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Appendix A: U.S. Patent No.

5,716,981
I USOO5716981A
United States Patent WI 1111 Patent Number: 5,716,981
Hunter et al. 1451 Date of Patent: Feb. 10, 1998

1541 ANTI-ANGIOGENIC COMPO!+ITIONS AND sg80~99 lhP95 Fearnot et al. ...._.......a............ 604d-265
METHODS OF USE 5283,928 111995 Scott a al. .................................. 623/l
5,403,858 4/1995 Bastard et al. _.. ..... ..-.....- .... 514449
5,407,683 40995 Shivdy ._-.. .. ._-....... ............. 424439
DS] Inventors: Wllhm L. Bunter; Lindmy S. 5,415.869 50995 Saanbia$er et d. . ........... . .-. 424450
Machan. both of Vancouver; A. Lmy 5.419.760 50995 Narciso, Jr .. . ............................. f&V8
Arsenault. Paris. all of Canada 5,439,446 WPPS Bany ....... ..” .- . ..” ............ ......
5.439.686 S/J%‘% Ikai CI al. L..._......._.............. 424/451
179 Assignee: Anglogenesis Technobgies, Inc. 5.441.745 iln9Ps l’resanr et al. . I.............” ........ 424450
vancouver. Canada 5;44i;724 911995 Helmus et al. ............. .._........ 424I426
5.449513 911995 Yokoyama et al. . . ............... 424n8.08
[21] AppL No.: 47833 5,457;113 1011995 Cul&xu~ el al. ............I .......... 514019
5$60,817 KY1995 Lpngley et al. .....“. ................. 424Mo8
[22] Filed: Jun. 7,1!w5 5.462.866 l(Y1995 Wang .............. . ......“. ............. 435074
5&x,450 1111995 Busoemi et al. .. .._.................. m.. 623/6
Related U.S. Application Data 5,464,650 11/1995 3ag et al. ... . ......................... 427R.3
5,473,oss w1995 Mongdli et al. ... . ...... ..“. ....... 53w329
[60] Divisim of Scz No. 417,160, Apr. 3, 1995, abaadooed, FORE?iGN PATEhT DOCUMENTS
which is a continw&on-in-part of Ser. No. 94,536, Jul. 19,
1993, abaadoaed. 375 520 Al 6/1990 Europan Pat. Off. .
Foreign ApplicPtion Priority Data 470 569Al 211992 bu‘q=a~ Pu. off. .
1301 566 24.5Al lo/1993 Enmpum PP off. .
id. 19, 1994 [wol wm . . ..... ... . . ..- PcwC494Q0373 567 816Al lUl993 Euroum Pat. off. .
627 226Al 211994 Eum&n Pat. off. .
[Sl] Int CL6 .. ... ... ..._I......... A61K 3lf335; A61M 29/00 679 373A2 1111995 E~lmpan Pat. off. .
[52] U.S. CL ... .. ... ... ... ....._.....514/449; 12&‘898; 526/304; wo90113332 1111990 QIIPb.
5281421; 604153; 604I2& 60W21; 604/%; wo 91/10424 70991 WIPO
6041269; 604’198; 623112 wo 91/11193 8/1991 WIPO.
[58] Weld of Search .. ... ... ... ..._..._.........w..604/53. 20. 21. wo 91112779 9/1991 WIPO.
WO 92f12717 ff.‘1992 WE’0 .
604f96.269; 128f898; 514/449; 606/198; WOW15286 9/1992 WPO
623/12;528/421; 52#304 wo93106792 411993 WIPO
w094/21308 911994 WIPO.
1561 References Cited wo 94Q4%1 1111994 WJPO I
wo95io3036 21199s WwO .
U.S. PATENT DWUMENTS w095/03795 2/1995 WIPO
4300,244 1111981 Bokms .....--..._...._e..........._..._ 1 3/l .4
CYl’HER PUBLICKl-iONS
4531,936 711985 ciodal “. ...-..................-- ....” ... 604149
4542,025 9f 1985 Tice et al. ....... .._....m......w........ 424fi% Walter et al., “Iutrrstitial Tax01 DcIivcrcd from a Biodegrad-
4580,568 4ll986 Gianlulco .... ........._“.............. 1281345 abie Polyma Implaut against Experimental Maliguaut
4,733,665 311988 edmm ..“^ .. ............... . .. ... .-. 1281343
4,739,762 40988 Pahu ..... ._........ . .I ... .._w..“. 1281413 Glioma.” Cancer Research 54~2207-2212. 1994.
4.776137 lo/1988 pahaz ..u........._...e................. 128/343 Bartoli et al.. “‘In vitro and in vivo antitumcral activity of
4.800.082. . 111989 G~~UUIOO. ._........_.....w.. .... .v. 12StM3 free. and encapsulated taxoL” Journal of hiic~naqpsuka-
4,824.436 441989 WW “. ...... . . ... . ...... .” ... 604153 tion 7(2): 191-197. 1990.
4.834.755 -5n9a9 sjbcwini et al. .-.- ._........... 623113 Bisset! aod Kaye. ‘Tax01 and Taxotere-Current Status and
4.955.878 9n990 see et al. ... ..... .. . . ““. . ... . ... 604/181 Future Prospects.” European Journal of Cancer 29A(9):
4;960,790 lam990 skna et al. .. . .. I.. .... ..*.--.. ..... 514M49 1228-1231, 1993.
4.989,601 2/1991 Mach&y et al. .-..... .. -.-. .... 12SI399 De Scheader et al.. %icwmpatiiility of polymer-coated
4990,155 2p91 Whff “.^ .- ...“. .. ...“. ...” ... . w191
ovasizedmetallicstentsirnplanMinwrmalpcrcinccoro-
s,o19,OsQ Y1991 Piadluk ... ..“. ... . ...“.“” .*. ... I .. 606w4
s,O49,132 911991 ShEa et al. ll.._..._ .. -- .. &XUOl
nary attexies.“Arhemckwsis 114: 10.5-114. 1995.
5.053.04s lo/1991 Pin&l& ........ ..... .” .... ........ ... . . 623n (List continucdon next page.)
5,059,166 10/1991 Fiscbdl u al. . . ..... .w_........... ..... 6oon
5059.178 1011991 Ya .“.“. .“. .. ..“. ... ..“.....” ..“. ... . . w101 Primary EkaminerGfihailen&a Kumar
5.059211 lo/1991 Slacketd. “...““I.“..“.““..... . @J&198 Attomq Agent, or Fima-Sccd aud Berry W
S.092.841 3/1992 spaas .. . ... ..“. ... “” .. .. ... “..I ... 604i96
S.lrn.417 4i1992 Palmtz ... .... ... .“.............“..“I 60&l% [57l ABSTRACT
s,171+217 12M92 Maldl et al. I.” ..“. .... “.“....... ... 6O‘w53
5,171262 131992 Mecolegclr . .. ............ ...“f...” ... 623/l The present invention provides compositions con@stig an
5.176.617 111993 Fii u d. .. . . ..... *........ .. 4wl.l anti-angiogcnic factor. and a polyrnaic carrier. Representa-
5jl33, 50993 slepipl et al. ... .. I._ .................. 62311 tive examples of anti-angiogcnic factors include Anti-
5234,456 s/l993 siivesaini ... . ..”. . .... .”...I.. ... 606/M Invasive Facfca. Rdinoic acids and daivatives thereof. and
spa.54 2f1994 sbapdand et al. .......... . .“. ...... 604m paclitaxel. Also provided are merttods for cmboiizing blood
5292~12 3f1994 sdr.xfa et al. .. ..“. .... ....“. .... 424b401 vessels. and elimiuating biliary. urethral, esophageal. and
5304,121 4Il994 Salvujinn .“. ....... ................ I .. . 604/53
s314.688 511994 Icau&Mn et al. ...“” .-.......... . 424423 trachcaubfonchial obstruuions.
$342348 8/1994 Kpplm I.._......._._..-__ ..... . @4.‘891.1
5342,621 al1994 B=Y ........ . ... . ........ “.“.” ..I .... w423 18 Claims, 75 Drawbg Sheets
5,716$81
Page 2

OTHER PUBLXXlONS Saoro et al.. ‘The Anti-‘hmor Agent, Taxol. Attenuates


Conttadile Activity in Rat Aatic Smooth Musck.” Life
Flay et al.. ‘Design of Biodegradable Polymer Systems for Sciences 56(7): PL, 157-161.1995.
Controlled Releaseof Bioactive Agents,” in El-Nokaly et al
(eds.). Polymeric Lklivery Systems: Pmperdes and AppLi- Schindia et al., “Biodegradable Polymers for Sustained
cations, American Cbemieal Society. Washington, DC.. Drug Delivery,” Contemp. Tbp. Polym Sci. 2: 25 l-89.1977.
1993. pp. 154-167. Sinko and Kolm. “Polymeric Drug Delivery Systems. An
Holland et aL. “Polymers fa Biodegradable Medical ovavietv.” in El-Nokaly et al. (eds). Polymeric Delivery
Devices. 1. The Potential of Polyesters as Controhd Mac- Systems: Pmperrits md Appiicatio~ American Chemical
romolecular Release Systems.” 3ownal of Contmlkd Society, Washington D.C.. 1993, pp. 18-41.
Rekmse 4: 155-180, 1986. Sollott et al.. ‘Tax01 Inhibits Neointimal Smooth Musck
J&L R. “Biodegradable Poly(LaUic Acid) and Poly(Lac- Cell Accumulation after Angioplasty in the Rat,” Joumf of
tide<o-Glycoiide) Polymers in Sustained Drug Deliveq.” CZinicuZInvestigatZm 95: 1869-1876. 1995.
Drug Develqment and Industrial Pharmacy 16(16): Sonnlcbsen and Relling, “Clinical Phamxieokinetics of
2353-2367.1990. PaclitaxeL” C&z. Phmmacokhet. 27(4): 256-269. 1994.
Kohkr and Goldspiel. “Evahation of New Drugs. Paclitaxel
Spencer and Faulds. “PaditaxeL A Review of its Pharma-
(Taxol).” Pburmacotkqy 14(l): 3-34.1994.
codynamic and Pbarmacokinetic Properties and Therapeutic
Kuhn. J.. “pharmacology and Pharmocokinetics of Pacli-
taxel.” Annals of Phamacothe~ 28: S15S17, 1994. E pne Treatment of Cawa.” Drugs 48(5):
hger. R. “1994 Wbitaker Lecture: Polymers for Dmg
Delivtsy and ‘IIssue Engineering.” Anna& of Biomedical Vainion& et aL. ‘Surgical Applications of Biodegradable
Engineering 23: 101-111. 1995. Polymers in Human Tissues.” Pmg. PoZym Sci. 14:
Pas&W.. “Synthetic Polymas in Modem Pharmacy.”Pmg. 679-716.1989.
Polym Sci. 14(S): 629-677. 1989. Yasumai et al.. ‘placement of Covered Self-Expanding
Rowinsky et al.. ‘Taxol: A Novel Investigational Antimi- Metallic Stats in the Common Bile Duct A Feasibility
emtubule Agent,” Jourmi of the National Gmcer Institute Study.” Jowml of biascukzr andlnterventional Radiology 4:
82: 1247-1259, 1990. 773-m. 1993.
U.S. Patent Feb. 10,1998 Sheet 1 of 75 5,716,981

D
P
U.S. Patent Feb. lO,l!J98 Sheet 3 of 75 5,716,981
US. Patent Feb. 10, 1998 Sheet 4 of 75 5,716,981

i .c
Feb. lo,1998 Sheet 5 of 75 5,716,981
U.S. Patent Feb. 10,1998 Sheet 6 of 75 5716,981
U.S. Patent Feb. 10, 1998 Sheet 7 of 75 5,716,981

Fig. 7
722.000
,=t
::
2 lq600 -
23 1.200-
s
$ 0.800-
is
t,
4 0.400-
%
2cg0.000 t
0 2 4
I I
6 8
I I
IO
I
12
I
14
IIWGHTOF SURAMll?IN 5Omg POLY
(ETHYLENE-VINYL
Acme) (mg)
Fig. 8
U.S. Patent Feb. 10,1998 sleet 8 of 75 5,716,981

20 40 60 80
SIZE RANGE (jdi DlAhlETER)

fig. 9

SUE RANGE (‘bf DIAMETER)

Fig. 10
U.S. Patent Feb. 10, 1998 Sheet 9 of 75 5716,981

0 eo 40 60 80 100
SIZE RANGE (phi DIAMETER)

poly (D,L lactic acid)

poly (ethylene-vinyl ocetote)


acetote)

0 2 4 s . s ’ tb
TM? (DAYS)

Fig. 12
U.S. Patent Feb. 10,1!298 Sheet 11 of 75 5,716,981

CALL

fig. 14A

STENT HOLLIING
THE BILKD~C
U.S. Patent Feb. 10,1998 Sheet 12 of 75 5716,981

80-

POLYMERBLENDRATIOOF EVA TO PLA

fig. 15f4
U.S. Patent Feb. 10,1998 Sheet 13 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 14 of 75 5716,981
U.S. Patent Feb. lO,l!W8 Sheet 15 of 75 $716,981

I I I I I I
0 10 20 30 40 50
TM? (DA YS)

Fig, 15D
,_( ,..
, ..
‘, i,,y;, -;.‘.~.~,;“.
.; :
..:: ‘f 1 ,,.,

,. ., ,‘, ‘.
.‘,‘,’ ‘, :. : :
._ ” :
U.S. Patent Feb. 10,19!N? Shea 18 of 75 5716,981

50-
+ 1% TAXOL
4 2% TAXOL
-+- 5% TAXOL
+ 10% TAXOL

40-

$ 30-
2Tc .

2
2
2
% 20.

10.

lb . io . io * 40 . 1
TIME (DAYS)

Fig. 16A
U.S. Patent Feb. l&l998 Sheet 19 of 75 5,716,981
U.S. Patent Feb. lo,1998 Sheet 20 of 75 5,716,981

400
RELEASE OF TAXOL FROM EVA FILMS
- I% TAXOL FILM
_ -o- 5% TAXOL FILM
-e 10% TAXOL FILM
4 20% TAXOL FILM T

1 A c I

0 I I I I I I I
2
TIME (DAYSj

fig. f7A
U.S. Patent Feb.10,1998 Sheet21 of75 5716,981

PERCENT TAXOL REMAINING IN FJLMS


+ I% TAXOL FILM
-Q- 5% TAXOL FILhi
-+- 10% THOL FILM
\l 4 20% TAXOL FILM
‘k
go- ‘.Q-
--o-,-
-.
+----&--+,-‘A,
---=k*=*
h

60

Fig. 17B
U.S. Patent Feb. 10, 1998 Sheet 22 of 75 5,716,981

SWELLING OF EVA/F127
FILMS WITH NO TAXOL l-
-a- EVA 100% / F127 0%
4 EVA 35% / Ff27 5%
0 / 0
+ EVA 75% / F127 25% T
// 1
/ /
/ / f
/ /
iE

0 20 40 60
TIME (DAYS)

Fig. 1X
U.S. Patent Feb. 10,19!B Sheet 23 of 75 5,716,981

Fig. 17D
U.S. Patent Feb. 10, 1998 Sheet 24 of 75 5,716,981

STRESS/STRAIN CURVE OF EVA AND


BLEND FILMS CUNTAININC HA/F 127
4 EVA 100% / F1.27 0%
-+- EVA 35% / F?27 5%
-+- BVA 75% / F127 25%

A
-- -4
* _---

0 10000 20000
STRAiN (g/7n?7t2)

Fig. 17E
U.S. Patent Feb. lo,1998 Sh& 25 of 75 5,716,981

44-

431
0 10 20 : 0

% MePEG

0 f0 30

% MePEG

fig. 183
US. Patent Feb. 10,19!W Sheet26 of 75 5,716,981

,:
. ^

0 10 20 30 40
% MePEG

Fig. 186

=O% MePEG
05% MePEG
l 10% MePEG
020% MePEG

0 0 0 0
U.S. Patent Feb. 10, 1998 Sheet 27 of 75 5,716,981

+ 0.8% MePEG
-p- 0.8% MePEG
+ 5% MePEG
4 70% MePEG
-+- 20% MePEG

0 2 4 6
TIME (DAYS)

fig. 18E
U.S. Patent Feb. 10,1998 Sheet 28 of 75 5,716,981

0 2 4 6 8
TM? (DA US)

Fig. 18F

u 1: 0.2% TAXOL
--m- 2: 0.2% TAXQL
-+- 3: 1% TAXOL
4 4: 10% TAXOL

0
0 i s s
TIME (DAYS)

Fig. 18G
U.S. Patent Feb. lo,1998 Sheet 29 of 75 5,716,981

---

% MePEG

Fig. 18H
U.S. Patent Feb. 10, 1998 Sheet 30 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 31 of 75 5716,981
U.S. Patent Feb. 10, 1998 Sheet 32 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 33 of 75 5,716,981
U.S. Patent Feb. 10,1998 Sheet 34 of 75 5,716,981

1.4 0
EFFECT OF TAXOL/PCL ’
ON TUMORGRWTH

*. .

l 8
0

l m

l l

:
0 I I i I
CONTROL PCL PCL, PCL,
102 TUOL 20% THOl,
U.S. Patent Feb. 10, 1998 Sheet 35 of 75

. .. .. i.......

_.,‘:,.. :
: ,.
;: :I’:.-
U.S. Patent Feb. 10,199s Sheet 36 of 75 5716,981

:,+,.,,:<::...y,...~... . . _. ..
_.._.., ._.. . . ..
U.S. Patent Feb. IO, 1998 Sheet 37 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 38 of 75 5,716,981
U.S. Patent Feb. 10,19!M Sheet 39 of 75 5,716,981
U.S. Patent Feb. 141998 sheet 40 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 41 of 75 5,716,981
U.S. Patent Feb. 141998 Sheet 42 of 75 5,716,981
CHEMILUMINESCENCE
(mV)
% INHIRITION
OF CHEMILUMINESCENCE
-NW&cno¶NIaCO;;
00000000000
I I I , , , ., , , ,
s

I
% INHIBITiONOF SUPEROXIDE
ANIONRELEASE SUPEROXIDE
ANION(NMOL/MIN)

-NWPulcn-wlcnCPZ;
0000000000
I I I1 I I I II 1
U.S. Patent Feb. 10, 1998 Sheet 45 of 75 5,716,981

g 800 -
7OO-
E
t3 600-
t2 soo-
s 4OO-
3
zz 300-
x 200-
100'
0 I I I I I 1
0 5 10 15 20 25 30

TjhdE(MINUTES)

Fig. 31A

TIME (IANUTES)

Fig. 318
% INHIBITION
Of ENZYMERELEASE MYELOPEROXIDOSE
(UNITS)

-NCc(PulmV(Dws
00000000000 0
> II,,,,, * , ,

;-
U.S. Patent Feb. 10,19!8 Sheet 47 of 75 5,716,981

TIME (MINUTES)

Fig. 33
120-

m 24 HOURS

20-

0-
C 1o-5 10-6 to-7 10-8
TAXOL,M

Fig. 34
U.S. Patent Feb. lO,l!W8 sfieet 48 of 75 5,716,981

C c lo+ to-' 10'" 10-5 .a-

TAXOL, M P

fig. 35
U.S. Patent Feb. 10,1998 Sheet 49 of 75 5,716,981
U.S. Patent Feb. 10, 1998 sheet 50 of 75 5,716,981
U.S. Patent Feb. 10, 1998 Sheet 51 of 75 5,716,981

TAX01 CONCENTRATION,
M
P

fig. 38
U.S. Patent Feb. 10, 1998 Sheet 52 of 75 5,716,981

40 - GELATIN-TAXOL-PC1
__o__ GELATIN-TAXOL-PCL
(LARGE)
35 LARGE(>ZOOpM)

30

25

SMALL(MO0p.M)
20

15

10

0 I 1 1 1 I , , , ,

0 1 2 3 4 5 6 7

TIME (DAYS)

fig. 39
U.S. Patent Feb. 10,WB Sheet 53 of 75 5,716,981

30 - TAXOL-PCL
- GELATIN-TAXOL-PCL(20:20:60)
- NoCf-TAXOL-PCL(20:20:60)
- GELATIN-NaCI-TAXOL-PCL (10:10:20:60)
25

20

0. I , I , , I ‘ 8 I

0 1 2 3 4 5 6 7

TIME (DAYS)

Fig. 40
U.S. Patent Feb. 10,1998 Sheet 54 of 75 5,716,981

120 - PEG20%-lmm
- PEG30%-lmm
- PEG30%-0.65mm
- PEG40751mm

i';!
g 80
+
m '
??&
44
Yf\
E;:9

0
0 5 10 15
*
m RELEASETIME, DAY

fig. 41
U.S. Patent Feb. 10, 1998 Sheet 55 of 75 5,716,981

-II -o- PCL 30%


--o-- PCL 20%
I-
-cr- PCL 10%
50 * PCL 5%
-0 --p- PCL 1%
II
40

30

20
fig. 42A
10

0
0 2. 4 6 8 10 12 14
TIME(DAYS)

80

60 -k- %remP30%
--+- %remPZO%
*a %remPlO%
--a--- %remP5%
----v- %remPl%

fig. 42B
U.S. Patent Feb. 10, 1998 Sheet 56 of 75 5,716,981
300-

250-

20% MePEG
0% MePEG

1
0 2 4 6 8 10 12 14
TIME (DAYS)

figi 43A

100
90
80
70
60
50
40 - 0% MePEG

30 --c- 20% MePEG

20
10
0
0 2 4 6 8 10 12 14

TIME (DAYS)

Fig. 43B
U.S. Patent Feb. 10,199s Sheet 57 of 75 5,716,981
49

‘-r
2 46-

ti
= 45-

44-

43 I I
0 10 1
20 30
% MePEG

fig. 44A

150-
150

1
I -I-

I I 1
00 10 20 30
% MePEC

Fig. 448
U.S. Patent Feb. lo,1998 Sheet 58 of 75

--p- TENSILESTRENGTH
250- -5

200- -4

150- -3

loo- -2

50- -1

0.-J
01 I I I 1 -0
5 10 15 20
% MePEG
fig. 45

lo-
10 - IRRADIATED
--D-
- NOT IRRAOIATED

0 I I I I
0 5 10 15 20
TIME(DAYS)

Fig. 46
U.S. Patent Feb. 10, 1998 Sheet 59 of 75 5,716,981
7000
1 I
6000

5000

4000
- 2O%MTX
3000
- lO%MTX
2000 - S%MTX
- 2%MTX
1000 - l%MTX

0
0 1 2 3 4 5 6 7
TIME(DAYS)

Fig. 47A

; 200 I& 1 * - 20%MTX


I - fO%MTX u
25 ,b - S%MTX
2 100 - Z%MTX
+ - l%MTX
I I9.
I i f
0 I I I I I I 1
0 1 2 3 4 5 6 7

TIME (DAYS)

fig. 47B
U.S. Patent Feb. 10, 1998 Sheet 60 of 75 5,716,981

PUREpcl THERMOPASTE
- l%MTX
- Z%MTX
100 - S%MTX
- lO%MTX
- 20%MTX

ml THERMOPASTE
1
WITH
20%MEPEC
- lO%MTX
- S%MTX
- ZO%MTX
-20 - l%MTX
- 2%MTX
i
-40 01 1I 2I 31 I I I 7
4 5 6 7

TIME(DAYS)
US. Patent Feb. 10, 1998 Sheet 61 of 75 5716,981

.:
.;
:’ :
._
-<::i:
U.S. Patent Feb. l&1998 Sheet 62 of 75 5,716,981
DIFFERENTIAL
VOLUME
7-r
Ii
6- I
I
I
5- I
I
II
4-
I
3- iI
1I
2- II
II
l- :
0 fI
I I I', I I I I ,I,', I+
0.5 0.6 0.8 1 . 2 3 4567810 20
PARTICLEDIAMETER (pm)
LC=O.429jfmuc=14.96jirn {too.os,\

i?@. 48

DIFFERENTIAL
VOLUME

t
0' I I I
I I I , I I I I-
0.6 1 2 4 6 a10 20 40 60 a0

PARTICLEDIAUETER(pm)
2.398% 8 1.762/m

fig. 49
U.S. Patent Feb. 10,1998 Sheet 63 of 75 5,716,981

--+-LOW PLA-1tOmin
0.2 _+_ LOW PIA- SOmin
1 - LOW/HIGHPDLIA
- LOW PLA/HIGH PLGA -8

I 1
12
TIME, DAY

fig. 50
U.S. Patent Feb. 10, 1998 sheet 64 of 75 $716,981

- op PMMA
- op PLA
- op PCL
- op EVA/PLA
- PMMA
- PLA
- PCL
- EVA/PLA

0 5 10 15 20 25 30 35
TOME(MINUTES)

