U.S. patent application number 11/129358 was filed with the patent office on 2005-11-17 for polymeric coupling agents and pharmaceutically-active polymers made therefrom.
Invention is credited to LaRonde, Frank J., Li, Mei, Santerre, Paul J..
Application Number | 20050255082 11/129358 |
Document ID | / |
Family ID | 35309655 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255082 |
Kind Code |
A1 |
Santerre, Paul J. ; et
al. |
November 17, 2005 |
Polymeric coupling agents and pharmaceutically-active polymers made
therefrom
Abstract
A pharmaceutically-active polymeric compound of the general
formula (I), Y-[Y.sub.n-LINK B-X].sub.m-LINK B (I) wherein (i) X is
a coupled biological coupling agent of the general formula (II)
Bio-LINK A-Bio (II) wherein Bio is a biologically active agent
fragment or precursor thereof linked to LINK A through a
hydrolysable covalent bond; and LINK A is a coupled central
flexible linear first segment of <2000 theoretical molecular
weight linked to each of said Bio fragments; (ii) Y is LINK
B-OLIGO; wherein (a) LINK B is a coupled second segment linking one
OLIGO to another OLIGO and an OLIGO to X or precursor thereof; and
(b) OLIGO is a short length of polymer segment having a molecular
weight of less than 5,000 and comprising less than 100 monomeric
repeating units; (iii) m is 1-40; and (iv) n is selected from 2-50.
The compounds are useful as biomaterials, particularly, providing
antibacterial activity in vivo. Also provided are biological
coupling agents useful as intermediates in the preparation of the
pharmaceutically-active polymeric compounds.
Inventors: |
Santerre, Paul J.; (Toronto,
CA) ; Li, Mei; (Toronto, CA) ; LaRonde, Frank
J.; (Toronto, CA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
35309655 |
Appl. No.: |
11/129358 |
Filed: |
May 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11129358 |
May 16, 2005 |
|
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10875550 |
Jun 25, 2004 |
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Current U.S.
Class: |
424/78.27 ;
424/78.3 |
Current CPC
Class: |
A61K 47/55 20170801;
A61K 47/59 20170801; A61L 2300/42 20130101; A61L 2300/604 20130101;
A61K 9/0024 20130101; A61K 47/60 20170801; C08G 18/4277 20130101;
A61L 2300/416 20130101; A61L 2300/41 20130101; A61K 47/56 20170801;
A61L 2300/406 20130101; C08G 18/73 20130101; A61L 27/54 20130101;
A61L 2300/45 20130101 |
Class at
Publication: |
424/078.27 ;
424/078.3 |
International
Class: |
A61K 031/785 |
Foreign Application Data
Date |
Code |
Application Number |
May 14, 2004 |
CA |
2,467,321 |
Claims
1. A pharmaceutically-active polymeric compound of the general
formula (I), Y-[Y.sub.n-LINK B-X].sub.m-LINK B (I) wherein (i) X is
a coupled biological coupling agent of the general formula (II)
Bio-LINK A-Bio (II) wherein Bio is a biologically active agent
fragment or precursor thereof linked to LINK A through a
hydrolysable covalent bond; and LINK A is a coupled central
flexible linear first segment of <2000 theoretical molecular
weight linked to each of said Bio fragments; (ii) Y is LINK
B-OLIGO; wherein (a) LINK B is a coupled second segment linking one
OLIGO to another OLIGO and an OLIGO to X or precursor thereof; and
(b) OLIGO is a short length of polymer segment having a molecular
weight of less than 5,000 and comprising less than 100 monomeric
repeating units; (iii) m is 1-40; and (iv) n is selected from
2-50.
2. A compound as defined in claim 1 wherein LINK A is linked to Bio
by carboxylic ester, amide or sulfonamide links.
3. A compound as defined in claim 1 wherein LINK A contains within
itself polyalkyl, polyethylene oxide, polyalkylene oxide,
polyamides, polyester, polyvinyl, polycarbonate, polyanhydrides or
polysiloxanes.
4. A compound as defined in claim 1 wherein LINK A has a molecular
weight selected from 60-700
5. A compound as defined in claim 1 wherein Y.sub.n has a
theoretical molecular weight of less than 15,000,
6. A compound as defined in claim 5 wherein Y.sub.n has a molecular
weight of <10,000.
7. A compound as defined in claim 6 wherein Y.sub.n has a molecular
weight of <5,000.
8. A compound as defined in claim 1 wherein LINK B has a molecular
weight selected from 60-2000.
9. A compound as defined in claim 8 wherein LINK B has a molecular
weight selected from 60-700.
10. A compound as defined in claim 1 wherein LINK B is linked to
other segments via urethanes, esters, ureas, sulfonamides,
carbonates, anhydrides or amides.
11. A compound as defined in claim 1 having at least two Bio
fragments or precursors thereof of different biological
activities.
12. A compound as defined in claim 1 wherein said Bio has a
molecular weight of <4000.
13. A compound as defined in claim 12 wherein said Bio has a
molecular weight of <2000.
14. A compound as defined in claim 1 wherein said Bio is a
biologically-active entity selected from an anti-coagulant, an
anti-inflammatory agent, an anti-proliferative agent, an
antimicrobial agent, an anti-thrombic agent and a hydrolysable
precursor fragment thereof.
15. A compound as defined in claim 1 having at least two of said
Bio entities and/or said hydrolysable precursor fragment
thereof.
16. A compound as defined in claim 14 wherein said Bio is an
antimicrobial agent or hydrolysable precursor fragment thereof.
17. A compound defined in claim 16 wherein said antimicrobial agent
is selected from Cipofloxacin and Norfloxacin.
18. A compound as defined in claim 14 wherein said anti-flammatory
agent is selected from Amfenac, Aceclofenac, Oxaceprol and
Enoxolone.
19. A compound as defined in claim 14 wherein said anti-thrombic
agent is selected from Bromofenac, Tirofiban and Lotrafiban.
20. A compound as defined in claim 14 wherein said
anti-proliferation agent is selected from Acivicin and Alkeren.
21. A biological coupling agent of the general formula (III)
PBio-LINK A-PBio (III) wherein PBio is a biologically active agent
fragment or precursor thereof linked to LINK A through a
hydrolysable covalent bond and having at least one functional group
to permit step growth polymerization; and LINK A is a coupled
central flexible linear first segment of <2000 theoretical
molecular weight linked to each of said PBio fragments.
22. A coupling agent as defined in claim 21 wherein LINK A is
linked to Bio by carboxylic ester, amide or sulfonamide links.
23. A coupling agent as defined in claim 21 wherein LINK A has a
molecular weight selected from 60-700.
24. A coupling agent as defined in claim 21 wherein said Bio is a
biologically-active entity selected from an anti-coagulant, an
anti-inflammatory agent, an anti- proliferative agent, an
antimicrobial agent, an anti-thrombic agent and a hydrolysable
precursor fragment thereof.
25. A coupling agent as defined in claim 24 wherein said
anti-microbial agent is selected from Cipofloxacin and
Norfloxacin.
26. A coupling agent as defined in claim 24 wherein said
antiflammatory agent is selected from Amfenac, Aceclofenac,
Oxacepro and Enoxolone.
27. A coupling as defined in claim 24 wherein said anti-thrombic
agent is selected from Oxaceprol, Bromofenac, Tirofiban and
Lotrafiban.
28. A coupling agent as defined in claim 24 wherein said
anti-proliferation agent is selected from Acivicin and Alkeren.
29. A pharmaceutically-active polymeric composition comprising a
pharmaceutically- active polymeric compound as defined in claim 1
in admixture with a compatible base polymer.
30. A composition as defined in claim 29 wherein said base polymer
is selected from the group consisting of polyurethanes,
polysulfones, polycarbonates, polyesters, polyethylene,
polypropylene, polystyrene, polysilicone, poly(acrylonitrile-
butadienestyrene), polybutadiene, polyisoprene,
polymethylmethacrylate, polyamine, polyvinylacetate,
polyacrylonitrile, polyvinyl chloride, polyethylene, terephthalate,
cellulose and other polysacharides.
31. A composition as defined in claim 29 wherein said base polymer
is a segmented polyurethane, a polyester, a polycarbonate,
polysaccharide or polyamide or polysilicone.
32. A composition as defined in claim 29 in the form of a shaped
article.
33. A shaped article as defined in claim 32 in the form of an
implantable medical device, self-supporting film, or fiber.
34. A compound as defined in claim 1 in the form of a shaped
article.
35. A shaped article as defined in claim 34 in the form of an
implantable medical device, self-supporting film or fiber.
Description
FIELD OF THE INVENTION
[0001] This invention relates to polymeric coupling agents as
intermediates, pharmaceutically-active polymers made therefrom,
composition comprising said polymers and shaped articles made
therefrom.
BACKGROUND TO THE INVENTION
[0002] It has become common to utilize implantable medical devices
for a wide variety of medical conditions, e.g., drug infusion and
haemodialysis access. However, medical device implantation often
comes along with the risk of infections (1), inflammation (2),
hyperplasia (3), coagulation (4). It is therefore important to
design such materials to provide enhanced biocompatibility.
Biocompatibility is defined as the ability of a material to perform
with an appropriate host response in a specific application. The
host relates to the environment in which the biomaterial is placed
and will vary from being blood, bone, cartilage, heart, brain, etc.
Despite the unique biomedical related benefits that any particular
group of polymers may possess, the materials themselves, once
incorporated into the biomedical device, may be inherently limited
in their performance because of their inability to satisfy all the
critical biocompatibility issues associated with the specific
application intended. For instance while one material may have
certain anti- coagulant features related to platelets it may not
address key features of the coagulation cascade, nor be able to
resist the colonization of bacteria. Another material may exhibit
anti-microbial function but may not be biostable for longterm
applications. The incorporation of multi-functional character in a
biomedical device is often a complicated and costly process which
almost always compromises one polymer property or biological
function over another, yet all blood and tissue contacting devices
can benefit from improved biocompatibility character. Clotting,
toxicity, inflammation, infection, immune response in even the
simplest devices can result in death or irreversible damage to the
patient. Since most blood and tissue material interactions occur at
the interface between the biological environment and the medical
device, the make-up of the outer molecular layer (at most the
sub-micron layer) of the polymeric material is relevant to the
biological interactions at the interface. This is a particularly
challenging problem for biodegradable polymer systems when a
continuous exposure of new surfaces through erosion of the bulk
polymer requires a continuous renewal of biocompatible moieties at
the surface.
[0003] Bioactive agents containing polymer coatings have been
developed to improve the biocompatibility of medical device
surfaces. Patnaik et al. (5) described a method of attaching
bioactive agents, such as heparin (an anti-coagulant) to polymeric
substrates via a hydrophilic, isocyanate/amine-terminated spacer in
order to provide a coating of the bio-active material on the
medical device. The investigator found that the bioactive agent's
activity was achieved when the spacer group had a molecular weight
of about 100-10,000 daltons. But most preferably that is of 4000
daltons. Unfortunately, such a material would only be applicable
for substrates which were not intended to under go biodegradation
and exchange with new tissue integration since the heparin in
limited to surface and does not form the bulk structure of the
polymer chains.
[0004] Another example of biomaterial design relates to infection
control. In the last decade, a number of strategies have been used
in attempts to solve problems such as those associated with medical
device infection. One approach is to provide a more biocompatible
implantable device to reduce the adhesion of bacteria. Silver
coated catheters have been used to prevent exit site infections
associated with chronic venous access (6) and peritoneal dialysis
(7). However, longterm studies have failed to demonstrate a
significant reduction in the number or severity of exit site
infections. In addition, bacterial resistance to silver can develop
over time and carries with it the risk of multiple antibiotic
resistances (8).
