U.S. patent application number 10/513982 was filed with the patent office on 2005-08-11 for compounds and method for coating surfaces in a haemocompatibe manner.
Invention is credited to Di Baise, Donato, Faust, Volker, Hoffmann, Erika, Hoffmann, Michael, Horres, Roland, Linssen, Marita Katarina.
Application Number | 20050176678 10/513982 |
Document ID | / |
Family ID | 29421502 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050176678 |
Kind Code |
A1 |
Horres, Roland ; et
al. |
August 11, 2005 |
Compounds and method for coating surfaces in a haemocompatibe
manner
Abstract
The invention concerns oligosaccharides and polysaccharides as
well as the use of these oligosaccharides and/or polysaccharides,
which contain the sugar unit N-acylglucosamine or
N-acylgalactosamine for the production of hemocompatible surfaces
as well as methods for the hemocompatible coating of surfaces with
said oligosaccharides and/or polysaccharides, which imitate the
common biosynthetic precursor substance of heparin, heparan
sulphates and chitosan. The invention further describes methods for
producing said oligosaccharides and/or polysaccharides and
discloses various possibilities of using hemocompatibly coated
surfaces. The invention relates particularly to the use of said
oligosaccharides and/or polysaccharides on stents with at least one
according to invention deposited hemocompatible coating, which
contains an antiproliferative, antiinflammatory and/or
antithrombotic active agent, methods for the preparation of said
stents as well as the use of said stents for the prevention of
restenosis.
Inventors: |
Horres, Roland; (Stolberg,
DE) ; Linssen, Marita Katarina; (Aachen, DE) ;
Hoffmann, Michael; (Eschweiler, DE) ; Hoffmann,
Erika; (Eschweiler, DE) ; Di Baise, Donato;
(Aachen, DE) ; Faust, Volker; (Aachen,
DE) |
Correspondence
Address: |
Gregory Turocy
Amin & Turocy
National City Center
1900 East 9th Street 24th Floor
Cleveland
OH
44114
US
|
Family ID: |
29421502 |
Appl. No.: |
10/513982 |
Filed: |
November 8, 2004 |
PCT Filed: |
April 15, 2003 |
PCT NO: |
PCT/DE03/01253 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60378676 |
May 9, 2002 |
|
|
|
Current U.S.
Class: |
514/54 ;
536/53 |
Current CPC
Class: |
A61K 31/13 20130101;
C08B 37/0075 20130101; A61P 7/02 20180101; C09D 105/08 20130101;
A61K 31/11 20130101; Y02A 50/475 20180101; A61K 31/21 20130101;
C08L 5/10 20130101; A61K 31/045 20130101; A61P 3/06 20180101; Y02A
50/30 20180101; A61K 31/12 20130101; A61K 31/727 20130101; A61P
37/06 20180101; A61L 33/08 20130101; A61K 31/28 20130101; A61K
31/722 20130101; A61P 29/00 20180101; A61K 31/075 20130101; A61P
35/00 20180101; A61L 31/16 20130101; A61K 31/33 20130101; A61L
31/10 20130101; A61P 19/06 20180101; A61K 31/095 20130101; A61L
33/08 20130101; C08L 5/10 20130101; A61L 31/10 20130101; C08L 5/10
20130101 |
Class at
Publication: |
514/054 ;
536/053 |
International
Class: |
A61K 031/728 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2002 |
DE |
102 21 055.1 |
Claims
1. Compounds of the general formulas Ia and Ib 5wherein n is an
integer between 4 and 1050, Y and Z, independently of each other,
represent the groups --CHO, --COCH.sub.3, --COC.sub.2H.sub.5,
--COC.sub.3H.sub.7, --COC.sub.4H.sub.9, --COC.sub.5H.sub.11,
--COCH(CH.sub.3).sub.2, --COCH.sub.2CH(CH.sub.3).sub.2,
--COCH(CH.sub.3)C.sub.2H.sub.5, --COC(CH.sub.3).sub.3,
--CH.sub.2COO--, --C.sub.2H.sub.4COO--, --C.sub.3H.sub.6COO--,
--C.sub.4H.sub.8COO--, and salts of said compounds.
2. The compounds of the general formula Ia according to claim 1,
wherein Y and Z, independently of each other, represent the groups
--COCH.sub.3, --COC.sub.2H.sub.5, --COC.sub.3H.sub.7,
--CH.sub.2COO--, --C.sub.2H.sub.4COO--, --C.sub.3H.sub.6COO--, as
well as salts of said compounds.
3. The compounds of the general formula Ib according to claim 1,
wherein Y represents the groups --COCH.sub.3, --COC.sub.2H.sub.5,
--COC.sub.3H.sub.7, as well as salts of said compounds.
4. A process for the production of the compounds of the general
formula Ia, characterized in that heparan sulphate and/or heparan
sulphate heparin is substantially entirely desulphated and
subsequently N-acylated via an acid.
5. A process for the production of the compounds of the general
formula Ib, characterized in that a) chitosan is partially
N-carboxyalkylated and then N-acylated, or b) chitosan is partially
N-acylated and then N-carboxyalkylated, or c) chitin is partially
deacetylated and then N-carboxyalkylated.
6. The process according to claim 5, characterized in that
substantially the half of the amino groups of chitosan or chitin
are acylated and the other half thereof are carboxyalkylated.
7. The compounds of the general formula Ia and/or Ib obtainable
according to a process according to claim 4.
8. The compounds of the general formula Ia and/or Ib according to
claim 1, characterized in, that the content of sulphate groups per
disaccharide unit is less than 0.005.
9. The compounds of the general formula Ia and/or Ib according to
claim 1, characterized in, that the content of free amino groups is
less than 1% with respect to all of the --NH--Y groups.
10. The compounds of the general formula Ia and/or Ib according to
claim 1, characterized in, that the sequence of the sugar units is
substantially alternating.
11. The compounds of the general formula Ia according to claim 1,
characterized in, that 45%-55% of all of the amino groups carry the
group Y and the remaining amino groups carry the group Z.
12. Use of the compounds of the general formula Ia and/or Ib for
the production of hemocompatible surfaces of medical devices.
13. Use according to claim 12, characterized in, that the the
compounds of the general formula Ia and/or Ib are bound covalently
to the surface.
14. Use of oligosaccharides and/or polysaccharides for the
hemocompatible coating of surfaces, characterized in, that of the
oligosaccharides and/or polysaccharides contain the sugar unit
N-acylglucosamine or N-acylgalactosamine between 40% and 60%, and
substantially the remaining sugar units have one carboxyl group per
sugar unit.
15. The use according to claim 14, characterized in that
substantially each second sugar unit of the oligosaccharides and/or
polysaccharides is N-acylglucosamine or N-acylgalactosamine.
16. The use according to claim 14, characterized in that in the
case of N-acylglucosamine N-acetylglucosamine and in the case of
N-acylgalactosamine N-acetylgalactosamine is concerned.
17. The use according to claim 14, characterized in that the
remaining sugar units are uronic acids.
18. The use according to claim 17, characterized in that the uronic
acids are substantially D-glucuronic acid and L-iduronic acid.
19. The use according to claim 12, characterized in that the
sequence of said sugar units is substantially alternating.
20. The use according to claim 14, characterized in that the
oligosaccharides and/or polysaccharides comprise substantially
desulphated and substantially N-acylated heparan sulphate as well
as partially N-carboxyalkylated and N-acylated chitosan.
21. Method for the hemocompatible coating of biological and/or
artificial surfaces of medical devices comprising the following
steps: a) providing a surface of a medical device and b) deposition
of at least one oligosaccharide and/or polysaccharide according to
claim 1 as hemocompatible layer on this surface and/or b')
deposition of a biostable layer on the surface of the medical
device or the hemocompatible layer.
22. Method according to claim 21, wherein the hemocompatible layer
or the biostable layer is coated via dipping or spraying method
with at least one biodegradable and/or biostabile layer which
comprises an active agent covalently and/or adhesively bound.
23. Method according to claim 21 comprising the further step c): c.
deposition of at least one active agent in and/or on the
hemocompatible layer or the biostable layer.
24. Method according to claim 23, wherein the at least one active
agent is implemented and/or deposited via dipping or spraying
methods on and/or in the hemocompatible layer or the biostable
layer and/or the at least one active agent is bound via covalent
and/or adhesive coupling to the hemocompatible layer or the
biostable layer.
25. Method according to claim 21 comprising the further step d): d)
deposition of at least one biodegradable layer and/or at least one
biostable layer on the hemocompatible layer or the active agent
layer, respectively, or d') deposition of at least one
oligosaccharide and/or polysaccharide according to one of the
compounds of general formula Ia and/or Ib as hemocompatible layer
on the biostable layer or the active agent layer, respectively.
26. Method according to claim 21 , comprising the further step e):
e) deposition of at least one active agent in and/or on the at
least one biodegradable and/or biostable layer or the
hemocompatible layer.
27. Method according to claim 26, wherein the at least one active
agent is deposited and/or implemented via dipping or spraying
methods on and/or in the at least one biodegradable and/or
biostable layer or the hemocompatible layer and/or the at least one
active agent is bound via covalent and/or adhesive coupling to the
at least one biodegradable and/or biostable layer or the
hemocompatible layer.
28. Method according to claim 25, wherein the biostable and/or
biodegradable layer is covalently and/or adhesively bound on the
surface of the medical device and the hemocompatible layer is
covalently bound to the biostable layer and covers it completely or
incompletely.
29. Method according to claim 21, characterized in, that the
hemocompatible layer comprises heparin of native origin of
regioselectively synthesized derivatives of different sulphation
coefficients and acylation coefficients in the molecular weight
range of the pentasaccharide, which is responsible for the
antithrombotic activity, up to the standard molecular weight of the
purchasable heparin of 13 kD, of heparansulphate and its
derivatives, oligo- and polysaccharides of the erythrocytic
glycocalix, desulphated and N-reac(et)ylated heparin,
N-carboxymethylated and/or partially N-ac(et)ylated chitosan as
well as mixtures of these substances.
30. Method according to claim 21, characterized in, that as
biodegradable substances for the biodegradable layer
polyvalerolactones, poly-.epsilon.-decalactones, polylactonic acid,
polyglycolic acid, polylactides, polyglycolides, copolymers of the
polylactides and polyglycolides, poly-.epsilon.-aprolactone,
polyhydroxybutanoic acid, polyhydroxybutyrates,
polyhydroxyvalerates, polyhydroxybutyrate-co-valera- tes,
poly(1,4-dioxane-2,3-diones), poly(1,3-dioxane-2-one),
poly-para-dioxanones, polyanhydrides as polymaleic anhydrides,
polyhydroxymethacrylates, fibrin, polycyanoacrylates,
polycaprolactonedimethylacrylates, poly-b-maleic acid,
polycaprolactonebutyl-acrylates, multiblock polymers such as from
oligocaprolactonedioles and oligodioxanonedioles, polyetherester
multiblock polymers such as PEG and poly(butyleneterephtalates),
polypivotolactones, polyglycolic acid trimethyl-carbonates,
polycaprolactone-glycolides, poly(g-ethylglutamate),
poly(DTH-iminocarbonate), poly(DTE-co-DT-carbonate),
poly(bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic
acid trimethyl-carbonates, polytrimethylcarbonates,
polyiminocarbonates, poly(N-vinyl)-pyrrolidone, polyvinylalcoholes,
polyesteramides, glycolated polyesters, polyphosphoesters,
polyphosphazenes, poly[p-carboxyphenoxy)propane],
polyhydroxypentane acid, polyanhydrides,
polyethyleneoxide-propyleneoxide, soft polyurethanes, polyurethanes
with amino acid rests in the backbone, polyetheresters such as
polyethyleneoxide, polyalkeneoxalates, polyorthoesters as well as
their copolymers, lipids, carrageenanes, fibrinogen, starch,
collagen, protein based polymers, polyamino acids, synthetic
polyamino acids, zein, modified zein, polyhydroxyalkanoates, pectic
acid, actinic acid, modified and non modified fibrin and casein,
carboxymethylsulphate, albumin, moreover hyaluronic acid, chitosane
and its derivatives, heparansulphates and its derivatives,
heparine, chondroitinesulphate, dextran, b-cyclodextrines,
copolymers with PEG and polypropyleneglycol, gummi arabicum, guar,
gelatine, collagen, collagen-N-Hydroxysuccinimide, lipids,
phospholipids, modifications and copolymers and/or mixtures of
these substances are used.
31. Method according to claim 21, characterized in, that as
biostable substances for the biostable layer polyacrylic acid and
polyacrylates such as polymethylmethacrylate,
polybutylmethacrylate, polyacrylamide, polyacrylonitriles,
polyamides, polyetheramides, polyethylenamine, polyimides,
polycarbonates, polycarbourethanes, polyvinylketones,
polyvinylhalogenides, polyvinylidenhalogenides, polyvinylethers,
polyisobutylenes, polyvinylaromates, polyvinylesters,
polyvinylpyrollidones, polyoxymethylenes, polytetramethyleneoxide,
polyethylene, polypropylene, polytetrafluoroethylene,
polyurethanes, polyetherurethanes, silicone-polyetherurethanes,
silicone-polyurethanes, silicone-polycarbonate-urethanes,
polyolefine elastomeres, polyisobutylenes, EPDM gums,
fluorosilicones, carboxymethylchitosanes,
polyaryletheretherketones, polyetheretherketones,
polyethylenterephthalat- e, polyvalerates, carboxymethylcellulose,
cellulose, rayon, rayontriacetates, cellulosenitrates,
celluloseacetates, hydroxyethylcellulose, cellulosebutyrates,
celluloseacetatebutyrates, ethylvinylacetate copolymers,
polysulphones, epoxy resins, ABS resins, EPDM gums, silicones such
as polysiloxanes, polydimethylsiloxanes, polyvinylhalogenes and
copolymers, celluloseethers, cellulosetriacetates, chitosanes and
copolymers and/or mixtures of these substances are used.
32. Method according to claim 21, characterized in, that the active
agents are chosen from the group which contains sirolimus
(rapamycin), everolimus, pimecrolimus, somatostatin, tacrolimus,
roxithromycin, dunaimycin, ascomycin, bafilomycin, erythromycin,
midecamycin, josamycin, concanamycin, clarithromycin,
troleandomycin, folimycin, cerivastatin, simvastatin, lovastatin,
fluvastatin, rosuvastatin, atorvastatin, pravastatin, pitavastatin,
vinblastine, vincristine, vindesine, vinorelbine, etoboside,
teniposide, nimustine, carmustine, lomustine, cyclophosphamide,
4-hydroxyoxycyclophosphamide, estramustine, melphalan, ifosfamide,
tropfosfamide, thymosin .alpha.-1, chlorambucil, bendamustine,
dacarbazine, busulfan, procarbazine, treosulfan, tremozolomide,
thiotepa, daunorubicin, doxorubicin, aclarubicin, epirubicin,
mitoxantrone, idarubicin, bleomycin, mitomycin, dactinomycin,
methotrexate, fludarabine, 2-methylthiazolidine-2,4-dicarboxylic
acid, tialin-Na (sodium salt of tialin),
fludarabine-5'-dihydrogenphosphate, cladribine, mercaptopurine,
thioguanine, cytarabine, fluorouracil, gemcitabine, capecitabine,
docetaxel, carboplatin, cisplatin, oxaliplatin, amsacrine,
irinotecan, topotecan, hydroxycarbamide, miltefosine, pentostatin,
aldesleukin, tretinoin, asparaginase, pegasparase, anastrozole,
exemestane, letrozole, formestane, aminoglutethemide, adriamycin,
azithromycin, spiramycin, cepharantin, dermicidin, smc
proliferation inhibitor-2w, epothilone A and B, mitoxantrone,
azathioprine, mycophenolatmofetil, c-myc-antisense,
b-myc-antisense, betulinic acid, camptothecin, PI-88 (sulphated
oligosaccharide), melanocyte stimulating hormon (a -MSH), activated
protein C, IL1-.beta. inhibitor, fumaric acid and its esters,
calcipotriol, tacalcitol, lapachol, .beta.-lapachone,
podophyllotoxin, betulin, podophyllic acid 2-ethylhydrazide,
molgramostim (rhuGM-CSF), peginterferon .alpha.-2b, lanograstim
(r-HuG-CSF), filgrastim, macrogol, dacarbazine, exemestan,
letrozol, goserelin, chephalomannin, basiliximab, trastuzumab,
daclizumab, selectin (cytokine antagonist), CETP inhibitor,
cadherines, cytokinin inhibitors, COX-2 inhibitor, NFkB,
angiopeptin, ciprofloxacin, camptothecin, fluroblastin, monoclonal
antibodies, which inhibit the muscle cell proliferation, bFGF
antagonists, probucol, prostaglandins, 1,11-dimethoxycanthin-6-one,
1-hydroxy-11-methoxycanthin-- 6-one, scopolectin, colchicine, NO
donors such as pentaerythritol tetranitrate and syndnoeimines,
S-nitrosoderivatives, tamoxifen, staurosporine, .beta.-estradiol,
.alpha.-estradiol, estriol, estrone, ethinylestradiol, fosfestrol,
medroxyprogesterone, estradiol cypionates, estradiol benzoates,
tranilast, kamebakaurin and other terpenoids, which are applied in
the therapy of cancer, verapamil, tyrosine kinase inhibitors
(tyrphostines), cyclosporine A, paclitaxel and derivatives thereof
such as 6-.alpha.-hydroxy-paclitaxel, baccatin, taxotere and
others, synthetically and from native sources obtained macrocyclic
oligomers of carbon suboxide (MCS) and derivatives thereof,
mofebutazone, acemetacin, diclofenac, lonazolac, dapsone,
o-carbamoylphenoxyacetic acid, lidocaine, ketoprofen, mefenamic
acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine,
hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol,
celecoxib, .beta.-sitosterin, ademetionine, myrtecaine,
polidocanol, nonivamide, levomenthol, benzocaine, aescin,
ellipticine, D-24851 (Calbiochem), colcemid, cytochalasin A-E,
indanocine, nocadazole, S 100 protein, bacitracin, vitronectin
receptor antagonists, azelastine, guanidyl cyclase stimulator
tissue inhibitor of metal proteinase-1 and -2, free nucleic acids,
nucleic acids incorporated into virus transmitters, DNA and RNA
fragments, plaminogen activator inhibitor-1, plasminogen activator
inhibitor-2, antisense oligonucleotides, VEGF inhibitors, IGF-1,
active agents from the group of antibiotics such as cefadroxil,
cefazolin, cefaclor, cefotixin, tobramycin, gentamycin, penicillins
such as dicloxacillin, oxacillin, sulfonamides, metronidazol,
antithrombotics such as argatroban, aspirin, abciximab, synthetic
antithrombin, bivalirudin, coumadin, enoxoparin, desulphated and
N-reacetylated heparin, tissue plasminogen activator, GpIIb/IIIa
platelet membrane receptor, factor X.alpha. inhibitor antibody,
heparin, hirudin, r-hirudin, PPACK, protamin, thialin-Na,
prourokinase, streptokinase, warfarin, urokinase, vasodilators such
as dipyramidole, trapidil, nitroprussides, PDGF antagonists such as
triazolopyrimidine and seramin, ACE inhibitors such as captopril,
cilazapril, lisinopril, enalapril, losartan, thioprotease
inhibitors, prostacyclin, vapiprost, interferon .alpha., .beta. and
.gamma., histamine antagonists, serotonin blockers, apoptosis
inhibitors, apoptosis regulators such as p65, NF-kB or Bcl-xL
antisense oligonucleotides, halofuginone, nifedipine, tocopherol,
vitamin B1,B2, B6 and B12, folic acid, tranirast, molsidomine, tea
polyphenols, epicatechin gallate, epigallocatechin gallate,
Boswellic acids and derivatives thereof, leflunomide, anakinra,
etanercept, sulfasalazine, etoposide, dicloxacillin, tetracycline,
triamcinolone, mutamycin, procainimid, retinoic acid, quinidine,
disopyrimide, flecainide, propafenone, sotolol, amidorone, natural
and synthetically obtained steroids such as bryophyllin A,
inotodiol, maquiroside A, ghalakinoside, mansonine, strebloside,
hydrocortisone, betamethasone, dexamethasone, non-steroidal
substances (NSAIDS) such as fenoporfen, ibuprofen, indomethacin,
naproxen, phenylbutazone and other antiviral agents such as
acyclovir, ganciclovir and zidovudine, antimycotics such as
clotrimazole, flucytosine, griseofulvin, ketoconazole, miconazole,
nystatin, terbinafine, antiprozoal agents such as chloroquine,
mefloquine, quinine, moreover natural terpenoids such as
hippocaesculin, barringtogenol-C21-angelate,
14-dehydroagrostistachin, agroskerin, agrostistachin,
17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic
acid, baccharinoids B1, B2, B3 and B7, tubeimoside, bruceanol A, B
and C, bruceantinoside C, yadanziosides N and P,
isodeoxyelephantopin, tomenphantopin A and B, coronarin A, B, C and
D, ursolic acid, hyptatic acid A, zeorin, iso-iridogermanal,
maytenfoliol, effusantin A, excisanin A and B, longikaurin B,
sculponeatin C, kamebaunin, leukamenin A and B,
13,18-dehydro-6-.alpha.-senecioyloxychapa- rrin, taxamairin A and
B, regenilol, triptolide, moreover cymarin, apocymarin,
aristolochic acid, anopterin, hydroxyanopterin, anemonin,
protoanemonin, berberine, cheliburin chloride, cictoxin,
sinococuline, bombrestatin A and B, cudraisoflavone A, curcumin,
dihydronitidine, nitidine chloride,
12-.beta.-hydroxypregnadien-3,20-dione, bilobol, ginkgol, ginkgolic
acid, helenalin, indicine, indicine-N-oxide, lasiocarpine,
inotodiol, glycoside la, podophyllotoxin, justicidin A and B,
larreatin, malloterin, mallotochromanol,
isobutyrylmallotochromanol, maquiroside A, marchantin A,
maytansine, lycoridicin, margetine, pancratistatin, liriodenine,
bisparthenolidine, oxoushinsunine, aristolactam-AII,
bisparthenolidine, periplocoside A, ghalakinoside, ursolic acid,
deoxypsorospermin, psycorubin, ricin A, sanguinarine, manwu wheat
acid, methylsorbifolin, melanocyte stimulating hormon (alpha-MSH),
sphatheliachromen, stizophyllin, mansonine, strebloside, akagerine,
dihydrousambaraensine, hydroxyusambarine, strychnopentamine,
strychnophylline, usambarine, usambarensine, berberine,
liriodenine, oxoushinsunine, daphnoretin, lariciresinol,
methoxylariciresinol, syringaresinol, umbelliferon, afromoson,
acetylvismione B, desacetylvismione A, vismione A and B.
