U.S. patent application number 10/694688 was filed with the patent office on 2004-05-06 for polymers with bioactive agents.
This patent application is currently assigned to Chienna B.V.. Invention is credited to Bezemer, Jeroen Mattijs, Blitterswijk, Clemens Antoni van, Feijen, Jan, Grijpma, Dirk Wybe.
Application Number | 20040086544 10/694688 |
Document ID | / |
Family ID | 8240692 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040086544 |
Kind Code |
A1 |
Bezemer, Jeroen Mattijs ; et
al. |
May 6, 2004 |
Polymers with bioactive agents
Abstract
The invention relates to a process for loading a polymer with
one or more bioactive agents, using a wet spinning technique. The
invention further relates to a polymer loaded with one or more
bioactive agents, obtainable by said process and to the use thereof
as a carrier for controlled drug release or as scaffold for tissue
engineering.
Inventors: |
Bezemer, Jeroen Mattijs;
(Utrecht, NL) ; Blitterswijk, Clemens Antoni van;
(Hekendorp, NL) ; Feijen, Jan; (Hengelo, NL)
; Grijpma, Dirk Wybe; (Enschede, NL) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109-9601
US
|
Assignee: |
Chienna B.V.
Bilthoven
NL
|
Family ID: |
8240692 |
Appl. No.: |
10/694688 |
Filed: |
October 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10694688 |
Oct 28, 2003 |
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09676648 |
Sep 29, 2000 |
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6685957 |
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Current U.S.
Class: |
424/423 ;
264/172.11; 514/19.3; 514/2.4; 514/3.3; 514/54; 514/7.6;
514/9.7 |
Current CPC
Class: |
C12N 5/0068 20130101;
A61K 9/70 20130101; Y10S 530/817 20130101; C12N 2533/30 20130101;
C07K 17/04 20130101 |
Class at
Publication: |
424/423 ;
264/172.11; 514/002; 514/054 |
International
Class: |
A61K 038/18; A61K
038/22; A61K 031/739; D01D 005/30 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 1999 |
EP |
99203195.5 |
Claims
What is claimed is:
1. A process for preparing a polymer loaded with one or more
bioactive agents comprising the steps of: a) providing a solution
of the polymer in a suitable first solvent; b) adding an aqueous
solution of the bioactive agent to the polymer solution to obtain a
water-in-oil emulsion; c) immersing the water-in-oil emulsion in a
suitable second solvent by injecting the emulsion through a nozzle
into the second solvent; d) allowing the first solvent to migrate
into the second solvent to obtain a solid, fibrous polymer loaded
with the bioactive agent.
2. A process according to claim 1, wherein the polymer is
biocompatible and biodegradable.
3. A process according to claim 2, wherein the polymer is an
amphiphilic block copolymer, comprising hydrophilic blocks and
hydrophobic blocks.
4. A process according to claim 3, wherein the polymer is a
copolymer of a polyalkylene glycol and an aromatic ester.
5. A process according to claim 1, wherein the bioactive agent is
chosen from the group of antimicrobial agents, such as
antibacterial and anti-fungal agents, anti-viral agents, anti-tumor
agents, immunogenic agents, lipids, lipopolysaccharides, hormones
and growth factors.
6. A process according to claim 1, wherein the bioactive agent is
chosen from the group of peptides, oligopeptides, polypeptides and
proteins.
7. A process according to claim 1, wherein the first solvent is
immiscible with water and miscible with the second solvent, and
wherein the polymer is essentially insoluble in the second
solvent.
8. A process according to claim 7, wherein the first solvent has a
greater solubility in the second solvent when the polymer is
dissolved in the first solvent.
9. A process according to claim 1, wherein the water-in-oil
emulsion is immersed into the second solvent by injecting through a
syringe or an extruder.
10. A bioactive agent loaded polymer obtainable by the method of
claim 1.
11. A bioactive agent loaded polymer obtainable by a process
according to claim 9.
12. A bioactive agent loaded polymer according to claim 10 wherein
said bioactive agent is a peptide, oligopeptide, polypeptide or
protein.
13. A process for bonding fibers according to claim 1 to form a
fibrous mesh, wherein the fibers are collected and are bonded
together by use of a suitable solvent mixture.
14. A fibrous mesh obtainable by a process according to claim
13.
15. The use of a bioactive agent loaded polymer, according to claim
10, as a carrier for controlled drug release or as a scaffold for
tissue engineering.
16. The use of a fibrous mesh according to claim 14 as a carrier
for controlled drug release or as a scaffold for tissue
engineering.
Description
[0001] The invention relates to a fibrous polymer loaded with one
or more bioactive agents and to a process for preparing the fibrous
polymer loaded with the bioactive agent or agents. The invention
further relates to the use of the polymer loaded with the bioactive
agent or agents as a scaffold for tissue engineering.
[0002] The development of biological substitutes, that can restore
or improve tissue function, is a rapidly evolving interdisciplinary
field in science. New tissues can be engineered from living cells
and three dimensional scaffolds. The function of the scaffold is to
provide structural integrity and space for growing tissue, and to
guide tissue formation. For this purpose, scaffolds are needed with
a high porosity and a high surface area. Ideally, the scaffold
delivers bioactive factors which modulate cellular behavior such as
proliferation, migration and adhesion. For example, it has been
shown that release of bone morphogenetic protein (rhBMP-2) from
biodegradable porous scaffolds stimulated growth of bone into the
scaffolds in vivo (see K. Whang et al., J. Biomed. Mater. Res. 42
(1998) 491-499).
[0003] Macroporous scaffolds for tissue engineering have been
fabricated by various techniques, including fiber bonding (see A.
G. Mikos et al., J. Biomed. Mater. Res. 27 (1993) 183-189), solvent
casting/salt-leaching (see A. G. Mikos et al., Biomaterials 14
(1993) 323-330), phase separation (see H. Lo et al., J. Biomed.