[0005] Since bacteria adhesion is a very complex process, complete
prevention of bacteria adhesion is difficult to achieve with only a
passive approach. There remains a need for local controlled drug
delivery. The advantages for the latter approach include 1) a high
and sustained local drug concentration can be achieved without the
systemic toxicity or side effects which would be experienced from
systemic doses sufficient to obtain similar local drug
concentration; 2) high local drug concentration can be attained,
even for agents that are rapidly metabolized or unstable when
employed systemically; 3) some forms of site-specific delivery have
the potential to establish and maintain local drug action, either
by preventing its efflux from the arterial wall or by using
vehicles or agents that have a prolonged duration of action; 4) it
gives the potential for designing a smart drug delivery system,
which can be triggered to start the release and/or modulate the
rate of release according to the infection status.
[0006] Methods for obtaining compositions which contain drugs and
polymers in a composite form to yield bioactive agent release
coatings are known. For example, Chudzik et al. (9) formulated a
coating composite that contained a bioactive agent (e.g. a drug)
and two polymers, i.e., poly(butyl methacrylate) and
poly(ethylene-co-vinyl acetate). The coating formed from the above
formulation provided good durability and flexibility as well as
significant drug release, which could be particularly adapted for
use with devices that undergo significant flexion and/or expansion
in the course of their delivery and/or use, such as stents and
catheters. These approaches have the benefit of localized delivery
at high drug concentration, but are unable to keep a sustained and
controlled release of drug for long periods. Ragheb et al.(10)
found a method for the controlled release of a bioactive agent from
polymer coatings. Wherein, two coating layers of polymer were
applied to a medical device. The first layer of the device is an
absorbent material such as parylene derivatives. Drug or bioactive
agent is deposited over at least a portion of this layer. The
second biocompatible polymer layer on top of the drug and the first
layer must be porous. The polymer is applied by vapor deposition or
by plasma deposition. Since the drug release mechanism is totally
controlled by porous sizes, making a suitable porous size
distribution in the second layer in order to satisfy the required
release model is often a technical challenge. As well, this type of
system requires multiple processing steps which increases
production cost and adds to the need for QA/QC steps.
[0007] In addition to the traditional diffusion-controlled delivery
systems described in the above references, there exist several more
sophisticated in situ drug delivery polymers which can alter the
efficacy of drugs by improving target delivery and changing the
control parameters of the delivery rate. These include
biodegradable hydrogels (11), polymeric liposomes (12),
bioresorbable polymers (13) and polymer drugs (14-16). Polymer
drugs contain covalently attached pharmaceutical agents on the
polymer chain as pendent groups, or even incorporated into the
polymer backbone. For example, Nathan et al (17) conjugated
penicillin V and cephradine as pendant antibiotics to
polyurethanes. Their work showed that hydrolytically labile pendant
drugs were cleaved and exhibited antimicrobial activities against
S. aureus, E. faecalis and S. pyogenes.
[0008] Ghosh et al. (18) coupled nalidixic acid, a quinolone
antibiotic, in a pendant manner to an active vinyl molecule. These
vinyl groups can then be polymerized to generate a polymer with
pendent antibiotics on each monomer. However, having such pendant
groups will dramatically alter the physical structure of the
polymer. A better strategy would be to have the drugs within the
linear backbone portion of the polymer. In in-vivo hydrolysis
studies they reported a 50% release of drug moieties over the first
100 hours. This quinolone drug has been shown to be effective
against gram negative bacteria in the treatment of urinary track
infections, however chemical modifications of the latter (e.g.
ciprofloxacin, norfloxacin and others) have a wider spectrum of
activity. More recent work on the conjugation of norfloxacin to
mannosylated dextran has been reported. This was driven in an
effort to increase the drug's uptake by cells, enabling them to
gain faster access to micro-organisms (19). The studies showed that
norfloxacin could be released from a drug/polymer conjugate by
enzyme media and in vivo studies, the drug/polymer conjugate was
effective against Mycobacterium tuberculosis residing in liver
(20). In the system, norfloxacin was attached pendant to sequences
of amino- acids which permitted its cleavage by the lysosomal
enzyme, cathepsin B.
[0009] Santerre (13a) describes the synthesis and use of novel
materials to which when added to polymers converts the surface to
have bioactive properties, while leaving the bulk properties of the
polymer virtually intact. Applications are targeted for the
biomedical field. These materials are oligomeric fluorinated
additives with pendant drugs that are delivered to the surface of
bulk polymers during processing by the migration of the fluorine
groups to the air/polymer interface. These materials can deliver a
large array of drugs, including anti-microbials, anti-coagulants
and anti-inflammatory agents, to the surface. However modification
is limited to the surface. This becomes a limitation in a
biodegradable polymer which may require sustained activity
throughout the bio-erosion process of the polymer.
[0010] Santerre and Mittleman (14) teach the synthesis of polymeric
materials using pharmacologically-active agents as one of the
co-monomers for polymers. Wherein, 1,6-diisocynatohexane and/or
1,12-diisocyanatododecane monomers or their oligomeric molecules
are reacted with the antimicrobial agent, ciprofloxacin, to form
drug polymers. The pharmacologically-active compounds provide
enhanced long term anti- inflammatory, anti-bacterial,
anti-microbial and/or anti-fungal activity. However, since the
reactivities of the carboxylic acid group and the secondary amine
group of ciprofloxacin with the isocyanate groups are different,
the reaction kinetics become challenging. As well, formulations
must be selective in order to minimize strong van der Waals
interactions between the drug components and hydrogen bonding
moieties of the polymer chains since this can delay the effective
release of drug. Hence, an improvement over the latter system are
biomonomers made up of the drugs and agents which, without being
bound by theory, would ensure a less restricted access of the drug
during hydrolysis of the polymer, as well as providing more uniform
chemical function for reaction with the isocyanate groups or other
monomer reagents.
PUBLICATIONS
[0011] (1) Mittelman, M W, "Adhesion to biomaterials" in Bacterial
Adhesion: Molecular and Ecological diversity, M Fletcher(ed)
89-127, 1996)
[0012] (2) John F. Burke, et al., "Applications of materials in
medicine and dentistry", in Biomaterials Science, 1996, Ch. 7, pp
283-297.
[0013] (3) Martin R. Bennett, Michael O'Sullivan, "Mechanism of
angioplasty and stent restenosis: implications for design of
rational therapy", Pharmacology & Therapeutics 91 (2001) pp
149-166.
[0014] (4) Eberhart, R. C., and C. P. Clagett, "Platelets,
catheters, and the vessel wall; catheter coatings, blood flow, and
biocompatibility", Seminars in Hematology, Vol. 28, No. 4, Suppl.
7, pp 42-48 (1991).
[0015] (5) U.S. Pat. No. 6,096,525--Patnaik, B K. Aug. 1, 2000
[0016] (6) Groeger J. S. et al., 1993, Ann. Surg. 218:206-210.
[0017] (7) Mittelman M. W., et. al., 1994. Ann. Conf. Peritoneal
Dialysis, Orlando. Fla.
[0018] (8) Silver S. et al., 1988, Ann. Rev. Microbiol.
42:717-743
[0019] (9) U.S. Pat. No. 6,344,035--Chudzik, et al. Feb. 5,
2002
[0020] (10) U.S. Pat. No. 6,299,604--Ragheb, et al. Oct. 9,
2001
[0021] (11) U.S. Pat. No. 6,703,037--Hubbell et al. Mar. 9,
2004
[0022] (12) Valerio D. et al. Biomaterials, 19:1877-1884 (1998)
[0023] (13) U.S. Pat. No. 4,916,193--Tang et al. and U.S. Pat. No.
4,994,071--MacGrego
[0024] (13a) U.S. Patent filed on Jun. 7, 2002, application Ser.
No. 10/162,084, Santerre, Paul J.
[0025] (14) U.S. Pat. No. 5,798,115--Santerre, Paul J. and
Mittleman, Marc W. Aug. 25, 1998.
[0026] (15) Modak S. M., Sampath, L., Fox, C. L., Benvenisty A.,
Nowygrod, R., Reemstmau, K. Surgery, Gynecology & Obstertrics,
164, 143-147 (1987).
[0027] (16) Bach, A.; Schmidt, H.; Bottiger, B.; Schreiber B.;
Bohrer, H.; Motsch, J.; Martin, E.; Sonntag, H. G., J. Antimicrob.
Chemother., 37, 315, (1996)
[0028] (17) Nathan, A.; Zalipsky, S.; Ertel, S. I.; Agarthos, S.
N.; Yarmush, M. L.; Kohn. J. Bioconjugate Chem. 1993, 4,
54-62.)
[0029] (18) Ghosh M. Progress in Biomedical polymers, Gebekin CG.
Et al (ed), Plenum press, New York, 1990, 335-345; Ghosh M.
Polymeric Materials, Science&Engineering 1988, 59: 790-793
[0030] (19) Coessens, V.; Schacht, E., Domurado, D. J. Controlled
Release 1997, 47 283-291
[0031] (20) Roseeuw, E.; Coessens V.; Schacht E., Vrooman B.;
Domurado, D.; Marchal G. J Mater. Sci: Mater. Med. 1999, 10,
743-746
[0032] (21) Hemmerich, K. J. Polymer materials selection for
radiation sterilized products, Medical Device & Diagnostic
Industry, February, 2000
[0033] (22) ISO 11137: Sterilization of health care
products-Requirements for validation and routine control-Radiation
sterilization.
SUMMARY OF THE INVENTION
[0034] Since the availability of drugs that can serve as commercial
monomers, specifically designed for the synthesis of the above drug
polymers or polymers to be used in composites are limited, there is
a need for custom synthesis methods of the drug precursors. Rather
than depending on the chemical function that common commercial
drugs inherently provide, it would be better provide monomers that
have similar multi- functional groups and preferably similar
di-functional groups for the synthesis of hydrolysable type
polymers. The current invention represents a group of novel diamine
or diol monomers that simultaneously incorporate the following
features: 1) they are synthesized under mild conditions for
coupling biological or pharmaceuticals or biocompatible components
together via a hydrolysable bond; 2) they contain selectively
reactive groups (di-functional or greater) (including amines
(secondary or primary) and hydroxyls) that could be used for
subsequent polymerization of polyesters, polyamides, polyurethanes,
polysulfonamides and many other classical step growth polymers; 3)
they contain selectively hydrolysable groups that permit the
release of defined degradation products consisting of biological,
pharmaceutical or biocompatible components; 4) their molecular
weights may vary depending on the molecular weight of the
pharmaceutical or biocompatible reagents to be as high as 4000, but
typically the molecular weights of the molecules will be preferably
less than 2000 in order for them to have good mobility of the
molecular segment once incorporated within the polymer, and have
good reactivity in the reaction polymerization solution; 5) they
provide a strategy for enhancing the introduction of important
biological, pharmaceutical or biocompatible reagents which
otherwise contain functional groups (such as shielded esters,
sulphonamides, amides and anhydrides) that would have poor
reactivity in hydrolytic reactions due to strong van der Waals or
hydrogen bonding between drug polymer backbones. 6) Since, these
molecules will have similar functional groups they will provide
consistent and more predictable reactivity in a classical step
growth polymerization. This invention describes the unique
synthesis pathways for the biomonomers, provides examples of their
use in the synthesis of polymers and defines methods of processing
said polymers for applications as biodegradable materials ranging
from biomedical to environmental related products.
[0035] It is an object of the present invention to provide
synthetic pathways of biological coupling agents/biomonomers
comprising, such as, anti-inflammatory, anti-bacterial,
anti-microbial and/or anti-fungal pharmaceuticals as biomonomer
precursors with good reactivity for step growth polymer
synthesis.