33. Method according to claim 21 one of the claims 21,
characterized in, that the deposition or the immobilisation of the
oligosaccharides and/or polysaccharides according to one the
general formula Ia and/or Ib is achieved via hydrophobic
interactions, van der Waals forces, electrostatic interactions,
hydrogen bonds, ionic interactions, cross-linking and/or covalent
bonding.
34. Medical device available by one method according to claim
21.
35. Medical device, wherein the surface of the medical device is
coated directly and/or via at least one interjacent biostable
and/or biodegradable layer and/or active agent layer with a
hemocompatible layer comprising at least one oligosaccharide and/or
polysaccharide according to claim 1.
36. Medical device according to claim 35, wherein under the
hemocompatible layer or between two hemocompatible layers at least
one biostable and/or biodegradable layer is present.
37. Medical device according to claim 35, wherein the
hemocompatible layer is coated completely and/or incompletely with
at least another, suprajacent biostable and/or biodegradable
layer.
38. Medical device according to claim 36, wherein at least one
active agent layer is present between the biostable and/or
biodegradable layer and the hemocompatible layer, which comprises
at least one antiproliferative, antiinflammatory and/or
antithrombotic active agent covalently and/or adhesively bound.
39. Medical device according to claim 35, wherein at least one
antiproliferative, antiinflammatory and/or antithrombotic active
agent is bound covalently and/or adhesively in and/or on the
hemocompatible layer and/or the biostable and/or biodegradable
layer.
40. Medical device according to claim 35, characterized in, that
the used active agents are chosen from the group, which contains
sirolimus (rapamycin), everolimus, pimecrolimus, somatostatin,
tacrolimus, roxithromycin, dunaimycin, ascomycin, bafilomycin,
erythromycin, midecamycin, josamycin, concanamycin, clarithromycin,
troleandomycin, folimycin, cerivastatin, simvastatin, lovastatin,
fluvastatin, rosuvastatin, atorvastatin, pravastatin, pitavastatin,
vinblastine, vincristine, vindesine, vinorelbine, etoboside,
teniposide, nimustine, carmustine, lomustine, cyclophosphamide,
4-hydroxyoxycyclophosphamide, estramustine, melphalan, ifosfamide,
tropfosfamide, chlorambucil, bendamustine, dacarbazine, busulfan,
procarbazine, treosulfan, thymosin .alpha.-1, tremozolomide,
thiotepa, tialin (2-methylthiazolidine-2,4-dica- rboxylic acid),
tialin-Na (sodium salt of tialin), aunorubicin, doxorubicin,
aclarubicin, epirubicin, mitoxantrone, idarubicin, bleomycin,
mitomycin, dactinomycin, methotrexate, fludarabine,
fludarabine-5'-dihydrogenphosphate, cladribine, mercaptopurine,
thioguanine, cytarabine, fluorouracil, gemcitabine, capecitabine,
docetaxel, carboplatin, cisplatin, oxaliplatin, amsacrine,
irinotecan, topotecan, hydroxycarbamide, miltefosine, pentostatin,
aldesleukin, tretinoin, asparaginase, pegasparase, anastrozole,
exemestane, letrozole, formestane, aminoglutethemide, adriamycin,
azithromycin, spiramycin, cepharantin, smc proliferation
inhibitor-2w, epothilone A and B, mitoxantrone, azathioprine,
mycophenolatmofetil, c-myc-antisense, b-myc-antisense, betulinic
acid, camptothecin, PI-88 (sulphated oligosaccharide), melanocyte
stimulating hormon (.alpha.-MSH), activated protein C, IL1-.beta.
inhibitor, fumaric acid and its esters, dermicidin, calcipotriol,
tacalcitol, lapachol, .beta.-lapachone, podophyllotoxin, betulin,
podophyllic acid 2-ethylhydrazide, molgramostim (rhuGM-CSF),
peginterferon .alpha.-2b, lanograstim (r-HuG-CSF), filgrastim,
macrogol, dacarbazine, letrozol, goserelin, chephalomannin,
trastuzumab, exemestan, basiliximab, daclizumab, selectin (cytokine
antagonist), CETP inhibitor, cadherines, cytokinin inhibitors,
COX-2 inhibitor, NFkB, angiopeptin, ciprofloxacin, camptothecin,
fluroblastin, monoclonal antibodies, which inhibit the muscle cell
proliferation, bFGF antagonists, probucol, prostaglandins,
1,11-dimethoxycanthin-6-one, 1-hydroxy-11-methoxycanthin-- 6-one,
scopolectin, colchicine, NO donors such as pentaerythritol
tetranitrate and syndnoeimines, S-nitrosoderivatives, tamoxifen,
staurosporine, .beta.-estradiol, .alpha.-estradiol, estriol,
estrone, ethinylestradiol, fosfestrol, medroxyprogesterone,
estradiol cypionates, estradiol benzoates, tranilast, kamebakaurin
and other terpenoids, which are applied in the therapy of cancer,
verapamil, tyrosine kinase inhibitors (tyrphostines), cyclosporine
A, paclitaxel and derivatives thereof such as
6-.alpha.-hydroxy-paclitaxel, baccatin, taxotere and others,
synthetically and from native sources obtained macrocyclic
oligomers of carbon suboxide (MCS) and derivatives thereof,
mofebutazone, acemetacin, diclofenac, lonazolac, dapsone,
o-carbamoylphenoxyacetic acid, lidocaine, ketoprofen, mefenamic
acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine,
hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol,
celecoxib, .beta.-sitosterin, ademetionine, myrtecaine,
polidocanol, nonivamide, levomenthol, benzocaine, aescin,
ellipticine, D-24851 (Calbiochem), colcemid, cytochalasin A-E,
indanocine, nocadazole, S 100 protein, bacitracin, vitronectin
receptor antagonists, azelastine, guanidyl cyclase stimulator
tissue inhibitor of metal proteinase-1 and -2, free nucleic acids,
nucleic acids incorporated into virus transmitters, DNA and RNA
fragments, plaminogen activator inhibitor-1, plasminogen activator
inhibitor-2, antisense oligonucleotides, VEGF inhibitors, IGF-1,
active agents from the group of antibiotics such as cefadroxil,
cefazolin, cefaclor, cefotixin, tobramycin, gentamycin, penicillins
such as dicloxacillin, oxacillin, sulfonamides, metronidazol,
antithrombotics such as argatroban, aspirin, abciximab, synthetic
antithrombin, bivalirudin, coumadin, enoxoparin, desulphated and
N-reacetylated heparin, tissue plasminogen activator, GpIIb/IIIa
platelet membrane receptor, factor X.sub.a inhibitor antibody,
heparin, hirudin, r-hirudin, PPACK, protamin, prourokinase,
streptokinase, warfarin, urokinase, vasodilators such as
dipyramidole, trapidil, nitroprussides, PDGF antagonists such as
triazolopyrimidine and seramin, ACE inhibitors such as captopril,
cilazapril, lisinopril, enalapril, losartan, thioprotease
inhibitors, prostacyclin, vapiprost, interferon a, .beta. and
.gamma., histamine antagonists, serotonin blockers, apoptosis
inhibitors, apoptosis regulators such as p65, NF-kB or Bcl-xL
antisense oligonucleotides, halofuginone, nifedipine, tocopherol,
tranirast, molsidomine, tea polyphenols, epicatechin gallate,
epigallocatechin gallate, Boswellic acids and derivatives thereof,
leflunomide, anakinra, etanercept, sulfasalazine, etoposide,
dicloxacillin, tetracycline, triamcinolone, mutamycin, procainimid,
retinoic acid, quinidine, disopyrimide, flecainide, propafenone,
sotolol, amidorone, natural and synthetically obtained steroids
such as bryophyllin A, inotodiol, maquiroside A, ghalakinoside,
mansonine, strebloside, hydrocortisone, betamethasone,
dexamethasone, non-steroidal substances (NSAIDS) such as
fenoporfen, ibuprofen, indomethacin, naproxen, phenylbutazone and
other antiviral agents such as acyclovir, ganciclovir and
zidovudine, antimycotics such as clotrimazole, flucytosine,
griseofulvin, ketoconazole, miconazole, nystatin, terbinafine,
antiprozoal agents such as chloroquine, mefloquine, quinine,
moreover natural terpenoids such as hippocaesculin,
barringtogenol-C21-angelate, 14-dehydroagrostistachin, agroskerin,
agrostistachin, 17-hydroxyagrostistachin, ovatodiolids,
4,7-oxycycloanisomelic acid, baccharinoids B1, B2, B3 and B7,
tubeimoside, bruceanol A, B and C, bruceantinoside C, yadanziosides
N and P, isodeoxyelephantopin, tomenphantopin A and B, coronarin A,
B, C and D, ursolic acid, hyptatic acid A, zeorin,
iso-iridogermanal, maytenfoliol, effusantin A, excisanin A and B,
longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B,
13,18-dehydro-6-.alpha.-senecioyloxychapa- rrin, taxamairin A and
B, regenilol, triptolide, moreover cymarin, apocymarin,
aristolochic acid, anopterin, hydroxyanopterin, anemonin,
protoanemonin, berberine, cheliburin chloride, cictoxin,
sinococuline, bombrestatin A and B, cudraisoflavone A, curcumin,
dihydronitidine, nitidine chloride,
12-.beta.-hydroxypregnadien-3,20-dione, bilobol, ginkgol, ginkgolic
acid, helenalin, indicine, indicine-N-oxide, lasiocarpine,
inotodiol, glycoside 1a, podophyllotoxin, justicidin A and B,
larreatin, malloterin, mallotochromanol,
isobutyrylmallotochromanol, maquiroside A, marchantin A,
maytansine, lycoridicin, margetine, pancratistatin, liriodenine,
bisparthenolidine, oxoushinsunine, aristolactam-AII,
bisparthenolidine, periplocoside A, ghalakinoside, ursolic acid,
deoxypsorospermin, psycorubin, ricin A, sanguinarine, manwu wheat
acid, methylsorbifolin, sphatheliachromen, stizophyllin, mansonine,
strebloside, akagerine, dihydrousambaraensine, hydroxyusambarine,
strychnopentamine, strychnophylline, usambarine, usambarensine,
berberine, liriodenine, oxoushinsunine, daphnoretin, lariciresinol,
methoxylariciresinol, syringaresinol, umbelliferon, afromoson,
acetylvismione B, desacetylvismione A, vismione A and B.
41. Medical device according to claim 40, characterized in, that in
the case of the used active agents tacrolimus, pimecrolimus, PI 88,
thymosin .alpha.-1, PETN, baccatine and its derivatives, docetaxel,
colchicin, paclitaxel and its derivatives, trapidil, .alpha.- and
.beta.-estradiol, dermicidin, tialin-sodium, simvastatin,
macrocyclic suboxide (MCS) and its derivatives, sirolimus,
tyrphostine, D24851, colchicin, fumaric acid and its esters,
activated protein C (aPC), interleukin 1.beta. inhibitors and
melanocyte stimulating hormon (.alpha.-MSH) as well as mixtures of
these active agents are concerned.
42. Medical device accordig to claim 34, characterized in, that the
medical device comprises prostheses, organs, vessels, aortas, heart
valves, tubes, organ spare parts, implants, fibers, hollow fibers,
stents, hollow needles, syringes, membranes, tinned goods, blood
containers, titrimetric plates, pacemakers, adsorbing media,
chromatography media, chromatography columns, dialyzers, connexion
parts, sensors, valves, centrifugal chambers, recuperators,
endoscopes, filters, pump chambers.
43. Medical device according to claim 42, characterised in, that
the medical device is a stent.
44. Stents according to claim 43, wherein the polymer is deposited
in amounts between 0.01 mg to 3 mg/layer, preferred between 0.20 mg
to 1 mg and especially preferred between 0.2 mg to 0.5
mg/layer.
45. Stent according to claim 43, characterized in, that the
antiproliferative, antiinflammatory and/or antithrombotic active
agent is comprised in a pharmaceutically active concentration of
0.001-10 mg per cm.sup.2 stent surface.
46. Use of the stent according to claim 43 for the prevention or
reduction of restenosis.
47. Use of the stent according to claim 1 for continuous release of
at least an antiproliferative, antiinflammatory and/or
antithrombotic active agent.
48. Use of the medical devices according to claim 34 for the direct
contact with blood.
49. Use of the medical devices according to claim 34 for prevention
or reduction of the adhesion of proteins on the coated surfaces of
the medical devices.
50. The use according to claim 48, characterized in that the
hemocompatibly coated surface of microtiter plates or other carrier
media used for diagnostic detection methods prevents or reduces the
unspecific deposition of proteins.
51. The use according to claim 48, characterized in that the
hemocompatibly coated surface of adsorber media or chromatography
media prevents or reduces the unspecific deposition of proteins.
Description
[0001] The invention relates to the use of oligo- and/or
polysaccharides containing the sugar building block
N-acylglucosamine and/or N-acylgalactosamine for the preparation of
hemocompatible surfaces, methods for the hemocompatible coating of
surfaces with said oligo- and/or polysaccharides as well as the use
of the hemocompatibly coated surfaces.
[0002] In the human body the blood gets in contact with surfaces
other than the internal face of natural blood vessels only in case
of an injury. Consequently the blood coagulation system is always
activated to reduce the bleeding and to prevent a life-threatening
loss of blood if blood gets in contact with foreign surfaces. Due
to the fact that an implant also represents a foreign surface all
patients receiving an implant which is permanently in contact with
blood are treated for the duration of the blood contact with drugs,
with so called anticoagulants which suppress the blood coagulation.
This is also true for patients applicated an extracorporeal
circulation such as hemodialysis patients. However this coagulation
suppressive medication is in some extent afflicted with
considerable side effects which range from loss of hair, nausea and
vomitus beyond thrombocytopenia, hemorrhagic skin necroses and
increased hemorrhagic diathesis up to side effects with a fatal
outcome such as cerebral hemorrhages.
[0003] Thus there is a demand for non-thrombogenic, hemocompatible
materials such as protheses, organ spareparts, membranes, cannulae,
tubes, blood containers, stents etc. which do not activate the
coagulation system in case of blood contact and do not cause
coagulation of the blood.
