CA2155005C

CA2155005C - Pharmaceutical compositions containing bactericidal permeability increasing protein and a surfactant

Info

Publication number: CA2155005C
Application number: CA002155005A
Authority: CA
Inventors: Weldon Courtney Mcgregor; James Stubstad; C. Paul Chang
Original assignee: Xoma Corp
Current assignee: Xoma Corp
Priority date: 1993-02-02
Filing date: 1994-02-02
Publication date: 1999-04-06
Anticipated expiration: 2014-02-02
Also published as: CA2155005A1; DK0682524T3; US5488034A; HK1014156A1; AU695125B2; EP0682524A1; DE69428521T2; JP3946246B2; US5955427A; US6066620A; WO1994017819A1; ES2164098T3; DE69428521D1; PT682524E; AU6133094A; ATE206308T1; EP0682524B1; CN1163264C; US6255284B1; CN1127992A

Abstract

Polypeptide pharmaceutical compositions having improved stability and resistance to aggregation, particle formation and precipitation comprising a polypeptide pharmaceutical and poloxamer surfactants alone, or in combination with polysorbate surfactants. Preferred polypeptides stabilized are bactericidal/permeability increasing (BPI) protein, biologically active fragments of BPI, biologically active analogs of BPI, and biologically active variants of BPI.

Description

- WO ~/17819 PCT~l0L~9 ~ t ~
""" ,.

PHARMAcEuTIcAL COMPOSITIONS CONTAINING BACTERICIDAL PERMEABILITY
INCREASING PROTEIN AND A SURFACTANT

BACKGROU~D OF THE nNVEN~nON

The present h,~e,l~n relates generally to pharm~eutirql Coll"~ûsiLions and more ~rifi~qlly to improved protein and polypeptide ph -~ ",~r~ C~l~ for use as l)ale.lt~ l drugs. Recent advances in the d~,~elul""~,n~
10 ' of genetic e.~e ;n~Y.. ;ng t~ S ~!ogy have made a wide variety of biologically active polypeptides available in s~rr~c;-~1ly large quqntiti~os for use as drugs.
Poly~ ides, however, can be subject to particulate fc,.~ ;o~ and loss of biological activity by a vanety of ~l~f...i,~,l and physical means inCl~l~ling del~lul~lion due to heating or r,~ng and by c~ ul.i to e~ ..lc pH or other 15 çllf....i-~l deg. -~<.I;~n.
Particulate formation and loss of ~ ogit~ql activity can also occur as a result of physical qgitqtion and intera~ions of poly~ ~.de molecules in solution and at the liquid-air interfaces within storage vials. It is believed that the polypeptide molecules adsorb to an air-liquid ;.~t---r;-~e, unfolding to present20 hyd.ù~)hobic groups to air with the hydlu~)hilic groups illl...f .~ed in the aqueous phase. Once so positioned at the surface, the pol~lide molecules are ~s~ G~ 1e to ag~lcE,-Iic n, particle formation and ~ I;on It is also believed that further co-~ lionql changes can occur in ~ es adsorbed to air-liquid and solid-liquid interfaces during cc,---~),essiun~ cion of the interfaces 25 such as occurs from agitation during l-a-,:,l,u-~tion or otherwise. Such agitation can cause the protein to entqngl~ ale~ form particles and uhimqt~-ly p.~c;pit~te with other adso~bed proteins.
- Particle formation due to surface denalu.alion can be somewhat controlled by a~ .iate sclc~;liûn of the ~ on~ of storage vials and by ,s .~

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...in;...;~;..g the air volume (hP~ lcp~~e) in those vials. In this regard, par~ially filled cc~ ;nr,.~ e~.ll the worst case for ~ dlion inrillce~ ~r~;r.;li~';nn.
Particle fo.-~ ;n~ can also be controlled by incoll,uldtion of surfactants into the protein COI~ ;Qg co~ on in order tO lower the surface S tension at the solntion-air int~Prf~ . Cldssic st~hiti7~tion of ~ c-eutir~l~ by svrf~ct~nt~ or cmlllcifiprs hads focused on the ~...pl.;~ h;~ nature of molecular groups cQn~;n;,~g both llydlolJhilic and llydlu~ obic ~lu~c,~,es within the surfactant mQkP~le. Thus, the art teaches that one can make a stable solution ofl.le mc~ s such as oil-in-water or water-in-oil by sPI~ .g an 10 ~)~un~pliate wl fa( t~nt as a compatibilizer. One Py~mrle is the stable çmlllcifi~qti~ln of soybean oil using pol~"; ---.,r 188 (PLURONIC F-68, BASF
Wyandotte Corp., P,...~ y, N~. Another ,- , le is the use of polyso,l~te 80 (1~V13EN 80, ICI ~r~l.o.rir~c, Inc., Wil...;~.g~.- DE) to emulsify oil-soluble vitamins A, E and K in -queouc solntion for -~ri ~; f inn via oral and vascular routes. Work by Krantz, et al., "Sugar ~l~ohnlc - XXvm. Toxicologic, Pl,a~",a~dynamic and Clinical Observations on TWEEN 80," Bull. of the School of Med., U. of MD., 36, 48 (1951) laid the groundwork leading to the listing of poly~.l,ate 80 as a drug ingredient for which USP/NF l~uil-"l,ents have been hl;$l~rd in U.S. Ph~-.-.~.rop~iq- XXII.
Of interest to the present invention is the work related to use of poly~,l~le 80 for stabili7qtion of an~ od~-based product fo~nnlqtiollc as ~es~ d in Levine, et al., J. P~ .al Sci. Technol., 45, 3, 16~165 (1991).
This work ~iis~los~l that the a nount of snrfartqnt .~uu~id for stabili~-q-ti~r was in excess of the theoretical ~ u~d to reduce surface tension. The work further showed that the need for excess snt~~tant beyond the theoretiral ,... could be all~i~uled to (1) the co~ n .c~llhcd to ~--~;nt~;n an intact ~ul~ e layer on a turbulent int~ ~e during random shaking; and (2) to surfactant loosely -q~soriqt~d with protein and bound to c~ ,r walls.

