24 0, 134 1.200 0.063 T24 1.203 0.145 POLY B-l. 5.HBV-20 TO POLY B-1.5.HBV-40 0.012 0.005 TO 0.012 T2 0.005 0.040 0.004 T2 0. T4 .040 0 004 0.058 0.026 T4 0. .024 T6 0 010 0.507 0.196 T6 0. T8 .147 0 262 0.948 0.128 T8 0. .438 0 T12 390 1.046 0.120 T12 1. .016 0 T24 102 1.201 0.129 T24 1. .153 0 143 Table A-6 (continued.) Table A-6 (continued.) TIME MEAN Afifin S.D TIME MEAN_A.660 s • D - POLY B-12,HBV-0 POLY B-12.HBV-5 TO 0. 012 0.005 TO 0.012 0.005 T2 0, 040 0.004 T2 0.040 0.004 T4 0.018 0.005 T4 0.025 0.005 T6 0 Oil 0.005 T6 0.016 0.006 T8 0 009 0.003 T8 0.011 0.005 T12 0 075 0.150 T12 0.010 0.005 T24 0 472 0.498 T24 0.196 0.361 POLY B-12.HBV-10 POLY B-12,HBV-20 TO 0 012 0.005 TO 012 005 T2 0 040 0.004 T2 040 004 T4 0 029 0.004 T4 030 006 T6 0.017 0.005 T6 017 ,006 T8 0.012 0.003 T8 013 .003 T12 0.024 0.051 T12 0.012 0 004 T24 0.201 0.352 T24 0.073 0 184 POLY B-12.HBV-40 TO 0. 012 005 T2 0.040 004 T4 0, 048 007 T6 0.022 ,006 T8 0.016 ,005 T12 0, 015 .006 T24 0 047 .085 Table A-7 The checkerboard assay results of ampicillin and honeybee venom verus kanamycin resistant S.. aureus . HME. MEAN A 660 S.D. TIME MEAN ARΛΠ S .D. AMP-0, HBV-0 AMP-0, HBV-2 TO 0.020 0.016 TO 0.020 0.016 T2 0.064 0.020 T2 0.064 0.020 T4 0.382 0.155 T4 0.150 0.134 T6 0.885 0.173 T6 0.533 0.286 T8 1.108 0.041 T8 0.937 0.207 T12 1.191 0.035 T12 1.167 0.038 T24 1.233 0.049 T24 1.217 0.041 AMP-0,HBV-4 AMP-0, HBV-8 TO 0.020 0, 016 TO 0.020 T2 0.064 0.016 0.020 T2 0.064 T4 0.038 0.020 0.021 T4 0.032 T6 0.040 0.019 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 TO , HBV-0 0.020 0.016 TO T2 0.020 .0.016 0.064 0.020 T4 T2 0.064 0.020 0.033 0.016 T6 T4 0.230 0.054 0.013 0.005 T8 T6 0.338 0.076 0.007 0.004 T8 T12 0.372 0.144 0.008 0.004 T24 T12 0.352 0.220 0.126 0.305 T24 0.461 0.139 AMP-0.05,HBV-2 TO AMP-0.05, ,HBV-4 0.020 0.016 T2 O 0.020 0.016 0.064 0.020 T4 T2 0.064 0.020 0.112 0.099 T6 T4 0.044 0.025 175 0.144 T8 T6 0.031 190 0.021 0.153 T12 T8 0.025 0.018 130 0.131 T24 T12 0.018 0.012 440 0.260 T24 0.581 0.239 AMP-0.05,HBV-8 TO AMP- 0.05, HBV-16 0.020 0.016 TO T2 0.020 0.016 0.064 0.020 T4 T2 0.064 0.020 0.025 0.013 T4 T6 0.035 0.016 0.013 0.009 T8 T6 0.013 0.004 0.008 0.005 T12 T8 0.008 0.004 0.010 0.0Q7 T24 T12 0.008 0.004 0.150 0.295 T24 0.011 0.002 81 Table A-7 (continued.) TIME IME MEAN_A660 S.D. AMP-0. 1,HBV-0 TO AMP-0. l.HBV-2 0.020 0.016 T2 TO 0 .020 0.064 0.016 0.020 T4 T2 0.064 0.379 0.020 0.430 T6 T4 0, 0.156 ,109 0.079 0.045 T8 T6 0.108 0.112 0.063 0.038 T12 T8 0. 0.050 075 0.011 0.037 T24 T12 0.053 0.037 0.023 0.009 T24 0.052 0.034 AMP-0.: L.HBV-4 TO AMP-0.1.HBV-8 0.020 0.016 T2 TO 0.064 0.020 0.020 0.016 T4 T2 0.045 0.064 0.020 0.029 T6 T4 0.030 0.030 0.018 0.022 T8 T6 0.023 0.015 0.016 0.008 T12 T8 0.018 0.010 0.004 0.012 T24 T12 0.044 0.008 0.004 0.102 T24 0.011 0.004 AMP-0.2, ,HBV-2 TO 0.020 AMP-0.2.HBV-4 0.016 T2 TO 0 0.064 020 0.020 0.016 T4 T2 0 0.071 064 0.052 0.020 T6 T4 0 0.062 047 0.047 0.029 T8 T6 0, 0.039 033 0.026 0.026 T12 T8 0, 0.018 024 0.011 0.018 T24 T12 0.017 0.017 0.010 0.010 T24 0.068 0.212 Table