[0004] EP-B-0 333 730 describes a method for preparation of
hemocompatible substrates by incorporation, adhesion and/or
modification and attachment of non-thrombogenic endothelial cell
surface polysaccharide (HS-I). The immobilization of this specific
endothelial cell surface proteoheparan sulphate HS I on biological
or artificial surfaces causes that suchlike coated surfaces become
blood compatible and suitable for the permanent blood contact.
However, it is disadvantageous that said process for the generation
of HS I requires the cultivation of endothelial cells, so that the
economical usability of said process is very limited because the
cultivation of endothelial cells is time consuming, and relatively
large amounts of cultivated endothelial cells are only available at
a considerably high cost.
[0005] It is the object of the present invention to provide
substances for the hemocompatible coating of surfaces as well as
methods for hemocompatible coating of surfaces and their use on
surfaces for the prevention or reduction of undesired
reactions.
[0006] Particularly it is the object of the present invention to
provide medical products, which allow a continuous controlled
ingrowth of the medical product--on the one hand by suppression of
the cellular reactions during the first days and weeks after the
implantation by means of the chosen active agents and active agent
combinations and on the other hand by providing an atrombogeneous
resp. inert resp. biocompatible surface, which guarantees, that
with decreasing of the active agent influence no reactions on the
present alien surface occur anymore, which also can lead to
complications on the long-term.
[0007] This object is solved by the technical teaching of the
independent claims of the present invention. Further advantageous
embodiments of the invention are evident from the dependent claims,
the description, the figures and the examples.
[0008] The present invention discloses polysaccharides of the
general formula Ia 1
[0009] as well as structurally very similar polysaccharides of the
general formula Ib 2
[0010] The polysaccharides according to formula Ia have molecular
weights from 2 kD to 400 kD, preferably from 5 kD to 150 kD, more
preferably from 10 kD to 100 kD, and particularly preferably from
30 kD to 80 kD. The polysaccharides according to formula Ib have
molecular weights from 2 kD to 15 kD, preferably from 4 kD to 13
kD, more preferably from 6 kD to 12 kD, and particularly preferably
from 8 kD to 11 kD. The variable n is an integer ranging from 4 to
1050. Preferably, n is an integer from 9 to 400, more preferably
from 14 to 260, and particularly preferably an integer between 19
and 210.
[0011] The general formulas Ia and Ib represent a disaccharide,
which is to be seen as a basic unit of the polysaccharide according
to invention and forms the polysaccharide by stringing together
said basic unit n times. Said basic unit comprising two sugar
molecules does not intend to suggest that the general formulas Ia
and Ib only relate to polysaccharides having an even number of
sugar molecules. Of course, the general formula Ia and the formula
Ib also comprise polysaccharides having an odd number of sugar
units. Hydroxy groups are present as terminal groups of the
oligosaccharides and polysaccharides, respectively.
[0012] The groups Y and Z, independently of each other, represent
the following chemical acyl or carboxyalkyl groups: --CHO,
--COCH.sub.3, --COC.sub.2H.sub.5, --COC.sub.3H.sub.7,
--COC.sub.4H.sub.9, --COC.sub.5H.sub.11, --COCH(CH.sub.3).sub.2,
--COCH.sub.2CH(CH.sub.3).sub- .2, --COCH(CH.sub.3)C.sub.2H.sub.5,
--COC(CH.sub.3).sub.3, --CH.sub.2COO.sup.-,
--C.sub.2H.sub.4COO.sup.-, --C.sub.3H.sub.6COO.sup.-- ,
--C.sub.4H.sub.8COO.sup.--.
[0013] Preferred are the acyl groups --COCH.sub.3,
--COC.sub.2H.sub.5, --COC.sub.3H.sub.7 and the carboxyalkyl groups
--CH.sub.2COO.sup.-, --C.sub.2H.sub.4COO.sup.-,
--C.sub.3H.sub.6COO.sup.-. More preferred are the acetyl and
propanoyl groups and the carboxymethyl and carboxyethyl groups.
Particularly preferred are the acetyl group and the carboxymethyl
group.
[0014] In addition, it is preferred that the group Y represents an
acyl group, and the group Z represents a carboxyalkyl group. It is
more preferred if Y is a group --COCH.sub.3, --COC.sub.2H.sub.5 or
--COC.sub.3H.sub.7 and in particular --COCH.sub.3. Moreover, it is
further preferred if Z is a carboxyethyl or carboxymethyl group,
the carboxymethyl group being particularly preferred.
[0015] The disaccharide basic unit shown by formula Ia comprises
each a substituent Y and a further group Z. This is to make clear
that the polysaccharide of the invention comprises two different
groups, namely Y and Z. It is important to point out here that the
general formula Ia should not only comprise polysaccharides
containing the groups Y and Z in a strictly alternating sequence,
which would result from stringing together the disaccharide basic
units, but also polysaccharides carrying the groups Y and Z in a
completely random sequence at the amino groups. Further, the
general formula Ia should also comprise polysaccharides containing
the groups Y and Z in different numbers. The ratios of the number
of Y groups to the number of X groups can be between 70%:30%,
preferably between 60%:40%, and particularly preferably between
45%:55%. Especially preferred are polysaccharides of the general
formula Ia carrying on substantially half of the amino groups the Y
residue and on the other half of the amino groups the Z residue in
a merely random distribution. The term "substantially half" means
exactly 50% in the most suitable case but should also comprise the
range from 45% to 55% and especially from 48% to 52% as well.
[0016] Preferred are the compounds of the general formula Ia,
wherein the groups Y and Z have the following meanings:
1 Y = --CHO and Z = --C.sub.2H.sub.4COO.sup.- Y = --CHO and Z =
--CH.sub.2COO.sup.- Y = --COCH.sub.3 and Z =
--C.sub.2H.sub.4COO.sup.- Y = --COCH.sub.3 and Z =
--CH.sub.2COO.sup.- Y = --COC.sub.2H.sub.5 and Z =
--C.sub.2H.sub.4COO.sup.- Y = --COC.sub.2H.sub.5 and Z =
--CH.sub.2COO.sup.-
[0017] Especially preferred are the compounds of the general
formula Ia, wherein the groups Y and Z have the following
meanings:
2 Y = --CHO and Z = --C.sub.2H.sub.4COO.sup.- Y = --COCH.sub.3 and
Z = --CH.sub.2COO.sup.-
[0018] Especially preferred are the compounds of the general
formula Ib, wherein Y is one of the following groups: --CHO,
--COCH.sub.3, --COC.sub.2H.sub.5 or --COC.sub.3H.sub.7. Further
preferred are the groups --CHO, --COCH.sub.3, --COC.sub.2H.sub.5
and especially preferred is the group --COCH.sub.3.
[0019] The compounds of the general formula Ib contain only a minor
amount of free amino groups. As with the ninhydrine test free amino
groups could not be detected anymore, it can be concluded due to
the sensitivity of this test, that less than 2%, preferred less
than 1% and especially preferred less than 0.5% of all of the
--NH--Y groups are present as free amino groups, i.e. at this low
percentage of the groups --NH--Y that Y represents hydrogen.
[0020] As the polysaccharides of the general formula Ia and Ib
contain carboxylate groups and amino groups, the general formulas
Ia and Ib also comprise alkali and alkaline earth metal salts of
the respective polysaccharides. Thus, alkali metal salts such as
the sodium salt, potassium salt, lithium salt or alkaline earth
metal salts such as the magnesium salt or calcium salt can be
mentioned. Further, with ammonia, primary, secondary, tertiary and
quaternary amines, pyridine and pyridine derivatives, ammonium
salts, preferably alkyl ammonium salts and pyridinium salts, can be
formed. The bases forming salts with the polysaccharides include
inorganic and organic bases such as NaOH, KOH, LiOH, CaCO.sub.3,
Fe(OH).sub.3, NH.sub.4OH, tetraalkyl ammonium hydroxides and
similar compounds.
[0021] Heparan sulphates are ubiquitous on cell surfaces of
mammals. Depending on the cell type, they are very different with
respect to molecular weight, degree of acetylation and degree of
sulfation. Liver heparan sulphate, for example, has a degree of
acetylation of approximately 50%, whereas the heparan sulphate from
the glycocalyx of endothelial cells can show a degree of
acetylation of up to 90% and more. Heparin only shows a very small
degree of acetylation of up to 5%. The degree of sulfation of liver
heparan sulphate and heparin is .about.2 per disaccharide unit, of
endothelial cell heparan sulphate nearly 0, and of heparan
sulphates from other cell types between 0 and 2 per disaccharide
unit.
[0022] The following illustration shows a tetrasaccharide unit of a
heparin or heparan sulphate with a random distribution of the
sulphate groups and a degree of sulfation of 2 per disaccharide
unit as typical for heparin: 3
[0023] All heparan sulphates have the process of biosynthesis in
common with heparin. In this case, first, the core protein with the
xylose-containing bond region is built up. It consists of the
xylose and two galactose residues connected therewith. To the last
one of the two galactose residues, glucuronic acid and
galactosamine are then alternately bonded to each other until the
respective chain length is achieved. Finally, a multistage
enzymatic modification of the common precursor polysaccharide of
all heparan sulphates and the heparin by sulfotransferases and
epimerases is effected, which, by means of their reactions of
varying completeness generate the broad spectrum of various heparan
sulphates up to heparin.
[0024] Heparin is built up alternately from D-glucosamine and
D-glucuronic acid resp. L-iduronic acid, wherein D-glucosamine and
D-glucuronic acid are linked in a .beta.-1,4-glycosidic manner
(resp. L-iduronic acid in an .alpha.-1,4-glycosidic manner) to the
disaccharide, which forms the heparin subunits. These subunits, in
turn, are linked to each other in a .beta.-1,4-glycosidic manner
and lead to the heparin. The position of the sulfonyl groups can
change. A tetrasaccharide unit contains an average of 4 to 5
sulfuric acid groups. Heparan sulphate, also referred to as
heparitin sulphate, contains, with the exception of liver heparan
sulphate, less N-- and O-bonded sulfonyl groups than heparin, but
more N-acetyl groups.
[0025] As evident from FIG. 3, the compounds of the general formula
Ia (see FIG. 3c as example) and the compounds of the general
formula Ib (see FIG. 3b as example) are structurally very similar
to the natural heparan sulphate of endothelial cells, but prevent
the initially described disadvantages in using endothelial cell
heparan sulphates.
[0026] For the antithrombotic activity a special pentasaccharide
unit is made responsible which can be found in commercial heparin
preparatives in about every 3rd molecule. Heparin preparations of
different antithrombotic activity can be produced by special
separation techniques. In highly active, for example by
antithrombin-III-affinitychromatography obtained preparations
("High-affinity"-heparin) this active sequence is found in every
heparin molecule, while in "No-affinity"-preparations no
characteristical pentasaccharide sequences and thus no active
inhibition of coagulation can be detected. Via interaction with
this pentasaccharide the activity of antithrombin III, an inhibitor
of the coagulation key factor thrombin, is essentially
exponentiated (bonding affinity increase up to the factor
2.times.10.sup.3) [Stiekema J. C. J.; Clin Nephrology 26, Suppl.
No. 1, S3-S8, (1986)].
[0027] The amino groups of the heparin are mostly N-sulphated or
N-acetylated. The most important O-sulphation positions are the C2
in the iduronic acid as well as the C6 and the C3 in the
glucosamine. For the activity of the pentasaccharide onto the
plasmatic coagulation basically the sulphate group on C6 is made
responsible, in smaller proportion also the other functional
groups.
[0028] Surfaces of medicinal implants coated with heparin or
heparansulphates are and remain only conditionally hemocompatible
by the coating. The heparin or heparansulphate which is added onto
the artificial surface loses partially in a drastic measure its
antithrombotic activity which is related to a restricted
interaction due to steric hindrence of the mentioned
pentasaccharide units with antithrombin III.
[0029] Because of the immobilisation of these polyanionic
substances a strong adsorption of plasma protein on the heparinated
surface is observed in all cases what eliminates on the one hand
the coagulation suppressing effect of heparin resp. of
heparansulphates and initialises on the other hand specific
coagulation processes by adherent and hereby tertiary structure
changing plasma proteins (e.g. albumin, fibrinogen, thrombin) and
thereon adherent platelets.
[0030] Thus a correlation exists on the one hand between the
limited interaction of the pentasaccharide units with antithrombin
III by immobilisation on the other hand depositions of plasma
proteins on the heparin-resp. heparansulphate layer on the
medicinal implant take place, which leads to the losses of the
antithrombotic properties of the coating and which can even turn
into the opposite, because the plasma protein adsorption that
occurs during a couple of seconds leads to the loss of the
anticoagulating surface and the adherent plasma proteins change
their tertiary structure, whereby a thrombonegeous surface arises.
Surprisingly it could be detected that the compounds of the general
formulas Ia and Ib, despite of the structural differences to the
heparin resp. heparansulphate, still show the hemocompatible
properties of heparin and additionally during the immobilisation of
the compounds of the general formulas Ia and Ib no noteworthy
depositions of plasma proteins which represent an initial step in
the activation of the coagulation cascade could be observed. The
hemocopatible properties of the compounds according to invention
still remain also after their immobilisation on artificial
surfaces.
[0031] Further on it is supposed that the sulphate groups of the
heparin resp. the heparansulphates are necessary for the
interaction with antithrombin III and impart thereby the heparin
resp. the heparansulphate the anticoagulatory effect. The inventive
compounds according to formula Ib as well as the compounds
according to formula Ia are not actively coagulation suppressive,
i.e. anticoagulative, due to an almost complete desulphation the
sulphate groups of the compounds of the general formulas Ib are
removed up to a low amount of below 0.2 sulphate groups per
disaccharide unit.
[0032] The compounds of the invention according to the general
formula Ib can be made from heparin or heparan sulphates by first
substantially entirely desulfating and then substantially entirely
N-acylating the polysaccharide. The term "substantially entirely
desulphated" refers to a degree of desulfation of more than 90%,
preferred more than 95%, and particularly preferred more than 98%.
The degree of desulfation can be determined according the so-called
ninhydrine test, which detects free amino groups. Desulfation is
effected to such an extent that a color reaction is no longer
obtained with DMMB (dimethylmethylene blue). This color test is
suitable for detecting sulphated polysaccharides; however, the
detection limit thereof is not known in the literature of the art.
The desulfation can, for example, be carried out by heating the
pyridinium salt in a solvent mixture. In particular, a mixture of
DMSO, 1,4-dioxane and methanol proved to be suitable.
[0033] Heparansulphates as well as heparin were desulphated via
total hydrolysis and subsequently reacylated. Thereafter the number
of sulphate groups per disaccharide unit (S/D) was determined by
13C-NMR. The following table 1 shows these results on the example
of heparin and desulphated, reacetylated heparin (Ac-heparin).
3TABLE 1 Distribution of functional groups per disaccharide unit on
the example of heparin and Ac-heparin as determined by
13C-NMR-measurements. 2-S 6-S 3-S NS N--Ac NH.sub.2 S/D Heparin
0.63 0.88 0.05 0.90 0.08 0.02 2.47 Ac-heparin 0.03 0 0 0 1.00 --
0.03 2-S, 3-S, 6-S: sulphate groups in position 2, 3, 6
respectively NS: sulphate groups on the amino groups N--Ac: acetyl
groups on the amino groups NH.sub.2: free amino groups S/D:
sulphate groups per disaccharide unit
[0034] A sulphate content of about 0.03 sulphate
groups/disaccharide unit (S/D) in case of Ac-heparin in comparison
with about 2.5 sulphate groups/disaccharide unit in case of heparin
was reproducibly obtained.
[0035] As described above the difference in the sulphate contents
of heparin resp. heparansulphates and the compounds of the general
formulas has a considerable influence on the activity adverse to
antithrombin III and the coagulatory effects of these
compounds.
[0036] The compounds of the general formulas Ia and Ib have a
content of sulphate groups per disaccharide unit of less than 0.2,
preferred less than 0.07, more preferred less than 0.05 and
especially preferred less than 0.03 sulphate groups per
disaccharide unit.
[0037] By the removal of the sulphate groups of heparin, to which
the active coagulation suppressive working mechanism is accredited
to, one receives for a surface refinement suitable hemocompatible,
coagulation inert oligo-resp. polysaccharide which on the one hand
has no active role in the coagulation process and which on the
other hand is not detected by the coagulation system as foreign
surface. This coating imitates successfully the nature given
highest standard of hemocompatibility and passivity against the
coagulation active components of the blood.
[0038] The examples 5 and 6 clarify, that surfaces, which are
coated with the compounds according to invention according to the
general formulas Ia and/or Ib, especially which are coated
covalently, result in a passivative, athrombogeneous,
hemocompatible, antiproliferative and/or antiinflammatory coating.
This is clearly proven by the example of the Ac-heparin.
[0039] The term "substantially entirely N-acylated" refers to an
N-acylating degree of more than 94%, preferably more than 97%, and
particularly preferably more than 98%. The acylation is effected in
such a complete manner that the ninhydrine detection of free amino
groups does no longer show any color reaction. As acylation agents,
carboxylic acid chlorides, bromides or anhydrides are used
preferably. Acetic acid anhydride, propionic acid anhydride,
butyric acid anhydride, acetic acid chloride, propionic acid
chloride or butyric acid chloride, for example, are suitable for
preparing the compounds according to the invention. Carboxylic acid
anhydrides are particularly suitable as acylation agents.
[0040] As the solvent, in particular for the carboxylic acid
anhydrides, deionized water is used, preferably together with a
cosolvent, which is added in an amount of 10 to 30 volume percent.
As cosolvents, methanol, ethanol, DMSO, DMF, acetone, dioxane, THF,
acetic acid ethyl ester and other polar solvents are suitable. If
carboxylic acid halides are used, polar water-free solvents such as
DMSO or DMF are preferably used.
[0041] As the solvent deionized water is used, preferably together
with a cosolvent, which is added in an amount of 10 to 30 volume
percent. As cosolvents, methanol, ethanol, DMSO, DMF, acetone,
dioxane, THF, acetic acid ethyl ester and other polar solvents are
suitable.
[0042] The compounds of the invention according to the general
formula Ia have a carboxylate group on half of the sugar molecules,
and a N-acyl group on the other half.
[0043] Such compounds can also be made from the polysaccharides
hyaluronic acid, dermatan sulphate, chondroitin sulphate.