* PLURONIC , TWEEN , TRITON , BRU AND ZONYL
referred to herein are registered trademark~.

WO g4/17819 PCT/USg4tO1239 ' 2155~05 Regulatory requirements limit the types and specific iclelltiti~.s of sllrfart~nt~ that can be incol~olaled into p~nlelal compositions for injection into ~ the human body. Generally accepted s~lrf~rt~ntc having a history of use and listed in the U.S. Ph~ ropoeia X~I include poloxamer and polysorbate polymers.
S However, either of these aione may provide less than complete stabilization for the ph~lla~eutir~l compositions when used at concentrations of 0.1% or lower.
Elevated concentrations of surf~ct~nt may pose increased risk of toxic effects, earlier onset of hemolysis, and observed changes in neutrophils and platelets, both of which are involved in blood complement activation. The highest safe concentration for poloxamer 188 in approved parenteral solutions is 2.7% when it is used in limited doses as a blood substitute and is diluted as much as 10 fold in the bloodstream. Similarly, polysorbate 80, approved in p~nleldl solutions for over 20 years, is rarely used in concentrations greater than 0.1 % in solution volumes of 100 mL or more. Krantz et al., supra, identifies the onset of 15 hemolysis in the dog for a polysoll~ale conrentr~tion of 0.1% at 90 minutes.
Neonatal deaths have been associated with the use of poly~oll ate 80 at concentrations of greater than 1 ~o. Accordingly, there exists a need in the art for pharrn~celltir~l compositions providing improved protein stability which comprise only those components which are regarded as safe and are included in parenterals20 approved by regulatory authorities for commercial use.

SUMMARY OF THE INVENTION
The present invention relates to ph~nn~ce~-tical compositions of polypeptides and is directed to the discovery that poloxamer surfactants and 25 combinations of poloxamer surfactants with polysorbate s~ ct~nt~ enhance the solubility/stability of bactericidaVpermeability increasing (BPI) protein, biologically active fragments of BPI, biologically active analogs of BPI, and biologically active variants of BPI (produced by either recombinant or nonrecombinant means) in aqueous solution. The invention particularly provides WO 94/1781g PCrlUS94/0123g 21550~

