Antimicrobial compositions comprising minocycline and degradation products of oxidized cellulose
11690937 · 2023-07-04
Assignee
Inventors
- Eve Montia (Rehovot, IL)
- Dwayne Looney (Flemington, NJ, US)
- Theresa Scheuble (Rockaway, NJ, US)
- Ronen Eavri (Binyamina, IL)
- Roi Mashiach (Kiryat-Ono, IL)
Cpc classification
A61L2300/404
HUMAN NECESSITIES
A61L31/16
HUMAN NECESSITIES
A01N1/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
International classification
A61L31/16
HUMAN NECESSITIES
A01N1/00
HUMAN NECESSITIES
A61L26/00
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
Abstract
Provided is an antimicrobial composition comprised of at least one degradation product of oxidized cellulose (OC), such as oxidized regenerate cellulose (ORC), and minocycline, methods of preparation thereof and uses thereof.
Claims
1. A method for the treatment of an infection in a subject in need thereof, the method comprising administering to the subject an antimicrobial composition comprising an extract of at least one degradation product of an oxidized regenerated cellulose (ORC) substrate in a solid form, and an antibiotic being minocycline, wherein the at least one degradation product comprises one or more members selected from the group consisting of a monosaccharide and an oligosaccharide, and wherein the carboxyl content of the ORC is about 12% to about 21%, by weight.
2. The method of claim 1, wherein the antimicrobial composition is devoid of an additional antibiotic.
3. The method of claim 1, wherein the minocycline is present in the antimicrobial composition at a concentration of from about 12.6 ng/ml.
4. The method of claim 1, wherein the antimicrobial composition further comprises at least one additional antibiotic.
5. The method of claim 1, wherein the substrate is in the form selected from the group consisting of a cloth, a pad, and a mesh, and wherein the minocycline is dispersed on and/or within the cloth, the pad, or the mesh.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Some embodiments of the invention are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments of the invention may be practiced.
(2) In the Figures:
(3)
(4)
(5)
(6)
(7)
(8)
DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
(9) The invention, in some embodiments thereof, relates to a composition comprising minocycline and at least one degradation product of OC, methods of preparation thereof and uses thereof. In some embodiments, the OC comprises ORC.
(10) The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description. Upon perusal of the description, one skilled in the art is able to implement the invention without undue effort or experimentation.
(11) Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description. The invention is capable of other embodiments or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting.
(12) As shown in the Examples below, the present inventors conducted as a primary stage a preliminary minimal inhibitory concentration (MIC) test for minocycline, in order to confirm the antibiotic effect of minocycline on the bacterial strains tested. Serial dilutions of the antibiotic were prepared and the MIC was determined as the dilution at which visible growth of bacteria was first seat. The MIC for S. aureus was found to be 0.25 μg/ml, which is consistent with the MIC values reported in the literature (0.06-0.5 μg/ml).
(13) Following establishment of the MIC, the present inventors studied the interaction between degradation products of ORC and minocycline by incubating the minocycline at a concentration equal to or lower than the MIC, in the presence or absence of degradation products of ORC. Upon addition of a small amount of degradation products of ORC to the growth medium, the antimicrobial activity of minocycline was significantly enhanced, resulting in an MIC of 0.05 μg/ml.
(14) Pads of dimensions of approximately 1×1.5 inch, weighing between 65-75 mg (70 mg on average) were used as a source of degradation products of ORC. The levels of degradation products of ORC obtained and used did not show intrinsic antimicrobial activity and the antimicrobial activity obtained with the combination could not be attributed to the effect of degradation products of ORC alone or to an additive effect of degradation products of ORC with minocycline.
(15) In addition, any acidifying effect of the ORC during and/or after degradation was buffered, such as by adding a buffering agent to the composition, thereby forming an isotonic non-acidic medium for the bacteria to grow in.
(16) When antibiotics other than minocycline were tested in combination with degradation products of ORC, no enhancement of antibiotic activity was seen. In fact, some tested antibiotics (such as gentamycin and rifampin) showed a reduction in the antibiotic activity as compared to the antibiotic alone, indicating interference between the degradation products of ORC and antibiotic.
(17) The synergistic effect between minocycline and degradation products of ORC persisted when additional antibiotics (rifampin and/or clindamycin), which did not themselves exhibit a synergistic effect with ORC, were added to the mixture of minocycline and degradation products of ORC. Moreover, the inclusion of such additional antibiotics resulted in a further increase of antibiotic activity, in addition to that seen with minocycline and degradation products of ORC. Use of such combinations of antibiotics provides a wide coverage of gram negative and gram-positive antibiotics.
(18) Minocycline-impregnated ORC pads were then prepared by immersing ORC pads in solutions of minocycline dissolved in methanol, followed by drying, in order to determine the best minocycline concentrations to be used as a source of product comprising minocycline and degradation products of ORC.