A-7 (continued.) TIME MEA ■ N- ■ ■ ϋO c ,11 o . u . TIME M - ■ EAN ϋD O AMP- 0.4 , HBV-0 AH P-O. TO ,HBV-2 0.020 0.016 TO T2 0.020 0.016 0.064 0.020 T2 T4 0.064 0.020 0.202 0.184 T4 T6 0.080 0.290 0.055 0.415 T6 T8 0.073 0.285 0.044 0.472 T8 T12 0.043 0.271 0.023 0.514 T24 T12 0.021 0.277 0.012 0.530 T24 0.020 0.012 AMP-0.4, , HBV-4 TO AMP- 0.4, HBV-8 0.020 0.016 T2 TO 0.020 0.064 0.016 0.020 T4 T2 0.064 0.044 0.020 0.026 T6 T4 0.030 0.028 0.019 0.016 T8 T6 0.015 0.021 0.008 0.011 T8 T12 0.008 0.014 0.005 0.006 T24 T12 0.008 0.011 0.005 0.003 T24 0.011 0.003 AMP-0. , HBV-16 TO 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 83 Table A- 8 The checkerboard assay results of kanamycin and honeybee venom verus kanamycin resistant S.. ajurreus . KANA-0, HBV-0 TO KANA-0 ,HBV-2 0.016 0 005 T2 TO 0.015 0.047 0.005 0.009 T4 T2 0 0.047 636 0.009 0.151 T6 T4 1 0.187 246 0.116 0.026 T8 T6 1 0.980 331 0.205 015 T12 T8 1 1.056 356 0.481 025 T24 T12 1 1.100 417 0.498 039 T24 1.418 0.020 . Table A-8 (continued.) HME MEAN A 660 s_ TIME MEAN KANA-10,HBV-0 TO KANA-1C 0.016 I.HBV-2 0.005 T2 TO 0.047 0.015 0.004 T4 0.009 T2 0.135 0.047 0.065 0.009 T6 T4 0.172 0.045 0.080 0.026 T8 T6 0.200 0.044 0.086 0.029 T12 T8 0.397 0.043 0.186 0.031 T24 T12 1.164 0.185 0.145 0.182 T24 1.056 0.412 85 Table A-8 (continued, HUE MEAN -ego S .D . TIME MEAN_A.660 S.D KANA-40,HBV-0 TO KANA-40.HBV-2 0 016 0.005 TO T2 0.015 0 004 0, 047 0.009 T2 T4 0.047 0, 009 0.116 0.057 T4 T6 0.048 021 0 ,146 0.069 T6 T8 0.047 021 0 161 0.075 T8 T12 0.043 020 0 184 0.084 T12 T24 0.049 022 0 697 0.396 T24 0.692 463 KANA-40.HBV-4 TO KANA-40.HBV- 0.015 0.004 TO T2 0.015 0 004 0.047 0.009 T4 T2 0.047 0 009 0..033 0.023 T6 T4 023 0 Oil 0.029 0.016 T6 T8 017 0 007 0.026 0.015 T8 T12 015 0 008 0.043 0.068 T24 T12 016 0 007 0.433 0.401 T24 0.113 0, 176 KANA-40.HBV-16 TO 0.015 0.004 T2 0.047 0.009 T4 0.023 0.011 T6 0.017 0.008 T8 0.016 0.008 T12 0.019 0.007 T24 0.023 0.009 Table A-9 The checkerboard assa _y, resu * lt • . « ε » W o J f. p (lu o i lyymmyyxx i inn B and honeybee venom verus kanamycin resistant JS.. aur 'eus HUE MEAN .Agfip s_ ∑IM MEAN_A660 S_ POLY B-0,HBV-0 TO POLY B-0 0.009 I.HBV-2 0.003 T2 TO 0.068 0.009 0.004 0.003 T4 T2 0.329 0.068 0.079 0.004 T6 T4 0.726 0.178 0.149 0.042 T8 T6 0.887 0.621 0.107 0.122 T12 T8 1.020 0.851 0.078 0.112 T24 T12 1.027 1.065 0.093 0.072 T24 1.106 0.083 POLY B-0,HBV-4 TO 0 POLY B-0 009 ,HBV-8 T2 0.003 0, TO 068 0.009 0.003 T4 0.004 0. T2 046 0.068 0.004 T6 0.019 0. T4 050 0.023 0.013 T8 0.020 0. T6 162 0.012 0.087 0.011 T12 0. T8 921 0.007 0.053 0.003 T24 T12 1.029 0.138 0.090 0.142 T24 1.038 0.068 POLY B-O.HBV-16 TO 0 POLY B-12 009 !.5,HBV-0 0 T2 ,003 0 TO 068 0.009 0 0.003 T4 004 0, T2 034 0.068 0.004 T6 0, 010 0. T4 013 0.266 0. 0.051 T8 004 0. T6 010 0.640 0. 0.120 T12 002 0.011 T8 0.826 0. T24 003 0.110 0. T12 142 0.976 0.268 0.095 T24 0.962 0.074 POLY B-12.5.HBV-2 TO 0.009 POLY B-12 •5.HBV-4 0 T2 .003 0.068 TO 0.009 0 T4 .004 0.003 T2 0.132 0.068 0, T6 .039 0.004 T4 0.490 0.035 0. T8 ,142 0.012 T6 0.742 0.024 0. T12 ,196 0.007 T8 1.027 0.039 0. 