Mater. Res. 30 (1996) 475-484) and emulsion freeze-drying (see K.
Whang et al., Polymer 36 (1995) 837-842). Often, the methods used
to prepare macroporous structures are not suitable for
incorporation of labile proteins and other bioactive compounds, due
to the high temperatures used, exposure to organic solvents, or the
need for removal of the porogens.
[0004] Recently, Whang et al. (see J. Biomed. Mater. Res. 42 (1998)
491-499) developed an emulsion freeze-drying process to overcome
these drawbacks in the incorporation of proteins into porous
matrices. This method consists of creating an emulsion from a
poly(lactide-co-glycolide) (PLG) solution in methylene chloride and
an aqueous protein solution. Subsequently, the emulsion is quenched
in liquid nitrogen, and methylene chloride and water are removed by
freeze-drying. The large pores in the resulting matrices are formed
by the dispersed water phase and since the proteins are also
dissolved in the water phase, this implies that the proteins are
located within the large interconnected pores. This might limit the
possibilities to obtain slow release of proteins. Furthermore, it
appeared that the type of protein influenced the ultimate structure
of the pores. In case of bovine serum albumin (BSA) loaded
scaffolds, the median pore size was 65 .mu.m, while incorporation
of rhBMP-2 resulted in a median pore size of only 9 .mu.m, which is
probably too small for optimal bone-ingrowth.
[0005] The present invention aims to provide a method for preparing
a fibrous polymer loaded with one or more bioactive agents.
Further, in particular in view of the application of polymers as
scaffold for tissue engineering, it is often desired to be able to
incorporate (bioactive) additives in a solid body that constitutes
the scaffold. For instance, the presence of growth factors may be
very much desired in order to enhance cell growth or
differentiation. As many of these bioactive additives are very
sensitive compounds, the need for working under mild conditions
becomes even more important. It is particularly desired that the
method can be performed under such mild conditions that the
biologic activity of the bioactive agent is essentially not
deteriorated during the carrying out of the method. Further, it is
desired that the bioactive agent can be homogeneously distributed
throughout the polymer.
[0006] Surprisingly, it has now been found that a wet spinning
technique is highly suitable for achieving the above goals.
Accordingly, the invention specifically relates to a process for
preparing a polymer loaded with one or more bioactive agents
comprising the steps of:
[0007] a) providing a solution of the polymer in a suitable first
solvent;
[0008] b) adding an aqueous solution of the bioactive agent or
agents to the polymer solution to obtain a water-in-oil
emulsion;
[0009] c) immersing the water-in-oil emulsion in a suitable second
solvent by injecting the emulsion through a nozzle into the second
solvent;
[0010] d) allowing the first solvent to migrate into the second
solvent to obtain a solid, fibrous polymer loaded with the
bioactive agent or agents.
[0011] The present process is carried out under mild conditions; no
high temperatures or extreme pH is required. As a result, the
stability and activity of the bioactive agent or agents is
essentially maintained during the process. Furthermore, it has been
found possible in a process according to the invention to obtain a
polymeric substrate in which the bioactive agent is homogeneously
distributed. Another advantage is that the present process yields a
fibrous product, which is believed to be a highly suitable form for
scaffolds in tissue engineering, enabling diffusion of nutrients
and waste materials to and from cells seeded on the scaffold and
mimicking natural fibrous tissues, such as muscle tissue.
Furthermore the present product may find advantageous application
in the field of surgical devices and aids, for instance as device
for controlled release of bioactive agents in vivo. Specific
examples of such devices are spacers that may be used to release an
antibiotic, such as gentamycin, in case of an infection, for
example when a revision hip implant is to be inserted in a patient,
or devices for the release of anti-conception agents.
[0012] The polymer which is loaded according to the present
invention may be any kind of polymer. Preferably, the polymer is a
biocompatible polymer, thus enabling the use of the polymer, loaded
with the bioactive agent, for pharmaceutical and/or biological
purposes. In the context of the present invention, the term
biocompatible is intended to refer to materials which may be
incorporated into a human or animal body substantially without
unacceptable responses of the human or animal. It is further
preferred that the polymer is a biodegradable polymer, which makes
the polymer loaded with bioactive agent(s) highly suitable for use
as a scaffold in tissue engineering. The term biodegradable refers
to materials which, after a certain period of time, are broken down
in a biological environment. Preferably, the rate of breakdown is
chosen similar or identical to the rate at which the body generates
autogenous tissue to replace an implant manufactured of the
biodegradable material.
[0013] Suitable examples of polymers to be loaded with one or more
bioactive agents in accordance with the invention are amphiphilic
block copolymers, comprising hydrophilic and hydrophobic blocks.
The hydrophilic component is preferably a polyalkylene glycol, such
as polyethylene glycol. The hydrophobic blocks may be chosen from a
variety of possibilities, including poly(lactide-co-glycolide),
poly(caprolactone), polybutylene terephtalate, poly(propylene
fumarate), and poly(anhydrides). Such block copolymers may be
diblock, triblock, multiblock or star-shaped block copolymers. It
has been found that the use of these polymers lead to very stable
emulsions, which beneficially affects the formation of the polymer
fibers.
[0014] A preferred class of polymers according to the invention, is
a copolymer of a polyalkylene glycol terephtalate and an aromatic
polyester. Preferably, the copolymer comprises 20-90 wt. %, more
preferably 40-70 wt. % of the polyalkylene glycol terephtalate, and
80-10 wt. %, more preferably 60-30 wt. % of the aromatic polyester.
A preferred type of copolymers according to the invention is formed
by the group of block copolymers.
[0015] The polyalkylene glycol terephtalate may have a weight
average molecular weight of about 150 to about 4000. Preferably,
the polyalkylene glycol terephtalate has a weight average molecular
weight of 200 to 1500. The aromatic polyester preferably has a
weight average molecular weight of from 200 to 5000, more
preferably from 250 to 4000. The weight average molecular weight of
the copolymer preferably lies between 10,000 and 300,000, more
preferably between 40,000 and 120,000.
