WO2009026544A1 - Ampicilline et poly(anhydride-amides) à base d'amoxicilline - Google Patents

Ampicilline et poly(anhydride-amides) à base d'amoxicilline Download PDF

Info

Publication number
WO2009026544A1
WO2009026544A1 PCT/US2008/074070 US2008074070W WO2009026544A1 WO 2009026544 A1 WO2009026544 A1 WO 2009026544A1 US 2008074070 W US2008074070 W US 2008074070W WO 2009026544 A1 WO2009026544 A1 WO 2009026544A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
polymer
formula
ampicillin
pharmaceutically acceptable
Prior art date
Application number
PCT/US2008/074070
Other languages
English (en)
Inventor
Kathryn E. Uhrich
Almudena Prudencio
Original Assignee
Rutgers, The State University Of New Jersey
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rutgers, The State University Of New Jersey filed Critical Rutgers, The State University Of New Jersey
Publication of WO2009026544A1 publication Critical patent/WO2009026544A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/21—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a nitrogen atom directly attached in position 6 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D499/44—Compounds with an amino radical acylated by carboxylic acids, attached in position 6
    • C07D499/48—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical
    • C07D499/58—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical substituted in alpha-position to the carboxamido radical
    • C07D499/64—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical substituted in alpha-position to the carboxamido radical by nitrogen atoms
    • C07D499/68—Compounds with an amino radical acylated by carboxylic acids, attached in position 6 with a carbon chain, substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, attached to the carboxamido radical substituted in alpha-position to the carboxamido radical by nitrogen atoms with aromatic rings as additional substituents on the carbon chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • Bacterial infections are a safety issue with medical implants, including stainless steel materials; such infections are extremely difficult to treat due to the development of resistant biof ⁇ lm networks that are not susceptible to traditional antibiotics (Patel R. Biofilms and antimicrobial resistance. Clin Orthop Relat Res, 2005, 437, 41-47). Such infections often lead to multiple revision surgeries, and in some cases, death (Campoccia D, et al., Biomaterials, 2006, 27, 2331- 2339).
  • bacterial infections are systemically treated with a course of antibiotics.
  • systemic treatments expose the entire body to the drug such that the implant site may not receive the dosage necessary to kill the bacteria (Burrows LL, Khoury AE. Infection of medical devices. In: Wnek GE, Bowlin GL., editor. Encyclopedia of Biomaterials and Biomedical Engineering. New York: Marcel Dekkar; 2004.839-848).
  • this lowered dose results in the development of resistant bacteria, and ultimately biof ⁇ lm formation (Hanssen AD, et al., Clin Orthop Relat Res, 2005, 437, 111-114).
  • Localized antibiotic therapy can also be used to treat bacterial infections associated with medical implants.
  • the most commonly studied localized antibiotic therapy is the physical admixture of antimicrobials within polymer matrices to treat bacterial infections (see von Eiff C, et al., Int J Artif Organs, 2005, 28, 1146-56; Garvin K, Feschuk, Clin Orthop Relat Res, 2005, 437, 105- 110; Gollwitzer H, et al., J Antimicrob Chemother, 2003, 51, 585-591 and Sharkawi T, et al., JBioact and Compat Pol, 2005, 20, 153-168).
  • the present invention provides compounds that have antibacterial properties. Accordingly there is provided a compound of the invention which is a compound of formula I:
  • n 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; or a salt thereof.
  • n 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and each R is independently H or OH; or a salt thereof.
  • each R is OH. In certain embodiments, each R is H.
  • one R is H and one R is OH.
  • n 4, 5, 6, 7 or 8.
  • n 6, 7 or 8.
  • n 8
  • the invention also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable diluent or carrier.
  • the invention also provides a medical device having a coating that comprises a compound of the invention. In one embodiment the invention also provides a medical device comprising a compound of the invention.
  • the medical device is an orthopedic implant. hi certain embodiments, the medical device is a knee joint or a hip joint.
  • the invention also provides a method to promote an anti-bacterial effect in an animal comprising administering a compound of the invention to the animal.
  • the antibacterial effect is against gram negative and/or gram positive bacteria.
  • the invention also provides a method to promote an antibacterial effect comprising contacting bacteria (e.g., in vivo or in vitro) with a compound of the invention.
  • the invention also provides a method to reduce biofilm formation on a surface comprising contacting the surface with a compound of the invention.
  • the compound of the invention is incorporated into a coating that is on the surface.
  • the invention also provides a compound of the invention for use in medical therapy.