for solubilization/stabilization of bactericidal/permeability increasing proteins which are biologically active amino~ l fragments of BPI or analogs and variants thereof. Amino-te.J~Iinal fragments of BPI, such as those design~ted rBPI23 or any amino-te....in~l fragment comprising from about the first 193 to S about the first 199 amino-tGlmi~lal amino acid residues of BPI, are believed to be particularly susceptible to loss of stability in a~ueous solution.
The present invention is directed in particular to the discovery that a combination of two specific types of s~ ct~nt~ provides a surprising improvement in protein stability to pharmaceutical compositions cOIllpa~c;d to either surfactant alone. Specifically, it has been found that a ph~nn~ce~ltir~l composition comprising the combination of a poloxamer (polyoxypropylene-polyoxyethylene block copolymer) snrfa~t~nt and polyso~l,a~e (polyoxyethylene sorbitan fatty acid ester) s~ t~nt provides improved stability and resi~t~nre toaggregation, particle formation and precipitation of protein pharmaceutical agents.
The combination of these two types of surf~t~nts provides improved stability andre~ict~nce to surface denalulalion~ aggregation, particle formation and pl~i~ alion coll,~ c;d with either s~ ,t~nt alone.
The poloxamer s~lrfa(~,t~nt col"po~ is preferably present in a concentration of from about 0.01% to about 1% by weight with a concentration of 0.1% to 0.2 % by weight being pl.,f~ d to stabilize protein solutions comprising less than or equal to 2 mg/mL. The polysorbate surfactant component is preferably present in a concentration of from about 0.0005% to about 1% by weight with a concentr~tion of 0.002 % by weight being plerelled. Most preferred is the combination comprising 0.1% to 0.2% by weight of poloxamer 188 and 0.002% by weight polysorbate 80. This combination is particularly useful for preventing particle formation of extremely degradation sensitive proteins such as bactericidal/permeability increasing protein (BPI) but is also useful for promoting the stability of other polypeptide pharmaceuticals. It is contemplated that the combination of poloxamer and polysorbate surfactants may WO 94/17819 215 5 0 0 5 pcTlus94lol23s . ,._ be used alone or in combination with additional surf~~,t~nt~. Moreover, the invention is not limited to a single poloxamer surf~rt~nt in combination with a ~ single polysorbate s--rf~-t~nt and can include one or more poloxamer surfactants in combination with one or more polysorbate surfact~ntc.
A further aspect of the invention relates to the discovery that a poloxamer sl-rf~ct~nt is particularly useful for the solubilization/stabilization of compositions compli~-ng an aqueous solution of BPI protein or biologically active fr~gments, analogs, or variants of BPI protein (produced by recombinant or nom~cco.,.binant means). The invention provides a method of solubilizing/
stabilizing such polypeptides by cont~tin~ the polypeptide with a poloxamer s--rf~t~nt. Without being bound by a theory of the invention, it is believed that poloxamer surf~st~nt~ stabilize BPI protein products not by a mechanism involving lowering the surface tension of the aqueous solution, but, at elevatedte,..peldtur~s, by stabilizing unfolded and partially unfolded BPI protein molecules and preventing precipitation of those molecules.
Preferred poloxamer surfactants are characterized by a HLB value greater than about 14 and a surface tension between 10 and 70 mN/m as measured in aqueous solution at room le.nyt;l~ulc and at a concentration of 0.1%. More prcrelled is a poloxamer surf~t~nt which has an HLB value between about 25 and 35 and has a surface tension between 30 and 52 mN/m as measured in aqueous solution at room telllyeldlurc and at a concentration of 0.1%. Most prcrel,cd is poloxamer 188 available commercially as PLURONIC F-68 (BASF
Wyandotte, P~iyy~ly, N.J.) which is characterized by a surface tension of 50 mN/m and by an HLB value of 29.
A pr~r~d polysoll,ate s~ t~nt preferably has a surface tension between 10 and 70 mN/m as measured in aqueous solution at room temperature and at a concentration of 0.1%. More preferably, the polysorbate surfactant is characterized by a hydrophilic/lipophilic balance (HI~B) value of about 15 and by a surface tension between 40 and 50 mN/m as measured in aqueous solution at WO 94/17819 PCT/US94/0123g 21S~ ~05 room te~ )el~lurG and at a concGI~ lion of 0.1%. Most plGrel~Gd is polysorbate 80 (sorbitan mono-9-octadeconoate) which is available commercially as TWEEN
80 (ICI Americas Inc., Wil...i~ on, Del.).

BRIEF DESCRIPIION OF THE DRAWINGS
Fig. 1 is a graph depicting survival results over time of an actinomycin-D se~ d mouse model;
Fig. 2 is a graph depicting survival results according to BPI dose 10 in an actinomycin-D mouse model; and Fig. 3 is a graph depicting turbidity measurements of various BPI
proteins with and without the plGrGllGd s~ ct~nt~ of the invention.
Fig. 4 is a graph depicting surface tension measurements of rBPI2~/~cys solutions with varying s--rf~ct~nt concentrations of polysoll,ate 8015(PS80) and poloxamer 188 (F68).
Fig. 5 is a series of graphs of dirrelGIllial sc~nning calorimetry results of rBPI2l~cys with various concentrations of the s~ çt~nt poloxamer 188 (F68).
Fig. 6 is another series of graphs of dirrGlGnlial sc~nning 20calorimetry results of rBPI2l~cys with various concentrations of poloxamer 188(F68) Fig. 7 is a plot of the dGn~lluldlion and precipitation lel"pGl~ul~s of rBPI2l~cys over varying concentrations of the s~lrfaçt~nt poloxamer 188 (F68~.
Fig. 8 is a series of graphs of dirrGrc"lial sc~nning calorimetry 25results of rBPI2l~cys with various concentrations of polysollJ-dte 80 (PS80) alone or in combination with 0.1% poloxamer 188 (F68) by weight.
Fig. 9 is a set of graphs of dirrelG,llial sc~nning calorimetry results of rBPI2l~cys with the surfactant polysorbate 80 (PS80) at two dirr~lc,ll concentrations .

~7~f~ ~

Fig. 10 is a set of graphs of differential scanning calorimetry results after a solution of rBPI21Acys and poloxamer 188 (F68) was heated to a temperature higher than the denaturation/unfolding temperature but lower than the precipitation temperature, and then was cooled down for repeat scanning.