(19) MIC of 0.0126 μg/ml was obtained in the presence of degradation products of ORC, as compared to 0.25 ng/ml in the absence of degradation products of ORC (Table 1), the MIC being at least 5.8-fold lower in the presence of degradation products of ORC than that in their absence. The MIC of minocycline together with degradation products of ORC, when prepared using degradation of ORC impregnated with minocycline was 12.60 ng/ml, the MIC being at least 19.8-fold lower in the presence of degradation products of ORC than that in their absence. The MIC of minocycline with at least one more antibiotic together with degradation products of ORC, when prepared using degradation of ORC impregnated with minocycline and the at least one more antibiotic was 200-fold lower of the MIC for minocycline alone. For example, 1.26 ng/ml minocycline in combination with 38.96 ng/ml clindamycin (total 10.22 ng/ml antibiotic loaded) together with ORC degradation products (from antibiotic-impregnated ORC) is a sufficient level of antibiotic to provide a high level of antibiotic activity.
EXAMPLES
(20) The present inventors aimed to identify a preferred antibiotic for use in the preparation of a product comprising an antibiotic and one or more degradation products of ORC, as well as to determine suitable concentrations of the selected antibiotic. A study was conducted in 3 stages as described below.
(21) Materials and Methods
(22) Materials
(23) Tryptic Soy broth (TSB) was acquired from Hy-Labs, Rehovot, Israel (Catalog no. (Cat #) BP266/1009).
(24) Mueller Hinton Broth II (MHBII) was acquired from Becton Dickinson, N.J., USA (Cat #BBL298268).
(25) Staphylococcus aureus (S. aureus), Strain #6538 was acquired from ATCC.
(26) ORC pads used were Surgicel® from Ethicon, Cat #1951.
(27) Phosphate buffer Saline (PBS) was acquired from Biological Industries Ltd., Beit Haemek, Israel (Cat #20-023-1A).
(28) 0.8/0.2 μm syringe filters were acquired from Pall Corporation, N.Y., USA (Cat #: 4658).
(29) Antibiotics were acquired from Tokyo Chemical Industry (TCI), Tokyo. Japan as follows: Minocycline (Cat #: M2288); Gentamycin (Cat #: G0383); Rifampin (Cat #: R0079); Clindamycin (Cat #: C2256); and Tetracycline (Cat #: 12525).
(30) Sterile water (SW) used was milli-Q purified water subjected to sterilization using a 0.2-micron cellulose acetate filter.
(31) Orbital shaker: Thermo Fisher sold under the trademark MaxQ™ 4450 Benchtop Orbital Shakers.
(32) Methods
(33) Preparation of Inoculum
(34) An isolated colony of S. aureus bacteria was picked from a fresh streak plate no more than 1 week old). The colony was transferred into 3-4 ml of TSB and incubated overnight in an orbital shaker at 35° C.-37° C.
(35) The following day, the turbidity of the actively-growing broth culture was adjusted with Calcium-adjusted Mueller Hinton broth II (MHBII) to be equivalent to that of a 0.5 McFarland standard (1×10.sup.8 CFU/ml). The broth culture was maintained under ambient conditions prior to use.
(36) No more than 15 minutes prior to use, the culture was further diluted 20-fold in MHBII to obtain a final 5×10.sup.6 CFU/ml stock suspension.
(37) Preparation of Extracts of Degradation Products of ORC
(38) ORC pads undergo hydrolyzation, resulting in degradation products, when immersed in an aqueous solution. At least some degradation products of ORC are present in the form of particles which might interfere with OD readings and provide inaccurate results. In order to avoid this, degradation products were obtained as extracts from the ORC pads as described below and used in the present study.
(39) a. Preparation of Blank Extract Comprising Degradation Products of ORC
(40) ORC pads which had not been impregnated with antibiotic (referred to herein as “blank ORC pads”), were subjected to extraction and degradation in PBS for three hours at ambient temperature. Extract obtained from these blank ORC pads is referred to herein as “blank extract of degradation products of ORC”.
(41) b. Preparation of Extracts of Degradation Products of Blank ORC in Antibiotic
(42) Blank ORC pads were subjected to extraction and degradation in a solution of antibiotic in PBS. Extracts of degradation products obtained from blank ORC pads in a solution of antibiotic in PBS is referred to herein as “extracts of degradation products of blank ORC in antibiotic”.
(43) Due to the acidity of ORC, which provides some antibiotic effect, the size of the pad used was small i.e. approximately 1×15 inch, weighing between 65-75 mg (70 mg on average) in order to prevent the ORC from affecting the final pH of live assay and therefore the growth of the bacteria.
(44) 15 ml tubes, each containing 3 ml of an antibiotic solution at a known concentration in sterile PBS, were prepared in duplicate.
(45) Blank ORC pads were subjected to extraction and degradation in the antibiotic solution by immersing the ORC pads in the antibiotic solution for 3-5 hours at ambient temperature on a roller mixer (Stuart. Cat #SRT9D).