  • the invention also provides the use of a compound of the invention for the manufacture of a medicament for producing an anti- bacterial effect in a mammal.
  • the invention also provides a compound of the invention, for the prophylactic or therapeutic treatment of a bacterial infection.
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or (II) or salts thereof.
  • the compounds, compositions and methods of the invention are useful for preventing biofilm formation and for treating antibiotic resistant bacterial infections, such as those infections associated with medical implants.
  • polymeric materials for biological applications such as medical devices (e.g., artificial organs, wound dressings, sutures, pacemakers, intravascular catheters, contact lenses, orthopedic implants) and drug delivery systems has evoked increasing interest.
  • medical devices e.g., artificial organs, wound dressings, sutures, pacemakers, intravascular catheters, contact lenses, orthopedic implants
  • drug delivery systems has evoked increasing interest.
  • Numerous polymeric biomaterials are implanted each year in humans, yet bacterial infections associated with these medical implants remain a significant issue. For example, nearly half of the two million nosocomial infections occurring in the United States are associated with medical implants.
  • the presence of a foreign body, such as a prosthetic implant provides a site for microbial adhesion and subsequent colonization, often leading to device-related infections.
  • Current clinical therapies to prevent biomaterial- related infections involve the lengthy administration of antibiotics at high systemic concentrations. Unfortunately, repeated failures of this therapeutic approach require device removal to ensure eradication of the infection and to avoid relapse.
  • degradable biomaterials were designed to release antimicrobials upon polymer degradation to locally prevent infections.
  • This work describes the synthesis of biodegradable antimicrobial-based polymers in which the antimicrobial drugs ampicillin and amoxicillin are chemically incorporated into the polymeric backbone by anhydride and amide bonds, rather than attached as a side group or physically admixed.
  • Antibacterial properties of the polymers were examined using gram-negative (Escherichia col ⁇ ) and gram- positive (Staphylococcus aureus) bacterial strains.
  • E. coli is the major cause of infections in biliary stents and urinary catheters, and S. aureus is responsible for infections associated with dental and orthopedic implants and fixation devices.
  • Antibiotic molecules (up to nearly 82% by weight) were incorporated into a polymeric backbone.
  • the polymers were prepared under mild conditions using solution polymerization methods. This milder polymerization method was chosen over typical melt-condensation processes because of the thermal instability of the antibiotics and polymer precursors. No glass transition temperatures (T g ) were observed for the polymers in the measured range from - 10 °C up to polymer decomposition ( ⁇ 200 °C).
  • T g glass transition temperatures
  • the antimicrobial-based polymers displayed enhanced solubility in organic solvents relative to the antibiotics alone. This factor is significant, as it determines processability of polymer into solvent-cast films, microspheres, and fibers that may be used as controlled release devices.
  • biodegradable antimicrobial-based poly(anhydride-amides) display promise as implants or coatings to reduce implant failure resulting from bacterial infections.
  • a salt of a compound of formula I or II can be useful as an intermediate for isolating or purifying a compound of formula I or ⁇ .
  • a compound of formula I or II as a pharmaceutically acceptable acid or base salt
  • pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the compounds of the invention are useful in a variety of applications where antibacterial activity is desired. Examples of such applications include, but are not limited to, medical, dental and cosmetic uses.
  • the compounds of the invention may be prepared in accordance with methods commonly employed in the field of synthetic chemistry. The compounds can be readily processed into pastes or solvent cast to yield films, coatings, microspheres and fibers with different geometric shapes for design of various medical implants.
  • the compounds of the invention can be incorporated into and/or released from medical implants such as, for example, vascular grafts and stents, bone plates, sutures, Implantable sensors, implantable drug delivery devices, orthopedic devices (e.g. replacement joints such as knees and hips), and stents for tissue regeneration.
  • the diacid compounds of formula (I) or (II) can be released by hydrolysis of a corresponding anhydride polymer.
  • the compounds of formula (I) or (II) can also be incorporated (e.g. dispersed) into the matrix of such an anhydride polymer or another polymer, and be released from that polymer over time (e.g. upon hydrolysis of the polymer).
  • Compounds of the invention can also be incorporated into oral formulations and into products such as skin moisturizers, cleansers, pads, plasters, lotions, creams, gels, ointments, solutions, shampoos, tanning products and lipsticks for topical application.