DETAILED DESCRIPTION
The present invention provides improved methods and materials for maintaining the stability of polypeptide pharmaceuticals and preventing surface denaturation of such biologically active polypeptides. Specifically, the invention relates to the discovery that a combination of two specific types of surfactant molecules provides synergistic improvements in stabilization from surface denaturation of polypeptide pharmaceuticals. The invention also relates to the discovery that poloxamer surfactants have unique properties in the solubilization/stabilization of BPI-related proteins. While specific embodiments of the invention are directed to stabilization of bactericidal/permeability increasing protein (BPI) and biologically active fragments and/or analogs or variants thereof which are particularly susceptible to denaturation and particle formation, the utility of the invention extends generally to all protein and polypeptide pharmaceuticals.
BPI and active fragments and analogs thereof useful with the present invention include recombinant produced proteins such as described in U.S. Patent No. 5,198,541. Co-owned, copending patent application Theofan et al., WO 93/23434, addresses BPI-Immunoglobulin fusion proteins which are variants of BPI protein comprising at the amino terminal a BPI protein or a biologically active fragment thereof, and retaining the same biological activity of BPI protein.
Particularly preferred BPI materials include recombinant produced polypeptides produced according to the method of co-owned and copending Theofan et al. WO 94/18323 and entitled "Stable Bactericidal/Permeability Increasing Protein Products and Pharmaceutical Compositions Containing ~.

CA 021~00~ 1998-10-26 the Same". A preferred BPI fragment is characterized by about 1 to 199 or about 1 to 193 of the amino-terminal amino acid residues of the mature human BPI molecule as set out in Gray et al., J. Biol. Chem., 264, 9505-9509 (1989) except that residue 185 is glutamic acid rather than lysine as specified in Gray. The recombinant expression product of DNA encoding BPI amino acids 1 to 199 has been designated rBPI23. The recombinant expression product of DNA encoding BPI amino acids 1 to 193 has been designated rBPI(1-193). A
preferred BPI fragment analog comprises the first 193 amino acid residues as set out in Gray except that residue 185 is glutamic acid rather than lysine and the cysteine at position 132 is replaced with a non-cysteine residue such as alanine. Such a protein is designated rBPI21~cys or rBPI(1-193)ala132.

Example 1 In this example, tests of various surfactant systems were conducted to determine their utility for surface stabilization of a polypeptide pharmaceutical (rBPI23). The rBPI23 was provided at a concentration of 1 mg/mL in citrate buffered saline (0.02 M citrate, 0.15 M NaC1, pH 5.0).
Various surfactants were then added to this preparation in order to determine their utility as stabilizers.
According to this test, rBPI23 [BR-1] characterized by about 1 to about 199 of the first 199 amino acids of the mature human BPI molecule and produced according to the methods of Theofan et al., W0 94/18323 was filled by hand to 5 mL in sealed sterile 6 mL molded glass vials (total capacity 8.4 mL, Wheaton) in the desired formulation buffer.
The vials to be tested were set horizontally on a flat bed shaker (S/P rotor V) and fixed to the shaker by tape. Vials were then shaken at 150 rpm at room temperature. At 0 hours, 2-4 hours, and 18 hours, 150~1 samples were withdrawn in a biosafety cabinet using a 1 mL syringe fitted with WO 94/17819 21 ~ r n n ~ PCT/US94/01239 U ~ ~

a 21 gauge needle. The starting, in process, and ending soluble rBPI23 concentrations were determined by an ion exch~n~e HPLC assay and visual observation of clou~linPss of the solution was also recorded. The results are shown below in Table 1 in which acceptable stability was dele.~ ~l by visual 5 inspection after the shake test.
Testing of protein ~ lions comprising single suRactants showed good results for use of octoxynol-9 (I~ITON X-100, Rohm & Haas), laureth-4, (BRU 30, ICI ~merir~c), poloxamer 403 (PLURONIC P123, BASF
Wyandotte) and telomere B monoether with polyethylene glycol (ZONYL FSO-10 100, E.I. DuPont de Nemours). While these surfact~ntc are capable of reducingsurface tensions to low levels, they are not included in approved par~nleldl ph~rm~ceutic~l~ due to suspected toxic effects or unknown biOcollll)a~ibility.
Testing of other surfactants as shown in Table 1 shows that surfact~nts producing a surface tension lower than 35 mN/m are capable of 15 stabilizing rBPI at surf~t~nt concentrations of 0.1% . This example further shows that both polysorbate 80 (TWEEN 80) and poloxamer 188 (PLURONIC F-68) were inc~p~hle of stabilizing the protein ~ ion alone under the shake test conditions employed. The incoll,ola~ion of polysorbate 80 did, however, have theeffect of clarifying a cloudy solution of BRIT 30 which is not readily water soluble 20 without the help of an additional solubilizer.

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TABLE 1 c~
Visual Observation rBPI23 Conc. by HPLC
(mg/mL) Surface Tension mN/m at Sur-0.1 % Conc.factant at Room Concen- Stability as Sur- Temp. intration in Determined byExp factant Water (w),Form. Visual No. Used Buffer (b)l Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection O
ZONYL 17(W) 0.100% ---- Clear 0.96 ---- 1.00 Stable 2 PS-80 41(b~ 0.100% ---- Cloudy 1.11 ---- 0.02 Unstable 3 BRL~ 30 27.5(b~ 0.500% Cloudy Cloudy 1.08 ---- 1.14 BRL~ 30 alone 1S cloudy.