[0036] It is a further object of the present invention to provide
biological polymers comprising said biological coupling
compounds/monomers with pharmaceutically active properties.
[0037] It is a further object of the present invention to provide
said polymer compounds alone or in admixture with a compatible
polymeric biomaterial or polymer composite biomaterials for
providing a shaped article having pharmaceutically active
properties.
[0038] It is a further object of the present invention to provide
said shaped article for use as a medical device, comprising a body
fluid and tissue contacting device in the biomedical sector, or for
use in the biotechnology sector to provide anti-infection, anti-
inflammatory properties.
[0039] It is a further object of the present invention to provide
said polymer compounds alone as a coating or in admixture with
either a base polyurethane, polysilicone, polyester,
polyethersulfone, polycarbonate, polyolefin or polyamide for use as
said medical devices in the biomedical sector, for improving
anti-infection, anti-inflammatory, antimicrobials,
anti-coagulation, anti-oxidation, anti-proliferation function.
[0040] It is a further object of the invention to provide processes
of manufacture of said biomonomers, polymers containing said
biomonomers, said admixtures and said shaped articles.
[0041] The invention, generally, provides the unique synthesis
pathways for covalently coupling biologicals or pharmaceuticals or
biocompatible components to both sides of a flexible diol or
diamine, such as but not limited to triethylene glycol or any other
kind of linear diol or diamine under mild conditions. Bioactive
agents must possess a reactive group such as a carboxylic acid,
sulfonate or phosphate group which can be conjugated to the
flexible diols or diamines by using a carbodiimide-mediated
reaction. Bioactive agents used in the coupling reaction must also
contain selectively reactive multi- functional and preferably
di-functional groups (including amines (secondary or primary) and
hydroxyls) that could be used later on for subsequent
polymerization of polyesters, polyamides, polyurethanes,
polysulfonamides and any other classical step growth polymer
pharmaceutic containing coupling agents/monomers.
[0042] The invention provides in one aspect, a biological coupling
agent (biomonomer) having a central portion comprising of flexible
i.e. not limiting chain dynamic movement such as do aromatic rings,
linear or aliphatic (saturated) segments of <2000 theoretical
molecular weight and hydrolysable linkages
[0043] Accordingly, the invention provides a biological coupling
agent of the general formula (III)
PBio-LINK A-PBio (III)
[0044] wherein PBio is a biologically active agent fragment or
precursor thereof linked to LINK A through a hydrolysable covalent
bond and having at least one functional group to permit step growth
polymerization; and LINK A is a coupled central flexible linear
first segment of <2000 theoretical molecular weight linked to
each of said PBio fragments.
[0045] By the term "biomonomers" in this specification and claims,
is meant compounds of the formulae (III) used in the synthesis of
the compounds of formula (I) through the use of the functional
group for step growth polymerization.
[0046] Most preferably each of the PBio fragments is limited to a
single functional group for use in step growth polymerization.
[0047] Thus, in a further aspect the invention provides a
pharmaceutically-active polymeric compound of the general formula
(I),
Y-[Y.sub.n-LINK B-X].sub.m-LINK B (I)
[0048] wherein (i) X is a coupled biological coupling agent of the
general formula (II)
Bio-LINK A-Bio (II)
[0049] wherein Bio is a biologically active agent fragment or
precursor thereof linked to LINK A through a hydrolysable covalent
bond; and LINK A is a coupled central flexible linear first segment
of <2000 theoretical molecular weight linked to each of said Bio
fragments;
[0050] (ii) Y is LINK B-OLIGO; wherein
[0051] (a) LINK B is a coupled second segment linking one OLIGO to
another OLIGO and an OLIGO to X or precursor thereof; and
[0052] (b) OLIGO is a short length of polymer segment having a
molecular weight of less than 5,000 and comprising less than 100
monomeric repeating units;
[0053] (iii) m is 1-40; and
[0054] (iv) n is selected from 2-50.
[0055] The invention provides in another aspect, a
pharmaceutically-active polymeric material having a backbone made
from said biomonomer. Such polymers comprise oligomeric segments of
<5,000 theoretical molecular weight and optional link segments,
herein denoted [link B] covalently coupled to the oligomeric
segment denoted herein [oligo] and the said biomonomer.
[0056] By the term "oligomeric segment" is meant a relatively short
length of a repeating unit or units, generally less than about 50
monomeric units and molecular weights less than 10,000 but
preferably <5000. Preferably, [oligo] is selected from the group
consisting of polyurethane, polyurea, polyamides, polyalkylene
oxide, polycarbonate, polyester, polylactone, polysilicone,
polyethersulfone, polyolefin, polyvinyl, polypeptide,
polysaccharide; and ether and amine linked segments thereof.
[0057] By the term "LINK A molecule" is meant a molecule covalently
coupling bioactive agents together in said biomonomer. Typically,
LINK A molecules can have molecular weights ranging from 60 to 2000
and preferably between 60 to 700, and have multi-functionality but
preferably di-functionality to permit coupling of two bioactive
agents. Preferably the LINK A molecules are synthesized from the
groups of precursor monomers selected from diols, diamines and/or
compounds containing both amine and hydroxyl groups, with or
without water solubility. Examples of typical LINK A precursors are
given in Table 1 but they are not limited to this list.
1 TABLE 1 Ethylene glycol Butane diol Hexane diol Hexamethylene
diol 1,5 pentanediol 2,2-dimethyl-1,3 propanediol 1,4-cyclohexane
diol 1,4-cyclohexanedimethanol Tri(ethylene glycol) Poly(ethylene
glycol), Mn: 100-2000 Poly(ethylene oxide) diamine, Mn: 100-2000
Lysine esters Silicone diols and diamines Polyether diols and
diamines Carbonate diols and diamines Dihydroxy vinyl derivatives
Dihydroxy diphenylsulfone Ethylene diamine Hexamethylene diamine
1,2-diamino-2 methylpropane 3,3,-diamino-N-methyldipropylamine 1,4
diaminobutane 1,7 diaminoheptane 1,8 diaminooctane
[0058] By the term "LINK B molecule" is meant a molecule covalently
coupling oligo units together to form the second coupling segments
within the central portion. Typically, LINK B molecules can have
molecular weights ranging from 60 to 2000 and preferably 60-700,
and have difunctionality to permit coupling of two oligo units.
Preferably the LINK B molecules are synthesized from diamines,
diisocyanates, disulfonic acids, dicarboxylic acids, diacid
chlorides and dialdehydes. Terminal hydroxyls, amines or carboxylic
acids on the oligo molecules can react with diamines to form
oligo-amides; react with diisocyanates to form oligo-urethanes,
oligo-ureas, oligo- amides; react with disulfonic acids to form
oligo-sulfonates, oligo-sulfonamides; react with dicarboxylic acids
to form oligo-esters, oligo-amides; react with diacid chlorides to
form oligo-esters, oligo-amides; and react with dialdehydes to form
oligo-acetal, oligo- imines.
[0059] By the term "pharmaceutical or biologically active agent",
or precursor thereof, is meant a molecule that can be coupled to
LINK A segment via hydrolysable covalent bonding. The molecule must
have some specific and intended pharmaceutical or biological
action. Typically the [Bio] unit has a molecular weight ranging
from 40 to 2000 for pharmaceuticals but may be higher for
biopharmaceuticals depending on the structure of the molecule.
Preferably, the Bio unit is selected from the group of anti-
inflammatory, anti-oxidant, anti-coagulant, anti-microbial
(including fluoroquinolones), cell receptor ligands and
bio-adhesive molecules, specifically oligo-peptides and oligo-
saccharides, oligonucleic acid sequences for DNA and gene sequence
bonding, and phospholipid head groups to provide cell membrane
mimics. The Bio component must have difunctional groups selected
from hydroxyl, amine, carboxylic acid or sulfonic acid so that
after coupling with Link A molecule, said biomonomer can react with
the secondary groups of oligomeric segment to form LINK B linkage.
The said secondary group may be protected during the reaction of
primary groups with the LINK A.
2TABLE 2 Typical Pharmaceutical Molecules Used For The Synthesis Of
BiomonomerCoupling Agents Pharmaceuticals Function Chemical
structures Norfloxacin Antimicrobial 1 Ciprofloxacin Antimicrobial
2 Amfenac Antiinflammatory 3 Aceclofenac Antiinflammatory 4
Oxaceprol.sup.1 Antiinflammatory 5 Enoxolone.sup.1 Antiinflammatory
6 Bromofenac Antithrombic 7 Tirofiban.sup.1 Antithrombic 8
Lotrafiban.sup.1 Antithrombic 9 Acivicin Antiproliferation 10
Alkeren Antiproliferation 11
[0060] This invention is of particular value to those
pharmacologically active compounds which are bioresponsive as
hereinabove defined to provide in vivo a pharmacological active
ingredient which has at least two functional groups but one of the
functional groups has low reactivity with diisocyanates to form
oligo-urethanes, or oligo-ureas, oligo-amides; react with
disulfonic acids to form oligo-sulfonates, oligo-sulfonamides;
react with dicarboxylic acids to form oligo-esters, oligo-amides;
react with diacid chlorides to form oligo-esters, oligo-amides; and
react with dialdehydes to form oligo- acetal, oligo-imines. Such a
pharmacological agent would include the fluoroquinolone family of
antibiotics, or anti-coagulants, anti-inflammatory or
anti-proliferative agents of the type listed in Table 2 above.
[0061] The present invention is of particular use wherein the
pharmacologically-active fragment is formed from the antibacterial
7-amino-1-cyclopropyl-4-oxo-1,4-dihydroquinoline and
naphthyridine-3-carboxylic acids described in U.S. Pat. No.
4,670,444. The most preferred antibacterial members of these
classes of compounds is
1-cyclopropyl-6-fluoro-1,4-dihyro-4-oxo-7-piperazine-quinoline-3-carboxyl-
ic acid and
1-ethyl-6-fluoro-1,4-dihyro-4-oxo-7-piperazine-quinoline-3-car-
boxylic acid having the generic name ciprofloxacin and norfloxacin,
respectively. Others of this class include sparfloxacin and
trovafloxacin.
[0062] Without being bound by theory, it is believed that the
presence of LINK A as hereindefined, allows of a satisfactory
"inter-bio distance" in the biologically-active polymer according
to the invention, which inter-bio distance facilitates hydrolysis
in vivo to release the biologically-active ingredient. LINK A
offers a range of hydrolysis rates by reason of chain length
variation and possibly, also, due to steric and conformational
variations resulting from the variations in chain length.
[0063] Prior art compounds not having LINK A chain length
variations but having LINK B chain lengths between the two
biological entities cannot provide this advantageous variations in
hydrolysis rates.
[0064] The present invention is of particular use wherein the
pharmacologically-active fragment is formed from the
anti-inflammatory
(2S,3S)-1-Acetyl-4-hydroxy-pyrrolidine-2-carboxylic acid having
generic name Oxaceprol and (2S4aS,6aS,6bR,8aR,10S, 12aS,12bR,
14bR)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydro-picene-2-carboxylic
acid having the generic name Enoxolone.
[0065] The present invention is of particular use wherein the
pharmacologically-active fragment is formed from the anti-thrombic
(S)-2-(butane-1-sulfonylamino)-3-[4-(4-piperidin-4-yl-butoxy)phenyl]-prop-
ionic acid having the generic name Tirofibanc and
[(S)-7-([4,4']bipiperidi-
nyl-1-carbonyl)-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-
-2-yl]-acetic acid having the generic name Lotrafiban.
[0066] The present invention is of particular use wherein the
pharmacologically-active fragment is formed from the
anti-neuplastic (.alpha.S,
5S)-.alpha.-amino-3-chloro-2-isoxazoleacetic-5-acetic acid having
the generic name Acivicin and 4-[Bis(2-chloroethyl)amino-]-L-pheny-
lalanine having the generic name Alkeren.