(46) The extracted fluid comprising degradation products of ORC was filtered using 0.8/0.2 μm syringe filters (Pall, Cat #4658) and mixed with calcium-adjusted MHBII to provide antibiotic-ORC test suspension.
(47) c. Preparation of Extract of Degradation Products of Antibiotic-Impregnated ORC
(48) Solutions comprising different concentrations of antibiotics to be tested were prepared in methanol.
(49) Blank ORC pads of dimensions 1×1.5 inch, weighing between 65-75 mg (70 mg on average), in duplicate, were immersed in the antibiotic solutions and then dried using a rotary evaporator to provide extract of degradation products of antibiotic-impregnated ORC pads.
(50) 15 ml test tubes containing 3 ml of sterile PBS were prepared, and the antibiotic-impregnated ORC pads were immersed in the tubes and incubated for 3-5 hours at ambient temperature on a roller mixer (Stuart. Cat #SRT9D).
(51) Each of the above extracts w as filtered out using a sterile 0.8/0.2 μm syringe filter into a clean 15 ml tube. These extraction fluids are referred to herein as “extract of degradation products of antibiotic-impregnated ORC”.
Example 1: Primary Stage Determination of Antibiotic MIC
(52) The purpose of this stage was to confirm antibiotic activity according to MIC levels reported in the literature, to establish specific activity for the antibiotic, and to identify the preferred antibiotic for further studies Antibiotics tested were minocycline, rifampin, clindamycin, gentamycin and tetracycline.
(53) MIC Measurement
(54) The method used in this stage was based on the classic Minimum Inhibitory Concentration (MIC) measurements, as described by the Clinical and Laboratory Standards Institute (CLSI) document M07-A9: “Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically”. While classic MIC measurements require visual inspection of plates for bacterial growth after 18 hours, growth was monitored in the present study according to optical density (OD) at 600 nm, using an ELISA reader (MultiSkan™) FC ELISA reader, Thermo Fisher Scientific, Mass., USA), thereby obtaining bacterial growth curves for tested antibiotic solutions.
(55) A 96-well assay plate layout (Corning Cat 35%) was prepared as follows:
(56) Columns 1 and 3-12: 100 μl of MHBII alone was added to each well.
(57) Row 2: 200 μl of a solution of antibiotic in MHBII at the highest concentration to be tested was added to each well.
(58) A series of 2-fold dilutions was performed, wherein 100 μl of solution of antibiotic was withdrawn from each well of column 2, transferred to the corresponding well of column 3, and mixed.
(59) The dilution was then repeated by transfer from column 3 to column 4, and so on, until column 11, when the 100 μl of solution of antibiotic withdrawn front each well of column 11 was discarded.
(60) The 5×10.sup.6 CFU/ml stock suspension which had been prepared in advance w as transferred into a reservoir and 10 μl of the stock suspension were added into each well of columns 2-11 using a multichannel pipette to obtain a final 5×10.sup.5 CFU/ml suspension.
(61) The plate was then incubated at 37° C. with shaking in the ELISA reader and the optical density at 600 nm was read every 20 minutes for a total of 18 hours.
(62) The average OD reading obtained for each antibiotic concentration was calculated and plotted against time to obtain a growth curve.
(63) The MIC was determined as the first antibiotic concentration at which some bacterial growth occurred by the end of the 18-hour period.
(64) Results
(65) Results are presented in Table 1 below
(66) TABLE-US-00001 TABLE 1 MIC values for MIC values Antibiotic Conc. S. aureus according found in the (μg/ml) to the literature present study Rifampin 0.004-0.015 0.015 Clindamycin 0.06-0.25 0.062 Minocycline 0.06-0.5 0.25 Gentamicin 0.12-1 0.25 Tetracycline 0.06-0.5 0.06
(67) As seen in Table 1, the MIC values obtained in the present study were consistent with those published by the scientific literature.
Example 2: Secondary Stage Determination of Interaction of Antibiotic with Degradation Product(s) of OC
(68) This stage comprised MIC measurements for several antibiotics, either alone or in combination with degradation products obtained from ORC, in order to identify any interference of enhancement of the antibiotic activity in the presence of degradation products of ORC as evidenced by changes in MIC levels for the antibiotic.
(69) MIC Measurement
(70) The preferred antibiotic concentrations, as established in the first stage, were used for antibiotics in combination with the degradation products of ORC pads.
(71) The various antibiotics tested and the concentrations used are shown in Table 2 below.
(72) TABLE-US-00002 TABLE 2 MIC determined Conc. 1 Conc. 2 Conc. 3 Conc. 4 Cone. 5 in Example Antibiotic (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml) 1(μg/ml) Minocycline 0.5 0.4 0.2 0.1 0.05 0.25 Rifampin 0.11 0.055 0.028 0.014 0.007 0.015 Clindamycin 0.25 0.125 0.062 0.031 N/A 0.062 Gentamicin 11 0.8 0.5 0.3 0.1 0.75 Tetracycline 0.4 0.2 0.1 0.05 0.025 0.06
(73) Blank extract of degradation products of ORC and extract in antibiotic of degradation products of ORC, prepared as described above, were diluted 10 folds in MHBII in sterile 15 ml capped tubes.