4 TRITON 32~h~ 0.100% Clear Clear 1.001.01 0.98 Stable PLUR 34.3(w) 0.100% Clear Clear 1.081.08 1.08 Stable P123 c~
6 BRIJ ---- 0.1%/ Clear Clear 1.191.21 1.17 Stable 30/PS-80 0.125 %

Visual Observation rBPI23 Conc. by HPLC
(mg/mL) Surface Tension mN/m at Sur-0.1% Conc. factant at Room Concen- Stability as Sur- Temp. in tration in Delellllined by Exp factantWater (w), Form. Visual No. UsedBuffer (b)' Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection c~
7 PLUR 46~b) 0.100% Clear Haze 1.23 1.22 0.95 Marginal o F-68 stability. c Slight haze, specks.
8 PLUR 44(b) 0.200% Clear Haze ---- ---- 1.04 Marginal F-68 Stability.
Slight haze with a few specks. y ~' ~

o ~
TABLE I
Visual Observation rBPI23 Conc. by HPLC
(mg/mL) Surface Tension mN/m at Sur-0.1% Conc. factant at Room Concen- Stability as Sur- Temp. in tration in Dele~ ed by Exp factant Water (w), Form. VisualNo. Used Buffer (b)' Buffer 3-4 hr 18 hr 0 hr3-4 hr 18 hrInspection 9 - PLUR 47(b) 0.1%/ Clear Clear 1.14 1.09Stable.
F-68/ 0.001% Crystal clear PS-80 with a few specks.
urface tensions with , ~ w are obtained from the sul~i _t r lul~r. Surface tensions with s, -.sc.i~)l b are obtained eA~,~. Iy u sing Wilhelny plate method.

WO 94/17819 21~ ~ ~ 0 5 PCT/US94/01239 .",~

F.~ 2 In this example, additional comparisons were carried out according - to the methods of Example 1 using various s~ rt~nt~ alone and in combination to stabili7e a rBPI23 ~l~alion. The results are shown below in Table 2 in S which acceptable stability was delGl,llined by visual inspection after the shake test.
The results, particularly those of t;,~yclill,ents 52-58 show the unexpected utility of the combination of poloxamer 188 and polysorbate 80 for stabilizing the rBPI23 composition at concentrations where either surfactant alone is incapable of equivalently stabilizing the m~t~.ri~l under the conditions of the test. The 10 experiments show that various combinations of concentrations of the two surfactants exhibit synergistic effects but that the plerel,ed combination specific to rBPI23 at 1 mg/mL concentration is that having 0.1 % by weight poloxamer 188 and 0.001 % by weight polysorbate 80 in citrate buffered saline (0.02 M citrate,0.15 NaCl, pH 5.0). The results with polysorbate 80 at concentrations lower than0.001 % produced prompt cloudiness after 18 hours of ~h~king, but with only a small loss of protein as determined by ion-exchange HPLC MA7C column (Bio-Rad, Hercules, CA). Nevertheless, the cloudiness is unacceptable for appearance and suggests lowered stability. Testing with polysorbate 80 at concentrations of0.005% and above all give good stability at up to 18 hours of shaking with little 20 sign of protein loss by HPLC. Nevertheless, these higher concentrations of polysorbate 80 may provide less stability during long-term storage at 4~C and atstress telllpel~lules of ambient room temperature or above.

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Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant Used Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection ZONYL 0.100% ---- Clear 0.96 ---- 1.00 Stable 2 PS-80 0.100% ---- Cloudy 1.11 ---- 0.02 Unstable 3 Dextran Sulfate 1 mg/mL ---- Cloudy ---- ---- 0.00 Unstable 4 Glycerol 10.0% ---- Cloudy 0.86 ---- 0.02 Unstable HSA 5.0% ---- Cloudy 0.92 ---- 0.00 Unstable 6 Control- ---- ---- Cloudy 1.13 ---- 0.03 Unstable 5 mL Fill Volullle 7 Control ---- ---- Clear 1.13 ---- 1.04 Stable. One 8.4 mL speck of pre- C
(complete) cipitate.
Fill Volume Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant Used Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 8 Control- ---- Cloudy Cloudy 1.16 0.21 0.00 Unstable ,_ S mL (partial) Fill Volume 9 T~ITON 0.500% Clear Clear 1.04 0.99 1.11 Stable c~

PS-80 0.500% Clear Cloudy 1.12 0.95 0.59 Unstable I l PLURONIC 0.500% Clear Clear 1.15 ---- 1.13 Stable 12 BRIJ 30 0.500% Cloudy Cloudy 1.08 ---- 1.14 BRLJ 3~1one is cloudy.
13 TRITON 0. lO0% Clear Clear 1.00 1.01 0.98 Stable c c~ o o ~ -Visua~ ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant UsedBuffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 14 'TRITON 0.010% Slt. Haze Cloudy 0.96 0.84 0.04 Unstable PLURONIC 0.100% Clear Clear 1.08 1.08 1.08 Stable ~,~

16 PLURONIC 0.100% Clear Clear 1.23 1.26 0.94 Stable 17 PLURONIC 0.050% Clear Slt. Haze 1.21 1.18 1.11 Unstable 18 PLURONIC 0.010% Cloudy Cloudy I .14 0.00 0.00 Unstable 19 BRIJ 30/ 0.1%/ Clear Clear 1.19 1.21 1.17 Stable PS-80 0.125%
BRL~ 30/ 0.075%/ Clear Clear 1.22 1.20 1.18 Stable ~
PS-80 0.094% O