[0067] The oligomeric polymeric segment preferably has a molecular
weight of <10,000; and more preferably, <5,000.
[0068] The term "theoretical molecular weight" in this
specification is the term given to the absolute molecular weight
that would result from the reaction of the reagents utilized to
synthesize any given bioactive polymers. As is well known in the
art, the actual measurement of the absolute molecular weight is
complicated by physical limitations in the molecular weight
analysis of polymers using gel permeation chromatography methods.
Hence, a polystyrene equivalent molecular weight is reported for
gel permeation chromatography measurements. Since many
pharmaceutically active compounds absorb light in the UV region,
the gel permeation chromatography technique also provides a method
to detect the distribution of pharmaceutically active compound
coupled within polymer chains.
[0069] The polymeric materials of use in the practice of the
invention have polystyrene equivalent molecular weights of chains
ranging from 2.times.10.sup.3 to 1.times.10.sup.6, and preferably
in the range of 2.times.10.sup.3 to 2.times.10.sup.5.
[0070] In a further aspect, the invention provides compositions of
polymers containing biomonomers alone or a base polymer in
admixture with polymers containing biomonomers, as hereinabove
defined, preferably in the form of a shaped article.
[0071] Examples of typical base polymers of use in admixture with
aforesaid bioactive polymers according to the invention, includes
polyurethanes, polysulfones, polycarbonates, polyesters,
polyethylene, polypropylene, polystyrene, polysilicone,
poly(acrylonitrile-butadienesty- rene), polyamide, polybutadiene,
polyisoprene, polymethylmethacrylate, polyvinylacetate,
polyacrylonitrile, polyvinyl chloride, polyethylene terephtahate,
cellulose and other polysacharides. Preferred polymers include
polyamides, polyurethanes, polysilicones, polysulfones,
polyolefins, polyesters, polyvinyl derivatives, polypeptide
derivatives and polysaccharide derivatives. More preferably, in the
case of biodegradable base polymers these would include segmented
polyurethanes, polyesters, polycarbonates, polysaccharides or
polyamides.
[0072] The polymers containing said biomonomers, or the admixed
compositions according to the invention may be used as a surface
covering for an article, or, most preferably, where the polymers or
admixtures are of a type capable of being formed into 1) a
self-supporting structural body, 2) a film; or 3) a fiber,
preferably woven or knit. The composition may comprise a surface or
in whole or in part of the article, preferably, a biomedical device
or device of general biotechnological use. In the case of the
former, the applications may include cardiac assist devices, tissue
engineering polymeric scaffolds and related devices, cardiac
replacement devices, cardiac septal patches, intra aortic balloons,
percutaneous cardiac assist devices, extra-corporeal circuits, A-V
fistual, dialysis components (tubing, filters, membranes, etc.),
aphoresis units, membrane oxygenator, cardiac by-pass components
(tubing, filters, etc.), pericardial sacs, contact lens, cochlear
ear implants, sutures, sewing rings, cannulas, contraceptives,
syringes, o- rings, bladders, penile implants, drug delivery
systems, drainage tubes, pacemaker lead insulators, heart valves,
blood bags, coatings for implantable wires, catheters, vascular
stents, angioplasty balloons and devices, bandages, heart massage
cups, tracheal tubes, mammary implant coatings, artificial ducts,
craniofacial and maxillofacial reconstruction applications,
ligaments, fallopian tubes. The applications of the latter include
the synthesis of bioresorbable polymers used in products that are
environmentally friendly (including but not limited to garbage
bags, bottles, containers, storage bags and devices, products which
could release reagents into the environment to control various
biological systems including control of insects, biologically
active pollutants, elimination of bacterial or viral agents,
promoting health related factors including enhancing the
nutritional value of drinking fluids and foods, or various
ointments and creams that are applied to biological systems
(including humans, animals and other).
[0073] In a preferred aspect, the invention provides an admixed
composition, as hereinabove defined, comprising in admixture either
a segmented polyurethane, a polyester, a polycarbonate,
polysaccharide, polyamide or polysilicone with a compatible polymer
containing said biomonomer.
[0074] The polymers containing said biomonomer, according to the
invention, are synthesized in a manner that they contain a polymer
segment, i.e. the [oligo] segments and said biomonomer in the
backbone of polymer containing biochemical function with either
inherent anti-coagulant, anti-inflammatory, anti-proliferation,
anti-oxidant, anti- microbial potential, cell receptor ligands,
e.g. peptide ligands and bio-adhesive molecules, e.g.
oligosaccharides, oligonucleic acid sequences for DNA and gene
sequence bonding, or a precursor of the bioactive component.
[0075] The in vivo pharmacological activity generated may be, for
example, anti- inflammatory, anti-bacterial, anti-microbial,
anti-proliferation, anti-fungal, but this invention is not limited
to such biological activities.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] In order that the invention may be better understood,
preferred embodiments will now be described by way of example only,
with reference to the accompanying drawings wherein:
[0077] Error! Not a valid heading level range.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0078] Synthesis of Biomonomers.
[0079] A description of the novel process for preparing the
biological coupling agents/biomonomers of production D is set forth
in Scheme A, where, R is CH.sub.2CH.sub.3 or cyclopropyl for
norfloxacin and ciprofloxacin, respectively. Typically, linkA
molecules have molecular weights ranging from 60 to 2000 and
preferably 60 to 700, and must have at least di-functionality to
permit coupling of at least two [Bio] units. The [Bio] unit has a
molecular weight <2000 but may be higher depending on the
structure of the molecule. Preferred [Bio] components include but
are not limited to the following categries and examples:
Anti-inflammatory: non-steroidal-Oxaceprol, steroidal Enoxolone;
antithrombotic: Tirofiban, Lotrafiban; anti-coagulant: heparin;
anti-proliferation: acivicin and alkeren; anti-microbial:
fluoroquinolones such as norfloxancin, ciprofloxacin, sparfloxacin
and trovafloxacin and other fluoroquinolones.
[0080] Scheme A provides a general synthetic procedure for
preparing the compounds of product D with formula (I). 12
[0081] In step A, a pharmaceutically active drug, such as
norfloxacin or ciprofloxacin (in the form of hydrochloride salt) is
reacted with protecting groups such as trityl halides in the
presence of triethylene amine to provide an intermediate with both
amine and carboxylic acid groups protected with a trityl group. It
is understood by those skilled in the art that other protecting
groups can be used as exemplified in this document's examples.
[0082] A suitable trityl halide is reacted with norfloxacin or
ciprofloxacin hydrochloride salt in a suitable solvent, such as
chloroform. Many other solvents may be needed depending on the
solubility of the selected protecting groups and the agents forming
the biomonomer. Suitable trityl halides include trityl chloride and
trityl bromide. A preferred trityl halide is trityl chloride. The
amount of trityl halide ranges from 2 to 4 molar equivalent of
norfloxacin/ciprofloxacin, a preferred amount is 2.2 molar
equivalents. Triethylamine is added to scavenge free HCl which is
generated as a by-product. A little excess amount of triethylamine
will avoid the deprotection of the N-triethylamine group in the
following selective hyrolyzation step. In the case of
ciprofloxacin, an excess molar amount of triethylene amine such as
2 to 4 times was added into reaction mixture. A preferred amount is
3 times. The reaction mixture is stirred for a period of time
ranging from 2-24 hours in a temperature range of 0.degree. C. to
60.degree. C. A preferred stirring time is 4 hours and a preferred
temperature is 25.degree. C. A homogenous solution is obtained.
Following this step, product A is left in the reaction solution for
the next step of the in-situ reaction. No isolation of the product
A is required during processing.
[0083] In step B, the reaction product of step A, such as
norfloxacin/ciprofloxacin with both amine and carboxylic acid
groups protected with trityl group, is selectively deprotected to
yield product B containing free carboxylic acid and N-triethylamine
groups.
[0084] For example, in step B, a large amount of methanol was added
into the reaction mixture of step A. The volume of methanol ranges
from equivalent to two times that of the solvent used in step A. A
preferred volume is 1.5 times that of the solvent volume. The
reaction mixture is stirred for 1-24 hrs in a temperature range
from 25.degree. C. to 60.degree. C. A preferred stirring time is 2
hrs and a preferred temperature is 50.degree. C. The selectively
deprotected fluroquinolone material is precipitated from the
reaction solution. Product B is recovered from the reaction zone by
filtration after the reaction mixture is cooled down to room
temperature. Product B is further purified from CHCl.sub.3/Methanol
(9:1) by standard recrystallization method.
[0085] In step C, the purified amine-protected fluroquinolone is
coupled to both sides of a diol or diamine (in this example,
triethylene glycol is used) containing a flexible and/or
water-soluble central portion.
[0086] For example, the purified amine-protected fluroquinolone
(Product B) is coupled to a tri(ethylene glycol) in the presence of
a suitable coupling agent such as
1-ethyl-3-(3-dimethylamino-propyl)carbodiimide herein denoted as
EDAC and an appropriate base such as 4-(dimethylamino)pyridine
herein denoted as DMAP as a catalyst. Other coupling reagents may
include various carbodiimides such as CMC
(1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide),
DCC(N,N'-dicyclohexyl-c- arbodiimide), DIC (Diisopropyl
carbodiimide) etc, but are not limited to these. The amount of diol
ranges from 0.3 to 0.5 molar equivalent of product B. A preferred
amount of diol is 0.475 molar equivalent of product B. The amount
of coupling agent EDAC ranges from 2 tol times molar equivalent of
product B. A preferred amount of EDAC is 8 times molar equivalent.
The amount of base DMAP can range from 0.1 to equal molar amount of
product B. A preferred amount is 0.5 molar equivalents. The
reaction was carried out in a suitable solvent such as
dichloromethane under a noble atmosphere such as nitrogen, argon.
Other solvents may be appropriate depending on their solubility
properties with product B and their potential reactivity with the
reagents. The reactants are typically stirred together for a period
of time ranging from 24 hours to 2 weeks at a temperature range
from 0.degree. C. to 50.degree. C. A preferred stirring time is one
week and a preferred temperature is 25.degree. C.
[0087] After the reaction is finished, solvent is removed by rotary
evaporator. The residues are washed with water several times to
remove soluble reagents such as EDAC. The solids are then dissolved
in chloroform. Product C in Scheme 1 is recovered from the solution
by standard extractive methods using chloroform as the extraction
solvent. Product C was isolated by column chromatography using a
developer made up of chloroform/methanol/ammonia hydroxide aqueous
solution (9.2:0.6:0.2). Product C is further purified with
recrystallization techniques from chloroform and methanol.
[0088] In step D, the N-trietylamine groups of the purified product
C are deprotected to yield the corresponding desired pharmaceutical
coupling agent/biomonomer.
[0089] For example, the appropriate product C is reacted with a
small amount of water in the presence of a small amount of weak
acid, such as trifluoroacetic acid, in a suitable organic solvent
such as dichloromethane. The amount of water can range from 1% to
10% volume percentage and a preferred amount is 1%. The amount of
trifluoroacetic acid is between 1% to 10% volume percent, with a
preferred amount being 2%. The reaction mixture is stirred within a
temperature range of 0.degree. C. to 50.degree. C. over a time
period of 2 to 24 hours. A preferred temperature is 25.degree. C.
and a preferred time period is 4 hours. Product D is precipitated
from reaction solution and collected by filtration. The product is
further purified by washing with CHCl.sub.3.
[0090] Use of Biomonomers in a Polymer Synthesis.