(74) The 5×10.sup.6 CFU/ml stock suspension which had been prepared in advance was transferred into a reservoir and 10 μl of the stock suspension were added into each well of columns 2-11 using a multichannel pipette to obtain a final 5×10.sup.5 CFU/ml suspension.
(75) TABLE-US-00003 TABLE 3 The plate layout used is shown in Table 3 below. 1 2 3 4 5 6 7 8 9 10 11 12 A SW SW SW SW SW SW SW SW SW SW SW SW B SW BLANK Ab Ab Ab Ab Ab Ab Ab Ab W/O SW Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. 1 + 1 + 1 + 1 + 1 1 1 1 ORC ORC ORC ORC C SW BLANK Ab Ab Ab Ab Ab Ab Ab Ab W/O SW Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. 2 + 2 + 2 + 2 + 2 2 2 2 ORC ORC ORC ORC D SW BLANK Ab Ab Ab Ab Ab Ab Ab Ab W/O SW Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. 3 + 3 + 3 + 3 + 3 3 3 3 ORC ORC ORC ORC E SW BLANK Ab Ab Ab Ab Ab Ab Ab Ab W/O SW Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. 4 + 4 + 4 + 4 + 4 4 4 4 ORC ORC ORC ORC F SW + ve Ab Ab Ab Ab Ab Ab Ab Ab + ve SW Ctrl. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Ctrl. 5 + 5 + 5 + 5 + 5 5 5 5 ORC ORC ORC ORC G SW + ve PBS- PBS- PBS- PBS- PBS PBS PBS PBS + ve SW Ctrl. ORC ORC ORC ORC Ctrl. H SW SW SW SW SW SW SW SW SW SW SW SW
(76) 150 μl sterile water (SW) was added to each of the border wells (i.e. columns 1 and 12, rows A and H) in order to prevent any drying effect for the duration of the experiment.
(77) Column 2, rows B to E were left as blanks, i.e. containing no antibiotics without bacteria, in order to provide a reference for the basic OD of the plate.
(78) Columns 3 to 6, rows B to F, were filled with the Blank ORC extracted in antibiotic at decreasing concentrations 1 to 5, diluted 10 folds in MHBII. Each sample tested was placed in 4 well repeats for statistical results.
(79) Columns 7 to 10, rows B to F, were filled with the antibiotic solution (AB) at decreasing concentrations 1 to 5, diluted 10 folds in MHBII Each sample tested was placed in 4 well repeats for statistical results.
(80) Columns 2 and 11, rows F and G, contained as a positive control (+ve) antibiotic alone, at low and high concentrations, prepared from a stock solution of the test antibiotic in PBS, diluted in sterile MHBII.
(81) Columns 3 to 6, row G, were filled with the blank extract of degradation products of ORC (PBS-ORC) diluted 10 folds in MHBII. These samples were used as a negative control in order to ascertain that the bacteria were not significantly affected by the degradation products of ORC alone and that any bacterial growth impairment detected was due to the effect of the antibiotic
(82) Columns 7 to 10, row G, were filled with PBS alone (diluted 10-fold in MHBII, as a further negative control.
(83) Column 11, rows B to E, contained a further negative control comprising with bacteria and no antibiotic (MHBII) providing the bacterial growth performance in these conditions without any antibacterial interference (W/O).
(84) At least 4 wells contained bacteria alone in MHBII in order to verify that no interference in bacterial growth occurred.
(85) The plate was incubated at 37° C. with shaking in the ELISA reader and the optical density at 600 nm was read every 20 minutes for a total of 18 hours.
(86) The average and standard deviation for OD readings was calculated for each time point of the wells using Excel. The average values were plotted against time to obtain a graph of the bacterial growth curves in the presence of each of the antibiotics tested.
(87) In some cases, the average OD was plotted for a single endpoint of 800 minutes for the various antibiotic treatments and controls. The endpoint of 800 minutes was selected as this was found to be the time at which the difference between the various antibiotic concentrations is greatest.
(88) The average values were also entered into the GraphPad Prism program (GraphPad Software Inc., Calif., USA) in order to calculate the Effective Time 50 (ET50) i.e. the time, in minutes, required for the antibiotic to induce a response halfway between the baseline and maximum, where the maximum is the bacterial growth in MHBII with PBS. This provides a quantifiable measure of the efficacy of the antibiotics tested and enables comparison of the various samples tested. It is noted that ET50 values of above 1650 are indicative of high antibiotic activity while ET50 values of greater than 2000 minutes were found to be imprecise and are therefore regarded as maximal growth inhibition. The range of 1000 to 1650 is indicative of positive antibiotic activity. In cases wherein there was such limited growth that an ET50 value could not be established, the result is indicated as N/A.