Fig. 51
U.S. Patent Feb. 10, 1998 Sheet 65 of 75 5,716,981

POLYCAPROLACTONE
g
w 100
x COATING

- PLASMA
50 - fl27+PLASMA
- F127
- UNCOATED

20 25 30

TIME(MINUTES)

fig. 52

150

POLYMETHYU(ETHACRYLATE
g
. g 100. \
0 5 COATING
5:
z" - PLASMA
f
B3 - Fl27tPlASMA
g so-
Y - Fl27
v - UNCOATED
1

O- I I I I I 1
0 5 10 15 20 25 30

TIMI (MINUTES)

Fig. 53
U.S. Patent Feb. 10, 1998 Sheet 66 of 75 5,716,981
150,

POLYIACTICACID

COATING

- PLASMA
- Fl27tPLASMA
50-
- F127
- UNCOATED

0 1
I 1 I . I I 1
0 5 10 15 20 25 30

TIME(wUTES)

fig. 54

75-

ACID/EVA

COATING

- PLASMA
- Fl27tPL4SMA
25-
- F127
- UNCOATED

0 f I I I I I 1
0 5 10 15 20 25 30

TIME(MWTES)
Fig. 55
U.S. Patent Feb. 10, 1998 Sbeet 67 of 75 54716,981
150

E
1 POLYCAPROLKTONE
MICROSPHERES

-
COATED

IgG
loo- II
0” 1 - F127/lgG
z5 - UNCOATED
Y
5i
; so-
5

0 ' I t I 1
0 5 10 15 20
TIME(MINUTES)
. fig. 56

150- POLYMETHYLMETHACRYLATE
MICROSPHERES
COATED-
z - IgG
k? - F127/lg6
3 loo-
- UNCOATED
I

r TI
1. I i

0 I I I
5 10 15 20

TIME(MWES)
fig. 57
U.S. Patent Feb. lO,l!W8 Sheet fit3 of 75 5,716,981

POLYLACTIC
ACID
MICROSPHERES
COATED

- IgG
- F127/lgG
- UNCOATED

TIME (MWKS)
fig. 58

POLYEvA/PL
MICROSPHERES
COATED

TIME (MWES)
Fig. 59
U.S. Patent Feb. lo,1998 Sheet 69 of 75 5716,981
U.S. Patent Feb. 10,1998 Sheet 70 of 75 5,716,981

100

P 90 4

f I I I I I I
0 1 2 3 4 5 6 7 8 9 10

DAYNUMBER

fig. 61
US. Patent Feb. 10,1998 Sheet 72 of 75 5,716,981

. . 2O%von
- lS%von
- lD%votl
I uvu
- S%van
23

“y

1
0 12 3 4 5 6 7 8 10

TIME (DAYS)

Rig. 63A

- 20%van
- 15%van
- lO%von
- 5%van

80 1 I 1 I 1 I I 1 I 1
I
0 12 3 4 5 6 7 8 9 10

TIME (DAYS)

Fig. 63B
U.S. Patent Feb. 10,199s Sheet 73 of 75 5,716,981
U.S. Patent Feb. IO, 1998 Sheet 74 of 75 5,716,981
U.S. Patent Feb. lo,1998 Sheet 75 of 75 $716,981

