CA1328634C - Method and compositions for solubilization and stabilization of polypeptides, especially proteins - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention provides a method for the solubilization and/or stabilization of polypeptides, especially proteins, by means of cyclodextrin selected from the group consisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl ant maltotriosyl derivatives of .beta.- and .gamma.-cyclodextrin. Solubilized ant/or stabilized compositions comprising a polypeptide, especially a protein, and the selected cyclodextrin are also described.

Description

- 132863~

METHOD AND COMPOSITIONS FOR SOLUBILIZATION
AND STABILIZATION OF POLYPEmDES, ~-ESPECL~LLY PROTEINS ~ ~

The present invention relates to a methot for the solubi1ization - ;
5 and/or stabilization of polypeptites, especially proteins, by means of selectet cyclotextrin derivatives. Solubilizet ant/or stabilizet compositions comprising a polypeptide, especially a protein, and selectet cyclodextrin derivatives are also described. ~ -Cyclodextrins are cyclic oligosaccharides. Tl}e most common cyclodextrins are ~-cyclodextrin, which is composed of a ring of six :
glucose residues; p-cyclodextrin, which is composed of a ring of seven glucose residues; and ~-cyclodextrin, which is composed of a ring of eight glucose units. The insite cavity of a qclode~ctrin is lipophilic, while the outside of the cyclodextrin is hydrophilic;` this combination of ~ ~ ;
15 properties has led to widespread study of the natural cyclodextrins, panicularly in connection with pharmaceuticals, and many inclusion complexes have been reported. p-Cyclotextrin has been of special interest becauge of ib cavity size, but it~ relatively low aqueous solubili1~ (about 1.8% w/v at 25C) ant aUendant nephrotoxicity have 20 ~ ~ limitet its use inthe pharmaceutical fielt. ~;
-- Attanpts to modify the properties of the natural cyclodextrins have resulted in the development of heptakis (2,6-di-0-methylj~-gclodextrin, heptakis (2,3,6-tri~methyl)-p-cyclodextrin, hydroxypropyl-p-gclotextrin, p-cyclodextrin-epichlorohydrin polymer and others. For 2~ ~ - a comprehensive~review of qclodextrins and their use in - ~ ~ pharmaceutical research, see Pitha et al, in Controlled Drug Delive~, ~ ;
et. S.D. Bruck, Vol. I, CRC Press, Boca Raton, Florida, pp. 125-148 :; ~ '' '' '-.
~ ''.'' :,-' 13~8~3~

(1983). For an even more recent overview, see Uekama et al, in CRC
Critical Reviews in Therapeutic~rug Carrier Sys~ems, Vol. 3 (1), 1-40 (1987); Uekama, in Topjçs in Pharmaceutical Sciences i987. eds. D.D.
Breimer ant P. Speiser, Elsevier Science Publishers B.V. (Biomedical S Division), 181-194 (1987); ant Pagington, Chemistry in Britain, pp. 455-458 (May 1987).
Inclusion complexes of ~-, p- or 7-cyclodextrin or their mixtures with ~ . . .
a variety of drugs have been describet by numerous parties ant various .:
advantages have been attributed to the complexes. These descriptions .::
include the following: ;

.
. .

. - .

-~ :' 1328~34 ~ :
. .. ~, .. .

-3- ~:
., :.--,-U5 ACll'tE
INV~rOR PAlENP NO INGREDIENI USE ADVANIAGE
' ' "'' Notb el al 4 C24 223 menthol htor anliphb~tic redut#d unplecunl methyl ~4euc cdor inaec od ~aliq1ale wel packiny effecl .
-,: , ,:i Szejlli el d 42211160 indomelh cin ~ti-inlbm- reduced uker live m to y, p~ efrect tecli~ durin~
pre~nrnq llay -hi et al 4 232 009 ~halo PGI~ hypotenrive incre sed A bil;ty analq~ utenne con-tr ction ctimubtiny blood pbteht .-~re~tion inhibiliny , ~ .
M~t umoto et al 4 351846 3 hydro~y- nd utenne contr c- ir~ed ct bility 3~ tioo timuhtin~
p~ndin :
,.,: :.
: . ., . ,:, ~m h~ d 1 4352 79~ ber~ychne nticonvul--nt incre#ed ~bility fum~te n~odihtive ~I trony cc;d pH
racter ~utric emptyin~ bleher bbod concentntion-tac irrll~lion impm~d hemolytic ~ctivily . .: :~' .

llp~ri 43~3992 ~ ~1 improved w ter cor~ colubility ina ed ~d~F~ the~peutic ~pon~e : ~bdic jo eye ,.: -~ ~ ~ e b~

~, ,: ~ : .
. .

4,407j'195 p_ nli lh~ enbanced , . .
~inoben~c . ~ic b;o~v ihbility -: -: : bdcodium :.: .
,~- ::. :::
: ~
:: :-~: ;''`'' . ~

-:

~328~34 , .

US ACllVE
INV~I~R P~NrNO INGREDD~ USE ~DV~NrAGE
~.
, Tutlbl 4,424,209 3,4-diisooulyr- ardi c yh~N-13 (4 contr ctility i obuty~oxy- gent phenyl)-l-mothyl-n-propyll-p-phonothybmine Tutlle 4,425,336 3~dihydroxy- cardi c c pable d or l N-13-(4-hydroxy- conlracliliq adminislralion phenyl)-1- agenl mothyl-n-propyl~p- ' :' phenolhylamine ' " ' Wagu et al 4,438,106 EPA and DHA deodorized (f~lty cids) aora~e stabk Mawda et aP 4,47~,~11 2-(2 lluor~ nti- reduced eye biphonyb1)pro- inlbmm-tory irribtbn, ~ -pionk cid ophth lmk higberconcon-or - lt tr tbn~, no dde ~troct bighb ~olubk, bng ~biliq, oxcelbnt phrmKolodal . , ''. .
orr~
. . .
,, ~, ' .
Shinoda el al 4,478,995 cid dditbn nti-ulcor oxcolknt vvater ult of (2' ~olubiliiy, good benzyb~ycar- bsorption in diges- -bon~l)phenyl tive tnct pod t nii-uke; ctivity dinoJnethl,1cyclo-l~le .' ,'~: .
, . ,:
: . .-kbya h d al 4,479,944 PGI~ analog fortn tmenl ot dabilizalion g-inst ~clero-i~, decompo ilbn or th~mbo~

- ~:

.',. ~
,' ' -:

1328~3~
: .

INV~IOR P~'IENTNO. INGRI~D~ USE ~DV~TJ~CE i.' p ; ."~
, llay~h d 1 4,479,g66 6,9-meth-no-rOr hypertenrion, incre~td r~bility PGI~ ~ cer br l throm-ou~ie ~nd the aht . , ., ' :, :
, ~lu~ el ~1 4,4g7,tO3 bnhcidinuttibidic rOr enh~nced vntler group cn~ibiotk rwinttdy ontery olubilily~nd rl~bilily, incre~ ed r te nnd ~mount of ~b orption ` - `
~:. :.'. '.', Mawdn ~1,499,0~5 pro tnghndintre ting noxi lulog of br in cells , :-.-: .

S~jth et ~1 4518,588 phendiline, ie eoron rydihtor improved w~ter olu-N(l-ohenyi c lcium bility, ccekr led ethl,1)-3,3- nbpni tnd incre- ed ~
diphonylpro- vivo ror~tion : ;
pp nuno or h h dir, olution ~I pH/
hydroehbride tempor~turt d pdrk ck - Sujtli d-nl ~ 4,52~,068 piperonyl ~ oUib hndiod te~ido patkidbi offoctc~t-llino olidi di~ vn in octlim~ro~dvrntor olu-ci~ u~d fun~bihty, inc~uod rido~ ~orptioo h veloaq of pntetrntion :' - --rembru~ ~-~oDe~ 4S55504 ~ r~li c c rdi c eltect high ;ueoue eolu- ~ -bbeatt';r~
- bla'y Uek~ma d i 4S6s,8~ pi pr~ren nthinfhm- improved rtability m tory, to oldbtion, ~Ige~ic, rreodom frombitter . . -ntipyretk tute, lee~ irrib-tine :- : ~ ~ ` . .. ..
. ~.
, , ~ ~ , .:.-:~ -, : . -132~3~
-U5 ACmE
INV~IOR r~rr No. INCREDIENI USE ~DV~ACE

Ued~ et ~1 4,57S,548 2-nilro ymelhyl- rorv scuhr non-vobtibpo~vder o-chb~pyridine di orderr vc vobtive oil ' Ohw l~i et ~1~ 4,598,070 trip mide nnli hypet- improved r,olub;liv tend~

' ''"~'';' Chiéd el ~1 4,6û3,123 piro~ m, ie nli-inll~m-hydro~y-2- m~tory, ~Igesic rnetbyl-N-2-pyridgl-2H-1,2-benzothi zine-3-cubo~mide-l,1- .
dioride Hue~nn et ~1 4i60e,366 mobenzo~minej ntiemeb'c, rtor~go ct bilily, ,.
io 1-12-(4- ~nlirpumodk betl b o ption melh~yben7,hy- Ihroudl di~tive 413-(411uoro- tr ct -bereoS~)propyll-piperaine Hir i d rl~ 4,669,696 polypeplido improving drug ~I rpldor~ by non- ~ -injection routes '.
: ' ;. '' Szejlli d ~1 4,623,641 PCI~ rnethyl ~nti-ulcer improqed rtorage ~ ~

:: . ,.
Ninpret 1 4,663,316 u~lur td nnlibiotic, enhulced A~bilily pho phon~ nlihngnl, ~inrt o~lion eont~inin~ ntitumor rntibioti, - includiny ~ ~ - pho photnenin -~

. . . ; .:: .

. : :
~: : ' .~:.
~; ~

132~3~ ~:
:
.

-7- .:

INV~I~R PAlENP NO. ~GREDIENI' USiE ADVANI'~GE

Fukazaw~ et ~1 4,675,395 hinokitiol b~e~ericiW, improvod m~ersolu- ~ -b~cterioet~tiebiiity, k-- odor ; ' Shimiw el ~1 4,728,509 2- mino 7- nli- lier~ic, impro~ nler olu-i opropyl-5- ~nli- bilily to iner~o o~o-SH-[II- inll mm lorY concenlr lhn to benzopyr no- the peulic levels 12,3-b]pyridine- in n~al dro,os snd 3 earboylic rcid eye drops ;~
Shihnd 4,728,510 milk b~lh impro red stabilily l al~ componenl prep~lion '~' `':
l~ad e~ sl 4,751,09!i ~ rl~me dipeplide sl-bilizadon rrom -swee~ener hydrolysis :. ;,-~ .
. ~
Tullb eko de~ribeJ u of 2,6-di~melh~1-p-eyclodlrin nd 2,3,6-lri~melhyl p- . ~ -cyehderlrin to form the inelwion eomple~ -~This may not be n inelu ion compk~, but dmply ~ phy ic l mi~lure ~ -~thir~ mutur~ uld/or n inelwir,~ nnpouDd, ~o imentore ~bo rnention prbr ho~n ~olubillly imprrN~ment- d eycbde~trin inelwionr ot b rbilurie eid derivnlinr, meterAmie eid, ind~lh cin nd ehlor mphenieol lThe imenton rerer to Ihir n ocelu ion~ compound, ~ -'rhe inventots nbo mention deriv ti ~e or ryebd~trin ~ eyclodtrir~ . ;
eontuniny d reh _ion produet for use in formin~ tbe ehtbr lc , . .
.:,.:

.