[0016] The weight average molecular weight may suitably be
determined by gel permeation chromatography (GPC). This technique,
which is known per se, may for instance be performed using
chloroform as a solvent and polystyrene as external standard.
Alternatively, a measure for the weight average molecular weight
may be obtained by using viscometry (see NEN-EN-ISO 1628-1). This
technique may for instance be performed at 25.degree. C. using
chloroform as a solvent. Preferably, the intrinsic viscosity of the
copolymer lies between 0.2289 and 1.3282 dL/g, which corresponds to
a weight average molecular weight between 10,000 and 200,000.
Likewise, the more preferred ranges for the weight average
molecular weight measured by GPC mentioned above can also be
expressed in terms of the intrinsic viscosity.
[0017] In a preferred embodiment, the polyalkylene glycol
terephtalate component has units of the formula --OLO--CO-Q-CO--,
wherein O represents oxygen, C represents carbon, L is a divalent
organic radical remaining after removal of terminal hydroxyl groups
from a poly(oxyalkylene)glycol, and Q is a divalent organic
radical.
[0018] Preferred polyalkylene glycol terephtalates are chosen from
the group of polyethylene glycol terephtalate, polypropylene glycol
terephtalate, and polybutylene glycol terephtalate and copolymers
thereof, such as poloxamers. A highly preferred polyalkylene glycol
terephtalate is polyethylene glycol terephtalate.
[0019] The terms alkylene and polyalkylene generally refer to any
isomeric structure, i.e. propylene comprises both 1,2-propylene and
1,3-propylene, butylene comprises 1,2-butylene, 1,3-butylene,
2,3-butylene, 1,2-isobutylene, 1,3-isobutylene and 1,4-isobutylene
(tetramethylene) and similarly for higher alkylene homologues. The
polyalkylene glycol terephtalate component is preferably terminated
with a dicarboxylic acid residue --CO-Q-CO--, if necessary to
provide a coupling to the polyester component. Group Q may be an
aromatic group having the same definition as R, or may be an
aliphatic group such as ethylene, propylene, butylene and the
like.
[0020] The polyester component preferably has units
--O-E-O--CO--R--CO--, wherein O represents oxygen, C represents
carbon, E is a substituted or unsubstituted alkylene or
oxydialkylene radical having from 2 to 8 carbon atoms, and R is a
substituted or unsubstituted divalent aromatic radical.
[0021] In a preferred embodiment, the polyester is chosen from the
group of polyethylene terephthalate, polypropylene terephthalate,
and polybutylene terephthalate. A highly preferred polyester is
polybutylene terephthalate.
[0022] The preparation of the copolymer will now be explained by
way of example for a polyethylene glycol terephtalate/polybutylene
terephthalate copolymer. Based on this description, the skilled
person will be able to prepare any desired copolymer within the
above described class. An alternative manner for preparing
polyalkylene glycol terephtalate/polyester copolymers is disclosed
in U.S. Pat. No. 3,908,201.
[0023] A polyethylene glycol terephtalate/polybutylene
terephthalate copolymer may be synthesized from a mixture of
dimethyl terephthalate, butanediol (in excess), polyethylene
glycol, an antioxidant and a catalyst. The mixture is placed in a
reaction vessel and heated to about 180.degree. C., and methanol is
distilled as transesterification proceeds. During the
transesterification, the ester bond with methyl is replaced with an
ester bond with butylene and/or the polyethyene glycol. After
transesterification, the temperature is raised slowly to about
245.degree. C., and a vacuum (finally less than 0.1 mbar) is
achieved. The excess butanediol is distilled off and a prepolymer
of butanediol terephthalate condenses with the polyethylene glycol
to form a polyethylene/polybutylene terephthalate copolymer. A
terephthalate moiety connects the polyethylene glycol units to the
polybutylene terephthalate units of the copolymer and thus such a
copolymer also is sometimes referred to as a polyethylene glycol
terephthalate/polybutylene terephthalate copolymer (PEGT/PBT
copolymer).
[0024] The bioactive agent which is to be loaded into the polymer
may be chosen from various groups of compounds. The term
"biologically active agent" or bioactive agent, as used herein,
includes an agent which provides a therapeutic or prophylactic
effect, a compound that affects or participates in tissue growth,
cell growth, cell differentiation, a compound that may be able to
invoke a biological action such as an immune response, or could
play any other role in one or more biological processes. Such
agents include, but are not limited to, antimicrobial agents
(including antibacterial and anti-fungal agents), anti-viral
agents, anti-tumor agents, hormones, immunogenic agents, growth
factors, lipids, lipopolysaccharides, and peptides, polypeptides
and proteins in general.
[0025] An important group of compounds that can be used for loading
a polymer according to the invention is formed by peptides and
proteins, of which in principle any kind may be incorporated
according to the present invention. Both peptides and proteins are
compounds that are built up out of amino acids, linked to one
another via an amide bond (or peptide bond). This bond is the
product of the joining of an amino group of one amino acid with a
carboxylic acid group of the other. Relatively small peptides may
be referred to by the number of amino acids (e.g. di-, tri-,
tetrapeptides). A peptide with a relatively small number of amide
bonds may also be called an oligopeptide, whereas a peptide with a
relatively high number may be called a polypeptide or protein. In
addition to being a polymer of amino acid residues, certain
proteins may further be characterized by the so called quaternary
structure, a conglomerate of a number of polypeptides that are not
necessarily chemically linked by amide bonds but are bonded by
forces generally known to the skilled professional, such as
electrostatic forces and vanderwaals forces. The term peptides,
proteins or mixtures thereof as used herein is to include all above
mentioned possibilities.
