U.S. patent application number 10/987771 was filed with the patent office on 2005-06-16 for 42-o-alkoxyalkyl rapamycin derivatives and compositions comprising same.
This patent application is currently assigned to Sun Biomedical, Ltd.. Invention is credited to Betts, Ronald E., Savage, Douglas R., Shulze, John E..
Application Number | 20050131008 10/987771 |
Document ID | / |
Family ID | 34552458 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050131008 |
Kind Code |
A1 |
Betts, Ronald E. ; et
al. |
June 16, 2005 |
42-O-alkoxyalkyl rapamycin derivatives and compositions comprising
same
Abstract
42-O-alkoxyalkyl derivatives of rapamycin having biological
activity are described. Compositions and delivery devices
comprising the 42-O-alkoxyalkyl rapamycin derivatives are also
disclosed.
Inventors: |
Betts, Ronald E.; (La Jolla,
CA) ; Savage, Douglas R.; (Del Mar, CA) ;
Shulze, John E.; (Rancho Santa Margarita, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
Sun Biomedical, Ltd.
|
Family ID: |
34552458 |
Appl. No.: |
10/987771 |
Filed: |
November 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10987771 |
Nov 12, 2004 |
|
|
|
10706055 |
Nov 12, 2003 |
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Current U.S.
Class: |
514/291 ;
540/456 |
Current CPC
Class: |
C07D 498/18 20130101;
A61P 37/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
514/291 ;
540/456 |
International
Class: |
A61K 031/4745; C07D
491/14 |
Claims
It is claimed:
1. A compound of the form: 6wherein R is R.sup.a--O--R.sup.b, where
R.sup.a is C.sub.2-6 alkylene and R.sup.b is C.sub.1-5 alkyl, and
where the number of carbon atoms in the sum of R.sup.a and R.sup.b
is 7 or fewer.
2. The compound according to claim 1, wherein R is of the form
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.mH, where n is from 2 to 6
and m is from 1 to 5.
3. The compound according to claim 2, wherein n is 2-5 and m is
1-4.
4. The compound according to claim 2, wherein n is 2 and m is 1 or
2.
5. The compound according to claim 2, wherein n is 2 and m is
1.
6. The compound according to claim 2, wherein n is 2 and m is
2.
7. The compound according to claim 1, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 6 or fewer.
8. The compound according to claim 1, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 5 or fewer.
9. The compound according to claim 1, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 4 or fewer.
10. A composition comprising a compound according to claim 1
together with a carrier.
11. The composition according to claim 10, wherein said carrier is
a pharmaceutical preparation having the form of an ointment or a
gel.
12. The composition according to claim 10, wherein said carrier is
comprised of polymer microparticles.
13. The composition according to claim 10, wherein said carrier is
a pharmaceutical preparation having the form of a liquid, tablet,
or suppository.
14. The composition according to claim 10 wherein said carrier is a
stent.
15. The composition according to claim 14 wherein said stent is
formed of metal or polymer.
16. The composition according to claim 15 wherein said stent is
formed of a biodegradable polymer.
17. The composition according to claim 15, wherein said stent is
metal and said compound is carried directly on the surface of the
stent.
18. The composition according to claim 15, where said compound is
carried in a polymer layer in contact with said stent.
19. The composition according to claim 16, where said compound is
carried in a polymer layer in contact with said stent.
20. A stent for use in treating restenosis, comprising an
expandable stent body; and carried on said stent body for release
therefrom at a controlled rate, a compound of the form 7wherein R
is R.sup.a--O--R.sup.b, where R.sup.a is C.sub.2-6 alkylene and
R.sup.b is C.sub.1-5 alkyl, and where the number of carbon atoms in
the sum of R.sup.a and R.sup.b is 7 or fewer.
21. The stent according to claim 20, wherein R is of the form
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.mH, where n is from 2 to 6
and m is from 1 to 5.
22. The stent according to claim 21, wherein n is 2-5 and m is
1-4.
23. The stent according to claim 21, wherein n is 2 and m is 1 or
2.
24. The stent according to claim 21, wherein n is 2 and m is 1.
25. The stent according to claim 20, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 6 or fewer.
26. The stent according to claim 20, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 5 or fewer.
27. The stent according to claim 20, wherein the number of carbon
atoms in the sum of R.sup.a and R.sup.b is 4 or fewer.
28. The stent according to claim 20, wherein said stent body is
comprised of metal or polymer.
29. The stent according to claim 28, wherein said stent body is
comprised of a biodegradable polymer.
30. The stent according to claim 20, wherein said stent further
includes a polymer layer in contact with said stent body and said
compound incorporated into said polymer layer.
31. The stent according to claim 30, wherein said polymer layer is
comprised of a biodegradable polymer.
32. The stent according to claim 30, further comprising a polymer
underlayer disposed between the stent body and the polymer
layer.
33. The stent according to claim 20, wherein said stent body has a
surface, and wherein said surface is treated to enhance adhesion of
said compound relative to a stent surface with no treatment.
34. The stent according to claim 33, wherein said stent surface is
treated with a nitric acid solution.
35. The stent according to claim 33, wherein said stent surface is
treated by a process selected from sand blasting, laser etching,
and chemical etching.
36. The stent according to claim 20, wherein said compound is
applied to the stent from a solution of the compound in an organic
solvent.
37. The stent according to claim 36, where a membrane is applied
over the compound to control bioavailability of the compound.
38. The stent according to claim 36, where a polymer underlayer is
in contact with the stent, and the compound/polymer film is in
contact with the polymer underlayer.
39. The stent according to claim 38, where the polymer underlayer
is polytetrafluoroethylene (Teflon) or poly(dichloro-para-xylylene)
(Parylene).
40. The stent of claim 37, where the membrane is a polymer
membrane.
41. The stent according to claim 36, wherein said solution is
applied to the stent by a technique selected from the group
consisting of brushing, spraying, dipping, and flowing.
42. The stent according to claim 36, wherein said compound forms a
glassy layer on the stent.
43. The stent according to claim 36, wherein said solution is
comprised of between about 2 and 60% by weight compound, remainder
solvent.
44. The stent according to claim 43, wherein said solvent is ethyl
acetate.
45. A method of treating a condition responsive to treatment by
rapamycin, comprising administering a compound according to claim
1.
46. A method of treating restenosis, comprising providing a stent
according to claim 20.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/706,055 filed Nov. 12, 2003, now pending,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to 42-O-alkoxyalkyl
derivatives of rapamycin and to compositions comprising
42-O-alkoxyalkyl rapamycin derivatives.
BACKGROUND OF THE INVENTION
[0003] Rapamycin is a macrocyclic triene compound that was
initially extracted from a streptomycete (Streptomyces
hygroscopicus) isolated from a soil sample from Easter Island
(Vezina et al., J. Antibiot. 28: 721 (1975); U.S. Pat. Nos.
3,929,992; 3,993,749). Rapamycin has the structure depicted in
Formula I: 1
[0004] Originally described for use as an antifungal agent (U.S.
Pat. No. 3,929,992), it has subsequently found to be an effective
agent for other conditions and disorders, including use in the
treatment of cancer and tumors (U.S. Pat. No. 4,885,171), use for
the prevention of experimental immunopathies (experimental allergic
encephalitis and adjuvant arthritis; Martel, R., Can. J. Physiol.,
55: 48 (1977)), inhibition of transplant rejection (U.S. Pat. No.
5,100,899), and inhibition of smooth muscle cell proliferation
(Morris, R., J. Heart Lung Transplant, 11 (pt. 2) (1992)).
[0005] The numbering convention for rapamycin has been recently
changed, and under the revised Chemical Abstracts nomenclature,
what was formerly the 40-position is now the 42-position and the
former 28-position is now the 31-position.
[0006] The utility of the compound as a pharmaceutical drug has
been restricted by its very low and variable bioavailability and
its high toxicity. Also, rapamycin is only very slightly soluble in
water, i.e., 20 micrograms per milliliter, making it difficult to
formulate into stable compositions suitable for in vivo delivery.
To overcome these problems, prodrugs and derivatives of the
compound have been synthesized. Water soluble prodrugs prepared by
derivatizing rapamycin positions 31 and 42 (formerly positions 28
and 40) of the rapamycin structure to form glycinate, propionate,
and pyrrolidino butyrate prodrugs have been described (U.S. Pat.
No. 4,650,803). The numerous derivatives of rapamycin described in
the art include monoacyl and diacyl derivatives (U.S. Pat. No.
4,316,885), acetal derivatives (U.S. Pat. No. 5,151,413), silyl
ethers (U.S. Pat. No. 5,120,842), hydroxyesters (U.S. Pat. No.
5,362,718), as well as alkyl, aryl, alkenyl, and alkynyl
derivatives (U.S. Pat. Nos. 5,665,772; 5,258,389; 6,384,046; WO
97/35575).
[0007] One of the shortcomings of many of the prodrugs and
derivatives of rapamycin is the complicated synthesis involved in
preparing the prodrug or derivative, where additional synthetic
steps are required to protect and deprotect certain positions.
Also, care must be taken in designing prodrugs and derivatives to
preserve activity of the compound and to not sterically hinder
positions necessary for protein binding or other cellular
interactions. Derivatives having a shorter overall chain length
and/or overall steric bulk (volume) in the chemical moiety attached
to the compound are less likely to produce steric hinderance of
binding sites. It would be desirable to design a derivative that
has a shorter chain length or smaller size in the attached
moiety.
[0008] One of the recent therapeutic uses of rapamycin and its
derivatives has been treatment of restenosis. Restenosis after
percutaneous transluminal coronary angioplasty (PTCA) remains one
of its major limitations (Hamon, M. et al., Drug Therapy, 4:
291-301 (1998)). The occurrence of restenosis after initial PTCA is
between 30 and 50%, despite initial success (Bauters, C. et al.,
Am. Coll. Cardiol., 20: 845-848 (1992); Bauters C. et al., Eur.
Heart J., 16: 3348 (1995)). Restenosis after PTCA is thought to be
a two component process of both intimal hyperplasia and vascular
remodeling, the former coming initially, the latter occurring later
in the process (Hoffman, R. et al., Circulation, 94: 1247-1254
(1996); Oesterle, S. et al., Am. Heart J., 136: 578-599
(1998)).
[0009] One strategy to eliminate or reduce restenosis is to limit
the process of vascular remodeling. This can be accomplished by
placing a stent in the lumen of the vessel after PTCA. Coronary
stents are small metal tubular implants that are being extensively
used to prevent acute reclosure or collapse of vessels following
angioplasty. Currently, stents are routinely placed in 70 to 80% of
all interventional cases.
[0010] In many cases this strategy works, however, the problem of
restenosis is yet to be fully understood or conquered (Hamon, M. et
al., Drug Therapy, 4: 291-301 (1998); Oesterle, S. et al., Am.
Heart J., 136: 578-599 (1998)) The injury caused by angioplasty and
stent placement often causes excessive healing response, including
thrombosis and rapid cell proliferation inside the stent, leading
to eventual reclosure of the vascular channel. There remains a need
to solve the eventual renarrowing of the lumen inside the stent
(i.e. restenosis) after angioplasty and stent placement experienced
by many patients.
SUMMARY OF THE INVENTION
[0011] Accordingly, it is an object of the invention to provide a
compound for treatment of conditions responsive to treatment by
rapamycin. More specifically, it is an object of the invention to
provide an ether-like derivative of rapamycin that possess
immunosuppressive, antifungal, anti-tumor, and/or anti-inflammatory
activity in vivo and therefore useful in the treatment of
transplantation rejection, infectious diseases, autoimmune
diseases, and conditions characterized by excessive cell
proliferation.