093 0.018 T24 T12 1.083 0.684 0. 063 0.171 T24 0.996 0.077 POLY B-12.5.HBV-8 TO 0 POLY B-12, ,009 .5.HBV-16 T2 0.003 0 TO 068 0.009 T4 0.004 0.003 0, T2 023 0.068 T6 0.012 0.004 0. T4 010 0.036 0.012 T8 0.005 0. T6 007 0.013 0.003 0.004 T12 0. T8 050 0.009 0.056 0.004 T24 0. T12 993 0.011 0.073 0.004 T24 0.161 0.202 Table A-9 (continued.) TIME MEAN ggp S.D. TIME MEAS_A660 S.D. POLY B-25,HBV-0 POLY B- -25.HBV-2 TO 0.009 0.003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.243 0.033 T4 0.123 0.037 T6 0.629 0.073 T6 0.375 0.130 T8 0.835 0.114 T8 0.619 0.228 T12 1.008 0.096 T12 1.994 0.088 T24 1.048 0.091 T24 1.075 0.046 POLY B-25.HBV-4 POLY B- -25.HBV-8 TO 0, 009 0.003 TO 0.009 0.003 T2 0, 068 0.004 T2 0.068 0.004 T4 0.034 0.013 T4 0.022 0.012 T6 0.018 0.007 T6 0.009 0.003 T8 0.024 0.012 T8 0.007 0.003 T12 0.489 0.198 T12 0.016 0.014 T24 0.973 0.093 T24 0.906 0.171 POLY B-25.HBV-16 POLY B- ■ 50, HBV-0 TO 0, 009 0, 003 TO 0.009 0.003 T2 0.068 0.004 T2 0.068 0.004 T4 0.035 0.013 T4 0.208 0.034 T6 0.015 0.008 T6 0.376 0.123 T8 0.009 0.003 T8 0.567 0.192 T12 0.Oil 0.004 T12 0.841 0.115 T24 0.178 0.291 T24 0.968 0.048 POLY B-50.HBV-2 POLY B- 50.HBV-4 TO 0.009 0 003 TO 0.009 0.003 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 TO 0.009 0.003 TO 0.009 T2 0.003 0.068 0.004 T2 0.068 T4 0.004 0.021 0.010 T4 0.038 T6 0.012 0.009 0.002 T6 0.015 T8 0.005 0.006 0.003 T8 0.011 T12 0.005 0.011 0.004 T12 0.012 T24 0.004 0.807 0.222 T24 0.023 0.028 Table A-9 (continued.) HME HEAN_A660 S_ TIME MEAN A660 S.D POLY B-100,HBV-0 TO 0.009 POLY B-100,HBV-2 0 003 T2 TO 0.068 0.009 0, 004 0.003 T4 T2 0.243 0.068 0.033 0.004 T6 T4 0.629 0.123 0.037 T8 0.073 T6 0.835 0.375 0.114 0.130 T12 T8 1.008 0.619 0.096 0.228 T24 T12 1.048 1.994 0.091 0.088 T24 1.075 0.046 POL B-100.HBV-4 TO 0.009 POLY B-100.HBV-8 0 003 T2 TO 0.068 0.009 0 004 0.003 T4 T2 0.034 0.068 0, T6 013 0.004 T4 0.018 0. 022 T8 007 0.012 T6 0.024 009 0.012 0.003 T12 T8 0.489 0. 007 198 0.003 T24 0.973 T12 016 0.093 0.014 T24 906 0.171 POLY B-100.HBV-16 TO 0 009 0 T2 003 0, 068 0 T4 004 0.042 0, T6 012 0.020 0, T8 006 0.015 0. T12 005 0.014 0. T24 004 0.106 0.232 Tablθ A'10 -elittin and out kanamycin nigie treatment model . 2 no treatment 2.96 2 4.16 3.77 me 1 i tt in - 50ng 3.89 3.39 2 2.79 2.58 polymyxin B - 2ug 2.88 3.88 2 3.0β 3.34 mel 50ng + pol 2ug 3.27 2.38 0.00 2.62 2.15 3 no treatment 2.34 3 2.62 2.51 melittin - 50ng 1.90 3.52 2.34 3 1.61 polymyxin B - 2ug 3.08 3.08 3.11 3 2.91 mel 50ng + pol 2ug 0.00 2.41 1.32 1.32 0.00 missing observation due to Inadequate blood s amp 1 e 91 Table A-12 Raw data for repeated treatments model. Exp. Treatment Loglø Bacteria/ml Blood mouse 1 mouse 2 mouse 3 mouse 4 1 no treatment 4.80 4.38 4.57 4.4 0 1 melittin - 50ng 4.04 4.74 5.07 4.62 ! polymyxin B - 2ug 5.04 4.56 3.48 0.00 1 mel 50ng + pol 2ug 0.00 0.00 3.30 0 .00 2 no treatment 4.67 4.36 4.45 4 4..4411 2 mel i tt in - 50ng 4.67 4.51 4.89 4.90 2 polymyxin B - 2ug 1.78 3.09 2.57 4.06 2 mel 50ng + pol 2ug 1.30 1.85 0.00 3.21 3 no treatment 