(89) In order to compare different antibiotic concentrations, the average OD at 800 minutes for each sample tested was plotted against the antibiotic concentration and the EC50 calculated i.e. the concentration of antibiotic which induces a response halfway between the baseline and maximum after a specified exposure time.
(90) Results
(91) a. Minocycline
(92) Results are shown in Table 4 below and in
(93) TABLE-US-00004 TABLE 4 Minocycline 0 μg/ml 0.05 μg/ml 0.1 μg/ml 0.2 μg/ml 0.4 μg/ml 0.5 μg/ml concentration − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC ET50 411.7 423.6 428.1 901.1 555.1 >2000 1771 >2000 >2000 >2000 >2000 >2000
(94) The results show that addition of degradation product(s) of ORC to minocycline resulted in a strong synergistic effect. The effect was greater at lower concentrations of minocycline, where the antibiotic activity of minocycline alone was low.
(95) For example, 0.05 μg/ml minocycline alone had no antibiotic effect alone (ET50 428.1 minutes, which is very similar to the ET50 value of 411.7 minutes obtained in the absence of minocycline). Following addition of ORC degradation product(s) to the minocycline solution, the ET50 was increased to 901 minutes.
(96) A similar effect was seen with minocycline concentrations of 0.1 μg/ml of (ET50 of 555 and over 2000 in the absence and presence, respectively, of ORC) and 0.2 μg/ml (ET50 of 1771 and over 2000 in the absence and presence, respectively, of ORC).
(97) Since 0.4 μg/ml of minocycline alone showed an ET50 of above 2000 minutes, such that maximal growth inhibition was shown, the addition of degradation product(s) of ORC to the antibiotic solution did not produce any increase in antibiotic effect.
(98) Degradation products of ORC alone, in the amount used, did not show any antibiotic effect.
(99) b. Rifampicin (Rifampin)
(100) Results are shown in Table 5 below and in
(101) TABLE-US-00005 TABLE 5 Rifampin 0 μg/ml 0.015 μg/ml 0.031 μg/ml 0.062 μg/ml 0.125 μg/ml 0.25 μg/ml Concentration − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC ET50 424.9 432.8 470.7 440.5 448.4 436.3 669.1 432.8 N/A 432.3 N/A 430.8
(102) The results show that degradation products of ORC interfere with the antibiotic activity of rifampin, such that no antibiotic activity was seen upon addition of degradation products of ORC to solutions of rifampin at concentrations which showed antibiotic activity when used alone.
(103) For example, at concentrations of 0.25 μg/ml and 0.125 μg/ml rifampin alone there was no growth at all (no ET50 values were therefore available). However, once degradation products of ORC were added to the rifampin solutions, the growth profile resembled that of the control (ET50 values of around 430 minutes). At a concentration of 0.062 μg/ml rifampin, delayed bacterial growth was seen (ET50 of 669.1), but the antibiotic activity was decreased by the addition of degradation products of ORC, reducing the ET50 value to 430 minutes.
(104) c. Clindamycin
(105) Results are shown in Table 6 below and in
(106) TABLE-US-00006 TABLE 6 Clindamycin 0 μg/ml 0.03125 μg/ml 0.0625 μg/ml 0.125 μg/ml 0.25 μg/ml concentration − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC ET50 650.2 648 1001 1049 1494 1432 >2000 >2000 N/A N/A
(107) The results show that addition of degradation products of ORC had no effect on the antibiotic activity of clindamycin at the clindamycin concentrations tested.
(108) d. Gentamycin
(109) Results are shown in Table 7 below and in
(110) TABLE-US-00007 TABLE 7 Gentamycin 0 μg/ml 0.1 μg/ml 0.3 μg/ml 0.5 μg/ml 0.8 μg/ml 1 μg/ml Conc. − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC ET50 414.1 420.7 509.4 423.7 1360 429.3 N/A 423.1 N/A 422.7 N/A 446.6
(111) The results show that addition of degradation products of ORC to solutions of gentamycin resulted in decreased antibiotic activity at all gentamycin concentrations tested.
(112) e. Tetracycline
(113) Results are shown in Table 8 below and in
(114) TABLE-US-00008 TABLE 8 Tetracycline 0 μg/ml 0.025 μg/ml 0.05 μg/ml 0.1 μg/ml 0.2 μg/ml 0.4 μg/ml Conc. − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC − ORC + ORC ET50 565.6 425.3 531.1 517.7 989.9 741.1 1889 1470 >2000 >2000 >2000 >2000
(115) The results show that addition of degradation products of ORC had no effect on the antibiotic activity of tetracycline at the tetracycline concentrations tested.