:.<
5,716:98 1
1 2
ANTI-ANGIOGENIC COMPOSITIONS AND Othershavesuggestedthe Useof antibodiesin the treat-
METFIODS OF USE meat of cancer.Briefly. antibodiesmay he developedwhich
recognizecertaincell surfaceantigensthatare eitherunique.
CROSS-REFERENCE ‘l-0 RELCl??D or mae pcvalent on cancerceils compared to rmmal c&s.
AF’FUWONS s These ~tiies, a “magic builets.” may be utilized either
alone a conjugatedwit& a toxin in o&r to specifically
This applicationis a division of U.S. patent applicati0n target and kill turn- cells GMlman. ‘Antibody Therapy.”
Ser. No. W417.160, filed Apr. 3, 1995. now abandoned; Frincip&s qf Cancer Bidwwpy. Oldham (ti). Raven
which is a contirmation-in-partof U.S. patent application Press.hi.. New Yort 1987).However.onedifkiarltvI is that
Ser.No. CRVW536, filed Jul. 19. 1993,now abandoned 1o most mormclonalantiies are of murinc origin. and thus
TECHMCALFIELD hypersensitivityagainstthe murine antibody may limit its
efficacy.particularlyafier repeatedtherapies.Commonside
The psent invention relates gencdly to coqositiotks effcetsin&de fever.sweatsaadchills. skin rashes.arthritis.
and methods for treating cancer and other angiogenic- and nervepalsies.
dcpeudentdiseases.and more sptcificahy. to compositions One additionaldifficulty of presentmethodsis that local
comprisingaoti-angiogenicfactors and polymeric cauias, IS recutrcnceandlocal diseasecontrol remainsa major chal-
stern.5which havebeen coatedwith such compositions.as leugein the treatmentof malignancy.In particular.a total of
well as methodfor utilixing thesestentsaud compositions. 630,ooO patientsannually (ia the U.S.) have localixed dis-
BACKGROUND OF THR INVRNTION ease(POevidenceof distantmetastaticspread)at the time of
1. presentation;this represents64% of al those patientsdiag-
Angiogenesis-dependent diseases(Le., those diseases nosedwith malignancy(this doesnot include nonmelanoma
which require or induce vasculargrowth) repaesenta sig- skin cancaror carcinomain situ). For the vast majority of
ni.6cantportion of all diseasesfor which medicaltreatment thesepatients,surgicalresectionof the diseaserepresentsthe
is sought.For example.canceris the secondltadiag cause greatestelm@ for a cureand indeed428.000will be cured
of deathin the united states.andaccountsfor over one&M 25 after the initial ttWment428.000. Unfcxtuaatcly,202.ooO
of the total matality. Briefly, canceris characterkd by the (or 32% of all patientswith lokalixcd disease)will relapse
uncontrolleddivision of a populationof cells which, most after the in&l treatment.of thosewho relapse.the number
typkdly. leadsto the formationof oneor moretumors.Such whowillrelapseduetolocalrecmrenceofthedisease
tumorsarealsocharacte&dbytheingrowthofvasculature amounts to 133.000 patients aapually (or 21% of ail those
which provide various factcrs that permit continuedtumce 3. with localizeddisease).The aumbtxwho will relapsedue to
growth. Although cancer is genaally mace readily diag- distantmetastases of thediseaseis 68.000patientsannually
nosedthan in the past. manyfcems, evenif detectedearly, (11% of all thosewith kxAized disease).Another 102.139
are still incurable. patients annually wiIl die as a direct result of an inability to
A varieryof methodsarepresentlyutilized to treat cancer, cord& the local growth of the disease.
including for example. various surgical procedures.If s Nowhere is this problem more evident than in breast
treated with surgery alone however, many patients cancer,which affeus 186.000womenannually in the U.S.
@arIiculatiy those with eertaia types of cancer. such as and whose mort&ty rate has remainedunchangedfa 50
bre.asLbrain. colon and hepatic cancer) will exprzience years. Surgical resection of the diseasethrough radical
recurrenceof the cancer.Tbaefore. in additionto surgery. tn@eUomy.modified radical mastectomy.or lumpectomy
many cancersare also treatedwith a combinationof t&a- 40 remains the mainstay of treatment for this condition.
pies involving cytotoxic chemotherapeuticdrugs (e.g.. UnfatunatelY.39% oftbose treatedwith lunmectomvalone
vinuistine. vinblastine,cisplatin.methotrexate.5-FU, etc.) will develcpbrecuuenceof the disease,and &prisir&y, so
and/a radiationtherapy.One di5culty with this appwacth will 25% of thosein which the resectionmargin is found to
however, is that radiothcrapeuticand chemotherapeutic be ckr of tumor histologically.As many as 90% of these
agents arc toxic to ncamaI tissues, and often aoatc life 45 local recanren ceswillocauwithin2cmoftheprcvious
thmtening sideeffects.In additiion,theseapproahesoften excisionsite.
have extremelyhigh failurckuission rates. Shihrly. in 1991.over 1l3,ooOdeathsand 238.600new
In addition to mgical, chum+ and radiation therapies, casesof liver meW&sis were npgtul in North America
othersbaveattempWtoutilkeanindivi~sownimmune alone. The mean survival time for patients with liver
systeminordertoeliminatecancerouscells.Fcrexample, x, memtascs is only 6.6 months once liver lesions have
some have suggested the use of bacterM ce viral compo developed.Non-surgicaltnatmUlt for hepatic m&stases
nentsas adjuvantsin adex to stimulatethe immune system include systemic chemotherapy. radiation.
to destroytuma cells. (SeegenerallyTrinciplea of Cancer chemoemhohxation.bepatic arterial chemotherapy,and
Biotherapy,”Oldham(ed), RavenPress,New Ya 1987.) iam radiation.However,despiteevidencethat such
Suchagentshavegermrallybeenuseful as adjuvantsand as 55 treatmentscan transientlydecreasethe size of the hcpatk
nonspecific.uimuhts in animaltuna models,but havenot lesions (e.g.. systemicchemotheqy and hepatic arterial
as of yet provedto be genaally effective in bumarm chemothempyinitiaUyreduceslesionsin H-2046. and 80%
Lymphoki.r4es have also beenutilized in the treatmentof of patients.respectively),the lesions invariably reoccur
cancer.Briegy. lymphokines are seueted by a variety of Surgicalrcscetionof liver mctastasesrepresentsthe only
cells, and generallyhave an efFecton specilic cells in the 60 possibilityfor a cure.but sucha procedureis possiblein only
gcllcratiotlof an immune response.Examplesof lympbolr- 5% of patientswith metastases.and in only H-208 of
iJJc!sinclude Intffleukins (IL)-1. -2. -3, and -4. as well as patientswith primaty bcpatic cancer.
colony stimuh% factors such as GCSF, GM-C!% and One methodthat haskfsi attemptedfor the treatmentof
M-W? Recently,one grouphas utilixed IL2 to sbmulak tumas with limited successis ttlaapcutic emboIimiocb
peripheralblood cells in orderto expandand producelarge 65 BrielIy, bloodvesselswhi& nourisha tumor aredcliiately
quantities of cells witida arc cytotoxic to tumor cells blockedbyhjeuion of an embolicmat&al into the vessels.
(Rosenberget al., N. EJsgLJ. Med 313:1485-1492,198s). A varie4yof mate&Is have been attemptedin this regard
5,716,981
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inciuding autologoussubstances suchasfat. blood clot and metal (e.g.. a vanadiumspecies.molyb&rium species.tung-
choppedmuscle fhcgrBellts.as well as altiflcial maaials sten species.titanium spcclcs.niobium species[x bintalum
such as WOOLcotton. steel balls. plastic ar glass b&s. spccles)which inhibits the form&on of new blood vessds.
tankdmnpowder.siliconecompounds,radioactiveparticles. witbin oneembodimentof the invention.the composition
StKile absorbaMegehlia sponge(st&pott. GtWam), oxi- 5 has an average size of 15 to 200 pm. within other
dized oeUulost (Ox@). steel coils, alcohoL lyophilixcd emlmlheats, the polymeric cania of the compositionhas
human dura mater (Lyodura), microlBriUar collagen a molecularweightranging from less than LOGI daltoosto
(Aviteac). collagen fibrils (Tachotop),polyvinyl alcohol lzrcata than 2amoo to 3oo.coo daltolls. withill vet other
sponge(PVN, Ivaloa). Barium-impregnated silicon spheres mbodimeats.the comqitions provided herein-may be
(Biss) anddetachableballoons.The sizz of l&r metastases10 klXtlCd blt0 fib With 8 lbi&eSS Of b&WCCB 100 p and
may be temporarily&~easal utilizbq such methods.but 2 mm, or thamologically active compositionswhich are
tumors typically respondby causing the growth of new liquid at OBC tunpahue (e.g.. above45” C.) sad solid or
blood vesselsinto the tumor. semi-solidat snother(e.g.,37OC.).
A relatedpoblcm to tumor formationis the devel~meat wil aaodla aspat of the presentinvcutioa methods
of caacerousblockageswhich i&ii the fiow of material 15 for embolizinga blood vesselsre provided comprisingthe
step of deliveringinto the vasd a therapeutically&ctive
through body passageways, suchas the bile ducts.uachca. amount of M anti-angiogeniccomposition (as dcsaibcd
esophagus.vasadatureand urethra One device, the stcnf above).such that the biti vessel is effectively occluded.
has been developedin order to hold open passageways Within one embodimentthe anti-aagiogeniccompositionis
which have been bloclad by tumors or other sukttanccs. delivacd to a blood vesselwhich nourishesa tumor.
Rqrcsentstive examplesof common stems iacludc the 20
within yet Moth+% aSpWr Of the PSCBt iDVClltiO0. StCDtS
Walkcat. Strecka steat. Gianmrcosteak and the Pahnaz
stent The major ~obkrn with steats.however,is that they are provided comprising a geacmlly tubular struc~urc. the
surface being coated with one cx mom anti-angiogcnic
do not prevent the iBgrowI of tumcr or iat3ammatory
mataial throughthe intastices of the steat.If this material compositions.within OthK as- of the prtscnt inveatioa.
reachesthe iasidc of a stcat and compromisesthe stent 25 methodsare providedfor expandingthe tureen of a body
lumen. it may result ia blockageof the body passageway passageway. comprising inserting a stent into the
into which it has bcca inserted.In additioa psencc of a passageway.the stmt having a gcncraily tubular skucture,
stcntin the body may iaduccrcactivcn inflammauuytissue the smfsoz of the skuclure being coated with an anti-
(e.g., blood vessels.fibroblasts.white blood cells) to eata aagiogcnicamposition as dcsa-iid above.such that the
the steat tureen.resultingia partial cc compktc dosure of 30 passagewayis expanded.within various embod&ats of
the stent. the iave~tion.methodsare providtd for eliminating bilisry
ohstructioas.comprisiag inserting a biliary stcnt into a
The presentinventionprovidc~compositionspad mcth- biliary passageway;for chmin&ng urethral obskuctions.
cds suitable for heating cancers.as well as other non- comprising insating a urethral stcnt into a urtthra; for
tumcrigenic angiogencsisdcpca&atdiseases,and fmtha eliminating esophagealobskuctions. comprising inserting
35
providesother relatedadvantages. an esophagealstem into an csopbagus;and for elimiaatiag
SUMMARY OF THE INVENTION kachcaVbroachial obskuctions. comprising inserting a
tmchcalibroadtialstentiuto the tracheaor bfonchi. In each
Briefly stated, the present iaventioa provides aati- of these embodiments.the dent has a generally tubular
angiogeniccompositions,as well as m&hods and dcviccs 40 StruUure.the surface of which is coated with an aati-
whkil utilize suchcompositioBsfor the treatmentof CancQ aagiogenicCOlBpO&OB as dcsaii above.
and other angiogeaesis-dcpcrideat diseases.Within one W&in antier aspectof the presentiavcntion. mcthcds
aspectof the presentinventioa impositions arc pvidcd are provided fCr treating tumor excision sites. coqaising
(anti-angiogeaic compositions)comprising (a) an anti- adminiskaingan anti-anglogcnlccompositionas dcscribcd
angiogenicfactor and(b) a polymuic c&a. A wide vroiety 45 above to the resaxioa margins of a tumor subsequeutto
of molc4ulesmay be utilized within the scopeof theFcseat excision. such that the local recurmaceof cancer aad the
iavcntioa BSanti-aagiogcaicfactors,induding for example formation of new blood vesscis at the site is i&Wed.
Anti-Invasive Fador. retiaoic acids and $hcir derivatives. wlthia yet Motba aspwt of the iaveatioa. mctbods for
paclitaxd including analoguesand dcrivativts thereof, keating coracal ocovascularizatJoll are provided, amlplis-
suramia. msuc Iahibitot of Metalloproteinnsc-1,Tiisuc so iag the step of administeringto a patient a tbcrapcutically
Inhibitor of MeUoproteinase-2. PiasminogeaActivakr cffccrive amouat of sa anti-angiogenic compo6itioa as
lnhiiitor-1 and Pla.uninogtoActivator Inhibitor-2, and dcsetihcd aboveto the coma. such that the formation of
lighter Y group” transitionmet&. Similarly~P wide variety blood vessels is inhibited. Within one embodiment.the
of polymeric carriers may be utilized. rcprcscntative anti-aaglogtmlccomposhion fuxtlux compiscs a topical
examplesof which include poly (cthyleac-vinyl acetate)SS corticostaoid.
(40% cross-linked).poly (D.L.lactic acid) oligomcrs and Withia anotha aspectof the present invention, methods
polymas. poiy (ihctic acid) oligotnus andJJO@IlKS, poly are provided for inhibiting angiogenesisin patients with
(glycolic acid). copolyn~~~of lactic acid and glycolic acid non-tumorigcaic,angiogenuisdcpcadcnt diseases,com-
poly (capolac&ne),poly (valr!rolactone). poly (anhydrides), prising adminiskriagto a patient a tbaaptuticaUy effective
t-apolymKsof poly (cqxolactone)or poly (lactic acid)with 60 amount of paclitaxel to a patient with a non-tumorigenic
jrolyethyleneglycol, sad blendsthereof. angiogeuesisdcpcndant disurst. such that the formationof
Within ccatainfrefand ca&o&mctits, the compositioas new blood vesselsis inhibited. Within other aspects.mcth-
comprlsca compouadwhich disruptsmicrotubulefuadion, ads an pcovidcd for cmbolixiug blood vessels in aon-
sucll as. for example.pacliuuctl. c.stramustiae. colchicjne, tumorigeBic,mgiogeaesis-depen&ntdiseases,comlxising
mcthokcxatc,curacin-A.cpothilone.vinblastineor BC!EV. 65 &lhmiBg to tilt vessela thcrapWticallyeffcctivc amountof
within otha preferredcmbo&unts. the compositiouscom- a compositionatmprising paclitaxcl. such that the blood
@se a polymeric aria and a lighter d gropu transition vessc.lis cffcctivcly occluded.
5,716,981
5 6
Within yet otheraspectsof thepresentinvention,methods image takenwith a stereomicroscope. of JivJng,unstained
areprovidedfor expandingthe lumenof a body passageway, cqiJlaries (1040x). FIG. 1C is a photographof a corrosion
comprisinginsetting a stent into the passageway, the stent castingwhich showsCAM miuovasculaturethat arc fed by
having a genemdlyMxJar structure.the surface of the Jarga. urtderJyingvessels.(arrows; 1300x). FIG. ID is a
structure being coated with a composition comprisJng s photographwhich depictsa 0.5 nunthickpJastic sectioncut
paclkaxel. such that the passagewayis expanded.Within transvasely through the CAM, and recorded at the light
variousembodiments of&beinvention.methodsareprovided micruscopeIed. This photogr;aphshowsthe composition
for eliminatingbJJiaryobstructions,compiisingJnsating a of the CA&I, including an outer double-layeredectoderm
bilhry stent into a bihary passageway;for ehminating (EC). a mesoderm@I) contai&g cqiharies (arrows) and
urethraJ0bstmctJons.com&Jng insating a urethraI stent 1. scat&redadventit& cells. and a single layered endoderm
into a urethra; for eEminating esophagealobstructions. (J3) (400x).FIG. I.3 is a photographat the eJectronmicro-
ampxising insuting an esophagealstentinto an esophagus; scopelevel (3500x) wherein typic& capibary structureis
and for eJimJnatin8tracbeaUhronchiaJ obstructions,com- presented showing thin-walled endothelial cells
@sing insettinga tracheal/bronchial dent into the &a&a or (arrowheads)and an associatedpen’cyte.
bronchi.Within eachof theseembodimentsthe stenthas a FIGS. 2A2B. 2C and 2D am a saics of digitized Jmages
genuaJJytubularstructure,the surfaceof the structurebeing t5 of four diffaent. unstainedCAMS taken after a 48 hour
coatedwith a compositioncoquJsJngpachtaxet exJxmre to digitized Images of four different living.
Within anotheraspectof the prcscntinvention.methods unstainedCAMS wac takenafter a 48 h exposureto 10~
are providedfor treatinga tumor excisionsite. comprisiig pacJitaxe1per 10 ml of methylceJJulose. The transparent
administuiog a compositioncomprisktgpacJJtaxe1 to the methyJcclh&se disk (+) conrainingpaeJitaxeJ is presenton
resectionmar@ of a tumor subse8utatto excision. such m eachC~bf and is positionedover a sing&w avas&ar zone
that the localrecurrenceof cancerandthe formationof new (A) wJth surroundingblood islands (Is). These avascular
blood vesselsat the site is itdriited. within orheraspects, areasextendbeyondthe disk and typically have a diameter
methodsare provided for treating neovasctdardiseasesof of 6 min. FJG. 2D illustratesthe typical “elbowing” effed
the eye,cotqxising adminJsterJng to a patient a thaapeuti- (arrowheads)of both .smaJJ and large vesselsbeing redi-
caJJyeffectiveamountof an anti-angiogenicfactor (suchas a rectal away from the peripheryof the avascularzone.
a compoundwhidt disrupts mJuotubuJefunction) to the
eye.su& that the fcrmation of new vesselsis inhibited. FIG. 3A is a photograph(Mag==x) which showsjust
WitJtJootheraspectsof the presentinvention.methodsare peripheral
(amowheada)
to the avascular zone. that capillaries
exhJbJtnumerousendothelialceJJsarrestedin
povided for treating in&mmatory a&ritJs. comprising
administeriogto a patienta therapeutically&ective amount mitosis. - (Ec); Mesoderm (M); Endoderm(Rn).
of an anti-angiogenicfactor (such as a compoundwhich x, FIG. 3B (Mag=tOOx)showsthat within the avascular zone
dhupts microtubulefunction).ora compositioncomprising proper the typical capiJJarystructurehas been cJiminated
an anti-angiogenicfador and a polymericcauier to a joJnr and there are numerous extravasatcd blood cells
Within preferredembodhnents,the anti-angiogedcfactor (arrowheads). FIG, 3C (Mag==x) showsthatJnthe centrat
may be a compoundwhich disruptsmicrotubulefunction area of the avascularzone. red blood ceJJsare disposed
suchas padJtaxe1.or an demerit from the lighter ‘d grollp’ ” throughoutthe mesoderm.
transition metals. such as a vanadiumspecies. FIGS. 3A. 333and X are a seriesof photogra@ of 0.5
Witbin yet aootha aspectof the invention.pharmaceuti- mm thick plastic sections transversely cut through a
al productsareprovided,contpisiog (a) a compoundwhich pachtaxei-treatedCAM at three differcot locations within
disrupts microtubuJefunction. in a container.and (b) a the avascular zone.
notice associated wJtJrthe containerin form prescrii by a 4o FXGS.4A. 4B and 4C arc series of electronmicrographs
goveromental agency regulating the manufacture.use. or which wae takenfrom locationssimilar to thatof FIGS. 3A.
sale of pharmaceuticals.whJch notice is reflective of 3B and 3C (respe&veJy)above. .
approval by the agency of a compoundwhich disrupts FIG. 4A (Mag=22OOx)shows a srnah capJJJarylying
mJcrotubuJe tirnctioe for humanor vetetimuy administra- subjacentto the ectodermaJJaya (Ec) posse&JagtJuee
tion to treat eon-tumcrigenicangiogenesisdependent dJa- 45 cndotheliatc&s arrestedJn mitosis (+). Severalother ceJI
easessuchas.for example.intlammatoq arthritisor neovas- typesinbotJtthtectodermandmesodermareaJsoarrested
cular diseasesof the eye.BriefJy,FederaJLaw requiresbrat io mitosis. FIG. 43 (Mag=Z.8OOx) shows the early yvasasiar
the useof a pharmaceutJcaJ agentJn the therapyof humans phase contains extravasatedblood cells subjacent to the
kc approvedby an ageney of the Federal govunmcm ectd, theseblood celtsarei&mixed with presumptive
Responsibilityfor enforcement(in the UssitedStates)is with m cndothellalcells (*) and their processes.DegratJvecellular
the Fooda6dIhug Administration,which issuesappqrJate vacuoles(arrowhead).FIG. 4C (Mag==2.8OOx) showsthatin
ngulationsfor securingsuchapJn0va.L detailedin 2I U.S.C. responseto pacJitaxe1.the ecto-me- iutuface has
~$301-392.Regulationfor bJoJogicaJ mataiaLscomprising beumepopdated with cellsin variousstagesof degradatJon
pducts madefrom the tissuesof animals.is also provided containittgdensevacuolesand granules(auowheads).
under42 U.S.C.$262.SJmiJarapprovalis requiredby most 35
countries,although.regulationsmay vary from country to FIG. 5 is a bar graph which depictsthe size distrJbutJon
country. of microspheresby number (5% poiy (ethylene-vinyl
acetate)with 10 mg sodiumsurammJnto 5% WA).
TJmc and other aspects of t&e presentinvention will
bwme evident upon referenceto the foJJowJngdetaJJed60 of FIG. 6 is a bar graphwhich depictsthe size distribution
microspheres by weight (5% poJy(ethylene-vinylantate)
dewiption and attacheddrawings. In addition, various
referencesareset forth beJowwhich de&be in moredetaiJ with 10 mg sodiumsuramininto 5% WA).
catain proccdurrs. devices or compositions, and arc thcrc- FIG. 7 is a graph which depicts the weight of ennrpsu-
fare incclrporated by referencein their entirq. lation of Sodium Suramin in 50 mg poly (ethylene-vinyl
-1.
BRIEF DRSCRR’TIONOF THE DRAWINGS 6s FIG. 8 is a graphwhich depictsthe percentof encapsu-
FIG. lA is a photographwhich shows a shell-lessegg lation of Sodium Suraminin 50 mg poly (ctJrylen*vinyJ
cultureon day 6. FIG. 13 is a digitized computerdispJayed acetate).
5,716,981
7 8
RG. 9 is a bar graph which depictsthe size distribution is unaffected.FIG. 19B is a photographof 20% paclitaxel-
by weight of 5% ELVAX microspherescontaining 10 mg loaded thamopaste on a CAM. Note the disnrptionof the
sodium suraminmadein 5% PVA conmining 10% NaCL Y8sculattlre when compartd to the smndiog tissalcs. The
FIG. 10 is a bar graphwhich depictsthe size distribution drug loadedpaste has blockedthe growth of the capihxies.
by wdght of 5% microspherescontaining 10 mg sodium 5 pausedregressionof the largervessels.andcreated a region
suramii madein 5% PVA coataioing10% NaCL of avascular@on the CAM assay.PlG. 19Cis a photograph
of 0.5% paditaxel-loadedtbemxmstc on a CAM (Man.-
FIG. 11 is a bar graph which depictsthe sire distribution 40x). B&ly. the paclitaxel&adcd thermopa& zisk
by numberof 5% microspherescontaining 10 mg sodium induwd an avascular wtle memrhg6mrniodiametaon
suraminmadein 5% PVA containing10% NaCL 1o the CAM. This avascuhrrregion was inducedby blocking
RG. 12 is a line graphwhich depictsthe time courseof new capWirygrowth andoccluding.disrupting.andregress-
sodium suraminrelease. ing the existing blood vessels found within the treated
PIG. 13 is an illustration of a representativeembod&mnt region. FIG. 19D is a photographof control (unloaded)
of hepatic tumor aribolizatiw. Thermopasteon a CAM. Briefiy. after a 2 day exposure.the
RG. 14 is an illustration of the insertion of a repcesen-1s
blood vesse1cu8anirationof the CAM (Mag=50x) treated
tative stent coatedwith an anti-angiogeniccomposition. with the contrcl pasteshowsncrmal Moodvesselorganira-
tion. Functional~easds arc locatedimmediatelyadjacentto
FIG. IsA is a graph which shows the c&t of the tileunlwdedpaste.
EV&PM polymer blend ratio upon aggregatiooof micro- FIGS. 2OA and 2oB are two photographsof a CAM
spheres.FIG. 158 is a scanningelectronmicrographwhich haviog a arrn~ treatedwith control(unloaded)thermq~ste.
showsthe size of “smaU”miuosphaes. FIG. 1sC (which 20
Briefly. in FlG. 20 A the centralwhite massis the tumor
indudes a magnitiedinset-labeUed WC-inset”) is a scan- tissue. Note the abundanceof blood vesselsentering dre
ning elexthonmiaograph which showsthe size of “large” twtm from theCAM in ali directions.Tire tumorinducesthe
microsphaes. PIG. MD is a graphwhich depictsthe time ingrowth of the host vasculaturethrough the productionof
course of in vitro pa&axe1 release from 0.6% wlv ‘kngiogeoic factors.” The tumor tissue expandsdistaIiy
paditaxel-loaded50~50E!VAzEXApolymer blend micr+ 25 along the blood vesselswhich supply it FIG. 2OB is an
spheresinto phosphatebufferedsaline (pH7.4) at 37’ C. undaside view of tireCAM show0in 20A. Briefly. this view
Opeo circles are “small” sized microspheres.and dosed demonstratesthe radial appearanceof the blood vessels
circles are “large” sized miuospheres.FIG. l5P is a pho- whidt eata the tumor like the spokesof a whcd. Note that
tograph of a CAM which shows the results of paditaxel the blood vesseldensityis greatain the vicinity of the tumor
30
releaseby microspheres(“MS”). FIG. l5P is a photograph thanit is in the surroundingnormalCAM tissue.FIGS. 2OC
simihu to that of SE al increase magnificati0~ andaODaretwophotograpbsofaCAMhavingatumar
PIG. 16A is a graph which showsrdease rate prorilts treated with 20% paditaxd-loadedtbermopastc.Briefly. in
tiom polycaproIactonemicrospberescontahliog 1%. 2%. FIG. ZOCthecentralwhite massis the tnmu tissue.Note the
5% or 10% paditaxd into phosphatebuffaed salineat 37O paucity of bloodvesselsin the vicinity of the tumor tissue.
35
C. RG. MB is a photographwhich shows a CAM treated The sustainedrelease of the angiogenesisinhibitor is
with control microsphaes.PIG. 16C is a photographwhich capableof ovacomingthe angiogenicstimulusproducedby
shows a CAM treated with 5% paclitaxeI loaded miuc+ the tumu. The tumor itself is poorly vascularhedand is
spheres. progressivdydtmeahg in size.RG. #ID is takenfrom the
PIGS. 17A and 17B. respectively.are two graphswhich 40 undaside of theCAM shownin MC. anddemonstrates the
