Abstract:
This invention is the design and synthesis of a caprolactone monomer which bears a pendant protected carboxyl group. This monomer has been copolymerized with caprolactone in varying ratios. After polymerization, the protecting group can be removed and an antibiotic can be attached as a new pendant group. The bioactivity of the antibiotic-bound poly(caprolactone) polymer is described.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Application No. 60/747,061 filed on May 11, 2006, entitled “Antibiotic-Bound Poly(Caprolactone) Polymer”. 
    
    
     STATEMENT OF GOVERNMENT INTEREST 
     This invention was made with Government support under Grant No. RO1 A1 51351 awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     FIELD OF INVENTION 
     This invention relates to attaching one or more bioactive molecules to the same polymer, and also for attachment before or after polymerization 
     BACKGROUND OF THE INVENTION 
     The delivery of water-insoluble drugs to targets within the human body is a challenge that presently places strict limitations on what drugs can be applied clinically. The need for methods which overcome this is of high priority in the development of new therapeutics for treatment of human disease. 
     The development of antibiotics for control of pathogenic bacteria has been of pressing need in this era of drugresistant infections. N-Methylthiolated b-lactams have been identified as a new family of antibacterial agents active against  Staphylococcus  bacteria, including methicillin-resistant  Staphylococcus aureus  (MRSA). (See Turos, E.; Konaklieva, M. I.; Ren, R. X. F.; Shi, H.; Gonzalez, J.; Dickey, S.; Lim, D.  Tetrahedron  2000, 56, 5571; Bart Heldreth, Timothy E. Long, Seyoung Jang, Suresh K. R. Guntireddygari, Edward Turos, Sonja Dickey, Daniel V. Lim, “N-Thiolated b-Lactam Antibacterials: Effects of the N-Organothio Substituent on anti-MRSA Activity,”  Bioorganic and Medicinal Chemistry  14, 3775-3784 (2006); and Edward Turos, Jeung-Yeop Shim, Yang Wang, Kerriann Greenhalgh, G. Suresh Kumar Reddy, Sonja Dickey, Daniel V. Lim, “Antibiotic-Conjugated Polyacrylate Nanoparticles: New Opportunities for Development of Anti-MRSA Agents,” Bioorganic and Medicinal Chemistry Letters 16, in press (2006); which are incorporated herein by reference). 
     The compounds have also displayed promising anticancer properties. These lactams exert their growth inhibitory effects on bacteria through a mode of action that is distinctively different to that of other b-lactam antibiotics, and possess structure-activity patterns unlike those already mapped for other b-lactam antibacterials such as the penicillins. One of the major limitations in the potential application of these N-thiolated b-lactam compounds, however, is their exceedingly low water solubility. 
     Drug delivery vehicles such as liposomes and gold nanoparticles have been developed to improve bioavailability, efficacy, and specificity of pharmaceutical compounds, particularly for anticancer agents, but nanoparticles have received surprisingly little attention in the antibiotic and infectious disease area. Some of the few notable examples have included antibiotic-encapsulated polymeric nanoparticles and liposomes, biodegradable nanospheres and surface-coated gold and silver nanoparticles. 
     SUMMARY OF INVENTION 
     In one embodiment, the invention provides an effective drug delivery platform that would enhance the water solubility of the lactams, without sacrificing inherent bioactivity. 
     In another embodiment, the invention provides for the development of antibacterial polyacrylate nanoparticles based on well-precedented emulsion polymerization procedures. 
     This invention addresses this need, and demonstrates the use of antibiotic-bound poly(caprolactone) polymers as anti-infective materials for biomedical applications in the prevention of bacterial infections. 
     In a first embodiment, the invention includes a functionalized compound comprising at least one caprolactone ring with an appended functional group. A plurality of methylene groups act as a spacer between the lactone ring and the functional group. In a preferred embodiment, the functional group is an antibiotic, such as a N-thiolated β-lactam. The functional group is preferably covalently bonded. 
     In an alternate embodiment, a method is provided for producing the functionalized compound of the previous embodiment. The method includes providing a at least one caprolactone ring with a protecting group spaced apart from the caprolactone ring by a plurality of methylene groups. The protecting group is then cleaved from the caprolactone ring and replaced by bonding a functional group to the caprolactone ring. Illustrative reagents for cleaving the protecting group include 10% pd/C as a catalyst in the presence of H 2(g)  and ethyl acetate (EtOAc). The protecting group is selected from the group consisting of alcohol protecting groups, amine protecting groups, carbonyl protecting groups and carboxyl protecting groups. In a preferred embodiment, the protecting group is selected from the group consisting of a benzel ester and a tert-butyl ester. As with the previous embodiment, the functional group is an antibiotic such as a N-thiolated β-lactam. 
     In another embodiment, the invention includes the compound represented by the formula: 
                                