.

: .

~ ':

Among the patents listed above, only Hirai et al U.S. Patent No.
4,659,696 appears to relate to proteins or polypeptides. It would appear from the claims of the Hirai et al patent, however, that an inclusion complex is not formed ant that what the invention provides is a pharmaceutical composition consisting essentially of a physical -mixture of a hydrophilic, physiologically active polypeptide and cyclodextrin, sait composition being a uniform mixture in dosage form.
The cyclode~trin may be ~-"~- or ~1-cyclotextrin, or a cyclotextrin derivative, exemplified by tri-O-methylcyclotextrin and triaminocyclodextrin, ~-cyclodextrin being particularly preferred. The physiologically active peptide may be thyrotropin releasing hormone, luteinizing hormone-releasing hormone, insulin, somatostatin, growth hormone, prolactin, adrenoconicotrophic hormone, melanocyte stimulating hormone, or many others. The composition is designed for nasal, vaginal or rectal preparation, i.e. a non-oral or non-injectable route of administration is used. :-Matsuyama et al, Dru~ Development a~dlndustrial Pharmacy, -13(15), 2687-2691 (1987), have reported on the thermodynamics of binding aromatic amino acids to ~-"~- and 1-cyclodextrin. Human serum albumin, which contains aromatic amino acids, was also tested as ;
a protein model. The authors suggest that cyclodextrins may protect peptides from enzymatic hydrolysis. Also, according to Sekisui Chemical Co., Ltd.'s Japanese Kokai Tokkyo Koho JP 59/104556 - -(84/104556), pub1ishet June 16, 1984, cyclized oligosaccharides such as p-cyclodextrin have been recently used in protein stabilizers which prevent denaturation of proteins and enzymes in blood. Human blood mixed with octylphenoxypoly(ethoxyethanol) and ~-cyclodextrin was maintained for 24 hours with little change.
Inclusion complexes of 2,6-di-O-methyl~-cyclodextrin with dibenzolbd]pyran derivatives and salts having analgesic, antiemetic and narcosis-potentiating activities have been described in Nogradi et al ; - i .: ..
.
- -.

1328~3~

U.S. Patent No. 4,599,327; incre ed water solubility and thus improved biological activity have been claimed for the complexes. A review of the pharmaceutical applications of such methylated cyclodextrins has been published by Uekama, Pharm. Int., March 1985, 61-65; see also ~:
S Pitha, Journal of ~Qç~jon Pheno~, 477485 (1984).
Cyclodextrin polymer has been reported by Fenyvesi et al, Chem.
pharm~ L ~ (2), 665-669 (1984) to improve the tissolution of furosemite. Improvements in the dissolution ant absorption of phenytoin using a water-soluble ~-cyclotextrin epichlorohydrin polymer have been described by Uekama et al, Int~national Journal of :
Pharma~çutics, ~, 3542 (1985). . -Hytroxypropyl~-cyclodextrin (HPBCD or HPCD) and its preparation by propylene oxide addition to p-cyclodextrin were described in Gramera et al United States Patent No. 3,459,731 nearly 20 years ago. Gramera et al also described the analogous preparation of hydroxyethyl~-cyclodextrin by ethylene oxide reaction with p-gclodextrin. Much more recently, Pitha and co-workers have described the improvet preparation of this gclotextrin terivative and its effects on the tissolution of various drug molecules. Pitha United States Patent No. 4,596,795, tatet June 24, 1986, tescribes inclusion complexes of sex hormones, particularly testosterone, progestersne, ant ~ -estratiol, with specific gclotextrins, preferably hydroxypropyl~-cyclotextrin ant poly7~-cyclodextrin. The complexes enable the sex horrnsnes to be successfully telivered ts the systemic circulatisn via the sublingual or buccal rsute; the effectiveness of this telivery is believed ~ -~- ts be due to ~the high tissolution power of hydrophilic terivatives sf c~clodextrins, the non-aggregated structure of their complexes with steroids, and their low toxicity ant irritancy of mouth tissue~. Success with other cyclodextrins, including poly~-cyclodextrin and - 30 hytrsxypropyl7-cyclsdextrin, have also been noted in the Pitha patent.
~ ~ See also Pitha et al, J. Pharm. Sci., Vsl. 74, No. 9, 987-990 (September : ,,' ' ' ~- :' ', ':.:
~ -. ;. , , . , . , ; ~ ~ . .. .. ~ ... .. .. . .... . .. :, ,.. .;. .. .

132863~

-10- ' 1985), concerning the same and related studies. Pitha et al also describe in the J. Pharm. ~ci. article the storage stability of tablets containing a testosterone-hydroxypropyl~-cyclodextrin complex and the lack of toxicity of the cyclodextrin itself, as well as the importance of S the amorphous nature of the cyclodextrin derivatives and their complexes with drugs in improving dissolution properties.
The improvet, optimized preparation and purification of hydroxypropyl~-cyclodextrin have been recently descr;bed by Pitha et al, International Journal of Pharrnaceutics 29, 73-82 (1986). In the same puUication, the authors have described increased water solubility , for 32 drugs in concentrated (40 to 50%) aqueous solutions of hydroxypropyl~-cyclodextrin; improved solubilization of acetaminophen, apomorphine, butylated hydroxytoluene, chlorthalidone, cholecalciferol, dexamethasone, dicumarol, digoxin, diphenylhydantoin, estradiol, estriol, ethinylestradio1-3-methyl ether, ethisterone, furosemide, hydroflumethiazide, indomethacin, iproniazid phosphate, 17-methyltestosterone, nitroglycerin, norethindrone, ouabain, oxprenolol, progesterone, retinal, retinoic acid (all trans and salt forms), retinol, spironolactone, sulpiride, testosterone ant theophylline was noted. The authors inticatet this to be sn extension of their earlier work with hytroxypropyl~-cyclotextrin, which was previously fount effective for oral atministration of the sex hormones to humans. Their later work reported in Pitha et al, International JQurnal Q Pharmaceutics 29, 73- - -;
82 (1986), has also been very recently tescribet in Pitha United States Patent NQ 4,727,064, tated February 23, 1988. That patent claims a composition containing an amorphous complex of cyclodextrin and a drug, and a method of producing a stabilizing amorphous complex of a drug and a mixture of cyclodextrins comprising (1) dissolving an intrinsically amorphous mixture of cyclodextrin derivatives which are water soluble and capable of forming inclusion complexes with drugs in ~ :
water; ant (2) solubilizing lipophilic drugs into aqueous media to form ~ ~;

~ -. , .. . .. ,; ;.. . ,, . . .,, , . .,.. , , ,.. , .. . . .. .. , . . ~ . :

132863~ ;

a solution and form a solubilized drug/cyclodextrin complex. Work with numerous drugs has been reported in the patent, but none involves ~
proteins or other peptides. The patent describes the preparation of ~`
various substituted amorphous cyclodextrins, including hydroxypropyl~
5 cyclodextrin and hydroxypropyl~-cyclodextrin, the latter by analogous condensation of propylene oxide with 7-cyclodextrin.
Uekama et al, CRC Critical Reviews in Ther~apeutic Dru~ Carrier Svstems, Vol. 3 (1), pp. 1-40 (1987), have described the characteristics of various cyclodextrins, including hydroxypropyl~-cyclodextrin. The 10 authors have presented data showing improved solubilization in water in the presence of 15 mg/mL of HPBCD for the drugs carmofur, diazepam, digitoxin, digoxin, flurbiprofen, indomethacin, isosorbide dinitrate, phenytoin, prednisolone, progesterone and testosterone. In a discussion of the metabolism and toxicity of cyclodextrins, Uekama et al -have inticated that cyclodextrins at low-moderate concentrations (usually 3-8% w/v and higher) cause hemolysis, and that the methylated cyclodextrins have higher hemolytic activity than the natural cyclode~trins. Hydroxypropyl~-cyclodextrin is said to cause hemolysis beginning at 4.5 mM. The authors have further indicated that parenteral administration of large toses of cyclodextrins shoult be avoitet, but 1hat ~-cyclotextrin ant hytroxypropyl~-cyclotextrin seem to be useful in trug solubilization for injections ant liquit preparations uset for mucous membranes." Use with~polypeptides such as pro1eins is not mentionet -JANSSEN PHARMACEUTICA N.V's International Patent Application No PCT/EP84/00417, published under International PuWication No WO85102767 on July 4, 198~, has described pharmaceutical compositions comprising inclusion compounds of drugs, which are unstable or only sparingly soluble in water, with partially etherifiedp~yclodextrin derivatives having hydroxyalkyl and optionally - ~
additional alkyl groups Among the cyclodextrin derivatives ~ ~ ;

:

.
.
- ~

1 3 ~ 8 ~ ~4 contemplated are hydroxypropyl~-cyclodextrin and hydroxyethyl~-cyclodextrin, while the drugs include non-steroidal anti-rheumatic -agents, steroids, cardiac glycosides and derivatives of benzodiazepine, ~
benzimidazole, piperidine, piperazine, imidazole and triazole. Preferred ~ -5 drugs include etomidate, ketoconazole, tubulazole, itraconazole, levocabastine and flunarizine. The pharmaceutical compositions of the invention include oral, parenteral and topical formulations, with 4 to 10% solutions of cyclodextrin terivatives being utilized to solubilize various drugs. Improved solubilities of indomethacin, digitoxin, `
progesterone, dexamethasone, hydrocortisone and diazepam using 10%
HPBCD are shown, and an injectable formulation of diazepam in 7% ;`~
HPBCD is specifically described. The relatively low cyclodextrin -concentrations used reflect a desire to avoid or minimize the hemolytic effects observed at higher cyclodextrin concentrations. -~
Carpenter et al, The Joumal of Pediatrics, 111, 507-512 (October 1987) tescribe intrawnous infusion of 2-hytroxypropyl~-cyclodextrin, prepared as a 5% solution in water, to treat sewre hypervitaminosis A.
It was found that, during infusion, circulating retinyl esters increased transiently, while total vitamin A excreted in the urine was enhanced after infusion. Thu9, intravenous infusion of 5% HPBCD was found to docreaso ~ vivo levels of the vitamin, presumably by complexing with the vitamin and removing some of the excess from the body. ;
Formulation of a particular dihydropyridine ~ pyridinium salt redox system for brain-specific delivery of estratiol "in a water-soluble hydroxy-cyclodextrin~ is reportet by Bodor and co-workers in Estes et al, "IJse of a Chemical Redox Syst-m for Brain Enhanced Delivery of ;
E~strati:ol Decreases Prostate Weight,~ in 8iolo3~cal Approaches to the -Controlled Delivery of Drugs, ed. R. L Juliano, Annals of the New - -York Academy of Sciences, Volumè ~07, 1987, 334-336.
JANSSEN PHARMACEUl~CA N.V.'s European Patent Application No. 86200334.0, published under EPO Publication No.
, -: ~ '., .:.''' '' ''-'., , 1328634 ::-: . .