[0012] It is another object of the invention to provide a
composition comprising a 42-O-alkoxyalkyl derivative of
rapamycin.
[0013] It is still another objective of the invention to provide
derivatives of rapamycin that are synthetically prepared with ease
relative to other rapamycin derivatives.
[0014] It is yet another objective of the invention to provide a
stent capable of eluting 42-O-alkoxyalkyl derivative of
rapamycin.
[0015] In one aspect, the invention includes a compound of the
form: 2
[0016] wherein R is R.sup.a--O--R.sup.b, where R.sup.a is C.sub.2-6
alkylene and R.sup.b is C.sub.1-5 alkyl, and where the number of
carbon atoms in the sum of R.sup.a and R.sup.b is 7 or fewer. In
various embodiments, R is of the form
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.mH, where n is from 2 to 6
and m is from 1 to 5; n is 2-5 and m is 1-4; n is 2 and m is 1 or
2; n is 2 and m is 1; and n is 2 and m is 2. In other embodiments,
the number of carbon atoms in the sum of R.sup.a and R.sup.b is 6
or fewer, preferably 5 or fewer, more preferably 4 or fewer.
[0017] In another aspect, the invention includes a composition
comprising a compound as described above, together with a carrier.
In various embodiments, the carrier is a pharmaceutical preparation
having the form of an ointment or a gel, polymer microparticles, or
a pharmaceutical preparation having the form of a liquid.
[0018] In a preferred embodiment, the carrier is a stent. The stent
can be formed of metal or polymer, including biodegradable
polymers. When the carrier is a stent, in one embodiment the drug
compound can be carried directly on the surface of the stent.
Alternatively, the compound is carried in a polymer layer in
contact with the stent.
[0019] In another aspect, the invention includes a stent for use in
treating restenosis. The stent is comprised of an expandable stent
body; and carried on the stent body for release therefrom at a
controlled rate, is a compound having the form given above. The
stent body can be formed of metal or polymer, including a
biodegradable polymer.
[0020] In one embodiment, the stent further includes a polymer
layer in contact with the stent body and the compound is
incorporated into the polymer layer. The polymer layer can be
comprised of a biodegradable polymer or a non-biodegradable
polymer.
[0021] In another embodiment, the stent includes a polymer
underlayer disposed between the stent body and the compound or
polymer layer.
[0022] In other embodiment, the surface of the stent body is
treated to enhance adhesion of the drug compound, relative to a
stent surface with no treatment. For example, in one embodiment,
the stent surface is treated with a nitric acid solution. In other
alternative embodiment, the stent surface is treated by a process
such as sand blasting, laser etching, or chemical etching.
[0023] In other embodiment, the compound is applied to the stent
surface from a solution of the compound in an organic solvent.
[0024] The stent can also include a membrane applied over the
compound to control bioavailability of the compound. For example, a
membrane formed of a polymer material and placed over the
drug-coated stent is contemplated to control the release rate of
the compound.
[0025] In another embodiment, the stent includes a polymer
underlayer in contact with the stent surface, and the
compound/polymer film is in contact with the polymer underlayer.
That is, the underlayer is disposed between the stent body and the
drug-laden polymer layer. Exemplary polymer underlayers are
polytetrafluoroethylene (Teflon) and poly(dichloro-para-xylylene)
(Parylene).
[0026] In one embodiment, the compound is applied to the stent body
in the form of a solution of the compound in an organic solvent.
The solution is applied to the stent by a technique selected from
brushing, spraying, dipping, and flowing. In a preferred
embodiment, application of the compound is done in such a way as to
form a glassy layer of compound on the stent surface. Solutions
comprised of between about 2 and 60% by weight compound, remainder
solvent, are preferred. An exemplary organic solvent is ethyl
acetate.
[0027] In yet another aspect, the invention includes a method of
treating a condition responsive to treatment by rapamycin, by
administering a compound having the form described above. The
compound can be administered in the form of a carrier, such as
those described above.
[0028] In another aspect, the invention includes a method of
treating restenosis, comprising providing a stent, as described
above.
[0029] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows a representative MS fragmentation pattern of a
42-O-alkoxyalkyl rapamycin derivative, 42-O-(2-ethoxyethyl)
rapamycin;
[0031] FIG. 2 is a plot showing cellular proliferation of human
smooth muscle cells in vitro, expressed as the percentage of growth
relative to control cells, as a function of molar drug
concentration of 42-O-(2-ethoxyethyl) rapamycin (squares),
rapamycin (triangles, circles), and 42-O-(hydroxy heptyl) rapamycin
(diamonds);
[0032] FIGS. 3A-3C illustrate an endovascular stent having a
metal-filament body, and formed in accordance with one embodiment
of the present invention, showing the stent in its contracted (FIG.
3A) and expanded (FIG. 3B, FIG. 3C) conditions in side view (FIG.
3B) and cross-sectional view (FIG. 3C);
[0033] FIG. 4 shows the in vitro release rate of drug, in .mu.g,
into an ethanol-water release medium as a function of time, in
hours, from six stents, each carrying a poly-dl-lactic acid layer
loaded with (42-O-(2-ethoxyethyl) rapamycin;
[0034] FIGS. 5A-5I are computer-generated photomicrographs of
histology slides for a pig implanted with three
42-O-(2-ethoxyethyl) rapamycin eluting stents, the stents placed in
the left anterior descending artery (FIGS. 5A-5C), the left
circumflex artery (FIGS. 5D-5F), and the right coronary artery
(FIGS. 5G-51);
[0035] FIGS. 6A-6I are computer-generated photomicrographs of
histology slides for a pig implanted with three
42-O-(2-ethoxyethyl) rapamycin eluting stents, the stents placed in
the left anterior descending artery (FIGS. 6A-6C), the left
circumflex artery (FIGS. 6D-6F), and the right coronary artery
(FIGS. 6G-61);
[0036] FIGS. 7A-7C show the right coronary artery of a control
animal 30 days after implant of a bare, metal stent, where FIG. 7A
shows an image of the stent in place in the artery and FIGS. 7B-7C
show cross-sectional views of the stent at two different
magnifications;
[0037] FIGS. 8A-8C show the left anterior descending artery of a
control animal 30 days after implant of a bare, metal stent, where
FIG. 8A shows an image of the stent in place in the artery and
FIGS. 8B-8C show cross-sectional views of the stent at two
different magnifications;
[0038] FIGS. 9A-9C show the left circumflex artery of a control
animal 30 days after implant of a bare, metal stent, where FIG. 9A
shows an image of the stent in place in the artery and FIGS. 9B-9C
show cross-sectional views of the stent at two different
magnifications;
[0039] FIG. 10 is a plot of late loss, in mm, as a function of
injury score for 42-O-(2-ethoxyethyl) rapamycin eluting stents
(solid line), 42-O-hydroxy heptyl rapamycin eluting stents (dashed
line), and control (bare metal) stents (dotted line), 30 days after
implantation in pig vessels; and
[0040] FIG. 11 is a plot of late loss, in mm, as a function of
injury score for 42-O-(2-ethoxyethyl rapamycin eluting stents
(solid line), 42-O-hydroxy heptyl rapamycin (i.e.
everolimus)-eluting stents (dashed line), and control (bare metal)
stents (dotted line), 30 days after implantation in pig
vessels.
[0041] FIG. 12 shows a graph on frequency of restenosis with
increasing stent lengths. The data were obtained from the Sirius
Trial for the sirolimus-eluting stent (Cypher) as reported in 2002
at the Trans Catheter Therapeutics Scientific Sessions in
Washington D.C.
DETAILED DESCRIPTION OF THE INVENTION
[0042] I. Definitions
[0043] "Rapamycin" as used herein intends a compound of the
structure: 3
[0044] This compound is also known in the art as `sirolimus`.
[0045] A "42-O-alkoxyalkyl rapamycin derivative" refers to a
compound where the hydroxyl group at carbon number 42 in the
rapamycin compound is modified with a moiety of the form
(CH.sub.2).sub.n--O-- (CH.sub.2).sub.mH, where n is two (2) or more
and m is one (1) or more. Recently, the numbering convention for
rapamycin has changed, and under the revised Chemical Abstracts
nomenclature, what was formerly the 40-position is now the
42-position.
[0046] "Everolimus" intends a compound of the structure: 4
[0047] where R is CH.sub.2CH.sub.2OH (hydroxy ethyl).
[0048] The compound referred to herein as "42-O-hydroxy heptyl
rapamycin" refers to the structure shown for everolimus where the R
group is of the form (CH.sub.2).sub.7OH.
[0049] An "efficacious amount" or an "effective amount" intends a
dosage sufficient to provide treatment for the disorder or disease
state being treated. This will vary depending on the patient, the
disease, and the treatment being effected, but is readily
determined using clinical markers particular to the disorder or
disease of concern. For example, a cross-sectional area measurement
of the amount of new cells (i.e. tissue) growth inside a stent
after implantation and injury of the vessel wall by overexpansion
of the stent with a balloon catheter provides a clinical marker for
restenosis. A reduction or stabilization of tissue volume after
application of a dosage of active drug at a tumor site provides a
clinical marker for tumor treatment. A clinical marker relating to
organ transplant or vascular graft surgery would be to monitor
organ function or to monitor continued patency (i.e. freedom from
reocclusion or renarrowing) of transplant allografts. For skin
wounds, a clinical marker would be to watch for a change in
inflammation markers of redness, granuloma formation, or fibrosis.
For an enlarged prostate, a clinical marker would be to monitor for
any reduction in recurrence of ureter blockage.
[0050] II. 42-O-alkoxyalkyl Rapamycin Derivative Compounds
[0051] In one aspect, the invention provides rapamycin derivatives,
specifically 42-O-(alkoxyalkyl) rapamycin derivatives of the form:
5
[0052] where R is R.sup.a--O--R.sup.b, where R.sup.a is C.sub.2-6
alkylene and R.sup.b is C.sub.1-5 alkyl, and where the number of
carbon atoms in the sum of R.sup.a and R.sup.b is 7 or fewer. As
used herein, "alkylene" refers to a divalent alkyl group,
e.g.--CH.sub.2CH.sub.2-- (ethylene). Preferably, R.sup.a is
C.sub.2-5 alkylene and R.sup.b is C.sub.1-4 alkyl, and the sum of
the carbon atoms in R.sup.a and R.sup.b is 7 or fewer, preferably 6
or fewer, more preferably 5 or fewer. In R.sup.a and R.sup.b,
alkylene or alkyl groups having three or more carbons may be either
linear or branched or cyclic. When each of R.sup.a and R.sup.b is
linear, R may be represented by
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.mH, where n is from 2 to 6
and m is from 1 to 5.
[0053] In selected embodiments, n is 24 and m is 14. Preferably, m
is 1 or 2. In further selected embodiments, n is 2 (i.e., R.sup.a
is ethylene) and m is 1 or 2 (i.e., R.sup.b is methyl or ethyl). In
a particularly preferred embodiment, each of n and m is 2 (i.e.,
R.sup.a is ethylene and R.sup.b is ethyl), to give the compound
42-O-(ethoxyethyl) rapamycin.
[0054] The rapamycin 42-O-(alkoxyalkyl) derivatives of the
invention may be prepared by reaction of the 42-hydroxyl group of
rapamycin with a compound of the form L-R.sup.a--O--R.sup.b, where
R.sup.a and R.sup.b are as defined above and L is a leaving group.
Suitable leaving groups include, for example, halogens, such as
bromo or iodo, and sulfonates, such as tosylate, mesylate, or
trifluoromethane sulfonate (triflate). The derivative is formed by
displacement of the leaving group by the 42-hydroxyl group of
rapamycin, as illustrated in Example 1. A representative product
ion scan of 42-O-(2-ethoxyethyl) rapamycin obtained using tandem
mass spectrometry (Sciex AP14000) is shown in FIG. 1.