Differencies to heparin and heparansulphate result from the
connection of the monosaccharides, which are here not present in a
1,4-glycosidic but 1,3-glycosidic connection. The disaccharides are
again connected to each other 1,4-glycosidically. In the case of
the also in the blood coagulation antithrombotically active
dermatan sulphates [Biochem. J 289, 313-330 (1993)] and the
chrondoitin sulphates N-acetylglucosamine is substituted by
N-acetylgalactosamine, which differ in the steric position of the
hydroxyl group at the C-atom 4.
[0044] Due to their related structure the polysaccharides are
assigned according to the present differencies as follows:
[0045] Type 1) Uronic Acid--Galactosamine Type (HexA-GaIN)n:
[0046] Hereto accounted are chondroitin sulphate and dermatan
sulphate. Typical for this group ist the .beta.-1,3-glycosidic
bonding of the uronic acid to the galactosamine. Galactosamine is
bound on its part .beta.-1,4-glycosidically to the next uronic
acid. Dermatan sulphate differs from chondroitin sulphate by a high
amount of another also in heparin and heparan sulphate occurring
uronic acid, the L-iduronic acid. The sulphation degree of
chondroitin sulphate is at 0.1 to 1.3 sulphate groups per
disaccharide. Dermatan sulphate has with 1.0 to 3.0 sulphate groups
per disaccharide an averagely higher sulphation degree as
chondroitin sulphate and thereby reaches heparin like values. The
amino groups are N-acetylated.
[0047] The desulphation and N-reacetylation leads here as in the
case of heparin and heparan sulphate to compounds, which are also
suitable for the use as athrombogeneous coating.
[0048] Type 2) Uronic Acid--Glucosamine Type (HexA-GIcN)n:
[0049] Hereto accounted are heparin, heparan sulphate and
hyaluronan. Heparin and heparan sulphate are solely
.beta.-1,4-bound, whilst in hyaluronan, which is also accounted to
this type, the monosaccharids D-glucuronic acid and D-glucosamine
are .beta.-1,3-bound monosaccharids. This polysaccharide has as
only polysaccharide no sulphate groups and is N-acetylated. The
molecular weight reaches in comparison to heparin and heparan
sulphate maximum values up to 8000 kD. The reduction of the chain
length and the maintaining of the acetyl groups resp. the
N-reacetylation leads to a structure, which is distinguishable from
the formula Ib only by the .beta.-1,3-glycosidic connection of the
monosaccharids.
[0050] Further compounds can be prepared also from chitin or
chitosan.
[0051] Chitin is a nitrogen-containing polysaccharide, the monomer
units of which consist of N-acetyl-D-glucosamine, which are linked
in a .beta.-1,4-glycosidic manner. This results in linear polymers
consisting of about 2,000 sugar units and having a molecular weight
of about 400,000 g/mol. Chitin shows a very poor solubility and is
almost insoluble in water, organic solvents and dilute acids or
dilute bases. Mixing with strong acids leads to hydrolysis, where
D-glucosamine and acetic acid are produced. The treatment with
strong bases, however, leads to chitosan and acetate.
[0052] Chitosan can easily be produced by the saponification of
chitin. Chitosan consists of .beta.-1,4-glycosidically linked
glucosamine (2-amino-2-deoxy-D-glucose). Chitosan is known for its
film forming properties, and is further used as a basic material
for ion exchangers and as an agent for reducing the cholesterol
level in the blood serum and for weight reduction.
[0053] The substances according to the invention of the general
formula Ia can be made from chitin by partially deacetylating
chitin by means of strong bases and then monocarboxyalkylating the
free amino groups (see FIG. 1). The deacetylation degree, i.e. the
amount of demasked primary amino groups, can be determined
volumetrically. The quantitative detection of the free amino groups
is effected by means of the ninhydrine reaction. Depending on the
way the experiment is carried out, deacetylation degrees of 20 to
80% can be obtained. Deacetylation degrees of 40 to 60% are
preferred, 45 to 55% are particularly preferred.
[0054] By this way of synthesis, polysaccharides can be obtained
the sugar units of which contain either an N-acetyl group or an
N-carboxyalkyl group in a merely random distribution.
[0055] Chitosan, which is easily accessible by the basic hydrolysis
of the N-acetyl groups of the chitin (see FIG. 1), equally serves
as a starting material for the synthesis of the polysaccharides
according to formula Ia.
[0056] Chitosan only has very few N-acetyl groups. Thus, the
compounds according to the invention can, on the one hand, be
obtained by carboxyalkyating substantially the half of the free
amino groups in a first step, and then acylating the remaining free
amino groups, or by first carrying out the acylation and then
reacting the remaining free amino groups with a suitable
carboxyalkylation agent. It is preferred if substantially the half
of the amino groups is acylated and the remaining half is
carboxyalkylated.
[0057] "Partially N-acylated chitosan" refers to an N-acylation
degree of 30-70%, preferably of 40-60%, and particularly preferably
of 45-55%. Particularly preferred are chitosan derivates carrying
the Y residue on substantially the half of the amino groups, and on
the other half of the amino groups the Z residue in a merely random
distribution. The term "substantially the half" means exactly 50%
in the most suitable case, but should also include the range of 45%
to 55%. The carboxyalkylation and acylation degrees can be
determined by means of 13C-NMR, for example (deviation tolerance
.+-.3%).
[0058] Due to the fact that in a first reaction step a certain
number of the free amino groups are acylated or carboxyalkylated,
this inevitably results in a completely random distribution of the
acyl groups resp. carboxyalkyl groups in the polysaccharide of the
general formula Ia. The formula Ia thus is only intended to show a
disaccharide unit of the polysaccharides according to the
invention, but not determine an alternating sequence of the acyl
groups and carboxyalkyl groups.
[0059] The following illustration shows a typical tetrasaccharide
unit of an N-carboxymethylated, N-acetylated chitosan: 4
[0060] The present invention describes the use of the compounds of
the general formulas Ia and/or Ib as well as salts of said
compounds for the coating, in particular a hemocompatible coating
of natural and/or artificial surfaces. "Hemocompatible" refers to
the property of the compounds according to the invention, which
means not to interact with the substances of the blood coagulation
system or the blood platelets and thus not to trigger the blood
coagulation cascade.
[0061] In addition, the invention discloses oligosaccharides and/or
polysaccharides for the hemocompatible coating of surfaces.
Preferred are polysaccharides within the molecular weight limits
mentioned above. One of the remarkable features of the
oligosaccharides and/or polysaccharides used is, that they contain
large amounts of the sugar unit N-acylglucosamine or
N-acylgalactosamine. This means that 40-60%, preferred 45-55% and
especially preferred 48-52% of the sugar units are
N-acylglucosamine or N-acylgalactosamine, and substantially the
remaining sugar units each have a carboxyl group. Thus, usually
more than 95%, preferably more than 98%, of the oligosaccharides
and/or polysaccharides consist of only two sugar units, one sugar
unit carrying a carboxyl group and the other one an N-acyl
group.
[0062] One sugar unit of the oligosaccharides and/or
polysaccharides is N-acylglucosamine resp. N-acylgalactosamine,
preferably N-acetylglucosamine resp. N-acetylgalactosamine, and the
other one is uronic acid, preferably glucuronic acid and iduronic
acid.
[0063] Preferred are oligosaccharides and/or polysaccharides
substantially consisting of the sugar glucosamine resp.
galactosamine, substantially the half of the sugar units carrying
an N-acyl group, preferably an N-acetyl group, and the other half
of the glucosamine units carrying a carboxyl group directly bonded
via the amino group or bonded via one or more methylenyl groups.
These carboxylic acid groups bonded to the amino group are
preferably carboxymethyl or carboxyethyl groups. Further are
preferred oligosaccharides and/or polysaccharides, wherein
substantially the half, i.e. 48-52%, preferred 49-51% and
especially preferred 49.5-50.5%, consists of N-acyl glucosamine
resp. N-acyl galactosamine, preferably of N-acetyl glucosamine or
N-acetyl galactosamine, and substantially the other half thereof
consists of an uronic acid, preferably glucuronic acid and iduronic
acid. Particularly preferred are oligosaccharides and/or
polysaccharides showing a substantially alternating sequence (i.e.
despite of the statistic deviation ratio in the case of the
alternating connection) of the two sugar units. The ratio of the
deviated connections should be under 1%, preferred under 0.1%.
[0064] Surprisingly, it has been shown that, for the uses according
to the invention, in particular substantially desulphated and
substantially N-acylated heparin as well as partially
N-carboxyalkylated and N-acylated chitosan as well as desulphated
and substantially N-acylated dermatan sulphate, chondroitin
sulphate and also chain length reduced hyaluronic acid are
especially suitable. In particular, N-acetylated heparin as well as
partially N-carboxymethylated and N-acetylated chitosan are
suitable for the hemocompatible coating.
[0065] The desulphation and acylation degrees defined by
"substantially" were already defined more above. The term
"substantially" is intended to make clear, that statistic
deviations have to be taken into consideration. A substantially
alternating sequence of the sugar units means, that as a rule two
equal sugar units are not bonded to each other, but does not
completely exclude such an erroneous linkage. Correspondingly,
"substantially the half" means nearly 50%, but permits slight
variations, because especially with biosynthetically produced
macromolecules, the most suitable case is never achieved, and
certain deviations have always to be taken into consideration as
enzymes do not work perfectly and catalysis usually involves a
certain rate of errors. In the case of natural heparin, however,
there is a strictly alternating sequence of N-acetyl glucosamine
and uronic acid units (instead of glucuronic).
[0066] Furthermore, a process for the hemocompatible coating of
surfaces is disclosed, which are intended for direct blood contact.
In said process, a natural and/or artificial surface is provided,
and the oligosaccharides and/or polysaccharides described above are
immobilized on said surface.
[0067] The immobilisation of the oligosaccharides and/or
polysaccharides on these surfaces can be achieved via hydrophobic
interactions, van der Waals forces, electrostatic interactions,
hydrogen bonds, ionic interactions, cross-linking of the
oligosaccharides and/or polysaccharides and/or by covalent bonding
onto the surface. Preferred is the covalent linkage of the
oligosaccharides and/or polysaccharides (side-on bonding), more
preferred the covalent single-point linkage (side-on bonding) and
especially preferred the covalent end-point linkage (end-on
bonding).
[0068] Any natural and/or artificial surfaces of medical products
can be used here such as surfaces of prostheses, organs, vessels,
aortas, cardiac valves, tubes, organ replacement parts, implants,
fibers, hollow fibers, stents, hypodermic needles, syringes,
membranes, conserves, blood containers, titer plates, pacemakers,
adsorber media, chromatography media, chromatography columns,
dialyzers, connection parts, sensors, ventiles, centrifuge
chambers, heat exchangers, endoscopes, filters, pump chambers as
well as other surfaces, which should have hemocompatible
properties. The term "medical products" is to be understood widely
and refers especially to the surfaces of such products, which come
into contact with blood shortly (e.g. endoscopes) or permanently
(e.g. stents).
[0069] In the following the coating methods according to invention
are described. Biological and/or artificial surfaces of medical
devices can be provided with a hemocompatible coating by means of
the following method:
[0070] a) providing a surface of a medical device and
[0071] b) deposition of at least one oligosaccharide and/or
polysaccharide according to formula Ia or Ib, and/or
[0072] at least one oligosaccharide and/or polysaccharide, which
contains between 40% and 60% the sugar unit N-acyl glucosamine or
N-acyl galactosamine and the remaining sugar units substantially
contain one carboxyl group per sugar unit.
[0073] "Deposition" shall refer to at least partial coating of a
surface with the corresponding compounds, wherein the compounds are
deposited and/or introduced and/or immobilized or anyhow anchored
on and/or in the subjacent surface.
[0074] Under "substantially the remaining sugar building units" is
to be understood, that 93% of the remaining sugar building units,
preferred 96% and especially preferred 98% of the remaining 60%-40%
of the sugar building units bear a carboxyl group.
[0075] An uncoated and/or non hemocompatible surface is preferably
provided. "non-hemocompatible" surfaces shall refer to such
surfaces that can activate the blood coagulatory system, thus are
more or less thrombogeneous.
[0076] An alternative embodiment comprises the steps:
[0077] a) providing surface of a medical device and
[0078] b') deposition of a biostable layer onto the surface of the
medical device or
[0079] a) providing surface of a medical device and
[0080] b) deposition of at least one oligosaccharide and/or
polysaccharide according to formula Ia or Ib, and/or
[0081] at least one oligosaccharide and/or polysaccharide, which
contains between 40% and 60% the sugar unit N-acyl glucosamine or
N-acyl galactosamine and the remaining sugar units substantially
contain one carboxyl group per sugar unit.
[0082] b') deposition of a biostable layer onto the surface of the
medical device and
[0083] d') deposition of a further hemocompatible layer of at least
one inventive oligosaccharide and/or polysaccharide according to
formula Ia or Ib, and/or
[0084] at least one oligosaccharide and/or polysaccharide, which
contains between 40% and 60% the sugar unit N-acyl glucosamine or
N-acyl galactosamine and the remaining sugar units substantially
contain one carboxyl group per sugar unit.
[0085] The last-mentioned embodiment makes sure, even in the case
of mechanical damage of the polymeric layer and therewith also of
the exterior hemocompatible layer, e.g. due to inappropriate
transport or complicated conditions during the implantation, that
the surface coating does not lose its characteristic of being blood
compatible.
[0086] Under "biological and artificial" surface is the combination
of an artificial medical device with an artificial part to be
understood, e.g. a pork heart with an artificial heart valve.
[0087] The single layers are deposited preferably by dipping or
spraying methods, whereas one can deposit at the same time with the
deposition of one layer also another or more active agents onto the
medical device surface, which is then implemented in the respective
layer covalently and/or adhesively bound. In this way one or more
active agents can be deposited at the same time with the deposition
of a hemocompatible layer onto the medical device.
[0088] The active agents as well as the substances, which can be
used for a biostable or biodegradable layer, are itemized more
below.
[0089] Onto this first biostable or hemocompatible layer it is then
possible in an additional non compulsory step c) to deposit an
active agent layer of one or more active agents. In a preferred
embodiment the active agent or agents are bound covalently on the
subjacent layer. Also the active agent is preferably deposited by
dipping or spraying methods.
[0090] After the step b) or the step c) an additional step d) can
follow, which comprises the deposition of at least one
biodegradable layer and/or at least one biostable layer onto the
hemocompatible layer resp. the active agent layer.
[0091] According to the alternative embodiment after step b') or
step c) a step d') can follow, which comprises the deposition or
immobilisation of at least one oligosaccharide and/or
polysaccharide according to invention according to formula Ia or Ib
and/or at least one oligosaccharide and/or polysaccharide, which
contains between 40% and 60% the sugar unit N-acyl glucosamine or
N-acyl galactosamine and the remaining sugar units substantially
contain one carboxyl group per sugar unit, as hemocompatible layer.
Preferably after step b') the step d') follows.
[0092] After step d) resp. d') the deposition of another active
agent layer of one or more active agents can take place into or
onto the subjacent biodegradable and/or biostable layer or the
hemocompatible layer.
[0093] Additionally to the deposited active agent layers the
biostable, biodegradable and/or hemocompatible layers can contain
further active agents, which were deposited together with the
biostable and/or biodegradable substances or the hemocompatible
oligosaccharides and/or polysaccharides on the medical device and
are contained in the respective layers.
[0094] According to a preferred embodiment the biostable layer is
covalently and/or adhesively bound on the surface of the medical
device and completely or incompletely covered with a hemocompatible
layer, which (preferably covalently) is bound to the biostable
layer.
[0095] Preferably the hemocompatible layer comprises heparin of
native origin of regioselectively synthesized derivatives of
different sulphation coefficients (sulphation degrees) and
acylation coefficients (acylation degrees) in the molecular weight
range of the pentasaccharide which is responsible for the
antithrombotic activity, up to the standard molecular weight of the
purchasable heparin of 13 kD, heparansulphate and its derivatives,
oligo- and polysaccharides of the erythrocytic glycocalix,
desulphated and N-reacetylated heparin, N-carboxymethylated and/or
partially N-acetylated chitosan as well as mixtures of these
substances.
[0096] Subject of the invention are also medical devices, which are
hemocompatibly coated according to one of the herein mentioned
methods.
[0097] Furthermore subject of the invention are medical devices,
whereas the surface of the medical devices is covered directly or
via at least one interjacent biostable and/or biodegradable layer
and/or active agent layer with a hemocompatible layer, which
consists of at least one oligosaccharide and/or polysaccharide,
which contains between 40% and 60% the sugar unit N-acyl
glucosamine or N-acyl galactosamine and the remaining sugar units
substantially contain one carboxyl group per sugar unit.
[0098] According to a preferred embodiment under the hemocompatible
layer of the afore-mentioned oligosaccharides and/or
polysaccharides is at least one biostable layer present, which is
additionally preferred covalently bound to the surface of the
medical device.
[0099] It is further preferred, if on the hemocompatible layer at
least one biostable and/or at least one biodegradable layer is
present, which covers the hemocompatible layer completely or
incompletely. Especially preferred is a biodegradable layer, which
covers the hemocompatible layer.
[0100] A further preferred embodiment contains between the
biostable lower layer and the subjacent hemocompatible layer an
active agent layer of at least one antiproliferative,
antiinflammatory and/or antithrombotic active agent, which is bound
covalently and/or adhesively to the hemocompatible layer.
Alternatively to the active agent layer or additionally to the
active agent layer the lower biostable and/or upper hemocompatible
layer can contain further active agents, which are deposited
preferably together with the deposition of the respective
layer.
[0101] Basically every layer, i.e. a biostable layer, a
biodegradable layer and a hemocompatible layer can contain one or
more antiproliferative, antiinflammatory and/or antithrombotic
active agents and moreover between the afore-mentioned layers
active agent layers of one or more active agents can be present.
Preferred are coating systems of two layers, a biostable and a
hemocompatible layer, whereas the hemocompatible layer is the
external layer and both layers can contain one or more active
agents. Further it is preferred if on or under the hemocompatible
layer an active agent layer of one or more active agents is
present. Also preferred are three-layer systems, which consist of a
biostable, biodegradable and hemocompatible layer. Thereby
preferably the lowest layer is a biostable layer. Additionally one
or two active agent layers are possible. It is also possible to
deposit two active agent layers directly above each other. In a
further preferred embodiment at least one active agent is bound
covalently on or in a layer.