  • Useful dosages of the compounds can be determined by comparing their in vitro activity, and in vivo activity of the compounds of the invention in animal models. Additionally, useful dosages can be determined by measuring the rate of hydrolysis for a given polymer under various physiological conditions.
  • the amount of a polymer required for use in treatment will vary not only with the particular polymer selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
  • the compounds of formula I or II can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed, hi addition, the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • useful dermatological compositions which can be used to deliver the compounds of formula I or ⁇ to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • DMF Dimethylformamide
  • acetic anhydride dichloromethane
  • diethyl ether diethyl ether
  • pyridine pyridine
  • triethylamine purchased from Aldrich (Milwaukee, WI). All other reagents and solvents were purchased from Fisher Scientific (Fair Lawn, NJ) and used as received. Triethylamine was dried over calcium hydride, all other reagents were used without further purification.
  • the ampicillin-based diacid (2) was prepared in DMF with pyridine as a base at 0°C.
  • the polymer (1) was synthesized using triphosgene as a coupling agent and triethylamine as an acid acceptor at 0 °C under nitrogen.
  • a 5 % (w/v) ampicillin-based poly(anhydride-amide) (1) solution was prepared in DMF for solvent-casting.
  • the polymer solution 150 ⁇ L was pipetted onto 316L stainless steel coupons (McMaster-Carr, cut to 10 x 30 mm by Rutgers Physics Department Machine Shop).
  • the solvent was evaporated using one of three methods: i) ambient conditions overnight; ii) heating samples to 60 °C for two hours; and iii) heating under vacuum to 60 °C for 45 minutes. Additionally, the ampicillin-based diacid precursor was precipitated onto stainless steel substrates for comparison to the polymer coatings. Peel tests were conducted using the x-cut method in American Standard
  • Two 10 mm X-cuts were scratched into coated coupons using a fresh razor blade.
  • Permacel P-99 tape was adhered to the surface and peeled away at a 180° angle after 90 seconds.
  • Each sample was examined for coating removal and ranked according to the following scale: 5A- no peeling or removal; 4A-trace peeling or removal; 3A-jagged removal along incisions; 2A-jagged removal along most of the incisions; 1 A-removal from most of the area of the X under the tape; and 0 A-removal beyond the area of the X.
  • Sessile contact angle measurements were completed using deionized water on a model 250 Rame-Hart goniometer (Mountain Lakes, NJ) with DROPimage software. One water drop was placed on each coating and measured in real time with a camera. The contact angle was measured as the average value from five water droplets with three angle measurements taken for each drop. The final contact angle value is the average of at least 15 measurements.
  • Staphylococcus aureus was obtained from ATCC (Manassas, VA). The coatings were exposed to the Staphylococcus aureus bacteria cells at the required density for biofilm formation (10 6 colony forming units (CFU)/mL) for 24 hours at 37 °C. Adherent bacteria cells were recovered and quantified compared to negative uncoated controls. In Vitro Degradation with Bacteria Present
  • the HPLC method was developed to separate the free ampicillin released from the ampicillin diacid at 210 nm on a Gemini Cl 8 column (15O x 4.6 mm, 5 ⁇ m, Phenomenex, Torrance, CA).
  • the method was a linear gradient with a mobile phase consisting of 20 mM monobasic potassium phosphate pH 4.0 and methanol.
  • Five point calibration curves were generated for both the ampicillin and the ampicillin diacid with concentrations ranging from 0.005 mg/mL to 0.5 mg/mL.
  • the degradation media was diluted in monobasic potassium phosphate, if needed, to calculate release within the calibration curve.
  • Cytotoxicity of the polymer coatings was analyzed by culturing cells in media containing degraded polymer. Individual ampicillin-based polymer- coated stainless steel plates were placed in 50 mL of cell culture media for 48 hours. Media consisted of Dulbecco's Modified Eagle's Medium (DMEM; Sigma, St. Louis, MO), 10% v/v fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), penicillin/streptomycin, and L-glutamine.
  • DMEM Dulbecco's Modified Eagle's Medium
  • L929 mouse areolar/adipose fibroblasts (Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey) were sustained in a cell culture incubator at 37°C and an atmosphere containing 5% CO 2. Confluent fibroblasts were removed from the cell culture flask via trypsinization. After media removal, trypsin (2 mg/mL, Sigma, St. Louis, MO) was placed on cells and incubated at 37°C for five minutes to allow cell detachment from the surface of the flask. Trypsin activity was ceased with addition of media and the solution centrifuged for 2 min at 2000 rpm (Thermo Electron 5682 3L GP, Franklin, MA).