[0026] Usually, the protein and/or peptide will be selected on the
basis of its biological activity. Depending on the type of polymer
chosen, the product obtainable by the present process is highly
suitable for controlled release of proteins and peptides. In a
preferred embodiment, the protein or peptide is a growth factor. A
growth factor is defined as a protein or peptide that has a
beneficial effect on the growth, proliferation and/or
differentiation of living cells. According to this embodiment, the
process of the invention provides a material that can
advantageously be used as a scaffold for tissue engineering,
wherein the growth factor is released from the polymer in a delayed
manner, thus providing a beneficial environment for tissue to grow
and/or differentiate.
[0027] Examples of preferred growth factors are Bone Morphogenetic
Proteins (BMP), epidermal growth factors, e.g. Epidermal Growth
Factor (EGF), fibroblast growth factors, e.g. basic Fibroblast
Growth Factor (bFGF), Nerve Growth Factor (NGF), Bone Derived
Growth Factor (BDGF), transforming growth factors, e.g.
Transforming Growth Factor-.beta.1 (TGF-.beta.1), and human Growth
Hormone (hGH).
[0028] Further examples of peptides or proteins or entities
comprising peptides or proteins which may advantageously be
contained in the loaded polymer include, but are not limited to,
immunogenic peptides or immunogenic proteins, which include, but
are not limited to, the following:
[0029] 1. Toxins: diphtheria toxin, tetanus toxin
[0030] 2. Viral surface antigens or parts of viruses: adenoviruses,
Epstein-Barr Virus, Hepatitis A Virus, Hepatitis B Virus, Herpes
viruses, HIV-1, HIV-2, HTLV-III, Influenza viruses, Japanese
encephalitis virus, Measles virus, Papilloma viruses,
Paramyxoviruses, Polio Virus, Rabies, Virus, Rubella Virus,
Vaccinia (Smallpox) viruses, Yellow Fever Virus
[0031] 3. Bacterial surface antigens or parts of bacteria:
Bordetella pertussis, Helicobacter pylori, Clostridium tetani,
Corynebacterium diphtheria, Escherichia coli, Haemophilus
influenza, Klebsiella species, Legionella pneumophila,
Mycobacterium bovis, Mycobacterium leprae, Mycrobacterium
tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis,
Proteus species, Pseudomonas aeruginosa, Salmonella species,
Shigella species, Staphylococcus aureus, Streptococcus pyogenes,
Vibrio cholera, Yersinia pestis
[0032] 4. Surface antigens of parasites causing disease or portions
of parasites: Plasmodium vivax--malaria, Plasmodium
falciparum--malaria, Plasmodium ovale--malaria, Plasmodium
malariae--malaria, Leishmania tropica--leishmaniasis, Leishmania
donovani, leishmaniasis, Leishmania branziliensis--leishmaniasis,
Trypanosoma rhodescense--sleeping sickness, Trypanosoma
gambiense--sleeping sickness, Trypanosoma cruzi--Chagas' disease,
Schistosoma mansoni--schistosomiasis, Schistosomoma
haematobium--schistomiasis, Schistosoma japonicum--shichtomiasis,
Trichinella spiralis--trichinosis, Stronglyloides
duodenale--hookworm, Ancyclostoma duodenale--hookworm, Necator
americanus--hookworm, Wucheria bancrofti--filariasis, Brugia
malaya--filariasis, Loa loa--filariasis, Dipetalonema
perstaris--filariasis, Dracuncula medinensis--filariasis,
Onchocerca volvulus--filariasis
[0033] 5. Immunoglobulins: IgG, IgA, IgM, Antirabies
immunoglobulin, Antivaccinia immunoglobulin
[0034] 6. Anititoxins: Botulinum antitoxin, diphtheria antitoxin,
gas gangrene antitoxin, tetanus antitoxin.
[0035] 7. Antigens which elicit an immune response against: Foot
and Mouth Disease, hormones and growth factors such as follicle
stimulating hormone, prolactin, angiogenin, epidermal growth
factor, calcitonin, erythropoietin, thyrotropic releasing hormone,
insulin, growth hormones, insulin-like growth factors 1 and 2,
skeletal growth factor, human chorionic gonadotropin, luteinizing
hormone, nerve growth factor, adrenocorticotropic hormone (ACTH),
luteinizing hormone releasing hormone (LHRH), parathyroid hormone
(PTH), thyrotropin releasing hormone (TRH), vasopressin,
cholecystokinin, and corticotropin releasing hormone; cytokines,
such as interferons, interleukins, colony stimulating factors, and
tumor necrosis factors: fibrinolytic enzymes, such as urokinase,
kidney plasminogen activator; and clotting factors, such as Protein
C, Factor VIII, Factor IX, Factor VII and Antithrombin III.
[0036] 8. Examples of other proteins or peptides: albumin, atrial
natriuretic factor, renin, superoxide dismutase,
.alpha..sub.1-antitrypsi- n, lung surfactant proteins, bacitracin,
bestatin, cydosporine, delta sleep-inducing peptide (DSIP),
endorphins, glucagon, gramicidin, melanocyte inhibiting factors,
neurotensin, oxytocin, somostatin, terprotide, serum thymide
factor, thymosin, DDAVP, dermorphin, Met-enkephalin, peptidoglycan,
satietin, thymopentin, fibrin degradation product,
des-enkephalin-.alpha.-endorphin, gonadotropin releasing hormone,
leuprolide, .alpha.-MSH, and metkephamid.
[0037] It is to be understood, however, that the scope of the
present invention is not limited to any specific peptides or
proteins.
[0038] Although, in view of the delicacy of proteins and peptides,
the present process is particularly useful for making polymers
loaded with proteins and peptides, it is of course also possible to
load a polymer with a substance other than a protein or peptide.
Such biologically active agents which may be incorporated include,
but are not limited to, non-peptide, non-protein drugs. It is
possible within the scope of the present invention to incorporate
drugs of a polymeric nature, but also to incorporate drugs of a
relatively small molecular weight of less than 1500, or even less
than 500.