[0055] Compounds of the form L-R.sup.a--O--R.sup.b are readily
prepared from the corresponding alkoxy alcohols
HO--R.sup.a--O--R.sup.b, by reaction with, for example,
trifluoromethylsulfonic (triflic) anhydride, generally in the
presence of an amine catalyst such as lutidine, as described in
Example 1 below.
[0056] Many such alkoxy alcohols are commercially available. For
example, the compounds 2-ethoxy ethanol, 2-methoxy ethanol,
1-methoxy-2-propanol, 3-ethoxy-1-propanol, 2-isopropoxy ethanol,
1-methoxy-2-butanol, 3-methoxy-1-butanol, 2-propoxy ethanol,
2-butoxy ethanol, 3-methoxy-3-methyl-1-butanol, 3-propoxy propanol,
1-tert-butoxy-2-propano- l, 3-butoxy propanol, and propylene glycol
butyl ether are all available from Aldrich Corporation. With
respect to selected embodiments above, the subject compounds in
which n is 2 (i.e., R.sup.a is ethylene) and m is 1-2 (i.e.,
R.sup.b is methyl or ethyl) can be prepared using 2-methoxy ethanol
and 2-ethoxy ethanol, respectively. Preparation of
42-O-(2-methoxyethyl) rapamycin is described in Example 2, and
preparation of 42-O-(2-ethoxyethyl) rapamycin is described in
Example 1.
[0057] Alkoxy alcohols HO--R.sup.a--O--R.sup.b are commercially
available or can also be prepared from the corresponding diols
HO--R.sup.a OH by etherification with R.sup.b, preferably using a
process which gives predominantly or exclusively a monoderivatized
product. For example, Shanzer (Tet Lett. 21 (2), 221-2, 1980)
describes a process for efficient monoalkylation of diols by
reaction of a stannoxane intermediate, derived from the diol, with
an alkyl halide. Martinelli et al. describe monotosylation of diols
by reaction with p-toluenesulfonyl chloride and triethylamine in
the presence of catalytic Bu.sub.2SnO (J. Am. Chem. Soc. 124 (14):
3578-3585, 2002). The monotosylate could then be treated with an
alkoxide to give the alkoxy alcohol. The cited procedures are most
suitable for 1,2-diols; that is, where R.sup.a represents a
two-carbon chain separating the hydroxyl groups (as in the
preferred derivatives). A method for monoderivatization of longer
diols is given by McDougal (J. Org. Chem., 51: 3388 (1986)). A high
yield of tert-butyldimethylsilyl monosilyated product
(TBS--ORa--OH) is obtained from symmetric diols. This protected
product can then be activated with a suitable leaving group such as
triflate by reaction with trifluoromethane sulfonic anhydride and a
suitable base such as 2,6-lutidine. Reaction of the triflate
product with the appropriate alcohol (R.sup.b--OH) and 2,6-lutidine
followed by hydrolysis of the silyl protecting group with acid will
afford the alkoxy alcohol. In the case of non-symmetrical diols,
the less hindered terminus will generally react preferentially. In
any case, any bis-alkoxy side product (e.g.
R.sup.bO--R.sup.a--OR.sup.b) can easily be separated from the
desired alkoxy alcohol (HO--R.sup.a--OR.sup.b) by standard
purification procedures, such as chromatography.
[0058] Potency of 42-O-(2-ethoxyethyl) rapamycin was tested in
vitro and compared to the potency of another rapamycin derivative,
42-O-hydroxy heptyl rapamycin, and to the potency of rapamycin. The
testing procedure is described in Example 3 and the results are
shown in FIG. 2.
[0059] 42-O-(2-ethoxyethyl) rapamycin (squares) derivative
effectively inhibited growth of smooth muscle cells over five
orders of magnitude concentration, for both porcine and human
cells, as seen in FIG. 2. It was also observed that
42-O-(2-ethoxyethyl) rapamycin inhibited cellular growth as
effectively as 42-O-hydroxy heptyl rapamycin (diamonds) or
rapamycin (triangles, circles).
[0060] III. Compositions Comprising a 42-O-Alkoxyalkyl Rapamycin
Derivative
[0061] In another aspect, the invention includes a composition
incorporating a 42-O-(alkoxyalkyl) rapamycin derivative compound. A
wide variety of compositions and formulations are contemplated and
several specific examples will be discussed in more detail below.
In general, the composition serves as a sort of drug reservoir
which contains and releases the compound after application or
deposition of the composition at a target site.
[0062] A. Polymer Particles
[0063] An exemplary composition is a formulation of polymer
particles that are suitable for placement in vivo via injection or
via transport and deposition using a device, such as a catheter.
The polymer particles can be microporous, macroporous, or
non-porous and can be formed of a polymer that is capable of
retaining the desired 42-O-alkoxyalkyl rapamycin derivative
compound.
[0064] Porous polymer particles have interconnected pores which
open to the particle surface for communication between the exterior
of the particle and the internal pore spaces. Exemplary particles
for formation of such macroporous reservoirs are described, for
example, in U.S. Pat. No. 5,135,740, incorporated by reference
herein. In brief, porous particles are formed, for example, by
suspension polymerization in a liquid-liquid system. In general, a
solution containing monomers and a polymerization catalyst is
formed that is immiscible with water. An inert solvent miscible
with the solution but immiscible with water is included in the
solution. The solution is then suspended in an aqueous solution,
which generally contains additives such as surfactants and
dispersants to promote the suspension or emulsion. Once the
suspension is established with discrete droplets of the desired
size, polymerization is effected, typically by activating the
reactants by either increased temperature or irradiation. Once
polymerization is complete, the resulting solid particles are
recovered from the suspension. The particles are solid, spherical,
porous structures, the polymer having formed around the inert
liquid, thereby forming the pore network. The inert solvent, which
served as a porogen, or pore-forming agent, occupies the pores of
the particles. The porogen is subsequently removed.
[0065] The macroporous particles can also be prepared by solvent
evaporation, from either a biodegradable or a non-degradable
polymer. For the solvent-evaporation process, the desired polymer
is dissolved in an organic solvent and the solution is then poured
over a layer of sodium chloride crystals of the desired particle
size (Mooney, et al., J. Biomed. Mater Res. 37: 413-420, (1997)).
The solvent is removed, generally by evaporation, and the resulting
solid polymer is immersed in water to leach out the sodium
chloride, yielding a porous polymer reservoir. Alternatively sodium
chloride crystals can be dispersed in the polymer solution by
stirring to obtain a uniform dispersion of the sodium chloride
crystals. The dispersion is then extruded dropwise into a
non-solvent for the polymer while stirring to precipitate the
polymer droplets around the sodium chloride crystals. The solid
polymer particles are collected by filtration or centrifugation and
then immersed in water to leach out the sodium chloride, yielding a
porous polymer reservoir. It will be appreciated that alternatives
to sodium chloride include any non-toxic water soluble salt or low
molecular weight water soluble polymer which can be leached out to
produce the desired porosity.
[0066] The porous particles can be loaded with one or more drugs by
including the compounds in the polymer during particle formation or
by loading the particles post-particle formation. Post-particle
loading can be done by, for example, dissolving the drug compound
in a solvent that acts to solvate the drug but that is a nonsolvent
for the polymer and mixing by stirring the particles and the drug
solution. The solution of drug is absorbed by the particles to give
a free flowing powder. The particles may then be treated for
solvent removal, as needed.
[0067] Another exemplary polymer particle composition is non-porous
particles, such as microcapsule and microparticles having the
compound contained or dispersed therein. Both microcapsules and
microparticles are well known in the pharmaceutical and drug
delivery industries (see, for example, Baker, R. W., CONTROLLED
RELEASE OF BIOLOGICALLY ACTIVE AGENTS, John Wiley & Sons, NY,
1987; Ranade V. and Hollinger, M., DRUG DELIVERY SYSTEMS, CRC
Press, 1996). Microcapsules typically refer to a reservoir of
active agent surrounded by a polymer membrane shell. A
microparticle typically refers to a monolithic system where the
therapeutic agent(s) is dispersed throughout the particle. There
are, however, many formulations falling between these two
definitions, such as agglomerates of microcapsules, and such
formulations would also be suitable for use herein.
[0068] Microcapsules and microparticles can be prepared from
biodegradable or non-biodegradable polymers. Microcapsules are
readily formed by a number of methods, including coacervation,
interfacial polymerization, solvent evaporation, and physical
encapsulation methods (, Baker, R. W., CONTROLLED RELEASE OF
BIOLOGICALLY ACTIVE AGENTS, John Wiley & Sons, NY, 1987).
Microparticles are prepared by numerous techniques known in the
art, one simple way being to merely grind a polymer film containing
dispersed therapeutic agent into a suitable size. Spray drying
particulate therapeutic agent from a polymer solution is another
approach. Specific procedures for encapsulation of biologically
active agents are disclosed in U.S. Pat. No. 4,675,189 and U.S.
patent application No. 20010033868, which are incorporated by
reference herein.
[0069] Polymers suitable for particle formation are numerous and
varied; the general selection criterion being a polymer capable of
carrying a 42-O-alkoxyalkyl rapamycin derivative compound.
Exemplary polymers include, but are not limited to, poly(d,
l-lactic acid), poly(l-lactic acid), poly(d-lactic acid),
methacrylate polymers, such as polybutyl methacrylate and the like,
ethylene vinyl alcohol (EVOH), .epsilon.-caprolactone, glycolide,
ethylvinyl hydroxylated acetate (EVA), polyvinyl alcohol (PVA),
polyethylene oxides (PEO), polyester amides, and co-polymers
thereof and mixtures thereof. These polymers all have a history of
safe and low inflammatory use in the systemic circulation.
Typically, between 20-70 weight percent of polymer will be combined
with between 30-80 weight percent of the 42-O-hydroxy alkyl
substituted rapamycin compound to form the polymer composition.
[0070] The particles, whether porous or non-porous, may vary widely
in size, from about 0.1 micron to about 100 microns in diameter,
preferably from about 0.5 microns to about 40 microns. The
particles can be administered as neat particles, or can be
formulated in a gel, paste, ointment, salve, or viscous liquid for
application at the target site.
[0071] In another embodiment, polymer particles are admixed with
the rapamycin derivative and the mixture is applied to the surface
of a stent. The polymer particles in this embodiment serve to bind
the drug into a coating material and to control the release rate of
drug from the coating. Particles formed of waxes, lipids, short
chain polymers, such as propane and butane, are suitable.
[0072] As exemplified by the polymer particles, the polymer
composition of the invention is one capable of being dispensed or
placed at the target site, for contact of the polymer composition
with the tissue at the target site. Those of skill in the art will
appreciate that polymer particles are merely one example of a
composition that achieves contact with the target tissue. Polymers
capable of carrying a load of a hydrophobic compound can be
formulated into films, patches, pastes, salves, or gels, all of
which can be placed or dispensed at the target site. For example, a
simple polymer patch prepared from a polymer loaded with the
42-O-alkoxyalkyl rapamycin derivative compound can be placed on the
surface of tissue in need of treatment. Such a tissue surface can
be a vessel, an organ, a tumor, or an injured or wounded body
surface.
[0073] B. Mucoadhesive Polymer Composition
[0074] In another embodiment, the composition is comprised of a
polymer substrate having mucoadhesive properties, for placement
adjacent mucosal tissue. Mucosal sites in the body include the
cul-de-sac of the eye, buccal cavity, nose, rectum, vagina,
periodontal pocket, intestines and colon. Mucoadhesive delivery
systems exhibit adhesion to mucosal tissues for administration of
the compound(s) contained within the mucoadhesive polymer.