  • Cells were seeded at a concentration of 100,000 cells/well in media containing polymer degradation media and analyzed after 24, 48, and 72 hours. All samples were studied in triplicate. A standard curve was generated with cells seeded at 25,000, 50,000, 100,000, and 500,000 cells/well in cell culture media without polymer, analyzed after 24 hours. Imaging was performed with a fluorescent microscope (Olympus 1X81,
  • Fluorescent intensity was quantified using a fluorescence plate reader (Cytofluor ® Series 4000, Applied Biosystems, Woodinville, CA) at 485 nm excitation and 530 nm emission. For the three days, live cell numbers were quantified against the standard curve using Microsoft Excel ®. Results and Discussion
  • the quality of the polymer coatings was further assessed by an ASTM peel test (ASTM D 3359. West Conshohocken, PA: ASTM International). The results from the qualitative peel test are provided in the following table.
  • Peel results showed trace coating removal along x-cut incisions, but overall, the coatings strongly adhere to the stainless steel substrates. These results may be due to a nonspecific chemical interaction between the steel and the coating, ultimately causing the coating to strongly adhere to the stainless steel surface without modification.
  • a jagged removal along the x-cut incisions was noted.
  • the vacuum and heat processed samples were typically more robust.
  • the contact angle of the polymer coatings was about 45°, regardless of the solvent evaporation method. These coatings have intermediate contact angles, which are amenable to degradation, yet not immediately solubilized in aqueous media.
  • the decomposition temperatures of the polymer coatings prepared by various processing methods were measured by TGA to ensure polymer stability during processing.
  • Polymer (1) displayed a slight increase in the decomposition temperature following the coating preparation, increasing to a Tj of about 210
  • the polymer does not decompose.
  • the hydrolytic degradation scheme for the polymer (1) is shown below.
  • the anhydride bonds are most labile, whereas the amide bonds in the diacid (2) intermediate are expected to hydrolyze more slowly and possibly require enzymatic degradation.
  • Polymer 1 was found to hydrolyze in a more controlled manner with more than 70 % of the ampicillin-based polymer (1) coating degraded in 60 hours. Additional release data was obtained from a Varian USP 7 Dissolution Instrument with the ability to separate degradation products by HPLC. Ln these studies, the free ampicillin drug was not detected because the degradation media was PBS at pH 7.4. The conditions were not amenable to amide bond hydrolysis and there was no ampicillin peak observed in the HPLC chromatogram (results not shown). Again, the diacid intermediate (2) was the final degradation product released from the coatings.
  • Staphylococcus aureus growth was monitored during exposure to diacid (2) and polymer (1) coatings, as well as uncoated stainless steel controls.
  • the bacteria cells were seeded to facilitate biofilm growth and were exposed to the coatings for 24 hours. The results of the study are shown in the following table.
  • the ampicillin-based diacid (2) demonstrated complete prevention of Staphylococcus aureus biofilm.
  • polymer 1 itself did not inhibit biofilm formation, indicating that the diacid 2 is a bioactive compounds.
  • No significant difference in bacterial growth was observed between the uncoated control and the polymer samples. Given that Staphylococcus aureus does not grow on the diacid (2) coating, over longer time periods, it was determined that the diacid will prevent bacteria cell growth and biofilm formation.
  • Fibroblasts were tested in media containing degraded polymer coatings through heat or through a heat and vacuum methods. Cell proliferation was quantified through 72 hours of culture. Fibroblast culture growth was not impeded by the use of polyampicillin in the cell culture medium. All samples showed positive growth throughout the duration of testing with neither polymer impeding cell proliferation and amplification.
  • Cell morphology was compared to cells cultured in the control, which contained media without any polymer present.
  • the cells displayed their natural, heterogeneous morphology. Generally, the fibroblasts readily attached and remained attached to the surface despite washing and showed characteristic spreading and extensions.
  • cells cultured in media containing polymer 1 coatings prepared with fast solvent evaporation (heat and vacuum) did not extend and spread fibers as frequently as the control and polymer 1 coatings prepared with slow solvent evaporation (heat). Nonetheless, cells showed stellate morphology through 72 hours, proving a low cytotoxicity to the polymer degradation products.
  • Ampicillin-based poly(anhydride-amide) coatings may improve current clinical therapies for bacterial infections.
  • the coatings have strong adhesion properties to 316L stainless steel, which have not been demonstrated in any other polymer system.
  • the in vitro degradation studies yielded the ampicillin-based diacid as the final degradation product, in PBS pH 7.4.
  • the polymer (1) itself was found to be inactive against bacteria growth, it ws found to degrade to the more hydrophilic diacid (2), which completely eradicated Staphylococcus aureus growth and biofilm formation.