(116) A summary of the results obtained for all the tested antibiotics is shown in
(117) As shown in the lower and upper panels of the middle of
(118) As shown in the upper panel at the right of
(119) As shown in
Example 3. Tertiary Stage-Determination of Antimicrobial Activity of Degradation Products of ORC Obtained from Extracts of Orc Pads Impregnated with a Single or Combination Antibiotic
(120) This stage comprised testing the antimicrobial activity of extract of degradation products of antibiotic-impregnated ORC pads in order to identify a suitable concentration range for single or multiple antibiotics in the antibiotic-impregnated ORC pads as a source of a product comprising antibiotic and degradation products of ORC for use in treatment of SSI.
(121) Determination of Antibiotic Impregnation Levels
(122) In order to determine the quantity of antibiotic impregnated into an ORC pad from which the extract of degradation products of antibiotic-impregnated ORC is to be obtained, the antibiotic was extracted out of the pad and the antibiotic levels were measured.
(123) Impregnation was carried out as described above in section “Preparation of antibiotic impregnated ORC extracts” under “Methods”. Briefly. ORC pads weighing on average 70 mg each were impregnated with the selected antibiotic.
(124) Samples of pads impregnated with rifampin, minocycline and clindamycin, as well as blank ORC pads (w/o impregnated antibiotics) were taken for antibiotic loading analysis. Each sample was weighed and placed in a 15 ml scintillation vial and 1 ml of methanol was added.
(125) The vials were hand shaken periodically and after 2 hours the methanol was decanted off for analysis (“1.sup.st extract”). A second 1 ml of methanol was added into the scintillation vial, and the process was repeated to obtain the “2.sup.nd extract”. A third 1 ml of methanol was added into the scintillation vial, and the process was repeated to obtain the “3.sup.rd extract”.
(126) The 1.sup.st extract, 2.sup.nd extract, and the 3.sup.rd extract were then analyzed by LC/UV/MS using an Agilent 1260 Series UPLC with photodiode array detector and Agilent 1100 MSD mass spectrometer.
(127) The concentration of the antibiotic in each extract of degradation products of antibiotic-impregnated ORC was estimated by using the calibration curve of corresponding standards (peak area at 350 nm for rifampicin; 425 and 458 peak area for clindamycin and minocycline, respectively).
(128) Results are presented in Table 9 below.
(129) TABLE-US-00009 TABLE 9 Total Antibiotic Impregnation impreg- Loading Antibiotic Conc. per ORC pad nation Solution (μg/g) level in Conc. 1.sup.st 2.sup.nd 3.sup.rd ORC Antibiotic (μg/ml) extract extract extract pad (μg/g) Minocycline 20 8.9 1.9 <1.4 10.8 Clindamycin 10 10.7 2.1 <1.4 12.8 Rifampin 200 409.2 139.0 79.9 628.2
(130) The results presented in Table 9 were then used to calculate the amount of antibiotic loaded on the rest of the antibiotic-impregnated ORC pads to be for production of extract of degradation products of antibiotic-impregnated ORC used in the present studies. A linear dilution of the antibiotic was used in order to prepare the loading solution, a simple linear equation can be assigned in turn in order to deduct the level of antibiotic loaded onto the rest of the antibiotic impregnated pads.
(131) ORC pads for production of degradation products of ORC were impregnated with a single antibiotic or with a combination of two antibiotics. The antibiotic loading onto each of the pads tested, calculated according to the loading levels described in Table 9, are depicted in Table 10 below.
(132) Table 10 shows ORC impregnation levels deduced according to the impregnation levels found in Table 9 above and their consequent concentrations in the following MIC experiments (see section b).
(133) TABLE-US-00010 TABLE 10 Impregnation Total Total Antibiotic Loading antibiotic Antibiotic concentration Solution impregnation impregnation in MIC Conc. level in ORC level in ORC experiment (μg/ml) pad (μg/g) pad (%) (ng/ml) Rifampin 200 628.2 0.06282 1465.8 150 471.15# 0.047115 1099.35 100 314.1 0.03141 732.9 50 157.05 0.015705 366.45 40 125.64 0.012564 293.16 30 94.23 0.009423 219.87 20 62.82 0.006282 146.58 10 31.41 0.003141 73.29 Clindamycin 20 25.6 0.00256 59.73 10 12.8 0.00128 29.87 5 6.4 0.00064 14.93 2.5 3.2 0.00032 7.47 3 3.84 0.000384 8.96 2 2.56 0.000256 5.97 1.5 1.92 0.000192 4.48 1 1.28 0.000128 2.98 0.5 0.64 0.000064 1.49 Minocycline 40 21.6 0.00216 50.4 20 10.8 0.00108 25.2 10 5.4 0.00054 12.6 5 2.7 0.00027 6.3 4 2.16 0.000216 5.04 3 1.62 0.000162 3.78 2 1.08 0.000108 2.52 1 0.54 0.000054 1.26
a. Antimicrobial Activity of Degradation Products of ORC Pads Impregnated with Different Concentrations of a Single Antibiotic
(134) ORC pads impregnated with a single antibiotic to be used for preparation of degradation products of ORC were tested for antimicrobial activity using the MIC measurement described in Example 2 above. Briefly, ORC pads weighing on average 70 mg each were impregnated with the selected antibiotic. Extracts of degradation products of antibiotic-impregnated ORC were prepared by immersion and incubation in 3 ml of sterile PBS for 3 hours at ambient temperature. The ORC degeneration products were then removed, and any remaining ORC particles were filtered out using a 0.8/0.4 μm syringe filter. This extraction fluid was then diluted 10-fold into Muller Hinton growth medium containing bacteria. Bacterial growth was monitored every 20 min for a total of 18 hours according to optical density (OD) at 600 nm, using an ELISA reader.