4.51 4.46 3.78 1.95 3 mel itt in - 50ng 4.99 4.43 4.61 4.41 3 polymyxin B - 2ug 3.20 3.26 3.95 3.16 3 mel 50ng + pol 2ug 3.80 3.24 3.22 3.46 4 no treatment 4.92 3.18 3.78 4.93 4 melittin - 50ng 5.14 4.18 4.28 4.76 4 pol myxin B - 2ug 3.35 3.51 3.51 3.89 4 mel 50ng + pol 2ug 2.60 3.68 3.51 2.23 5 no treatment 3.53 4.30 4.46 4.08 5 mel itt in - 50ng 4.08 4.76 4.32 4.45 5 polymyxin - 2ug 4.43 2.94 3.34 3.72 5 mel 50ng + pol 2ug 2.34 3.41 3.05 2.93 93 Haberman, E. J972. Bee and wasp venoms: The biochemist! y and pharmacology of the r peptides and enzyme mc »• •" viewed. Science 177:314-322. Haberman, E. and J. Oontsch. .1967. Scqυon/ analy t* dι-: ' - melittiπs aus den tryptischen und p pH t. f.π spalt:stuckeπ. Huppe-Seylπr 'ε Z. Physiol. Chem. 3-i'.'.z ' 7-'.> Han e, [• }. ' . C. Methfessel, H. U. Wilm eπ, F. kat,-, ..uu-i- and G. Bohuiπi. 1903. I it »1. it. tin and a In-Miiic U ■ modified trichotoxin form alampthicin-type mul ti- :.l al' * pores. Biochim. Biophys. Acta 727:100- -.1.14. Lauterwein , J., C. Bosch, L. R. Brown and . HuMi ic . 1979. Physioch emical studies of the protein- 1 ip id in ¬ teractions in πielittiπ-containing micelles. Biochim. Biophys. Acta 556:244-264. Lauterwein, J. , L. R. Brown a d . Wuthri.ch. I^ u. High-resolution 3 H-HNR studies of monomeric melittin in aqueous solution. Biochim. Biophys. Acta 622:219-230. Lowry, 0. H. , N. J. Rosenbrough, A. L Farr and \i. .1. Randall. 1951. Protein measurement with the fotin Phenol reagent. J. Biol. Chem. 193:265-275. Moellering, R. C., C. Weπnersteπ and A. N. Weinberg. 1971. Studies of antibiotic synergism against enteroco ci . J. Lab. Clin. Med. 77:821-827. Mollay, C. and G. Kre.il. .1973. Fluorometr.ic measurements on the interaction of melittin with lecithin. Biouhini. Biophys. Acta 316:196-203. Mulfinger, L. M. , Λ. W. Npritoii, If. W. txt al n i ι- and !:. A. Wilson. 1906. Λ qu.-i.l it t.i t- ;m quant i 1 «-ι t i vr> .,π l* *.i - of proteins found in ve pid vr>πom*:>. J. Λl -i y ι.:J iιι. Immuπol . 77 : 681- t.:J . Oriel, S. and F. Markwardt. 1955. Invest. j ciaL.inπs on 1h'* bactericidal properties of bee venom (Tr ami] a ted l il.lπ, in German). Pharmazie 10:743-746. A r.ti HI.-1.IMJ in Chemical Abstracts. J956. 50: 1229c. Schmidt-Laπge, w. 19 J. The bactericidal action nl b'.-* venom (Translated title, in German). MUΠCIUMIH-Γ Mediziriische Wooheπschr i f t 80 : 35-93<.. Sebek, 0. K. 1980. Antibiotics: volume 1 : IIK-I .ιiιi--,ιn nl ' .n ϊάmQT±. u. Gottlieb and f. IK Miaw (<* " d:.. Springer-Ve lag, New York. pp.142-14^. 94 Tu, A. T. 1977a. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc., New York, London, Sydney, and Toronto. pp. 1-16. Tu, A. T. 1977b. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc , New York, London, Sydney, and Toronto, pp. 501-512. Tu, A.T. 1977c. Venoms: chemistry and molecular biology. John Wiley and Sons, Inc., New York, London, Sydney, and Toronto, pp . 505-509. Volk, W. A. 1978a. Essentials of medical microbiology. 0. May and J. Frazier (eds.). J. P. Lippincott Company, Phila. , New York, San Jose and Toronto, pp.121-122. Volk, W. A. 1978b. Essentials of medical microbiology. 