  • Example 2 Poly(anhydride-amides) that include thermally sensitive ⁇ -lactam antibiotics were prepared by solution polymerization.
  • Biodegradable antimicrobial-based polymers in which antimicrobial drugs, namely ampicillin and amoxicillin, were chemically incorporated into the polymeric backbone by anhydride and amide bonds were designed to locally prevent infections associated, e.g., with medical devices, by controlled hydrolytic degradation.
  • the in vitro degradation of the polymer into bioactive products was measured and the antibacterial properties examined using gram-negative (Escherichia col ⁇ ) and gram-positive (Staphylococcus aureus) bacteria.
  • the cytotoxicity of the polymers and bioactive degradation products were screened using fibroblast cells.
  • the polymer degradation products demonstrated antibacterial activity with no cytotoxic effects on fibroblasts.
  • the biodegradable antimicrobial-based poly(anhydride-amides) display promise as implants or implant coatings to reduce implant failure resulting from bacterial infections.
  • antimicrobial-based poly(anhydride-amides), 4 comprised of either ampicillin (a) or amoxicillin (b).
  • the polymer precursor or diacid (3) was synthesized by direct coupling of the antibiotic (1) (ampicillin, a, or amoxicillin, b) with sebacoyl chloride (2) in dimethylformamide (DMF) containing pyridine.
  • the antibiotic (1) ampicillin, a, or amoxicillin, b
  • sebacoyl chloride (2) in dimethylformamide (DMF) containing pyridine.
  • DMF dimethylformamide
  • pyridine acts as acid acceptor by neutralizing the HCl by-product.
  • the 1 H and 13 C NMR spectra clearly indicate that the pure products are obtained.
  • the polymers could not be generated via melt-condensation conditions typically used to generate drug-containing polyanhydrides. Therefore, the diacids were polymerized under mild conditions in methylene chloride at 0 0 C using triphosgene (1/3 equivalents) as a coupling agent and triethylamine as acid acceptor to form the poly(anhydride-amide) (4) in nearly quantitative yields.
  • the chemical incorporation of the ⁇ -lactam antibiotics, ampicillin Ia and amoxicillin Ib, into polymeric backbones yielded polymers with drug loadings of nearly 82 % by weight.
  • poly(anhydride-amides) synthesized from these antimicrobials ranged from 14,400-24,800 Da. These moderate molecular weights are similar to other poly(anhydride-ester) molecular weights obtained by melt-polymerization methods.
  • Polymers 4 displayed no glass transition temperatures (T g ) from -10 °C to the temperatures just below decomposition (-200 "C).
  • the ampicillin-based poly(anhydride-amide) (4a) displayed a higher decomposition temperature (T d ) of 235 °C than the amoxicillin-based polymer (4b) with T d 190 °C.
  • the antimicrobial- containing polymers (4) were relatively more soluble in organic solvents than the free antibiotic (1); this characteristic is particularly significant for polymer processability.
  • S. aureus (gram-positive) was the most susceptible organism; test compounds showed more potent activity against gram- positive than gram-negative bacteria.
  • the slightly smaller inhibition zones on the plates containing E. coli compared to those with S. aureus reflects the difficulty for the test compounds to diffuse across the extra outer membrane present in the gram-negative E. coli.
  • the diacids 3 themselves exhibited an inhibitory effect on the bacteria, displaying inhibition zones with diameters in the range of 10-15 mm for E. coli and 31-34 mm for & aureus. This phenomenon is notable because the NMR and in vitro degradation studies indicated that free antibiotic (1) was not formed from diacid 3 in media alone. Inhibition zones were not necessarily expected for the polymers (4), given that their large size may not allow for diffusion through the bacterial membrane. However, small inhibition zones were observed surrounding the polymer samples. These results indicate that the hydrolysis of the polymer (4) into the diacids (3) is sufficient to generate inhibitory concentration of diacid.
  • Ampicillin and amoxicillin were obtained from MP Biomedicals (Solon, OH).
  • Dimethylformamide (DMF), dimethylacetamide (DMA, anhydrous), pyridine, triethylamine, acetic anhydride, methylene chloride (anhydrous), and diethyl ether were purchased from Fisher (Fair Lawn, NJ). All other fine chemicals and solvents were obtained from Aldrich (Milwaukee, WI).
  • Triethylamine was dried over calcium hydride (CaH 2 ) and all other reagents were used with no further purification.
  • Weight-averaged molecular weights (M w ) and polydispersity indices (PDI) were determined by gel permeation chromatography (GPC) on a Waters liquid chromatography system consisting of a Waters 410 refractive index detector, a Waters 510 LC pump, and an ISS 200 advanced sample processor.
  • GPC gel permeation chromatography
  • An IBM Thinkcenter computer running Waters Empower software was used for data collection and processing. Polymers were dissolved (5 mg/mL) in dimethylformamide (DMF) containing 0.1 trifluoroacetic acid (TFA) and filtered through 0.45 ⁇ m poly(tetrafluoroethylene) (PTFE) syringe filters (Whatman, Clifton, NJ) before elution.
  • DMF dimethylformamide