show the rdase of paditaxel from EVA films. and the Dixon of blood flow into the tumor whencomparui to
pacent paclitaxl rcmaimngin thosesamefilms ova time. c~ntml tumor tissue. Note that the Mood vcssct dcosity is
FIG. 17C is a graphwhich showsthe swell@ of EVA/P127 rcduccdinthevicinityofthtturnor~disspiaserthanthat
iilms with DO paditaxel ow time. FIG. 17D is a graph of the normal surroundingCAM tissue.
whidl shows the swdlitlg of Evmpall80 films with no 4s RG. 2L4 is a graphwhich showsthe effectof pacliuuel/
paclitaxel over time. FIG. 17E is a Bnph which depictsa PCL on tumorgrowth, FIGS. 21B and 21C arc two photo-
stressvs. strain curve for variousEVA/FU7 blends. graphs which show the effect of control. 10%. and 20%
RGS. 18A and 18B arc two gra$s which show the paditaxel-loadedthm-mopaste on tumor growth.
mdting point of PCUhlepEo polymerblendsas a function FIG. 2% is a photographof synotium from a PBS
of % MePEG in the formulation (MA), and the percent so injectedjoint. PIG. ZZB is a photographof synoviumfrom
inacastintimentededforpcL.pasteat6ooC.tobeingto a microsphereinjected joint. FlG. 2X is a photographof
solidify as a function of the amount of MePEG in the cattihsgetirn joints injected with PBS. andFTG.22D is a
formulation (18B). FIG. 18C is a graph whic!b depicts the photographof cartilage from joints injected with micro
softoessof varying PUfMePEG polymer blends.FIG. 18D spheres.
is a graphwhich showsthe percentweight changeova time 55 PIG. 23A is a photograph of a 03% Paclitaxel Oph-
for polymer blendsof variousMcPE!Gconcentrations.FIG. thalmic Drcq Suspcnsioa00 a CAM (Mag.=32x). The
18Eisagraphwhichdepictsrheratcofpaditaxdrrlew plastic ring was usedto localizethe drug treatmentto the
over time from various polymer blends loaded with 1% CAM. Note the lack of blood vessels kated within and
paclitaxel. FIGS. 18F and 18G are graphswhich depict the imu&iaWy adjacentto the ring. The functional blood
elfea of varying quantitiesof paditaxel on the total amount a vessels bordaing the avasw zoac are definedby their
of pa&axe1 releasedfrom a 20% McPEG/XL Mend.F’lG. “elbowing” morphologyaway fom the dmg source. FIG.
ISHisa~whichdcpiasttrc~~ofMePEGonthc 23B is a pbotogmpbof a control (unloaded)ophthalmic
tensile streogthof a MePEGKL po!yrner. Drou Susnensi~ on a CAM Na~32x). Note the normal
PIG. 19A is a photographwhich showscootroi (unloaded) org&a&o of the CAM blood v&s& hodthe abundance
thamopaste on a CAM. Note that both large vesselsand 65 of functional vessels locatedwitbln the ring.
.smaUvessels(cxpibriea) arefoundimmediatelyadjacentto FIG. 24A is a photogmphof a 2.5% Paclitaxel-Loaded
the paste.Blood Bow in the arcaaroundand underthe paste Stcnt Coating (Mw26x). Briefiy, the blood ~cssels sur-
$716,981
9 10
rouodingthe avascularzonem moiphologicailyredirected CFPD crystals (50 rnghl,. Bffcct of pa&axe1 at (0) no
awayfrom the paclitaxclscurce;this producesan avascular pad&cl, (‘) 28 p&f, (A) Control (cells atone), (A) Control
zone which can measureup to 6 mm in diamucz The (ce&andpaclitiuclat28pM);n3.
disrupted vascular remnants which represent vascular FIG. 34 is a graphwhich depictsproliferationof synovio-
regressioncaa be seenwithin the avascularzone.FIG. 24B 5 cytes at vaious concentrationsof paciitaxel.
is a control (unloaded)Stent Coatiag (Mag=26x).B&fly. FIG. 35 is a bar graphwhich depicts the cytatoxicity of
the blood vessds of the CAM are found immediately paditaxcl at various coaccntrations to pro&rating synovio-
adjacentto the stult anddo not illustrate ahoy1~aph010gicd cytec
alta;ltionS. FIGS. 36A. 36B. and 36C arephotographsof a seriesof
FIG. 25 is a photographof a control stint. Briefly, this 10
gels which show the effcu of various conczntrationsof
image shows the longib~dinaiorientationof a nylon stent paclitaxelon c-FOSexpression.
incorporatedwithin gliosarcoma tissue of the rat liver. FlGS. 37A and 37B are photographsof a seriesof gels
Ingrowthwithin the nylon stmt is evident. which showtheeffectof variousconcentrationsof paclitaxel
FIG. 26 is a photographof a control stcnt. Briefly, this on collagemscexpression.
imagealso iuustratcstumor ingrowth WithiJlthe tureenof 15 FIG. 38 is a bar graph which depicts the effects of
the nylon stent. paditaxel on viability of namal chondrocytcsin vitro.
FIG. 27 is a photograph of a lung. Briefly. in additionto FIG. 39 is a gap41which showsthe parentage of padi-
largeliver tumors.metastasisto the lung is amunon. Such taxel rdease basedupon gdatinixcd-pditaxcl of either a
mctastas.zsarc evident by the presenceof small white 20 large (7200 pm) or SmalJ (2100 pm) size.
lobole seenthroughoutthe lung, FIG. 40 ls a graph which showsthe effectof gelatinand/or
FTG. #IA is a photographof Suramin and Cortisone scdium chlorideon the rfkdsc of paclitaxtl from FTL.
Acetate on a CAM (Map*). Briefly. this imageshowsan FIG. 41 is a graph which shows the release of paclitaxd
avascularmne treatedwith 20 pg of suraminand70 pg of from PDLLA-PEG-PDUA cylinderscontaining20% padi-
cortisoneacetatein 0.5% ~nethylcelklosc.Note the blood 25 UXCL
vesselsiocakd at tbc puipbuy of the avascularzonewhich FIG. 42A is a graph which depicts the time course of
arebeingrcdireded awayfrom the drug source.FIG. 28B is paditaxei releasefrom 2.5 mg pellets of PU. F’IG.423 is
a photographwhich showsthe vasculardetail of the dkcted a graphwhich showsthe percentpaclita~elremainingin the
rcgirm at a highu magnilication(Mag==2Ox). Ncte the avaa-
atlar regionsand the typical uclbowing”effectof the blood 30 pdlct, over time.
vesselsbordcriagthe avascularzone. FXG. 43Ais a graph w&b showsthe cl&t of MePEGon
FIG. 29A is a graphwhich shoWsthe chemiluminescencc
paditaxcl rdcasc from PCL paste lea&d with 20% pacli-
taxel. FXG. 43B is a graphwhich showsthe percentpacli-
response of neutrophils (Sx106 cells/ml) to plasma taxel -g io the pdl#. OVCItime.
qJsonizedCFPD crystals(50 q/ml). Effect of paclitaxd at
(@&no p~litaxel. (.) 4.5 JIM.(A.) 14 m. (A) 28 @t (0) 46 35 FIGS. 44A and 4dB are graphs which show the effect of
:n=. various amccntions of MePE!Gin PCL in terms of
melting point (44A) and time to solidify (44B).
FIG. 29B is a gqh which showsthe time coursecon-
centrationdependenceof paclitaxel inhibition of plasma FIG. 45 is a graph which shows the effect of MePEG
cpsonizcdCPPD crystal inducedswtrq~hil chcmilurnine~ immpmdion into FCL on the tensile strengthand time to
ceoa. 40 fail of the polymer.
FIG. 30A is a graph which shows superoxidt anion FIG. 44 is a graphwhich showsthe effectof irradiationoo
productionby ncutrophils(5x10* c&s/ml) in responseto paditaxel release.
plasma opsonizcd CPPD uystals (50 mglml). Effect of FIGS.47A, B. C. D sod E show tbc c&‘ed of MTX release
paclhaxelat (0) no pa&tax& (‘) 28 pM. (A) Control (cells from PCL ovcztime.
45 FlG. 48 is a graphof partide diameter(pm) &term&d
alone);n=3. FIG. 30B is a graphicwhich showsthe time
courseconcentrationdependenceof paditaxd inhibition of by a Co&a@ LS130 Partide Size Analysis.
plam opsonized CPF’D uystal induced neuhqhil supa- PIG.49isagraphofpartidediamder()un)dckrmined
oxide anion production: n 3. by a CoultacQ Is130 Farticlc Size Analysis.
FIG. 3lAis a graphwhich showsthe d~cm&tminesocr~~e
50
FIG. 50 is a graphwhich showspaclita~elreleasefrom
responseof ncutrophils (5x10” ccWml) iu responseto variouspolymuic fcXmulations.
plasmaopsonizcdzymozan(1 m&l). EiRct of paditaxe1at FIG. 51 is a gtl?pbwhich shows the effect of plasma
(0) no drug, p) 28 pM; x1=3. FIG. 31B is a graph which opsonlzationof polymuic microspheresOPtit chemilumi-
showsplasmaops5nizedzymosaninducedncutrophilsuper- neLesceMxresponseof neutr (20 m&d microsphcrcsip
oxide anion production. Effect of paciitaxel at (0) no 55 0.5 ml of cells (cont. 5x1F cells/ml) to PCL microsphcres.
pacli~cl. p) 28 pM. (A) Control (cells alone);II 3. FIG. 52 is a graph which showsthe dfcu of precoating
FIG. 32A is a graph which shows mydopcroxidase plasma #-2% @Ironic Fl27 on the dlemilumirlesceBcc
rekase from neutrophils(Sxld c&/ml) in responseto mpoose of neutrophils(5x106 ccll&lll) to PCL micro-
plasma opsonizedCPPD crystals (SO r@ml). Effc& of sphacs.
paclitaxelat (0) no pad&ax& (‘) 28 pM. (A) Con&d (cells 60 FIG. 53 is a graphwhich showstbt dku of prccoating
alone).(A) Control (cells witb paclitaxel at 28 JIM); n=3. plasma if-2% plurotic Fl27 on the chemiluminesccnce
FIG. 32B is a graph whid~ shows the co~~centration rcsponscof ncutropbils{5x10* cc.lWml) to P&WA micro-
&pendence of paclitaxc.l inhibition of myclopcroxidase spheres.
releasefrom ncutrophilsin responseto plasmaopsonized FIG. 54 is a graph which shows the effect of pccoating
CPPD aystals; n 3. 65 plasma i&2% plumoic Fl2’7 03 the chcmihukxence
FlG.33isagrap which showslysozymc rckase from responseof tsmtr@& (5x10* CWml) to PLA micro-
mteophib (5x10 /ml) in responseto plasma opsoniizd sphacs.
5.716,981
11 12
FIG. 55 is a graph which showsthe effed of precoatiug sampleto he testedmy be readily datamid by visual-
plasma %2% pluronic Fl27 on the chemiluminescen~ imtion of the chick chorioallantoicmembranein theregion
respouseof neuhcphils(Sx106cellsIn@toEvAL~miao- surroundingtbt methylcellulosedisk Inhibition of vascular
spheres. growth may also be detedned quantitatively.for example.
FIG. 56 is a graph which shows the effect of precoating 5 by detaminiog the numberand size of blood vesselssur-
IgG (2 mghl). or 2% pluronicF127 thenIgG (2 m@nl) on roundingthe methyl cellulosedisk ascomparedto a conmi
the cbemiiuminncence responseof neutrophilsto PCL methyl celbtlast disk. Although anti-augiogeuicfactors as
microspbaes. desaii herein are consideredto inhibit the formation of
FIG. 57 is a graph which showsthe effect of prccoating new blood vesselsif they do so in merely a statistica~y
ZgG (2 rngfml), or 2% pluronicFl27 thenI@ (2 @ml) on 10 significantmanna. as comparedto a control, within prc-
the &emiluminescenceresponseof neutropbilsto PMMA fenad aspectssuch anti-angiogenicfactorswill completely
miaosphaes. inhibit theformationof new blood vessels.as weU asreduce
the sizeand numberof previously existing vessels.
FIG. 58 is a graph which showsthe effect of ~cecoating
JgG (2 mghnl), or 2% pluronicF127 thenIgG (2 mglml) on 15 ln additionto the CAM Way describedabove.a variety
the chemiluminescenceresponseof aeutrophilsto WA of o&a assaysmay alsobe utilized to &amine the efticacy
miaosphacs. of anti-angiogenicfactorsin viva. including for example.
mousemodelswhich havebeendevelopedfor this purpose
FIG. 59 is a graph which showsthe effed of ~ecoating (seeRoberstonet aL. Cizncer.Res. JL:1339-1344.1991).In
IgG (2 tug/ml). or 2% pluronic F127thenIgG (2 mglmi) on addition.a variety of qsentative in vivo assaysrelating
the cbemiluminescenctresponse of neutfophib to JXlkPLA 20 to variousaspec&of the inventionsdescribedherein have
miCKOsphaeS.
also beendcwibcd in more detail below in Examples5 to
FIG. 68 is a photographof 10% methotrexate(WILY’) 7. and 17 to 19.
loadedmiuospberesmadefrom a 5050 ratio of PLA:GA As noted above.the mnt inventionprovides compo-
(Iv 0.78). sitions comprising an anti-angiogenicfactor. and a poty-
FIG. 61 is a graph which depicts the releaseof 10% 2s
maic carrier. Briefly, a wide variety of anti-angiogeuic
loadedvanadyi sulfate from FCL. factas may be readily utilizd within the context of the
FfG. 62 is a photographof hyalurouicacid miaospheres presentinvention. Representativeexamplesinclude Anti-
containingvanadiumsulfate. Invasive Factor, retinoic acid and derivatives thaeof.
FTG.63A is a graphwhich depictsthe &ease of organic paditaxd. Suamin,Xssuelnhibitw of Metalloproteinase-1.
vanadatefrom PCL. FIG. 63B depicts the percentageof 30 TissueInhibitor of Metalloproteinase-2. PlasmiuogenActi-
aganic vanadateremainingova a time course. vator Inhibitr~-1, PlasminogenActivator Inhibitor-2. and
FIG. 64 is a photographshowingpoly D.L. lactic acid various fm of the lighta “d group”transition metals.
microspherescontainingwganic vanadate. Theseandotba anti-augiogenicfaaorSwill be discusstdin
FIGS. 65A and 6SBarephotographsof control(uncoated)3s mae detail below.
stentswhich show typical epithelialingrowtbseenat beth 8 BrielIy.Anti-InvasiveFactor,or “‘AIF’ which is prepared
weeks (A) and at 16 weeks (B). Indentationsof the stent from extractsof cartilage,containsconstituentswhich are
tines (t) and narrowing of the lumen (lu) are &own. There responstblefor i&Wing the growth of new blood vessels.
is progressiveepithelial overgrowthof the stentsurfaceova l&se constituentscon4aisc a family of 7 low molecular
this time hy fibrous and inflammatory tissue. 40 weight wins (dO.ooO daltoos) (Kuettner and Pauli,
FIGS. 66A. 66B. 66C. and 66D are a seriesof photo. “‘Inhiiition of oe.ovasculariration
by a cartilage factor” in
graphswhich show control and paciitaxe&oatedWary Devclqineat ofthe Varcdar Sy+rn. Pitman tiooks (CIBA
stents. FIG. 66A illustrates the cbliteratioo of tbe ‘stent FoundationSymposiumIOO),pp. 163-173.1983).including
lumen by the processof benigncpithelial o~agrowtb.~At a varietyof proteinswbicb have inhibitory effects againsta
higha magnidcation(66B). the fibrous and intlsmu#ory 15 variety of proteases (Eisentein et al. Am. J. Pothol.
tissue is evident with little luminal spaceremain&. like 81337-345.1975; Langa et al.. Science193:70-n. 1976:
paclitaxel-treatedhi&By dna remains patent (66c). At and Horton et al., S&ICC lQQ:l342-1345, 1418). AIF
higher nqnification, ncmal biliary tract epitbelium # suitablefor use within thepresentinventionmay be readily
presentwith only minimal alterationof the mucosallining prepared utiiizing techniques known in the art (e.g..
by the mated stent tines (t). so
E!isenteinet ai, sup: Kuettna apdPauli,supra;andLanger
et aL.SW). purified constituentsof AIF suchas Cacti@*
DEAEEDDEXRXFIT ON OFTHE Derived Inhibitor (“CDP’) (SW Moses et at.. Science
RWENTlON 248:1408-1410,1990) may aisb be readily preparedand
As notedahove. the presentinventionpvides metheds utilized within the context of the presentinvention.
and compasitions which utilize anti-angiogenicfactors. 5s Retinoicacidsaltertbemetabolismof extracellularmatrix
Briefly, witbin tbe context of tbe prcseatinventioa anti- eomponeuts,reMting in the inhibition of angiogeneds.
angiogcnicfactors should be UrldcIstoWJto include any Addition of poiine analogs.ang$ostaticsteroids,or hcpariu
prcrdn. peptide.CbemicaLor othermolecule,wbicb ads to may be utilized in OrdM to synagisticaliy increase the
inhibit vascular&row&.A vanity of methodsmay benadily anti-angfogeniceffectof mic acid.Retinoic acid,as
utiliml to determinethe anti-angiogenicactivity of a given 60 well as daivativa thereofwbicb may alsobe utilized in the
facts. including for example,chick cbtioallantoic mem- contut of the presentiovention, may be readily &&ted
bane (“CAM” j assays.Briefly. as describedin man?&tail from commerc+l sources,in&ding for example. Sigma
below ia Examples2A and2C. a portion of the shell from Chemid Co. (+RX25).
a fleshly fatilized chicka~ egg is removed,and a methyl Pa&axe1 is a highly dcrivarizeddimoid (Wani et aL,
cellulose disk containing a sampleof the anti-angiogenic65 f. Am Ckcm.Sot. 93:2325,1971)which has been obtained
factor to be testedis placedon the membrane.After several from tite harvested and dried bark of Taucs bm@dia
days (e.g., 48 hours), iubiiition of vasculargrowth by the (Pacific Yew.) and Tamnyces Andrrmrac and Endophytk
5,?16.981
13 14
Fungus of the Pacific Yew (Stierle et al.. Science and vaaadyi Sdfate inChIding vanadyl sulfatehydratessuch
60~214-216.1993). Generally.paclitaxel acts to stabilize as vanadyl &fate mono- and trihydratcs.
microtubularstructuresby bindingtubuknto form abnormal Representativeexamplesof tungstenaud molybdenum
mitotic spindles.“Paclitaxel”(which shouldhe understood c0mp~exe.s ah itdude 0x0 complexes.Suitable0x0 Nng-
herein to include anaioguesand dcrivativcs such as. far 5 sten complexesinclude tungstateand tungstenoxide com-
example.TAXOL@.TAXCYIHRII@,l@desacetyIanalogues plexes. Suitabletungstate(i.e.. WO,*3 complexesinclude
of paclitaxel and 3Ndesbettzoyl-3%t-butoxy carbonyl ammoniumutngstate(i.e.. (NH&WOJ. calcium tongstate
analoguesof paclitaxel)may be readily preparedutilizing (i.e., CaWO,). sodium tungstate dihydrate (i.e..
techniquesknown to thoseskilled in the art (seealso WO Na,WO,.2H,O). and tuugstic add (i.c. H,WO,). Suitable
94I07882. WO 94107881.WO 94lO7880.WO 94/1)7876,10 tungstenoxidesincludetunp;steo(IV) oxide (i.e.. WO,) and
WO 93/23555.WO 93/10076,U.S. Pat. Nos. 5294.637, tun&en (VT) oxide (i.e.. WOa). Suitable0x0 molybdkwn
5983.253. 5279.949. X274.137. 5.202.448. 5.200534. complexes include molybdate. molybdenum oxide. and
~kU29, and HP 590267).or obtainedfroma variety of molybdenylcomplexes.Suitablemdybdate (i.e.. MOO,“)
complexes include ammonium molybdate (i.e.. (,NH,)
ammercial sources.includfngfor example.Sigma Chemi- &io04) andits hydrates.sodiummolybdatc(it.. Na,MoO,)
cal Co.. St. Louis. Miss. (T7402---fromTearsbrcvtifoli). I5 and its hydrates.and potassiummolybdate (i.e., I&MOO>
Suramir~is a polysulfo~atcd naphthylurca compound that and its hydrates. Suitable molybdenum oxides -include
is typicaUy used as a trypanocidalagent Briefly, Suramia mofybdenum(VK) oxide (i.e.. MoO,). molybdenum(VI)
blocks the speciiic cell surfacebinding of various growth oxide (i.e., MOO,). andmolybdlc acid. Suitablemolybdenyl
factors such as platelet derived growth factor C’PDGF”), (i.e..MOO,*+) cornmexesinclude.for examnle,molvbdenvl
epidermalgrowth factor (‘EGF”). transforminggrowth fac- 20 tktyiace6natc. Other suitable tungstenand molybdenum
tor (‘TGF-r). insulin-like growth factor (“IGF-I”). and complexesinclude hydroxo derivativesderived from. for
fibrobiast growth factor C’fIPGF”).Suraminmay be pe- example.glycerol. tartaric acid. and sugars.
pared in accordancewith known techniques.or readily A wide variety of other anti-angiogenicfactors may also
obtainedfrom a variety of commercialsources.includingfor be utilized within the context of the present iuveation.
exampleMobay Chdcd Co.. New York. (seeGagliardiet 25 Representativeexampiesinclude Platelet Factor 4 (Sigma
al.. CancerRcs. 525073-5075.1992;andCoffey.Jr.,et al.. Chemical Co.. #Fl385); Rotamine Sulphate (Clupeine)
J. of Cc& Phys. 132:143-148. 1987). (Sigma Chemical Co.. rCP4505);SulphatedChitin Duiva-
tives @reparedfrom queencrab shells). (Sigma Chemical
Tissue Inhibitor of Metalloproteiuases-1(LTIMP”) is Co.. K3641; Murata et al.. Cancer Res. SEX?-26, 1991);
secreted by endothelialcells which also secreteMTPases. SuiphatedPolysaccharidePeptidoglycaaCompkx (SP-PG)
TlMP is glycosylatedand has a molecularweight of 28.5 30 (the function of this compoundmay be euhancedby the
kDa. TIMP-1 regulatesangiogenesis by bindingto activated presenceof steroidssuchas estrogen,and tamoxifea citrate):
metalloproteinases.thereby suppressingthe invasion of Staurospotine(SigmaChemical Co.. +&i400); Modulators
blood vesselsinto the extraccllularmatrix. ‘Issue Inhibitor of Matrix Metabolism. including for example.proline ana-
of Metallcproteinases-2 (‘TIMP-2”) may also be utilized to logs { [(L-azetidine-2-carboxylic acid (LACA) (Sigma
inhibit angiogenesis. Briefly. TIMP-2 is a 21 kDa nongly- 35 Chemical Co.. #A0760)). CishydroxyproIine, hL-3.4-
axylated protein which b&is to mewoproteiuasesin both dehydropsoline (SigmaChemicalCo.. #DO265).Thiaproline
the active and latent.procnzymefcrms. Both TIM&l and (Sigma Chemical Co.. aO631)J. a.a-dipyridyl (Sigma
TIMP-2 may be obtainedfrom commacial sourcessuchas Chemical Co.. #D750S), ~aminopropionitrile fumarate
Syoqen. Boulder.Colo. (Sigma ChemicalCo.. #A3134)1]: MDL 27032 (4propyl-
PlasminogenActivator Inhibitor-l (PA) is a 50 kDa 5-(4-pyridinyI)-2(3H)-oxazoloae; Merioo Merrel Dow
40
glycoproteiuwhich is presentin bloodplatelets,andcanalso ResearchhtiNte); Methotrexate(Sigma Chemical Co..
#A6770; Hirata et aI.. Arthritis and Rheumatism
be synthesizedby endothelialcells audmusclecells. PAI- 32X%5-1073.1989); Mitoxantrone(Polverini and Novak.
inhibits t-PA and urokinaseplasminogenactivetar at the Biochem. Biophys. Res. Comm 140:901-907): Heparin
basolatcralsite of the endothelium.aud additionallyregu- 45 (Folkttm, B&Y. Phar: 34905-909. 1985; Sigma Chemical
lates the fibrinoiysis process. Plasminogen Activator Co., #P8754); Intetferoos (e.g., Sigma Chemical Co..
Inhibitor-2 (PAI-2) is generallyfound only in the blood #13265); 2 MauoglobuIin-serum(Sigma Chemical Co..
unducertain circumstances such as in pregnancy, and in the WM7151); ChIMP-3 (Pavloff et al.. J, Bia. Chrm.
presenceof tumors.Briefly. PAI- is a 56 kDa proteinwhich 267:17321-17326.1992); Chymostatin (Sigma Cbcmical
is secretedby mouocytesandmacrophages. It is believedto so Co., uc7268: Tomkinson et al., Biochem J. 286:475-480.
regulate fibrinolytic acfivity,andin particularinhibits uroki- 1992); ~clodextrin TetradecasuIfate(Sigma Chemical
nase plasminogeo activator and tissue plasminogen Co.. UC4767): Hponemycin; C.amptothccin;Fumaglllln
activator, thereby preventing filxinolysis. (SfgmaChemicalCo., #F6771;CanadianPatentNo. 2.024.
Lighter “d group” traasition metals inclu&. for example, 306;Iogberet aL. Nararc348:555-557,199O);Gold Sodium
vanadium,molybdenum mngstcn.titanium, niobium. and 5s Thiomalate(WV”; Sigma:G4022:Matsubaraand-ZfI. J.
tantalum species.Such transitionmetal speciesmay form Ciin. invest. 79:1440-1446. 1987); (D-Penicillamine
transition metal complexes.Suitable complexes of the (“CDPR Sigma Chemfcal Co.. #P487S or P5OOqHCl));
above-mentioned transitionmetal speciesinclude 0x0 tran- pl-anticolla~enase-serum: a2-antiplasmin (Sigma Chat
sition metal complexes. C0.:A0914: Holmes et al.. J. Biol. Chem. 262(4)
Relxeseutativeexamplesof vanadium complexesinclude 60 :1659-1664;1987);Bisantrenc(National CancerInstitut&
oxo vanadium complexes such OS vanadate and vanadyl Lobenzarit disodium (N-(2)-carboxyphenyl-4-
complexes.Suitablevanadate complexes include mctawna- chloroaothronilfcacid disodium or ‘YICA”; Takeudri et al.
date(it.. VOs-) andorthovanadate (i.c, VO,‘-) complexes AgenrsActions36:3%316.1992);Tbalidomidc;Augostat.k
such as, for example. ammonium metavanadate(i.e., steroi& AOM-1470; carboxymiminolmidazo1e; metallopro
NH,VOs). sodiummetavauadate (i.e.. NaVO,), and sodium 6s teinase inhibitors such as BB94 and the pcptide
catt~ovamdate (k. Na.,VO,,).Suitablevauadyl(i.e.. VO*+) CDPGYIGSR-NH,(SEQUHNCHID NO. 1) (Iwaki Glass.
complexesinclude. fcf example,vanadyl acetylacetoaate Tokyo. Japan).
5.716.981
15 16
Althcugh the above aoti-angi~genicfactors have been G-CSE epidamal gmvtb factor. transfamiug growth fsc-
jxovided for the purposesof ilhMMion. it shouldbe under- tars.@&a and beta,TNF. aud antagonists of vascularepi-
stood that the present invention is not so limited. In thelial growth factor. endothehaigrowth factor. acidic or
pmticular. althoughastain imti-angiogenicfactors xc spe- basicfibroblastgrowth factors.andplateletdervivedgrowth
cifically referredto above.the presentinvention shouldbe 5 factor); inhibitorsof the Ps receptor(e.g..heparin);protease
understoodto include smalogues. derivativesandcoujugates and collagenaseinhibitors (e.g..TIMPs. discussedabove);
of such anti-angiogenlcfactors. Fn example. padibrxel nihovasc&lators (e.g.. isosorbide dir&ate); anti-mitotic
should be understoodto refes to not only the commoo agents(e.g..colchicine.anthracydineaand otherantibiotics,
chemicallyavailableform of paclitaxel,but analogues(e.g.. folate antagonists and other anti-metabolites, vinca
taxotue. as noted above) and paclitnxel conjugates(e.g.. 10 alkaloids. nltrosoureas. DNA alkylating agents. topoi-
paditaxd-FEG. pactitaxel-dextran,or paclitruel-xylos). someraseinhlbitcxs. purine antagouistsand analogs.pyri-
Aoti-angiogeniccompositionsof the presentinvention midioc aotagonistsand analogs.alkyl sulfonates):immune-
may additionallycomprisea wide varim of compoundsin suppressive agents (e.g.. adrenocorticosteroids,
additionto the anti-angiogenicfactor andpolymeric carrier. cydospaine); sense or antisenseoligonucieotides(e.g..
For example.anti-angiogeniccompositionsof tie present 15 DNA. RNA. nudeic acid anatogues(e.g., peptidenucleic
invention may also. witbio cat&o embodhents of the adds) or any combinationsof these): and i&&hors of
inveotioll, also comprise one or more aotiiotics anti- transuiption factor activity (e.g.. lighter d grouptransition
idumnaties. anti-vital agents.anti-fungalagentsand/or metals).
anti-protozoalagents.Representativeexamplesof antibiot- Anti-angiogeniccompositionsof the presentinvention
ics included within the compositions described herein 20 may alsoamprise additionalingredientssuchassurfactants
include: penicillins; cepbalosporinssuch as cefadroxil, (tither hydrophilic or hydrophobic;seaExample 13). anti-
cefazolin. cefaclor; aminoglycosidessuch as gcntamynh neoplasticcc chemo~c agents(eg.. 5-fluorouxacil.