[0091] The pharmaceutically active polymers are synthesized in a
traditional stepwise polymerization manner as are well known in the
art. A multi-functional LINK B molecule and a multi-functional
oligo molecule are reacted to form a prepolymer. The prepolymer
chain is extended with said biomonomer to yield a polymer
containing the biomonomers. Non-biological extenders such as an
ethylene diamine, butane diol, ethylene glycol and others may also
be used. The linkB molecule is preferably, but not so limited, to
be di-functional in nature, in order to favour the formation of a
linear polymer containing biomonomers. Preferred linkB molecules
for biomedical and biotechnology applications are diisocyanates:
for example, 2,4 toluene diisocyanate; 2,6 toluene diisocyanate;
methylene bis(p-phenyl)diisocyanate; lysine diisocyanato esters;
1,6 hexane diisocyanate; 1,12 dodecane diisocyanate; bis-methylene
di(cyclohexyl isocyanate); trimethyl-1,6 diisocyanatohexane,
dicarboxylic acids, di-acid chlorides, disulfonyl chlorides or
others. The oligo component is preferably, but not so limited,
difunctional, in order to favor the formation of a linear polymer
containing said biomonomers. Preferred oligo components are
terminal diamine and diol reagents of: for example, polycarbonate,
polysiloxanes, polydimethylsiloxanes; polyethylene-butylene
co-polymers; polybutadienes; polyesters including
polycaprolactones, polylactic acid, and other polyesters;
polyurethane/sulfone co-polymer; polyurethanes; polyamides;
including oligopeptides (polyalanine, polyglycine or copolymers of
amino-acids) and polyureas; polyalkylene oxides and specifically
polypropylene oxide, polyethylene oxide and polytetramethylene
oxide. The molecular weights of the [oligo] groups are less than
10,000, but preferably have molecular weights of less than 5000.
Synthesis of the prepolymers to the bioactive polymer can be
carried out by classical urethane/urea reactions using the desired
combination of reagents but with the excess amount of linkB
molecules in order to end-cap the prepolymer with linkb molecule.
When the prepolymer with desired chain length is reached, said
biomonomer is added to extend the prepolymer chain giving a final
bioactive polymer. Alternatively the biomonomers may be substituted
for inclusion as the oligo groups.
[0092] Bioactive polymers can be synthesized with different
components and stoichometry. Prior to synthesis, the LINK B
molecules are, preferably, vacuum distilled to remove residual
moisture. The biomonomers are desiccated to remove all moisture.
Oligo components are degassed overnight to remove residual moisture
and low molecular weight organics.
[0093] While reactants can be reacted in the absence of solvents if
practical, it is preferable to use organic solvents compatible with
the chemical nature of the reagents, in order to have good control
over the characteristics of the final product. Typical organic
solvents include, for example, dimethylacetamide, acetone,
tetrahydrofuran, ether, chloroform, dimethylsulfoxide and
dimethylformamide. A preferred reaction solvent is
dimethylsulfoxide (DMSO, Aldrich Chemical Company, Milwaukee,
Wis.).
[0094] In view of the low reaction activity of some diisocyanates,
e.g. DDI and THDI, with oligo precursor diols, a catalyst is
preferred for the synthesis. Typical catalysts are similar to those
used in the synthesis of urethane chemistry and, include,
dibutyltin dilaurate, stannous octoate, N,N'
diethylcyclohexylamine, N-methylmorpholine, 1,4 diazo (2,2,2)
bicyclo-octane and zirconium complexes such as Zr tetrakis
(2,4-pentanedionato) complex.
[0095] In the first step of the preparation of a prepolymer, for
example, the linkB molecules are added to the oligo component and,
optionally, catalyst to provide the prepolymer of the bioactive
polymer. The reaction mixture is stirred at a temperature of
60.degree. C. for a suitable time period, which depends on the
reaction components and the stoichiometry. Alternate temperatures
can range between 25.degree. C. to 110.degree. C. Subsequently,
said biomonomer is added to the prepolymer and, generally, the
mixture is allowed to react overnight. The reaction is terminated
with methanol and the product is precipitated in ether or a mixture
of distilled water with ether or other suitable solvents. The
precipitate is dissolved in a suitable solvent, such as acetone and
precipitated in ether or a mixture of distilled water with ether
again. This process was repeated 3 times in order to remove any
residual catalyst compound. Following washing, the product is dried
under vacuum at 40.degree. C.
[0096] Alternatively, the biomonomers can be used to make
polyamides using classical reactions such as those described
below.
[0097] Fabrication of Product:
[0098] The pharmaceutical polymers containing biomonomers are
either used alone or admixed with suitable amounts of base polymers
in the fabrication of article products. If admixed in a blend, then
suitable polymers may include polyurethane, polyester or other base
polymers. Product may be formed by; 1) compounding methods for
subsequent extrusion or injection molding or articles; 2)
co-dissolving of base polymer with bioactive polymer into a solvent
of common compatibility for subsequent casting of an article in a
mold or for spinning fibers to fabricate an article; 3) wetting the
surface of an article with a solution of bioactive polymer or a
blend in solvent of common compatibility with a polyurethane or
other polymer to which the bioactive polymer solution is being
applied; or 4) in admixture with a curable polyurethane, for
example, 2 part curing system such as a veneer. All of the above
processes can be used with the pure polymer, containing the
biomonomer groups or with blends of said polymer and common
biomedical polymers.
[0099] The invention, thus, provides the ability to synthesize a
range of novel polymeric materials possessing intramolecular
properties of pharmaceutical or biological nature. When said
polymers are used alone or in admixture with, for example, a
polyurethane, the bioactive polymer provides the composite having
better pharmaceutical function, particularly for use in medical
devices, promoting cell function and regulation, tissue
integration, pro-active blood compatibility and specifically
anti-coagulant/platelet function, biostability function,
anti-microbial function and anti-inflammatory function, or for use
in the biotechnology sector for biological activity.
[0100] The application for these materials include the synthesis of
bioresorbable polymers used in medical device products that require
the delivery of biologicals, pharmaceuticals or the release of
biocompatible materials upon biodegradation within or in contact
with a biological body (human or animal). This includes the
manufacturing of products in the form of films (cast or heat
formed), fibres (solvent or melt spun), formed into composite
materials (polymers combined in any form with ceramics, metals or
other polymers) of any shape, injection molded, compression molded,
extruded products. Such product can include but are not limited to:
cardiac assist devices, tissue engineering polymeric scaffolds and
related devices, cardiac replacement devices, cardiac septal
patches, intra aortic balloons, percutaneous cardiac assist
devices, extra-corporeal circuits, A-V fistual, dialysis components
(tubing, filters, membranes, etc.), aphoresis units, membrane
oxygenator, cardiac by-pass components (tubing, filters, etc.),
pericardial sacs, contact lens, cochlear ear implants, sutures,
sewing rings, cannulas, contraceptives, syringes, o-rings,
bladders, penile implants, drug delivery systems, drainage tubes,
pacemaker lead insulators, heart valves, blood bags, coatings for
implantable wires, catheters, vascular stents, angioplasty balloons
and devices, bandages, heart massage cups, tracheal tubes, mammary
implant coatings, artificial ducts, craniofacial and maxillofacial
reconstruction applications, ligaments, fallopian tubes.
[0101] Other non-medical applications may include of bioresorbable
polymers used in products that are environmentally friendly
(including but not limited to garbage bags, bottles, containers,
storage bags and devices, products which could release reagents
into the environment to control various biological systems
including control of insects, biologically active pollutants,
elimination of bacterial or viral agents, promoting health related
factors including enhancing the nutritional value of drinking
fluids and foods, or various ointments and creams that are applied
to biological systems (including humans, animals and other). In
these examples, the following acronyms are used.
[0102] NORF (Norfloxacin)
[0103] CIPRO (Ciprofloxacin)
[0104] OC (Oxaceprol)
[0105] POC (Protected Oxaceprol)
[0106] TF (Tirofiban)
[0107] PTF (Protected Tirofiban)
[0108] AK (Alkeren)
[0109] PAK (Protected Alkeren)
[0110] AF (Amfenac)
[0111] AV (Acivicin)
[0112] BF (Bromfenac)
[0113] TEG (Triethylene glycol)
[0114] HDL (1,6-Hexanediol)
[0115] HDA (1,6-Hexanediamine)
[0116] TrCl (Trityl Chloride)
[0117] DMAP (4-(dimethylamino)pyridine)
[0118] EDAC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)
[0119] TEA (Triethylene amine)
[0120] TFA (Trifluoroacetic acid)
[0121] THDI (trimethyl-1,6 diisocyanatohexane)
[0122] PCL polycarprolactone diol
[0123] AC (Adipoyl Chloride)
[0124] THDI/PCL/TEG (segmented polyurethane)
[0125] DBTL (dibutyltin dilaurate)
[0126] DCM (Dichloromethane)
[0127] DMF (dimethylformamide)
[0128] TLC (thin layer chromatography)
[0129] CC (Column chromatography)
[0130] Where appropriate all isocyanate reactions were catalysed
with DBTL (dibutyltin dilaurate).
[0131] Nuclear magnetic resonance was used to identify the
structure of the biomonomer.
[0132] Mass spectroscopy was used to confirm the molar mass of the
synthesized biomonomer.
[0133] Gel permeation chromatography was used to define the
distribution of [Bio] the moiety within the drug polymer and to
estimate relative molecular weights of the polymer.
[0134] Characterization of tin residues located at the surface of
the drug polymer coatings was demonstrated using X-ray
photoelectron spectroscopy (measuring chemical composition) at 90
degree. Elimination of tin residues is important for biological
applications since the latter is toxic.
[0135] In vitro evaluation of antimicrobial release and
biodegradation were performed in order to assess the rates of
degradation for the different antimicrobial polymer formulations
and determines periods of efficacy. In these studies the polymers
are incubated with enzyme and the solution is recovered for
separation of degradation products. Hydrolytic enzymes related to
monocyte macrophages, specifically cholesterol esterase, and
neutrophils (elastase), with in a pH 7 phosphate buffered saline
solution may be used for in vitro tests over a 10-week time frame.
Degradation products may be characterized using High Performance
Liquid Chromatography (HPLC), combined with mass spectroscopy.
[0136] Minimum inhibitory concentration (MIC) assays were used to
evaluate the antimicrobial activity of incubating solutions
obtained from drug polymer biodegradation studies against P.
aeruginosa. Turbidity of each culture was recorded to evaluate the
inhibitory properties of degradation solution of drug polymers.
[0137] Sterilization stability of drug polymers was estimated after
drug polymers were sterilized by .gamma.-radiation sterilization
(radiation dose: 25 Kgy), a standard method in the medical device
field. GPC measurements were carried on with these samples before
and after they were radiated and after a time period of 1 to 4
weeks.
[0138] Biocompatibility study of the drug polymers was also
performed in order to assess the biocompatibility of control and
drug polymers with mammalian cells. In this study, HeLa cells were
cultured directly onto the polyurethane polymers films and
incubated at 37.degree. C. for 24 hours. Cell viability was
measured by staining for succinate dehydrogenase.
[0139] In vivo animal studies are performed on substrates, devices
or articles according to the invention formed in whole or in part
of bioactive polymers. The articles containing either bioactive
polymer or non-bioactive control polymer were implanted in the
peritonitis of male rats accompanied with an inoculation of P.
aeurogniosa bacteria. The articles were explanted after rats were
housed for 1 week. The effect of the antimicrobial polymer was
evaluated.
EXAMPLES
[0140] The following examples illustrate the preparation of
biomonomers and bioresponsive pharmacologically active polymers
according to the invention.
Example 1
[0141] NORF-TEG-NORF and CIPRO-TEG-CIPRO are examples of
antimicrobial drug containing biomonomers according to the
invention. The example shows the use of a single drug or
combination of drugs. The conditions of synthesis for this reaction
are as follows.