[0039] Examples of non-peptide, non-protein drugs which may be
incorporated include, but are not limited to, the following:
[0040] 1. Anti-tumor agents: altretamin, fluorouracil, amsacrin,
hydroxycarbamide, asparaginase, ifosfamid, bleomycin, lomustin,
busulfan, melphalan, chlorambucil, mercaptopurin, chlormethin,
methotrexate, cisplatin, mitomycin, cyclophosphamide, procarbazin,
cytarabin, teniposid, dacarbazin, thiotepa, dactinomycin,
tioguanin, daunorubicin, treosulphan, doxorubicin, tiophosphamide,
estramucin, vinblastine, etoglucide, vincristine, etoposid,
vindesin.
[0041] 2. Anitimicrobial agents
[0042] 2.1 Antibiotics
[0043] Penicillins: ampicillin, nafcillin, amoxicillin, oxacillin,
azlocillin, penicillin G, carbenicillin, penicillin V,
dicloxacillin, phenethicillin, floxacillin, piperacillin,
mecillinam, sulbenicillin, methicillin, ticarcillin,
mezlocillin
[0044] Cephalosporins: cefaclor, cephalothin, cefadroxil,
cephapirin, cefamandole, cephradine, cefatrizine, cefsulodine,
cefazolin, ceftazidim, ceforanide, ceftriaxon, cefoxitin,
cefuroxime, cephacetrile, latamoxef, cephalexin
[0045] Aminoglycosides: amikacin, neomycin, dibekacyn, kanamycin,
gentamycin, netilmycin, kanamycin, tobramycin
[0046] Macrolides: amphotericin B, novobiocin, bacitracin,
nystatin, clindamycin, polymyxins, colistin, rovamycin,
erythromycin, spectinomycin, lincomycin, vancomycin
[0047] Tetracyclines: chlortetracycline, oxytetracycline,
demeclocycline, rolitetracycline, doxycycline, tetracycline,
minocycline Other antibiotics: chloramphenicol, rifamycin,
rifampicin, thiamphenicol
[0048] 2.2 Chemotherapeutic agents
[0049] Sulfonamides: sulfadiazine, sulfamethizol, sulfadimethoxin,
sulfamethoxazole, sulfadimidin, sulfamethoxypyridazine,
sulfafurazole, sulfaphenazol, sulfalene, sulfisomidin,
sulfamerazine, sulfisoxazole, trimethoprim with sulfamethoxazole or
sulfametrole
[0050] Urinary tract antiseptics: methanamine,
quinolones(norfloxacin, cinoxacin), nalidixic acid, nitro-compounds
(nitrofurantoine, nifurtoinol), oxolinic acid
[0051] Anaerobic infections: metronidazole
[0052] 3. Drugs for tuberculosis: aminosalicyclic acid, isoniazide,
cycloserine, rifampicine, ethambutol, tiocarlide, ethionamide,
viomycin
[0053] 4. Drugs for leprosy: amithiozone, rifampicine, clofazimine,
sodium sulfoxone, diaminodiphenylsulfone (DDS, dapsone)
[0054] 5. Antifungal agents: amphotericin B, ketoconazole,
clotrimazole, miconazole, econazole, natamycin, flucytosine,
nystatine, griseofulvin
[0055] 6. Antiviral agents: aciclovir, idoxuridine, amantidine,
methisazone, cytarabine, vidarabine, ganciclovir
[0056] 7. Chemotherapy of amebiasis: chloroquine, iodoquinol,
clioquinol, metronidazole, dehydroemetine, paromomycin, diloxanide,
furoatetinidazole, emetine
[0057] 8. Anti-malarial agents: chloroquine, pyrimethamine,
hydroxychloroquine, quinine, mefloquine, sulfadoxine/pyrimethamine,
pentamidine, sodium suramin, primaquine, trimethoprim,
proguanil
[0058] b 9. Anti-helminthiasis agents: antimony potassium tartrate,
niridazole, antimony sodium dimercaptosuccinate, oxamniquine,
bephenium, piperazine, dichlorophen, praziquantel,
diethylcarbamazine, pyrantel parmoate, hycanthone, pyrivium
pamoate, levamisole, stibophen, mebendazole, tetramisole,
metrifonate, thiobendazole, niclosamide
[0059] 10. Anti-inflammatory agents: acetylsalicyclic acid,
mefenamic acid, aclofenac, naproxen, azopropanone, niflumic acid,
benzydamine, oxyphenbutazone, diclofenac, piroxicam, fenoprofen,
pirprofen, flurbiprofen, sodium salicyclate, ibuprofensulindac,
indomethacin, tiaprofenic acid, ketoprofen, tolmetin
[0060] 11. Anti-gout agents: colchicine, allopurinol
[0061] 12. Centrally acting (opoid) analgesics: alfentanil,
methadone, bezitramide, morphine, buprenorfine, nicomorphine,
butorfanol, pentazocine, codeine, pethidine, dextromoramide,
piritranide, dextropropoxyphene, sufentanil, fentanyl
[0062] 13. Local anesthetics: articaine, mepivacaine, bupivacaine,
prilocaine, etidocaine, procaine, lidocaine, tetracaine
[0063] 14. Drugs for Parkinson's disease: amantidine,
diphenhydramine, apomorphine, ethopropazine, benztropine mesylate,
lergotril, biperiden, levodopa, bromocriptine, lisuride, carbidopa,
metixen, chlorphenoxamine, orphenadrine, cycrimine, procyclidine,
dexetimide, trihexyphenidyl
[0064] 15. Centrally active muscle relaxants: baclofen,
carisoprodol, chlormezanone, chlorzoxazone, cyclobenzaprine,
dantrolene, diazepam, febarbamate, mefenoxalone, mephenesin,
metoxalone, methocarbamol, tolperisone
[0065] 16. Hormones and hormone antago7nistics
[0066] 16.1 Corticosteroids
[0067] 16.1.1 Mineralocorticosteroids: cortisol,
desoxycorticosterone, flurohydrocortisone
[0068] 16.1.2 Glucocorticosteroids: beclomethasone, betamethasone,
cortisone, dexamethasone, fluocinolone, fluocinonide,
fluocortolone, fluorometholone, fluprednisolone, flurandrenolide,
halcinonide, hydrocortisone, medrysone, methylprednisolone,
paramethasone, prednisolone, prednisone, triamcinolone
(acetonide)
[0069] 16.2 Androgents
[0070] 16.2.1 Androgenic steroids used in therapy: danazole,
fluoxymesterone, mesterolone, methyltestosterone, testosterone and
salts thereof
[0071] 16.2.2 Anabolic steroids used in therapy: calusterone,
nandrolone and salts thereof, dromostanolone, oxandrolone,
ethylestrenol, oxymetholone, methandriol, stanozolol
methandrostenolone, testolactone
[0072] 16.2.3 Antiandrogens: cyproterone acetate