  • TFA trifluoroacetic acid
  • PTFE poly(tetrafluoroethylene)
  • Sessile-drop contact angles of phosphate buffer solution on polymer disks were measured using an automated Rame-hart goniometer (Model 250, Netcong, NJ) with DROPimage Advanced software on a Dell Dimension 3000 computer. Angles were measured on three different disks for each polymer and an averaged value was taken.
  • Diacid Synthesis (3) Diacids (3) were prepared by reaction of the appropriate antimicrobial 1 with sebacoyl chloride (2) in the presence of a base (pyridine). The preparation of 3a is provided as an example. Ampicillin (Ia) (3.5 g, 10 mmol) was added to DMF (100 mL) and pyridine (4.0 mL, 50 mmol) to afford a suspension. Sebacoyl chloride (2) (1.1 mL, 5.2 mmol) dissolved in dimethylformamide (10 mL) was added dropwise over 5 minutes to the stirring reaction mixture at 0 °C to afford a clear solution.
  • Ampicillin (Ia) 3.5 g, 10 mmol
  • DMF 100 mL
  • pyridine 4.0 mL, 50 mmol
  • the reaction was stirred for 2 h at 0 °C, poured over water (400 mL) and acidified to pH ⁇ 2 using 0.1 N hydrochloric acid solution while stirring.
  • the white solid (diacid, 3) that formed was isolated by vacuum filtration, washed with water (3x100 mL) and dried overnight under vacuum at room temperature.
  • Polymer pellets were prepared by pressing ground polymers (14.0 ⁇ 0.5 mg) into 6 mm diameter x 0.5 mm thick disks in an IR pellet die (International Crystal Laboratories, Garf ⁇ eld, NJ) with a bench-top hydraulic press (Carver model M, Wabash, IN). A pressure of 5,000 psi was applied for 5 min at room temperature. No change in polymer color was observed upon applying pressure.
  • Degradation media consisted of phosphate buffer solution (PBS) containing 0.1 M potassium hydrogen phosphate and 0.1 M potassium dihydrogen phosphate.
  • PBS phosphate buffer solution
  • the pH was adjusted to 7.4 with 1 M sodium hydroxide and/or 1 N hydrochloric acid solutions and pH-measurements were performed on an Accumet® ARl 5 pH-meter (Fisher Scientific, Fair Lawn, NJ).
  • diacids were dissolved in deuterated dimethylsulfoxide
  • Antimicrobial Assays Polymers and Bacterial Strain. The inhibition zone test of polymers and their degradation products against the bacteria
  • Bacteria inocula were suspended in nutrient broth (EMD Chemicals, Gibbstwon, NJ) and culture turbidity adjusted to match that of a 0.5 McFarland standard (see below) to give a bacterial count of approximately 10 colony forming units/mL.
  • McFarland 0.5 turbidity standard was prepared according to the protocol described by the National Committee for Clinical Laboratory Standards (NCCLS) (Performance standards for antimicrobial susceptibility testing, hi National Comittee for Clinical Laboratory Standards (NCCLS); Eds.; document M100-S9: Wayne, PA, 1999; 21). Briefly, 0.5 mL of a 1.175% (wt/v) barium chloride dihydrate (BaCl 2 -2H 2 O) (Aldrich, Milwaukee, WI) solution was added to 99.5 mL of 1% (v/v) sulfuric acid (Fisher, Fair Lawn, NJ). The McFarland standard was stored at room temperature in the dark.
  • NCCLS National Committee for Clinical Laboratory Standards
  • BaCl 2 -2H 2 O Aldrich, Milwaukee, WI
  • Disk Diffusion Method The Mueller-Hinton II agar plate surface was inoculated with the broth culture of Escherichia coli and Staphylococcus aureus using a cotton swab (Fisher, Fair Lawn, NJ). Sterile paper disks (6 mm-diameter, Becton Dickinson, Sparks, MD) impregnated with 20 ⁇ L of the test solutions were placed and gently pressed down onto the plates using sterile forceps. Only four disks were placed on each plate to minimize overlap of the inhibition zones. The plates were incubated (Precision Scientific Incubator, South Plainfield, NJ) at 37 °C for 24 h under aerobic conditions. After incubation, the plates were examined and inhibition zones surrounding the disks were measured in millimeters using a ruler. All determinations were carried out in triplicate.
  • Cytotoxicity Studies Polymer Samples. Cytotoxicity of the polymers was analyzed by culturing cells in media containing the polymer. To prepare the polymer stock solution, the polymers were first sterilized by UV irradiation for 300 s and dissolved in DMSO to 40 mg/niL. The stock solution was then diluted with cell culture medium to three different final concentrations (1, 0.1 and 0.01 mg/mL). Cell culture media consisted of Dulbecco's Modified Eagle's Medium (Sigma, St. Louis, MO) with 10 % v/v fetal bovine serum, 1 % v/v glutamine, and 50 LVmL of penicillin and streptomycin. Media containing DMSO at the concentration used to prepare the polymer solutions was used as control.
  • Dulbecco's Modified Eagle's Medium Sigma, St. Louis, MO
  • Media containing DMSO at the concentration used to prepare the polymer solutions was used as control.
  • Calcein AM is a cell membrane permeable fluorescent marker only for live cell cytoplasms (Cutler, S., Garcia, A. Biomaterials 2003, 24, 1759-1770).
  • Calcein AM staining solution (8 ⁇ M, Molecular Probe, Carlsbad, CA) for 40 min at 4 °C. Fluorescent intensity was measured using a fluorescence plate reader
  • Fibroblast growth in polymer solutions was statistically compared to controls at each time point in triplicate. Student's /-test was performed using