[0102] Preferably used in the methods of coating and on the medical
devices as active agents are tacrolimus, pimecrolimus, PI88,
thymosin .alpha.-1, PETN (pentaerythritol tetranitrate), baccatin
and its derivatives, docetaxel, colchicin, paclitaxel and its
derivatives, trapidil, .alpha.- and .beta.-estradiol, dermicidin,
tialin (2-methylthiazolidine-2,4-dicarboxylic acid), tialin-sodium
(sodium salt of tialin), simvastatine, macrocyclic suboxide (MCS)
and its derivatives, sirolimus, tyrphostines, D24851, colchicin,
fumaric acid and fumaric acid esters, activated protein C (aPC),
interleucine-1.beta. inhibitors, and melanocyte-stimulating hormone
(.alpha.-MSH) as well as mixtures of these active agents.
[0103] The natural and/or artificial surfaces of the medical
devices, which are coated according to the herein described methods
with a hemocompatible layer of the inventive oligosaccharide and/or
polysaccharide resp. the oligosaccharides and/or polysaccharides
which contain between 40% and 60% the sugar unit N-acyl glucosamine
or N-acyl galactosamine and the remaining sugar units substantially
contain one carboxyl group per sugar unit, are especially suitable
as implants and organ replacement parts, respectively, which are in
direct contact with the blood circuit and the blood. The medical
devices coated according to invention are especially suitable, but
not only, for the direct and permanent blood contact, but show
surprisingly also the characteristic to reduce or even to prevent
the adhesion of proteins onto suchlike coated surfaces.
[0104] The adhesion of plasma proteins on foreign surfaces which
come in contact with blood is an essential and initial step for the
further events concerning the recognition and the implementing
action of the blood system.
[0105] This is for example important in the in vitro diagnostics
from body fluids. Thus the deposition of the inventive coating
prevents or at least reduces for example the unspecific adhesion of
proteins on micro-titer plates or other support mediums which are
used for diagnostic detection methods, that disturb the generally
sensitive test reactions and can lead to a falsification of the
analysis result.
[0106] By use of the coating according to invention on adsorption
media or chromatography media the unspecific adhesion of proteins
is also prevented or reduced, whereby better separations can be
achieved and products of greater purity can be generated.
[0107] Especially stents are coated according to the inventive
methods. The implantation of stents using balloon dilatation of
occluded vessels increasingly established in the last years.
Although stents decrease the risk of a renewed vessel occlusion
they are until now not capable of preventing such restenoses
completely.
[0108] An exact conceptual description of restenosis cannot be
found in the technical literature. The most commonly used
morphologic definition of the restenosis is the one which defines
the restenosis after a successful PTA (percutaneous transluminal
angioplasty) as a reduction of the vessel diameter to less than 50%
of the normal one. This is an empirically defined value of which
the hemodynamic relevance and its relation to clinical pathology
lacks of a massive scientific basis. In practical experience the
clinical aggravation of a patient is often viewed as a sign for a
restenosis of the formerly treated vessel segment.
[0109] There are three different reasons for the restenosis caused
by the stent:
[0110] a.) During the first period after the implantation the stent
surface is in direct contact with the blood and an acute thrombosis
can occur which again occludes the vessel due to the now present
foreign surface.
[0111] b.) The implantation of the stent generates vessel injuries,
which induce inflammation reactions, which play an important role
for the recovery process during the first seven days. The herein
concurrent processes are among others connected with the release of
growth factors, which initiate an increased proliferation of the
smooth muscle cells which rapidly leads to a renewed occlusion of
the vessel, because of uncontrolled growth.
[0112] c.) After a couple of weeks the stent starts to grow into
the tissue of the blood vessel. This means that the stent is
surrounded totally by smooth muscle cells and has no contact to the
blood. This cicatrization can be too distinctive (neointima
hyperplasia) and may lead to not only a coverage of the stent
surface but to the occlusion of the total interior space of the
stent.
[0113] It was tried vainly to solve the problem of restenosis by
the coating of the stents with heparin (J. Whorle et al., European
Heart Journal 2001, 22, 1808-1816). Heparin addresses as anti
coagulant only the first mentioned cause and is moreover able to
unfold its total effect only in solution. This first problem is
meanwhile almost totally avoidable medicamentously by application
of anti-coagulants. The second and third problem is intended now to
be solved by inhibiting the growth of the smooth muscle cells
locally on the stent. This is carried out by e.g. radioactive
stents or stents which contain pharmaceutically active agents.
[0114] U.S. Pat. No. 5,891,108 discloses for example a hollow
moulded stent, which can contain pharmaceutical active agents in
its interior, that can be released throughout a various number of
outlets in the stent. Whereas EP-A-1 127 582 describes a stent that
shows ditches of 0.1-1 mm depth and 7-15 mm length on its surface
which are suitable for the implementation of an active agent. These
active agent reservoirs release similarly to the outlets in the
hollow stent the contained pharmaceutically active agent in a
punctually high concentration and over a relatively long period of
time which however leads to the fact that the smooth muscle cells
are not anymore or only very delayed capable of enclosing the
stent. As a consequence the stent is much longer exposed to the
blood, what leads again to increased vessel occlusions by
thromboses (Liistro F., Colombo A., Late acute thrombosis after
Paclitaxel eluting stent implantation. Heart 2001, 86,
262-264).
[0115] One approach to this problem is represented by the
phosphorylcholine coating of biocompatibles (WO 0101957), as here
phosphorylcholine, a component of the erythrocyte cell membrane,
shall create a non thrombogeneous surface as a component of the
coated non biodegradable polymer layer on the stent. Dependent of
its molecular weight, thereby the active agent is absorbed by the
polymer containing phosphorylcholine layer or adsorbed on the
surface.
[0116] The stents according to invention are coated with a
hemocompatible layer and feature one or more additional layers
which at least comprise an antiproliferative and/or
antiinflammatory and if needed an antithrombotic active agent.
[0117] The hemocompatible coating of a stent provides the required
blood compatibility and the active agent (or active agent
combination) which is distributed homogeneously over the total
surface of the stent provides that the covering of the stent
surface with cells especially smooth muscle and endothelial cells
takes place in a controlled way. Thus no rapid population and
overgrowth with cells takes place on the stent surface which could
lead to a restenosis whereas the covering of the stent surface with
cells is also not completely prevented by a high medicament
concentration which involves the risk of thrombosis.
[0118] Thus the incorporation of active agents guarantees that the
active agent or the active agent combination which is bound
covalently and/or adhesively to the subjacent layer and/or
implemented covalently and/or adhesively into the layer is released
continuously and in small doses so that the population of the stent
surface by cells is not inhibited however an overgrowth is
prevented. This combination of both effects awards the ability to
the inventive stent to grow rapidly into the vessel wall and
reduces both the risk of restenosis and the risk of thrombosis. The
release of one or more active agents spans over a period from 1 to
12 months, preferably 1 to 3 months after implantation.
[0119] Antiproliferative substances, antiphlogistic as well as
antithrombotic compounds are used as active agents. Preferably
cytostatics, macrolide antibiotics and/or statins are used as
antiproliferative active agents. Applyable antiproliferative active
agents are sirolimus (rapamycin), everolimus, pimecrolimus,
somatostatin, tacrolimus, roxithromycin, dunaimycin, ascomycin,
bafilomycin, erythromycin, midecamycin, josamycin, concanamycin,
clarithromycin, troleandomycin, folimycin, cerivastatin,
simvastatin, lovastatin, fluvastatin, rosuvastatin, atorvastatin,
pravastatin, pitavastatin, vinblastine, vincristine, vindesine,
vinorelbine, etoposide, teniposide, nimustine, carmustine,
lomustine, cyclophosphamide, 4-hydroxycyclophosphamide,
estramustine, melphalan, betulinic acid, camptothecin, lapachol,
.beta.-lapachone, podophyllotoxin, betulin, trofosfamide,
podophyllic acid 2-ethylhydrazide, ifosfamide, chlorambucil,
bendamustine, dacarbazine, busulfan, procarbazine, treosulfan,
temozolomide, melanocyte-stimulating hormon (.alpha.-MSH),
thiotepa, daunorubicin, doxorubicin, aclarubicin, epirubicin,
mitoxantrone, idarubicin, bleomycin, mitomycin, dactinomycin,
methotrexate, fludarabine, fludarabine-5'-dihydrogenphosphate,
mofebutazone, acemetacin, diclofenac, lonazolac, dapsone,
o-carbamoylphenoxyacetic acid, lidocaine, ketoprofen, mefenamic
acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine,
hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol,
celecoxib, .beta.-sitosterin, ademetionine, myrtecaine,
polidocanol, nonivamide, levomenthol, benzocaine, aescin,
cladribine, mercaptopurine, thioguanine, cytarabine, fluorouracil,
gemcitabine, capecitabine, docetaxel, carboplatin, cisplatin,
oxaliplatin, amsacrine, irinotecan, topotecan, hydroxycarbamide,
miltefosine, pentostatin, aldesleukin, tretinoin, asparaginase,
trastuzumab, exemestane, letrozole, goserelin, chephalomannin,
pegaspargase, anastrozole, exemestane, letrozole, formestane,
aminoglutethimide, adriamycin, azithromycin, spiramycin,
cepharantin, smc proliferation inhibitor-2w, epothilone A and B,
mitoxantrone, azathioprine, mycophenolatmofetil, c-myc-antisense,
b-myc-antisense, selectin (cytokine antagonist), CETP inhibitor,
cadherines, cytokinin inhibitors, COX-2 inhibitor, NFkB,
angiopeptin, ciprofloxacin, camptothecin, fluroblastin, monoclonal
antibodies, which inhibit the muscle cell proliferation, bFGF
antagonists, probucol, prostaglandins, folic acid and derivatives,
vitamines of the B-series, vitamine D-derivatives such as
calcipotriol and tacalcitol, D24851, fumaric acid and its
derivatives such as dimethylfumarate, IL-1.beta. inhibitor,
colchicine, NO donors such as pentaerythritol tetranitrate and
syndnoeimines, S-nitrosoderivatives, tamoxifen, staurosporin,
.beta.-estradiol, .alpha.-estradiol, estrone, estriol,
ethinylestradiol, fosfestrol, medroxyprogesterone, estradiol
cypionates, estradiol benzoates, tranilast, kamebakaurin and other
terpenoids, which are applied in the therapy of cancer, verapamil,
tyrosine kinase inhibitors (tyrphostines), cyclosporine A,
paclitaxel and derivatives thereof (6-.alpha.-hydroxy-paclitaxel,
baccatin, and others), synthetically produced as well as from
native sources obtained macrocyclic oligomers of carbon suboxide
(MCS) and derivatives thereof, molgramostim (rhuGM-CSF),
peginterferon .alpha.-2b, lenograstim (r-HuG-CSF), filgrastim,
macrogol, dacarbazine, letrozol, goserelin, chephalomannin,
trastuzumab, exemestan, basiliximab, daclizumab, ellipticine,
D-24851 (Calbiochem), colcemid, cytochalasin A-E, indanocine,
nocodazole, S 100 protein, PI-88, melanocyte stimulating hormon
(.alpha.-MSH),bacitracin, vitronectin receptor antagonists,
azelastine, guanidyl cyclase stimulator tissue inhibitor of metal
proteinase-1 and -2, free nucleic acids, nucleic acids incorporated
into virus transmitters, DNA and RNA fragments, plasminogen
activator inhibitor-1, plasminogen activator inhibitor-2,
interleucine-1.beta. inhibitors, antisense oligonucleotides, VEGF
inhibitors, called IGF-1.
[0120] From the group of antibiotics furthermore cefadroxil,
cefazolin, cefaclor, cefotixin, tobramycin, gentamycin are used.
Positive influence on the postoperative phase have also the
penicillins such as dicloxacillin, oxacillin, sulfonamides,
metronidazol, antithrombotics such as argatroban, aspirin,
abciximab, synthetic antithrombin, bivalirudin, coumadin,
dermicidin, enoxaparin, hemoparin, tissue plasminogen activator,
GpIIb/IIIa platelet membrane receptor, factor X.sub.a inhibitor,
activated protein C, dermicidin, antibodies, heparin, hirudin,
r-hirudin, PPACK, protamin, prourokinase, streptokinase, warfarin,
urokinase, vasodilators such as dipyramidole, trapidil,
nitroprussides, PDGF antagonists such as triazolopyrimidine and
seramin, ACE inhibitors such as captopril, cilazapril, lisinopril,
enalapril, losartan, thiol protease inhibitors, caspase inhibitors,
apoptosis inhibitors, apoptosis regulators such as p65 NF-kB and
Bcl-xL antisense oligonucleotides and prostacyclin, vapiprost,
.alpha., .beta. and .gamma. interferon, histamine antagonists,
serotonin blockers, halofuginone, nifedipine, tocopherol,
tranirast, molsidomine, tea polyphenols, epicatechin gallate,
epigallocatechin gallate, Boswellic acids and derivatives thereof,
leflunomide, anakinra, etanercept, sulfasalazine, etoposide,
dicloxacillin, tetracycline, triamcinolone, mutamycin, procainamid,
retinoic acid, quinidine, disopyramide, flecainide, propafenone,
sotalol, amidorone. Further active agents are steroids
(hydrocortisone, betamethasone, dexamethasone), non-steroidal
substances (NSAIDS) such as fenoprofen, ibuprofen, indomethacin,
naproxen, phenylbutazone and others. Antiviral agents such as
acyclovir, ganciclovir and zidovudine are also applyable. Different
antimycotics are used in this area. Examples are clotrimazole,
flucytosine, griseofulvin, ketoconazole, miconazole, nystatin,
terbinafine. Antiprozoal agents such as chloroquine, mefloquine,
quinine are effective active agents in equal measure, moreover
natural terpenoids such as hippocaesculin,
barringtogenol-C21-angelate, 14-dehydroagrostistachin, agroskerin,
agrostistachin, 17-hydroxyagrostistachin, ovatodiolids,
4,7-oxycycloanisomelic acid, baccharinoids B1, B2, B3, tubeimoside,
bruceanol A, B, C, bruceantinoside C, yadanziosides N and P,
isodeoxyelephantopin, tomenphantopin A and B, coronarin A, B, C and
D, ursolic acid, hyptatic acid A, zeorin, iso-iridogermanal,
maytenfoliol, effusantin A, excisanin A and B, longikaurin B,
sculponeatin C, kamebaunin, leukamenin A and B,
13,18-dehydro-6-.alpha.-senecioyloxychapa- rrin,
1,11-dimethoxycanthin-6-one, 1-hydroxy-11-methoxycanthin-6-one,
scopoletin, taxamairin A and B, regenilol, triptolide, moreover
cymarin, apocymarin, aristolochic acid, anopterin,
hydroxyanopterin, anemonin, protoanemonin, berberine, cheliburin
chloride, cictoxin, sinococuline, bombrestatin A and B,
cudraisoflavone A, curcumin, dihydronitidine, nitidine chloride,
12-.beta.-hydroxypregnadien-3,20-dione, bilobol, ginkgol, ginkgolic
acid, helenalin, indicine, indicine-N-oxide, lasiocarpine,
inotodiol, glycoside 1a, podophyllotoxin, justicidin A and B,
larreatin, malloterin, mallotochromanol,
isobutyrylmallotochromanol, maquiroside A, marchantin A,
maytansine, lycoridicin, margetine, pancratistatin, liriodenine,
oxoushinsunine, aristolactam-AII, bisparthenolidine, periplocoside
A, ghalakinoside, ursolic acid, deoxypsorospermin, psychorubin,
ricin A, sanguinarine, manwu wheat acid, methylsorbifolin,
sphatheliachromen, stizophyllin, mansonine, strebloside, akagerine,
dihydrousambarensine, hydroxyusambarine, strychnopentamine,
strychnophylline, usambarine, usambarensine, berberine,
liriodenine, oxoushinsunine, daphnoretin, lariciresinol,
methoxylariciresinol, syringaresinol, umbelliferon, afromoson,
acetylvismione B, desacetylvismione A, vismione A and B, further
natural terpenoids such as hippocaesculin,
14-dehydroagrostistachin, agroskerin, agrostistachin,
17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic
acid, yadanziosides N and P, isodeoxyelephantopin, tomenphantopin A
and B, coronarin A, B, C and D, ursolic acid, hyptatic acid A,
zeorin, iso-iridogermanal, maytenfoliol, effusantin A, excisanin A
and B, longikaurin B, sculponeatin.
[0121] The active agents are used separately or combined in the
same or a different concentration. Especially preferred are active
agents which feature also immunosuppressive properties besides
their antiproliferative effect. Suchlike active agents are
erythromycin, midecamycin, tacrolimus, sirolimus, paclitaxel and
josamycin. Furthermore preferred is a combination of several
antiproliferatively acting substances or of antiproliferative
active agents with immunosuppressive active agents.
[0122] Preferred for the present invention are tacrolimus,
pimecrolimus, PI88, thymosin .alpha.-1, PETN (pentaerythritol
tetranitrate), baccatin and its derivatives, docetaxel, colchicin,
paclitaxel and its derivatives, trapidil, .alpha.- and
.beta.-estradiol, dermicidin, simvastatine, macrocyclic suboxide
(MCS) and its derivatives, sirolimus, tyrphostines, D24851,
colchicin, fumaric acid and fumaric acid esters, activated protein
C (aPC), interleucine-1.beta. inhibitors and melanocyte-stimulating
hormone (.alpha.-MSH) and tialin
(2-methylthiazolidine-2,4-dicarboxylic acid) as well as tialin-Na
(sodium salt of tialin).
[0123] The active agent is preferably contained in a pharmaceutical
active concentration from 0.001-10 mg per cm.sup.2 stent surface
and per active agent layer or active agent containing layer.
Additional active agents can be contained in a similar
concentration in the same or in other layers.
[0124] The medical devices coated according to invention,
especially the stents coated according to invention, can release
the active agent or the active agents continuously and controlled
and are suitable for the prevention or reduction of restenosis (see
FIG. 6).
[0125] The hemocompatible layer which covers directly the stent
preferably comprises heparin of native origin as well as
synthetically obtained derivatives with different sulphation
coefficients (sulphation degrees) and acylation coefficients
(acylation degrees) in the molecular weight range of the
pentasaccharide which is responsible for the antithrombotic
activity up to the standard molecular weight of the purchasable
heparin, as well as heparan sulphates and derivatives thereof,
oligo- and polysaccharides of the erythrocyte glycocalix, which
imitate in a perfect way the athrombogeneous surface of the
erythrocytes, since contrary to phosphorylcholine, here the actual
contact between blood and erythrocyte surface takes place,
completely desulphated and N-reacetylated heparin, desulphated and
N-reacetylated heparin, N-carboxymethylated and/or partially
N-acetylated chitosan, chitosan and/or mixtures of these
substances. These stents with a hemocompatible coating are prepared
by providing conventional normally non coated stents and by
preferably covalent deposition of a hemocompatible layer which
permanently masks the surface of the implant after the release of
the active agent and thus, after the decay of the active agent's
influence and the degradation of the matrix.