and tobramycin; sulfonamidessuch as sulfamethoxa~ol~ virwaihe, vinblastine,cisl%atin.doxyrubicin.adriamycin.
and metronidaxole. Representative examples of anti- or tamodfen), radioactive agents (e.g., Cu-64. Q-67.
inflammatories include: steroids such as prednisonc. 25 Ga-68. 2.89. Ku-97. Tc-99m Rh-105. Pd-109, In-111.
prednisolone.hydrocortisone.adrenccorticotropic hormone. 1-123. I-125, I-131, Re-186. Re-188. Au-198. Au-199.
and sulfasalazioe; and oon-staoidal a&-lngammatory Pb-203,At-211. Pt+212 and Bi-212) or toxins (e.g.. &in.
drugs (“NSAJDS”) such as aspirin. ibuprofen. nqxoxen. abrin, diphtheriatoxin. cholera toxin. geloain. pokeweed
fenoprofen,indomethacln.and phenylbutazone.Relxesen- antiviral protein, ttitin, Shigella toxin. and Pseudomonas
tative examples of antiviral agents include acyclovir. 30 exotoxinA). .
gaocidovir. xidovudiue. Representativeexamplesof anti- As noted above. anti-angiogeniccompositionsof the
fungal agentsiocludeznystatin.ketoconazole.griseofulvia preseotinventionco@ an anti-augiogeuicfactor aad a
flucytosint. micooaxole. clotrimazole. Representatfve polymezic cania. I0 addition to the.wide array of anti-
examples of antiprotozoal agents include: pentatuldine aogiogenicfactors and other compoundsdiscussedabove,
isethionate.quinine. chloroquine.and mc5oqttine. 3.5 anti-aogiogeniccompoaitioosof the presentinvention are
Aoti-angiogcnic compositionsof the presentinvention provided io a wide variety of polymeric car&s. including
may also contain one or mcxe hormooessuch as thyroid for example both biodegradableand non-biodegradable
hormone.estrogen.progesterone.ccatisoneand/or growth compositions. Representative cxamplcs of biode~dable
hormone. other biologically active molecules such as compositionsinclude albumin. gelatin. starch. cellulose,
insulio. as well as TH1 (e.g., Merleukins-2, -l2, and -15. 40 dextmns,polysaabarides, 6ogeo. poly (DL lactide).
gamma ioterfaoo) or Tr,2 (e.g.. Interleuhin~ and -10) poly (DL-&tide-coglycoli&). poly (glycoli&). poly
cytokioe5. (hydroxybutyrate), poly (alkykarbooate) and poly
Within certainprefared embodimentsof the invention. (os?hoaters)(seegenerally.Ill&n, L. David& S. S. (eds.)
aoti-angiogeoiccompositions are pr0vided Which Contain ‘Tolyrmxs in controlled Drug Delivery” Wright. Bristol.
oneor Inorecompoundswhich disruptmiuotubuk function. 45 1987; Arshady. 1. Com&d Release 17:i-22. 1991;Pitt
Representative examplesof suchcompoundsincludepacli- ht. J. Phm 59:173-l%. 1990;Hollandet al., 3. C’cmtmlkd
taxel (discussed above), estramustine (available from Reimse 4:155-0180, 1986). Representativeexamplesof
Sigma: Wang and Steams CMcrr Res. 48:6262-6271. nondegradable polymersinclude EVA copolymers.silicone
1988).epothilone.cumcin-A. col&icine. meth0trexiu.e. vln- ~bber and poly (methylmethactylate).Particularly pre-
blastine and 4-tat-butyl-[~2chIoroctbyl)unido)~~~ 50 ferred polymeric carriers include poly (ethylene-vinyl
(‘%BCEW). acetate&W%cross-linked),poly (DMactic acid)otigomers
hti-aogiogenic compositionsof the presentinvention andpolymers.poly (L-lactic acid) oligomersandpolymas,
may alsocontaina wide variety of othercompcnmds, lndud- poly (glycelic acid). qx+urs of lactic add andglycolic
iog for example:a-adreoagic blocking agents.angiotensin acid, poly (caprolactooe). poly (valerolactone),
II receptor ~tagouists aod receptor antagonists for 55 poIyaohydrides,copolymas of poly (cqXolactone)or poly
histamioe.serotonio,endothc~, i&bitom of the sodium/ (lactic acid) with polyethylene(#ycol and blendsthereof.
hydrogen anfiPerta (e.g.. amllcxide and lts derivatives); Polymericeauiersmay be fashionedin a varietyof forms.
agentsthat modulate intracellular cd+ trampa? such as induding for example.rod-shapeddevices,pellets.slabs.or
I+e (e.g., diltfazem. nifedipine. vempamil) or T-type capsules(see. e.g., Goodell et al.. Am 3. Hosp. Phann
Ca + channelbfockas (e.g., nmiloride),calmodulinatttagct 60 43:1454-1461,1986,Langa et al.. “controlled releaseof
nists (e.g.. H,) and inhibits of the sodium/caWrmanti- macromoleatlesfrom polymas”. in BiomediccIpolymers
porta (e.g.,amilaidc); apl inhibitots (for tyrosinekinases, Pdymen~c materials and phanmceuticals for biomedical
urotein kinase C. mvosin light chain kittase, Ca2+/
L
WC, ~dbesg,EP.,N~~k(eds.)AcademicRess,W
calmQdulinkinaseD;&inldna;eH);Mti~&(~g. 1%137, 1980; Rhine et al., J. Pliam 5%. 695?65-270.
amytriptyline. fluoxetioe, LWOX@ and PAXTL@);cytok- 63 1980;Brown et al., 1. Pham sci. 72:1181-1185.1983;and
ine and/or growth factors. as well as their respective Bawa et al.. J. ConrmucdRelease 1:259-267.1985). Antl-
receptors.(e.g.. tbc interleukins, a. p cx ~IFN. GM-CSl? aogiogenickctors may be linked hy ocdusion ia the mafri-
5,716,981
17 18
ces of the polymer.bound by covalentlinkages.cr cncap. to 100 pm thiclc Such fihns are preferablyflexible with a
sulated in microcapsules. Within certain preferred good tensile strength (e.g.. greater than 50. preferably
embodimentsof the invention. anti-angiogeniccomposi- greaterthan 100. and mate preferably greater than 150 or
tions are provided in aon-capstdat formulations such as 200N/cm2), goodadhesiveprop&es (i.e..readilyadheresto
microspl~aes(rangingfrom nanometersto micrometersin s moist d wet surfaces).and has controlkd pameability.
size),pastes.threadsof various size. films and sprays. Representativeexamplesof suchfilms areset forth belowin
Preferably.anti-angiogeniccompositionsof the present the Examples(set e.g.. Example l3).
invention (whicfi comprise one a more antI-angiogenk Representativeexamplesof the incomomtion of anti-
fadm. and a polymeric carrk) are fashionedin a manner angiogenicfactors such as those describedabove into a
appqxiate to the intendeduse.WQhincertainaspectsof the 10 polymctic cauias is desuii in more detail below in
presentioveatioa the anti-sogiogeniccompositionshould Examples 3.4 Md 8-15.
be biocompstiblc.and release oae or more anti-angiogcnic POL~CURRIERS FORTHERELEASE
factors over a periodof severaldaysto months.For exampk,
“quiclt release”or “burst- anti-angiogeniccompositionsare OF HYDROPHOBICCOMPOUNDS
providedthat releasegreeterthan IO%, 20%. oc 25% (w/v) IS W&i0 furrhu aspectsof thepfueot inveotioo.polymeric
of an anti-angiogenicfactor (e.g..paclitaxel)over a p&d cactias are provided which are adaptedto contain and
of 7 to 10 days.Such“quick &case” compositionsshould, releasea hydrophobiccompound.the carriercontainingthe
within cerrainembodiments.be capableof rekasing che- hydrophobiccompound in combination with a carbohydrate,
mothaqmtic levels (where applicable)of a desiredanti- protcio a poIypeptidc Within cectaio embodiments.the
angiogenicfactor Wlthin other embodimtnts.“low rekase” 20 polymeric anicr conrainsa comphes regions.pockets.or
anti-angiogcniccompositionsareprovided that rekase kss granules of one or more hydrophobic compounds.For
than 1% (w/v) of an anti-angiogenicfactor over a period of example,whhio one embcdmeotof the invention, hydro-
7 to 10 days. Further.anti-angiogeniccompositionsof the phobic compoundsmay be inccapcearedwithin a matrix
presentiovcntioa should pzfcrably be stable fa several which con- the hydrophobiccompouad followed by
monthsandcapableof bcingproducedandmaintainedunda 2s incorporation of the mati within the polymeric cmia. A
stuik conditions. variety of matricescan be utilized in this regard,including
Within certain aspectsof the presentinvention, anti- for example, carbohydratesand polysaccharidessuch as
angiogenic compositionsmay be fashioned in any sixe starch, cellulose.dextran.methylcelhrlose.aud hyaluronic
ranging from 50 mo to 500 pm. dcpendiog up00 the par- acid. proteinsor polypeptidcssuchas albumin.collagenaad
ticular use.Fcr example.whenusedfor thepurposeof tumcn 30 gelatin (see e.g.. Example 31). Within alternative
embolization(asdiscussedbelow),it is generallypmfembk embodbwnts,hydrophobiccompouOdsmay be contained
to fashionthe anti-angiogeniccompositionin microsphcres witbin a hydrophobiccore.andthis core containedwithin a
of between15 and500 pm preferablybctweeo15 and 200 hydrophilic s&R. For example.as describedin &ample 38.
pm, and most preferably. between 25 and 150 pm. paditaxel may be in-ted into a hydrophobiccore
Alternatively. such compositions may also be readily 3s (e.g.. of thepoly D.Llactic add-FE0 or MeFEG aggregate)
appliedss a “spray*. which solidifiesinto a film or coating. which has a hydrophihcshell.
Such spraysmay be preparedfrom microspheresof a wide A wide variety of hydrophobic compoundsmay be
amy of sixes.includingfor example,from 0.1 pm to 3 pm. released from the poiyme@ccan&s de&bed above.
fiomlO~to3O~andfrom3O~to1oO~(see induding fcr exampIe: certain hydrophobic compounds
Example8). 40 whiti disrupt microtubulefunction such as paclitaxcl and
Anti-angiogeolc compositionsmay also be prepared estramustine;hydrophobicproteins such as my&in basic
given,thc disclosureprovidedherein,for a variety of other protein. peoteolipidproctios of CNS myelin. hydrophobic
applications.For example.for administrationto the come& cell wall protein, podns, membraneproreios (EMBO 1.
the aoti-angiogcnicfactors of the presentinventionmay be 4s 12(9):3409-3415.1993). myelin oligodeodrocyteglycoprc+
ioaxporatedinto moco-adhesive polymus (e.g.,polyaaylic tein (“MOO”) (Biockm andhfol. BioL in!. 30(5):945-958,
acidssudt as(CARBOpoLO. dextton,polyme&aaylate,oc 1993, P27 Cancer Rcs. 53(1?):4096-4101, 1913.
starch (see LcMmg and Robinson,3. of CoMoilcd RCL bsUuioopsio. human surfactantprotein (“HSB”: 1. Bioi.
5:223. lQ88)), a nanometer-sizedmicrospheres (see Ckm. 268(15):1116&-11166. 1993). and SP-B or SP-C
generally.Kreuu J. Control&d Release l&169-176,1991; so (Biuchhica ct Biophpica Acta 1105(1):16X-169.1992).
Couvreurand Vauthia. J. Comviled Relcarc 17:187-198, ARTERLGEMBOLZWl-ION
1991). In addition to the compositionsdesaibcd above. the
Anti-aogiogeniccompositionsof the presentinvention preseatiovenrho alsoprovidesa vat&y of methodswhich
mayalsobeprrpgledinavarietyof’paste” orgelforms.Prr utilize the above-descrii anti-angiogeniccompositions.In
example, within one embod&znt of the inventiw, anti- 5s particulpt, within ooe aspectof tbe present invention metb-
angiogcniccompositionsare provided which are liquid at ads am providedfor embolizinga blood vessel.comprising
one tunpnaarrr (e.g.,temperaturegreaterthan 37OC., such tbe step of delivering into the vessei a &rapeuticslly
as 40’ C.. 45’ C.. 50’ C., 55’ C. a 60” C.), and solid oc effective aolouor of an anti4ulgiogeniccompositiotl (as
semi-solid at another temperature(e.g., ambient body dcscrihcdabove),such that the blood vesselis cffcctively
tempemtute.or any temperaturelower than 37OC.}. such 60 occluded. Thaapeutically effective amounts s&able for
‘yhernropastes” may be readily made given the disdwure ocdudiog blood vesselsmay be readily detmminedgiven
pvided herein (see.e.g.. Examples10 and 14). the disdosureprovidedbelow,aedas describedia Example
within yet other aspects of the invention, the anti- 6. Wirhin a proticolatly prefm rmbdment, the aoti-
aogiogeolccompositionsof the presentinvention may be angiogcniccompositionis deliveredto a blood vesselwhkh
formed as a filoL Preferably,such8lms are genaally less 6s nourishes a tumor (seeFIG. 13).
than$4.3.2, or 1. mm thi&. morepreferablylessthan0.75 Briefly. the& are a oumlsr of c&d& shwions (e.g..
mm or 0.5 mm thick, and mostprefemblyless than 500 pm bleeding,tumordevelopment)whereit is desirableto reduce
5,716,981
19 20
cc abolish the Mood supply to an orgao or region. As through the catbcter, until flow is obsuvcd to cease. Occlu-
&saii in greater &tail below. this may be accomplished sion may be confirm4 by npeating the angiogram.
by iojutiog aoti-angiogenic compositions of the present FiubboWoo the.rapygeoaally rcsuhs in the distriiutioo
invention into a desired blood vessel through a salcctivcly Of composi~oos cootaidg anti-angiogenic faders through-
JxJsitioncd catheter (see Jm. l3). The compositioo travels 5 out the intmZic42s of the tumcx or vascular mass to be
via Uxe blood stream until it becomes wedged in the treated The physical bulk of the cmb0lic particles &gging
vasctdauuc. thereby physically (CT cbemicaJJy) occluding the arterial Jumeo results in the occlusion Of the blood
the blood vessd. The reduced or abolished blood flow to the Supply. In add&ion to this affect. the presence of an anti-
sekcted area ma&s in infarction (dJ death doe to an aogiogeoic facto@) prevents the formation of new blood
inadequate supply of oxygen and outrknts) or reduced blood 10 Vessds to supply the tumor Q vascular mass. enhancing the
loss from a damaged vessel. devJmlizing effect of cutting off the blood suppiy.
For use in anbo~oo thcqy. anti-aogiogenic camp Therefcrc. it sbould be evident that a wide variety of
sitions of the present invention are preferably non-toxic. tumors may he embolized utilizing the compositiolts of the
thrombogeoic. easy to inject down vascuk catheters.radio- present ioventioo. Briefly. tumors arc tvpically divided into
op;lquc, rapid and permanent io efkct. sterile. attd readily 1s two dasscs: benign and maJJguant Jn a benign tumor the
available in different shapes or sizes at the time of the ceJls retain their dilfercntlatcd features and do not divide in
prmdore. Jo addition. the compc&tJons pr&rabJy result in a compJctcly uncootrokd manner. Jn addition. the tumor is
the slow (id&y. over a paiod of scvadl we& to months) Jocalkud and oonmctastatic. Jn a malignant tomor. the cells
rchse of an anti-angiogenk factor. PartJcuJarJypreferred become oodBumtiatcd do not respond to the body’s
anti-at~giogeoiccompositioos shouJdhave a predictable size 20 growth and hormonal signals. and multiply in an uncon-
of U-200 pm after being injected into the vascular system trokd manob; the tmncr is invasive and capable of spread-
Referably. they should not clump Jnto Jargcr particles eJtJuz ing to distant sites (mctastasiziog).
in soJution cr once injecttd. In additiw. pcfaabJe compo- Withiit we aspect of the present invention. mctastascs
sitions should not change shape or physical prop&es (sccoo~ almoM) of the liver may be treated utihzing
dutiog stcrage prior to use. 25
embolhtioo timapy. BridJy. a catheter is inserted via the
EhboJkdoo therapy may be utiJized in at least three fcmcd oc brad&d artery and advanced mto the hcpatic
prindpaJ ways to assist JII the manage~ot of o&asmsz (1) artuy by steeting it through the art&al system under
&iinitive lrcattocot of tumors (USWQ bcuign); (2) for fluoroscopic guidance. The catheter is advanced into the
pxpemtive emboJizatioo; and (3) for paJJJativeembohxa- hepatic attfdal tree as far as necessary to aJJow cowlem
tioo. Briefly, benign tumors may sometimes be succc&tdJy 30 blockage of the blood vessds supplying the tumor(s), whiJe
treated by embdization therapy ,alooc. ExampJes of such spat&g as many of the artuiaJ branches supplying ttormaJ
tumors include simple tumors of vascular origin (e.g.. structrnw aspossible. WalJy this will be a segmental branch
hacmangiomas). endocrine tumors such as parathyroid of the hspatic artuy. but it couJd be that the entire hcpatic
adenomas. and benign bone tumors.
35 artery distal to the origin of the gasuoduodenaJartcry or
For other tumors. (e.g.. renal adenocsrcinoma), preoJx!ra- even multiple separate arteries. will need to be bJockzd
tivc embolizatioo may be employed hours or days before depending on the &cot of tumor and its individual blood
surgical nzxctioo in ox-da to reduce operative blood loss. supply. Once the d&red catheter position is achieved, the
shorten the duration of the operation, and reduce the risk of axtuy is cmboJizcd by injecting anti-angiogenic corn@-
dissunination of viable malignant cells by surgicaJman@- 40 tiotls (as desaibed above) through the attd catheter uotil
Jation of the tumor. Many tumors may he SOC~MUJI~ flow in the artery to be MocJmd ceases.preferably even after
cmbolizcd prcopcrativeJy. iocJudiog fee cxam~rJenasopha- observation for 5 miuutcs. OcclusJoo of the attay may be
ryngcal ttmmrs. gJomus jug&u tumors. merdogiomas. coofirmed by JnjcqtJug m&opaque contrast Uuough the
chexnodectomas.and vagaJ neuromas. cathcta and demonstrating by fiuoroscopy or X-ray fihn that
E?mboJJm,tioomayakobcutihxedasaprimatymodcof 4s the vessel which previously %IJcd with contrast no longer
beatmat for JnopcrabJt tuahgnancks. in c&r to extend the docsso.Thcsameproce&cmaybcrcpcatedwJthcach
sumivaJ time of patients with advanced disease.HmboJJm- feuiingatterytobcoccludcd
tion may produce a marked itnpverntnt in the quality of As ooted above, both benign and malignant tumors may
Jife of patients with ruahgoant tumors by alleviating be cmbolizul utiJi2iogkolnpositions of the present inveo-
unpksaut symptoms such as bJccdhrg. veuous obstruction 50 tion. Rcpmscotative exan@es of benign hcpatic tumors
and tracheaJcom~xcssJon.The greatest benefit from paJiia- include Hepatocellular Adcnoma. Cavernous
tive tumor cmboiization. however. may be seen iu patients HacmangJoma, and Focal Nodular Hypu@asia. Other
suffering from the humcraJ effects of mahgnant &ocrinc benign tumors, which arc more ram and often do not have
tumors. whaein mctastascs from carchtoid tumcrs and other clioical maoifcstatiws. may also be tawcd. These include
endocrine ncopJasms such as JnsuJinomasand gJucagooo- 55 Bile JIuct Adcnomas, BJJe Duct Cystadcnomas, J%romas.
mas may k slow growing. and yet cause great distress by Lipomas, Lciomyomas, Mesotheliomas. Teratomas.
vi!tue of the endocaine syncJromeswhich they product. Myxomas, and Nodular Jtegeocxative Hypgplasia.
In general. uoboJizati00 thempy utiwog anti-aogio@ic Maligoaot Hcpatic Tumas arc gcncraJJysubdivkicd into
compositioos of the present inveodon is typicnuy paformad tw0catcgosJes:gKimaryandsccotldary.primarytumcrsarlsc
in a sindar manner. regardless of the site. BrJcBy. attgJog- 6(1 , dinxtJy from the tissue Jn which they are found. Thus, a
rapJ~y(a road map of the blood vesscJs) of the area to be JKimalylivtrtumaisdaivcxJoriginaltyfromthculls
embolizcd is first perfarmed by injecting radiopaque coo- wbicb male up the liver tissue (such as hepatocytes and
trast through a catheter inserted into 00 attay (rr veio biliary cdJs). Rc~rcacntatJve el‘ramples of pimary kpatJc
(depcndingoothesitctobecmbdized)Isrmx-Myistalen. maligoands wJti& may be treated by art&J embokatJoo
The carbeta may be insuted citha pacutaneousJy or by 63 include HepatocellularcarcJnoma. ChoJangiocarcinoma.
surgery. The blood vessel is then embohxed by mfhming AogJosarcoma. Cystadcoocarcinoma, Squamous CclJ
anti-aogiogeoic compositions of the present invention Carcinoma. and HcpatobJastoma.
5,716,981
21 22
A secondarytumor. or metastasis.is a tumor which USE OF ANTI-ANGIOGENK COMPGSITIONS
originatedelsewhere in the body but has now spreadto a AS COATINGS FOR SIENIS
distant organ.The commonroutesfor metastasisare dlred
growth into adjacentstructures,spreadthroughthe Vascular As noted above. the present invention also provides
a lymphatic systems.and trackingalong tissueplanesand 5 steots. comprising a generally tubular structure (which
body spaces (peritoneal fluid. cerebrospinalfluid. etc.). iocludeafor example.spiral shapes).the surfaceof which is
Secondatyhepatic tumors m one of the most common coatedwitb a compositionas desuii above. Briefly, a
causesof deathIn the cancerpatientandareby far andaway steotis a scaffolding.usuallycylindrical in shape.that may
the most commonform of llver tumor. Although virtually be insettedinto a body passageway(e.g., bile ducts) or a
any malignancycan metastasizeto the liver. tumorswhich 1o portion Of a body passageway,which has been narrowed.
are most likely to spreadto the liver Include:cancerof the irregularly c~ntured.obstrucZe&or occludedby a disease
stomach,colon. and pancreas;melanoma:tumors of the process(e.g.. ingrowth by a tumor) in order to prevent
lung. oropharynx. and bladder; Hodgkin’s and non- closureorreclosureof the passageway. Stcntsact by physi-
Hodgkin’s lymphoma; tumors of the breast, ovary. and ally holding openthe walls of the bodypassageinto which
prostate.Each one of the above-named primarytumorshas rJ they arc kc&d.
numerousdifferent tumor types which may be treatedby A variety of stcntsmay be utilixed within the contextof
arterial embolization(for example,therearc over 32 difkr- the presentinvcntio~. including for uample. esophageal
em types of ovarian cancer). sttnts. vascularstems.biliaty stems.pancreaticstems.ure-
As noted above, embolization therapy utilhing anti- teric and urethral stents. lacrimal stents.Eustachiantube
auglogenlccompositionsof the presentinventionmay also M stems.fallopian tube stentsand tracheaYtxonchialsteots.
be~pr;led to a variety of otherclinical situationswhereit is - Stats miy be readily obtained from commacial sources.
&sired to occludeblood vessels.Within one aspectof the or constructedin accordancewith well-known techniques.
present invention. attuiovenous malformati00 may be. RepreseMative samples of stemsincludethosedesaii in
treated by administrationof one of the above-descrii U.S. Pat No. 4.768523.entitled“HydrogelAdhesive:”U.S.
compositions. Briefly. arteriovenous malformations 25 Pat.No. 4.776337,entitledTxpandable IntraluminalGraft,
(vascular malformations) refers to a group of diseases and Method andApparatusfor Implanting and Expandable
wherein at least one (and most typically. many)abnormal SnWuminalGraft;” U.S.Pat.No. 5.041.126entitled“Endo-
communicationsbetweenarteriesand veins occur,resulting vascularStem and INkay System:”U.S. Pat. No. 5.052,
in a local tumor-likemasscomposedpredominantlyof blood 998 entitled ‘%dwelIing Stent and Method of Use;” U.S.
vessels.Suchdiseasemay be eithercongenitalor acquired.sc pat No. 5.064.435 entitled “Self-Ekpanding ProsthesisHav-
Within one embodimentof the invention. an arterio ing StableAxial Length:”U.S. Pat. No. 5.089606. entitled
venousmalformationmay be treatedby insertinga catheter ‘Water-insol&Ie PolysaccbarideHydrogelFoam for Mcdi-
via the femoral or brachial attay. and advancingit into tbe CalAppIkations;”U.S. Pat.No. 5.147370.entitled “Nitinoi
feeding artery underfluoroscopicguidance.The catbeta is Stat for Hollow Body Conduits;”U.S. Pat No. 5176.626.
Feferably advancedas far as necessaryt5 allow complete 35 entit3ed”Ipdweliing Stent;”U.S. Pat No. 5.213580.entitied
blockage of the blood vessels supplying the vascular “Biodegradablepolymeric EndoIuminal Sealing Process;”
maIformation.while sparingasmanyof thearterialbra&es and U.S. Pat. No. 5328.471. entitled“Method andAppara-
supplyingnormalstructuresaspossible(ideallythis will be tus for Treatmeat of Focal Diseasein Hollow Tubular
a single artery.but most oftenmultiple separatexrterieamay Orgaosand Other‘IIssueLumens.”
needto be occluded,dependingon the extent of the vascular 40 Stats may be coatedwith anti-angiogeaiccompositions
malformation sod its individual blood supply). Once the or anti-angiogenicfadccs of the present invention in a
desired catheterposition is achieved,eachartery may be varidy of manners.iocluding for example:(a) by directly
embolized utilixing anti-angiogeniccompositionsof the af6xingto the stentan anti-angiogeniccomposition(e.g.,by
grescntinvention. either sprayingthe stent with a polymer/drug Elm. or by
Within anotkr aspectof theinventioo.embol&tion may 45 dippiog the stent into a polymer/drug solution). (b) by
tc accomphshd in order to treat conditionsof excessive coating the stcnt with a substance such as a hydrogelwhich
bleeding. For example. menorrhagia(excessivebkeding will in tmn absorb the anti-angiopic composition (or
with mcmtmatioo) may be readily treatedby embolization anti-aogiogeoicfactor above), (c) by interweaving anti-
of uterine ties. Briefly. the uterine arteries are branches aogiogedccompositioncoatedthread (a the polymer itself
of the iotemaI iliac artuies bihaally. Within one embodi- 50 formed into a thread)into the stentstructure.(d) by iamting
ment of the invention, a catheter may be insuted via the the stent iDto a sleeveor mesh which is comprisedof or
femoral cr bra&al arkxy. and advanced into each utaine coated with an anti-angiogeniccomposition. or (e) con-
attery by steering it through the ark&l system unda structingthe stem itself with an anti-angiogeniccomposi-
fluoroscopicguidance.The cathetershould be advanced as tion. Within preferred cadmdhents of the invention, the
far as necessaryto allow completeblockageof the blood 5s composition should&rnly adhereto the steotduring storage
vesselsto the utcxus.while sparfogasmanyart&al branches and at the time of insertion, and should not be dislodged
that arise from the uterine attery and supply normalstruc- from the stent when the diameteris expandedfrom its
lures aspossible.SdealIya singleuterinearteryon eachside collapsedsizeto its full expansionsire.The anti-angiogenic
may be unbolixed,but occasionallymultiple separateatter- composition should also preferably not degrade during