0197571 on October 15, 1986, describes 7-cyclodextrin derivatives which are 7-cyclodextrin substituted with C,-C" alkyl, hydroxy C,-Ca alkyl, carboxy C,-C~ alkyl or C,-C~ alkyloxycarbonyl C,-C~, alkyl or mixed ethers thereo Among the specific derivatives named are hydroxypropyl7-cyclodextrin ant hydroxyethyl7-cyclotextrin.
Compositions comprising the cyclotextrin derivatives ant a drug are also dcscribed. See also corresponding ~iiller United States Patent No. 4,764,604, datet August 16, 1988.
The inclusion characteristics of yet other derivatized cyclodextrins have also been described in the literature. Studies of branched cyclodextrins which are glucosyl and maltosyl derivatives of ~ - and 7- ~ ~ ~
cydodextrin and their inclusion complexes with drugs have recently ~ ~ -been reported. Uekama, in~ic~n Pharmaceutical Sciences 198?, eds. D. D. Breimer and P. Speiser, Elsevier Science Publishers B. V.
(Biomedical Division), 181-194 (1987), has described the effects on bio-pharmaceutical properties of maltosyl and glucosyl cyclodextrin derivatives, including enhanced drug absorption. In that publication, Uekama again indicated that the natural cyclodextrins caused hemolysis in the order of 7 ~ . In the case of chemically modified cydodextrins, the order changed to hytroxyethyl~ ~ maltosyl~ c hytroxypropyl~ ~ p ~ methyl~. ~
Koizumi et al, ~;hem~har~, 35 (8), 3413-3418 (1987), have ; - -reported on inclusion complexes of poorly water-soluble drugs witb glucosyl gclodextrins, namely 6-O~-D-glucosyl~-CD (G, ~-CD), 6-O-~-D-glucosyl~-CD (G,~-CD) and 6A, 6D-di-O~-D-glucosyl~-CD (2G,--CD).
Oka~da et al, Chem. Pha~. Bull., 36 (6), 2176-2185 (1988), have -reported on the inclusion complexes of poorly water-soluble drugs with maltosyl cydodextrins, namely 6-O~-malto~l~-CD (G2~-CD), 6-O~-maltosyl~-CD (G2~-CD), 6-O~-maltosyl7-CD (G27-CD), 6-O~-maltotriosyl~-CD (G~-CD), 6-O~-maltotriosyl~-CD (G3~-CD) and :
, .
:~ ' . ' '' .. ..

. ~
132~634 :~

6-O~-maltotriosyl7-CD (G37-CD).
Yamamoto et al, in Internationa~ Lournal of Pharmaceutics 49, 163-171 (1989), have described physicochemical properties of branched ~-cyclodextrins such as glucosyl~-cyclodextrin, maltosyl~-cyclodextrin and di-maltosyl~-cyclodextrin, and their inclusion characteristics. Those authors report that the branched,~-cyclodextrins are better solubilizers for poorly water-soluble drugs and have less hemolytic activity than,s-cyclodextrin itself, and they suggest that glucosyl~-cyclodextrin and maltosyl~-cyclodextrin may be especially useful in parenteral preparations. -The patent literature reflects much recent work on the branched cyclodextrins carried out by Japanese workers, as discussed below.
Japanese Kokai 63-135402 IOKUYAMA SODA KK), published June 7, 1988, describes compositions consisting of maltosyl~-cyclodextrin and at least one of digitoxin, nifedipine, flulubiprophene, isosorbide nitrate, phenytoin, progesterone or testosterone. The compositions have improved water solubility and reduced erythrocyte -destruction, are safe for humans and can be used as injections, eye drops, syrups, and for topical and mucous membrane application.
Japanese Kokai 62-281855 (DAIKIN KOGYO KK), published ;~
December 7, 1987, describes stable, water-soluble inclusion compounds of maltosyl~-cyclodextrin with a variety of vitamins and hormones, e.g.
steroid hormones such as prednisolone, hydrocortisone and estriol. -~
These lipophilic vitamins and hormones can thus be used as aqueous -~
solutions.
- - Japanese Kokai 63-036793 (NIKKEN CHEM KK), published -February 17, 1988, tescribes the preparation of dimaltosyl~
cyclodextrin and its general use in medicines.
Japanese Kokai 62-106901 (NIKKEN CHEM KK), published May -18, 1987, describes the preparation of diglucosyl~s-cyclodextrin and its general use for pharmaceuticals.
. ;. .
. ~..."
,~ - , ,, , ' , .. .
. :~
-: . . - -' :. : . . , . - . . . : : ' 1328~3~ `

Japanese Kokai 6~-236802 (NIKKEN CHEM KK), published October 22, 1986, describes the preparation of maltosyl7-cyclodextrin and its general use with drugs.
Japanese Kokai 61-197602 (NIKXEN CHEM KK), published 5 September 1, 1986, describes the preparation of maltosyl~-cyclodextrin and its expected use in medicines. ~ -Japanese Kokai 61-070996 (NIKKEN CHEM KK), published April 11, 1986, describes the preparation of maltosyl~-cyclodextrin and its general use in pharmaceuticals.
Japanese Kokai 63-027440 (SANRAKU OCEAN), published February 5, 1988, describes compositions containing a water-insoluble or slightly soluble drug together with glucosylated branched `
cyclodextrin. Among the drugs mentioned are steroid hormones. ~-Japanese Kokai 62-164701 (SHOKUHIN SANGYO BIO), lS published July 21, 1987, describes 1he preparation of diglucosyl~-cyclodextrin and its general use in medicine.
Japanese Kokai 62-003795 (TOKUYAMA SODA KKj, published -January 9, 1987, describes production of glucose and ; ~
maltoligosaccharite (W glucose units) derivatives of ~-"9- and 7- i -20 ~ cyclotextrin and their use a9 9tabilizer9 for pharmaceuticals.
In recent years, there has been a tremendous increase in the number of products and potential products containing polypeptides, ~
especialb proteins, intended for therapeutic or diagnostic or analytical ~ ~ -we. Typically, the proteins or other peptides are formulated in -25 aqueow solution for injection or for use in diagnostic or analytical applications. Unfortunately, proteins often suffer from solubility and/or stabilib problems. Some proteins, for example, are not water-soluble.
~- ~ Others are soluble in water but still suffer from stability problems caused by protein degradation/denaturation, dimerization andior poly-30 ~ meri~ation, and the like, ar~y of which may lead to inactivation. This : - :' . ~-. -132863~

seriously limits shelf-life and often imposes low-temperature storage requirements and restrictions on mechanical movement.
There are numerous causes of protein/peptide instability or degradation, induding covalent bond reactions, such as hydrolysis, and the tenaturation process. In the case of denaturation, the conformation ;
or three-dimensional structure of the protein is disturbet and the protein unfolds from its usual globular structure. Rather than refolding to its natural conformation, hydrophobic interaction may cause ~:
clumping of molecules together, or aggregation, or it may cwse ~ ;
refolding to an unnatural conformation. Either of these end results entails diminution or loss of biological activity. For a summary of the cawes of protein instability, see Wang et al, Journal of Parenteral Science and Technoloev. Technical Report No. 10, "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", Supplement Volume 42, Number 25, 1988, pp. S3-S26.
- The Wang et al publication referenced above also summarizes the art's use of various excipients to stabilize parenteral formulations of proteins and other peptides, including use of serum albumin, amino acids, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinylpyrrolidone, hydrolyzed gelatin ant ammonium sulfate, including agents used as cryopratectants, which prevent or lessen denaturation under freezing conditions or Iyophilization, such as carbohydrates, alcohols, polyvinylpyrrolidone and glutamic acid. Examples of excipients proposed by the art for stabilizing specific proteins are described in Hayashi ef al United States Patent No. 4,457,916, issued July 3, 1984, KYOWA HAK~CO KOGYO CO, LTD.'s European Patent Publication :
; No. 0123291 and E. L DUPONT DE NEMOURS AND COMPANY'S - -. ~ . .
European Patent Publication No. 0231132.
30~ ~ The Hayashi et al patent describes a method for stabilizing Tumor Necrosis Factor (IN~;) by adding a stabilizing agent selected from a , .. - ;, .

: .. .. .
.

1328~3~ ~

nonionic surfactant, at least one substance selected from D-glucose, D-galactose, D-xylose, D-glucuronic acid, a salt of D-glucuronic acid, trehalose, a dextran and a hydroxyethyl starch and mixtures thereof.
The resultant a~,ueous solution or powder containing TNF is said to be 5 capable of prolonged storage without loss of activity and stable on freezing, thawing, Iyophilization, heat-treatment or the like.
European Patent Publication No. 0123291, publishet October 31, 1984, tescribes a methot for stabilizing interferon imolving adtition of an alkali metal salt or magnesium chloride to interferon, optionally 10 including addition of serum albumin and freeze-drying to further enhance stability. The patent publication also describes a method for , stabilizing interferon by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulfuric acid, starch, ~ -glycogen, insulin, textrin and alginic acid salt.
European Patent Publication No. 0231132, published August 5, 1987, relates to a recombinant interleukin-2 (1I~2) composition ant process for making it. In discussing the prior art, the European publication refers to earlier European Patent Publication No. 0133767 as disclosing that gamma-interferon, obtainable from human leukocytes, 20 can be stabilized by adti1ion of albumin ant/or a sugar. The sugars can inclute monosaccharides, tisaccharides, sugar alcohols and mixtures thereof, e.g~ glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol and xylitol. Also, European Patent Publication No. -0231132 acknowletges that protein (chymotlypsinogen and 25 ribonuclease) stabiliza9On using glycerol is reported by Gekko et al, Biochen~j~y j~Q, 46774686 (1981). European No. 0231132 itself relates ` ~
to recombinant human IL,2 compositions comprising water, ~ ~-recombinant interleukin-2 and preferably a polyol. Suitable polyols are -said to be water-miscible or water-soluble, for example polyhydroxy ~: :
30 ~ alcohols, monosaccharides and disaccharides induting mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, glucose, ~ ` '' ' 1328634 ~
-18- ~ -fructose, arabinose, mannose, maltose and sucrose. The polyol is said to increase the activity of the IL,2.
Despite the foregoing, there remains a clear and pressing need in this art for a more effective and generally applicable method and 5 compositions for the solubi1ization and stabilization of polypeptides such as proteins. -.
The present imention provides a method and compositions for stabilizing and/or solubilizing polypeptides, especially proteins, by means of cyclodextrin selected from the group consisting of -10 hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl - ` ~ -derivatives of ~- and 7-cyclodextrin. Thus, in one aspect, the invention ~:
provides a method for solubilizing and/or stabilizing a polypeptide, said -method comprising combining said polypeptide with an effective solubilizing and/or stabiiizing amount of cyclodextrin selected from the IS group consisting of hydroxypropyl, hydroxyethyl, glucosyl, malfosyl and maltotriosyl derivativ of p- and ~-cyclodextrin. Preferably, an aqueous ^ --solution comprising said cyclodextrin is employed. In another aspect, ;
the present imention provides a composition, especially an aqueous composition, comprising a polypeptide ant an effectiw solubilizing 20 and/or stabilizing amount of cyclotextrin selectet from the group con-sisting of hydroxypropyl, hytroxyethyl, glucosyl, maltosyl and maltotdosyl terivatives of ~- and ~r-cyclodextrin. In still another aspect, the invention provides a Iyophilized composition comprising a -polypeptite and an effective stabilizing amount of cyclodextrin selected 25 from the group considing of hydro~ypropyl, hydroxyethyl, glucosyl, - `
maltosyl and maltotriosyl derivatives of ~- and ~1-cyclodextrin.
Preferably, the ~oregoing methots and compositions are directed taward -improvements in biologically useful protein formulations.
~ .' ''' `''' '';' ~.''' '' ; ' ''' ',". ' ' '' ' '' ' ' " ''" ' 1328~4 ; ~ ~
:
-19- . .
The objects and advantages of the present invention will be apparent from the following detailed description and accompanying drawings, in which:
FIG. 1 is a plot of fluorimeter units as an intication of initial light S scattering for samples of interleukin-2 (II~2) in increasing - -concentrations (percent w/v) of hydro~ypropyl~9-cyclodextrin (HPBCD);
FIG. 2 is a plot of fluorimeter units as an intication of initial light scattering for samples of HPBCD formulations of 11,2 (1 mg/mL) with sucrose bulking;
FIG. 3 is a bar graph depicting the results of an ultracentrifugation assay of samples of 1L,2 formulated with HPBCD as compared to unformulated 1L-2; and - . -FIG. 4 is a semi-logarithmic plot depicting the biological activity of - samples of 1L,2 containing increasing concentrations of HPBCD.