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne, sous un aspect, un composé antibactérien de formule II dans laquelle n vaut 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ou 12 ; R est H ou OH ; ou un sel de celui-ci. Sous d'autres aspects, l'invention propose également des dispositifs qui comprennent des composés ainsi que des procédés de production d'un effet antibactérien avec le composé ou son sel.
PCT/US2008/074070 2007-08-22 2008-08-22 Ampicilline et poly(anhydride-amides) à base d'amoxicilline WO2009026544A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95735907P 2007-08-22 2007-08-22
US60/957,359 2007-08-22

Publications (1)

Publication Number Publication Date
WO2009026544A1 true WO2009026544A1 (fr) 2009-02-26

Family

ID=40378699

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/074070 WO2009026544A1 (fr) 2007-08-22 2008-08-22 Ampicilline et poly(anhydride-amides) à base d'amoxicilline

Country Status (1)

Country Link
WO (1) WO2009026544A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7985415B2 (en) 1997-09-10 2011-07-26 Rutgers, The State University Of New Jersey Medical devices employing novel polymers
US8221790B2 (en) 2000-07-27 2012-07-17 Rutgers, The State University Of New Jersey Therapeutic polyesters and polyamides
US8263060B2 (en) 2005-05-23 2012-09-11 Rutgers, The State University Of New Jersey Fast degrading polymers
US8361453B2 (en) 2006-06-06 2013-01-29 Rutgers, The State University Of New Jersey Iodinated polymers
US8747832B2 (en) 2007-04-12 2014-06-10 Rutgers, The State University Of New Jersey Biodegradable polyanhydrides with natural bioactive molecules
US9108070B2 (en) 2006-09-13 2015-08-18 Polymerix Corporation Active agents and their oligomers and polymers
US9144579B2 (en) 2012-08-17 2015-09-29 Rutgers, The State University Of New Jersey Polyesters and methods of use thereof
US9387250B2 (en) 2013-03-15 2016-07-12 Rutgers, The State University Of New Jersey Therapeutic compositions for bone repair
US9782432B2 (en) 2012-10-25 2017-10-10 Rutgers, The State University Of New Jersey Polymers and methods thereof for wound healing
US9862672B2 (en) 2013-05-29 2018-01-09 Rutgers, The State University Of New Jersey Antioxidant-based poly(anhydride-esters)
US10023521B2 (en) 2014-06-13 2018-07-17 Rutgers, The State University Of New Jersey Process and intermediates for preparing poly(anhydride-esters)
US10251869B2 (en) * 2014-04-23 2019-04-09 Case Western Reserve University Compositions and methods of inhibiting metallo-β-lactamases
US10543162B2 (en) 2015-04-10 2020-01-28 Rutgers, The State University Of New Jersey Kojic acid polymers

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050100516A1 (en) * 2003-11-07 2005-05-12 Formulated Solutions, Llc Hydroalcohol based self-tanning composition
US20060013851A1 (en) * 2002-02-07 2006-01-19 Giroux Karen J Therapeutic polyanhydride compounds for drug delivery

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060013851A1 (en) * 2002-02-07 2006-01-19 Giroux Karen J Therapeutic polyanhydride compounds for drug delivery
US20050100516A1 (en) * 2003-11-07 2005-05-12 Formulated Solutions, Llc Hydroalcohol based self-tanning composition