0. May and J. Frazier (eds.). J. P. Lippincott Company, Phila., New York, San Jose and Toronto, pp.122-126. 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. Volk, W A. 1978d. Essentials of medical microbiology. C. May and J. Frazier (eds.). J. P. Lippincott Company Phila., New York, San Jose and Toronto, pp.133-135. Yunes, R. A. 1982. A circular dichroism study of the structure of Apis melifera melittin. Arch. Biochem. Biophys. 216(2) :559-565. SUBSTITUTESHEET 95 ADDITIONAL BIBLIOGRAPHY Goodman, M. G. and W. 0. Weigle. Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on membrane-directed versus intracellular activators. J. Al¬ lergy Clin. Immunol. 74:418-425, 1984. Kondo, E. and K Kanai. 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. Kondo, E. Melittin-stimulated anti ycobacterial activity of the membrane fraction isolated from phagocytes of guinea pigs. Japan. J. Med. Sci. Biol. 39:21-24, 1986. Somerfield, S. D. , J. Stach, C. Mraz, F. gervais, and E. Ska ene. Bee venom melittin blocks neutrophil Oz production. Inflammation 10:175-182, 1986. Mulfinger, L. M. The synergistic activities of honey bee venom with antibiotics. Unpublished M. S. thesis, Penn¬ sylvania State University, 1986 (contained in U. S. Patent Appln. S. N. 096,628). Lowry, 0. H., N. J. Rosenbrough, A. L. Fa , and R. J.Randall. Protein measurement with the folin phenol re¬ agent. J. Biol. Chem. 193:265-275. 1951. Ra achandran, J. and Virginia Lee. Preparation and proper¬ ties of the o-nitrophenyl sulfenyl derivative of ACTH: an inhibitor of the lipolytic action of the hormone. Biochem. Biophys. Res. Com. 38(3) :507-512. 1970. Scoffone, E., A. Fontana, and R.Rocchi. Sulfenyl halides as modifying reagents for polypeptides and proteins. I. Modifi¬ cation of tryptophan residues. Biochemistry Z(3) -.971-979. 1968. Couch, T. and A. Benton. The effect of the venom of the honey bee, Apis melli fera L., on the adreηocortical response of the adult male rat. Toxicon 10:55-62. 1972. SUBSTITUTESHEET

Claims: 96 CLAIMS : 1. A composition for the treatment of an infection in a mammal which comprises: an antibiotic agent having activity against said infection; and a second agent selected from the group consisting of at least one Hymenoptera venom, at least one active protein component of a Hymenoptera venom, at least one polypeptide component of a Hymenoptera venom, at least one analogue or chemically modified derivative of one active protein component of Hymenoptera venom, at least one analogue or chemically modified derivative of one polypeptide component of 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. 97 2. The composition 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 composition of claim 2 wherein 10 the second agent is selected from the group consisting of honeybee venom, bumblebee venom, yellow jacket venom, 15 bald faced hornet venom, active protein components of said venoms, active polypeptide components of said venoms, analogues or chemically modified derivatives of melittin, bombilitin, mastoporan and crabolin,and 20 mixtures thereof. 