[0075] A variety of polymeric compositions are used in mucosal
delivery formulations. Of particular interest for use with the
42-O-alkoxyalkyl rapamycin derivative compounds are mucoadhesives
having a combination of hydrophilic and hydrophobic properties.
Adhesives which are a combination of pectin, gelatin, and sodium
carboxymethyl cellulose in a tacky hydrocarbon polymer, for
adhering to the oral mucosa, are exemplary. Other mucoadhesives
that have hydrophilic and hydrophobic domains include, for example,
copolymers of poly(methyl vinyl ether/maleic anhydride) and
gelatin, dispersed in an ointment base, such as mineral oil
containing dispersed polyethylene (U.S. Pat. No. 4,948,580).
Another hydrophilic/hydrophobic system is described in U.S. Pat.
No. 5,413,792 where a paste-like preparation of a
polyorganosiloxane and a water soluble polymeric material is
disclosed.
[0076] In the present invention, a polymer composition comprised of
a mucoadhesive polymer substrate and a 42-O-alkoxyalkyl rapamycin
derivative compound is contemplated. The mucoadhesive polymer
composition is formulated into a delivery system suitable for
placement adjacent a mucosal surface. The compound when placed
adjacent the mucosal tissue elutes from the polymer composition
into the tissue. The delivery system can take the form of a patch
for placement on the surface of tissue to be treated. The tissue
can be an organ, a vessel, a tumor, or any body surface needing
treatment.
[0077] C. Conventional Drug Delivery Compositions: Liquid,
Ointment, Gel, Patch
[0078] The 42-O-alkoxyalkyl derivative compounds, in other
embodiments, are formulated into pharmaceutical preparations of
solid, semisolid, or liquid form. Such preparations are well known
in the art, where the active ingredient is mixed with an organic or
inorganic carrier or excipient suitable for external, enteral, or
parenteral administration. Carriers and excipients for preparation
of tablets, pellets, capsules, suppositories, solutions, emulsions,
suspensions, are well known and include, but are not limited to,
water, glucose, lactose, gum acacia, gelatin, mannitol, starch,
magnesium trisilicate, talc, keratin, colloidal silica, urea, and
the like. Stabilizing agents and thickening agents may also be
used.
[0079] Liquid formulations are comprised of one or more
42-O-alkoxyalkyl derivative compounds admixed with a liquid carrier
or excipient. Such a formulation can include other components, such
as stabilizers, as desired. When admixed with the carrier, the
42-O-alkoxyalkyl derivative compound may be present in a dissolved
state or in a suspended state. Components, particularly organic
solvents, to enhance solubility of the 42-O-alkoxyalkyl derivative
compound can be added.
[0080] Preparation of gels or ointments is also contemplated.
Thickening agents added to a liquid preparation containing a
42-O-alkoxyalkyl derivative compound is a simplified approach to
gel preparation. Components to enhance transport of the active
compound across skin, mucosa, and cell membranes in general, can be
included in the gel or ointment.
[0081] The 42-O-alkoxyalkyl derivative compounds can also be
formulated into topical patches for application to a body surface,
including the skin and mucosal body surfaces. Preparation of such
topical patches is well known in the drug delivery field.
[0082] D. Endovascular Stent
[0083] Another exemplary carrier for use in the composition of the
present invention is a stent. Endovascular stents are typically
cylindrically-shaped devices capable of radial expansion. When
placed in a body lumen, a stent in its expanded condition exerts a
radial pressure on the lumen wall to counter any tendency of the
lumen to close. Stents have found a particular use in maintaining
vessel patency following angioplasty, e.g., in preventing
restenosis of the vessel. In this application, a stent is inserted
into a damaged vessel by mounting the stent on a balloon catheter
and advancing the catheter to the desired location in the patient's
body, inflating the balloon to expand the stent, and then deflating
the balloon and removing the catheter. The stent in its expanded
condition in the vessel exerts a radial pressure on the vessel wall
at the lesion site, to counter any tendency of the vessel to close.
"Self-expanding" stents are also known, made from spring material,
mesh tubes, or shape-memory alloys, these devices are typically
mounted on a catheter shaft surrounded by a sheath that constrains
the expansion of the spring elements of the stent until the stent
is positioned at the lesion site. Retraction of the sheath portion
allows the stent to expand and contact the vessel lumen.
[0084] 1. Stent Geometry
[0085] In this embodiment of the invention, the stent carries one
or more 42-O-alkoxyalkyl rapamycin derivative compounds. The
compound can be carried on the external surface of the stent, for
direct contact with a lumen wall when the stent is deployed in a
lumen, on the internal stent surface, or on both the internal and
external stent surfaces. The compound(s) could also be carried on
only select portions of the stent surface to obtain more localized
therapeutic effects (see for example co-owned U.S. application Ser.
No. 10/133,814 and PCT application no. PCT/US03/12750, which are
incorporated herein by reference). The compound can be carried
directly on the stent surface or can be incorporated into a polymer
that is carried by the stent, as will be described below.
[0086] Numerous stent geometries and configurations are known in
the art, and any of the geometries are suitable for use herein. The
basic requirements of the stent geometry are (1) that it be
expandable upon deployment at a vascular injury site, and (2) that
it is suitable for receiving a coating of drug, or for carrying a
drug-containing coating, on its surface, for delivering drug into
the lumen in which the stent is placed. Preferably, the stent body
also has a lattice or mesh structure, allowing viable endothelial
cells in the stent "windows" to grow over and encapsulate the stent
struts which are supporting the vessel lumen. The stent body can be
formed of metal or polymer, including biodegradable polymers.
[0087] One preferred stent configuration is shown in FIGS. 3 and 4
where an endovascular stent suitable for carrying a
42-O-alkoxyalkyl derivative is illustrated. Shown in FIGS. 3A-3B is
a stent 20 in a contracted state (FIG. 3A) and an expanded state
(FIG. 3B). The stent includes a structural member or body 22 having
an outer surface for holding, directly or indirectly, and releasing
the 42-O-alkoxyalkyl rapamycin derivative compound, as will be
described further below. The stent body is formed of a plurality of
linked tubular members, such as members 24, 26. Each member is
comprised of a filament that has an expandable zig-zag, sawtooth,
or sinusoidal wave configuration. The members are linked by axial
links, such as links 28, 30 joining the peaks and troughs of
adjacent members. This construction allows the stent to be expanded
from a contracted condition, shown in FIG. 3A, to an expanded
condition, shown in FIG. 3B, with little or no change in the length
of the stent. At the same time, the relatively infrequent links
between peaks and troughs of adjacent tubular members allows the
stent to accommodate axial bending and flexing. This feature may be
particularly important when the stent is being delivered to a
vascular site in its contracted state, as in or on a catheter. The
stent has a typical contracted-state diameter (FIG. 3A) of between
0.5-2 mm, more preferably 0.71 to 1.65 mm, and a length of between
5-100 mm. In its expanded state, shown in FIG. 3B, the stent
diameter is at least twice and up to 8-9 times that of the stent in
its contracted state. Thus, a stent with a contracted diameter of
between 0.7 to 1.5 mm may expand radially to a selected expanded
state of between 2-8 mm or more.
[0088] FIG. 3C shows the stent of FIGS. 3A-3B in cross-sectional
view. The view is taken through stent tubular member 24, with
adjacent tubular members visible. The stent body, and more
specifically each tubular member, is coated with drug or with a
polymer-drug composition, for release of drug from the stent at a
target site. As will be more fully described below, the drug or
drug-polymer layer is applied to the external surface of the stent,
and can be deposited to achieve a uniform deposition thickess or a
non-uniform deposition thickness. FIG. 3C illustrates the
embodiment where the drug-polymer layer 32 is applied non-uniformly
so that the external stent suface has a thicker drug or
drug-polymer coating than the internal stent surface.
[0089] 2. Drug Coating
[0090] The stent serves as a carrier for a 42-O-alkoxyalkyl
derivative compound, which, as noted above, can be coated directly
onto the stent or can be incorporated into a polymer matrix that is
carried on the stent. Whether the drug is applied directly to the
stent surface or incorporated into a polymer film on the stent
surface, it is desirable that the 42-O-alkoxyalkyl rapamycin
derivative compound be released from the stent over an at least a
several week period, typically 4-8 weeks, and optionally over a
2-3-month period or more. Two methods for loading the drug on a
stent are discussed below.
[0091] a. Direct Surface Attachment
[0092] The drug is coated directly onto the stent by, for example,
applying a solution of drug to the stent surface by dip coating,
spray coating, brush coating, or dip/spin coating, and allowing the
solvent to evaporate to leave a film of drug on the stent surface.
In studies performed in support of the invention, a
42-O-alkoxyalkyl rapamycin derivative, 42-O-(2-ethoxyethyl)
rapamycin, was applied directly to the surface of a metal stent
from an ethyl acetate solution. The drug solution was painted on
the stent surface and the solvent was removed by evaporation to
leave a film of 42-O-(2-ethoxyethyl) rapamycin on the stent. The
drug film was sufficiently adherent to permit catheter implantation
of the stents into pigs and retention of the drug on the stent
surface for release. A membrane can be optionally applied over the
drug film to change the drug release characteristic. Polymer
membranes can be formed by dip coating or spray coating, as is well
known in the art. The membrane can also be applied to the stent
surface by a vapor deposition process or a plasma polymerization
process. In one exemplary embodiment, a polytetraflyoroethylene
(Teflon@) or parylene film is formed by vapor deposition as is well
known in the art, to modify the drug release rate from the stent.
Other suitable polymers and nonpolymers for formation of diffusion
controlling membranes include polyimides (via vapor deposition or
by solvent coating), fluorinated polymers, silicones (vapor/plasma
or deposition), polyketones (PEEK, etc.), polyether imides,
vapor/plasma deposited polyacrylates, plasma polymerized
polyethyleneoxide (PEO), and amorphous carbon.
[0093] The surface of the stent can be treated prior to application
of the drug, to enhance adhesion of the drug and/or to increase
surface area for retention of a higher drug load. In one
embodiment, the stent surface is physically treated. Physical
treatment of the surface can include roughening with sand paper or
by sand or glass microbead blasting. Etching of the stent surface
with a laser can also be done to alter the stent surface. Such
physical treatment methods achieve an increased stent surface area,
for example, by forming microscopic pores, undulations, pockets,
grooves, or channels in the stent surface. This technique can also
serve to enhance adhesion of drug to the stent surface.
[0094] In another embodiment, the stent surface is chemically
treated to create a rougher surface or an activated surface, both
serving to enhance and increase drug retention. For example, a
stent surface can undergo a process of passivation, where a heated
nitric acid solution creates an oxide on the stent surface to
promote adhesion and/or to prepare the surface for subsequent
organosilane attachment. Stent surfaces can also be chemically
activated to promote attachment of a drug.
[0095] It will be appreciated that application of the drug directly
to the stent surface can be done for stent bodies formed of metal
or polymer. Surface treatments prior to application of the drug are
also suitable for both polymer-based and metal-based stent bodies.
The treatment conditions can be tailored according to the material
from which the stent is formed.
[0096] In another embodiment of the invention, the 42-O-alkoxyalkyl
rapamycin derivative is applied to the stent resulting in a glassy
layer. "Glassy" as used herein refers to the physical state of the
material where the layer is visually transparent or translucent and
is non-crystalline. A glassy layer of the derivative compounds
described herein is achieved, for example, by applying to the stent
surface a concentrated solution of a compound in an organic
solvent. Removal of the solvent yields a film of drug, where the
film has few or no crystalline drug domains. Such films having a
thin profile may be applied to the stent surface before or after
reduction in diameter (i.e. crimping) as required to mount the
device on the delivery catheter.