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7985415B2 (en) 1997-09-10 2011-07-26 Rutgers, The State University Of New Jersey Medical devices employing novel polymers
US8221790B2 (en) 2000-07-27 2012-07-17 Rutgers, The State University Of New Jersey Therapeutic polyesters and polyamides
US8241668B2 (en) 2000-07-27 2012-08-14 Rutgers, The State University Of New Jersey Therapeutic polyesters and polyamides
US8263060B2 (en) 2005-05-23 2012-09-11 Rutgers, The State University Of New Jersey Fast degrading polymers
US8361453B2 (en) 2006-06-06 2013-01-29 Rutgers, The State University Of New Jersey Iodinated polymers
US9108070B2 (en) 2006-09-13 2015-08-18 Polymerix Corporation Active agents and their oligomers and polymers
US10092578B2 (en) 2006-09-13 2018-10-09 Polymerix Corporation Active agents and their oligomers and polymers
US8747832B2 (en) 2007-04-12 2014-06-10 Rutgers, The State University Of New Jersey Biodegradable polyanhydrides with natural bioactive molecules
US9144579B2 (en) 2012-08-17 2015-09-29 Rutgers, The State University Of New Jersey Polyesters and methods of use thereof
US9782432B2 (en) 2012-10-25 2017-10-10 Rutgers, The State University Of New Jersey Polymers and methods thereof for wound healing
US9387250B2 (en) 2013-03-15 2016-07-12 Rutgers, The State University Of New Jersey Therapeutic compositions for bone repair
US9862672B2 (en) 2013-05-29 2018-01-09 Rutgers, The State University Of New Jersey Antioxidant-based poly(anhydride-esters)
US10251869B2 (en) * 2014-04-23 2019-04-09 Case Western Reserve University Compositions and methods of inhibiting metallo-β-lactamases
US10023521B2 (en) 2014-06-13 2018-07-17 Rutgers, The State University Of New Jersey Process and intermediates for preparing poly(anhydride-esters)
US10543162B2 (en) 2015-04-10 2020-01-28 Rutgers, The State University Of New Jersey Kojic acid polymers

Similar Documents

Publication Publication Date Title
WO2009026544A1 (fr) Ampicilline et poly(anhydride-amides) à base d'amoxicilline
Weyell et al. Tailor-made material characteristics of bacterial cellulose for drug delivery applications in dentistry
US8361453B2 (en) Iodinated polymers
CN105778126B (zh) 一种京尼平交联生物凝胶及其制备方法与应用
US7772352B2 (en) Bioabsorbable and biocompatible polyurethanes and polyamides for medical devices
US8263763B2 (en) Chemically modified polyaminosaccharide by a hydrocarbyl sultone compound
US7411031B2 (en) Synthesis of polyanhydrides
US20120277852A1 (en) Coating compositions, methods and coated devices
CN1418116A (zh) 聚磷腈衍生物
CN102159623A (zh) 用于人医药和/或医学技术的可生物降解和/或可吸收性硅凝胶材料的生产中的含有至少一种治疗活性成分的硅溶胶材料
Li et al. Preparation of gentamicin-loaded electrospun coating on titanium implants and a study of their properties in vitro
KR101820306B1 (ko) 갈산-키토산 결합체 및 이를 이용한 창상치료용 조성물
CZ258598A3 (cs) Inhibitor neovaskularizace
Leśniak-Ziółkowska et al. Antibacterial and cytocompatible coatings based on poly (adipic anhydride) for a Ti alloy surface
Raj et al. Drug loaded chitosan/aloe vera nanocomposite on Ti for orthopedic applications
CN104784103B (zh) 基于寡聚氨基酸两亲分子可注射抗菌水凝胶
Song et al. Chitosan/poly (3-hydroxy butyric acid-co-3-hydroxy valeric acid) electrospun nanofibers with cephradine for superficial incisional skin wound infection management
Zhang et al. An antibacterial and biocompatible piperazine polymer
Márquez et al. Incorporation of biguanide compounds into poly (GL)-b-poly (GL-co-TMC-co-CL)-b-poly (GL) monofilament surgical sutures
Ismail et al. Mechanical and antibacterial activities study of gellan gum/virgin coconut oil film embedded norfloxacin
US11376345B2 (en) Hydrogel composition and its uses
Mo et al. Facile fabrication of chitosan/polysialic acid composite hydrogels crosslinked with tannic acid to enhance wound healing after cesarean section
RU2815508C1 (ru) Биологически активная полимерная медицинская композиция
JPH07277981A (ja) 徐放性抗腫瘍製剤
EP4047012A1 (fr) Peptoïde cationique antimicrobien et copolymères peptidiques n-substitués, préparation et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08798525

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08798525

Country of ref document: EP

Kind code of ref document: A1