[0126] The conventional stents which can be coated according to the
inventive methods, consist of stainless steel, nitinol or other
metals and alloys or of synthetic polymers.
[0127] Another preferred embodiment of the stents according to
invention shows a coating which consists of at least two layers.
Multiple layer systems are used as well. In such multiple layer
systems the layer which is directly deposited on the stent is
labelled first layer. Labelled second layer is that layer which is
deposited on the first layer, etc.
[0128] According to the two layer design the first layer consists
of a hemocompatible layer which is substantially covered completely
by a biodegradable layer which comprises at least an
antiproliferative, antiphlogistic and/or antithrombotic active
agent bound covalently and/or adhesively. Also applied are active
agent combinations which mutually facilitate and replenish
themselves.
[0129] As biodegradable substances for the external layer can be
used: polyvalerolactones, poly-.epsilon.-decalactones, polylactonic
acid, polyglycolic acid, polylactides, polyglycolides, copolymers
of the polylactides and polyglycolides,
poly-.epsilon.-caprolactone, polyhydroxybutanoic acid,
polyhydroxybutyrates, polyhydroxyvalerates,
polyhydroxybutyrate-co-valerates, poly(1,4-dioxane-2,3-diones),
poly(1,3-dioxane-2-ones), poly-p-dioxanones, polyanhydrides such as
polymaleic anhydrides, polyhydroxymethacrylates, fibrin,
polycyanoacrylates, polycaprolactonedimethylacrylates,
poly-b-maleic acid, polycaprolactonebutyl-acrylates, multiblock
polymers such as from oligocaprolactonedioles and
oligodioxanonedioles, polyether ester multiblock polymers such as
PEG and polybutyleneterephtalate, polypivotolactones, polyglycolic
acid trimethyl-carbonates, polycaprolactone-glycolides,
poly-g-ethylglutamate, poly(DTH-iminocarbonate),
poly(DTE-co-DT-carbonate), poly(bisphenol-A-iminocarbonate),
polyorthoesters, polyglycolic acid trimethyl-carbonates,
polytrimethylcarbonates, polyiminocarbonates,
poly(N-vinyl)-pyrrolidone, polyvinylalcoholes, polyesteramides,
glycolated polyesters, polyphosphoesters, polyphosphazenes,
poly[p-carboxyphenoxy)propane], polyhydroxypentanoic acid,
polyanhydrides, polyethyleneoxide-propyleneoxide, soft
polyurethanes, polyurethanes with amino acid residues in the
backbone, polyether esters such as polyethyleneoxide,
polyalkeneoxalates, polyorthoesters as well as copolymers thereof,
lipids, carrageenanes, fibrinogen, starch, collagen, protein based
polymers, polyamino acids, synthetic polyamino acids, zein,
modified zein, polyhydroxyalkanoates, pectic acid, actinic acid,
modified and non modified fibrin and casein, carboxymethylsulphate,
albumin, moreover hyaluronic acid, heparan sulphate, heparin,
chondroitinesulphate, dextran, b-cyclodextrines, copolymers with
PEG and polypropyleneglycol, gummi arabicum, guar, gelatine,
collagen, collagen-N-hydroxysuccinimide, lipids, phospholipids,
modifications and copolymers and/or mixtures of the afore mentioned
substances.
[0130] The layer and layers respectively which contain the active
agent is slowly degradated by components of the blood such that the
active agent is released of the external layer according to the
degradation velocity or resolves itself from the matrix according
to its elution behavior. The first hemocompatible layer guarantees
the required blood compatibility of the stent once the
biodegradable layer is degradated. This biological degradation of
the external layer and the corresponding release of the active
agent reduces strongly an ongrowth of cells only for a certain
period of time and an aimed controlled adhesion is enabled where
the external layer has been already widely degradated. The
biological degradation of the external layer spans advantageously
from 1 to 36 months, preferably from 1 to 6 months, especially
preferred from 1 to 2 months. It was shown that suchlike stents
prevent or at least very strongly reduce restenosis. In this period
of time the important healing processes take place. Finally the
hemocompatible layer remains as athrombogeneous surface and masks
the foreign surface in such a way that no life-threatening reaction
can occur anymore.
[0131] The amounts of polymer deposited on the surfaces of the
medical devices, preferably stents, are between 0.01 mg to 3
mg/layer, preferred between 0.20 mg to 1 mg/layer and especially
preferred between 0.2 mg to 0.5 mg/layer.
[0132] Suchlike stents are preparable via a method for the
hemocompatible coating of stents the basis of which is formed by
the following principle:
[0133] a) providing a non coated stent,
[0134] b) deposition of a preferred covalently bound hemocompatible
layer,
[0135] c) diffusion of active agent into the hemocompatible layer,
or
[0136] c') substantially complete coating of the hemocompatible
layer via dipping or spraying method with at least one active
agent, or
[0137] c") substantially complete coating and/or incomplete coating
of the hemocompatible layer via dipping or spraying method with at
least one biodegradable and/or biostable layer which comprises at
least one active agent and/or represents the active agent
itself.
[0138] The principle of coating offers a big range of variation
concerning the contrived requirements for the active agent and is
separable into different coating types, which can be combined also
among themselves.
[0139] Coating Principle I:
[0140] a) providing a non coated stent,
[0141] b) deposition of a hemocompatible layer,
[0142] c) deposition of an active agent or an active agent
combination on the hemocompatible layer without a matrix,
[0143] d) deposition of an active agent or an active agent
combination on the hemocompatible layer without a matrix and
substantially complete and/or incomplete coating of the layers with
a biodegradable and/or biostable material for diffusion
control.
[0144] Coating Principle II:
[0145] a) providing a non coated stent,
[0146] b) deposition of a hemocompatible layer,
[0147] c) substantially complete coating and/or incomplete coating
of the hemocompatible layer with at least one biodegradable and/or
biostable layer which comprises at least one active agent bound
covalently and/or adhesively to the hemocompatible layer,
[0148] d) substantially complete coating of the hemocompatible
layer with at least one biodegradable and/or biostable layer which
comprises at least one active agent bound covalently and/or
adhesively to the matrix and another biodegradable and/or biostable
layer without an active agent as diffusion barrier which covers the
subjacent layer completely and/or partially.
[0149] Coating Pprinciple III:
[0150] a) providing a non coated stent,
[0151] b) deposition of a hemocompatible layer,
[0152] c) substantially complete coating of the hemocompatible
layer with at least one biodegradable and/or biostable layer which
comprises at least one active agent bound covalently and/or
adhesively,
[0153] d) deposition of an active agent or an active agent
combination bound covalently and/or adhesively to the subjacent
layer,
[0154] e) substantially complete coating of the hemocompatible
layer with at least one biodegradable and/or biostable layer which
comprises at least one active agent bound covalently and/or
adhesively, deposition of an active agent or an active agent
combination and another biodegradable and/or biostable layer
without an active agent as diffusion barrier which covers the
subjacent layer completely and/or partially.
[0155] Coating Principle IV:
[0156] a) providing a non coated stent,
[0157] b) deposition of a hemocompatible layer,
[0158] c) substantially complete and/or incomplete coating of the
hemocompatible layer with at least two biodegradable and/or
biostable layers which comprise covalently and/or adhesively at
least one active agent in a different concentration per layer,
[0159] d) substantially complete and/or incomplete coating of the
hemocompatible layer with at least two biodegradable and/or
biostable layers which comprise at least one active agent bound
covalently and/or adhesively in a different concentration per layer
and at least another biodegradable and/or biostable layer without
an active agent as diffusion barrier which covers the subjacent
layer completely and/or partially,
[0160] e) substantially complete and/or incomplete coating of the
hemocompatible layer with at least one biodegradable and/or
biostable layer which comprises at least one active agent and/or at
least another active agent of the same group or from another group
of complementary properties in the same or different concentrations
in a covalent and/or adhesive form,
[0161] f) substantially complete and/or incomplete coating of the
hemocompatible layer with at least two biodegradable and/or
biostable layers which comprise at least one active agent and/or at
least another active agent of the same group or from another group
of complementary properties in the same or different concentrations
and at least another biodegradable and/or biostable layer without
an active agent as diffusion barrier which covers the subjacent
layer completely and/or partially,
[0162] g) substantially complete coating of the hemocompatible
layer with at least two biodegradable and/or biostable layers which
comprise covalently and/or adhesively at least one active agent in
the same and/or different concentrations and another biodegradable
and/or biostable layer without an active agent as diffusion barrier
which covers the subjacent layer completely or also just partially
and whereas that layer is covered by an active agent layer which
consists of at least one active agent bound covalently to the
subjacent matrix and/or adhesively without a support material.
[0163] Another advantageous embodiment is represented by a stent
with an at least three layered coating, whereas the first layer
covers the surface of the stent with the hemocompatible layer, the
second layer contains the active agent and is not biodegradable and
is covered by a third hemocompatible layer. The external layer
provides the stent herein the necessary blood compatibility and the
second layer serves as an active agent reservoir. The active agent
which is if needed covalently bound to the matrix via a
hydrolysis-weak bonding and/or added in a solvent dissolved matrix
which is required for the coating method, is thus released from the
second layer continuously and in small concentrations and diffuses
uninhibited through the external hemocompatible layer. This layer
assembly also yields the result that the population of the stent
surface with cells is not prevented but is reduced to an ideal
degree. The first layer offers a risk minimization for eventually
occurring damages of the coated stent surface during the
implantation e.g. by abrasions through the present plaque or during
the prearrangement e.g. during the crimping. A second security
guarantee results from the fact that even a bio-stable polymer is
degradated in the body over a more or less long period of time
which at least partially uncovers the stent surface. Combinations
especially with biodegradable material as described in the coating
principles are possible, too.
[0164] Suchlike stents can be prepared by providing a conventional
stent, depositing a hemocompatible first layer on its surface,
depositing a non biodegradable layer which at least comprises one
active agent as well as combinations with other active agents from
other groups bound covalently and/or adhesively and coating of this
layer substantially completely with another hemocompatible
layer.
[0165] Substances which come into question for the biostable layer
are all of the consistent materials used in medical science.
Thereto are accounted: polyacrylic acid and polyacrylates such as
polymethylmethacrylate, polybutyl methacrylate, polyacrylamide,
polyacrylonitriles, polyamides, polyetheramides, polyethylenamine,
polyimides, polycarbonates, polycarbourethanes, polyvinylketones,
polyvinylhalogenides, polyvinylidenhalogenides, polyvinyl ethers,
polyvinylaromates, polyvinyl esters, polyvinylpyrrolidones,
polyoxymethylenes, polyethylene, polypropylene,
polytetrafluoroethylene, polyurethanes, polyolefin elastomers,
polyisobutylenes, EPDM gums, fluorosilicones, carboxymethyl
chitosan, polyethylenterephthalate, polyvalerates,
carboxymethylcellulose, cellulose, rayon, rayon triacetates,
cellulose nitrates, cellulose acetates, hydroxyethylcellulose,
cellulosebutyrates, celluloseacetatebutyrates, ethylvinylacetate
copolymers, polysulphones, epoxy resins, ABS resins, EPDM gums,
silicones such as polysiloxanes, polyvinylhalogenes and copolymers,
cellulose ethers, cellulose triacetates, chitosan and copolymers
and/or mixtures of these afore-mentioned substances.
[0166] In case of multi layer systems the newly deposited layer
covers the subjacent layer substantially completely. The term
"substantially" means in this context, that at least the stent
surface, which comes into contact with the vessel wall, is covered
completely resp. at least 90%, preferred 95% and especially
preferred at least 98% of the stent surface are covered.
[0167] The stents according to invention solve both the problem of
acute thrombosis and the problem of neointima hyperplasia after a
stent implantation. In addition the stents according to invention
are well suitable due to their coating whether as single layer or
as multi layer system especially for the continuous release of one
or more antiproliferative and/or immunosuppressive active agents.
Due to this feature of aimed continuous active agent release in a
required amount the coated stents according to invention prevent
almost completely the danger of restenosis.
[0168] In addition, according to the described process, any plastic
surfaces can be coated with a hemocompatible layer of the
oligosaccharides and/or polysaccharides. As plastics, synthetic
polymers as well as biopolymers are suitable, comprising, for
example, the monomers ethene, vinyl acetate, methacrylic acid,
vinylcarbazole, trifluoroethylene, propene, butene, methylpentene,
isobutene, styrene, chlorostyrene, aminostyrene, acrylonitrile,
butadiene, acrylic ester, divinylbenzene, isoprene, vinyl chloride,
vinyl alcohol, vinylpyridine, vinylpyrrolidone,
tetrafluoroethylene, trifluorochloroethene, vinyl fluoride,
hexafluoroisobutene, acrylic acid, acrolein, acrylamide,
methacrylamide, maleic acid, hydroxymethyl methacrylic acid, methyl
methacrylic acid, maleic acid anhydride, methacrylic acid
anhydride, methacrylonitrile, fluorstyrene, fluoranilide,
3,4-isothiocyanatostyrene, allyl alcohol, sulfonic acid,
methallylsulfonic acid, diallyl phthalic acid, cyanoacrylic acid,
dimethylaminoethylmethacrylic acid, lauryl methacrylic acid,
acetaminophenylethoxymethacrylic acid, glycol dimethacrylic acid,
2-hydroxyethyl methacrylic acid, formaldehyde, fluoral, chloral,
ethylene oxide, tetrahydrofuran, propylene oxide, allyl glycidyl
ether, epichlorohydrin, glycerin, trimethylpropane, pentaerythrite,
sorbite, phthalic acid, succinic acid, fumaric acid, adipinic acid,
thiophene, ethyleneimine, hexamethylene adipamide, hexamethylene
sebacamide, hexamethylene dodecane diamide, aminobenzamide,
phenylene diamine, amide hydrazides, dimethyl piperazine,
benzimidazole, tetraaminobenzene, pyrones, .epsilon.-caprolactam,
isophthalic acid, glutaminic acid, leucine, phenyl alanine, valine,
lysine, urea, diisocyanate, thiourea and others or mixtures of the
above mentioned monomers. Furthermore, the following polymers can
be considered: silicones, cellulose and cellulose derivatives,
oils, polycarbonates, polyurethane, agarose, polysaccharides,
dextranes, starch, chitin, glycosamino glycans, gelatin, collagen
I-XII and other proteins.
DESCRIPTION OF THE FIGURES
[0169] FIG. 1 shows a disaccharide structure fragment of chitin
which can be transformed into chitosan by basic hydrolysis, or into
the compounds of the general formula Ia by partial deacetylation
and subsequent N-carboxyalkylation.
[0170] FIG. 2 shows a disaccharide structure fragment of chitosan,
which can be transformed into the compounds of the general formula
Ia by partial N-acylation and subsequent N-carboxyalkylation or by
partial N-carboxyalkylation and subsequent N-acylation.
[0171] FIG. 3 shows a tetrasaccharide unit of a heparin or heparan
sulphate with a random distribution of the sulphate groups and a
degree of sulfation of 2 per disaccharide unit as typical for
heparin (FIG. 3a). For comparison of the structural similarities
FIG. 3b shows an example of a compound according to the general
formula Ib and FIG. 3c shows a section with a typical structure for
a N-carboxymethylated, partially N-acetylated chitosan.
[0172] FIG. 4 shows the influence of an into a PVC-tube expanded,
surface modified stainless steel coronary stent on the platelet
loss (PLT-loss). An uncoated stainless steel coronary stent was
measured (uncoated) as reference. As zero value the level of the
platelet loss in case of the PVC-tube without stainless steel
coronary stent was set.
[0173] Thereby SH1 is a with heparin covalently coated stent, SH2
is a with chondroitinsulphate coated stent; SH3 is a stent coated
with polysaccharides gained from the erythrocytic glycocalix and
SH4 is a with Ac-heparin covalently coated stainless steel coronary
stent.
[0174] FIG. 5 shows a schematic presentation of the restenosis rate
of with completely desulphated and N-reacetylated heparin
(Ac-heparin) covalently coated stents and with oligo- and
polysaccharides of the erythrocytic glycocalix coated stents in
comparison to the uncoated stent and with polyacrylic acid (PAS)
coated stents after 4 weeks of implantation time in pork.
[0175] FIG. 6: Quantitative coronary angiography:
[0176] Images of the cross sections through the stent containing
vessel-segment of one with Ac-heparin coated stent (a.) and as
comparison of one uncoated (unco.) stent (b.). After four weeks in
the animal experiment (pork) a clear difference in the thicknesses
of the formed neointimas can be observed.
[0177] FIG. 7: Elution plot of paclitaxel from the stent (without
support medium).
[0178] FIG. 8: Elution diagram of paclitaxel embedded into
PLGA-matrix.
[0179] FIG. 9: Elution diagram of paclitaxel embedded into
PLGA-matrix and of a layer of undiluted paclitaxel which covers the
basis coating completely.
[0180] FIG. 10: Elution diagram of a hydrophilic active agent
embedded into the matrix and of a suprajacent active agent free
polymer (topcoat) which covers the basis coating completely for
diffusion control.
[0181] FIG. 11: Elution diagram of colchicine from PLGA-matrix.
[0182] FIG. 12: Elution diagram of simvastatin from
PLGA-matrix.
[0183] FIG. 13: Elution diagram of a statin from the matrix with
polystyrene which completely covers the basis coating as diffusion
controlling layer.
[0184] FIG. 14: Comparison of the thrombocyte number (platelet
number) in the blood after Chandler loop between coated (coat.) and
non coated (unco.) stent as regards the empty tube (control), the
platelet number of freshly extracted blood (donor) and after the
storage of 60 min in the syringe (syringe 60).
[0185] FIG. 15: Comparison of the platelet factor 4 concentration
in the freshly extracted blood (donor), in the empty tube (control)
after 60 minutes and non coated stents (unco.) with coated (coat.)
stent.
[0186] FIG. 16: Comparing diagram to the activated complement
factor C5a in the freshly extracted blood (donor), in the empty
tube (control) after 60 minutes and non coated (unco.) stents with
coated (coat.) stent.