or a pharmaceutically acceptable salt or ester thereof, wherein is selected from the group consisting of water-insoluble drugs, antibiotics, lactams, β-lactams, N-thiolated β-lactams and protecting groups. The protecting group is selected from the group consisting of alcohol protecting groups, amine protecting groups, carbonyl protecting groups and carboxyl protecting groups.
 
     In yet another embodiment, the invention includes the compound represented by the formula: 
                                
or a pharmaceutically acceptable salt or ester thereof.
 
     In another embodiment, the invention includes the compound represented by the formula: 
                                
or a pharmaceutically acceptable salt or ester thereof.
 
     In still another embodiment, the invention includes the compound represented by the formula: 
                                
or a pharmaceutically acceptable salt or ester thereof.
 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a fuller understanding of the nature and objects of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: 
         FIG. 1  shows the polymerization of a caprolactone monomer to polycaprolactone (PCL). 
         FIG. 2  shows the functionalized caprolactone polymer can be used for covalent binding of drug molecules. 
         FIG. 3  shows examples of functionalized caprolactones from the literature. 
         FIG. 4  shows the functionalized lactone of present invention. 
         FIG. 5A  shows drug appendage before polymerization. 
         FIG. 5B  shows drug appendage after polymerization. 
         FIG. 6  shows lactone resynthesis. 
         FIG. 7  shows ylide synthesis. 
         FIG. 8  is an image showing results of biological testing of the lactam-containing monomer vs. MSSA. 
         FIG. 9  demonstrates the anti-Bacillus activity of the β-lactam containing copolymer. 
         FIG. 10  shows the invention employing an alternate carboxyl protecting group. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part hereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. 
     N-thiolated β-lactams 
     New family of anti-MRSA and anti-Bacillus agents that have recently been reported by our laboratory. Extensive SAR studies have shown that changing the N-thioalkyl substituent has a large effect on the bioactivity and that changes at the other positions of the ring exert a more subtle effect. Recent research in our group has been focused on covalent attachment of these and other antibiotics to polymers for drug delivery and for new biomaterials. 
     
       
                 
         
             
             
         
      
     