The polypeptides intented for use according to the present invention are any polypeptides which a:re biologically or industrially useful. A Wologically useful polypeptide in accord with this invention is any po1ypeptide which can be employed in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of 20 ~ desirable physical or mental tevelopment and conditions in man or animals. Polypeptides, especially proteins, for use in human and/or ~ -- veterinary medicinc or in diagnosis (~ vivo or in vitro) are of particular interest. Industrially useful polypeptides are those employed analytically, in production or which are otherwise useful in chemical ;:
industry.
Amino acids are organic compounds that have at least one amino NH2) group ant 0 least one carboxyl (-COOH) group. Oligopeptides àre amino acid oligomers which are formed by reacting amino and --- carboxyl groups from different amino acids to release a water molecule ~ -:: ~

1328~3~ ~

and form a peptide linkage. Polypeptides are amino acid polymers which are formed in the same way. Generally, oligopeptides have between 2 and 20 peptide linkages whereas polypeptides have more.
Proteins are very large polypeptides. The amino acids which make up S the polypeptides of the present invention may be natural or synthetic, ;-may have the amino group attached to any noncarbonyl carbon atom (e.g., to the~"~,7,~ or~ carbon as measured from the~-COOH group ~ ~
of the amino acit), may exist as enantiomers (e.g. D- or L-) or - ;-diasteriomers, may have blocked carboxyl or amino groups and may :I0 have side chains which are hydrophobic, hydrophilic, acidic, basic or ::
neutral. The synthetic polypeptides may contain reversed peptide Iinkages, in which case they must contain at least one diamine and one dicarboxylic acid monomer. ^
The polypeptides intended for use in the methods and compositions IS of this invention include molecules to which nonpeptide prosthetic groups, such as carbohytrates, hemes and fatty acids, have been attached. The polypeptides include molecules made by living organisms or cells, molecul made by synthetic organic chemistry and molecules which are synthe1ically modified biological products. They may have an amino acit sequence identical to that of a natural substance or one .
altered by techniques such as site-directed mutagenesis In addition to the covalent (primary) structure, the polypeptides may possess unique conformations (combinations of secondary, tertiary and quaternary structure) which affect their biological function, aqueous solubility and ability to interact with the cyclodextrins The polypeptides may haw many biological functions. They may act as enymes; enzyme inhibitors; antibodies; antigens; transporters of --electrons, oxygen, metal ions, or small organic molecules; ionophores;
- antibiotics; mitogens; hormones; growth regulators; neurotransmitters; ;
30 cell surface recognition proteins; cell chemotactic factors; and cytotoxins Ihey may also be receptors, agonists, antagonists of the ' '' '.
' 1328634 :

following: ionophores, antibiotics, mitogens, hormones, neurotransmitters, growth regulators, cell surface recognition proteins, cell chemotactic factors and cytotoxins.
Uses for some of the preferred polypeptites of the invention -5 include: immunization (as vaccine adjuvants), ~ vitro diagnostics (to increase the solubility/stability or lower the nonspecific binding of antigens or antibodies) and ~ vivo diagnostics and therapeutics (to increase the solubility/stability of therapeutic and diagnostic polypeptides). Preferred therapeutic targets of these polypeptides are 10 cancers, such as melanoma, renal cell carcinoma, myeloma, leukemia, breast cancer, colorectal cancer, Iymphoma, neuroblastoma, astrocytoma and glioma; auto-immune diseases, such as diabetes mellitus, rheumatoid arthritis and multiple sclerosis; immunodeficiency diseases; ;and infectious tiseases.
Among the polypeptides contemplated by the present invention are therapeutically useful polypeptides such as anti-sera, anti-toxins and -antigens. Anti-ser may include, for example, antirabies, antivenin (black widow spider venom), hepatitis B immune globulin, tetanus immune globulin, intravenous immune globulin, pertussis immune globulin ant rabia immune globulin. Anti-toxins may include, for -example, those for tiphtheria and tetanus; Rho(D) immune globulin;
serum components, such as 5% normal human serum albumin, 5%
plasma protein fraction, 20% normal human serum albumin, 25% -` -normal human serum albumin, factor n, factor Vl~, factor vm, factor IX, factor X and Xa, aritithrombin m, transferrin, haptoglobin, fibro-nectin, gamma globulins, protein C, protein S ant thrombin; toxoids, such as tiphtheria and tetanus; vaccines, including attenuated vaccines (such as those for cholera, influenza, meningitis, Yersinia pest;s or plague, pneumonia, poliomyelitis, rabies, typhoid and staphyloccocus) and live vaccines (such as those for poliomyelitis, measles, rubella and mumps); growth factoa, hormones and like bioactive peptides, as :.~''" .
-:

:~.,.. ~ ..

illustrated by ~-1-antitrypsin, atrial natriuretic factor (diuretic), calcitonin, calmodulin, choriogonadotropin (~ and p), colony stimulating factor, corticotropin releasing factor, ~-endorphin, endothelial cell -growth supplement, epitermal growth &ctor, erythropoietin, fibroblast 5 growth factor, fibronectin, follicle stimulating hormone, granulocy1e colony stimulating factor, growth hormone (somatotropin), growth hor none releasing factor (somatoliberin), insulin, insulin-like growth factor (somatometin), an interferon (typically ~"~, ~), an interleukin (typically 1, 2, 3, 4), lutropin, Iymphotoxin, macrophage derived growth :
factor, macrophage inhibiting factor, macrophage stimulating factor, ~ -;
megakaryocyte stimulating factor, nerve growth factor, pancreatic entorphin, parathyroid hormone, platelet derived growth factor, relaxin, secretin, skeletal growth factor, superoxide tismutase, thymic hormone factor, thymic factor, thymopoeitin, thyrotropin, tissue plasminogen 15 activator, transforming growth factor (~ and ,~), tumor necrosis factor, tumor angiogenisis factor, vasoactive intestinal polypeptite and wound angiogenesis factor; immunosuppressives, such as RhO (D) ISG and IVGG's; thrombolytics such as urokinase, streptokinase ant tissue plasminogen activdor; ant antigens such as Rhus all (poison ivy), Rhus 20 tox poison ivy-polyvalent ant staphage Iysate (staphyloccocus Iysate).
Other polypeptides contemplated by this invention are polypeptites specifically intended for veterinary use, including vaccines, animal growth factors ant bovine interferons ant interleukin-2. 111ustrative vaccines inc}ude: bovine vaccines, for example those for anthrax, 25 clostridium (mul1iple species), pasteurella, leptospira pomona, bovine diarrhea, brucillosis, parainfluenza, 3-respiratory syncytial virus, tetanus, vesicular stomatitis and staphylococcus; canine vaccines, for example those for bordetella, coronavirus, distemper, parvovirus, parainfluenza and rabies; equine vaccines, for exarnple those for anthrax, ;
30 encephalomyelitis, iniluenza, tetanus, rabies and streptococcus-strangles;

'~' .', ' 1 3 2 8 ~

feline vaccines, such as those for leukemia, pneumonitis-chlamydia and rabies; ovine vaccines, for example those for anthrax, blacl~leg, bluetongue, enterotoxemia, tetanus and vi~riosis; ant porcine vaccines, for example those for anthrax, enterotoxemia, dysenteq, erysipelas, - -5 leptospirosis, parvovirus, pseudorabies, tetanus and rotavirus.
Yet other polypeptides contemplated by this invention have use in ~
immunology. These include monoclonal antiboties, polyclonal ~ -antibodies (unconjugated), second antibodies (alkaline phosphatase conjugated), immunoglobulin screening and isotyping kits, protein A
products and immunoassay reagents. Among the monoclonal antibodies ~ -are those approved for use in diagnostic kits, for example IgE, peroxidase-anti-peroxidase conjugated, human chorionic gonadotropin, ~
T cell, ferritin conjugated, carcinogenic embryonic antigen (CEA), : -OKT-II, anti-rabies, human growth hormone, Total T4, prolactin, 125I- -IgE, UCG, thyroid stimulating hormone, chlamydia, gentamicin and rubella. Other useful monoclonal antibodies include monoclonal antibodies for human cell surface antigens, monoclonal antibodies for murine cell surface antigens, monoclonal antibodies to compiement and bloot proteins, monoclonal antibodies to immunoglobulins (human), monoclonal antibodies to neurological antigens, monoclonal antibodies to tumor markers, monoclonal antibodies to cell components, Epstein Barr virus antigens, human Iymphocyte antigen (HLA) typing, hematology antiboties, leucocyte antibodies, bacterial antigens, parasitic antigens, T-cell Iymphotropic virus (EIIV-m) and cyloskeletal antibodies. Useful polyclonal antibodies ~unconjugated) include affinity purified antibodies to immunoglobulins, antibodies to plant viruses, antisera to human isoenz~nnes and chromatographically purified antibodies. Useful alkaline phoThatase conjugated second antibodies include affinity purified antibodies to immunoglobulins, an~ibodies to -30 plant viruses, biotin-conjugated antibodies, fluore~cein-isothiocyanate- - -conjugated antibodies (~:ll'C), gold-conjugated antibodies, peroxidase--, :, ' ~ .