(135) Tables 11a summarizes the results obtained by the used degradation products of single antibiotic-impregnated ORC pads according to their ET50 as described in Example 2. The results resemble those of the secondary stage MIC see Example 2, and the synergistic effect of minocycline is even more pronounced when the antibiotic is impregnated onto the ORC, such that degradation products of antibiotic-impregnated ORC pads are obtained.
(136) TABLE-US-00011 TABLE 11a Antibiotic conc. in % conc. in impregnation antibiotic MIC fluid on ORC ng/ml ET50 Minocycline 40 μg/ml I 0.00216 50.40 >2000 20 μg/ml I 0.00108 25.20 >2000 10 μg/ml II 0.00054 12.60 2000 5 μg/ml II 0.00027 6.30 1145 0 0 0 819.4 Rifampin 200 μg/ml 0.06282 1465.8 >2000 150 μg/ml 0.047115 1099.35 >2000 100 μg/ml 0.03141 732.9 1396 50 μg/ml 0.015705 366.45 643 0 712.5 Clindamycin 20 μg/ml 0.00256 59.73 >2000 10 μg/ml 0.00128 29.87 >2000 5 μg/ml 0.00064 14.93 1425 2.5 μg/ml 0.00032 7.47 975.9 0 0 0 663.2
(137) The MIC result presented in Table 11b below shows the reduction in the minocycline levels required in order to achieve antimicrobial activity. It is apparent that the interference of degradation products of ORC with Rifampin activity remains, generating an MIC which was 50 times higher than that of the primary stage MIC. The MIC of Clindamycin was four times lower in the ORC degradation products from the impregnated ORC. However, the MIC of Minocycline was 20 times lower than that of the 1.sup.st stage MIC, further establishing the synergistic effect of minocycline and degradation products of ORC.
(138) The results showed that the impregnation process did not impair the synergism of minocycline and degradation products of ORC described above, and that rifampin, the activity of which was detected as impaired, was also impaired once impregnated onto the ORC. These results provide further proof of the synergism between degradation products of ORC and Minocycline.
(139) TABLE-US-00012 TABLE 11b In-house Antibiotic-impregnated Fold reduction (primary)MIC ORC (tertiary) MIC in MIC Antibiotic# (μg/ml) # (μg/ml)# concentration Minocycline# 0.254# 0.0126# 19.85# Rifampin# 0.015# 0.733# 0.019# Clindamycin# 0.0624# 0.015# 4.15#
c. Antimicrobial Activity of Degradation Products of ORC Pads Impregnated with Different Concentrations of a Combination of Two Antibiotics
(140) Since a synergistic effect was observed between degradation products of ORC and minocycline, minocycline was used in all combination studies. Combinations of: 1—minocycline and clindamycin; or 2—minocycline and rifampin were studied, as shown in Table 12 below.
(141) TABLE-US-00013 TABLE 12 Minocycline ng/ml Rifampin ng/ml Total (ng/ml) ET50 0.00 0.00 0.00 409 1.26 0.00 1.26 585.4 2.52 0.00 2.52 799.9 5.04 0.00 5.04 >2000 6.30 0.00 6.30 >2000 0.00 73.29 73.29 406.8 1.26 73.29 74.55 606.8 6.30 73.29 79.59 1264 0.00 146.58 146.58 442.3 2.52 146.58 149.10 881.2 5.04 146.58 151.62 1992 0.00 293.16 293.16 450.4 2.52 293.16 295.68 1482 5.04 293.16 298.20 >2000 0.00 366.45 366.45 418.1 1.26 366.45 367.71 >2000 6.30 366.45 372.75 >2000 Minocycline ng/ml Clindamycin ng/ml Total ng/ml ET50 0.00 0.00 0.00 434.8 1.26 0.00 1.26 637.6 2.52 0.00 2.52 1072 5.04 0.00 5.04 >2000 6.30 0.00 6.30 >2000 0.00 1.49 1.49 448.7 1.26 1.49 2.75 734.5 6.30 1.49 7.79 >2000 0.00 2.99 2.99 485.1 2.52 2.99 5.51 1227 5.04 2.99 8.03 >2000 0.00 5.97 5.97 521.1 2.52 5.97 8.49 1488 5.04 5.97 11.01 >2000 0.00 8.96 8.96 579.8 1.26 8.96 10.22 1650 6.30 8.96 15.26 >2000
(142) Since the study included many possible combinations of two antibiotics, each with their own different concentration range, a matrix of possible antibiotic concentrations was designed, and degradation products of ORC were prepared accordingly (see Table 10). Since all the possible combinations could not be fitted into one 96 well plate, a series of MIC experiments were carried out. In order to compare between the different plates and obtain results which can provide information regarding the activity of the all antibiotics and concentrations tested, ET50 values (as described in Example 2) were used, which provided a quantitative measurement of the antibiotic activity which could be used to compare results from different assay plates.