[0073] 16.3 Estrogens
[0074] 16.3.1 Estrogenic steroids used it therapy:
diethylstilbestrol, estradiol, estriol, ethinylestradiol,
mestranol, quinestrol
[0075] 16.3.2 Anti-estrogens: chlorotrianisene, clomiphene,
ethamoxytriphetol, nafoxidine, tamoxifen
[0076] 16.4 Progestins: allylestrenol, desogestrel, dimethisterone,
dydrogesterone, ethinylestrenol, ethisterone, ethynadiol diacetate,
etynodiol, hydroxyprogesterone, levonorgestrel, lynestrenol,
medroxyprogesterone, megestrol acetate, norethindrone,
norethisterone, norethynodrel, norgestrel, progesterone
[0077] 17. Thyroid drugs
[0078] 17.1 Thyroid drugs used in therapy: levothyronine,
liothyronine
[0079] 17.2 Anti-thyroid drugs used in therapy: carbimazole,
methimazole, methylthiouracil, propylthiouracil
[0080] When a hydrophobic drug, such as, for example, a steroid
hormone is incorporated, preferably at least one hydrophobic
antioxidant is present. Hydrophobic antioxidants which may be
employed include, but are not limited to, tocopherols, such as
.alpha.-tocopherol, .beta.-tocopherol, .gamma.-tocopherol,
.delta.-tocopherol, .epsilon.-tocopherol, .zeta..sub.1-tocopherol,
.zeta..sub.2 -tocopherol, and .eta.-tocopherol; and 1-ascorbic acid
6-palmitate. Such hydrophobic antioxidants retard the degradation
of the copolymer and retard the release of the biologically active
agent. Thus, the use of a hydrophobic or lipophilic antioxidant is
applicable particularly to the formation of loaded polymers which
include drugs which tend to be released quickly, such as, for
example, drug molecules having a molecular weight less than 500.
The hydrophobic antioxidant(s) may be present in the loaded polymer
in an amount of from about 0.1 wt. % to about 10 wt. % of the total
weight of the polymer, preferably from about 0.5 wt. % to about 2
wt. %.
[0081] When the loaded polymer includes a hydrophilic drug, such as
an aminoglycoside, the loaded polymer may also include, in addition
to a hydrophobic antioxidant, a hydrophobic molecule such as
cholesterol, ergosterol, lithocholic acid, cholic acid, dinosterol,
betuline, or oleanolic acid, which may be employed in order to
retard the release rate of the agent from the copolymer. Such
hydrophobic molecules prevent water penetration into the loaded
polymer, but do not compromise the degradability of the polymer
matrix. In addition, such molecules have melting points from
150.degree. C. to 200.degree. C. or decreases the polymer matrix
diffusion coefficient for the biologically active agent, such as
drug molecule, to be released. Thus, such hydrophobic molecules
provide for a more sustained release of a biologically active agent
from the polymer matrix. The at least one hydrophobic molecule may
be present in the loaded polymer in an amount of from about 0.1 wt.
% to about 20 wt. %, preferably from 1.0 wt. % to 5.0 wt. %.
[0082] It is noted that, for the preparation of the water-in-oil
emulsion according to the invention, it is necessary that a
hydrophobic bioactive agent dissolves at least slightly in water,
preferably at least to such an extent that the resultant loaded
polymer comprises an amount of the bioactive agent which is
sufficient to achieve a desired effect in vivo. If necessary, a
surfactant may be added to the aqueous solution of the bioactive
agent in order to achieve that a minimal desired amount of the
bioactive agent is incorporated into the polymer. Examples of such
surfactants are well known to the skilled artisan and may be used
in amounts which can easily be optimized by the artisan based on
his normal knowledge of the art. Specific examples of suitable
surfactants include, but are not limited to, poly(vinyl)alcohol,
Span 80, Tween and Pluronics.
[0083] The invention further requires the use of two solvents which
are chosen to complement each other's action in the present
process. The first solvent is to be chosen such that it is
immiscible with water. In addition, the polymer which is to be
loaded with bioactive agent(s) should be soluble in the first
solvent. The second solvent is to be chosen such that the polymer
is not soluble in it. Also, the first solvent is to be well
miscible with the second solvent. Preferably, the first solvent
mixes better with the second solvent than that the polymer
dissolves in the first solvent. This ensures that, upon immersion
of the water-in-oil emulsion in the second solvent, the first
solvent will substantially completely migrate into the second
solvent. Further preferred is that both solvents are immiscible
with water. This makes it possible to prevent that the bioactive
agent, which is processed in an aqueous solution, comes into
contact with an organic solvent, which might be harmful to
bioactive agent. Depending on the nature of the polymer to be
loaded, the skilled person will be able to select suitable
solvents. By way of example, good results have been obtained by
using chloroform as the first solvent, and hexane as the second
solvent when the polymer is polyethylene glycol
terephtalate/polybutylene terephthalate copolymer.
[0084] In a first step of the present process, a solution is
provided of the polymer in the first solvent. The concentration of
this solution is not critical. On the one hand, it is important
that all of the polymer dissolves. On the other hand, it is
preferred that the amount of the first solvent used is kept as
small as possible in order to keep the process efficient.