[0097] In this embodiment where the drug is applied directly to the
surface of the stent, it is additionally contemplated to apply over
the drug layer a membrane that serves to control release of the
drug, thereby controlling its bioavailability. A membrane prepared
from any natural or synthetic compound is contemplated, with a
preferred type of membrane composed of a polymer. A wide range of
polymers can be selected according to the desired release of drug
from the stent. The polymer can be pre-formed into a membrane that
is placed over the drug-coated stent in the form of a sheath, or
the polymer membrane can be formed directly over the drug layer by
a process selected to not disturb or disrupt the drug layer.
[0098] b. Polymer Coating
[0099] An alternative method of applying the drug to the stent is
to incorporate the drug into a polymer film that is formed on the
stent surface or is carried on the stent. Typically, a solution of
polymer and drug in a solvent is prepared and then applied to the
stent. The solution is applied to all or a portion of the stent
surfaces. After solvent evaporation, a polymer film containing drug
remains on the stent surface. A preferred coating method is
described in co-owned U.S. application Ser. No. 10/133,814 and PCT
application no. PCT/US03/12750, incorporated by reference herein.
In this method, a drug-containing solution is applied directly to
the stent surface by flowing the drug from a moveable pressurized
column, where the motion of the column is computer controlled. It
is also possible to apply the drug-containing solution via
spraying.
[0100] Yet another method of applying the polymer/drug/solvent
mixture to the stent include or brushing, dipping, or rolling the
mixture on to the stent surface from a suitable applicator, such as
a brush, dipping fixture, or petrie dish containing a thin liquid
layer of the mixture.
[0101] Polymers for use in this embodiment may be any biocompatible
polymer material from which entrapped compound can be released by
diffusion and/or released by erosion of the polymer matrix. Two
well-known non-erodable polymers for the coating substrate are
polymethylmethacrylate and ethylene vinyl alcohol. Methods for
preparing these polymers in a form suitable for application to a
stent body are described for example, in U.S. 2001/0027340A1 and
WO00/145763, both documents incorporated herein by reference. Other
non-erodable polymers suitable for use in the invention include
polyacrylates and their copolymers (PMMA, PMMA/PEG copolymers,
polyacrylamides, etc.), silicones (polydimethly siloxanes etc),
fluorinated polymers (PTFE etc.), poly vinyl acetate, poly vinyl
alcohol and its copolymers, polyolefins and copolymers (with e.g.
styrene, etc.), nondegradable polyurethanes (e.g. urethanes
containing siloxane or carbonate soft segments), polyamides,
hydroxyapatite, phosphorylcholines, polysulfones, polyketones,
polyvinypyrrolidone, polystyrene, and ABS, polyvinyls-chloride,
halide, polycarbonate. Bioerodable polymers are also suitable for
use, and exemplary polymers include poly-l-lactide, poly-dl-lactide
polymers, and polyglycolic acid-polylactic acid co-polymers,
polyglycolides (PGA, poly(lactide-co-glycolide), polycaprolactone,
polydioxanone, polyanhydrides, polyorthoesters, polycyanoacrylates,
polyphosphazenes, polyglutamates, polyhydroxbutyric acid (PHB),
polyhydroxyvaleric acid (PHV), poly (PHB-co-PHV), polysaccharides
(cellulose, dextran, chitin, etc.), proteins (fibrin, casein,
etc.), natural polymers (gelatin, hyaluronans, etc.), poly(DTH
iminocarbonate), polytrimethylene carbonate, polyethylene imine,
tyrosines (pseudo amino acids), polyrotaxanes, acrylate-based
polymers or copolymers (including hydrogels), polyphosphoesters,
degradable polyurethanes, poly(ether-ester) copolymers (e.g. PEO
and its copolymers with PBT, etc.), poly ester/ether amides, and
polyalkylene oxalates. Depending on the polymer selected and the
properties of the drug, the polymer may contain up to 80% by dry
weight of the active compound distributed within the polymer
substrate. Generally, the polymer film can contain between about
35-80% dry weight active compound and 20-65% percent by dry weight
of the polymer.
[0102] The thickness of the drug-laden polymer film is typically
between about 3 microns and 30 microns, depending on the nature of
the polymer matrix material forming the coating and the relative
amounts of polymer matrix and active compound. Ideally, the coating
is made as thin as possible, e.g., 15 microns or less, to minimize
the stent profile in the vessel at the injury site. The coating
should also be relatively uniform in thickness across the upper
(outer) surfaces, to promote even distribution of released drug at
the target site. A cross-sectional view of a stent coated with a
drug-polymer matrix is shown in FIG. 3C, where the stent tubular
member 24 is coated with a drug-polymer layer 32. Here the external
stent surface has a slightly thicker drug-polymer layer than the
internal stent surface.
[0103] In another embodiment, a polymer underlayer is applied to
the stent surface, prior to formation of the drug-laden polymer
matrix. The purpose of the underlayer is to help bond the
drug-laden polymer coating to the stent-body, that is, to help
stabilize the coating on the surface of the stent. This is
particularly valuable where a high percentage of the compound, e.g.
between 35-80 weight percent compound, is present in the polymer
matrix. Suitable polymer underlayers can be formed of
poly(dl-lactic acid), poly(l-lactic acid), poly(d-lactic acid),
ethylene vinyl alcohol (EVOH), .epsilon.-caprolactone, ethylvinyl
hydroxylated acetate (EVA), polyvinyl alcohol (PVA), polyethylene
oxides (PEO), paryLAST, parylene (poly(dichloro-para-xylylene)),
silicone, polytetrafluoroethylene (TEFLON.TM.) and other
fluoropolymers, and co-polymers thereof and mixtures thereof. The
underlayer can be deposited from a solvent-based solution, by
plasma-coating, or by other coating or deposition processes (see,
for example, U.S. Pat. No. 6,299,604). The underlayer may have a
typical thickness between 1-5 microns.
[0104] Based on the foregoing, the various configurations of stents
contemplated for use as a carrier for a 42-O-alkoxyalkyl rapamycin
derivative can be appreciated. Stents formed of a metal that carry
the drug directly on the metallic surface or in a polymer film
applied to the metal surface directly or in combination with a
polymer underlayer are contemplated. Stents formed of a polymer,
biodegradable or non-biodegradable, that carry the drug directly on
or in the stent surface or in a polymer film applied to the stent
surface directly or in combination with a polymer underlayer are
also contemplated. Thus, it is also within the scope of the present
invention to produce a completely bioerodable stent by forming the
stent body of a bioerodable polymer and the drug-laden polymer
matrix of a bioerodable polymer.
[0105] Also contemplated is the use of a second bioactive agent
effective for treating the disease or disorder of concern or to
treat any anticipated secondary conditions that might arise. For
example, if the 42-O-alkoxyalkyl rapamycin derivative is
administered for treatment of restenosis, a second compound to
minimize blood-related events, such as clotting or thrombosis, that
may be stimulated by the original vascular injury or the presence
of the stent, or to improve vascular healing at the injury site can
be included. Exemplary second agents include anti-platelet,
fibrinolytic, or thrombolytic agents in soluble crystalline form or
nitric-oxide (NO) donors which stimulate endothelial cell healing
and control smooth muscle cell growth. Exemplary anti-platelet,
fibrinolytic, or thrombolytic agents are heparin, aspirin, hirudin,
ticlopidine, eptifibatide, urokinase, streptokinase, tissue
plasminogen activator (TPA), or mixtures thereof. If the
42-O-alkoxyalkyl rapamycin derivative is intended for use as an
anti-neoplastic agent, a second agent commonly used for
chemotherapy of neoplastic diseases can be included. Exemplary
second chemotherapeutic agents include paclitaxel, platinum
compounds, cytarabine, 5-fluorouracil, teniposide, etoposide,
methotrexate, doxorubicin, and the like. The amount of second-agent
included in the stent coating will be determined by the period over
which the agent will need to provide therapeutic benefit. The
second agent may be included in the coating formulation that is
applied to the stent-body or may be applied directly to the stent
surface.
[0106] IV. Methods of Use
[0107] The 42-O-alkoxyalkyl rapamycin-derivative compounds are
intended for use in treating any condition responsive to rapamycin,
everolimus, Abbott ABT 578, tacrolimus, paclitaxel, any of the
class of compounds commonly known as macrocyclic trienes or
macrocyclic lactones, and/or other anticancer agents. This includes
any condition associated with wound healing, such as post-surgical
procedures involving a vessel or an organ transplant procedure,
neoplastic diseases, where, for example, the polymer composition is
placed directly at a site of cancer, such as a solid tumor.
Inflammation and infection are also conditions treatable with the
42-O-alkoxy-alkyl derivatives. The compounds can also be used for
vascular treatment methods, and specifically for restenosis.
[0108] With respect to treatment of vascular injury or
inflammation, the risk and/or extent of restenosis in a patient who
has received localized vascular injury, or who is at risk of
vascular occlusion, can be minimized using a composition comprising
a 42-O-alkoxyalkyl rapamycin derivative compound. Typically the
vascular injury is produced during an angiographic procedure to
open a partially occluded vessel, such as a coronary or peripheral
vascular artery, but may also be created by more chronic
inflammatory conditions, such as atherosclerosis. In the
angiographic procedure, a balloon catheter is placed at the
treatment site, and a distal-end balloon is inflated and deflated
one or more times to force the narrowed or occluded vessel open.
This vessel expansion, particularly involving surface trauma at the
vessel wall where plaque may be dislodged, often produces enough
localized injury that the vessel responds over time by cell
proliferation and reocclusion. Not surprisingly, the occurrence or
severity of restenosis is often related to the extent of vessel
stretching involved in the angiographic procedure. Particularly
where overstretching is 35% or more, restenosis occurs with high
frequency and often with substantial severity, often causing
vascular occlusion.
[0109] In studies conducted in support of the invention, stents
coated with 42-O-(2-ethoxyethyl) rapamycin were prepared and
inserted with a catheter into test animals. The stent used in the
studies was a commercially available "S-Stent.TM." that is a highly
flexible, corrugated ring stent laser cut from a stainless steel
hypotube. Each corrugated ring has a total of six serially
connected, S-shaped segments. There are two bend joints within each
S-shaped segment that allow the stent to expand during deployment.
These bend joints have undercuts that increase flexibility and
reduce the expansion forces required while deploying the stent and
obtaining good vessel wall apposition.
[0110] Successive rings in the stent are connected by two short
links, with the successive pairs of these links oriented in 90
degree quadrature around the circumference of successive rings
(QUADRATURE LINKS.TM.). The links are believed to increase the
longitudinal flexibility of the stent, while maintaining high hoop
strength. Importantly, the mechanical design of the stent combines
a repeating "S" symmetry and very short segment lengths to provide
remarkable flexibility and high vessel wall support in both
straight and curved vessels. The flexibility of the S-Stent allows
it to be easily placed into tortuous coronary vessels, and enhances
conformability with the blood vessel after implantation.
[0111] The S-Stent was specifically designed with a uniform and
repeating pattern in order to achieve the objective of uniform drug
distribution in the vessel wall as drug is released from the stent
struts. The expansion characteristics of the stent insure that
minimum stresses are applied to the coating during expansion, as a
further attempt to eliminate tearing or cracking of the coating
during deployment.