[0187] FIG. 17: Schematic presentation of the %-diameter restenosis
rate of with completely desulphated and N-reacetylated heparin
(Ac-heparin) covalently coated stents and with a 2.sup.nd layer of
poly(D,L-lactide-co-glycolide) in comparison to the uncoated stent
(after 12 weeks of implantation time in the pork). The
chronological progression of the stenosis formation in the case of
PLGA is shown, whereas "%-diameter restenosis rate" represents the
diameter of the vessel related percentually to the initial state
directly after implantation of the stent (post). For the
experiments six to nine month old domestic porks were used, the
vessel diameter was measured before (pre) and after the
implantation of the stent (Post) via intravascular ultrasound
(IVUS). After one week (1 WoFUP), one month (4 WoFUP), six weeks (6
WoFUP) and after three months (12 WoFUP) the stented areas were
examined respectively via coronary angiography and with
intravascular ultrasound (IVUS). The obtained data show an
unexpected amazingly positive effect, which is due to the coating
beyond doubt. Although the values of stenosis after three months
hardly differ for the uncoated stent and for the coated stent, the
reaction of the vessel wall towards the PLGA-coated stent is
substantially smoother. After one week the stenosis value lies with
6% significantly below the value of the uncoated implants with
10.4%. The masking of the metal surface results after four weeks
even with 10% (an increase of 33%) in a more than factor two lower
stenosis rate than the uncoated stent, which reaches after this
period of time its maximum value of 22.6% (an increase of 54%). The
coated stent shows a maximum after six weeks with only 12.33%.
After 12 weeks the values of both systems equal each other with
approx. 11%.
[0188] FIG. 18: Pictures of the quantitative coronary angiography
of the animal experiments respectively to FIG. 17 after 1 week, 4
weeks, 6 weeks and 3 months of hemocompatibly supplied PLGA-coated
stents in the pig.
EXAMPLES
Example 1
[0189] Preparation of desulphated reacetylated heparin:
[0190] 100 ml of amberlite IR-122 cation exchange resin were filled
into a column having a diameter of 2 cm, transformed into the
H.sup.+-form with 400 ml 3M HCl and washed with distilled water
until the eluate was free from chloride and pH neutral. 1 g of
sodium heparin was dissolved in 10 ml of water, put onto the
cation-exchange column and eluted with 400 ml of water. The eluate
was allowed to drop into a receiver with 0.7 g of pyridine and
subsequently titrated with pyridine to pH 6 and freeze-dried.
[0191] 0.9 g of heparin pyridinium salt were added to 90 ml of a
6/3/1 mixture of DMSO/1,4-dioxane/methanol (v/v/v) in a round
bottomed flask with reflux cooler and heated to 90.degree. C. for
24 hours. Then, 823 mg of pyridinium chloride were added and
heating to 90.degree. C. was effected for further 70 hours.
Subsequently, dilution was carried out with 100 ml of water, and
titration to pH 9 with dilute soda lye was effected. The
desulphated heparin was dialyzed against water and
freeze-dried.
[0192] 100 mg of the desulphated heparin were dissolved in 10 ml of
water, cooled to 0.degree. C. and mixed with 1.5 ml of methanol
under stirring. To the solution, 4 ml of Dowex 1.times.4
anion-exchange resin in the OH.sup.--form and subsequently 150
.mu.l of acetic acid anhydride were added and stirred for 2 hours
at 4.degree. C. After that, the resin is filtrated, and the
solution is dialyzed against water and freeze-dried.
Example 2
[0193] N-carboxymethylated, Partially N-acetylated chitosan:
[0194] In 150 ml 0.1 N HCl, 2 g of chitosan were dissolved and
boiled under nitrogen for 24 hours under reflux. After cooling to
room temperature, the pH of the solution was adjusted to 5.8 with 2
N NaOH. The solution was dialyzed against demineralized water and
freeze-dried.
[0195] 1 g of the chitosan partially hydrolyzed this way was
dissolved in 100 ml of a 1% acetic acid. After adding 100 ml of
methanol, 605 .mu.l of acetic acid anhydride dissolved in 30 ml of
methanol were added and stirred for 40 minutes at room temperature.
The product was precipitated by pouring into a mixture of 140 ml of
methanol and 60 ml of a 25% NH.sub.3 solution. It was filtrated,
washed with methanol and diethyl ether and dried under vacuum over
night.
[0196] 1 g of the partially hydrolyzed and partially N-acetylated
chitosan was suspended in 50 ml of water. After adding 0.57 g of
glyoxylic acid monohydrate, the chitosan derivative dissolved
within the next 45 minutes. The pH value of the solution was
adjusted to 12 with 2 N NaOH. A solution of 0.4 g of sodium
cyanoboron hydride in as few water as possible was added and
stirred for 45 minutes. The product was precipitated in 400 ml of
ethanol, filtrated, washed with ethanol and dried in vacuum over
night.
Example 3
[0197] Synthesis of desulphated N-propionylated heparin:
[0198] 100 ml amberlite IR-122 cation exchange resin were added
into a column of 2 cm diameter, with 400 ml 3M HCl in the
H.sup.+-form converted and rinsed with distilled water, until the
eluate was free of chloride and pH neutral. 1 g sodium-heparin was
dissolved in 10 ml water, added onto the cation exchange column and
eluted with 400 ml of water. The eluate was added dropvise into a
receiver with 0.7 g pyridine and afterwards titrated with pyridine
to pH 6 and freeze-dried.
[0199] 0.9 g heparin-pyridinium-salt were added in a round flask
with a reflux condenser with 90 ml of a 6/3/1 mixture of
DMSO/1,4-dioxan/methano- l (v/v/v) and heated for 24 hours to
90.degree. C. Then 823 mg pyridiniumchloride were added and heated
additional 70 hours to 90.degree. C. Afterwards it was diluted with
100 ml of water and titrated with dilute sodium hydroxide to pH 9.
The desulphated heparin was dialyzed contra water and
freeze-dried.
[0200] 100 mg of the desulphated heparin were solved in 10 ml of
water, cooled to 0.degree. C. and added with 1.5 ml methanol under
stirring. To this solution were added 4 ml dowex 1.times.4 anion
exchange resin in the OH.sup.--form and afterwards 192 .mu.l of
propionic anhydride and stirred for 2 hours at 4.degree. C. Then
the resin was removed by filtration and the solution was dialyzed
contra water and freeze-dried.
Example 4
[0201] N-carboxymethylated, Partially N-propionylated chitosan:
[0202] In 150 ml 0.1 N HCl, 2 g of chitosan were dissolved and
boiled under nitrogen for 24 hours under reflux. After cooling to
room temperature, the pH of the solution was adjusted to 5.8 with 2
N NaOH. The solution was dialyzed against demineralized water and
freeze-dried.
[0203] 1 g of the chitosan partially hydrolyzed this way was
dissolved in 100 ml of a 1% acetic acid. After adding 100 ml of
methanol, 772 .mu.l of propionic acid anhydride dissolved in 30 ml
of methanol were added and stirred for 40 minutes at room
temperature. The product was precipitated by pouring into a mixture
of 140 ml of methanol and 60 ml of a 25% NH.sub.3 solution. It was
filtrated, washed with methanol and diethyl ether and dried under
vacuum over night.
[0204] 1 g of the partially hydrolyzed and partially N-acetylated
chitosan was suspended in 50 ml of water. After adding 0.57 g of
glyoxylic acid monohydrate, the chitosan derivative dissolved
within the next 45 minutes. The pH value of the solution was
adjusted to 12 with 2 N NaOH. A solution of 0.4 g of sodium
cyanoboron hydride in as few water as possible was added and
stirred for 45 minutes. The product was precipitated in 400 ml of
ethanol, filtrated, washed with ethanol and dried in vacuum over
night.
Example 5
[0205] Hemocompatibility Measurements of Compounds According to the
General Formula Ia and Ib by ISO 10933-4 (in Vitro
Measurements):
[0206] For the measurement of the hemocompatibility of the
compounds according to formulas Ia and Ib cellulose membranes,
silicon tubes and stainless steel stents were covalently coated
with a compound according to formula Ia and Ib and tested contra
heparin as well as contra the corresponding, in the single tests
utilised uncoated material surfaces.
[0207] 5.1. Cellulose Membranes (cuprophan) Coated With
desulphated, reacetylated heparin (Ac-heparin)
[0208] For the examination of the coagulatory physiologic
interactions between citrated whole blood and the Ac-heparin-resp.
heparin-coated cuprophan membranes the open perfusion system of the
Sakariassen-modified Baumgartner-chamber is used [Sakariassen K. S.
et al.; J. Lab. Clin. Med. 102: 522-535 (1983)]. The chamber is
composed of four building parts plus conical nipples and threaded
joints and is manufactured of polymethylmethacrylate and allows the
parallel investigation of two modified membranes, so that in every
run already a statistic coverage is included. The construction of
this chamber permits quasi_laminar perfusion conditions.
[0209] After 5 minutes of perfusion at 37.degree. C. the membranes
are extracted and after fixation of the adherent platelets the
platelet occupancy is measured. The respective results are set into
relation to the highly thrombogeneous subendothelial matrix as
negative standard with a 100% platelet occupancy. The adhesion of
the platelets takes place secondary before the formation of the
plasma protein layer on the foreign material. The plasma protein
fibrinogen acts as cofactor of the platelet aggregation. The such
induced activation of the platelets results in the bonding of
several coagulation associated plasma proteins, such as
vitronectin, fibronectin and von Willebrand-factor on the platelet
surface. By their influence finally the irreversible aggregation of
the platelets occurs. The platelet occupancy presents because of
the described interactions an accepted quantum for the
thrombogenity of surfaces in case of the foreign surface contact of
blood. From this fact the consequence arises: the lower the
platelet occupancy is on the perfunded surface the higher is the
hemocompatibility of the examined surface to be judged.
[0210] The results of the examined heparin-coated and
Ac-heparin-coated membranes show clearly the improvement of the
hemocompatibility of the foreign surface through the coating with
Ac-heparin. Heparin-coated membranes show a 45-65% platelet
occupancy, whilst Ac-heparin-coated surfaces show values from 0-5%
(reference to subendothelial matrix with 100% platelet
occupancy).
[0211] The adhesion of the platelets on the Ac-heparinated surface
is extremely aggravated due to the absent, for the activation of
platelets essential plasma proteins. Unlike to this the
heparin-coated surface with the immediately incipient plasma
protein adsorption offers optimal preconditions for activation,
deposition and aggregation of platelets, and ultimately the blood
reacts with the corresponding defense mechanisms to the inserted
foreign surface by activation of the coagulation cascade.
Ac-heparin fulfills by far superior than heparin the requirements
to the hemocompatibility of the foreign surface.
[0212] The interaction of plasma protein adsorption and platelet
occupancy as direct quantum for the thrombogenity of a surface, in
dependence of the to the blood offered coating, is made clear
especially well by this in-vitro test. Thus the utilisation of
covalently bound heparin as antithrombotic operant surface is only
strongly limited or not possible at all. The interactions of
immobilised heparin with blood revert themselves here into the
undesired opposite--the heparin-coated surface gets
thrombogeneous.
[0213] Obviously the outstanding importance of heparin as an
antithrombotic is not transferable to covalently immobilised
heparin. In the systemic application in dissolved form it can fully
unfold its properties. But if heparin is not covalently
immobilised, its antithrombotic properties, if at all, is only
short-lived. Different is the Ac-heparin ("No-affinity"-heparin),
that due to the desulphation and N-reacetylation in fact totally
loses the active antithrombotic properties of the initial molecule,
but acquires in return distinctive athrombogeneous properties, that
are demonstrably founded in the passivity versus antithrombin III
and the missing affinity towards coagulation initiating processes
and remain after covalent bonding.
[0214] Thereby Ac-heparin and thus the compounds of the general
formulas Ia and Ib in total are optimally suitable for the
camouflage of foreign surfaces in contact with the coagulation
system.
[0215] 5.2. Immobilisation on Silicone
[0216] Through a 1 m long silicon tube with 3 mm inside diameter
100 ml of a mixture of ethanol/water 1/1 (v/v) was pumped in a
circular motion for 30 minutes at 40.degree. C. Then 2 ml
3-(triethoxysilyl)-propylamine were added and pumped in a circular
motion for additional 15 hours at 40.degree. C. Afterwards it was
rinsed in each case for 2 hours with 100 ml ethanol/water and 100
ml water.
[0217] 3 mg of the deacetylated and reacetylated heparin
(Ac-heparin) were dissolved at 4.degree. C. in 30 ml 0.1 M
MES_buffer pH 4.75 and mixed with 30 mg CME-CDI
(N-cyclohexyl-N'-(2-morpholinoethyl
)carbodiimidemethyl-p-toluenesulphonate). This solution was pumped
in a circular motion for 15 hours at 4.degree. C. through the tube.
Afterwards it was rinsed with water, 4 M NaCl solution and water in
each case for 2 hours.
[0218] 5.3 Determination of the Platelet Number (EN30993-4)
[0219] In a 1 m long silicone tube with 3 mm inside diameter two 2
cm long formfitting glass tubes were placed. Then the tube was
closed with a shrinkable tubing to a circle and filled under
exclusion of air via syringes with a 0.154 M NaCl solution. In
doing so one syringe was used to fill in the solution and the other
syringe was used to remove the air. The solution was exchanged
under exclusion of air (bleb-free) with the two syringes against
citrated whole blood of a healthy test person.
[0220] Then the recess holes of the syringes were closed by pushing
the glass tubes over them and the tube was clamp taut into a
dialysis pump. The blood was pumped for 10 minutes with a flow rate
of 150 ml/min. The platelet content of the blood was measured
before and after the perfusion with a Coulter counter. For uncoated
silicone tubes the platelet loss was of 10%. In contrast to it the
loss was in silicon tubes, which were coated according to example
5.2, in average at 0% (number of experiments: n=3).
[0221] Also in this dynamic test system it is shown, that the
activation of platelets on an Ac-heparin coated surface is reduced.
Simultaneously it can be recorded, that the immobilisation of
heparin executes a negative effect on the hemocompatibility of the
utilised surface. Against it Ac-heparin shows, in accordance to its
passive nature, no effects in contact with the platelets.
[0222] 5.4 Whole Blood Experiments on 316 LVM Stainless Steel
Coronary Stents
[0223] In line with the biocompatibility experiments 31 mm long 316
LVM stainless steel stents were covalently coated with Ac-heparin.
In case of a total surface of 2 cm.sup.2 and a occupancy
coefficient of about 20 pm/cm.sup.2 stent surface the charging of
such a stent is about 0.35 .mu.g Ac-heparin. As comparison: in case
of thrombosis prophylaxis the usual daily application rate of
heparin is in contrast 20-30 mg and thus would correspond to the at
least 60.000 times the value.
[0224] These experiments were carried out at the Intitute of
Physiology at the RWTH Aachen with the established hemodynamic
Chandler loop-system [A. Henseler, B. Oedekoven, C. Andersson, K.
Mottaghy; KARDIOTECHNIK 3 (1999)]. Coated and uncoated stents were
expanded and tested in PVC tubes (medical grade PVC) with 600 mm
length and 4 mm inside diameter. The results of these experiments
confirm the according to the silicone tubes discussed experiments.
The initially to the stent attributed platelet loss in the
perfusate of 50% is reduced by the refinement of the stent surface
with Ac-heparin by more than 80%.
[0225] The influence of in the tube expanded, surface modified
coronary stents to the platelet loss is evaluated in further
Chandler tests during a 45 minute whole blood perfusion. For this
primarily the stent-free PVC tube is analysed, the outcome of this
is the zero value. The empty tube shows an average platelet loss of
27.4% regarding to the donor blood at a standard aberration of
solely 3.6%. This base value underlied different surface modified
stents are expanded in the PVC tubes and are analysed under
analogous conditions on the by them caused platelet loss.
[0226] It occurs also in this case, that the stent covered surface,
which solely accounts for about 0.84% of the total test surface,
causes a significant and reproducable effect on the platelet
content. According to the empty tube (base value) the analysis of
the polished, chemically not surface coated stent yields an
additional average platelet loss of 22.7%. Therewith causes this
compared to the PVC empty tube less than 1% measurable foreign
surface an approximately comparable platelet loss. A direct result
is that the medicinal stainless steel 316 LVM used as stent
material induces an about 100 times stronger platelet damage
compared to a medical grade PVC surface, although this test surface
only accounts for 0.84% of the total surface.
[0227] The analysed surface coatings on the stainless steel
coronary stents show to be able to reduce very clearly the enormous
dimension of the stent induced platelet damage (see FIG. 4). As
most effective proved with 81.5% the Ac-heparin (SH4).
[0228] If the effects of the Ac-heparin-coated stents on the
platelet loss are considered, then good congruent values result.
The correlation of the platelet loss in the perfusate resp. the
adhesion of the platelets to the offered surfaces show the
reliability of the measurements.
[0229] 5.4.1 Covalent Hemocompatible Coating of Stents
[0230] Not expanded stents of medicinal stainless steel LVM 316
were degreased in the ultrasonic bath for 15 minutes with acetone
and ethanol and dried at 100.degree. C. in the drying closet. Then
they were dipped for 5 minutes into a 2% solution of
3_aminopropyltriethoxysilane in a mixture of ethanol/water
(50/50:(v/v)) and then dried for 5 minutes at 100.degree. C.
Afterwards the stents were washed with demineralised water over
night.
[0231] 3 mg desulphated and reacetylated heparin were dissolved at
4.degree. C. in 30 ml 0.1 M MES-buffer
(2-(N-morpholino)ethanesulphonic acid) pH 4.75 and mixed with 30 mg
N-cyclohexyl-N'-(2-morpholinoethyl)car-
bodiimide-methyl-p-toluenesulphonate. In this solution 10 stents
were stirred for 15 hours at 4.degree. C. Then they were rinsed
with water, 4 M NaCl solution and water in each case for 2
hours.