[0085] Of the polymer solution, a water-in-oil emulsion is prepared
by mixing it with an aqueous solution of the bioactive agent(s),.
Under certain circumstances, it may be desired to add conventional
stabilizers for enhancing the stability of the water-in-oil
emulsion. Typical examples of such stabilizers include proteins
such as albumin or casein, Pluronics and Span 80. It is, however,
preferred that such stabilizers are not used.
[0086] The amount of bioactive agent(s), in the aqueous solution
will be chosen such that a desired amount of these bioactive agents
is eventually incorporated into the polymer. Depending on the type
of polymer and the nature of the bioactive agent(s), the amount of
incorporated agent may vary. For proteins and peptides, for
example, it has been found that various proteins and peptides can
be incorporated into the polymer in concentrations up to 10 wt. %,
based on the weight of the loaded polymer. When using particularly
hydrophilic bioactive agents, such as the protein leuprolide, it
has even been found possible to incorporate the agent into the
polymer in a concentration of up to 50 wt. %, based on the weight
of the loaded polymer. The lower limit of the amount of bioactive
agent(s), is not critical and will depend on the activity of the
bioactive agent(s), and on the envisaged application of bioactive
agent loaded polymer. In the case of proteins and peptides
typically, at least 0.01 wt. %, based on the weight of the loaded
polymer, of protein and/or peptide will be incorporated.
[0087] The amount of water used for preparing the aqueous bioactive
agent solution will be at least so high as to enable an efficient
dissolution of the bioactive agent without employing unduly harsh
conditions that might adversely affect the stability and/or
biological activity of the bioactive agent. The upper limit of the
amount of water used will depend on the rate at which the bioactive
agent is to be released from the polymer in a final, envisaged
application of the bioactive agent loaded polymer. It has been
found that the use of larger amounts of water, leads to higher
release rates of the polymer. Typically, the aqueous solution of
the bioactive agent(s) will comprise between 0.001 and 10 wt. % of
bioactive agent(s), based on the weight of the solution. In
practice, the amount of bioactive agents in the solution will
depend on the solubility of the bioactive agents and on the
stability of the water-in-oil emulsion.
[0088] The obtained water-in-oil emulsion is next immersed in the
second solvent by injection through a nozzle. The diameter and
shape of the nozzle can be varied to obtain fibers of different
thickness and shape. The injection itself will usually be driven by
a pressure by virtue of which the emulsion is transported through
the nozzle into the second solvent. The injection may for instance
be accomplished by use of a syringe or an extruder. The amount of
the second solvent is not critical. It should be at least
sufficient for the emulsion to be completely immersed in it and to
allow a substantially complete migration of the first solvent from
the emulsion into the second solvent. The upper limit will
generally chosen on the basis of economic considerations.
[0089] Upon immersion of the emulsion into the second solvent, due
to the specific selection of the first and second solvents, the
first solvent will migrate from the emulsion into the second
solvent. In practice, it may often be observed that first exchange
of the first and second solvents takes place, before the first
solvent will migrate into the second solvent. This may have the
effect that the polymer fibers are provided with a porosity. This
phenomenon and how it may be controlled to obtain a desired
porosity has been described by P. van de Witte, "Polylactide
membranes. Correlation between phase transitions and morphology",
PhD thesis, University of Twente, Enschede, 1994.
[0090] As a result, the polymer, which does not dissolve in the
second solvent, will solidify thereby incorporating the bioactive
agent(s). Finally, the solid loaded polymer may be removed from the
mixture of first and second solvents in any conventional manner and
may eventually be dried.
[0091] In a preferred embodiment, the obtained fibers may be formed
into a fibrous mesh by collecting the fibers in a mold, and bonding
them together by use of a suitable solvent mixture. This mixture
should comprise at least one solvent in which the polymer dissolves
and at least one solvent in which the polymer does not dissolve.
Preferably, a mixture is used of the above described first and
second solvents. The second solvent will generally be present in an
amount exceeding that of the first solvent, in order to avoid the
risk of any of the polymer dissolving in the solvent mixture.
Preferably, the volumetric ratio of the first solvent to the second
solvent lies between 1:1 and 1:3.
[0092] It will be understood that the invention also encompasses a
bioactive agent loaded polymer obtainable by the process as set
forth herein above. Said polymer loaded with one or more bioactive
agents may be used in biological, pharmaceutical and surgical
applications, wherein a (controlled) release of a bioactive
agent(s) from a polymeric substrate is desired. Examples of such
applications include, but are not limited to, carriers for
controlled drug release and scaffolds for tissue engineering.
[0093] The invention will now be elucidated by the following,
non-restrictive examples.
EXAMPLES
Materials and Methods
Materials
[0094] Poly(ethylene glycol)terephthalate/poly(butylene
terephthalate) multiblock copolymers (PEG/PBT) were obtained from
IsoTis BV, Bilthoven, The Netherlands. The copolymers contained 30
wt % PBT and the PEG segment length was 1000 g/mole
(1000PEG70PBT30). Phosphate buffered saline (PBS), pH 7.4 was
purchased from NPBI (Emmercompascuum, The Netherlands). Bovine
serum albumin (BSA, heat shock fractionate, fraction V powder
minimum 98%) was purchased from Sigma Chem. Corp. (St. Louis, USA).
All solvents used were of analytical grade.