[0112] The stent has moderate radiopacity that is sufficient for
locating the stent in most vessels during deployment when using a
high resolution angiography system. The sizes of stents range from
4 mm to 60 mm in length, from 1 mm to 12 mm in diameter when fully
expanded and are provided premounted on a catheter delivery system
that employs an expandable polymeric balloon. The working length of
the delivery catheter is approximately 142 cm and has a shaft
diameter of 2.9 French. This catheter will accept a 0.014" guide
wire. The working length of the delivery balloon is as required to
match the length of the stent. There are radiopaque marker bands on
the delivery catheter that are located under the shoulders of the
balloon to aid in the placement of the stent.
[0113] S-stents were prepared as described in Example 4 by
preparing a solution of 42-O-(2-ethoxyethyl) rapamycin and
poly-1-lactic acid polymer in acetone. The drug-polymer solution
was applied to the external surface of the stent using a
microprocessor-controlled syringe pump to precision dispense the
solution. This method of applying a solution to a stent is
described in co-owned PCT application number PCT/US03/12750, the
disclosure of which is incorporated by reference herein.
[0114] The in vitro release rate of drug from the stents into an
unstirred, ethanol-water bath at 37.degree. C. was measured and the
results are shown in FIG. 4. The six curves in FIG. 4 correspond to
six stents carrying a poly-dl-lactic acid polymer loaded with
42-O-(2-ethoxyethyl) rapamycin. Each stent held between about
224-235 .mu.g of drug, with an average drug load of 230 .mu.g. The
polymer-drug coated stents were sterilized at 25 KGy or at 27.5 KGy
prior to measuring the release rate. The elution curves show the
controlled release of drug from the polymer layer carried on the
stent, with less than half of the drug load released into the bath
at 96 hours.
[0115] After preparation of the stents having a polymer-drug
coating, they were implanted, as described in Example 5, into pigs
for a thirty day period. The swine model was chosen as the
experimental species for this study since the size of the heart and
great vessels allows for a technically feasible device evaluation.
Additionally, the size of the pig coronary artery is comparable to
humans and allows for usage of standard clinical devices. Also, the
pig is an excellent model for coronary vascular evaluations and
therapy models and is close to human vascular responses in many
ways. Also, the relative size of the animals allows accurate
visualization using standard angiographic equipment. Swine are the
most appropriate large animal model for restenosis, as they most
closely resemble human vascular reactivity (Gravanis et. al., JACC,
April 1993).
[0116] As described in Example 5, nine stents eluting
42-O-(2-ethoxyethyl) rapamycin from a polymer layer of poly-lactic
acid were implanted in pigs and six control, bare metal stents were
implanted. As comparative controls, 18 stents eluting everolimus
and 12 stents eluting 42-O-hydroxy heptyl rapamycin were implanted.
Quantitative coronary angiography was performed to measure the
vessel lumen diameter immediately before and after stent placement
and also 30 days later just prior to the follow-up angiogram taken
at animal sacrifice. (Bell et al., Cathet. Cardiovasc. Diagn., 40:
66-74 (1997)). Percent stenosis (% stenosis) was determined from
the measurements as:
% Stenosis=[(RVD-MLD)/RVD].times.100
[0117] where MLD is minimum lumen diameter and RVD is distal and
proximal reference vessel diameter. The stent to artery ratio was
also calculated from these measurements. The angiographic percent
stenosis was calculated from the measurements of minimum lumen
diameter obtained at the follow-up angiogram.
[0118] As described in Example 5, after the 30 day study period,
the stented vessels segments were processed for routine histology,
sectioned, and stained. FIGS. 5 and 6 show exemplary results from
the study. FIGS. 5A-5I are computer-generated photomicrographs of
histology slides for a pig implanted with three
42-O-(2-ethoxyethyl) rapamycin eluting stents, the stents placed in
the left anterior descending artery (FIGS. 5A-5C), the left
circumflex artery (FIGS. 5D-5F), and the right coronary artery
(FIGS. 5G-5I). FIGS. 5A, 5D, and 5G are views showing the stent in
place in the vessel. FIGS. 5B, 5E, and 5H are cross-sectional views
of the vessel, with the stent struts in cross-sectional view
visible. FIGS. 5C, 5F, and 5I show the vessel-cross section at a
higher magnification, with the neoinitmal response, or lack
thereof, visible.
[0119] FIGS. 6A-6I are computer-generated photomicrographs of
histology slides for a second exemplary pig implanted with three
42-O-(2-ethoxyethyl) rapamycin eluting stents, the stents placed in
the left anterior descending artery (FIGS. 6A-6C), the left
circumflex artery (FIGS. 6D-6F), and the right coronary artery
(FIGS. 6G-6I). FIGS. 6A, 6D, and 6G are views showing the stent in
place in the vessel. FIGS. 6B, 6E, and 6H are cross-sectional views
of the vessel, with the stent struts in cross-sectional view
visible. FIGS. 6C, 6F, and 61 show the vessel-cross section at a
higher magnification, with the neoinitmal response, or lack
thereof, visible.
[0120] FIGS. 7-9 correspond to photomicrographs for control, bare
metal stents placed in vessels for 30 days. FIGS. 7A-7C show the
right coronary artery of a control animal 30 days after implant of
a bare, metal stent, where FIG. 7A shows an image of the stent in
place in the artery and FIGS. 7B-7C show cross-sectional views of
the stent at two different magnifications.
[0121] FIGS. 8A-8C show the left anterior descending artery of a
control animal 30 days after implant of a bare, metal stent, where
FIG. 8A shows an image of the stent in place in the artery and
FIGS. 8B-8C show cross-sectional views of the stent at two
different magnifications.
[0122] FIGS. 9A-9C show the left circumflex artery of a control
animal 30 days after implant of a bare, metal stent, where FIG. 9A
shows an image of the stent in place in the artery and FIGS. 9B-9C
show cross-sectional views of the stent at two different
magnifications.
[0123] The photographs in FIGS. 5-9 permit determination via
planimetry of the area of new tissue inside the stent after 30 days
in vivo. The average thickness of new tissue formed inside the
stent for each implant was determined and plotted against an injury
score, also determined after the 30 day test period. A
least-squares linear regression analysis of the data provides a
sensitive method to compare the therapeutic benefit of different
stent compositions.
[0124] The degree of vascular injury was also quantified by
assigning an "injury score" based on the amount and length of tear
of the different wall structures. The degree of injury was
calculated as follows:
[0125] 0=intact internal elastic lamina
[0126] 1=ruptured internal elastic lamina with exposure to
superficial medial layers (minor injury)
[0127] 2=ruptured internal elastic lamina with exposure to deeper
medial layers (medial dissection)
[0128] 3=ruptured external elastic lamina with exposure to the
adventitia.
[0129] The mean injury score for each arterial segment was
calculated by dividing the sum of injury scores at each stent strut
site by the total number of stent struts in the proximal, middle,
and distal stent sections using the method described by Schwartz et
al. (J. Am. Coll. Cardiol., 19: 267-274 (1992)).
[0130] After analysis of the late loss and injury scores, the
average late loss as a function of injury score was plotted for the
test stents in each animal. FIG. 10 shows a plot of late loss, in
mm, as a function of injury score for 42-O-(2-ethoxyethyl)
rapamycin eluting stents (solid line), 42-O-hydroxy heptyl
rapamycin eluting stents (dashed line), and control (bare metal)
stents (dotted line). The stents were in place in the vessel for 30
days. Stents eluting 42-O-(2-ethoxyethyl) rapamycin showed a
neointimal response to injury of 0.83 mm/injury score increment
(i.e., slope of the regression line) and an intercept of the zero
injury of 0.126 mm. Stents eluting 42-O-hydroxy heptyl rapamycin
had a neointimal response to injury of 0.126 mm/injury score
increment (i.e., slope of the regression line) and an intercept of
the zero injury of 0.130 mm. The bare metal stents showed a
neointimal response to injury of 0.165 mm/injury score increment
(i.e., slope of the regression line) and an intercept of the zero
injury of 0.165 mm. Thus, stents eluting 42-O-(2-ethoxyethyl)
rapamycin had a lower neointimal thickness at 30 days (i.e. late
loss) than stents eluting 42-O-hydroxy heptyl rapamycin or than the
control metal stents.
[0131] FIG. 11 is a similar plot, showing average late loss, in mm,
as a function of injury score for 42-O-(2-ethoxyethyl) rapamycin
eluting stents (solid line), everolimus-eluting stents (dashed
line), and control (bare metal) stents (dotted line), 30 days after
implantation in pig vessels. Again, it is apparent that stents
eluting 42-O-(2-ethoxyethyl) rapamycin offer a therapeutic effect
when compared to bare, metal stents.
[0132] In another study, stents carrying 42-O-(2-ethoxyethyl)
rapamycin in the form of a polymer (poly-dl-lactic acid) coating
were prepared for insertion into pigs with a vessel overstretch
injury to the coronary artery. Comparative and control stents
includes bare metal stents, stents having a polymer coating of
poly-dl-lactic acid absent drug, stents carrying rapamycin in a
poly-dl-lactic acid polymer coating, and stents carrying everolimus
in a poly-dl-lactic acid polymer coating. The stents were inserted
into vessels which seriously injured (averaging approximately 36%
overstretch injury of the vessel) using an angioplasty balloon. The
controlled overstretching using the balloon cathether caused severe
tearing and stretching of the vessel's intimal and medial layers,
resulting in exuberant restenosis at 28 days post implant. In this
way, it was possible to assess the relative effectiveness of the
various test compounds presented to the vessels on the same metal
stent/polymer platform. At the time of insertion, the extent of
overstretch was recorded as a percent balloon/artery (B/A)
ratio.
[0133] Twenty-eight days after insertion of the stents, various
parameters were evaluated, including mean lumen late loss,
neointimal area, injury score, and percent diameter stenosis were
determined. For two of the test groups, the control group with a
bare metal stent and a comparative control group having a stent
carrying a polymer coating loaded with 325 .mu.g everolimus, data
was collected at a 6 month time point. The results are shown in
Table 1.
1TABLE 1 Stent length Drug load Polymer coat B/A ratio Mean lumen
Neointimal area Average Injury Diameter stenosis.sup.2 Test Group
(mm) (.mu.g) (.mu.g) (%) loss (mm) (mm.sup.2) score.sup.1 % bare
stent 28 days 18.7 -- -- 1.33 1.69 5.89 1.9 72.0 6 months 18.7 --
-- 1.19 0.40 2.68 1.26 32.7 polymer-coated stent 18.7 -- 1300 1.36
2.10 5.82 2.11 70.0 rapamycin - high dose 18.7 325 1300 1.39 1.07
3.75 2.10 55.0 rapamycin - low dose 18.7 180 1300 1.42 0.99 2.80
1.90 43.0 everolimus - high dose 28 days 18.7 325 1300 1.37 0.84
3.54 1.89 38.0 6 months 18.7 325 1300 1.31 1.15 4.18 2.67 68.5
everoliums - low dose 18.7 180 1300 1.36 1.54 3.41 2.10 53.0
everolimus - med. dose 18.7 275 640-780.sup.3 1.36 0.85 2.97 2.13
45.0 42-O-(ethoxyethyl) 15 225 225 1.19 0.62 1.30 1.29 14.8
rapamycin .sup.1injury scores quantify the degree of vascular
injury based on the amount, length, and depth of tear and is scored
using the scale of 1, 2, 3, given above. .sup.2a lower score
indicates higher efficacy .sup.326% drug to polymer ratio
[0134] The data in Table 2 shows that while the drug-carrying
stents performed better than the bare metal stent or the
polymer-coated stent, the stent carrying 42-O-(2-ethoxyethyl)
rapamycin resulted in the lowest mean lumen late loss and percent
diameter stenosis. Thus 42-O-(2-ethoxyethyl) rapamycin demonstrated
superiority for suppression of cell proliferation when directly
compared to rapamycin and other known rapamycin derivatives in the
pig coronary artery overstretch injury model.