     As used herein, the term “drug” to any therapeutic or prophylactic agent other than food which is used in the prevention, diagnosis, alleviation, treatment, or cure of disease in man or animal. 
     As used herein, the term “antibiotic” refers to any natural, synthetic, and semi-synthetic compound that has been identified as possessing antibacterial, antifungal, antiviral, or antiparasitic activity. 
     Polycaprolactones 
     Polycaprolactone (PCL) is a biodegradable polyester which can be prepared by ring opening polymerization of ε-caprolactone using a catalyst such as stannous octanoate, as shown in  FIG. 1 . PCL is degraded by hydrolysis of its ester linkages in physiological conditions (such as in the human body) and is therefore useful as a biomaterial. PCL has been approved by the Food and Drug Administration (FDA) for use in the human body as (for example) a suture (sold under the brand name Monocryl™ or generically). In particular, PCL has been used in long term implantable devices, owing to its degradation which is relatively slow. (See V. R. Sinha, K. Bansal, R. Kaushik, R. Kumria and A. Trehan; Poly-ε-caprolactone microspheres and nanospheres: an overview,  International Journal of Pharmaceutics , Volume 278, Issue 1, 18 Jun. 2004, Pages 1-23; which is incorporated herein by reference.) 
     The characteristics of PCL make is useful as a delivery mechanism for antibiotics. For example PCL is biodegradable (bulk hydrolysis of ester bonds), the byproducts of degradation are non-toxic (biocompatible), it is FDA approved and displays high permeability to many drugs. Therefore, the invention provides a functionalized caprolactone polymer can be used for covalent binding of drug molecules ( FIG. 2 ). 
     The functionalized caprolactones of the prior art (Detrembleur et al Macromolecules, 2000, 33, 14-18 and Trollsas et at Macromolecules, 2000, 33, 4619-4627) are shown in  FIG. 3 . In contrast, the functionalized lactone  10  of present invention is shown in  FIG. 4 . Lactone  10  comprises at least one lactone ring  12 , functional group  16  and at least one spacer  14 . As it can be seen, functional group  16  is placed away from site of polymerization. Moreover, an additional methylene spacer  14  between functional group  16  and lactone ring  12  enhances further functionalization. 
     In another embodiment, the invention provides a method of producing an antibiotic-conjugated functionalized caprolactone ( FIG. 5A  and  FIG. 5B ). In Step 1 includes providing a caprolactone comprising lactone ring  12 , methylene spacer  14  and protecting group  18 . In Step 2, protecting group  18  is cleaved, preferrably under mild conditions. Finally, in Step 3, drug of interest  20  is covalently bonded to the finished compound.  FIG. 5A  illustrates the method of appending a drug of interest to the functionalized lactone before polymerization.  FIG. 5B  illustrates the method of appending a drug of interest to the functionalized lactone after polymerization. Lactone retrosynthesis is shown in  FIG. 6  and Ylide synthesis is shown in  FIG. 7 . 
     Example 1 
     The following represents an embodiment of the invention wherein 1,4-dioxaspiro[4.5]decan-8-one is used to synthesize a functionalized lactone bearing a pendent benzyl ester as the protecting group. 
     
       
                 
         
             
             
         
      
     
     Next the functionalized lactone is coupled with an antibiotic, here N-thiolated β-lactam, after deprotection of benzyl ester.  FIG. 8  shows the results of biological testing of the lactam-containing monomer vs. MSSA. 
     
       
                 
         
             
             
         
      
     
     Example 2 
     In another embodiment, the invention provides a PCL derived from copolymerization of a functionalized lactone monomer with caprolactone. Copolymers containing 10%, 15%, 20%, 25% and 30% of the substituted lactone were prepared and characterized by TLC,  1 H, NMR,  13 C NMR and MALDI-TOF. The Copolymers displayed low molecular, between about 1000 and 4000. 
     
       
                 
         
             
             
         
      
     
     Deprotection of the copolymer and coupling with the antibiotic, β-lactam, is shown below and was achieved using the following reagents and conditions: (a) cat. 10% Pd/C, H 2(g) , EtOAc, 12-24 h. (b) 15 EDCl, cat. DMAP, dry CH 2 Cl 2 , 12 h. rt. The antibiotic activity of the completed PCL against  Bacillus  is shown in  FIG. 9 . 
     
       
                 
         
             
             
         
      
     
     Example 3 
     In yet another embodiment, the functionalized lactone comprises a carboxy protecting group, as shown in  FIG. 10 . Ylide synthesis is shown below using the reagents and conditions: (a) PPh 3 , C 6 H 6 , 12 h, rt; (b) 20% NaOH (aq) , 5 h, rt. 
     
       
                 
         
             
             
         
      
     
     Example 4 
     The following demonstrates the synthesis of lactone bearing pendant tert-butyl ester using the reagents and conditions: (a) 1.5 eq. ylide C 6 H 6 . 12 h, reflux, (b) H 2(g) , cat. 10% Pd?C, EtOAC, 24 h; (c) 0.1 eq. I 2 , dry acetone, 1 hr, rt; (d) 1.5mCPBA CHCl 3 , 3 h, reflux. 
     
       
                 
         
             
             
         
      
     
     Copolymerization with caprolactone was achieved as shown below. Copolymers containing 10%, and 20% of the substituted lactone were prepared and characterized by TLC,  1 H NMR and  13 C NMR. 
     
       
                 
         
             
             
         
      
     
     Functional caprolactone monomers have been synthesized and characterized. The monomers were further copolymerized with caprolactone. Caprolactone monomers and polymers with covalently bound N-thiolated β-lactams have been prepared and shown to possess bioactivity against MSSA and  Bacillis  respectively. 
     It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
     It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.