132863~

conjugated antibodies, rhodamine conjugated antibodies and iodine-conjugated antibodies. Polypeptide immunoassay reagents include EL~ :grade enzymes, enzyme-antibody complexes, reagents for immunology, enzyme linked immunosorbent æsays (ELISA) for use æ standards or 5 controls, immunoelectrophoresis (lEP) assays, radioimmunoassays (RLA) for use æ standards or controls, nephelometry for use as stantarts or controls, nuclear antigens and coatings for kit tubes and `plate wells.
Other polypeptides contemplated by the present invention ;nclude 10 polypeptides useful in cell biology/biochemistry, for example in serum-free cultures (æ supplements and reagents for cell culture), in - -glycoprotein and carbohydrate research (endoglycosidoses, exoglycositoses, enzymes for carbohydrate research, enzymes for analysis of glycoprotein oligosaccharides), æ molecular weight markers -15 (calibration proteins, e.g. for gel permeation chromatography, subunit proteins), proteases (for use in bloot research, protein sequencing, tissue tigestion and coll harvest, total digestion of proteins and immobilized proteases), cell surface recognition proteins (adenosine and snalogs, cyclic nucleotides, phosphoinositites), phospholipases ant : -bioluminescence assay reagents.
Yet other polypeptites contemplated for use herein are polypeptites of particular interest in the fielt of molecular biology, incluting various enzymes ant reagents. The enzymes can include ;
labelling enzytnes, modifying enymes, nucleases, polymeræes, sequencing enzymes and restriction enzymes. The reagents can include - -inhibitors, antibiotics and misc:ellaneous other reagents. -Prefemd polypeptides for use in accort with the instant invention :
inclute growth regulators. Among the preferred growth regulators are hematopoietic factors, which affect the maturation and proliferation of blood cells in Iymphoid tissue and bone marrow; cytokines, which generally influence euka~otic cell growth; ant Iymphokines, which -. ' ' . .
.: :-:
,- ~', ~

13~863~

- affect Iymphocyte growth. Specific polypeptides which are growth regulators or Iymphokines are: interleukin 1, 2, 3 and 4; ~
and ~ interferons; granulocyte colony stimulating factor (G^CSF);
granulocyte - macrophage CSF(GM-CSF); macrophage CSF(m-CSF); -S megakaryocyte CSF; multi CSF or IIr3 (also known as BPA, HCGF, MCGF ant PS~;); erythropoietin; Iymphotoxin; tumor necrosis factor ('INF, also known as cachectin); ~ and ~g transforming growth factor (TGF); platelet derivet growth factor (PDGF); epidermal growth factor (EGF); nerve growth factor (NGF); insulin-like growth factor I and lI
(IGF I is also called somatomedin C); growth hormone (GF, also called- ~
somatotropin); ant growth hormone releasing factor (GHRF, also ~`
called somatoliberin). See Clark et al, `'The Human Hematopoietic CO1OQY Stimu1ating Factors," Science, 1æ9-1237 (June 5, 1987);
Taniguchi, "Regulation of Cytokine Expression," Ann. Rev. Imm., 6, 439-464 (1988); and Watson et al, Molecular Biolo~v of the Gene, Vol.
Il, 4th d., Benjamin/Cummings Publishing (1987).
Other preferred polypeptides for use in accordance with thiS
invention are fusion proteins. Fusion proteins are covalently linked proteins or portions of proteins. TQUS, proteins or active fragments 20 thereof having different purposes can be linked to provide a fused mole~ule having characteristics of both. Generally speaking, one of the proteins/protein fragments is a "s~eker" and the other an "actor" or "destroyer". Illustrative such fusion proteins are pseudomonas exotoxin linked to IIr2, diphtheria toxin linked to Ik2 or either toxin 1inked to `` 25 other proteins, or 1inkages between other proteins such as those - preferred proteins described in the preceding paragraph, or portions thereof. See United States Patent Nos. 4,545,985; 4,468,382; and 4,67~,382.
Fusion proteins of IL-2 linked with toxin as described above are ~ -~
designed to kil1 cell5 with IL-2 receptors, thus find use in preventing graft-versus-host rejection. Other fusion proteins have varying utilities, , ..
~ : . .
. `' '' `'' ~328634 depending on the proteins or portions thereof which are combined.
Thus, for example, Substance P and a toxin form a fusion protein which can be used to relieve chronic pain, while ~-melanocyte-stimulating hormone (MS~) and diphtheria toxin form a fusion protein designed to kill melanoma cells.
Yet other preferred polypeptides for we herein are muteins, which are mutationally a1teret proteins. Preferred muteins are muteins of the -preferred growth regulators and fusion proteins identified in the precediDg paragraphs, and typically have the same purpose as the corresponting unaltered protei_s. Especially preferred muteins include ~ -II,2 muteins, described for example in United States Patent Nos.
4,752,585 and 4,518,584, incorporated by reference herein in their ~;
entireties and relied upon; and muteins of ~-interferon, described for example in United States Patent Nos. 4,737,462 and 4,588,585. - ;
}5 The cyc10dextrins contemp1ated for use herein are hydroxypropyl, : ~ -hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of ~-cyclodextrin and the corresponding derivatives of ~-cyclodextrin. The ~-hydroxyalkyl groupings may contain one or more hydroxyl groups, e.g.
hydroxypropyl (2-hydroxypropyl, 3-hydroxypropyl), dihydroxypropyl and ,~
the like. The glucosyl, maltosyl and maltotriosyl derivatives may - -contain one or more sugar residues, e.g. glucosyl or diglucosyl, maltosyl :
or timaltosy1. Various mixtures ~?f the cyclodextrin derivatives may be used as well, e.g. a mixture of maltosyl and dimaltosyl derivatives. :
Specific cyc10dextrin deriva ives for use herein include hydroxypropyl~-cyclodextrin (HPCD or HPBCD), hydroxyethyl~-cyclodextrin (HEBCD), hydroxypropyl7-cyclodextrin (HPGCD), hydroxyethyl7- -cyclodextrin (HEGCD), dihydroxypropyl~-cyclodextrin (2HPBCD), ucosyl~-cyclodextrin (G1~-CD or G,BCD), diglucosyl~-cyclodextrin (2G,~-CD or 2G1BCD), maltosyl7~-cyclodextrin (G2~-CD or G2BCD), i ~ 30 maltosyl7-cyclodextrin (G27-CD or G2GCD), maltotriosyl~-cyclodextrin (G3~-CD or G3BCD), maltotriosyl~-cyclodextrin (G37-CD
.

~ .- -~i :

~328634 or G3GCD) and dimaltosyl~-cyclodextrin (2C~2~-CD or 2G2BCD), and mixtures thereof such as maltosyl~-cyclodextrin/dimaltosyl~-cyclodextrin.
Hydroxypropyl~-cyclodextrin for use in the methods of the present invention is commercially available. Alternatively, it may be prepared by known methods, especially by use of the optimized procedure of Pitha et al, Internatjo~ Journa~l of P~la.~çutic~s, ~9, 73-82 (1986).
The following is a typical procedure using the Pitha et al method:
31 g of sodium hydroxide were dissolved in 250 mL of water.
Then, 100 g of ,~-cyclodextrin were added and the solvent was warrned ~,~
to effect solution. The flask was cooled and 50 mL of propylene oxide ;
were added. The flask was fitted with a dry ice/acetone condenser . `
during the addit;on. The solution was allowed to come to room temperature ant was stirred for 72 hours. The solution was then neutralized with concentrated hydrochloric acid and diluted with water. - -The solvent was removed ~ vacuo, leaving a syrup which was taken up in ethanol. After stirring for 30 minutes at room temperature, the sodium chloride produced was removed by filtration The filter cake was washed with ethanol and the combined ethanol layers were reduced ~ vacuo The residue was dissolved in water and dialyzed in cellulose acetato (#7, 38 mm, 4 6 mL/cm, molecular weight cut off = 1000, F;sher Scientific) After S hours at O~C, the solution was removed from ;~
the tialysis tubing and freeze-dried. The resulting solid was suspended in acdone ant stirred owrnigbt. The filtered solid was resuspended in - ~
acetone and stirred for 24 hours The solid was collected by fltration . -and dissolved in 200 rnL of water and then Iyophilized 75 grams of purifed hydrolypropyl~-cyclodextrin were obtained. The degree of substitution was calculated by NMR and by comparison with an authentic sarnple.

3 0 ~ ~The Pitha et al method for preparation of HPBCD by condensation of propylene oxide with,8-cyclodextrin in alkaline aqueous solution : .
: . :
..:-, :
.-:

-:

~32863~ :

unfortunately suffers from disadvantages, particularly in purification of the product. After completion of the condensation, the reaction -mixture is neutralized with hydrochloric acid, water ;s evaporated under vacuum and the syrupy residue is dissolved in ethanol to precipitate S sodium chloride, the main by-product of the reaction. After filtration, ethanol is evaporated under vacuum and the residue is dissolved in j :
water and dialyzet to remove the remaining sodium chloride and polymerization products of propylene oxide. During dialysis, part of the hydroxypropyl~-cyctodextrin goes through the membrane and is lost.
The dialysate is then freeze-dried, twice stirred in acetone and washed ;
to remove the remaining polymerization products. Final1y, hydroxypropyl~yclodextrin is freeze-dried again. The second freeze-drying is necessary because the product after washing with acetone is not homogeneous.
To overcome these difficulties with the Pitha et al process, a new `:
method has been developed by Maciej Smulkowski of the University of Florida, Gainesville, Florida for the synthesis of HPBCD. This new ~ ~ -methot involves removal of sodium hydroxide from the reaction mixture by an ion exchange resin (H'); as a result, several time-consuming steps of Pitha et al's purification can be avoided. Moreover, the amount of sodium hydroxide used by Pitha et al (7 equivalents for one of p-cyclodextrin) can be decreased to 2 equivalents of sodium hydroxide per cyclodextrin molecule, and still produce a product wifh the appropriate NMR and optical rotation. ;
According to the new method"B-cyclodextrin is first condensed with propylene oxide in alkaline solution, sodium hydroxide is removed on an ion exchange column (Dowex*SOW-X8, H~ form), the eluate is evaporated under vacuum to one-half of the original volume, the remaining solution is freeze-dried, the resulting white solid is washed with acetone and freeze-dried again, then subjected to grinding and sieving. Possible modifications of this method include: (1) use of the ionic exchange resin for neutralization in the reaction flask, with ~ .
*Trade mark ~' ' .

-2g-filtration of the resin and washing on the filter funnel; (2) use of calcium, magnesium, lithium or potassim hydroxite to dissolve the cyclodextrin; l3) removal of hytroxides after the reaction by saturating the reaction mixture with carbon tioxide or neutralization with sulfuric 5 acid in place of the ion exchange resin; (4) use of even less sodium hydroxide (between 1 and 2 equivalents); ant (5) elimination of the secont freeze-trying.
The following is a typical procedure using the new, improvet method:
50 g of,~-cyclodextrin were dissolved in a solution of 3.53 g of :
sodium hytroxide in 75 mL of water and treated with 29 mL of ~ -propylene oxide at 0~C. The reaction mixture was maintained for 5 hours at that temperature, then was kept at room temperature for 42 ;
hours. At the end of that time, the reaction mixture was passed through the Dowex 50W-X8 column (H~ form), the column was washed ~ -with water and the eluate was evaporated under vacuum to a volume of 100 mL then freeze-dried. The resulting white solid was washed with acetone to give 51 g of HPCD, with the same degree of substitution t4.7) and NMR a~ the HPCD prepared by the Pitha et al method.
20 Residue on ignition was 0.0%. Optical rotation also was identical to that of the Pitha d al product.
Condensation of 25 g of p-cyclodextrin us;ng 7.71 g of sodium hydroxide gave similar results.
A further improvement in the new, improwd HPBCD synthesis 25 utilizes activatet carbon for purification of the solution prior to the last freeze-trying. Thus, when the aqueous solution from the Dowex 50 ionic exchange column was treatet w;th activated carbon, most of the polymerization protucts were remowd without loss of HPBCD, and the filtrate after only one washing with ethyl acetate was ready for fina1 30 freeze-drying. In this way, only one free ~-drying w~ required.