[0142] In step A, of NORF(1.3 g, 4 mmol)/ or CIPRO hydrochloride
salt (4 mmol) were reacted with trityl chloride (2.7 g, 8.8 mmol)
and TEA(0.6 ml, 8 mmol) (Aldrich, 99%)/or 12 mmol of TEA in the
case of CIPRO in 40 ml of CHCl.sub.3 for four hours at room
temperature. A clear solution was obtained.
[0143] In step B, 40 ml of methanol was added into the above clear
solution. The mixture was heated to 50.degree. C. and stirred for
one hour; a precipitate appeared in the solution. After the
reaction mixture was cooled down to room temperature, precipitates
were collected by filtration. The precipitate was further purified
from CHCl.sub.3/methanol. 3.4 mmol of Product B were obtained.
Yield was usually greater than 85%.
[0144] In step C, Product B (20 mmol), TEG (1.44 g, 9.5 mmol), DMAP
(1.24 g, 10 mmol) were dissolved in 100 ml DCM. EDAC (31 g, 160
mmol) was then added into the reaction system. The reaction mixture
was stirred at room temperature under a nitrogen atmosphere for one
week. After reaction was finished, DCM was removed by rotary
evaporator. The residues were washed with de-ionized water several
times to remove soluble reagents such as the by-product of urea.
The solids were then dissolved in chloroform and washed with
de-ionized water again. The crude product of the reaction was
recovered from the solution by extraction. Product C was isolated
by column chromatograph using the developer of
chloroform/methanol/ammonia hydroxide aqueous solution
(9.2:0.6:0.2). Product C is further purified with recrystallization
technique from chloroform and methanol. Product C can be obtained
with a yield of 85%.
[0145] In step D, the purified product C (5.4 g, 4.4 mmol) was
dissolved in chloroform containing one volume percent of water and
1 volume percent of trifluoroacetic acid. The reaction solution was
stirred at room temperature for 4 hrs. White precipitates that were
produced in the reaction were collected by filtration and purified
by washing with chloroform. Following washing Product D, i.e. the
biomonomer was dried in vacuum oven for 24 hours at a temperature
of 40.degree. C. The pure Product D i.e. said biomonomer can be
obtained with a yield of 95%.
[0146] .sup.1H NMR of NORF-TEG-NORF: (400 MHz, DMSO). .delta.: 9.33
(bs, 2H, NH), 8.52 (s, 2H, H.sup.2, ar), 7.66 (d, 2H, J=13.6 Hz,
H.sup.5, ar), 7.01 (d, 2H, J=7.2 Hz, H.sup.8, ar), 4.33 (q, 4H,
J=6.8 Hz, N--CH.sub.2--CH.sub.3), 4.26 (t, 4H, J=4.8 Hz,
CO.sub.2CH.sub.2), 3.71 (t, 4H, J=4.8 Hz, CO.sub.2CH.sub.2CH.sub.2)
3.48-3.28 (m, 16H,piperazine), 1.33 (t, 6H, J=6.8 Hz,
NCH.sub.2CH.sub.3).
[0147] [FIG. 1]
[0148] .sup.13C NMR of NORF-TEG-NORF: (400 MHz, DMSO). .delta.:
171.9, 164.7, 159.3, 159.0, 153.8, 151.4, 149.0, 143.4, 143.3,
136.4, 123.4, 122.0, 119.0, 116.0, 112.4, 109.4, 106.6, 70.4, 68.9,
63.6, 48.6, 47.1, 43.1, 43.0, 14.6, [FIG. 2]
[0149] ES-MS of NORF-TEG-NORF (m/z, %) (Positive mode): Calculated
for mass C.sub.38H.sub.46F.sub.2N.sub.6O.sub.8: 752 amu, found 753,
377 (M+2H).sup.+. [FIG. 3]
[0150] .sup.1H NMR of CIPRO-TEG-CIPRO: (400 MHz, DMSO). .delta.:
9.16 (bs, 2H, NH--R), 8.30 (s, 2H, H.sup.2, ar), 7.49 (d, 2H,
J=13.2 Hz, H.sup.5, ar), 7.34 (d, 2H, J=7.6 Hz, H.sup.8, ar), 4.25
(t, 4H, J=5.2 Hz, N--CH(CH.sub.2).sub.2); 3.73 (t, 4H, J=4.4 Hz,
CO.sub.2CH.sub.2), 3.46-3.30 (m, 16H, piperazine), 1.22 (q, 4H,
J=6.4 Hz, CH(CH.sub.2 CH.sub.2)), 1.07 (m, 4H,
CH(CH.sub.2CH.sub.2)). [FIG. 4]
[0151] .sup.13C NMR of CIPRO-TEG-CIPRO: (400 MHz, DMSO). .delta.:
171.9, 164.1, 158.7, 153.9, 151.5, 148.4, 143.0, 142.9, 138.1,
122.6, 122.5, 111.9, 111.7, 109.2, 107.0, 79.6, 70.5, 70.4, 68.9,
63.7, 47.0, 43.2, 35.3, 7.9. [FIG. 5]
[0152] ES-MS of CIPRO-TEG-CIPRO (m/z, %) (Positive mode):
Calculated for mass C.sub.40H.sub.46F.sub.2N.sub.6O.sub.8: 776 amu,
found 777 (M+H.sup.+); 389 (M+2H).sup.+. [FIG. 6]
Example 2
[0153] CIPRO-HDL-CIPRO is example of biomonomer according to the
invention and different from example 1 by the introduction of a
hydrophobic link A molecule rather than hydrophilic link A
molecule. The conditions of synthesis for this reaction are as
follows.
[0154] The reaction conditions for selectively protecting amine
groups of CIPRO are the same as the step A and B in Example 1.
[0155] In step C, Product B (20 mmol), HDL (9.5 mmol), DMAP (1.24
g, 10 mmol) were dissolved in 100 ml DCM. EDAC (31 g, 160 mmol) was
then added into reaction system. The reaction mixture was stirred
at room temperature under a nitrogen atmosphere for one week. After
the reaction was finished, DCM was removed by rotary evaporator.
The residues were washed with de-ionized water several times to
remove soluble reagents such as the by-product of urea. The solids
were then dissolved in chloroform and washed with de-ionized water
again. The crude product of the reaction was recovered from the
solution by extraction. Product C was isolated by column
chromatography using the developer of chloroform/methanol/ammonia
hydroxide aqueous solution (9.2:0.6:0.2). Product C is further
purified with a recrystallization technique from chloroform and
methanol.
[0156] In step D, the purified product C (4 mmol) was dissolved in
chloroform containing one volume percent of water and 1 volume
percent of trifluoroacetic acid. The reaction solution was stirred
at room temperature for 4 hrs. White precipitates produced in the
reaction were collected by filtration and purified by washing with
chloroform. Following washing Product D, i.e. the biomonomer was
dried in vacuum oven for 24 hours at a temperature of 40.degree.
C.
Example 3
[0157] NORF-HDA-NORF is example of biomonomer according to the
invention and different from example 1 in that a diamine is used to
generate an amide rather than ester linkage in the biomonomer. The
conditions of synthesis for this reaction are as follows.
[0158] The reaction conditions for selectively protecting amine
groups of NORF are the same as the step A and B in Example 1.
[0159] In step C, Product B (20 mmol), HDA (9.5 mmol), DMAP (1.24
g, 10 mmol) were dissolved in 100 ml DCM. EDAC (31 g, 160 mmol) was
then added into reaction system. The reaction mixture was stirred
at room temperature under a nitrogen atmosphere for one week. After
the reaction was finished, DCM was removed by rotary evaporator.
The residues were washed with de-ionized water several times to
remove soluble reagents such as the by-product of urea. The solids
were then dissolved in chloroform and washed with de-ionized water
again. The crude product of the reaction was recovered from the
solution by extraction. Product C was isolated by column
chromatography using the developer of chloroform/methanol/ammonia
hydroxyl aqueous solution (9.2:0.6:0.2). Product C is further
purified with recrystallization technique from chloroform and
methanol.
[0160] In step D, the purified product C (4 mmol) was dissolved in
chloroform containing one volume percent of water and 1 volume
percent of trifluoroacetic acid. The reaction solution was stirred
at room temperature for 4 hrs. White precipitates produced in the
reaction were collected by filtration and purified by washing with
chloroform. Following washing Product D, i.e. the biomonomer was
dried in vacuum oven for 24 hours at a temperature of 40.degree.
C.
Example 4
[0161] OC-TEG-OC is an example of anti-inflammatory drug containing
biomonomer according to the invention. The biomonomer was
synthesized using Oxaceprol (OC), by reacting the carboxylic acid
with the hydroxyl of TEG and leaving the hydroxyl for subsequent
use in the polymerization. The conditions of synthesis for this
reaction are as follows.
[0162] In step A, OC (11.55 mmol) was reacted with
t-butyldimethylsilyl chloride (28.87 mmol) and
1,8-diazabicylco[5.4.0]undec-7-ene (30.03 mmol) in 4 ml of
acetonitrile at 0.degree. C. during the addition of the base and
then overnight at ambient temperature. A precipitate developed
during the progress of the reaction The precipitate was
filtered.
[0163] In step B, the filtrate was treated with water (10 ml) and
extracted with n-pentane (2.times.5 ml). The solvent for the
aqueous portion was removed under reduced atmosphere. The residue
was dissolved in methanol (10 mL), tetrahydrofuran (5 mL), water (5
mL) and then treated with 2N aqueous sodium hydroxide (8 mL). The
reaction mixture was stirred for 1.5 h at room temperature,
adjusted to a pH=3 with 1N HCl, concentrated and filtered. The
precipitate obtained was recrystallized from water and afforded
pure 4 (2.79 g, 84%).
[0164] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta.: 4.87 (bs, 1H,
CO.sub.2H), 4.61 (dd, 1H, J=8.0 Hz, 6.4 Hz, CHCO.sub.2H), 4.48 (p,
1H, J=4.4 Hz, CHOSi), 3.67 (dd, 1H, J=10.4 Hz, 4.8 Hz, CHHN), 3.36
(dd, 1H, J=10.4 Hz, 6.0 Hz, CHHN), 2.36 (dt, 2H, J=13.2 Hz, 5.2 Hz,
2H, CH.sub.2CHCO.sub.2H), 2.12 (s, 3H, COCH.sub.3), 0.86 (s, 9H,
C(CH.sub.3).sub.3), 0.08 (s, 3H, SiCH.sub.3), 0.07 (s, 3H,
SiCH.sub.3). [FIG. 7]
[0165] .sup.13C NMR: (400 MHz, CDCl.sub.3) 6:172.7, 172.3, 70.0,
58.3, 56.2, 37.1, 25.6, 22.2, 17.9, -4.8, -5.0. [FIG. 8]
[0166] ES-MS (m/z, %) (Negative mode): Calculated for mass
C.sub.13H.sub.25NO.sub.4Si: 287 amu, found 286.1. [FIG. 9]
[0167] In step C, Product B (3.48 mmol), TEG (1.58 mmol), DMAP
(0.16 mmol) were dissolved in DCM (5 ml). EDAC (3.95 mmol) was then
added into the reaction solution cooled to 0.degree. C. The
resulting solution was stirred for 1 h at 0.degree. C., the cooling
was removed, and the mixture was stirred for 5 days at ambient
temperature. The solvent was removed under reduced pressure. Water
(20 mL) and the system was extracted with pentane (3.times.10 mL).
The combined pentane extracts where dried using sodium sulphate,
filtered, and the solvent removed under reduced pressure. This
produced 0.74 g (67%) of the desired product.