(143) Table 12 presents ET50 results for combinations of minocycline with rifampin or minocycline with clindamycin at different concentrations. The results show that 6.3 ng/ml minocycline in combination with 366.45 ng/ml rifampin (total 372.75 ng/ml antibiotic loaded), as well as 1.26 ng/ml minocycline in combination with 38.96 ng/ml clindamycin (total 10.22 ng/ml antibiotic loaded) is a sufficient level of antibiotics which provides a great antibiotic activity. In addition, a combination of 2.52 ng/ml minocycline with 293.16 ng/ml rifampin as well as 2.52 ng/ml minocycline with 0.5.97 ng/ml of clindamycin also provided a high level of antibiotic activity.
(144) The primary stage study confirmed that no false positive antibacterial activity occurred, that the baseline antibacterial activity of the reagents and bacterial strains used was correct. The MIC obtained for S. aureus using a specific antibiotic alone was found to be consistent with values published in known literature.
(145) In the secondary stage, it was shown that although it is reported that ORC itself has some intrinsic antimicrobial activity (Dineen P. “The effect of oxidized regenerated cellulose on experimental infected splenotomies” Journal of Surgical Research 1977; 23: 114-116); the amount of ORC degradation products used in the present study was ineffective in providing bacterial growth inhibition as determined her MIC studies. The selected antibiotics were shown to exhibit different activity levels in the presence of degradation products of ORC. Specifically, rifampin and gentamycin activity was impaired by degradation products of ORC and MIC was increased; clindamycin and tetracycline activity showed no change in the presence of ORC and no change in MIC. However, surprisingly, only minocycline MIC was reduced when mixed with degradation products of ORC.
(146) In the tertiary stage, degradation products of ORC pads impregnated with selected antibiotics including minocycline, rifampin and/or clindamycin were used. MIC results were found to be consistent with those of the secondary stage.
(147) The levels of clindamycin required to be impregnated onto the ORC pad in order to achieve an inhibition of growth with degradation products of ORC were similar to those of the first stage MIC, i.e. there was no change of clindamycin antimicrobial activity. The levels of rifampin required to be impregnated onto the ORC pad used for production of degradation products of ORC in order to achieve an inhibition of growth were about 50 times higher than those of the first stage MIC i.e. the rifampin activity was impaired even when impregnated onto the ORC pad. Finally, the levels of minocycline required to be impregnated onto the ORC pad in order to achieve an inhibitory effect were about 20 times lower than those of the first stage MIC.
(148) It was further found that when minocycline is impregnated onto an ORC pad in combination with an additional antibiotic, the ET50 was reduced for both minocycline and the additional antibiotic, i.e. either rifampin or clindamycin. As little as 1.26 ng/ml minocycline, together with 366.5 ng/ml rifampin or 8.9 ng/rill clindamycin were found to be sufficient for growth inhibition of S. aureus. The MIC for these antibiotics when used as single antibiotics in the absence of ORC (first stage MIC results) was found to be 250 ng/ml for minocycline, 62 ng/ml for clindamycin and 15 ng/ml for rifampin. These results prove a 200-fold reduction of the MIC for minocycline, and 6.8-fold reduction for clindamycin. Although the MIC for rifampin was higher than that seen as a single antibiotic, it remained less than that of the second stage MIC. Interestingly, even though minocycline is an antibiotic from the tetracycline family of antibiotics, the activity enhancement was restricted to minocycline and was not demonstrated for other members of the family (tetracycline).
(149) The results demonstrate a synergistic effect for minocycline and degradation products of ORC. Degradation products obtained from a hemostatic ORC pad impregnated with a low concentration of minocycline are therefore able to provide a high level of antibiotic activity. The recommended antibiotic concentration for minocycline alone in an ORC pad is in the range of from 2.7 μg to 21.6 μg per gram ORC. When minocycline is used in combination with rifampin or clindamycin, the recommended antibiotic concentrations are from 0.5 μg to 2.7 μg minocycline per gram ORC together with from 31.4 μg to 157 μg rifampin per gram ORC or from 0.6 μg to 3.8 μg clindamycin per gram ORC.