~ " -'. ' ',,-."' ,., ~ .
: - ' 132863~ ~

Crystallization of the final product instead of freeze-drying is also ;-possible, at least on a small scale.
The product from the modified new process (using activated carbon) appears to be superior to that of the original new process and 5 the Pitha et al process. First, the product is snow white and produces a colorless aqueous solution, whereas solutions of the earlier products were yellow. Secontly, the product is not oily, which may be due to removal of more highly substituted, less soluble, oily cyclodextrins.
HPBCD can be prepared in varying degrees of substitution, such as 10 5 or 7. Typically, the foregoing procedure is used to produce HPBCD
(ASDS 7). The mass spectra for the isomeric mixture of HPBCD ~
centers around 7 degrees of substitution. This spectra is obtained by ~-"softly" ionizing the sample using fast atom bombardment. The ~ -~
generatet spectra is similar to those previously reported (obtained by -15 Californium-252 plasma desorption) in both the symmetry of the isomeric distribution and the numerical spread of the isomers formed.
Hytroxyethyl~-cyclotextrin (HEBCD) can be prepared analogously to HPBCD, utiliung the improved procedure detailed above but substituting an equivalent quantity of ethylene oxide for the propylene 20 oxide there employed.
The synthesis of 2-hydroxypropyl7-cyclotextrin (HPGCD) similarly us the same basic procedure as for HPBCD, substituting 7-cyclodextrin for the,~-cyclodextrin starting material. However, because 7-cyclodextrin contains eight glucose residues compared to seven for,~-25 cyclodextrin, the arnount of propylene oxide used can be reduced in ~- ~ order to lower the tegree of substitution Use of 0 75 mole of propylene oxide per 0 077 mole of 1-cyclodextrin (-20% excess considering 8 OH groups) affords HPGCD with a degree of substitution of 8, while use of Q56 mole of propylene oxide (-10~o less than -equivslent) gives a degree of substitution of about 7. -Hydroxyethyl7-cyclodextrin (HEGCD) can be prepared similarly to HPGCD as described in the preceding paragraph, simply using an ~.
- . , ' .

1328634 ~

equivalent quantity of ethylene oxide in place of the propylene oxide.
Thus, the hydroxyalkyl cyclodextrins intended for use herein can be prepared by procedures described by Pitha et al or variations thereof.
Obtained by these procedures, the cyclodextrins are intrinsicalb -5 amorphous mixtures. The importance of the amorphous nature of the cyclodextrins is described by Pitha et al,.J. Pharm. Sci., Vol. 74, No. 9, 987-990 (September 1985) and in Pitha U.S. Patent No. 4,727,064. The atvantages of the amorphous nature of these materials are more ~ ~ -pronouncet at higher concentrations of cyclodextrin.
For use with polypeptides, it is highly desirable to use cyclodextrin which is relatiwly free of impurities, i.e. oxidizing agents, peroxides, `
propylene oxide or other chemicals used to prepare the cyclodextrins, because oxidation can render the polypeptide less effective. Preferably, -the impurity lewl will be below 100~M peroxide or its equivalent.
The other cyclodextrins intended for use in the present invention, -i.e. the glucosyl, maltosyl and maltotriosyl derivatives of p- and 7-cyclodextrin, are branched cyclodextrins which are highly soluble in water as comparet to the parent cyclodextdns. These ~branched cyclodextrins can be producet by microbiological processes from the 20 parent cyclodextrins. Glucosyl-p-cydotex~trins can be obtainet from the mother l;quor of a large-scale ~-cyclotextrin synthesis with Bacillus ohbensis cyclomaltotextrin glucanotransferase; see Koizumi et al, Chem. Pharm. Bull., 35 (8), 3413-3418 (1987j and reference cited therein. Maltogyl and maltotriosyl p- and 7-cyclodextrins can be 25 prepared from the-parent cyclodextrin ant maltose or maltotriose through the reverse action of heudomonas isoamylase or e~e!la ;
aeroge~ pullulanase, while glucosyl7-cyclotextrin can be prepared by ~ ~
- enzymatic hydrolysis of maltosyl-7-cyclodextrin; see Okada et al, Chem. :-Pharm, ~., 36 (6), 2176-2185 (1988) and references cited therein.
30 The preparlltion of maltosyl~-qclodextrin by reacting maltose with,~

,-.

1 3 2 8 6 3 ~

cyclodextrin in the presence of pullulanase is also described in Japanese Kokai 61-287902, published December 18, 1986, and Japanese Kokai 61-197602, published September 1, 1986. A moYture of maltosyl~-cyc}odextrin ant various dimaltosyl~-cyclodextrins may be conveniently employed. See also Kainuma et al United States Patent No. 4,668,626, issued May 26, 1987.
Polypeptites such as proteins present unique solubility and stability problems as comparet to other trug molecules. These are a reflection of the unique structure of the polypeptides. Proteins, for example, are very large molecules of high molecular weight having numerous hydrophobic and hydrophilic regions within a single molecule.
Moreover, the three-timensional structure or conformation of proteins is critical to their activity and can be easily affected detrimentally by the protein's environment, an unfavorable emironment leading to aggregation. Thus, the solubilization and stabilization of these complex molecules is tifficult to accomplish ant the ability of an untried agent to achieve solubilization/stabilization, and to do so without appreciable loss of activity, cannot be readily predicted. However, the present inventors have teterminet that a selected group of cyclotextrins, i.e.
the hytroxypropyl, hytroxyethyl, glucosyl, maltosyl ant maltotriosyl terivatives of ~- ant ~1-cyclotextrin (the hytroxypropyl ant hytroxyethyl derivatives being preferably preparet in amorphous form as tescribed hereinabove) are well-suitet for use in solubilizing/stabilizing the polypeptites tefinet hereinabove, especially the proteins. The ~;
25 following examples illustrate the solubilization/stabilization results which can be achievet in accord with the present invention. These ~ -exampl are not intentet to limit the invention in any manner whatsoever.

.

`'~`" '' ' '." .',' ' .'.'.';.' f ",, '."- ' ',', " , ', ' '; ' ' ., ",'",'''' ' ,".' "' ' "'.' ' `.' `. ' . ,;, . :' ' ' , . -- .',', , 1328634 ~
~:

E~AMp~ -Preliminarv Testin~:
Preliminary studies on the use of a representative cyclodextrin -encompassed by the present invention, i.e. hydroxypropyl79-cyclodextrin S (HPBCD), as a stabilizer for interleukin-2 (IL-2) were conducted by first assessing whether a HPBCD-formulated solution of IL-2 could be Iyophilized to produce a clear solution upon reconstitution. This has provet to be a good first test for IL-2 formulation screening because very few agents give a clear IL-2 solution after Iyophilization and reconstitution.
First, IL-2 was formulated with HPBCD such that the final concentrations of 25.0 and 12.5 per cent HPBCD were present in 1 mL
of a 1 mg/mL IL,2 solution. These were Iyophilized in an LSL ~yolab -Iyophil;zer using the following cycle: primary drying at -30~C for 18 hours, temperature ramped up to 1~C at a rate of lZ/minute, followet by secondary drying at 15C for 20 hours, all steps being carried out under a vacuum of 50 microns. The Iyophilized ~
preparations were reconstituted with 1 mL of water for injection (WFI). -:
On visual observation, the solutions were found to be clear. Addition-ally, they gave light scattering numbers of 61 ant 66 when analyzed by a light scattering assay for assessing clarity of solutions. This assay deternunes the light scattering value by using a fluorimeter to measure the amount of light (510 nm) scattered from a sample at 90 degrees.
Usually, a ~alue of 500 or below represents a good and clear solution, a value in the range 500 - 1000 represents a marginal solution, and - scattering values > 1000 are obtained from turbid and hence unacceptable solutions.
Next, to determine the minimum HPBCD level needed to give a clear solution upon reconstitution, a range of concent~ations of HPBCD -(û.5-25~o w/v) were formulated with ~2 (1 mg), 1 or 2% sucrose, and 10 mM citrate buffer in a total volume of 1 mL per vial (the excipient). ;
:'.:

13286~
:~ , These were Iyophilized as before, and reconstituted with 1 mL of WFI.
A light scattering assay was used to measure the clarity of the resultant solution. As shown in FIG. 1 by the very low fluorimeter values ~30-60), a clear solution of IL-2 was obtained from all samples across the ~ -S tested range. Moreover, Westem blot analysis of the samples used for the light scattering analysis showed no appreciable increase in dimers when II,2 was Iyophilized with HPBCD.

EX~1PI,E 2 Further ~eht Scattç~ing Testing:
All IL,2 formulations were Iyophilized in the usual manner as described above. The Iyophilized plugs were reconstituted with water and characterized as follows: --Following the experiments detailed in Example 1, lower concentrations of HPBCD were tested in an attempt to determine the ; ~-minimum level of the excipient needed to stabilize a 1 mg/mL IL-2 `
formulation. Also, samples were bulked with sucrose to obtain more homogeneous plugs. Ligbt scattering values were obtained as in Example l. The results are depictet in FlG. 2. Curves 1 and 3 in the figure represcnt the light scattering values for formulation samples without correction for excipient scattering, while curves 2 and 4 show these values after exapient scattering corrections (obtained by subtracting the values for tbe control formulation witbout Il,2). An -increase in scattering values, indicating a deease in the clarity of the - ~ solution, is seen when the concentration of HPBCD in the formulation dropped below 0.2 ~o. On a molecular level, tbis translates to a mini-mum of 20 molecules of HPBCD per molecule of II,2. See Table 1 below.

,.

A ,', ,~

1328~3~ ::

T~BLE I
MOLAR RATIOS OF }I~ÇD AND IL-2 IN
SELECTED FO~MULA~IONS ~1.2) , % HPBCD in formulalion Mole H~CD/Mole IL-2 0.05 4.87 0.10 0.20 19A7 050 48.68 2.00 194.72 1. These ~alues have been calculated rom a HPBCD
molecular weight d 1540, assuming the average degree of p-cydodextrin subslitulion to be - 7.
2. IL-2 concentration is 1 mg/mL in Ihe formulalions.

Ultracentrifugation Assay:
. . .
Ultracentrifugation is a simple method of detecting the presence of ; -;
high molecular weight aggregates and oligomers. Ultracentrifugation -5~ was performet in a Beckman L8-70 using a type 70.1 Ti rotor. Flve milliliters of the ssmple formulation were spun at 105,000 x g for 1 ~;
hour. l~o 8upe was measuret by absorbance at 280 nm. Said `
absorb8nce: was 1hen compared to that prior to centrifugation.
Reconstituted samples of 1L-2 formulated with HPBCD were tested by -10 this assay. Samples were preparet similarly to those in Example 2 and the results are presented in F~G. 3. As seen in the hgure, unfor~
mulated II~2, before ~lyophilizationj contains ~ 93% of the protein in the supernatant by this assay, indicating that II,2 is nearly completely ~; unaggregated in solution. II~2 formulated with 05 or 2.0 per cent HPBD with læ or 2% sucrose behavet similarly to the unfonnulated ~ -2. These data indicate ~hat the protein was present in an unaggregated form when the HPBCD formulations were reconstituted. - --. -. ~-:. ~,: .
: , .

1 32~63~

Bioactivity Assav:
Formulations of IL,2 containing 0.05 to 25.00% HPBCD were Iyophilized and then reconstituted as described in Example 2.
S Bioactivity of IL,2 samples was measured by a HT-2 Cell Proliferation assay using the MTT stain. This assay has been adapted from the II~2 .
assay described by Gillis et al (1978) in.JQ~LQf Immur~
(Volume 120), pages 2027-2032. As seen from FIG. 4, IL.2 remained ~ -fully biologically active when formulated with HPBCD.