ies may need to be blocked dependingon the individual 60 storage.pior to imetion. or when warmed to body tem-
blood supply.Oncethe desiredcatheterPositionis achieved pasture after expansioninside the body. In addition, it
each artuy may be embolizedby administrationof the should preferably coat the stent smoothly and evenly. with
anti-angiogeniccompositionsas desuibedabove. a uniform dishibution of angiogcnesis inhibitor. while not
In a like manner,arterial embolizationmay be accom- changing the stat contour. Within preferred embodiments
plished in a variety of other conditions. including for 6S of the invention, the anti-angiogenic composition should
example. for acute bleeding. vasahr abacmalities. central provide a uoifum. predictable,prolonged releaseof the
nervous system disorders. and hypeqlenism. anti-angiogenic f&&or into the tissue surrounding the stent
5716,981
23 24
onceit hasbeendeployedFor vascularstents.in additionto affect the bib duct (e.g.. adenomaof the biliary system).
the abve pputies. the compositionshouldnot renderthe and.in rxc cases.squamouscell carcinomasof thebile duct
sum thrombogcnic(causingblood clots to fcam). or cause and adenocarcinomas of the gallbladder.may also cause
significantturbulencein blood flow (mire than the stent compressionof thebiliary treesod therefore.result in bilk-y
itself would be expectedto causeif it was uncoated). 5 obstruction.
Within amther aspectof the presentinvention. methods Compaession of the biliary bee is most commonly due to
areprovidedfor expandingthe lumenof a body passageway, tumus of the liver and pancreaswhich compressand
cmprising insating a stentinto the passageway.the stent tbaefcre obstrud the ducts. Most of the tumors from the
having a generally tubular structure.the surface of the pancreasarisefrom cells of the pancreaticduds This is a
highly fatal form of 4Xllccz(5% of au cancerdeaths;26,ooo
stsucturc.being coaufl with all Mti-MgiogeRic composition 10 new casesper ymr in the U.S.) with an averageof 6 months
(or. an antioangiogcnicfactor alone),spchthat the passage- survivd and a 1 yearsurvival rate of onIy I#%. When these
way is expanded.A variety of emb&ments are described tumus are located in the bead of the pancreasthey tit+
belowwhereinthelumenof a body passageway is expanded quentiy causebiliary obstruc#ion.and this detractssign&
in order to eliminate a biliary. esophageal.tracbeaU caatiyfromtbcqualityoflifeofth:patient.\krhile~typcs
bronchial,urethrai or vasculx obsimction. Yn addition. a IS of panczeatictauncasare genaally rekred to as “car&oma
representativeexampleis describedin more &tail belowin of the pancreas”tbae are histologic subtypesincluding:
EwRple 7. adenocarcin~ adenosquamouscarcinoma cystadeno-
Generally. stentsareinse.rtedin a similar fashionregard- uuchoma. tmd acinx cell aminoma. Hcpatic tumors.as
less of the site or the disease bdng treated Brieffy. a discussalabove.may also causecompessiooof the biliacy
preinsertion examination. usually a diagnostic imaging 20 tree. and thereforecauseobatnrctionof the bii ducts.
pmtdure. endoscopy.or direct visualimtion at the time of Within oneembcdimcntof the invention.a biiiary stentis
surgay,isgenaallyfirst~~inff&todctamiaethc iirst insertedinfo a b&try passagewayin ooe of several
apjqdse positioning fa stent inscztion.A guidewireis ways: from the top end by inscztinaa needlethrouahthe
then advanced through the lesion or proposed site of ttbdominalwall aoil lbrou& the liv& (a pacutaneou~tran-
sheuaticcholanpiaaramor TK”): from the bottomendbv
insution. and over this is passeda deIivety catheterwhich 2s cafdating thebiTe dud throngh an endoscopeinserteh
allows a stent in its coilapscd form to be insertedQpicaliy. tbmgb the anouh.smd. ar$ duodenum(an CRdoscopiC
stelltsarc capableof beiugcomprzssed.so that they can be retrogradecfiolangiogramoc “ERCP”); n by directincision
inscrtcdthroughtiny cavities via small cathtiffs. and then during a surgicalpmccxbe. A mscrtion examination.
expamledto a larger diameteronce they are at the desired FTC. ERCl? oc direct visualization at Uie time of surgay
30
locatioa. Once expanded the stent physicaLlyfcsces the shouldgendy be paformed to determiaethe appropriatt
wails of thepassageway apartandholdsthan open.As such. position for stud insertion.A guidewire is then advanced
theyarecapableof insertionvia a smallopening.andyet arc through the lesion. andover this ad&very catheteris passed
still able to hold opena largedknetcz cavity ocpassageway. to allow the stentto be insertedin its collapsedform. If the
The stent may be self-expanding(e.g.. the W&tent and diagnosticexam was a ETC. the guidewire and delivery
Gimurco sum). balloonexpandable(e.g..the Palmazstcnt 35 catheteris in+tai via the abdominalwail. while if the
andStredterstent).or implantedby a changein temature otigiml cxam,wasan ERcPthe swlt maybe placedvia the
(e.g.,the Nitinol stmt). mouth. The stent is then positioned under radiologic.
Stentsare typically maneuveredinto place underradio- cndosmpic. or direct visual control taking particular care to
logic or clireu visual como~ taking pxtiallar arc to place 40 placeit preciselyaauss the uarrowingin the biie duct..The
the stentprecisely acrossthe natTowingin the organbeing delivay catheteris thenremovedleaving the stentstanding
treated.The d&my caQUa is then remove& leaving the as a sc&olding which holds the bile duct open.A further
stent s-cling on its own as a scaffold.A post insertion cholaagbgrammay be pafamed to documentthatthe stent
examination.usually an x-ray. is often utilized to confirm is appmp&ely positioned.
appropriatepositioniDg. IS widlin yet Imotherembodimentof the invention.nztbcds
Within a prefured emkdiment of theinvention,methods areprovidedfor e&min&lg esophagealobstructions.com-
iireprovidedfor eliminatingbilirpy obstructions,comprising prisiig ins* an esopbagedstentinto an esophagus, the
iwuting a biliary stentinto a bitbuy passageway,the stent stenthaving a genaally tubular structure.the surfaat of the
having a generally tubular sbuctum. the surface of the Structurebeing coatedwith an anti-angiogcniccomposition
structure being coated with a composition as &scribed so as desaibedabove,such that the tsophag~ obi5trwtionis
above,suchthat thebiliary ObsmctioDis e4imbtd Bray. . *
alumnat& BrMy. the esophaguiis the hollow tubewhich
tumor overgmvth of the common bile dud resultsin pro- transportsfood and liquids from the mouth to the stomach.
gressivecbolestaticjaundice which is incompatible with Cancerof the esophagusor invasion by canax arising in
life. Gtnerally.tbe bihy systemwhich drainsbile from the adjacentorgans(e.g..cancerof the stomachor lung) results
Jiveri#otheduodenumismo.stoftenobstruuedby(l)a ss in the inability to swabw food u saliva. Within this
tumorcomposcd of bile duct ceils (ch0lMgioaucin0mii). (2) embodimat, a preinsertionexamination. usually a barium
a tumor which invades the bile duct (e.g., pancreatic swallow or endoqy should generally be performedin
carcinoma). or (3) a tumor wblch cxms extrinsic pressure orderto determhe the appropiate position for stentinsff-
andcompressesthe bile dud (e.g.. enlargedlymph nodes). tiOR. A catbCtt2 ff CnC&ScOpe may thaw k: p~&iODtd
Both#naatybiliarytumcrs,aswellasothertnmors 60 uirough the mouth, Ed a guidcwirc is advancui through the
which causecompressiwof the biliary tree may be treated blockage. A stent delivay catbeta is passedover the
lltwng the stem demibcd berein. one examge of pri- guide-wireunder radiologic or cndosoopic cmlrol, and a
mxybiliary~areadenoarrcinomss ,(whidt are also stentis placedprecisdy aaoas the narrowingin the esopha-
died KM&in tumorswhenfound at the bifurcation of the gus.Apost insatiou cxxninatio~ usuallya bariumswallow
commonbcpatic duct).Thesetumors arealso mfeued to as 6.5 x-ray, may be tlbifircd to co- llpppiate positioning.
bilhy c.aminomas. cboiedorbolMgiocardRomas. or ackno- Wa other anbodimentsof the invention.methodsare
carcinomasof the biliary system Benign tumors which provided for elimkting tmcheaYbiQnchialobstructions,
5716,981
25 26
comprisinginsting a traciteal/bronchialsteminto the tra- into the appropriateblood vesselby steeringit through the
chea or bronchi, the stent having a generally tubular vascularsystem u&x fluoroscopicguidance.A stent may
structure. the surface of which is coated with an anti- then be positioned across the vascular stenosis.A post
angiogeniccompositionas descriid above.such that the insertionanglogrammay also be utilized in orderto con&m
tracheal/bronchialobstnrction is eliminated. Brkfly. the 5 -opriate posidoningV
tracheaandbronchiaretubeswhich carry air fromthe mouth
and nose to the lungs. Blockageof the tracheaby cancer, USE OF ANTI-ANGIOGENIC COhSPOSITIONS
invasionby cancerarisingin adjacentorgans(e.g..cancerof IN SURGICALPRC~CED~RE~
the lung), or collapse of the tracheaor bronchi due to As noted above, anti-angiogeniccompositionsmay be
chondromalacia (weakeningof the cartilagerings) resultsin 10 utilized in a wide variety of surgical procedures.For
inability to breathe. Within this embodiieot of the example.within one aspect of the present invention an
invention. preinsertion examinatio5. usually an edosccpy. anti-mgiogeniccompositions(in the fotm of. for example.
should generallybe performedin order to determinethe asprayorfilm)maybeutilixedtoccatorsprayanareaprior
appropriakposition for stentinsertion.A cathcta n endo- to removalof a tumor.in orderto isolatenormalsurrounding
scopeis thenpositionedthroughthe mouth.and a guidewire ts tissuesfrommalignanttissue.andlorto prwent the spreadof
advancedthroughthe blockage.A delivery catheteris then diseaseto suxoundingtissues.Within other aspectsof the
passedoverthe guidewirein orderto allow a collqsed stent presentinvention.anti-arigiogenicCompositions (e.g.,in the
to be inserted The stent is placed under radiologic or form of a spray) may be deliveredvia endoscopicproce-
endoscopiccontrol in order to placeit preciselyacrossthe dmes in orda to coat tumors. or inhibit angiogenesisin a
narrowing. ‘Ihe delivery cathetermay then be nmoved 20 desired locale. Within yet other aspectsof the Fesent
leaving the stentstandingas a scaffoldon its owm A post invention. surgical mesheswhich have been coated with
insextionexamination.usually a bronchoscopymay be uti- anti-angiogeniccampositionsaf the presentinvention may
lized to confine apprcpriatepositioning. be utibd in any procedurewhereina surgicalmeshmight
Within anotherembodimentof the invention.methodsare be utilized. For example.within one embodimentof the
m&d for ewhg mthraf obstru&ons.ccmpi&g 25 invention a s~rgifal mesh ladenedwith an anti-angiogenic
insertinga urethralstem into a urethra.the stcnt having a compositionmay be utilized during abdominalcancerresec-
generallytubularstructure.the surfaceof thestructurebeing tion surgery(eg., subsequentto colon resection)in cederto
coated with an anti-angiogeniccompositionas descrlki provide supportto the stnrctme.and to releasean amountof
above, such that the urethral obstruction is eliminated. the anti-angiogenicfactor.
Briefly. the urethrais the tube which drains the bladder 30 Within further aspectsof the presentinvention.methods
through the penis. Extrinsic nanowing of the urethraas it are provided for treating tumor excision sites.comprising
passesthrough the prostate.due to hypatrophy of the administing an anti-angiogeniccompositionas described
prostate.occursin virtually everymanovertheageof 60 and above to the reseuion margins of a tumor subsequentto
causesprogressivediiliarlty with urination. Within this excision, such that the local recurrenceof cancerand the
embodiment.a preinsertionexamination,usuallyan endos- 35 formation of new blood vessels at the site is inhibited.
copy or urethrogramshouldgenerallyfirst be performedin Within one embodiment of the invention. the anti-
order to determine the appropriate position for stent angiogenic composition(s) (or anti-angiogenicfactor(s)
insertion.which is abovetheexternalurinarysphincterat the alone) are administereddireU.ly to the tumor excision site
lower end. and close to flush with the bladderneck at the (e.g.. appliedby swabbing.brushing or otherwisecoating
upper end. An endoscopeor catheteris thee positioned *o the resectionmatglnsof the tumor with the anti-angiogenic
throughthe penile openingand a guidewireadvancedinto composition(s) or factor(s)). Alternatively, the anti-
the bladder.A &livery catheteris then gassedover the angiogeniccomposition(s)or factor(s)may be incorporated
guidewire in order to allow stent insertion.The delivery into known surgicalpastesprior to administration.Within
catheteris thenremoved.and,the stentexpandedinto place. particularly prefmed embodimentsof the invention. the
A post insertionexaminatim. usualiy endoscopyoh r&o- 45 anti-angiogeniccompositions are applied after hepatic
gradeurethrogralumay be utilized to confirm appropriate reseetionsfar malignancy,and after nwosurgicaJ opera-
position. tions.
Wlthln anotherembodimentof the invention.methodsare Within one aspect of the present invention. anti-
providedfor eliminatingvascularobstructions,comprising ~giogcnic compositions (as described above) may be
insertinga vascularstentinto a bloodvessel,the stenthaving M administeredto the resectionmargin of a wide variety of
a generallytubular structure.the surfaceof the sttucture tumors, in&ding for example; breast colon. brain and
being coated with an anti-angiogenic composition as hepaticbunens.F%rexample,within oneembodhent of the
desaihedabove.suchthat the vascularobsttuctionis elimi- invention, anti-angiogeniccompositionsmay be adminis-
Wed. Briefly. stemsmay beplacedin a wide arrayof blood tered to the site of a ncuroIogical tumor subsequentto
vessels,both arteriesandveins,to &xeventremrent stenosis55 excision,suchthat the formationof newblood vesselsat the
at the site of failed angioplasties.to treat natrowingsthat site are inhibited. Briefly. the brain is highly functionally
would likely fail if treatedwith angioj&asty,andto tmatpost localiwi; i.e., eachspecificanatomicaltegion is specialized
surgicalnauowings(e.g..dialysisgraft stenosis).Repen- to cany out a specltk function. Thereforeit is the location
tative examplesof suitablesitesincludethe iliac, ret& and of brainpathology thatis ofkenmoreimporU& thanthe type.
coronary Meries. the superiorvena cava, and in dialysis 60 A relatively small lesion in a hey area can be far man
grafts. Within one embodiment.angiographyis ti per- devastatingthan a much larger lesion in a less important
formedin orderto localizethe sitefor placementof the stent. arei. Similarty, a lesion on the surfaceof the brain may bc
This is typically accomplishedby injecting radiopque con- easytares& surgically,while the sametumor locateddeep
trastthroughacathctcr~scrtcdiotoananayavcin~s in the kin may not (one would have to art through too
x-ray is taken. A catbeta may then be inserted eitha 65 my vital structuresto reachit). Also. evenbenigntumors
percutaneouslycc by surgeryinto the femoral artery,bra- ~bedangcrousfarseverslnasons:theymaygrowiaa
&id artery. femoral vein. or brachial vein, and advanced key are-nand causesignificant damage;even though they
5,716.981
27 28
would be cured by surgical resectionthis may not be leadiagto erosionsandfragmentationof the caMagetissue.
possible; and firrally, if left uncheckedthey can cause Eventually t&e is exosionof tie subchondtalbone with
iaueascd iatracmaial pressure.The skall is an endosed fibrous ankylosis and ultimately bony ankylosis. of the
spaceincapableof expansion.Therefore.if somethingis involved joint
growlag in one kation, somcthiogelse must be being 5 lt is geoadly believed.but not conclusivelyproven.that
compressedin another location-the result is increased RA is an autoimmunedisease.and that many different
pressureio the skull or maeasedintracranialpressure.If arthriogCoicstimuli activate the immune respoosein the
such a condition is left untmated.vital Structures can be immuaogeneticallysusceptiblehost.Both exogenousinfec-
comprcssaLnsultiog in death. The incidence of CNS tious agents (Ebstcin-Barr Virus. Rubella virus.
(centralnervoussystem)malignanciesis g-16 per 100.0tK~.IO Cytome@z$rus. Hapes Viis, FItman T-cell Lymphotro-
The prognosisof primary maligoancyof the brain is dismal. pit Vi& Mycopksma. and others)and endogcaouspm
with a mediansurvivalof lessthanoneyear.evenfollowing teins (collageo. protooglycans.altered immunoglobulins)
surgical resection.These tumors. especially ghomas.are have betto implicatedas the causativeagentwhich triggers
pz&minantly a local diseasewhich recur within 2 ceati- aa inamnopriatehost immune response.Regardlessof the
meters of the original focus of diseaseafter surgical 15 incitiag agent.autoimmunityplays a role in the progression
removal of the disease.Ja particular.the relevantantigenis ingested
Represeotativeexamplesof brain tumors which may be by antigen-presenting cells (ma~ophagesor dendriticceils
treatedutiliring the compositionsand methods descrfbed in the synovial mcmbmnc).processedand presentedto T
herein include Glial Tumors (such as Anaplastic lymphocytes.The T cells initiate a cellularimmuneresponse
Astrocytoma. Gliobiastoma Multiform. Pilocytic 20 and stimulate the prolifcratioo and difFerentiationof B
A%%ncycoma. oligodcndmglioma. EpMdymomat Myxo- lymphocytesiato plasma cells. The end result is the pro-
papillary Epcodymoma,Subependymoma. Choroid Plexus duction of an excessiveinqxopriate immune response
Papilloma); Neuron Tumors (e.g.. Neuroblastoma. directedagainstthe host tissues [e.g.. anti&odiesdirect&
Gaaglioneuroblastoma. Ganglioneuroma. and againstm II collagen.antiies directedagainstthe Fc
Mcdulloblastoma~ Pincai Gland TIunors(e.g.. Pinoobias- 25 portion of autologcusIgG (called “Rheumatoid Facta”)].
toma and Piacocytoma); Mcnigtal Tumors (e.g., This fiather amplifiesthe immuneresponseandhastensthe
Menio@oma, Meaingeal Hemaogiopcricytoma. Meoiagcal dcstruUi00 of the autilage tissue. Once this cascadeis
Sarcoma);Tumors of Nerve SheathCells (e.g., Schwan- ioitiated nnmmus mcdiatas of cartilage desrructionare
noma (Neurolemmoma)and Ncurofibroma);Lrltomas responsiblefcr the progressionof rheumatoidartkitis.
(e.g..Hodgkin’sand Non-Hodgkin’sLymphoma(ineluding 30 Thus. within one asped of the presentinvention.methods
numeroussubtypes.both primary and secondary);MaIfa- arefrovidedfatreatingor~cnt.ingintIammatory arthritis
mative Tumors (e.g.. Craniopbaryogioma.Epidermoid (e.g.,rheumatoidarthritis) comprisingthe stepof adminis-
Cysts.Dermoid Cysts and Colloid Cysts): and Metastatic taiog to a patienta tbera~euticabyeffective amountof an
Tumors(which an be derivedfrom virtually any tumor.the aoti-angiogeoicfactor or anti-angiogeuiccompositionto a
most coamoo be& from lung, bm4sLmclaaoma.kidney. 35 joint. Witbin a pefcrrcd embo&nent of the invention,
and gastrointestinaltract tumors). anti-angiogenic factors (including anti-aogiogenic
compositions.as described above) may be administered
dimctly by intra-articularinjecsioo.asa surgicalpaste.a as
lnhmmatory arthritis is a serioushealth problems in an oral agent (e.g.. containing the anti-angiogenicfactor
developedcountries,partia&uly giventhe increasingoum- 40 thalidanidc). Onerepresentativeexampleof sucha method
bcr of agedindividuals.For example,ooc form of infiam- is set forth in mcxc detail below in Example 19.
matoryarthritis.meumatoidarthritis (RA) is a multisystem As utilized within the contextof tbe presentinvention.it
dKooic, relapsiag.ioflammatorydiseaseof uokoowocause. should be understoodthat efficatiousadmkshation of the
Although many organsCM be aifcctcd, RA is basically a aoti-aogiogenicfactors and compositionsdascriid herein
severeform of chronic synovitis that sometimesleads to 45 may be asssstd io several ways, including: (1) by prcvcnt-
d&raction aad ankylosis of affectedjoiots (taken from iog a lesscaing the pathological and/or dioical symptoms
Robbins Patfrolopical Basis of Discare. bv R. S. Cotma. V. associated with rheumatoidarthritis: (2) by dowureguiating
l&oar, aod S. ‘i. Robbins.*W.B. Saunkrs Co.. 1989). the.white blood cell ruponse which initiates the iofiamma-
Pathologicallythe diseaseis charauaizcd by a marked tory cascadeand results in synovitis. swelhng.pain. and
thickeniogof tbc synovial membranewbidt forms vikus 50 tissuedestmtioa; (3) by fnhibiting the “tumor-likenprolif-
jxojccrioas that extend into the joint space,multiiayuing of eration of synoviocytesthat leads to the developmentof a
the synoviocyteliaiog (syaoviccytc proliferation),ittfiltra- locally invasive and destructive pannus tissue: (4) by
tion of the synovial memkane with white blood cells decreasing the production/aczivity of matrix mcta&jmtcin-
(roauophagcs.lymphocytes, plasma celts, and lymphoid ases produced by white blood cells. syooviocytes,
follicles; called an “iniiammatapysyuovitis”), and deposi- 55 chmdrocytes,and codothelialcells, which degrade the car-
tion of fibrin with cellular necrosiswithin the syaovimn Ihe tilage matrix and result in iucversible destructiouof the
tissueformedas a result of this processis calledpannusand articdar cartihge; and (5) by inhibiting blood vesselfa-
eveotuallythe pannus grows to fill the joint splcc. The mation which providesthe framewak and nutrientsneces-
paoousdevelopsan extensivenetworkaboew blood vessels sary for the growth and developmentof the pannustissue.
throughthe processof aagiogellesiawhich is csscntiaito the 60 Furthermore.the anti-angiogcnicfactors cc compositions
cvolntion of the synovitis. Releaseof digestive enzymes should not be toxic to normal cbondrocytesat tbcsapeutic
{matrix lnetall~s (e.g.. collagcnase,stromeiysia)l lCXl%EWhOftheSCSpU3SWiUbedisCIJ~edinmoFtde~
and other mediate of the i&unxnatory vss (e.g., below.
hydrogenperoxide, snpaoxides. lysosomalenzymes,and
gwxiw%sof arachadonicacid metabolism)firornthe cells of 65 k Inflammatay Response
the paanustissueleadsto the progmive dcstructlooof the Neutmphilsarefound in abundance in the synovial fluid,
cartilagetissue.Tbc panuusinvadesthe art&&r cartilage bat only ia small numbersin the syuoti membraneitself.
5,716,981
29 30
h is estimatedthat mom than 1 billioo neutrophilsentera growth of a suppcrkg Vasculature(i.e. angi0genesi.s).
AIJ
moderately inflamed rheumatoid knee joint each day these tidings are suggestiveof a tissue in which DO&
(Holingswath et al.. 1%7) and remain there becauseno growth regulationas beenlost.
pathwayexistsby which they canleavetbejoint Thesecells Witbin one embodknt. inhibition of synovkyte pro-
releasereactivefke radicalsand lys~somalenzymeswbid, 5 lifaation may be determinedby, fff example.analysisof
degra& the cattllagetissue.Other PMN productssuch as SH-tbymidineincarporation into synoviocytes.(it in vitro
pstagladins and leukotrienesaugmentthe inflammatory synovioqte proliferation. Sudh methodsarc illustratedin
responseand n&t more inflammatoryozlls into thejoint mue detail below in Example23.
tiSSW.
Lymphocytes.panicularly T cells. are present~JIabun- IO c. Ma&ix Metallopoteinases(MMP)
dance in the diseasedsynovial tissue. Activated T cells Irreparable degradationof the cartilage extracellular
producea varietyof lymphokinesandcoOpuatewith B ceils mat& is beIievedto be largely dueto the enzymaticaction
to pduce autoantibodies.T ceils productsresult in the Of matrix metaU0pr0t&aseson the componentsof the
activationmauopbages.a cell which is thoughtto havean cartilage matrix. Although mmur~us other enzymesare
importat role in the patbokgy of the disease.The mac- I.5 likely iW0lVed in the developmentof RA. coliagenase
raphagesproducea variety destructivelysosomalenzymes, CAMP-1)and strometysi~~ (MM’-3) play an importantrole
~osfaglaodins. and moaokines and are also capableob (Vhxdti U al., 1994)in diseaseprogression.Theseenxymes
stimulating angiogenesis.One of the more iq0rUnt arecapableof degradingtype 11collagen and proteoglycans
monokim secretedby macrophages is Cl. Briefiy. It1 is respecdvel~ the 2 major exfncellular componentsof carti-
known to: stimulatesyathesisandreleaseOf collagenase by 20 lage tissue.Qtokines suchas IL-l. cpidermalgrowthfactor
synovicqtes and synovial fibroblasts,inhibit prOteOglycan (E@). platelet-derivedgrowshfactor. and tuma necrosis
synthesis by drondmcytes. activate osteoclasts,induce facta areall potentstimulatorsof collagenaseaad str~mel-
changesin the endotbeliumof the syn~vial vasculatute ysin production.As descrii ab0ve.numerOusceli types
[stimulationof endothelialproductionof plasminogenacti- found in the arthritic joint (white blood ceils, synovi~cytes.
vator ande~lonysh’mulatingfactor, expressionof leukocyte 23 endothdial cells. and cbondr~cytes)are capableof synthe-
adhesionmolecules,promOtion of pr0c0agulantactivity sizing and seeming MMP!S.