4. The composition of claim 2 wherein the antibiotic agent comprises ampicillin and the second agent is honey bee venom. 5. The composition of claim 2 wherein 2 the antibiotic agent comprises ampicillin and the second agent is melittin. 98 6. The composition of claim 2 wherein the antibiotic agent comprises kanamycin and the second agent is honeybee venom. 7. The composition of claim 2 wherein the antibiotic agent comprises kanamycin and the second agent is melittin. 8. The composition of claim 2 wherein the antibiotic agent comprises polymixin B and the second agent is honeybee venom. 9. The composition of claim 2 wherein the antibiotic agent comprises polymixin B and the second agent is melittin. 10. The composition of claim 2 wherein the antibiotic agent comprises rifampicin and the second agent is honey bee venom. 11. The composition of claim 2 wherein the antibiotic agent comprises rifampicin and the second agent is 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 Hymenoptera venom, at least one active protein component of a Hymenoptera venom, 99 at least one polypeptide component of a Hymenoptera venom, at least one analogue or chemically modified derivative of one active protein component of Hymenoptera venom, at least one analogue or chemically modified derivative of one polypeptide component of 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 selected from the group consisting of honeybee venom, bumblebee venom, yellow jacket venom, bald faced hornet venom, active protein components of said venoms, active polypeptide components of said venoms. 100 analogues or chemically modified derivatives of melittin, bombilitin, mastoporan and crabolin, and mixtures thereof. 15. The dosage unit of claim 13 wherein the antibiotic agent comprises ampicillin and the second agent is honey bee venom. 16. The dosage unit of claim 13 wherein the antibiotic agent comprises ampicillin and the second agent is melittin. 17. The dosage unit of claim 13 wherein the antibiotic agent comprises kanamycin and the second agent is honeybee venom. 18. The dosage unit of claim 13 wherein the antibiotic agent comprises kanamycin and the second agent is melittin. 19. The dosage unit of claim 13 wherein the antibiotic agent comprises polymixin B and the second agent is honeybee venom. 20. The dosage unit of claim 13 wherein the antibiotic agent comprises polymixin B and the second agent is melittin. 21. The dosage unit of claim 13 wherein the antibiotic agent comprises rifampicin and the second agent is honey bee venom. 22. The dosage unit of claim 13 wherein the antibiotic agent comprises rifampicin and the second agent is melittin.

Inventors: BENTON ALLEN W, LOWENSTEIN HENNING, MULFINGER LORRAINE M, BENTON, ALLEN, W., LOWENSTEIN, HENNING, MULFINGER, LORRAINE, M.
Applicants: VESPA LAB INC, VESPA LABORATORIES, INC.
Application year: 1989
Main classification: A61K35
IPC codes: A61K31, A61K35, A61K38, A61K45, A61P31, A61P35