10 Western Blot Analysis:
T assess protein purity, Western blots were run on a number of IL,2 formulations which were combined with various concentrations of HPBCD, Iyophilizet and then reconstitutet with water as tescribed :~
before. Samples tested includet the following HPBCD formulations of II,2: 0.05% HPBCD with 1% sucrose, 0.1% HPBCD with 1% sucrose, -0,2% HPBCD with 1% sucrose, 05% HPBCD with 1% sucrose, 2.0%
HPBCD with 1% sucrose, 1% ~-cyclodextrin with 1% sucrose, 0.5%
HPBCD with 2~o~9ucrose ant 2.0% HPBCD with 2% sucrose.
Unformulatet IL 2 was also testet. The HPBCD ant ~-cyclodextrin 20 ; formulations were testet after 1 week at 37"C. There was a slight amount of dimer in the HPBCD formulations after Iyophilization ant subsequont rehytration, similar to the amoun~ seen in unformulated II,2, but the results for the HPBGD îormulations indicatet abow were all co~teret to be excellent. IL,2 formulated w;th unmotifiet,~-25 c3~clodextrin, which gaw a somewhat clear solution after reconstitutionof the Iyophilizet plug (scattering number - 300), exhibited a darker timer bant in the Westem than the HPBCD forrnulations.

, : -.. ~

1328~3~
... . . .

-37- ~:
EX~1PLE S
. ..
HPBCD was formulated with selected polypep!ides as described . :
previousb (Il,2, tumor necrosis factor or 'INF, macrophage colony :-stimulating factor or m-CSF, insulin ant human growth hormone) and - -the formulations were stored under various conditions of time and . ~
temperature. Light scattering values (determined as tescribed before) . :
and visual appearance were noted for each formulation. The results :. .
are tabulated below~
,~. :.`, ~' T~IE 2, P~r.~ ~ '``
CI~RJlY OFHP13CD/PRal'ElN FORMULATIONS
Proiein Fonnubtion r Stor ge Sc llerin~ nce IL-2 10 m~/mL 4 weeks 4 C l~ophilized 66 ckar ~ -10 mM N~ cilr le, pH 65 2 0% HPBCD, 2 0% ucr~e came 4 weeb 37' C l~ophilized 90 ck r - ~ -cau~o 4weeh 25' C Ibuid 26 clear a me 4 weeb 3t C liguid 402 p~rlku-htec lNF o~c m~/mL 4weeb 4 C bophilr~od 96 cle r 10 mM N cil~le, pH 65 05% HPI~CD, 10% wc~e 4w~eb 37' C bophilkod U cle~r .. ... . . .
m C8F I 0$/mL 4 weeb ~ 4' C baphilktd 34 cle r 211%HPBCD,20%wcrl~ae ' ~ .
U ~ h ulin 10~ 4weeb 4 C ~aphilized 26 cle r , -(13Ovine10 mM Na dtr te, pH 6S
pu~ 015 NHa ~ .-. ~' ,~ , - -Ik, 0.63% L~eHa : -, - ' , Si~ma)1D% HPLCD, 10% u~o }lumaD 10m~mL 4weeb 4' C ~p~-- 490 cli~hlb ~
-- Growth -10 mM N cilr~ h~
Hormone-~0% }~CD,-2~0% nu~e (S~ % M~itoi -~
}lum~ Growth Horne, 6~n Si~-w~ obldned - ~ ~ophilized product which - , reconsli~ l~y iuaom F~ liGH with 3% HPBCD reduoed lhe - h~nesa ~
'Wllhoul corrcclk~ ror the e~pienl control. .^ :: . :

h ~ ~

',' "''' ' ' ' ~ '~'','.
'' ~,'' ".

132863~
.

TA~ RT 11 Cl ARllY OF PROTEIN FORMULAnONS WITHOUT STA~ILIZER
Protein Formublion Type Saltering Appeu nce IL-2 10 mg/mL Iyophilized S58 cu~pended 10 mM N~ citr le, pH 65 p nkuhler, liquid 26 p~nicul~ler begin ~ -lo ppe r on - ~- -slor~8e t r~om bmper lure ~rler 2 hour~
TNF' Q2S m~/mL Iyophilized 26 cku 10 mM N cilr le, pH 65 m-CSF 10 m~/mL Iyophilized 46 cb r 10 mM N cilr le, pH 65 In-ulin 10 m~/mL Iyophilized - Iyophilized plug p naealic, 10 mM N~ cilr le, pH 65 does nol dissohre si2ma) ' ~, .
' Allhough TNF olulior wu ck r upr~n recon tilulion, Ihe olulion rormed 3-5%
dimcr in 4 we~ks at 37 C, wher~u wilh TNF hrmubted with HPB~D, no apprhble din~er (~ 02%) fiormed in the ri me tirne ' ' .''", ' -'. ' "
Bioactivity of selected samples was measured by known procedures.
II,2 bioactivity was ddermined as described before. TNF activi~r was measured using sn ~ yj~:Q cell cytotoxicity assay utilizing a TNF
sensitive mouse I, 929 fibroblast target cell line. This assay has been S adapted from techniques discussed by J, M, Ostrove ant G, E, Gifford in Proc, Soc. E KY, Biol, Med, .~Q: 354-358 (1979) and M. R, Ruff and ~ -G. E. Giffort in Inf. Immun. 31: 380-385 (1981). l~e m-CSF bioassay was perfor ned by using an NFS-60 murine retrovirus-induced myeloid leukemia cell line, The pertinent references for the CSF assay are: J. ~ -10 ~ Ihle et al., Advanc in Oncolo~y, Vol, 4, pp, 95-137, G, R~ein ed.
~- ~ (Raven Press, NY) and J. C. Lee et al., J. Immunol. 128: 2393-2398 (1982).
~: . : ~ .

1 3 2 ~ 6 3 ~

-39~
T~BLE 3 ~:
BIOACTIVITY OF HPBCD/PROTEIN FORMULATIONS

SPECIFIC ACTIVITY SPECIFIC ACTIVI~
BEFORE FORMULATION IN FORMULATION ~-PROTEIN _ U/MG . FORM~,~TION U~M~
IL-214 x 108 IL-2 1 mg/mL8.93 x 106 HPBCD 2%
Suaose 2% : :`~. ` -10mM Cilrate pH 65 ~:; .
TNF15.6x 108 TNF0.25 mg/mL265 x 106 :
HPBCD 0S% ~ . : ~ -:
Suaose 1% :.: ~: -10mM Citrate pH 65 ; :
m-CSF89 x 107 m-CSF 1 mg/mL4.93 x 107 -HPBCD 2%
Suaose 2%
10 mM Citrate pH 65 '.' ., ''';

The data for the HPBCD/protein formulations are similar to the ~ :
values for unformulated 11r2~ TNF and m-CSF, indicating that~these proteins retain their full biological activity after formulation with .
HPBCD.
S Selectet 9ample9 formulatet as specifiet in TABLE 2 were also assesset for timer forrnation as an indicat;on of protein denatura1;on.
SDS-PAGE gels were silver stainet to enhance detail for the detsction ~ -of d;mer in very small quant;ties. Normal Comassie or Fast Green - ~ stains to not normally show dimer formation at these low levels. There ~ ,. ~ .... ..
10 was no substantial timer formation in IIr2 after 12 weeks at 4~C, in INF after 10 weeks at 4~C and in human growth hormone after 7 weeks at 4'C. m-CSF-showed essentially 1he sarne composition on SDS-PAOE after 7 weeks of storage at 4~C as it displayet at the beginning of the study. m-CSF exists as a dimer in solution and hence 15 runs as a dimer. Therefore, these results represent no change in the ., . ... ..... ... . .

132~63~

:", ~ .
nature of the protein after formulation with HPBCD. Insulin exhibited :
slight dimer formation but the amount was stil1 very small. . ~ .
, EXAMPLE 6 ;: ~
: .
Fourth derhrative ultraviolet spectroscopy (4D-UV) is a technique :
S which can asscss emironmental changes of the aromatic amino acids thus denoting possible conformational changes. No tifference in the fourth derivatiw spectrum of the protein was noted for IL,2 formulatet with HPBCD as compared with unformulated IL~2, 1 mg/mL ~e HPBCD formulations contained~
. -Ik2 HPBCD Suqose 1 mg/mL ~ O ~% 05% 10 mM atrate pH 65 1 mg/mL 05% 05% 10 mM citrate pH 65 1 mg/mL 2.0% 2.0~o 10 mM atrate pH 6.5 : :

;'~,~ ~' '.

:: ~ : - : : -. .

-: : .
, : , .
, --" , . ~ - , - , 1 3 2 8 6 3 ~
:

The formulation of protein and cyclodextrin in accord with the present invention may be carried out as a separate operation utilizing purified protein as the starting material, or the formulation with -~
cyclodextrin may be incorporated into purification of the protein. By ~i ~
S way of example, there is described below a forrnulation of a selected -peptide, IL,2, with a selected cyclodextrin, HPBCD, which is carried out turing the purification process:
Thus, the protein starting material may be recombinant 1I,2 -present in the product in a gel filtration pool which is frequently the- ~ -10 final step of protein purification. A concentratet HPBCD solution may be atdet at this point in an appropriate amount to form a complex with IL,2 and stabilize it in solution. The concentrations of HPBCD
and IL,2 are adjusted, if desired, and typically a bulbng agent is added. -The bulbng agent is present in the solution at a level of about O.S to : --10% by weight, preferably 1 to 5% by weight. Preferred bulking agents :~ -are water-soluble, stable and do not react with IL~2, for example sucrose, lactose, trehalose, maltose, mannitol, sorbitol, starches, starch ~ ;
hydrolysates, microcrystalline celluloses and albumin such as human serum albumin. The bulbng agent protects the protein during ;
20 Iyophilization, gives a more homogeneous Iyophilizet product and also makes the freeze-driet protuct, when placet in unit dosage containers, easily seen by the naket eye.
After atding the buJking agent, the unit dosage amounts (i.e.
volumes that will provide about 0.01 to 2 mg, preferably 0.2 to 1.0 mg, IL-2 per dose~ o~ the solution are dispensed into containers, the - containers are capped with a slotted stopper, and the contents are -~ Iyophilized using conventional freeze-drying conditions and apparatus. -~
The Iyophilized, sterile product may consist of a mixture of (1) recombinant I1~2, (2) bulking agent (e.g. sucrose), (3) HPBCD and (4) a small amoud of buffer that will provide a near physiological pH ~ :
when the mi~nure is reconstituted. Ihe recombinant IL,2 will typically :,: ' '.
: .
, . .