(Wider et al.. 1991)]. and act as a chemoattractantfa In p&iferatiog rheumatoidsynovial tissue. collageoase
lymphocytes and neutrophils.
and stromeJysinbecomethe major gene productsof the
WW one embodiment.d0wnregulationof the white paanusandmay compriseas much as 2% of the messenger
blood cell response,or inhibition of the intlammat~ry 30 RNAs producedby the synovialfibroblasts(Brinkerboffand
response.may be assessed by determinationof the effectOf Auble. 1990).Increasedlevels of collagenaseand stromel-
the anti-angiogenicfactahor anti-angiogeniccomp0sitionon yain arepresentin the cartilageOfpatientswith RA and the
the responseof neutr~philsstimulatedwith ~psonixedCPPD level of cnxymeactivity in the joint correlateswetl with the
crystals or opsonizedzyr~san.Such methodsare illustrated Severityof the lesion (Mattel-Pelletieret al.. 1993;Wala-
95
in more detail below in Example22. kovitis et al.. 1992). Because these enzymes are fundamental
to the pathology of RA and result in irreversrblecartilage
B. SynoviocyteHype&&a damage.many thaapeutic strategieshave beendevisedto
During the developmentOf RA, the synovial lining cells inhibit their dfects.
become activated by products of intlammarionOr through 40 Numerousnaturallypresentinhibitors of MMP activity
phagoeytosisof Immune complexes.Several subtypesof havebeenidentifiedandnamed‘TlMPS” far Xssue Lnhibi-
synovial Lining cells have beenidentified and all Of them tars of Metalloproteinases. Many of theseproteininhibitors
becomeintensely activatedand undeqo excessivebyper- bind with the active MhiPs to f0rm 1:l noncovalentcom-
plasia and growth when stimulated.As_ the .synovial tissue plexeswhich inactivatethe MMP enxymes.The TlMPs are
organizesto form a pannus,the number of synovi~cytes.45 producedlocally by dtondrOcytesand synovial fibroblasts
blood vessels.connectivetissueelements,andi&unmatory and are likely responsibleffp the normal regulation of
cells increasesto form a mass100 timeaits original size.In conneUive tissue degradation. It is thought that much of tire
many ways. the synovitls in rlmmatold artbiitis behaves damaget0tbecarGagemaMxisduet0al0calimbaIance
much like a 10cakd neoplasia(Harris. 1990). In fact between MMP and TR@ activity. This is probablydue to
culturedrheumatoidsynovial cells developthe pbenotypic y) inueasedproduction of metaLtoprOteinases while the pro-
cbarauerlstics of anchorage-independent gr~wtlr usually duction of TiMPs remainsat a normal a c0nstantlevel
associatedwith ne0plasticcells if they given suflicient (Vincetti et al.. 1994).To overcomethis. thaapeutic strat-
plat&tde.rived growth factor (L@atis et al., 1989). Xn egieshavebeendesignedto addexogenousTIMPs (e.g..the
additioa the synoviocytesalso produce large amountsof cltanially modified tetracyclinemolearles.collagensub
eollagenase,stromelysin.prostagbdins, and Interl&in.l. 5s strate analogucs) or to upregulate TLMP production
The tumor-like prolifaation of tbe cells of the synovial (retin~ids, transf0uning growth faaa 0, E-6. IL 1 1.
connectivetissuestroma(synoviocytes.fibrcblast-lilrecells oncostatinM) in an elfcatto restorethe enxymaticbalaace.
and ncovascular tissue) produces a pannus with maay fea- Howeverthis approachhas yet to translateinto signiksnt
hues of a 10caliud maliguancy.SuppOrtingthis tumcs clirdud results.
analogy are several findings: the paunus expresseshigb 40 An alternativeappoacbis to inhibit Or downregulatethe
levels of oncaproteinssuch as c-myc and c-fos, produces production of the MbXPsto restore a ncnmal balanceof
metatloproseinases to facilitate surrwnding tissueinvasion, activity. Naturally okxlming compouruisfrNF& all-tram
expresscytoskeletdmarkerscharactaistic of poorly diffa- retinoic acid) and syntheticcompounds(retin~ids,glucoc~r-
entiatedmesencbymaftissue(e.g.,vimentin); synoviocytes ticoid hormones)have beettdemonstratedta inlubit MMP
in vitro grow rapidly. do not cOntactinhibit, fwm fOei and 65 activity by suppressing tmscri#ion and synthesisOf these
can be grown under anch0rag4ndependentconditionsin pmteins. A post-transcriptionalmethodof blocking MME’
soft agarose;and pannustissueis capableof inducing the releasecOuldalso be expectedto result in a decreasein the
5,716,981
31 32
amount of MAE’ produced and an improved balance SW23
ascornedaeovasadarization
(illcludiagcornealgratl
betweenhfMP and 7XMP activity ia the joint neovasc&rirdtion). comprisingthe stepof administeringto
Within one embodiment a decrease in the productionoc a patient a therapeutically eifeetive amount of an anti-
activity of MMP’S may be determinedby. for utanlple. anglogeniccomposidoIl(as dtsaibut above)to the cornea
analysisof IL1 induced coJlagenase expression.One such 5 sudt that theformationof bioodvesselsis inhibit& Briefly,
methodis illustratedin more &tail be10win Example24. the corneais a time which MlrmauyIadts blcmdvessels.In
certain pathological conditions bowever, capillaries may
D. Angiogenesis extendinto the corneafrom the pericorncalvascularplexus
of thehmbus.Whenthe corneabecomesvasculsrixed.it also
The developmentof an extensivenetwark of new blood 10 bea-mes doudbd resulting in a dcdine in the patient’s
vesselsis essentialto the developmentof the synovitis visual acuity.Visualloss may hecomecompleteif the cornea
pescnt in rheumatoidarthritis (Harris, 1990:Folkmanet al.. completelyopacimm.
1989;Sano et ah. 1990). Severallocal mediatorssuch as Blood vesselscan enterthe corneain a variety of patterns
piatdctdexivedgrowth factor (PDGF),TGF-j3.andfibroblsst and depths. depending upon the poccss which incites the
growthfacta 0 are likely responsiblefcr the induction 1S newaac&&&on. These patternshave beentraditionally
andpapetuationof neovascuM&ion within the synovium. defmedby *thdmologisu in the following types:pannus
Pamus tissnt composedof new capillaries and syaovial trachomatosus.pannus leprosus.pannus phyictenulosus.
connectivetissueinvades and destroysthe attic&r carti- pannusdegeneradvus. and glaucomatouspannus.The ccr-
kge. The migradngangiogenicvesselsthemselvesprodncc oealstromamay alsobe invadedby branchesof the anterior
and secreteinaeased levels of metaUoprot&amssuch as 20 ciliary artery (called interstidal vascularization) which
cdage~~c and stromelysin capableof degradingthe car- causesseveraldistinct clinical lesions: tamiual loops. a
tilage matrix (Caseet al. 1989).The newly formedvessels Yxush-iike”pattern.an umbelform. a lattice form inters&
are also quite Ye&y” with gaps present between the tial arcades (from cpisderal vessels), and aberrant irregular
miaova6ada.rendothelialcelts. This facilitates the exuda- vessds.
tion of plasmapcteins into the synovium(which increases75 A wi& variety of disorders can result in corneal
swelling). cnhanccsWBCs movementfrom the c+culation newascukrization. including for cxamp~e.cormal i&c-
into the pannustissue (which increasesinflammation).and tions (e.g. trachoma.herpessimplexkeratitis.leishmaniasis
kads to the paivascursr accumulationof mononuclear and onchoces&sis).immunological processes(e.g.. graft
i.nfiammatorycells (wilda et aL. 1991). rejection and Stevens-Johnson’syndrome).
s alkali bums.
In sltmmaty,the etldo#dial tissle plays an impomintrole 30 trauma i&mmadon (of any cause).toxic and nutritional
in the developmentof this diseaseby expressingthe neec+ &ficiency states,and as a complicationof wearing contact
say surfacereceptorsto atlow intlammatciy cells to Leave lenses.
the circulation and enter the developingpannus.seucting While the catweof comeal neovascularixation may vary,
pteolytic enzymes cap&k of degradingthe cartilage tire responseof the corneatu the insult and the subsequent
matrix, and proliferating to form the new vessels 35 vascltkr ingrowthis similar regxdltss of Thecause.Briefly.
(angiogenesis) required for the pannustissueto inueasein thefocatioDoftheinjuryappuuYtobeofimpatanceasonty
size and invadeadjacenttissues. thoselesionssituatedwithin a critical distanceof the limbus
Within one embodiment,inhihitiw of uew blood vessel will incite an angiogenicresponse.This is likely due to the
formation may he readily determinedin a variety of asays. fact that the angiogenicfactas responsi& for eliciting the
including the CAh4 assay describedabove and within 40 vascularinvasion are createdat the site of the lesion. and
J&ample2. must diffuse to the site of the nearestblood vessels(the
limbus) in orderto exut their effect.Pasta certain distance
NEOVASCUL.ARDISEASESOFTTiE EYE from the limbus. this would no longer be possibleand the
limbic endotheliumwould not be induced to grow into the
As noted above. the present invention also provides cornea Scvaal angiogenicfacta are likely involved in this
methodsfcr treatingneovaseutardiseasesof the eye,in&d- 45 prccess.many of which xc productsof the infiammatay
ing for example.comeal neovas&ar%Gon, neovascuisr response.Indeed.neovasadadzadon of the canea appears
glaucoma proliferative diabetic retinopathy. retrolental to only occur in as6ociadonwith an inflammatory cell
fibrdlasia andmacular degenaation. infiltrate. and the degreeof angiogermisis proportional to
Briefly, corneaIneovasadadzadonas a resultof injury to so the Went of the iaflanunatmy reaction. Corncal edema
the antaia segmentis a significant causeOf deapscd fttrtha facilit&s blood vessel ingrowth by looseningthe
visual acuity and blindness. and a majar risk factor frr corn4 stromal frtunewwk and px&ling a pathway of
rejectionof comealallografts.As describedby Burga et ah. “least resistance”through which the capillariescan grow.
Lab. Invest. 48:16%180. 1983, corneai angiogenesis Following the initial intlammatq reaction, capillary
Involves three phases:a lxe-vascularlatent period. active 55 growthiutothecomeaprocce&inthesamemannaasit
neovascuMMcm, and vascularmaturationandregression occursin othertissues.The ncumaliyquiescentendothelial
The identity andme&a&m of variousangiogenicfactarr, celts of the limbic capilkrks andvenulesare &m&ted to
induding elementsof the inffammstoryresponse.such as divide andmigrate.The endc4heJial cells pjeet away from
leukocytes.platelets.cytokine5,andeicosanoids,a uniden- their vesselsof &gin. digest the surroundingbasement
tified plasmaconstituentshave yet to bt revealed. 60 membraneandthe tissuethroughwhich they will travel. and
cuualtiy lm clinically 6atisfactorytherapy exists fu migratetowardsthe sourceof the angiogenicstimulus.The
inhibition of comeal neovaaarlarimtioncr regressionof blind endedsproutsacquire a huuen and theu anastomose
existingcorncalnew vessels.Topicalcorticostesoidsappear toget@ to form c.apiUy loops. The end result is the
to havesomedinical utility, presumablyby limiting stromal estabWtment of a vascular pkxus within the anneal
inaammatic4l. 65 stroma.
Thus, within one aspectof the presentinventice methods #mti-angiogcnicfaucrs and compositionsof the present
are provided fa; treating neovascuiardiseasesof the eye invention are useful by blocking the stimulatoryeffectsof
5716,981
33 34
angiogenesispromoters. reducing endothelial cell division, particular,aboutone thhd of the patientswith this disord~
decreasing endotbelial cell migration. and impairing the have diabeticretinopathyand28% havecentratretinal vein
activity of the proteolytic enzymessecretedby the endot- occlusion.0th~ causesin&de chronic retinal detachment.
helium. end-stageglaucoma.carotid artery obstructive disease. ret-
Within particularly preferred embodiments of the roltntal fitroplasia. sicldecell anemia.intraoatlar tumors.
invention. an anti-angiogenicfactor may be preparedfor and carotid CBV~~OUS fistulas. XII its early stages. neovas-
topical administrationin saline(combiuedwith any of the culat glaucoma nay be diagnosed by high magnification
presnvaiivta and anthnicrob4alagentsCoJnmonIyused in slitbmp biomiuoscopy where it reveals small. dilated.
ocularprepmtions). andadministeredin eyedropform The dimrgahtd capillarits (which leak fluorescein) on the
anti-angiogcnicfactor sohrtionor suspensionmay be prc- surfsc of the iris. Later gonioscopy demonstrates progres-
pared in its pure form and administeredseveraltimes daily.
Alternatively. anti-angiogeniccompositions.preparedas sive oblit~ation of the anterior chamba angle by fibrovas-
dtscrii above.may also be administereddirectly to the cnlar bands. While the anttrioc dmmb~ angle is still open.
comea.within preferredembodhnents,the anti-angiogenic consuvative therapies may be of assistaoce.However.once
composition is preparedwith a muco-adhesivepolym~ the angleclosessmgicalinttrvtotion is requiredin order to
which binds to cornea Within further embodiments,the alltviatt the pressure.
anti-angiogenicfactors or anti-angiogeniccompositions Therefcxe.within one embodimentof the invention anti-
may be utilized as an adjunct to conventionalsteroid angiogtnic factors (either alone cr in an anti-angiogenic
rn-PY. composition. as &scaibed above) may be administered
Topical therapy may also be useful prophyWkaUy in topicahy to the eye in order to treat early forms of neovas-
mrneal lesions which are known to havea high probability culat glaucoma
of inducing an angiogenic response(such as chemical Within other embodiments of the invention, anti-
bums).In theseinstancesthe treatment.li$eiy in combina- angiogenicannpositionsmay be implantedby injection of
tion with steroids.may be instiMed immediatelyto help the compoeEtioninto Ute m&m of the anterim chamber
preventsubsequentcomplications. angle.This pvides a sustainedlocahxedincreaseof anti-
Within Otha embodiments.the anti-angiogeniccomposi- angiogenicfactor,andpevents blood vessel growth into the
tions describedabove may be injected directly into the atta llnplaPted or injected anti-angbgtnic compositions
comeal stroma by an ophthalmologistwdcr microsoopic which are placedbetweenthe advancingcapillaries of the
guidance.Tht preferredsite of injection may vary with the iris and the anteriorchamberangle can “defend” the open
morphology of the individual lesion. but the goal of the anglefkom mvasc~on. As capillarieswill not grow
administrationwould be to place the cmnposition at the s” within a significant radius of the anti-angiogenic
advanciag front of the vasculahue (i.e.. interspersed compositioa patency of the angle amld be maintained
between the blood vesselsasrdthe normal cornea).In most Within 0thK anbodimcets.the anti-angiogeaiccomposition
casesthis would involve perilimbic cornealinjection to may also bt placed in any location such that the anti-
“protect”the cornta from the advancingbloodvessels.This ?= angiogenicfactor is continuoustYreleasedinto the aqueous
methodmay alsobt utilized shottlyaft~ a comealinsult in =-I httmor.This would increasethe anti-angiogenicfaaor con-
a&r to prcphylauiczdlyprevent corneal neovascularira- certhationwithirt thebwna. which in Uri-nbathesthe surface
tion. In this situationthe materialcould be injectedin tbe of the iris and its abnormal @lbuiess. tbtreby providing
pedimhic cane.43 inte~-~ptrsed
betweentht corneallesion aootha mechanismby which to deliv~ the medication.
aadits Undesiredpotentialhmbic blood supply.Suchme& 4. These*erapeutic modalitiesmaY alSobe UsefulPophYhc-
cdsmayalsobeutihxedinasimilarfasbiontoprevent tically and in combinationwith existing treatments.
capillary invasion of transphUnedcorneas.Ia a sustained. within amber aspwl of the presentinvention, methods
releaseforminjections might only bertquirtd 2-3 timaspa are providedfa; treating proliferative diabetic retinopatby.
year.Asteroid could also be addedto the injection solution ccmqxisingthe step of administeringto a patient a them-
to reducehflamnation resultingfrom the injection its&. ,5 gxmticallyefkU.ive amountof an anti-angiogenicco-i-
Within anotheraspectof the presentinvention,methods tion to tht eyes,such that the formation of blood vesselsis
are provided for treating neovascuk glaucoma. comprising inbibi@i
the step of administeringto a patient a thhbapoutically Briegy, tirepathologyof diabeticretinopathyis thoughtto
tffective amoUntof an anti-angiogeniccompositionto the bt similar to thatdescribedabovefor neovascularglaucoma.
eye. such that tb.e formation of blood vesselsis inhibited. ;u) In pattiadat, backgromrddiabeti~ retinopathyis Meved to
Briefly, neovasc~larglaucomais a pathologicalcondition convat to pJ-olifKauvediabulc Jeutlopatllylmdtr the iJJflu-
wherein new capillaries develop in the iris of the eye. ‘Ihe enct of retinal hypoxia. Generaliy. neovasarlar tissue
a&genesis Usuallycaiginatesfrom vesselslocatedat the sproutafrom the optic mxvc (usually within 10 mm of the
pupillaq margin.and progressesaaoss the root of the his tdgt), and fkwn the surface of the rttina ia regions what
and into the trabecu3a.r meshwork FJtihsts and O&K 55 t&UC pafuSiOn is pear. tidy the C%piEarieSgrow
connectivetissueelementsare associatedwith the capiuary bttweta the inner limiting znembmneof the rttina and the
growth and a tibrovascular membrartedevelops which posmior surface of tht vitrtous. Evenmally. the vessels
!paia aaossthe antaior surfaceof thehia. EventuallyU&s grow into tht vitteous and throughthe inner limiting mem-
tissuertaches the anttrior chamberangle whae it forms braoe.As the vitttous cormacts.traction is applied to the
synechiae.Thesesync&& ia ttxn coalesce,scar.and con- 60 vease&,oftenresulting in Waring of the vesselstmd blind-
tract to uhimattly close off cbeanteria cbambcrat&. Tbt ing of thevitreousdueto hemorrhage. Fibroustractionfrom
scar formation prevents adequate drainage of aqueous stxrrhg in the rttina may alsoprodUceretinaldefacbmertt
humor throughthe angleand into the trabectrlarmeshwork The conventionnlthtxapy of choice ia pametinalphoto-
resulting in an increasein intraoa.darpresmre that may coagulatiar~ to dtatast retinal tissue, andtherebydecrease
result in blindness. 65 rttiml oxygendemands.AlthoUghinitially effectivt. thereis
Neovasculatglaucomagenerallyoccursasa complication a high relapst rate wllh ntw ltsions faming in othexparts
of diseasesio which retinal ischemiais predominant.In of the retina Complications of this therapy include a
5.716,981
35 36
deereasein peaipheralvision of up to 50% of patients in orderto thesapcutieally treat a cancer or tumor. For
mechanicalabrasionsof the cxmlca.laser-inducedcatarau example,anti-angiogenicfactas or compositionsdescribed
formation. acute glaucoma.and stimuMion of subretiual llmin may be fcXml&ed for topical delivery. in order to
ncovasculargrowth (which can result in loss of vision). As treat cancerssuch as skin cancer.head and neck tumas.
a result. this prccedureis performedonly when severalrisk 5 breast tumors. and Kaposi’s sarcoma.Within yet 0th~~
factas are present.and the risk-benefitratio is clearly in aspects.tbc anti-aagiogeoicfactors a compositionspro
favor of intervention. vi&d haeiu may be utilized to treat superficialforms of
Tllaefae. witlliu particularly preferredcmbodhents of bladdercancerby, for example.intravesicaladministration.
the inventioll. pliferative diabetic lctinopatlly may bc IO addition to cancer, however, nmcrous etbcr non-
treatedby injection Of an anti-angiogenicfactor(s)(or auti- 10 tumor&& angiogenesis-dependent diseaseswhich are
angiogeniccomposition)into the aqueoushumor a the cbmcteked by the abnormalgrowth of blood vesselsmay
vitrcolls, in adcr to blaease the Iocal coIlaoaation of also be mated with Ihe aoti-angiogenicfactors or compo-
anti-angiogeaic facta in tbe retiua Preferably.this treat- sitionsof the presentinvention. Rqxesenuuiveexamplesof
ment shouldbe initiated Mor to the acquisitionof severe such non-tumorigenic angiogenesis-dependentdiseases
&case rquiring photoabagulation.WIthin other emhodi- 1s indude byput+& scarsaud k&ids. proliferativediabetic
meritsof the invention.&erics whirh feed the neovascular retinopathy (discussed above). rheumatoid arthritis
lesions may be embolized (utilizing anti-angiogenic (diawssedabove).artaioveoousmalfcrmations(discussed
compositions,as desdi above) above), atherosdrrotic plaques. delayed wound healing.
Within anotheraspectof the presentinvention. methods hemophilic joints, nonunion fractures. Osier-Weber
areprovidedfa treatingretrolentalfibrobiasia.comprising 2~ syndrome, psoriasis. pyogenic granuloma. sclemderma.
the step of administeringto a patient a tbempeuticaby ~IWOIM.m~nanhagia(di~~ussd &we) and vascularadhe-
dfcetive amount of a0 anti-fmgiogcuicfactor (a anti- sions.
angiogcIliccomposition)to the eye, suchthat tbc fcxmation Fa example,within one aspect of the presentinvention
of blood vesselsis iubibited. methodsare provided for treating hypertrophicscars and
Bridfy. retrolentali&oblasia is a conditionocaming in keloids. commisiag the step of administuing one of the
prematureinfants who receive oxygentherapy.The periph- abovedescrii anti-angiogeniccorm~~itionsto a &per-
ml retillal v-. particularly 011the tempcral side, trophic scar a keloid.
does not lxmme fully formed until the end of fetal life. Briefly. healingof wounds and scarformation occursin
Excessiveoxygen(evenlevels which would be physiologic tbrce pbascs:inflammation. proliferation. and maturation.
at term) and the formation of oxygen free radicals are The first phase. inflammation, occurs in responseto an
thought to be importmt by causing damageto the blood injury which is severeenoughto hmak the skin. During this
vesselsof the immatumretina.Thesevesselsconstrict,and phase.which lasts 3 to 4 days. blood and tissue fluid form
tbcn becomesmluumlly oblita7!tcdon exposutcto oxygtu. II adhesivecoagulumandfibrinousnetworkwhich servesto
As a result. the peripheralretina fails to vascu.la&eand bind the wound surfaoestogether.This is then followed by
rctiual ischemiaensues.In re:sponse to the iscbemia.neovas- a proliferativepbascin which thereis ingrowthof capillaries
culari&on is inducedat the junction of the namaJ andthe and conncdivetissuefrom the woundedges.andclosureof
isdmnic retiua. the skin defect, l%tally, onw capillary and fibroblastic
III 7.5%of the easestbcsc vcssclsregressspoutancously. proliferation has cursed, the maturation process begins
However.in the remaiGg 25% thereis continuedcapillary whereinthe scar contractsand becomesless cellular. less
growth, cxxmction of the fibrovasatlar component.and vascular,and appearsflat and white. This fmal phasemay
traction oo both the vcssclsand the retina.This resuhsin take between 6 and 12 xnontbs.
vitreous hemorrhageand/or retinal detachmentwhich can If too much COMC&VCtissueis producedandthe wound ’*
lead to blindness. Neovascular anglwzlosurc glaucoma is rcmaiaspcmistcntlycdlular. the scarmay becomered and ~
also a c~mplicatioa of this condition raiscd.IfthescarnmaiEIh witbin the bo~darics of the
As it is often impossibleto determinewhich caseswill ~woullditisrdaredtoasahypntrophicscar,butif
spont~ccusly resolve and which will progressin severity, it extendsbeyondthe original scaraudinto the stxounding
conventionaltreatment(i.e.. surgery)is generallyinitiated tissue. the lesion is refured to as a keloid. Hypatro@
only in patieotswith establisheddiseaseand a well devcl- scarsandkel~ids am producedduring the secondand third
cped pathology.This “wait and see”approachprecludes phasesof scar fcrmation. Several woundsare particularly
early intavention, andallows the progressionof diseasein proneto excessiveendotbelialand t&mblasticproliferation,
the25% who follow a complicatedcourse.Therefore,within induding buns, open wounds. and infected wounds.With
one cmboddent of tbe invention, toplcal administmtiooof hypatrophic scars,some degreeof matmtioo oocursand
anti-aogiogeoicfactors(or anti-angiogcniccompositions.as grsdualimprovementoccurs.In the caseof keloidshoweva.
described above) may kc accomplished in infants which are an actualtumor is producedwhich filIl becomequite large.
at high ri*fa developing this condition in an attemptto cut Spoutaneousimgovemcnt in SW&easesrarely occurs.
down on the incidenceof progressionof ntroiental fibro- l%crefaG withio one cndmdbcnt of the peaent iuven-
plasia within otbff embodhats. inhavitruws injections tioa eitherauti-augiogenicfactas alone.a anti-aagiogenic
an&or intmoadar implantsof an anti-angiogeniccomposi- compodions as &scribed above.arc directly injcctcd into a
tion may hc lItaid. such me#ods arc patticularlypreferred bypertropaicscara keioid, in ader to preventthe progms-
in casesof establisheddisease,in crda to reducethe need sion of these lesions. Tlke frequency of injections will
for surgery. depend upon the rckasc kinuics of the pOrrma used (if
present).and the clinical response.This therapyis of par-
UrHEiR -c USES OF AmI- ticular value in the prophylactic trcatmnt of conditions
ANOIOOB’N’ICCOMPOSITlONS wbicb are known to result io tke dcvclopmcntof hype
Anti-angiogcnicfactors and compositionsof the present tropbic scars and keloids (e.g., burns), and is preferably
inventionmay be utilized in a variety of additionalmethods initiated after the proliferative phase has bad time to
5,716,981
37 38