[0168] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta.: 4.87 (m, 2H,
CHCO.sub.2), 4.26 (m, 2H, CHOSi), 4.05 (m, 2H, CHHN), 3.74-3.31 (m,
4H), 3.31 (m, 2H, CHHN), 2.13 (m, 4H, CH.sub.2CHCO.sub.2), 2.12 (s,
6H, COCH.sub.3), 2.0 (m, 4H), 1.18 (m, 4H), 0.81 (s, 18H,
C(CH.sub.3).sub.3), -0.003 (s, 6H, SiCH.sub.3), -0.03 (s, 6H,
SiCH.sub.3). [FIG. 10]
[0169] .sup.13C NMR: (400 MHz, CDCl.sub.3) .delta.: 172.3, 171.0,
72.5, 70.6, 70.4, 64.0, 60.3, 57.5, 57.3, 55.9, 54.3, 52.1, 40.4,
38.3, 34.0, 26.6, 22.1, 20.9, 17.8, 14.1, -4.8, -5.0. [FIG. 11]
[0170] ES-MS (m/z, %) (Positive mode): Calculated for mass
C.sub.32H.sub.60N.sub.2O.sub.10Si.sub.2: 688 amu, found 689.3.
[FIG. 12]
[0171] In step D, the purified product C (0.7 mmol) was dissolved
in THF (5 ml). The resulting solution was cooled to 0.degree. C.
before the addition of tetra n-butyl ammonium fluoride (x ml, 1.4
mmol). The resulting solution was stirred at 0.degree. C. for 5 min
before the removal of the ice bath and continued stirring for an
additional 40 min at ambient temperature. The solvent was removed
at reduced atmosphere and the residue was treated with water and
the pH of the solution was adjusted to 3 upon which a precipitate
resulted. The precipitate was filtered to produce the desired
product.
Example 5
[0172] TF-TEG-TF is an example of anti-thrombic drug containing
biomonomer according to the invention. The biomonomer is
synthesized using tirofiban (TF), reacting the carboxylic acid with
the hydroxyl of TEG and leaving the amines for subsequent use in
the polymerization. The conditions for synthesis for this reaction
are as follows.
[0173] In step A, TF(4 mmol) is reacted with trityl chloride (8.8
mmol) and TEA (8 mmol) (Aldrich, 99%) in 40 ml of CHCl.sub.3 for
four hours at room temperature. A clear solution is obtained.
[0174] In step B, 40 ml of methanol is added into the above clear
solution. The mixture is heated to 50.degree. C. and stirred for
one hour, a lot of precipitates appeared in the solution. After the
reaction mixture is cooled down to room temperature, precipitates
were collected by filtration. They were further purified from
CHCl.sub.3/methanol. 3.4 mmol of Product B were obtained.
[0175] In step C, Product B (20 mmol), TEG (9.5 mmol), DMAP (1.24
g, 10 mmol) were dissolved in 100 ml DCM. EDAC (31 g, 160 mmol) is
added into the reaction system. The reaction mixture is stirred at
room temperature under a nitrogen atmosphere for one week. After
reaction is finished, DCM was removed by rotary evaporator. The
residues were washed with de-ionized water several times to remove
soluble reagents such as the by-product of urea. The solids were
then dissolved in chloroform and washed with de- ionized water
again. The crude product of the reaction is recovered from the
solution by extraction. Product C was isolated by column
chromatography using the developer of chloroform/methanol/ammonia
hydroxide aqueous solution (9.2:0.6:0.2). Product C is further
purified with recrystallization technique from chloroform and
methanol.
[0176] In step D, the purified product C (4 mmol) is dissolved in
chloroform containing one volume percent of water and 1 volume
percent of trifluoroacetic acid. The reaction solution is stirred
at room temperature for 4 hrs. White precipitates produced in the
reaction were collected by filtration and purified by washing with
chloroform. Following washing Product D, i.e. the biomonomer is
dried in vacuum oven for 24 hours at a temperature of 40.degree.
C.
Example 6
[0177] AK-TEG-AK is an example of anti-proliferation drug
containing biomonomer according to the invention. The biomonomer
was synthesized using Alkeren (AK), reacting the carboxylic acid
with the hydroxyl of TEG and leaving the amines for subsequent use
in the polymerization. The conditions for synthesis for this
reaction are as follows.
[0178] In step A, AK (0.32 mmol) was reacted with di-tert-butyl
carbonate (0.5 mmol) and TEA(0.32 mmol) (Aldrich, 99%) in THF (4
ml). The suspension was cooled to 0.degree. C. before the addition
of the anhydride. Dimethylformamide (0.9 ml) was added to
homogenize the reaction mixture. The solution was stirred for 2
hours at 0.degree. C., and thereafter overnight at ambient
temperature. The solution is then evaporated under reduced pressure
and the yellowish oily residue obtained is redissolved in a 5%
aqueous solution of sodium bicarbonate (3 ml). The solution is
washed with petroleum ether (3.times.3 ml) and the aqueous phase
was acidified to a pH of 3 with a 1N hydrochloric acid solution.
The mixture was extracted with ethyl acetate (3.times.3 ml). The
organic phases were dried over anhydrous sodium sulfate, filtered
and then evaporated under reduced pressure. The residue is
dissolved in a mixture of hexane, ethyl acetate, and acetic acid
(20:10:1) (3 ml). It is subsequently purified by chromatography on
a silica column. This produced 115 g (86%) of the desired product
(R.sub.f=0.49).
[0179] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta.: 11.02 (bs, 1H,
CO.sub.2H), 7.08 (d, 2H, J=5.4 Hz, Ar--H), 6.64 (d, 2H, J=5.4 Hz,
Ar--H), 4.97 (d, 1H, J=5 Hz, NH), 4.57 (m, 1H, CHCO.sub.2H),
3.72-3.59 (m, 8H, CH.sub.2CH.sub.2Cl), 3.12-2.98 (m, 2H,
CH.sub.2CH), 1.42 (s, 9H, C(CH.sub.3).sub.3). [FIG. 13]
[0180] .sup.13C NMR: (400 MHz, CDCl.sub.3) .delta.:177.3,
176.6155.4, 144.9, 130.7, 112.3, 80.2, 54.4, 53.6, 40.3, 28.3,
20.8. [FIG. 14]
[0181] ES-MS (m/z, %) (Positive mode): Calculated for mass
C.sub.18H.sub.26Cl.sub.2N.sub.2O.sub.4: 404 amu, found 405.1. [FIG.
15]
[0182] In step C, Product B (0.20 mmol), TEG (0.09 mmol), DMAP
(0.009 mmol) were dissolved in DCM (2 ml). To this stirring
solution at 0.degree. C. was added EDC-HCl (0.22 mmol) in
dichloromethane (1 ml) dropwise over 10 min. The resulting solution
was stirred for 1 h at 0.degree. C., the cooling was removed, and
the mixture was stirred for 3 days at ambient temperature. The
progress of the reaction was monitored by thin layer
chromatography. Once complete disappearance of starting material
was observed, the reaction solvent was removed under reduced
atmosphere. The product was purified by column chromatography
(R.sub.f=0.93) eluting with chloroform:methanol (9:1). This
produced 0.8 g (73%) of the desired product.
[0183] .sup.1H NMR: (400 MHz, CDCl.sub.3) .delta.: 7.03 (d, 4H,
J=5.4 Hz, Ar--H), 6.64 (d, 4H, J=5.4 Hz, Ar--H), 4.98 (d, 2H, J=5.0
Hz, NH), 4.55 (m, 2H, CHCO.sub.2H), 4.28 (m, 4H, CO.sub.2CH.sub.2)
3.72-3.59 (m, 24H, CH.sub.2CH.sub.2Cl, OCH.sub.2CH.sub.2OCH.sub.2),
3.06-2.95 (m, 4H, CH.sub.2CH), 1.42 (s, 18H, C(CH.sub.3).sub.3).
[FIG. 16]
[0184] .sup.13C NMR: (400 MHz, CDCl.sub.3) 6:177.3, 171.9, 155.0,
144.6, 130.7, 112.5, 79.8, 70.6, 69.0, 64.3, 54.4, 53.7, 40.2,
37.1, 28.3. [FIG. 17]
[0185] ES-MS (m/z, %) (Positive mode): Calculated for mass
C.sub.42H.sub.62Cl.sub.4N.sub.4O.sub.10: 922 amu, found 923.2.
[FIG. 18]
[0186] In step D, the purified product C (2 mmol) was dissolved in
chloroform containing one volume percent of water and 1 volume
percent of trifluoroacetic acid. The reaction solution was stirred
at room temperature for 2 hrs. White precipitates produced in the
reaction were collected by filtration and purified by washing with
chloroform. Following washing Product D, i.e. the biomonomer was
dried in vacuum oven for 24 hours at a temperature of 40.degree.
C.
Example 7
[0187] THDI/PCL/NORF is an example of pharmaceutically active
polyurethane containing 15% of drugs according to the invention.
The conditions of synthesis for this reaction are as follows.
[0188] 1.5 grams of PCL are reacted with 0.27 grams of THDI in the
presence of 0.06 ml of the catalyst, dibutyltin dilaurate, in a
nitrogen atmosphere with in dimethylsulfoxide (DMSO) (10 mL) for
one hour. The reaction temperature is maintained between
60-70.degree. C. 0.32 grams of NORF-TEG-NORF is dissolved in 5 ml
DMSO was then added into reaction system. The reaction is keep at
60-70.degree. C. for 5 hours and then at room temperature for
overnight. Reaction is finally stopped with 1 ml of methanol. The
final drug polymer is precipitated in a mixture of ether/water (50
v/v %). The precipitated polymer is then dissolved in acetone and
precipitated in ether again. This washing procedure is repeated
three times.
[0189] Norfloxacin is the only component in the drug polymer which
has a strong detectable absorbance at 280 nm in the UV range.
Hence, its presence can be detected using a UV detector. FIG. 5
super-imposes the UV chromatogram for the drug polymer with its
universal gel permeation chromatography (GPC) curves using a
universal refractive index detector. Similar data is shown for a
Ciprofloxacin polymer in FIG. 6. The latter detects the presence of
all molecules because it has a dependence on mass of material
present, eluting out of the GPC column at a specific time. Hence, a
comparison of the two signals shows that the distribution of
norfloxacin is identical to the distribution of actual molecular
weight chains, meaning that there was no preferential coupling of
norfloxacin/ciprofloxacin to low versus high molecular weight
chains or vice-versa; implies that the coupling of
norfloxacin/ciprofloxacin was uniform.
Example 8
[0190] AC/CIPRO is an example of pharmaceutically active polyamide
containing antimicrobial drug Ciprofloxacin according to the
invention. It differs from example 1 in that it is not a
polyurethane and shows the versatility for the use of the
biomonomers in a range of step growth polymerizations. The
conditions for this synthesis are a common polyamide interfacial
polycondensation reaction. They are described as follows:
[0191] A solution of 3.88 g (5 mmol) of CIPRO-TEG-CIPRO and 1.06 g
(10 mmol) of sodium carbonate in 30 ml of water was cooled in an
ice bath for 15 min before addition of as the water phase to a 150
ml flask containing a stir bar. A organic solution containing 0.915
g of adipoyl chloride (AC, 5 mmol) in 20 ml of methylene chloride
was added slowly into the water phase under vigorously stirring.
The organic solution has been previously cooled in an ice bath for
15 min. Immediately after addition of the organic phase, an
additional 5 ml of methylene chloride was used to rinse the
original acid chloride container and transfer the solvent to
reaction flask. The polymerization medium was stirred at maximum
speed for an additional 5 min. The resulting polymer was collected
by filtration. The polymer was then washed with water for at least
3 times. It was then washed with acetone twice. The product was
vacuum-dried at 40.degree. C. for 24 hours.