~, ~

o ~3 Visual ObservationConc. by HPLC
(mg/mL) Seability as Surfactant Detennined Exp Conc. in Form. by Visual No. Surfactant UsedBuffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hrInspection 21 BRIJ 30/ 0.03%/ Slt. Haze Cloudy 1.20 1.05 0.41Unstable PS-80 0.038% _, 22 BRIJ 30/ 0.01%/ Ctoudy Cloudy 1.14 0.48 0.00Unstable '~
PS-80 0.013 % O
23 PLURONIC 0.100% Clear Slt. Haze 1.23 1.22 0.95Marginal ~
F68 Stability 24 PLURONIC 0.100% Clear Slt. Haze ---- ---- 1.00Marginal F68 Stability PLURONIC 0.150% Clear Slt. Haze ---- ---- 1.06Marginal F68 Stability 26 PLURONIC 0.200% Clear Slt. Haze ---- ---- 1.04Marginal F68 Stability v-' o ~-Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant UsedBuffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 27 PLURONIC 0.300% Clear Slt. Haze ---- ---- 1.10 Stability 28 PLURONIC 0.500% Clear Slt. Haze ---- 1-08 Stabiglity 29 PLURONIC 0.070% Clear Clear Stability BRIJ 30/ 0.05%/ Clear Clear 1.04 1.01 1.01 Stable PS 80 0.063%
31 PLURF68/ 0.1~/ Clear Clear 1.05 1.06 1.10 Stable PS-80 0. 1 %
32 PLUR F68/ 0.1%/ Clear Clear 1.05 1.05 1.03 StableBRIJ 30 0.03% c 33 PLUR F68/ 0.1%/ Clear Clear 1.06 1.04 1.05 Stable BRIJ30 0.01%

I i Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant Used Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 34 PLURONIC 0.100% Cloudy C1Oudy 1.07 0.87 0.56 Unstable ,_ PLURONIC 0.100% Cloudy Cloudy 1.04 0.77 0.39 Unstable cn~

36 PLURONIC 0.100% Clear Cloudy 1.04 0.87 0.55 Unstable c 37 PLURONIC 0.100% Clear Clear 1.06 1.04 0.98 Marginal F127 Stability 38 PLUR F68/ 0.075%/ Clear Clear 1.12 ---- 1.11 Stable BRIJ 30 0.01 %
39 PLUR F68/ 0.05 %/ Clear Clear 1.12 ---- 1.09 Stable C
BRIJ 30 0.01 % ~, s~ ~

O ~D

Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant Used Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection PLUR F68/ 0.025~/ Clear Clear 1.10 ---- 1.04 Stable BRIJ30 0.01~ ~
41 PLUR F68/ 0.01 ~/ Cloudy Cloudy 1.07 ---- 0.64 Unstable O
BRIJ 30 0.01 ~
42 PLURONIC 0.100% Clear Clear 1.12 ---- 0.93 Marginal F127 Stability 43 PLURONIC 0.075 ~ Clear Clear 1.10 ---- 0.61 Unstable 44 PLURONIC 0.050~ Clear Slt. Haze 1.09 ---- 0.20 Unstable PLURONIC 0.025~ Slt. Haze Cloudy 1.07 ---- 0.00 Unstable F127 c 46 PLURONIC 0.010~ Cloudy Cloudy 1.06 ---- 0.00 Unstable ~o Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant UsedBuffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 47 PLUR F68/ 0.05%/ Clear Clear 1.04 ---- 1.01 Stable ~, BRIJ 30 0.01%
48 PLUR F68/ 0.05%/ Clear Clear 1.01 ---- 1.01 Stable ~t BRIJ 30 0.008%
49 PLUR F68/ 0.05%/ Clear Clear 1.00 ---- 1.03 Stable BRLl 30 0.005%
PLUR F68/ 0.03%/ Clear Clear 1.06 ---- 0.99 Marginal BRIJ 30 0.008% Stability Sl PLUR F68/ 0.03%/ Clear Cloudy 1.01 ---- 0.79 Unstable BRIJ 30 0.005%
52 PLUR F68/ 0.1%/ Clear Clear 1.14 ---- 1.11 Stable. c PS-80 0.05% A few specks. .p 'o o ~

Visual ObservationConc. by HPLC
(mg/mL) Stability as Surfactant Determined Exp Conc. in Form. by Visual No. Surfactant Used Buffer 3-4 hr 18 hr 0 hr 3-4 hr 18 hr Inspection 53 PLUR F68/ 0.1 %/ Clear Clear 1.14 ---- 1.11 Stable.
PS-80 0.01% A few specks.

54 PLUR F68/ 0.1%/ Clear Clear 1.15 ---- 1.10 Stable.
PS-80 0.005% A few specks.

PLUR F68/ 0.1%/ Clear Clear 1.14 ---- 1.09 Stable.
PS-80 0.001% A few specks.