132863~
~.

constitute about 0.015% to 3.85% by weight of the mixture, more preferably about Q4% to 2.0% of the mixture. HPBCD will typically constitute about 0.2% to 10% by weight of the mixture;
The Iyophilizet mixture may be reconstituted by injecting a 5 conventional parenteral aqueous injection liquit such as water for injection, Ringer's injection, dextrose injection, dextrose and salt ~-injection, or the like, or aqueous HPBCD solution into the vial. The amount of injection liquid added to the vial will typically be in the range of 1 to 10 mL preferably 1 to 2 mL for intramuscular or -10 subcutaneous injections, with larger volumes usable for intravenous inJections.
The reconstituted formulation is suitable for parenteral ~ ;
administration to humans or other mammals to provide IL-2 therapy ~ ~ -thereto. Such therapy is appropriate for a variety of ~ ~ ~
immunomodulatory indications such as T cell mutagenesis, induction of ~ -cytotoxic T cells, augmentation of natural killer cell activity, induction - -of IFN-gamma, restoration or enhancement of cellular immunity (e.g., treatment of immune deficient conditions) and augmentation of cell mediated anti-tumor activity.
Generally speaking, when formulation with cyclodextrin occurs as part of the polypeptide purification process, the cyclodextrin will be introducet as an aqueous solution comprising at least Q01% (w/v) cyclodextrin (e.g. HPIICD), more preferably from about Q1% (w/v) to about 60% (w/v) cyclodextrin (e.g. HPBCD). Most preferably, and ~ - -especially when the polypepffde is IL~2, the range is irom about 0.2%
(wiv) to about 60% (w/v) cyclodextrin.
When an alreaty purified polypeptide is to be formulated in accord with the present invention, the pslypeptide (typically Iyophilized) may be simply dissolved by mixing with an aqueous cyclodextrin (e.g.
HPBCD) solution of the selected concentration, the aqueous ~-cyclodextrin solution generally comprising at least 0.01% (w/v) ,' . ' ~.
. -:
;, , 132863~ ~

~3-cyclodextrin, e.g. HPBCD, more preferably from about 0.1% (w/v) to `~
about 60~o (w/v) cyclodextrin, e.g. HPBCD. Most preferably, the range :
is from 0.2æ (w/v) to about 60% (w/v) cyclodextrin, particularly when . ^. . ~-IL,2 is the polypeptide. The amount of cyclodextrin (e.g. HPBCD) used will vary with the particular polypeptide selected, but will in any event be an effective shbilizing and/or solubilizing amount. A
formuJation prepared in this manner could be administered in this ` ^
form, or Iyophilized ant subsequently reconstituted. In a Iyophilized `
composition, the weight ratio of cyclodextrin to polypeptide will ~ -generalb be between about 1:1 to 200:1, preferably between about 2:1 ~.
to 50:1. .~'`^''` ' ` ' As an altemative to the above, in selected situations the already purifiet polypeptide may be in liquid fomm (already dissolved in an appropriate aqueous medium) and the selected cyclodextrin may be added in dry form in a sufficient stabilizing amount. `
The drug formulations prepared in accord with the present imention can be used to treat a variety of conditions, depending upon - :
the pharmacological nature of the polypeptide selected for administration.
Preferably, the parenteral drug formulations prepared in accordance w;th the present imention comprise the selected polypeptide and an `
effective solubilizing and/or stabilizing amount of the selected cyclodextrin in an aqueow media Preferably, the formulations are :
substantially free of organic solvent, are sterile and pyrogen-free, and are prepared in accord with accepted pharmaceutical procedures, for example as described in Remin~ton's Pharmaceutical Sciences, sevènteenth etition, ed. Alfonso R. Gennaro, Mack Publishing ^ Company, Easton, PA (1985),1518-1552. The aqueous sterile injection ~;
solutions may further contain antioxidants, buffers, bacteriostats, isotonicity adjusters and like additions acceptable for parenleral formulations. Various unit dose and multi-dose containers, e.g. sealed 1328~34 ampules and vials, may be used, as is well-known in the art. The essential ingredients of the sterile parenteral formulations, i.e. the selected polypeptide, water and selectet cyclodextrin, may be presented in a variety of ways, just so long as the solution ultimately administered S to the patient contains the appropriate amounts of the essential ingre- -dients. Thus, for example, the polypeptite/cyclodextrin/water formulation may be presentet in a unit tose or multi-dose container, ready for injection. As another example, a concentrated solution of polypeptide/cyclotextrin/water may be presented in a separate container from a tiluting liquid (water or cyclotextrin/water) tesigned `
so that the contents can be combinet to give a formulation containing appropriate amounts for injection. As anotber alternative, the ~-polypeptite or, preferably, a polypeptite/cyclotextrin combination may be provitet in a freeze-triet contition in one container, while a separate container contains diluting liquid ~water or cyclotextrin/water.
tepending on the arnount of cyclotextrin in the other container), again designet so ~hat the contents can be combinet to give a formulation containing the appropriate amounts of the essential ingretients. In any ~ event, the contents of each container will be sterile.
In general terms, the therapeutic dossge ranges for administrstion of polypeptides in the parentersl formulstions described herein will be the ssme as those cbarscteristicslly uset for parenteral stministration of the polypeptite. Naturslly, such therapeutic dosage ranges will va ~ with the size ant species of the pstient, the contition for which the formulation is atministeret, the type of psrentersl atministration -~
employet ant the like. The quantity of given dossge form neeted to teliver the desiret dose of actiw ingretient will of course tepent upon -the concentration of the peptite in the parenteral formulstion.
While~many polypeptides are atministered by injection, some may ~ -be given orally. These could be stabilizet with the selected cyclodextrin in accort with this imention, then formulatet as tablets, capsules or the ~.... .
:
~ -~''.'' - . .

- 1328634 ~ ~
-45- `
like in the usual manner, with well-known carrier materials. See Remin~ton's Pharmaceutical Sciences, referred to hereinabove.
Diagnostics for ~ vivo use formulated accorting to this invention ~
will be prepared in the same manner as the therapeutics described ` ~ .
5 above, except that the unit dosage forms will contain a diagnostically effective amount rather than a therapeutically effective amount of the ` -selectet polypeptite. On the other hant, ~ vitro tiagnostics need not be pharmaceutically acceptable. Thus, ~ vitro tiagnostic components can, for example, be protected from exogenous contamination by the 10 addition of bacteriostatic or fungistatic agents which are prohibited for vivo applications. Other ingredients in the i1} vitro tiagnostics, e.g. ~ -chromogens, likewise need not meet the requirements for materials `
present in pharmaceutical formulations.
While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various ~;modifications, wbstitutions, omissions, and changes may be made without departing from the spirit thereo Accordingly, it is intended that the scope of the present invention be limited solely by the scope of ;
the fdlowing claims.

'"'.'':~'''..' , .

- ' ,.' :' , ~,.

" '~:

Claims (68)

1. A method for solubilizing and/or stabilizing a polypeptide, said method comprising combining said polypeptide with an effective solubilizing and/or stabilizing amount of cyclodextrin selected from the group consisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of .beta.- and .gamma.-cyclodextrin.

2. A method according to Claim 1, wherein said cyclotextrin is in an aqueous solution.

3. A method according to Claim 2, wherein said aqueous solution comprises at least 0.1% (w/v) of cyclodextrin.

4. A method according to Claim 3, wherein said aqueous solution comprises from about 0.2% (w/v) to about 60% (w/v) of cyclodextrin.

5. A method according to Claim 1, wherein said cyclodextrin is hydroxypropyl-.beta.-cydodextrin.

6. A method according to Claim 5, wherein said hydroxypropyl-.beta.-cyclodextrin is in an aqueous solution.

7. A method according to Claim 6, wherein said aqueous solution comprises at least 0.1% (w/v) of hydroxypropyl-.beta.-cyclodextrin.

8. A method according to Claim 7, wherein said aqueous solution comprises from about 0.2% (w/v) to about 60% (w/v) of hytroxypropyl-.beta.-cyclodextrin.

9. A method according to Claim 2, wherein said polypeptide is a protein.

10. A method according to Claim 9, wherein said protein is superoxide dismutase.

11. A method according to Claim 9, wherein said protein is insulin.

12. A method according to Claim 9, wherein said protein is tissue plasminogen activator.

13. A method according to Claim 9, wherein said protein is erythropoietin.

14. A method according to Claim 9, wherein said protein is an interferon.

15. A method according to Claim 14, wherein said interferon is .beta.-interferon.

16. A method according to Claim 9, wherein said protein is growth hormone.

17. A method according to Claim 9, wherein said protein is epidermal growth factor.

18. A method according to Claim 9, wherein said protein is an interleukin.

19. A method according to Claim 18, wherein said interleukin is IL-2.

20. A method according to Claim 9, wherein said protein is tumor necrosis factor.

21. A method, according to Claim 9, wherein said protein is atrial natriuretic factor.

22. A method according to Claim 9, wherein said protein is a colony stimulating factor.

23. A method according to Claim 2, comprising lyophilizing the aqueous solution containing said polypeptide ant said cyclodextrin.

24. A composition comprising a polypeptide and an effective solubilizing ant/or stabilizing amount of cyclodextrin selected from the group consisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of .beta.- and .gamma.-cyclodextrin.

25. An aqueous composition according to Claim 24.

26. A composition according to Claim 25, comprising at least 0.1%
(w/v) of cyclodextrin.

27. A composition according to Claim 26, comprising from about 0.2% (w/v) to about 60% (w/v) of cyclodextrin.

28. A composition according to Claim 25, wherein said cyclodextrin is hydroxypropyl-.beta.-cyclodextrin.

29. A composition according to Claim 28, comprising at least 0.1%
(w/v) of hydroxypropyl-.beta.-cydotextrin.

30. A composition according to Claim 29, comprising from about 0.2% (w/v) to about 60% (w/v) of hydroxypropyl-.beta.-cyclotextrin.

31. A composition according to Claim 25, wherein said polypeptide is a protein.

32. A composition according to Claim 31, wherein said protein is superoxite dismutase.

33. A composition according to Claim 31, wherein said protein is insulin.

34. A composition according to Claim 31, wherein said protein is tissue plasminogen activator.

35. A composition according to Claim 31, wherein said protein is erythropoietin.

36. A composition according to Claim 31, wherein said protein is an interferon.

37. A composition according to Claim 36, wherein said interferon is .beta.-interferon.

38. A composition according to Claim 31, wherein said protein is growth hormone.

39. A composition according to Claim 31, wherein said protein is epidermal growth factor.

40. A composition according to Claim 31, wherein said protein is an interleukin.

41. A composition according to Claim 40, wherein said interleukin is IL-2.

42. A composition according to Claim 31, wherein said protein is tumor necrosis factor.

43. A composition according to Claim 31, wherein said protein is atrial natriuretic factor.

44. A composition according to Claim 31, wherein said protein is a colony stimulating factor.

45. A composition according to Claim 31, intended for human or veterinary administration.

46. A composition according to Claim 31, intended for in vitro use.

47. A lyophilized composition comprising a polypeptide and an effective stabilizing amount of cyclodextrin selected from the group consisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivdives of .beta.- and .gamma.-cyclodextrin.

48. A composition according to Claim 47, comprising a cyclodextrin to polypeptide weight ratio of from about 1:1 to about 200:1.

49. A composition according to Claim 48, comprising a cyclodextrin to polypeptide weight ratio of from about 2:1 to about 50:1.

50. A composition according to Claim 47, wherein said cyclodextrin is hydroxypropyl-.beta.-cyclotextrin.

51. A composition according to Claim 50, comprising a cyclodextrin to polypeptite weight ratio of from about 1:1 to about 200:1.

52. A composition according to Claim 51, comprising a cyclodextrin to polypeptide weight ratio of from about 2:1 to about 50:1.

53. A composition according to Claim 47, wherein said polypeptide is a protein.

54. A composition according to Claim 53, wherein said protein is superoxide dismutase.

55. A composition according to Claim 53, wherein said protein is insulin.

56. A composition according to Claim 53, wherein said protein is tissue plasminogen activator.

57. A composition according to Claim 53, wherein said protein is elgthropoietin.

58. A composition according to Claim 53, wherein said protein is an interferon.

59. A composition according to Claim 58, wherein said interferon is .beta.-interferon.

60. A composition according to Claim 53, wherein said protein is growth hormone.

61. A composition according to Claim 53, wherein said protein is epidermal growth factor.

62. A composition according to Claim 53, wherein said protein is an interleukin.

63. A composition according to Claim 62, wherein said interleukin is IL-2.

64. A composition according to Claim 53, wherein said protein is tumor necrosis factor.

65. A composition according to Claim 53, wherein said protein is atrial natriuretic factor.

66. A composition according to Claim 53, wherein said protein is a colony stimulating factor.

67. A composition according to Claim 47, intended for human or veterinary administration.

68. A composition according to Claim 47, intended for in vitro use.