[0135] From the foregoing, it can be appreciated how various
feature and objects of the invention are achieved. 42-O-alkoxyalkyl
derivatives of rapamycin, as exemplified by 42-O-(2-ethoxyethyl)
rapamycin, have in vitro potency similar to or greater than the
potency of rapamycin and of another rapamycin derivative,
42-O-hydroxy heptyl rapamycin. The in vivo activity of
42-O-alkoxyalkyl rapamycin derivatives was illustrated using stents
coated with the drug and placed in the vessel of an animal for
inhibition of restenosis. The 42-O-alkoxyalkyl rapamycin
derivatives can be formulated into preparations suitable for
topical, parenteral, and local administration for use in treating
any condition responsive to rapamycin, everolimus cancer agents, or
other macrocyclic lactones.
V. EXAMPLES
[0136] The following examples further illustrate the invention
described herein and are in no way intended to limit the scope of
the invention.
Example 1
Preparation of 42-O-(2-ethoxylethyl) Rapamycin (Biolimus A9)
[0137] A. Synthesis of 2-ethoxyethanol Triflate
[0138] To a stirred, cooled (0.degree. C.) solution of 4.28 g
2-ethoxyethanol (Aldrich Chemical) and 10.14 g 2,6-lutidine in 160
mL CH.sub.2Cl.sub.2 under nitrogen was slowly added 19.74 g
trifluoromethanesulfonic (triflic) anhydride. The mixture was
washed with four portions of 200 mL brine and the organic solution
dried over anhydrous sodium sulfate, filtered and concentrated. The
residue was purified by flash chromatography on silica gel, 200-400
mesh (75:25 hexanes-ethyl ether (v/v)) to afford the triflate of
2-ethoxyethanol: light yellow liquid, TLC Rf=0.47 using
hexanes-ethyl ether 75:25 (v/v).
[0139] B. Reaction of 2-ethoxyethanol Triflate with Rapamycin
[0140] To a stirred solution of 1 g rapamycin, and 7.66 g
2,6-lutidine in 14.65 mL toluene held at 60.degree. C. was added
5.81 g 2-ethoxyethanol triflate. Stirring was continued for 90
minutes after which 50 mL ethyl acetate was added to the reaction
and the solution was washed with 50 mL 1 M HCl. The organic
material was washed with D.I. water until pH of wash solution was
neutral and the organic solution was dried over anhydrous sodium
sulfate, filtered, and concentrated. The residue was purified by
flash chromatography on silica gel, 200-400 mesh (40:60
hexane-ethyl acetate (v/v) to give 210 mg 42-O-(2-ethoxyethyl)
rapamycin. TLC Rf=0.41 using 40:60 hexane-ethyl acetate (v/v). MS
(ESI) m/z 1008.5 C.sub.55H.sub.87NNaO.sub.14. A mass spectrum of
the title compound is shown in FIG. 1.
[0141] The chemical structure of 42-O-(2-ethoxyethyl) rapamycin was
further verified by mass spectrometric tandem quadrupole
experiments (CAD experiments, collisionally activated
dissociation). These studies were done on a Thermo Finnigan, LCQ
Advantage quadrupole ion trap mass spectrometer equipped with an
electrospray ionization source. Direct infusion of the sample in
methanol was done at a flow rate of 2.5 .mu.L/min from a syringe.
CAD experiments were carried out after obtaining maximum signal
intensity. Helium was used as the collision gas. Collision energy
was tuned during the MS/MS experiments to obtain the full range of
fragments. The fragmentation patterns indicated the presence of the
ion pair 1008.5.fwdarw.417.5. These results are consistent with the
chemical structure of 42-O-(2-ethoxyethyl) rapamycin given
above.
[0142] Purity of the product was determined by HPLC. A Zorbax
SB--C18 HPCL system was used, with a 4.6 mm ID.times.250 mm (5
.mu.m) column. A step gradient solvent system was utilized
consisting of 100% (10% methanol-water), one minute; 50% (10%
methanol-water)/50% methanol, one minute; 25%(10%
methanol-water)/75% methanol, one minute; 100% methanol. A flow
rate of 1.0 mL was used. Column temperature was 55.degree. C. 2.0
.mu.g of 42-O-(2-ethoxyethyl) rapamycin was injected onto the
column in a volume of 20 .mu.L methanol. Detection by UV at 278 nm.
Purity was 98.7% (average of three runs; SD=0.2).
Example 2
Preparation of 42-O-(2-methoxyethyl) Rapamvcin
[0143] A. Synthesis of 2-methoxyethanol Triflate
[0144] To a stirred, cooled (0.degree. C.) solution of 1.80 g
2-methoxyethanol (Aldrich Chemical) and 5.07 g 2,6-lutidine in 80
mL CH2CL2 under nitrogen was slowly added 9.87 g
trifluoromethanesulfonic (triflic) anhydride. The mixture was
washed with five portions of 100 mL brine and the organic solution
dried over anhydrous sodium sulfate, filtered and concentrated. The
residue was purified by flash column chromatography on silica gel
200-400 mesh (75:25 hexane-ethyl ether (v/v)) to afford the
triflate of 2-methoxyethanol: light brown liquid, TLC=0.61 using
the same solvent system as above.
[0145] B. Reaction of 2-methoxyethanol Triflate with Rapamycin
[0146] A mixture of 8.0 mg rapamycin, 52.9 .mu.L lutidine, and 34
.mu.L 2-methoxyethanol triflate was held at 60.degree. C. in a 1.5
mL microcentrifuge tube (Laboratory Plastics) for 1.5 hours. 150
.mu.L ethyl acetate and 150 .mu.L 1 M HCl was added and the
solutions mixed by vigorous shaking. Separation and direct
preparative TLC of the organic layer (40:60 hexanes/ethyl acetate
(v/v)) resulted in 2.3 mg 42-O-(2-methoxy ethyl) rapamycin. MS
(ESI) MIZ 994.6 C.sub.54H.sub.85NNaO.sub.14. MS/MS
995.fwdarw.403.
Example 3
In Vitro Potency of 42-O-(2-ethoxyethyl) Rapamycin Derivative
[0147] Smooth muscle cell cultures were subjected to increasing
doses of 42-O-(2-ethoxyethyl) rapamycin, 42-O-(hydroxy heptyl)
rapamycin, and rapamycin over 8 or 9 orders of magnitude
concentration in the culture medium. The ability of the cell
culture to reproduce was assessed after drug exposure by addition
of a colored reagent which causes a color change in the surviving
cells, followed by cell cytometry. The ability of the cells to
migrate was assessed when cells from the culture moved through a
porous membrane barrier adjacent the culture, and again by staining
and cell cytometry. The results of these tests are shown in FIG. 2
for human smooth muscle cells. The results show that the
42-O-(2-ethoxyethyl) rapamycin (squares) and 42-O-(hydroxy heptyl)
rapamycin (diamonds) have similar potency in growth suppression of
smooth muscle cells over 5 orders of magnitude concentration, for
both porcine and human cells.
Example 4
Preparation of Stents Containing 42-O-(2-ethoxyethyl) Rapamycin
[0148] 100 mg poly(dl-lactide) was dissolved into 2 mL acetone at
room temperature. 5 mg 42-O-(2-ethoxyethyl) rapamycin was placed in
a vial and 100 .mu.L lactide solution added. A
microprocessor-controlled syringe pump was used to precision
dispense 4.5 .mu.L of the drug containing poly-dl-lactide solution
to the outer surface of a metal S-Stent (available from Biosensors
International Inc, Newport Beach, Calif.). Evaporation of the
solvent resulted in a uniform, drug containing single polymer layer
on the stent.
[0149] Comparative stents containing everolimus or 42-O-- hydroxy
heptyl rapamycin were prepared identically.
Example 5
In vivo Testing of Stents Carrying 42-O-(2-ethoxyethyl)
Rapamycin
[0150] A. Animal Model
[0151] Six out-bred juvenile swine weighing between 30-40 kg were
obtained. The animals were housed and quarantined for a minimum of
three days prior to entering into the study. All animals were
examined and housed in facility-approved pens under sanitary
conditions. A nutritionally balanced, standard pig chow was fed to
the study animals, and water was provided ad libitum.
[0152] B. Stent Placement
[0153] Three days prior to insertion of stents each animal received
650 mg aspirin, 500 mg ticlopidine daily, and 120 mg verapamil
daily. Aspirin (325 mg) daily was given throughout the duration of
the study. The animals were fasted twelve hours prior to stent
placement.
[0154] For stent placement, each animal was immobilized with an
intramuscular injection of 0.5 mg/kg acepromazine, 20 mg/kg
ketamine, and 0.05 mg/kg atropine. An intravenous cathether was
placed in an ear vein, and anesthesia was induced with 5-8 mg/kg
thiopental. The animal was intubated and ventilated; anesthesia was
maintained with inhaled 1-2% isoflurane. A loading dose of
intravenous bretylium tosylate (10 mg/kg) was administered for
anti-arrhythmic therapy.
[0155] The surgical site was shaved, cleaned, and draped. An
incision was made for blunt dissection of the tissue place above
the access artery (either the right and/or left carotid artery or
the right and/or left femoral artery). The distal and proximal
segments of the artery were secured with suture; the distal vessel
was ligated. An arteriotomy was performed and an introducer sheath
placed in the artery.
[0156] A guiding catheter was placed into the sheath and advanced
under fluoroscopic guidance into the coronary arteries. After
placement of the guide catheter, angiographic images of the vessel
were obtained to identify a suitable location for stent deployment.
The tip of the guiding catheter was included in the captured images
to facilitate quantitative coronary angiography (QCA) measurements.
A 0.014" guide wire was used to deliver stents to pre-determined
sites selected from the left anterior descending (LAD), left
circumflex (LCX), and/or right coronary (RCA) arteries. Stents were
typically placed in up to three coronary arteries, with only one
stent placed in any one artery. When needed, additional balloon
inflations were made to assure positive apposition of the stent
against the vessel wall. The final stent diameter was selected to
create an "overstretch injury" of 20%.+-.10% over mean vessel
diameter (MLD). Following stent deployment, additional angiographic
images of the treated vessel segments were obtained in the same
orientation as the initial images.
[0157] Intravenous bretylium tosylate (10 mg/kg) was administered
at the end of the procedure for anti-arrhythmic therapy. Following
the procedure, the catheters were removed, the proximal vessel
ligated with O-silk, and the arterial cutdown site repaired in a
three-layer fashion. The facia and subcutaneous layers were closed
with a running suture using 2-0 monocryl. The animal was allowed to
recover.
[0158] Nine stents eluting 42-O-(2-ethoxyethyl) rapamycin from a
polymer layer of poly-lactic acid were implanted in pigs and six
control, bare metal stents were implanted. As comparative controls,
18 stents eluting everolimus and 12 stents eluting 42-O-hydroxy
heptyl rapamycin were implanted. The results are shown in FIGS.
5-9.
[0159] C. Monitoring of Test Animals
[0160] At the end of the study, the animals were euthanized and a
thoracotomy was performed. The coronary arteries were perfused with
at least one liter of formalin infused into the coronary arteries.
A cardiectomy was then performed and the vessels were visually
inspected for any external or internal trauma. The stented vessels
were removed from the heart, stored in sealed laboratory bottles of
new 10% formaldehyde solution, and packaged for histology
preparation.
[0161] Quantitative angiography was performed to measure the vessel
diameter immediately before and after stent placement and also at
follow-up (i.e. animal sacrifice). The stent to artery (i.e. B/A or
Balloon/Artery) ratio was calculated from these measurements. The
angiographic percent stenosis was calculated from the measurements
of minimum lumen diameter obtained at the follow-up angiogram.