Example 9
[0192] Gamma irradiation is a popular and well-established process
for sterilizing polymer-based medical devices (21). It has been
known, however, that this technique can lead to significant
alterations in the materials being treated. High-energy radiation
produces ionization and excitation in polymer molecules. The
stabilization process of the irradiated polymer results in physical
and chemical cross-linking or chain scission, which occurs during,
immediately after, or even days, weeks after irradiation. In this
example, NF and CP polymers are dissolved in a suitable solvent
such as chloroform at 10%. The films are cast in a suitable holder
such as Teflon mold and placed in a 60.degree. C. air flowing oven
to dry. The dried films are sterilized by gamma radiation. The dose
shall be capable of achieving the pre-selected sterility assurance
level (22). One of two approaches shall be taken in selecting the
sterilization dose: (a) selection of sterilization dose using
either 1) bioburden information, or 2) information obtained by
incremental dosing; b) Selection of a sterilization dose of 25 Kgy
following substantiation of the appropriateness of this dose. Each
sample had twelve films (N=3) to be sterilized by Gamma radiation.
Resultant chemical changes can be detected at different time points
as follow: a) No sterile (3); b) Immediately after irradiation (3);
c) Two weeks after irradiation (3); d) 1 month after irradiation
(3). After Gamma sterilization, the films are analyzed by GPC to
detect the change in the number-averaged molecular weight (Mn),
weight-averaged molecular weight (Mw), and polydispersity (Mw/Mn)
of polymer chains before and after radiation. The results are
listed in Table 3. It shows that no obvious physical and chemical
changes happened to the drug polymers after radiation
sterilization.
3TABLE 3 Mn, Mw and Polydispersity of drug polymers before and
after Radiation Samples Mn g/mol Mw g/mol PI THDI/PCL/NF: A: 3.2
.times. 10.sup.4 6.9 .times. 10.sup.4 2.1 B: 3.2 .times. 10.sup.4
6.2 .times. 10.sup.4 2.1 C: 3.0 .times. 10.sup.4 6.4 .times.
10.sup.4 2.1 Right after radiation A: 3.0 .times. 10.sup.4 6.2
.times. 10.sup.4 2.0 B: 2.9 .times. 10.sup.4 6.2 .times. 10.sup.4
2.0 C: 3.2 .times. 10.sup.4 6.3 .times. 10.sup.4 2.0 1 week after
radiation A: 2.9 .times. 10.sup.4 6.0 .times. 10.sup.4 2.1 B: 3.1
.times. 10.sup.4 6.7 .times. 10.sup.4 2.2 C: 2.8 .times. 10.sup.4
6.0 .times. 10.sup.4 2.1 2 weeks after radiation A: 2.9 .times.
10.sup.4 6.0 .times. 10.sup.4 2.1 B: 3.0 .times. 10.sup.4 6.4
.times. 10.sup.4 2.1 C: 3.0 .times. 10.sup.4 6.3 .times. 10.sup.4
2.1 1 month after radiation A: 2.8 .times. 10.sup.4 6.1 .times.
10.sup.4 2.1 B: 2.8 .times. 10.sup.4 5.8 .times. 10.sup.4 2.1 C:
2.8 .times. 10.sup.4 5.9 .times. 10.sup.4 2.1 THDI/PCL/CP: A: 2.1
.times. 10.sup.4 3.4 .times. 10.sup.4 1.6 B: 2.1 .times. 10.sup.4
3.3 .times. 10.sup.4 1.6 C: 2.1 .times. 10.sup.4 3.3 .times.
10.sup.4 1.6 Right after radiation A: 2.1 .times. 10.sup.4 3.4
.times. 10.sup.4 1.6 B: 2.3 .times. 10.sup.4 3.6 .times. 10.sup.4
1.6 C: 2.3 .times. 10.sup.4 3.7 .times. 10.sup.4 1.6 1 week after
radiation A: 2.3 .times. 10.sup.4 4.0 .times. 10.sup.4 1.6 B: 2.2
.times. 10.sup.4 3.6 .times. 10.sup.4 1.6 C: 2.2 .times. 10.sup.4
3.7 .times. 10.sup.4 1.6 2 weeks after radiation A: 2.2 .times.
10.sup.4 3.7 .times. 10.sup.4 1.7 B: 2.2 .times. 10.sup.4 3.6
.times. 10.sup.4 1.7 C: 2.2 .times. 10.sup.4 3.9 .times. 10.sup.4
1.7 1 month after radiation A: 2.1 .times. 10.sup.4 3.4 .times.
10.sup.4 1.6 B: 2.1 .times. 10.sup.4 3.6 .times. 10.sup.4 1.7 C:
2.1 .times. 10.sup.4 3.5 .times. 10.sup.4 1.7
Example 10
[0193] This example shows the in vitro cytotoxicity of a
non-bioactive control polymer, NF and CP polymers with mammalian
cell lines using a direct contact method. In this method, 1 ml of
polymer DMSO solutions containing 1 mg/ml, 3 mg/ml and 5 mg/ml,
respectively, of control or drug polymer is loaded on each
Millipore 0.45 .mu.m filter that is set on top of agar in a Petri
dish. These dishes are then incubated at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 for 24 hours. After the
solvent is diffused into agar, these filters with polymers loaded
on it are transferred into a new Petri dish containing solidified
agar. HeLa cells are seeded onto these filters. The dishes are
incubated at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 for 48 hours. Cells are stained with succinic
dehydrogenase staining buffer. The stained areas on the filters
show the cytotoxicity of materials. FIG. 7 show the scanned
pictures of stained cells that are seeded on the filters loaded
with different amounts of control, NF and CP polymers. There are no
unstained areas in each filter. The results show that the control
polymer and bioactive polymers have good biocompatibility with
mammalian cells.
Example 11
[0194] NF polymer was used to evaluate the ability of a hydrolytic
enzyme to degrade the material and preferentially release drug. NF
polymer was coated onto small glass cylinders, and then incubated
in the presence and absence of hydrolytic enzyme (i.e. cholesterol
esterase) for up to 10 weeks at 37.degree. C. At each week interval
the incubation solution was removed from NF polymer and fresh
enzyme solution was added. The incubation solutions were assayed
via high pressure liquid chromatography (HPLC). Standard solutions
of pure norfloxacin were run through an HPLC system to get
calibration curve of this system. Norfloxacin concentration in the
incubated solution was determined by comparison of drug peak area
of incubation solution to calibration curve. FIG. 8 shows the
released norfloxacin from NF polymer in the presence and absence of
cholesterol esterase. In the presence of CE, there is an obvious
release of Norfloxacin 10 weeks. However in the absence of CE,
there only is some release of drug in the first 6 weeks and it is
lower than that of the enzyme incubated samples throughout the
experiment.
[0195] The same NF polymer incubation solutions assayed via HPLC
were also evaluated for antimicrobial activity using a biological
assay. A macro-dilution minimum inhibitory concentration (MIC)
assay was employed to determine the concentration of antimicrobial
(norfloxacin) that would inhibit the growth of a pathogen often
associated with device-related infections, Pseudomonas aeruginosa.
The MIC for this organism and norfloxacin was determined to be 0.8
mu g/mL. Incubation solutions from both enzyme and buffer control
treatment of NF polymer were used in a biological assay matrix that
was designed to estimate the concentration of norfloxacin as a
function of incubation time and treatment. The data are presented
in Table 4. Anti-microbial activity was not detected in the NF
polymer exposed to buffer (control) incubation solution after 2
weeks. However, the enzyme-treated NF polymers released clinically
significant levels (>MIC levels) of antibiotic over a 10 week
incubation period. These biological assay data show a significant
correlation with the HPLC data described above. The results of
these experiments demonstrate that the antibiotic agent is released
from NF polymer under enzymatic activation, and that the antibiotic
has antimicrobial activity against a clinically significant
bacterium. Furthermore, clinically significant concentrations
(i.e., MIC level) of the antibiotic are released over an extended
period of time, 10 weeks.
4TABLE 4 MIC Assay for antibacterial activity of degraded NF
polymer solutions Samples containing drugs greater than or less
than MIC level sample No 1 2 3 4 5 6 7 8 solution Time CE CE CE CE
buffer buffer buffer buffer 1 week > > > > > >
> > 2 weeks > > > > > > > > 3 weeks
> > > > < > < < 4 weeks > > > >
< > < < 6 weeks > > > > < < < <
8 weeks > > > > < < < < 10 weeks > >
> > < < < <
Example 13
[0196] In vivo animal studies are performed on formed coupons made
of control and CP polymer with a dimension of 1.times.2 cm.sup.2.
The coupons were implanted in the peritoneal cavity of male rats.
The coupons were explanted after rats were housed for 1 week. The
experimental conditions according to the invention are as
follows:
[0197] For implantation, 5 male Sprague-Dawley rats (250-300 g)
were used for every group of experiment. After they were
anesthetised, a 2 cm laparotomy incision was made in the abdomen.
The omentum and gubernaculum tissues were resected as they tend to
envelop the coupon. Then either a control coupon or a CP coupon
(1.times.2 cm.sup.2) was implanted in the abdominal cavity. The
incision was closed in two layers. After animals were housed for 1
week (rats were monitored daily), coupons were explanted from rats.
Gross observations were made including adhesion, abscess,
inflammation, and encapsulation. It was found that no adhesion,
abscess and inflammation associated with CP polymer coupons, but
there was obvious adhesion, abscess and serious inflammation
associated with implanted control polymer coupons. Coupons were
retrieved with sterile surgical instruments. A swab was taken of
the peritoneal cavity. Coupons were rinsed in PBS buffer to remove
non-adherent cells and placed in sterile tubes for further bacteria
culture. Bacteria counts obtained from cultures of control and CP
coupons are shown in FIG. 9. Clearly, CP coupons show an
antimicrobial effect, yielding significantly lower colony forming
units (CFUs).
Example 12
[0198] Examples of biomedical articles that integrate the bioactive
polymers to the polymers using described methods 1, 2, 3 below
include, for example, the following articles that are in whole or
in part made of polyurethane components, namely, cardiac assist
devices, tissue engineering polymeric scaffolds and related
devices, cardiac replacement devices, cardiac septal patches, intra
aortic balloons, percutaneous cardiac assist devices,
extra-corporeal circuits, A-V fistual, dialysis components (tubing,
filters, membranes, etc.), aphoresis units, membrane oxygenator,
cardiac by-pass components (tubing, filters, etc.), pericardial
sacs, contact lens, cochlear ear implants, sutures, sewing rings,
cannulas, contraceptives, syringes, o-rings, bladders, penile
implants, drug delivery systems, drainage tubes, pacemaker lead
insulators, heart valves, blood bags, coatings for implantable
wires, catheters, vascular stents, angioplasty balloons and
devices, bandages, heart massage cups, tracheal tubes, mammary
implant coatings, artificial ducts, craniofacial and maxillofacial
reconstruction applications, ligaments, fallopian tubes, biosensors
and bio-diagnostic substrates.
[0199] Non-biomedical articles fabricated by hereinbefore method 1)
include, for example, extruded health care products, bio-reactor
catalysis beds or affinity chromatography column packings, or a
biosensor and bio-diagnostic substrates.
[0200] Non-medical applications that are exemplified by method 2)
include fibre membranes for water purification.
[0201] Non-medical applications of the type exemplified by method
3) include varnishes with biological function for aseptic
surfaces.
[0202] Although this disclosure has described and illustrated
certain preferred embodiments of the invention, it is to be
understood that the invention is not restricted to those particular
embodiments. Rather, the invention includes all embodiments which
are functional or mechanical equivalence of the specific
embodiments and features that have been described and
illustrated.
* * * * *