Preparation of Bioactiue Agent Loaded PEG/PBT Fiber Meshes
[0095] The bioactive agent in this example was a protein. Protein
loaded PEG/PBT fibers were prepared from water-in-oil emulsions. To
produce such emulsions, 3 or 3.5 ml of a protein solution in PBS
(containing 25 mg/ml BSA) was emulsified in a solution of 2 g
PEG/PBT in 14 ml CHCl.sub.3 using ultra-turrax-mixing (30 s at 20.5
krpm, Ika Labortechnik T25). Subsequently, the emulsion was poured
into a 20 ml glass syringe (Becton Dickinson Multifit) equipped
with a 0.4 mm needle (Neolus Terumo 12G.times.1.5"). The emulsion
was pushed through the needle into a beaker containing 2 l hexane
at a speed of 0.5 ml/min. by means of a perfusion pump (Secura E,
B. Braun). The hexane bath was stirred at 300 rpm. to prevent
premature sticking of the fibers. After fiber formation was
completed, the fibers were collected and transferred to a glass
mold of the desired shape (cylindrical, 5 cm diameter and 1.7 cm
height). To bond the fibers, a mixture of hexane and CHCl.sub.3
(7:3, 3:2, or 1:1, v/v) was introduced into the mold. After drying
overnight under atmospheric conditions, the fiber structures were
freeze dried for 3 days, and stored at -40.degree. C.
Scanning Electron Microscopy (SEM)
[0096] A Hitachi S-800 field emission SEM was used to evaluate the
surface characteristics and internal structure of fibrous
scaffolds. The devices were cut in liquid nitrogen and mounted on a
substrate holder. Samples were sputter-coated with a thin gold
layer.
In vitro Release of Bioactive Agent
[0097] Protein loaded fiber meshes (approximately 40 mg) were
incubated in 5 ml PBS (pH 7.4). Vials were continuously shaken at
37.degree. C. and samples were taken at various time points.
Protein content was determined using a standard Coomassie Blue
assay (Pierce). Buffer was refreshed after sampling.
Results and Discussion
Matrix Characterization
[0098] Bonded fiber meshes, containing BSA, were prepared in a
three-step procedure. First, a water-in-oil emulsion was formed
from an aqueous protein solution and a polymer solution in
CHCl.sub.3. The second step involves wet spinning of the w/o
emulsion into a hexane bath. Hexane is miscible with CHCl.sub.3,
but is a non-solvent for the PEG/PBT copolymers. Consequently,
extrusion of the fibers into hexane results in solidification of
the fibers, due to exchange of solvent and non-solvent. Since
hexane is not miscible with water, contact between the incorporated
proteins and hexane is prevented as much as possible.
[0099] FIG. 1 shows scanning electron micrographs of a
cross-section of the obtained protein loaded PEG/PBT fibers
(magnification is 500.times. (A) or 2000.times. (B)). The fiber
cross-section was not circular (FIG. 1a). This is probably caused
by the shape of the needle. The surface of the fibers was porous,
whereas the interior of the fibers seemed to be dense. This is in
contrast with the morphology of protein loaded PEG/PBT matrices,
prepared by immersion precipitation of w/o emulsion droplets in
hexane. These structures showed a porous internal morphology (data
not shown). Probably, the morphology of the fibers was changed
during the fiber bonding step in the solvent/non-solvent mixture.
Furthermore, it cannot be excluded that the internal structure of
the fibers as shown in FIG. 1 was affected by the cutting
procedure, used to obtain cross-sections for scanning electron
microscopy.
[0100] In order to be used for tissue engineering applications,
fiber meshes must often be configured in a certain shape and
immobilized. This can be achieved by collecting the fibers in a
mold, followed by bonding in a solvent-non-solvent mixture. The
efficiency of this fiber bonding process was dependent on the
solvent to non-solvent ratio of the CHCl.sub.3/hexane mixture.
Immersion of the fiber meshes in mixtures with a CHCl.sub.3 to
hexane ratio of 3:7 (v/v) did not result in stable structures.
Improved bonding was obtained for devices immersed in a
solvent/non-solvent mixture with a composition of 2:3. Such bonded
fiber structures were stable for several days in PBS buffer at
37.degree. C. in a shaking bath. A bonded fiber mesh, prepared by
immersion in CHCl.sub.3/hexane 1:1, remained intact for over 50
days of continuously shaking at 37.degree. C. Solvent/non-solvent
mixtures containing over 50% (v/v) CHCl.sub.3 could not be used,
since the fibers dissolved in such mixtures.
[0101] FIG. 2 shows scanning electron micrographs of the structure
of the obtained protein loaded PEG/PBT fiber meshes (cross-section
(A, C) and surface morphology (B, D) of fibers, bonded in a mixture
of CHCl.sub.3 and hexane with a volume ratio 3:7 (A, B) or 1:1 (C,
D)).
[0102] As shown in FIG. 2D, confluency was observed for the
structures bonded in CHCl.sub.3/hexane 1:1 (v/v), whereas such
connections were scarcely found for meshes immersed in
solvent/non-solvent mixtures with a composition of 3:7 or 2:3 (FIG.
2B).
Bioactiue Agent Release from Bonded Fiber Meshes
[0103] Two different fiber meshes were selected to study bioactive
agent release in phosphate buffered saline (PBS). The fibrous
structures were bonded in a mixture of hexane and CHCl.sub.3 of
volume ratio 1:1. In order to modulate the bioactive agent release
rate, the composition of the w/o emulsion which was used to produce
the loaded fibers, was varied. Previous experiments have shown that
the water content in the w/o emulsion is a powerful tool to
manipulate the release rate of high molecular weight proteins. In
the present, devices were prepared from emulsions which contained a
protein as the bioactive agent, in a concentration of 1.5 or 1.75
ml of a protein solution per g of polymer, respectively.
[0104] The total protein release from the bonded fiber meshes is
presented in FIG. 3 (protein release from PEG/PBT fiber meshes,
bonded in a mixture of hexane and CHCl.sub.3 (1:1, v/v): the
devices were prepared from emulsions which contained 1.5 (open
symbols) or 1.75 ml (closed symbols) protein solution per g of
polymer (n=3; .+-.s.d.).). For both devices, a relatively large
amount of protein was released during the first hours of incubation
in the buffer. Thereafter, a slow release was observed, for a
period longer than 10 days. A higher protein release rate was found
for the device prepared from the emulsion which contained the
highest water content.
* * * * *