progress(apgcoximately14 d8YS after the initial injury). but intraocufarly. intranasaJJy.Jntradermahy.sublingually,
beforehypertrophicscar or keJoiddeveJqmtent. oraJJy.topically. iotravesicaJJy.intrathecaJJy.topically.
As notedabove.wJthJnyet anotheraspectof the present intravenously, intraperitoneally. intracranially,
invention. vascular grafts are provided comprisJnga syn- JntramnscuJarJy. SubartaneousJy. or even directly into a
tbetic tube. the surfaceof which Js coated~4th an anti- 5 tuma or diseasesite. O&r rgesentative routesof admin-
angiogeniccompositionas desaibedabove.Briefly, vascu- istrationindude gastmscopy.&XP andcoionoscopy.which
lar grafts are synthetictubes. usuaIJymade of Ihcsmn or do not re@re fnU operatingprocedurtsandhospitaJizatioa
Gokex. hated surgically to bypass attcriat blockages, but may require the presenceof medJc.aJ personnel.
mastfrquently from theaortato thefemoraJ.cc the femocaJ The anti-angiogenicfactors. anti-angiogenk composi-
to the poplitcaJattery.A majccproblemwhich partJc&arly t* tiom andpharmacautJcaJ composJtions providedhereinmay
complicatesfemoral-popJiteaJ bypassgraftsis the famation be placedwidtin containers.alongwith packagingmat&d
of a subendothelialscar-Jikereactionin the bloodvesselwaJJ which providesinstructionsregardhrgthe useof suchmate-
called aeoiraimal hyperplasia.which natrows the lumen rials. GeneraLly,such instructionswiJJ include a tangible
within and adjacentto eitherendof the graf&andwhich can expressiondescribingthe reagentconcentration.as weJJas
be progressive.A graft coated with a containing ant& ts within certain embodiments.rdadve asnountsof excipient
angiogenicfactors (or anti-angiogenk compositions,as ingredfentscx dfJuents(e.g.. water. saJ.ineor PBS) which
desuibedabove)may be utilJzedto limit the fcnmatJonof may be necessaryto reconstitutethe anti-angiogenicfactor.
neointimalhypexpJasia at either end of the gratt. The graft anti-angiogeniccomposition. or pJuumaceuticaJ composJ-
may tben be surgicaflyplacedby conventionalbypasste&- don.
niques. 20 The following examplesareofferedby way of illustration.
Anti-angiogeniccompositionsof the presentJnvention and not by way of Jimitation.
may ako be utilized in a variety of ctJtczmanners.For
example.they may be incorporatedinto sllrgicaJsuturesin
ader to preventstitch granulomas.implantedJn the uterus
(in the same manner as an IUD) for the treatment of 25 Example1
t&orrhagia ox as a form of female birth control. admitus- Preparationof Anti-InvasiveFactor
tered as either a paitooeal Iavage fluid or for perJtc+teaJ
JmpJamation in the h‘eatmentof endometriosis.attachedto a The shonldcr girdle and skuJifrom a dogfishis excised
monodonalantibodydirected against activated endo&eJJaJ30 thenscrapedwJtJta scalpe1in orda to removeall muscleand
celts as a form of systemicchemotherapy.or utiJizedJn associatedcottnectiVetissuefrom the cartilage.The cartilage
diagnosticimagingwhenattached.toa radioactivelyJabeled is thenbomogenizcd with a tissuegrinda. and extractedby
monocloaalantibodywhichrcoogaizesadivatodendothelial continuousstirring at room temperaturefor 2 to 5 daysin a
CdlS. solution contaJnJng2.OM guanhlhtm hydrochloride and
o.mM ME!s at pH 6.0.
KRMtTLtU’IONANDAD-ON 33 Afta 2 to 5 days.the cartiJageextractis passedthrough
As noted above. anti-angiogeniccouqositions of the gauzenettingin orderto removethe Jargerconstituents.The
Hesentinvention may be formuJatedin a variety of forms filkak is then passedthroughanAmicon u.ltratJJtratJon unit
(e.g.. miaosphaes. pastes.tihns cc sprays).Fur&r. the which utilizes spiral-woundcartsidges.with a molecular
compositionsof the presentinvention may be fonnuJatedto 40 weight cutofTof 100.000.The &rate (containJ.ng proteins
containmore than one anti-angiogenicfactor, to contaJna wiUi a moJecuJar weight of lessthan100.000drdtons)is then
variety of additional compounds,to have cataJa physical diabzed anainst 0.02M MES buffer I& 6) with an Amicon
pperties (e.g.. ehstidty, a pariictdar meJtingpoJnt or a UJ&ti%n ,unJtwhich retains~xo&s ‘titJt a molecular
specifiedreleaserate). WsUdncertain emJ3odJments of the Weight Of greater than 3.000 daJtons. UtiJixingthis method,
Jnvention.compositionsmay be combined Jn order to low molecular weight proteins and constituents are
achievea &sired effca (e.g..sevemJpreparationsof mJab ” removed.as well as excessiveamountsof guanbiiumHCL
spbaeamay be combinedin orderto achieveboth a quick The diaiysate is concentratedto a JinaJcmcenkation 9
aad a slow or prolonged release of one or mare anti- WeJ.
angiogenicfactor). ExampJe2
Anti-angiogenicfactors and compositionsof tJtepresent so
inventionmay be admJnJstered eJtheralone.cr in combkut- Analysis of Various Agentsfor Anti-Angiogenic
tion with phatmaceuticaUyor physiologicahyacceptabJe Activity
carrier. cxcipJent.sor d&tents. GeneraJJy.such carxders
shouldbe nontoxicto recipientsat the dosagesand concen- A. CbJckCborJoaJJantoJc Membrane(“cam”)
kationsemployed.GrdJnatiJy.the preparationof suchcom- 55 Assays
positions eataS comJ%aingthe therapeutic agent with Fatilizcd domesticchick embryoswae incubatedfor 3
buffas. antioxidantssuch as ascorbicacid, low molectdar dayspiar to she&lessarlturing. In this pcedsxe. the egg
weight(less than about 10 residues)poJyJqtides, wins. contentswere emptkd by removmgthe shelllocatedaround
amino acids. carbohydratesincluding ghzcose.sucroseor the air space.The iatuior sheJJmembranewasthen severed
dexkitts. d~elatingagentssuch as EMi% glutathioneand 60 andthe oppositeendof the sheJJwasperforatedto allow the
o&r stabilizersand excipients.NeutraJbnffued saJJnebt contentsof the egg to gentty slideout from the bluntedend.
dine mixed with nonspecificserumaJbumJn are exen&ary The egg contentswerecmptie.dinto round-bottomstuihacd
appriqdte diluents. glassbowls and coveredw&Jrpetri dish covers.‘Thesewere
As notedabove,anti-angiogenicfauors. anti-angiogenic thenplacedinto an incubatorat 90% relative humidity and
compositions.or phanm~ceuticalcompositionsprovhled 65 3% CO, and incubatedfcr 3 days.
hereJnmay be preparedfm admJnJstration by a variety of Pa&axe1 {Sigma St. Louis, Mich) was mixed at con-
different routes. JncludJng for example intrart.icuJarly, centrationsof 1, 5. 10. 30 ug per JO mJ aJicJuotof 0.5%
5.716981
39 40
aqueousmc#yIcchulose. since pa&tax& is insolublem dermallayercontainingcapillaries which lie subjaant to the
wata, glassbeadswere usedto poduce fine par&& Ten ectodum adventitialcek and an inna. singk end-
miaolita aliquotsof this sohltionwa dried on parafhmfor cell layer (FIG. ID). At the clcctronmicroscopiclevel. the
1hourformingdisks2mmindiamucr.Thcdricddisks typical structuraldetailsof the CAM capillariesare dcmon-
containing pachtaxel wen then CarefuRyplaced at the 5 strated.T&&xlly, thesevesselslie in close assockdionwith
growing edgeof ea& CAM at day 6 of incubation Controls theinnacclilayaofcccodam(FlG. lE)
wae Wed by placing paclitaxcl-free methylcellulose Alla 48 hoursc~posttrcto pachtaxelat concwatrations of
disksontbeCAMsovn~esamctimtcanst.Aftna2biy 0.25,O.!il,5.~0. Q 30 ug, eachCAM was examinedunder
exposure(day 8 of incubation)the vas&ahnc was exam- living c0nditbns with a storeomicroscopcequippedwith a
ined with the aid of a stcreomicroscopc.
Liposyn II. a white 10 Vidodco3npUU hltcrfacc in otdcr to evaluate the cfkts on
opaque solution, was iajaztcd into the CAM to inmasc the
visibility of the vascular details. The vasculature of angiogemsis.This imaging setupwas usedat a magnifica-
unstained.living embryoswae imagedusing a zdss stem tion of 160timeswhich permittedthe &ct visualizationof
omiffoscopc which was intcrfaoxl with a video camera bloodcellswithin the capillaries:therebybloodflow in areas
@age-MTJ Inc., Michigan City. Ind). These video signals of interestcould be easily assessedand recorded,For this
were thendisplayedat 160timesmagnificationandcaptmcd 1s study. the inhibition of angiogencais was d&ted as an area
using an image analysis system(Vidas, Kontrun: Etching. of the CAM lacking a capUary network and vascularblood
Gummy). Image negatives were tbca made on a graphics flow. Throughoutthe expcimtllrs. avascularzones wue
recorder (MO&I 3ooo; Matrix Instlumen~. orangeburg. assessed 011a 4 point avasculargradient(TableI). This scale
N.Y.). represults the degreeof ovcrau inhibition with maximal
The membranes of the 8 day-old shell-lessembryowere 20 inhibition rqxesentedas a 3 on the avasculargradientscale
flcnnicd with 2% glutmld&yde in O.lM Na cacodylatc Paclitaxel was vay consistent aud induced a maximal
buffer; additional fixative was injected under the CAhf. avasculsrzone(6 m m in diameter or a 3 on the avascuhtrc
Afta 10 minutesin situ. the CAM wasremovedandplaced gradientscale)within 48 hours dependingon its concentra-
into fresh fixative for 2 hcurs at room temprrat~. The tiO0.
tissue was then washed overnight in cacodylate buffer 25
containing6% suaose.The areasof i&rest WEXC postfixed TABLE I
in 1% osmiumtetroxidefor 1.5 hoursat 4“ C. The tissues
wcre thendehydratedin a gradedseriesof ethanols.solvent AVASCULAR GJtADm
exchangedwith propyleneoxide, and embeddedin Spun o-ncwnfdvpnrhrity
resin. Thin sectionswere cut with a diamondknife. plaocd 30
on coppergrids. stained.and examinedin a Joel 12OODI
dwtron miao5copc. Similarly. 0.5 mm sedlons wac cut
and stainedwith toluencblue for fight microscopy.
At day 11 of development.chick embryoswere usedfor The dose-dependent.experimentaldata of the CSectsof
the ccuosion casting technique.Macox resin (Ted P&a. 35 paciitaxelat dW@entconcentrationsare shownin Table IL
IJK.. R&ding. Calif.) wasinjectedinto the CAM wscuktm
using a 3O-gaugehypodumic needle.The castingmaterial TABLE I[
consistedof 2.5 gramsof Mctcox CL-2B polymer and0.05
gramsof catalyst(55% benzoylpaoxide) having a 5 minute
pcdymaizationtime. Afta injection.theplasticwasallowed
to sit iu situ for au hour at room temperatureand then
overnightin an oveuat 65’C. The CAM was thenplacedin
50% aqueoussolution of sodium hydroxide to digestall
crganic components. The plastic castswere washedcxten-
siv&y in distilled wata. air-dried, coated with gold/
paLl~x&&&mand&wed with the Philips SOlB scanning
Resrjs of the abovecxpgimentsareshownin FIGS.1-4.
Briefly. the genaal featuresof the namal chick shell-less
egg culture are shownin FIG. IA. At day 6 of incubation, so
the embryo is centrally positioacdto a radial& expanding TABLE III
networkof blood vcsscLs;the CAM developsadjacentto the
embryo.Thesegrowing vesselslie dose to the surfaceand
are readily visible making this system an ideahzcdmodelfor
ttle study of angiogenesis.Living, uustainedcap&ty net-
warks of the CAM can be imaged nouinvaaivclywith a
sterwmiuoscope.FIG. lB illustrateaswb a vascularareain
which the cellular blood ckmcnts within ca@hrics wcxc
xtxaded with the USCof a videofcompuuzinterface.The
3-dimcusionalzr&tecturc of suchCAM capillary networks
is shownby the corrusionca5ungmethodand vkwed ill the
scanning electron microscope(FIG. 1C). These castings
revealedunderlyingvesselswhich projecttoward tbc CAM qpii p8cmaxd-tr~ CAMS are also shown with Ibe
surface what they form a single layer of anastomotic transparent mdbylcclUosc disk antrally positioned ova
capiharies. 65 the avascularZOI~Cmeasuring6 mm in diam&r. At a slightly
Transversesectionsthrough the CAM show an outer highermagnificati~ the per&by of aucb avascular zones
cctodam consistingof a doublecell layer. a broadermeso- is dearly evident (FTG. ZC): the SulTwnding ftmuiooal
5,716,98 1
41 42
vesselswere often tcdirected away from the source of avascularzone at 48 hours illustratedin PIGS. 3A and 4A.
naclitaxel(PIGS. ZC and 2D). SU& aneular redirect&z of Also, we observedin the revasculatizationprocessinto the
blood flow was nevu observedundK;ormal conditi&. avascularwne previously observedIt has beenfound that
Anotherfeatureof the effectsof paclitaxelwasthe ftxmation the avascularwne formed by heparinand angiostaticste-
of Moodislandswithin the avascularzonerepresentingthe S roids becamerevascukiud 60 hours after application.In
aggregationof blood cells. one study.pacIitaxel-treatedavascuiartones did not revas-
The associated morphological altorations of the wlarize for at least7 days&a applicationimplying a more
paclitK.cl-treatedCAM arereadily anuatentat both thelight potent long-termeffect
&d eledron microscopic levels: Fb; the convenience-of Example3
presentation.threedistinct phasesof genKaltransitionfmm 10
the normaI to the avascular state are shown. Near the 3Wapsulation of Suxamin
puiphery of the avascularZODCthe CAM is hallmarkedby One milliliter of 5% EXVAX (poly(ethylene-vinyl
an abundanceof mitotic cells within all three germlayers acetate)cross-linked with 5% vinyl acetate)in did&.
(FIGS.3A and4A). This enhancedmitotic division wasalso romethane(“m is mixed with a fixed weight of sub-
a consistentckrvatioo for capillary cndothelial cells, 15 miaon groundsodiumsuramln.This mixtureis injectedinto
Howeva. the endothelialcells remainedjunctioWy intact 5 ml of 5% polyvinyl Alcohol (‘TVA”) in waterin a 30 ml
with no extravasation of blood cells. With further Batbottomedtesttube.TubescontainingdiffKent weightsof
degradatioathe CAM is CharactKizedby the tIrcak&wn the drug arethen suspendedin a multi-samplewater bath at
and dissolution of capillaries (PIGS. 3B and 4B). The 4o”for 90 minuteswith automatedstirring.The mixturesare
presumptiveend&dial cells. typically arrestedin mitosis, 20 removed and microspheresamplestakenfor size analysis.
still maintain a closespatialrelationshipwith bloodcellsand Tubes are centrifuged at 10tM g for 5 min. The PVA
lie subjacentto the ectode?m,howeva. thesecells am not supernatant is removed and saved for analysis
(n~encapsulateddrug).The tnicrosphexes arethen washed
junctionaIIyLink& The most centralportionof the avascu- (vatexed) in 5 ml ofwata andrecenttifuged.The5 ml wash
Iar zonewas characterlizbd by a thickenedecM~mal and is savedfar analysis(surfacebounddntg). Microspheresare
endodermallayer (PIGS.X and 4C).Althoughtheselayers 25 then welted in 50 ul.of methanol,andktexed ih 1 ml of
were thickened.the cellular junctions remainedintact and DCM to dissoIvethe ELVAX. Ilte microsuheresare thea
the layersmaintainedtheir structuralcharacnuistics. within warmedto 40”C.. and 5 ml of 50”C water& slowly added
the mesaterm. scatteredmitotically arrestedcells were with Stirring.This pimcahm resultsin the immediateevapo-
abundant; these celk did not exhii the endothelialall ration of DCM, thereby causing the releaseof sodium
pohrization observedin the formK phase.Also, throughout30 sttraminintotheSmlofwater
this avascularregion. degenaatingcells were commona5 All sampleswere assayedfor drug contentby quantifi-
noted by the electron dense vacuolesand cellular debris cation of ff~orcscencc. Briefly. sodium sutamin absorbs
(FIG. 4C). uvh-is with a lambda mar. of 312 nm. This absorptionis
In summaty,this study demonstratedthat 48 hoursaftea hnearintheOto 100ug/mlrangeiabothwata8nd5%WA.
paclitaxclapplicationto the CAM. angicgenesiswasinhib- 35 Sodiumsuramb ako ffuorescesstrongly with an excitation
ited- The blood vesselinhibition formedan avascularzone maximumat312muandemissionmaximumat400mu
which wasrepresentedby threetransitionalphasesof pacli- This flucreswnceis quantitiablein the 0 to 2.5uglml range.
taxel’s elfect.The ceniral. most affectedarea of the avas- The resultsof theseexperimentsis shownin PIGS. 5-11.
cular zonecontaineddisruptedcapillarieswith extravasated Resultsare shownin FIGS. 5-10. Briefly. the size distritu-
red blood cells: this indicated that intercellularjunctions 40 tion of mkzospheresby number(PIG. 5) a by weight (PIG.
betweenendotbelial cells were absent.The cells of the 6) appearsto’be unaifectedby inclusion of the drug in the
endcdermandectodermmaintainedtheir intercelIuhujunc- DCM. Goodyieldsof microspheresin the 20 to 6Opmrange
tions and therefore these germ layers remained intact; may be obtaiqq3.
however.they were slightly thiclrcocd.As the nomudvas- The encapsulationof suramktis very low (~1%)(seeFlG.
cular amawas approached.the blood vesselsretainedtbelr 45 8). Howeveras Sheweight of drug is increasedin the DCM
junctioaal complexesaad therefore also remained intad. At
thetotal amountof drug empdatad increasedalthoughthe
8 eacapdation deueascd.As is showain PIG. ?,50 ug of
the paiphcry of the paclitaxel-treatedzone,futthcr blood
vessel growth was inhibited which was evident by the
typed direchg or “elbowing”effectof the bloodvessels is shownin PIG. 9 (size distriibntionby weigh& Encapsu-
(FIG. 2D). xl lation of sodim suraminin 5% PVA containinn10% NaCl
Paclitaxel-treatedavascularwns alsorevealedan abun- is shownin PIGS.10 and 11 (size distributiou cwcight, and
danceof cells arrestedin mitosis in all threegerm layersof number.respectively).
the CAM; this was uniqueto paclitaxel since no prwious To assesssuratnin and cc&one acetate as potential
study has illustrated such an eveat. By being arrestedin anti-*ogenic agents,each agent was mixed with 0.5%
mitosis. cndothelial cells could not undergotheir normal s5 methylcelluIoseand applied the dried disks containingthe
metabolic functions involved in angiogeoesis. In agentonto the developingbIood vesselsof the 6-&y old
comparison.the avascular zone fcxmed by suramin and CAM. A combioationtreatmentof suramin (70 pg) with
cortisoneacetatedo not producemitotically arrest& cells in cmtiswe acetate(20 fig) was successfulin inhibiig angio-
the CAM; they only preventedfurtha blood vesselgrowth genesiswhentestedon the CAh4for 48 hours.The resulting
into the treatedarea.Therefore,eventhoughtheseagentsare 60 avasctdarregion measured6 mm in diametK and revealed
anti-MgIogenic,there are many points in which the an@ an absenceof blood flow and the appcamnceof sparseblood
genesispsncessmay be targeted. isIan& (FIGS. 28A and 28B).
The effectsof paditaxel OVK the 48 hour durationwere Example4
also ObsCrVCd. During this paid of obseaw.tionit was
noticedthat inhibition of angiogenesisoccursas e&y as 9 65 BncapsuIationof Paclitaxel
hours aftet application. Histological sectionsrevealed a Five hundredmiczogramsof eitherpa&axe1 or bacc&n
similarmorphologyas seenin thelirst transitionphaseof the (a paclitaxel analog.avaiIablehpm Inflazymephatmaceu-
5,716,981
43 44
ticals Inc., Vancower. British Columbia. Cenada) art dis- The abdcminal wound is closed with 6.0 resorptible suture
sowed in 1 ml of a SO50 ELVAX:poly-l-lactic acid mixture with skin clips. and the anesthetic terminated The rat is
in dcm Micmspheres are then prepared in a dissolution returned to the animal care facility to have a staadard diet for
machine (Six-spindle dissolution tester, VaaderKaap. vaa 14 days, at which time each tumor &posit will measure 1 an
Kell LadustriesInc.. U.S.A.) in triplicate at 200 rpm, 42’ C.. in &meter. The same procdure is repeated using Westar
for 3 hours. ?vlicrospheresso pateparedart washed twice in rats and a Colon Cancer cell line (Radiologic Oncology Lab.
wata aad sired on the micrcscope. M. D. Aadersoa Houston. Tex.). Ia this instance, 3 weeks
Determiaation of pa&axe1 encapsutafion is undertaken are required post-injection for the tumor deposits to measure
in a uv/vis assay (uv/vis lambda max. at 237 mm. fluores- lCXtilldiamacrCa&
cence assayat excitation 237, emission at 325 mn; Pluores- i%ftK 2 w 3 We&S, depending on the rat species, the same
cence results are presented in square brackets [ 1). Utilixiag general anesthetic prooedme is followed and a midline
the procednres dcscxibcd above. 58 pg (+I-12 pp) 175 pg abdominal iadsion ls pesfonaed. The duodenum is flipped
(+/-25 pg)] of pa&axe1 may be encapsulated from a total and the gastroduodeaal artery is identified aad mobilized,
SO0 pg of starting material. This represents 12% (M-2.4%) Ties arc placed above and below a cutdowa site oa the
[15% (t&S%)] of the orijJinai weight. cf 1.2% (+-I-&.25%) mk#wrtion of the gasiroduodenal artery (GDA). and 0.038
[IS% (+/-OS%)J by weight of the polymer. After 18 hours inch polyethylene tubing is iatroduced in a retrograde fat&-
of htmbliag in aa oven et 37’ C., 103% (+/-lo%) [6% ion into the artery using an operatiag microscope. The tie
(+/-5.6%)] of the total peclitaxel had heen released from the below the iasertion point will ligate the artery, while the one
microsphaes. above wiil fu the catheter in place. Aagiography is per-
For baccatin. NO+/-15 pg [83-M-231 & of bacc&ia caa formed by iajectiag 0.5 ml of 60% rediopaque coatrast
be encapsuiatedfrom a total of 500 w start@ matmial.This rnatcriaI through the catheter as an x-ray is taken. The
repseats 1 20% (d-3%) [17% (i/-S%) of the origiaal hepatic artery is then embolixed by refluxiag particles mea-
weight of baccatia. and 2% (+03%) [1.7% (+/-OS%)] by suring 15-200 )un through the gastroduodeaal artery cath-
weight of the polymer. Afta 18 hours of tnmblitrg ia an oven eter until flow. obsaved via the ope~atiag miaosccpe. is
at 37O C.. 55% (M-136) 160% (if-23%)] of the baczatia is seen to cease for at least 30 seconds. Occlusion of the
released &om the microspheres. hepatic artery is coaiirmed by repeating an angiogram
through the GDA catheter. Utikiag this procedure. one-half
Fxample 5 of the rats receive U-200 pm particks of polymer alone.
and the 0th~ half receive 15-200 p particks of the
AMIysis of surgical Paste Coateiaing Aati- 30 polymer-anti-angiogenic factor composition. The upper
Angiogenic Compositions GDAligatureistightenedtooccludctht GDAas thecatheter
Fisk rats weighing approximately 300 grams arc is withdrawn to ensure hemostasis. aad the hepatic artery
anestbelimi, aad a 1 cm transverse upper abdominal iaci- (although cmbdized) is left intact. The atxlomen is closed
Sian is made.Tw*tenths of a milliliter of saline coataiaiag with 6.0 absorbable suture and surgical clips.
lx 106 live 9 L gliosarcoma cells (eluted immediately prior Tbc rats are subsequently sacrificed at 2.7.14.21 and 84
to use from tissue culture) ere injected into 2 of the 5 hepatic days post-emboliration in order to determine efscacy of the
Iobts by piercing a 27 gauge needte 1 cm through the liver aati-angiogenic factor. Briefly. genemf anesthetic is given,
capsule. The abdominal wound is closai with 6.0 nsorptible aad utihxiag aseptic precantions. a midline incision per-
suture end skin clips aad the GA tumiaated. formed- Tbe GDA is mobilized again. and after placing a
After 2 we&s, the tumor deposits will measure approxi- ligature near the junction of the GDA and the hepatic attuy
matciylemAtthistime.bothhepatichuwrsarereseckd (i.e.. well above the site of the previous cutdown). a 0.038-
and the bare matgin of the liver is packed with a hemostatic inch polyethylene tubing is inserted via cutdown of the
agent. The rats are divided into two grcnrps:half is admia- vessel aad augiography is perfamed The rat is thea eutha-
istued polymeric canier alone. aad the other haif receives nixed by inject@ Euthanyi iuto the dorsal vein of the tail.
an anti-angiogeuic composition. Once euthanasia is confirm&, the liver is removed en bloc
aIoag with the stomach. spleen md both lungs.
Rats KC sacrificed 2.7.14.21 aad 84 days post hepatic
resection. la particular. the rats are cuthanized by i@ecting Histologic analysis is performed on a prepam slide
IWhanyl into the dorsal vein of the t&L The Iha, splcea. stained with bematoxylia and eosia (“H aad E”) staia.
and both lungs are removed. and histologic analysis is Briefly, the luags am sectioned nt 1 cm intervals to assess
performediaordert.ostudythetumcesforevideaceof passage of embolic materhI through the hepatic veins and
anti-angiogenic activity. into the right side of ckufatioa. The stomach and spleen arc
also sectioned in crdex to assessiaadvc&at immobiioa
Ejrample 6 fromrellux of particles into the C&X accessof the collaterel
circulatloR
Embolia3tion of F&t Artf!ries
Fii rats weighing approximately 300 grams are ants- Example 7
thetized Utilixiag aseptic proadures, a 1 cm transverse lkaasplaatatioa of Bitiery Steats ia Bats
abdomdl iacision is made. aad the liver identified
!iC&tths of a milEEta of saline containing 1 million live 60 General anesthetic is administ~ed to 300 gram Fkha
9 L gliosarcome cells (ehlted imme&mly prior fromtissue rats. A 1 cm traasvcrse iacision is thea made ia the upper
culture) is injected into each of the 5 hcpatic lobes by abdomen, aad the liver ideatificd In the most supafwial
piacing a 27 gauge needle 1 cm throogh the liver capsalt. lobe,01mlafsalineoon~gl~oa~sof9L
One-tenth of a millilk of acamel saliae is injected iato the glloarcoma c&s (elutcd thm tissue culture immedkkiy
needle as it is withdrawn to ensure that tbKC is DO spillage 65 priortouse)isinjccltdviaangaugc~etoadepmof
of celb into the pcrltoneal cavity. A pledger of g&ram is 1 cm iato the liver capsule. Hemostasis is achieved 8ftK
placed on each of the puncture sites to ensure hemostasis. removaI of the aeedle by placiag a pledger of gelfoam at the