56 PLUR F68/ 0.1%/ Clear Cloudy 1.12 1.09 1.02 Unstable PS-80 0.0005 %
57 PLURF68/ 0.1%/ Slt. Haze Cloudy 1.09 1.09 1.02 UnstablePS-80 0.0001 % c 58 PLUR F68/ 0.05%/ Clear Cloudy 1.08 1.00 0.72 Unstable PS-80 0.001 %

- i 23 '~
Example 3 In this example, a study was conducted to compare the efficacy of rBPI23 formulated with and without the preferred formulation of the invention in an actinomycin-D sensitized mouse model according to Pieroni et al., Proc. Soc. Exp.
Biol. & Med.; 133, 790 (1970). According to this example, ICR mice were ~m; nl stered an intravenous injection of actinomycin-D (800 ~g/kg). Immediately thereafter, groups of 15 mice each received an injection of one of several doses of rBPI23 [BR-1] characterized by about 1 to about 199 of the first 199 amino acids of the mature human BPI
molecule and produced according to the methods o-f Theofan et al., WO 94/18323 at 1 mg/mL in citrate buffered saline (0.2 M citrate, 0.15 M NaC1, pH 5.0). The mouse injections were at dosages of 0.03, 0.1, 1.0 and 3.0 mg/kg. As a control, some animals received the formulation buffer with or without the poloxamer and polysorbate surfactants. Deaths were recorded over seven days.
The results are shown in Figures 1 and 2. Figure 1 shows the number of mice surviving on each study day in the buffer and 3.0 mg/kg rBPI23 treatment groups. For both buffer groups (with or without poloxamer and polysorbate surfactants), mortality was 80~ overall. In contrast, rBPI23 in the presence of excipients was even more potent than either buffer or rBPI23 without excipients. Figure 2 summarizes the data for the different dose groups at day 7 (final survivors). Beginning at the 0.1 mg/kg dose level, rBPI23 formulated with the preferred surfactant formulations provided significantly greater protection to the lethal effects of LPS (P~0.05 or better) than did ~BPI23in the absence of added excipients.
A

_ 24 Example 4 ~ 7 ~ 5 In this example, experiments were conducted to determine the turbidity of various rBPI-containing pharmaceutical compositions with and without the preferred surfactant formulation of the invention. In this context, turbidity refers to the tendency of pharmaceutical compositions to engage in unfolding (i.e., loss of tertiary protein structure) and/or particle formulation (interactions between individual proteins to form larger (~ 10 ~m) particles). The pharmaceutical compositions tested contain-ed either rBPI(1-l99)ala132, rBPI(1-193)ala132 or various samples of rBPI23 produced according to co-owned and co-pending WO 94/18323 in either a citrate buffer (20 mM sodium citrate/150 mM sodium chloride, pH 5.0) or a citrate buffer containing 0.1~ poloxamer 188 and 0.002~ polysorbate 80.
Samples were analyzed to determine their resistance to turbidity over time at increasing temperature and at pH 7Ø
Prior to analysis, all samples were diluted to a concentration of 0.1 mg/mL in 50 mM potassium phosphate at pH 7Ø Turbidity measurements were obtained by placing samples in quartz cuvettes for use in a Shimadzu W-160 W-Vis spectrophotometer equipped with a temperature-controlled cuvette holder attached to a recirculating water bath. Upon equilibrating the cuvette holder at 57~C, absorbance at 280 nm was measured to confirm that samples had been diluted to the proper concentration. Following this, the absorbance of samples at 350 nm was measured every 2 minutes for 1 hour to determine the change in absorbance over time.
Results are presented in Figure 3 showing a lower rate of change in turbidity (i.e., a lower rate of increase in absorbance over time), indicating increased stability against the formation of particles. As shown in Figure 3, the addition of the preferred combination of surfactants resulted in increased stability (resistance to particle formation) of all compositions tested. Moreover, the rBPI(1-l99)ala132 and rBPI(1-193)ala132 exhibited greatly improved resistance to particle formation relative to wild-type compositions [rBPI23].

.~.