Histologic measurements were made from sections from the native
vessel proximal and distal to the stents as well as the proximal,
middle, and distal portions of the stents. The mean injury score
for each arterial segment was calculated by dividing the sum of
injury scores at each stent strut site by the total number of stent
struts in the proximal, middle, and distal stent sections using the
method described by Schwartz et al. (J. Am. Coll. Cardiol., 19:
267-274 (1992)).
[0162] Neointimal thickness was measured at the native vessel
proximal and distal to the stents and at the proximal, middle, and
distal portions within the stents. The neointima, media, and total
vessel cross sectional areas of each mid-stent section were
measured with digital histomorphometry to determine the neointimal
area and percent area stenosis defined as [(intimal area/native
lumen area).times.100]; where the native lumen area is the area
delineated by the internal elastic lamina. Data were expressed as
the mean.+-.standard deviation and as the maximum percent cross
sectional area narrowing for each specimen. Statistical analysis of
the histologic and angiographic data were accomplished using
analysis of variance (ANOVA). A p<0.05 was considered
statistically significant.
[0163] QCA measurements were made according to the teaching of Bell
et al., (Cathet. Cardiovasc. Diagn., 40: 66-74 (1997)). Percent
stenosis (% stenosis) was determined by:
% Stenosis=[(RVD-MLD)/RVD].times.100
[0164] where MLD is minimum lumen diameter and RVD is distal and
proximal reference vessel diameter.
[0165] Histomorphometric analysis included lumen cross sectional
area, internal elastic lamina (IEL) and/or stent area, neointimal
area, medial area, adventitial area, percent in-stent stenosis,
intimal thickness at each stent strut, and injury score at each
stent strut.
[0166] The degree of vascular injury was also quantified by
assigning an "injury score" based on the amount and length of tear
of the different wall structures. The degree of injury was
calculated as follows:
[0167] 0=intact internal elastic lamina
[0168] 1=ruptured internal elastic lamina with exposure to
superficial medial layers (minor injury)
[0169] 2=ruptured internal elastic lamina with exposure to deeper
medial layers (medial dissection)
[0170] 3=ruptured external elastic lamina with exposure to the
adventitia.
[0171] The mean injury score for each arterial segment was
calculated by dividing the sum of injury scores at each stent strut
site by the total number of stent struts in the proximal, middle,
and distal stent sections using the method described by Schwartz et
al. (J. Am. Coll. Cardiol., 19: 267-274 (1992)).
[0172] D. Histology
[0173] The stented segments were processed for routine histology,
sectioned, and stained following standard histology lab protocols
as described by Isner et. al (Biochemical and Biophysical Research
Communications, 235: 311-316 (1997)). Hematoxylin and eosin stain,
elastic and connective tissue stain were performed in alternate
fashion on serial sections through the length of the stent
(distal/proximal reference vessel proximal/middle/distal
stent).
[0174] Photographs of histology slides for animals treated with
42-O-(2-ethoxyethyl) rapamycin eluting stents are shown in FIGS.
5A-5I for one test animal having three stents placed in the left
anterior descending artery (FIGS. 5A-5C), the left circumflex
artery (FIGS. 5D-5F), and the right coronary artery (FIGS. 5G-5I);
and in FIGS. 6A-6I for a second test animal having three stents
placed in the left anterior descending artery (FIGS. 6A-6C), the
left circumflex artery (FIGS. 6D-6F), and the right coronary artery
(FIGS. 6G-6I).
[0175] Photographs of histology slides of vessels taken from six
control animals having an implanted bare metal stent for 30 days
are shown in FIGS. 7-9.
Example 6
Human Trial Using Biolimus A9-eluting Stent
[0176] A. Organization of the Clinical Trials
[0177] A 6-month clinical trial was carried out to determine the
safety and effectiveness of a coronary artery drug eluting stent
constructed in accordance with the present invention and formulated
to release 42-O-(2-ethoxylethyl) rapamycin (Biolimus A9), eluted
from a bioabsorbable PLA polymer-coated stent, in de novo coronary
lesions. The drug:polymer ratio in the stent coating was 1:1 by
weight. The coated stent was crimped on a balloon ptca catheter,
e-beam sterilized, delivered to the lesion site in the patient's
coronary artery, and expanded under fluoroscopy in a
catheterization laboratory to achieve implant of the stent in the
patients' artery.
[0178] The 120-patient double-blinded, randomized trial was
conducted at Siegburg Heart Center and Bruderkrankenhaus Trier, and
at Institute Dante Pazzanese of Cardiology, a medical research
hospital located in So Paulo, Brazil. The Harvard Cardiovascular
Research Institute (Boston) and the Cardiovascular Research
Foundation (New York) served as data and angiographic core
laboratories for the study, and the Stanford Cardiovascular
Research Institute (Stanford, Calif.) served as the IntraVascular
Ultrasound (IVUS) core laboratory.
[0179] Unlike currently approved drug eluting stents that use a
permanent polymer coating to release the anti-stenosis drug and
which remain inside the patient's coronary artery for life, the
Biolimus A9 stent used in these trials has a bioabsorbable coating
that dissolves during drug release into all-natural products (i.e.,
polylactic acid or "PLA"). The natural product is then metabolized
and excreted by the body as carbon dioxide and water.
[0180] B. Results of the Clinical Trials
[0181] The results of the clinical trials can be summarized as
follows. The angiographic analysis indicated a low restenosis rate
(3.9% vs. 7.7%, P=0.4) and decreased Late Loss inside the stent
(0.26 vs. 0.74, P<0.001) in the Biolimus A9 stent group compared
with the bare metal stent control group. "Late Loss" refers to the
thickness of the scar tissue, or "neointima", that forms inside the
stent. The formation of scar tissue after bare metal stent implant
in a significant proportion of bare metal stent implant cases
reduces blood flow to the heart and can reduce the long term
effectiveness of the stent.
[0182] No restenosis occurred at the proximal or distal edges of
the stent in either drug eluting stent or bare metal stent
(control) group. By Intravascular ultrasound (IVUS) analysis, the %
neointimal volume (2.6% vs. 23.5%, P<0.001) was significantly
lower in the Biolimus A9 stent group compared with the bare metal
stent control group. "Neointimal volume" is another method
measuring the total volume, rather than the thickness of neointima
formed inside a stent.
[0183] It was therefore concluded that the Biolimus A9-eluting
stent, eluting the rapamycin derivative Biolimus A9 from a
bioabsorbable PLA polymer coating, demonstrated significantly
reduced neointimal hyperplasia when compared to bare metal control
stents in this clinical trial. A low adverse event rate (5%) and
the absence of cardiac deaths further showed that this new stent is
safe for use in humans.
Example 7
Comparison of Performance of Biolimus A9-eluting Stent vs.
Sirolimus-Eluting Stent
[0184] The performance of a Biolimus A9 stent was compared with
that of a sirolimus-eluting stent. The data used in this comparison
were the Biolimus A9 clinical trial data presented in Example 6 and
clinical data from the literature on a sirolimus stent currently
marketed as the sirolimus-eluting Stent ("Cypher").
[0185] The results of the RAVEL First-in-Man trial of the Cypher
stent, eluting sirolimus (rapamycin) from a permanent polymer
coating, were reported by the Clinique Pasteur, Paris, France, at
the Euro PCR Scientific Sessions in April 2001. Table 2 below shows
the data from the RAVEL trial:
2TABLE 2 RAVELivus in-stent analysis at 6-months follow-up:
Sirolimus (N = 48) Control (N = 46) EEM volume 280 280 NS
(mm.sup.3) Stent volume (mm.sup.3) 131 132 NS Neointimal volume 2
37 <.0001 (mm.sup.3) Lumen volume 129 95 <.0001 (mm.sup.3) %
Volume 1.4 28.6 <.0001 obstruction Incomplete 20 4 <.015
apposition %
[0186] The Ravel Trial studied patients with shorter coronary
lesions, averaging 9.56 mm in length, and the longest
sirolimus-eluting stent implanted in the Ravel Trial was 15 mm. By
comparison the average lesion length in the Biolimus-A9 eluting
stent trial was 15.7 mm, and stent lengths of 14, 18, and 28 mm
were implanted. The average vessel diameters were similar in the
two trials (3.0 mm). The first four parameters cited in Table 2
were similar in the Biolimus A9-eluting stent after adjustment for
the differences in average implanted stent lengths. The % Volume
obstruction for the Biolimus A9 eluting stent was 2.6%, not
statistically different from the results reported above. However,
the Incomplete Apposition % in the Biolimus A9-eluting stent trial
was significantly lower (13%) compared to the sirolimus-eluting
stent. The Incomplete Apposition in the Biolimus A9-eluting
(BioMATRIX) stent was stratified by the IVUS Core Lab analysis as
shown in the Table 3.
3TABLE 3 Stent Incomplete Apposition BioMATRIX stent Total Stent
body:Stent edge Baseline 10 (13%) 2:8 (n = 75) 6-month Follow-up
12* (17%) 4:9** (n = 69) Persistent 10 (14%) 2:8 Resolved 0 (0%)
0:0 Late SIA 2 (3%) 2:0 *no baseline IVUS with one case **one case
has edge SIA (persistant) and body SIA (late acquired)
[0187] The much lower rate of Incomplete Apposition as seen in the
Biolimus A9-eluting stent trial was the result of a much lower Late
Stent Incomplete Appositon Rate (LSIA) in the Biolimus-eluting
Stent vs. the Sirolimus-eluting stent (3% vs.about.10%).
[0188] In fact, the incidence of Late Incomplete Stent Apposition
in the Biolimus A9-eluting stent was comparable to the historical
average seen by the Stanford Core Lab for bare metal stents, and as
was observed in the bare metal control stent used in the Biolimus
A9 eluting stent trial (the historical rate is 3%). This data
establishes that the Biolimus A9 eluting stent is more
biocompatible than the sirolimus eluting stent, causing a lower
incidence of aneurism formation in the vessel walls surrounding
stent. While there is currently no known clinical sequelae
associated with "Late Stent Incomplete Appositions," cardiologists
prefer a low incidence rate of this phenomenon when comparing
stents because it suggests the possibility of a localized tissue
reaction to the stent and vessel wall aneurism formation in some
patients which may or may not lead to some, as yet unknown, long
term complication such as vessel wall failure.
[0189] From these data, it was concluded that the Biolimus A9 drug
was superior to the drug sirolimus in the prevention of vessel wall
aneurism formation and Late Incomplete Appositions.
[0190] A further study (The Sirius Trial) of the sirolimus-eluting
stent (Cypher) was reported in September 2002 at the Trans Catheter
Therapeutics Scientific Sessions in Washington D.C. FIG. 12 shows
the data of the Sirius Trial presented at that meeting.
[0191] FIG. 12 establishes the relationship for the
sirolimus-eluting stent between restenosis rate in the implanted
vessel segment vs. stent length. Theses data were compared to the
results of the Biolimus A9-eluting stent with the result that at
each stent length the restenosis rate for the Biolimus-eluting
stent was in the range of 2-6% lower, indicating better efficacy of
the Biolimus-eluting stent. On average for all stent lengths
implanted in the range of 8-28 mm, the average in-lesion restenosis
rate of the Biolimus A9-eluting stent was 3.9%, with an average
implanted stent length in the Biolimus A9-eluting stent trial of
19.7 mm. The following stent lengths were implanted:
4 Stent length % of Total vessels 8 mm 1.3% 14 mm 44.1% 18 mm 32.5%
28 mm 22.1%
[0192] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention.
* * * * *