[0232] 5.4.2 Determination of the Glucosamine Content of the Coated
Stents by HPLC
[0233] Hydrolysis: the coated stents are given in small hydrolysis
tubes and are abandoned with 3 ml 3 M HCl for exactly one minute at
room temperature. The metal probes are removed and the tubes are
incubated after sealing for 16 hours in the drying closet at
100.degree. C. Then they are allowed to cool down, evaporated three
times until dryness and taken up in 1 ml degassed and filtered
water and measured contra an also hydrolysated standard in the
HPLC:
4 desulphat. + desulphat. + desulphat. + reacet. reacet. reacet.
sample heparin area heparin heparin stent area [g/sample]
[cm.sup.2] [g/cm.sup.2] [pmol/cm.sup.2] 1 129.021 2.70647E-07 0.74
3.65739E-07 41.92 2 125.615 2.63502E-07 0.74 3.56084E-07 40.82 3
98.244 1.93072E-07 0.74 2.60908E-07 29.91 4 105.455 2.07243E-07
0.74 2.80058E-07 32.10 5 119.061 2.33982E-07 0.74 3.16192E-07 36.24
6 129.202 2.53911E-07 0.74 3.43124E-07 39.33 7 125.766 2.53957E-07
0.74 3.43185E-07 39.34
Example 6
[0234] In Vivo Examination of Coated Coronary Stents (FIG. 5)
[0235] 6.1. In Vivo Examinations of Coronary Stents Coated with
Ac-heparin
[0236] Due to the data on hemocompatibility, which Ac-heparin
yielded in the in-vitro experiments, the suitability of the
Ac-heparin surface as athrombogeneous coating of metal stents was
discussed in vivo (animal experiment). The target of the
experiments was primarily to evaluate the influence of the
Ac-heparin coating on the stent induced vessel reaction. Besides
the registration of possible thrombotic events the relevant
parameters for restenotic processes like neointima area, vessel
lumen and stenosis degree were recorded.
[0237] The animal experiments were carried out at the Inselspital
Bern (Switzerland) under the direction of Prof. Hess an accredited
cardiologist. For the examinations 6-9 month old domestic porks
were used, one for the validation of stents for a long time
established and approved animal model.
[0238] As expected in these experiments neither acute, subacute nor
late acute thrombotic events were registered, what may be assessed
as proof for the athrombogeneous properties of Ac-heparin.
[0239] After four weeks the animals were dispatched (euthanized),
the stented coronary artery segments extracted and
histomorphometrically analysed.
[0240] Indications to a possible acute or subchronic toxicity,
allergisation reactions or ulterior irritations as consequence of
the implantation of Ac-heparin coated stents are not observed
during the complete experimental phase, especially in the
histologic examination. During the stent implantation as well as
the follow-up coronary-angiographic data sets were ascertained,
which permit an interpretation with regard to the vessel reaction
to the stent implantation.
[0241] The difference between the uncoated control stent and the
Ac-heparin coated stent is unambiguous. The generation of a
distinct neointima layer is in case of the uncoated control stent
very well observable. Already after four weeks the proliferation
promotional effect of the uncoated stent surface on the surrounding
tissue occurs in such a degree, that ultimately the danger of the
vessel occlusion in the stent area is given.
[0242] Contrary in case of the Ac-heparin coated stents a clearly
thinner neointima layer is observed, which argues for a well
modulated ingrowth of the stent under maintenance of a wide, free
vessel lumen.
[0243] The detailed histomorphometric and coronary angiographic
data substantiate this conclusion, as it can be observed
congruently, that via the Ac-heparin coating (SH4) the neointima
hyperplasia ("restenosis") was repressed by about 17-20% in
comparison to the uncoated control stent. This result is unexpected
and remarkably at the same time. Surely it is not demanded of an
athrombogeneous surface to have an influence also on processes that
lead to a neointima hyperplasia, i.e. to prevent restenoses, in
addition to the preposition of hemocompatible characteristics.
[0244] On the one hand with a dense, permanent occupancy of the
stent surface with Ac-heparin a direct cell contact to the metal
surface is prevented. As in technical literature the emission of
certain metal ions into the implant close tissue is discussed as
one probable reason of restenosis, an anti-restenoic potency could
be founded by one of the coating caused prevention of a direct
metal contact.
[0245] On the other hand such a positive side effect is plausible,
because on a passive, athrombogenenous stent surface with the
absence of a platelet aggregation also the proliferative effects of
the thereby released growth factors are to be missed. Thus an
important, starting from the lumen side, stimulus of the neointimal
proliferation is omitted.
Example 7
[0246] In Vivo Experiments of Coronary Stents Coated with
Covalently Bound Ac-heparin an a Second Suprajacent Layer of PLGA
50/50
[0247] 7.1 Hemocompatibility of the Used Matrix in Vitro
[0248] For evaluation of the hemocompatibility of PLGA 50/50
stainless steel stents were coated with the polymer and tested at
the Intitute of Physiology of the RWTH Aachen by means of in vitro
whole blood tests. The used established and standardized Chandler
loop system is a dynamic closed tube system, which operates without
the use of a pump. Within the scope of the hemocompatibility
experiments whole blood, platelet factor 4 (PF4) and complement
factor 5a (C5a) were determined (see FIG. 14-16). For reasons of
comparison the uncoated stent was included.
[0249] Carrying Out of the Experiment:
[0250] Donor blood is taken up into 1.5 U/ml of heparin. The stents
are introduced into PVC tubes (I.D. 3.5 mm, L=95 cm) and fixed via
balloon catheter. The 4 tubes (K3-K6) and two empty tubes (L1, L2)
are filled in each case with 7.5 ml isotonic sodium chloride
solution and rotated for 15 minutes at 5 r/min at 37.degree. C. in
the Chandler loop. The completely emptied tubes are filled
carefully with heparinated donor blood (7.5 ml) and rotated for 60
min at 5 r/min. Accordingly to the anticoagulants samples are taken
in monovettes and sample jars respectively (PF4-CTAD, C5a-EDTA,
BB-EDTA) and processed.
[0251] The determination of the platelet number shows no
significant difference between the empty control tubes, the coated
and non coated stents. The released PF4 is in case of the coated
and non coated tubes at the same level. The determination of the
activated complement factor 5 (C5a) shows in case of the coated
stents a smaller activation as in case of the non coated
stents.
[0252] 7.2 Hemocompatibility of the Used Matrix in Vivo
[0253] The aim of the experiments was primarily to evaluate the
influence of the PLGA-coating towards the stent induced vessel
reaction. For the experiments 28 six to nine month old domestic
porks were used. The stented areas were examined after one week (1
WoFUP), one month (4 WoFUP), six weeks (6 WoFUP) and after three
months (12 WoFUP). The obtained data show an unexpected amazingly
positive effect, which is due to the the presence of PLGA 50/50
beyond doubt. Although the values of stenosis after three months
hardly differ for the uncoated stent and for the coated stent, the
reaction of the vessel wall towards the PLGA-coated stent is
substantially smoother. After one week the stenosis value lies with
6% significantly below the value of the uncoated implants with
10.4%. The masking of the metal surface results after four weeks
even with 10% (an increase of 33%) in a more than factor two lower
stenosis rate than the uncoated stent, which reaches after this
period of time its maximum value of 22.6% (an increase of 54%). The
coated stent shows a maximum after six weeks with only 12.33%.
After 12 weeks the values of both systems equal each other with
approx. 11% (FIG. 16).
[0254] The data for the restenotic processes were determined via
quantitative coronary angiography (QCA) and intravascular ultra
sound examinations (IVUS).
Example 8
[0255] Experiments Concerning the Coating of Surfaces with
Tacrolimus:
[0256] Pre-Experiments with toluidine Blue:
[0257] First pre-experiments are carried out with toluidine blue
(Aldrich) since tacrolimus can be detected chemically quite
difficult.
[0258] Chemicals:
5 stainless steel tubes LVM 316 2.5 cm in length, 2 mm in diameter
polylactide Fluka, Lot. 398555/123500, HNo. 0409 toluidine blue
Aldrich, Lot. 19,816-1, HNo. 0430 PBS-buffer pH 7.4 14.24 g
Na.sub.2HPO.sub.4, 2.72 g NaH.sub.2PO.sub.4, 9 g NaCl
[0259] Realization:
[0260] The stent is weighed out on the analytical balance and the
weight is noted. In a small hydrolysis tube 0.5 g polylactide are
dissolved in 2 ml of CHCl.sub.3. Therefore, it is heated to
65.degree. C. in the water bath. The solution is cooled down in the
freezing compartment. Thereto are added 200 .mu.g toluidine blue in
200 .mu.l of CHCl.sub.3. The stent is dipped into this solution.
After a couple of minutes the stent is taken out of the solution
with tweezers and moved within the fume hood until the solvent
evaporates. Then the stent is dipped in for a second time. After
air drying the stent is freeze dried for about 10 min. After the
drying the stent is balanced again. The amount of the immobilized
polylactide with toluidine blue is measured from the weight
difference (sample 1).
[0261] This experiment is repeated another time with the same
solution (sample 2).
[0262] For sample 3 the dipping solution (1.93 ml) which results
from experiment 1 (sample 1) and experiment 2 (sample 2) is mixed
with 0.825 mg toluidine blue in 0.825 ml of CHCl.sub.3 and 250 mg
polylactide. The polylactide is dissolved during heating. Then a
stent is dipped into it two times as described above.
[0263] Results:
[0264] The untreated stents had a weight of 176.0 mg and 180.9 mg.
After dipping into the polylactide solution the stents balanced
200.9 and 205.2 mg.
[0265] The dipping solution contains 500 mg polylactide and 200
.mu.g toluidine blue. The bound amount of toluidine blue can be
measured for the samples 1 and 2 from this ratio. In case of sample
3 2.755 ml solution contain 1 mg toluidine blue and 638.6 mg
polylactide (initial weight-consumption sample 1+2; approx. 50 mg).
Here two stents are given into one preparation to obtain higher
absorptions. As the dipping solution was very viscous which yielded
a very thick coating it was diluted from 2.625 ml with chloroform
to 4 ml.
[0266] Concentrations in the Dipping Solution:
6 sample volume (ml) c (polylactide mg/ml) c (toluidine blue
.mu.g/ml) 1 2.2 227.3 90.9 2 2.2 227.3 90.9 3 2.755 231.8 363.0 4
4.0 134.5 212.5
[0267] Weight of the Tubes and the Resultant Measured Coating:
7 sample net weight total weight PL & toluidine blue Toluidine
blue 1 176.0 mg 200.9 mg 24.9 mg 9.96 .mu.g 2 180.9 mg 205.2 mg
24.3 mg 9.72 .mu.g 3 317.2 mg 410.8 mg 93.6 mg 135.73 .mu.g 4 180.8
mg 194.8 mg 14.8 mg 23.38 .mu.g
Example 9
[0268] Elution Behavior of the Coatings with Different
Concentrations:
[0269] As pre-experiment a UV-Vis spectra of a toluidine blue
solution in ethanol is taken (c=0.1 mg/ml) and the absorption
maximum is determined. The toluidine blue concentration in the
solution is measured at an absorption maximum of 627 nm. Thereto a
calibration curve is generated.
[0270] A stent is hung into a beaker with 25 ml of physiological
sodium chloride solution in a phosphate buffer pH 7.4 (14.24 g
NaH.sub.2PO.sub.4, 2.72 g K.sub.2HPO.sub.4 and 9 g NaCl) and
stirred gently at room temperature. After 0.5, 1, 2, 3, 6, 24, 48
and 120 hours, each time a sample of 3 ml is taken, measured
spectroscopically and given back into the preparation.
8 c c c c time/ (ng/ (ng/ (ng/ (ng/ h abs. s1 ml) abs. s2 ml) abs.
s3 ml) abs. s4 ml) 0 0.0002 0 -0.0002 0 0.0036 0 0.0063 0 0.5
-0.0011 0 0.0011 6.4 0.0095 29.2 0.0125 30.7 1 0.0003 0.5 0.0014
7.9 0.0164 63.3 0.0121 28.7 2 0.0007 2.5 0.0008 5.0 0.0256 108.9
0.0131 33.7 3 -0.0004 0 0.0006 4.0 0.0294 127.7 0.0136 36.1 6
0.0013 5.4 0.0015 8.4 0.0333 147.0 0.0142 39.1 24 0.0017 7.4 0.0020
10.8 0.0527 246.0 0.0239 176 48/96 0.0024 10.9 0.0033 17.3 0.1096
524.8 0.0147 41.6 120 0.0017 7.4 0.0038 19.8 0.1110 531.7 0.0161
48.5
[0271] Absorption of the samples after different periods of time.
For measuring of the concentration the cuvette difference (abs. at
T=0) is subtracted from the measured value.
[0272] After 12 and 13 days respectively the experiment was
terminated. On all of the stents after the expiration of the
experiment a coating was still present. For determining the amounts
of toluidine blue and polylactide respectively which were
dissolved, the stents were rinsed with water and ethanol and then
freeze dried during 1 h for balancing them afterwards.
9 final initial PL + diss. PL + S. weight weight Tb Tb diss. Tb.
rem. Tb. 1 196.5 200.9 mg 24.9 mg 4.4 mg 1.76 .mu.g 8.2 .mu.g 2
199.4 205.2 24.3 mg 5.8 mg 2.32 .mu.g 3.48 .mu.g 3 385.4 410.8 93.6
mg 25.4 mg 36.83 .mu.g 98.8 .mu.g 4 191.3 194.8 14.8 mg 3.5 mg 5.52
.mu.g 17.86 .mu.g
[0273] In case of concentrations of 90 .mu.g toluidine blue per ml
dipping solution the released amounts of toluidine blue are so low
that the absorptions are at the detection limit of the
spectrometer. In case of a concentration of 200 .mu.g/ml the values
are after a couple of hours in the measurable area. It is
recommended for the measurement to place two samples into a beaker
(elution jar) to yield higher absorptions. In case of the highest
polylactide/toluidine blue concentration a saturation effect seems
to appear. While the elution ratio in case of the thinner samples
has an almost linear trajectory. On all of the stents the coating
can still be detected after several days.
[0274] After approx. 2 weeks the bound toluidine blue dissolved in
average from about 1/4-1/5. Hence it results that the samples still
would have eluted toluidine blue for approx. 8 to 10 weeks.
[0275] The dipping solution may not be too thick and should be
cooled so that the chloroform cannot evaporate too fast during the
extraction as else the thickness of the coating becomes too large
and inhomogeneous. Here the polylactide concentration in sample 4
(134 mg/ml) seems to be sufficient, above all in case of higher
concentrations the solution becomes extremely viscous and the
polylactide is only very difficult to dissolve.
Example 10
[0276] Coating of the Stents Via the Spraying Method:
[0277] The according to example 1 and example 2 pre-prepared not
expanded stents are balanced and horizontally hung onto a thin
metal bar (d=0.2 mm) which is stuck on the rotation axis of the
rotation and feed equipment and rotates with 28 r/min. The stents
are fixed in such way, that the interior of the stents does not
touch the bar. At a feeding amplitude of 2.2 cm and a feeding
velocity of 4 cm/s and a distance of 6 cm between stent and spray
nozzle, the stent is sprayed with the respective spray solution.
After the drying (about 15 minutes) at room temperature and
proximately in the fume hood over night it is balanced again.
Example 11
[0278] Coating of the Stents with Pure Matrix:
[0279] Preparation of the Spray Solution:
[0280] 176 mg polylactide is balanced and replenished with
chloroform to 20 g.
[0281] The stents are sprayed in each case with 3 ml of the
spraying solution, balanced before and after the spraying and the
yielding layer thickness is determined by measuring under the
microscope 100-times magnified.
10 layer stent No. before coating after coating weight of coating
thickness 1 0.0193 g 0.0205 g 1.2 mg 10.4 .mu.m 2 0.0193 g 0.0205 g
1.2 mg 10.4 .mu.m 3 0.0204 g 0.0216 g 1.2 mg 10.4 .mu.m 4 0.0206 g
0.0217 g 1.1 mg 10.4 .mu.m
Example 12
[0282] Coating of the Stents with Pure Active Agent:
[0283] Preparation of the spray solution: 44 mg taxol are dissolved
in 6 g chloroform.
[0284] The stents are balanced before and after the spraying.
11 stent No. before coating after coating weight of coating 1
0.0194 g 0.0197 g 0.30 mg
Example 13
[0285] Determination of the Elution Behaviour in PBS-Buffer:
[0286] Each stent placed in a sufficiently small flask, 2 ml
PBS-buffer is added, sealed with parafilm and incubated in the
drying closet at 37.degree. C. After expiry of the chosen time
intervals in each case the supernatant is depipetted and its UV
absorption at 306 nm is measured.
Example 14
[0287] Coating of the Hemocompatibly Equipped Stents with an Active
Agent Loaded Matrix (FIG. 7):
[0288] Spray solution: Polylactide RG502/taxol--solution is
replenished from 145.2 mg polylactide and 48.4 mg taxol to 22 g
with chloroform.
12 weight weight weight of active layer spray before weight of
active agent thick- stent solution (g) after (g) coating agent
.mu.g/mm.sup.2 ness 1 0.8 ml 0.02180 0.02215 0.35 mg 146 .mu.g 1.97
7.9 .mu.m 2 0.8 ml 0.02105 0.02142 0.37 mg 154 .mu.g 2.08 6.7 .mu.m
3 0.8 ml 0.02247 0.02285 0.38 mg 158 .mu.g 2.14 9.8 .mu.m 4 0.8 ml
0.02395 0.02432 0.37 mg 154 .mu.g 2.08 11.0 .mu.m 5 0.8 ml 0.02247
0.02286 0.39 mg 163 .mu.g 2.20 9.1 .mu.m 6 0.8 ml 0.02442 0.02482
0.40 mg 167 .mu.g 2.26 12.2 .mu.m
Example 15
[0289] Coating of the Stents with an Active Agent Loaded Matrix and
an Active Agent as Topcoat (FIG. 8):
[0290] Basis coat: 19.8 mg polylactide and 6.6. mg taxol are
replenished with chloroform to 3 g.
[0291] Topcoat: 8.8 mg taxol are replenished with chloroform to 2
g.
13 weight weight weight of active layer spray before weight of
active agent thick- stent solution (g) after (g) coating agent
.mu.g/mm.sup.2 ness 1 0.85 ml 0.0235 0.0238 0.30 mg 131 .mu.g 1.56
9.7 .mu.m 2 0.85 ml 0.0260 0.0264 0.40 mg 175 .mu.g 2.09 10.1
.mu.m
Example 16
[0292] Coating of the Stents with a Polylactide which Contains a
Hydrophilic Active Agent and with an Active Agent Free Matrix as
Topcoat (FIG. 9):
[0293] Spray Solutions:
[0294] Basis coating: 22 mg polylactide and 22 mg hydrophilic
active agent are balanced and replenished with chloroform to 5
g.
[0295] Topcoat: 22 mg polylactide and 22 mg polystyrene are
balanced and replenished with chloroform to 5 g.
14 weight of weight of spray solution before coating after coating
coating active agent 0.85 ml 0.0135 g 0.0143 g 0.8 mg 200 .mu.g
* * * * *