~,~

Example 5 In this example surface tension measurements were made of polysorbate and poloxamer s~ t~nt~ or combinations of the two in solutions of the BPI protein product rBPI2l~cys according to the procedure set out in the S Kr;iss Digital Tensiometer KlOST Users ~nu~l, Chapter 4: MP~ nng with the Plate. A decrease in surface tension indic~tes an increase in the surface activity of the s~ ct~nt, which has conventionally been thought to be the mech~ni~m by which s~ et~nt~ stabilize proteins. These procedures established that poloxamer surfactants provide advantageous results by a dirrt;lc,l~ and unexpected 10 mechanism.
Specifically, a 2 mg/mL solution of unformulated rBPI2,/\cys (lot 30216) was diluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 5.0 rendering a 1 mg/mL solution. 15 mL of this solution was placed into a 50 mL
glass beaker co..~ g a mini stir bar. Surf~ct~nt~ poloxamer 188, polysorbate 80, or combinations of both were added incrPmPnt~lly up to 0.10%. Before each surface tension mea~u~n,e,ll, the pl~li.. plate was heated above the reducing zone (blue flame) of a gas burner until the plate just began to glow red. The pl~tinllm plate was heated for about 10 to 15 seconds while turning the plate from side to side and then suspended back into the instrument. Each addition of 20 surfactant was gently mixed using a m~gnptic stirrer and the solution was allowed to stand for 2 mil~ules on the thermostat vessel equilibrated at 4.6~C. The value for the surface tension was read after five ..~ es.
The first part of this t;~l~e.;.l-ent evaluated the surface activity of the surfactants alone in buffer. Using the citrate saline buffer (20 mM sodium citrate, 150 mM sodium chloride, pH 5.0) as the baseline, sl-rfact~nts were added incrementally. Figure 4 is a plot of surface tension dependence on surfactant concentrations; the corresponding data is presented in Table 3. The open squaresrepresent the citrate saline buffer in varying concentrations of poloxamer 188 while the closed circles represent the same buffer in varying concentrations of 2l~aos polysorbate 80. The citrate-saline buffer solution alone had a surface tension of about 75 mNlm at 4.6~C, similar to H2O. With increasing concentrations of surfactants, the buffer solution showed decreasing surface tension. With 0.10%
poloxamer 188, the surface tension of the solution was 55 mN/m. On the other hand, with 0.10% polysorbate 80, the surface tension of the solution was 45 mNlm. The decrease in surface tension infli( ~tPS an increase in the surface activity of the s~ t~nt i.e., the lower the surface tension, the higher the surface activity. The results inflir~te that polysollalt; 80 is more surface active thanpoloxarner 188.
In the second part of the experiment, the surface activity of rBPI
~cys in the presence of s~ ct~nt~ was ev~ t~l The results show that rBPI2l ~cys at 1 mglmL in citrate saline buffer, pH 5.0, is surface active with a surface tension of about 54 mNlm at 4.6~C. The addition of polysorbate 80 (PS80) alone up to 0.0005 % did not change the surface tension of rBPI2, ~cys solution either(Figure 4, closed triangles). At concentrations of polysorbate 80 excee~ling 0.0005 %, the surface tension of rBPI2l Acys follows that of buffer with PS80 alone (no BPI), in which the surface tension of the solution decreases as the concentration of polysorbate 80 is gradually increased. For buffer with PS80 alone, the surface tension of 54 mNlm was reached when the PS80 concentration was increased from 0.0005 % . These results in-lic~te that when PS80 concentration is less than 0.0005 %, the surface activity of the solution is domin~t~d by rBPI2lAcys. On the other hand, at PS80 concentration above 0.0005 %, the surface activity of the solution is modulated by polysorbate 80 the addition of poloxamer 188 (F68) alone to rBPI2l ~cys up to 0.10% did not change the surface activity of rBPI2,/\cys solution signif1c~ntly (Figure 4, open triangles).

2 3 4 s ~ 7 ~ g . ~
~68 .Bt~r . % PX8~1 ~uffer ~ F68 . ~ %P~ . ~P~O ~Cy9 +F~;g . +P~U . +F~ . . +O.I~F6~ +
iml~l/,n~. ~m~ ml ~mN/Il~) . .+P~O . ~mN/in~
~ . ~ilJN~iii~ . : .
I 0.00000 75.4 0.0000075. I0.0000054.2 0.00000 53.7 0.00000 54.9 2 0.00001 74.9 0.0000166.8 0.0000154.7 0.00001 53.4 0.00001 55.0 3 0.00003 74.3 0.0000260.0 0.0000254.2 0.00002 53.3 0.00002 53.2 4 0.00005 68.2 0.0000360.0 0.0000354.9 0.00003 53.9 0.00003 53.3 0.00007 65.9 0.0000560.0 0.0000454.8 0.00004 53.9 0.00004 52.8 r~

6 0.00010 64.0 0.0000757.4 0.0000555.0 0.00005 53.5 0.00005 52.4 2!;~

7 0.00013 65.8 0.0001056.6 0.0000655.2 0.00006 53.5 0.00006 53.3 0 8 . 0.00015 65.4 0.0001557.2 0.0000755.4 0.00007 53.4 0.00007 53.6 C
9 0.00017 66.5 0.0002056.7 0.0000854.8 0.00008 53.8 0.00008 53.8 9 0.00020 65.7 0.0005055.6 0.0000955.0 0.00010 53.4 0.00009 53.2 ~-I l 0.00023 66.0 0.0007055.3 0.0001054.9 0.00020 53.5 0.00010 53.5 12 0.00027 64.4 0.0010054.2 0.0003055.3 0.00030 53.2 0.00020 53.2 13 0.00030 63.8 0.0030052.7 0.0005054.5 0.00050 52.3 0.00030 53.0 14 0.00033 64.1 0.0070049.2 0.0007055.5 0.00070 51.5 0.00050 52.0 0 00037 63.1 0.0100048.3 0.0010054.9 0.00100 51.0 0.00070 51.2 V
16 0.00040 64.2 0.0300046.5 0.0050054.9 0.00200 50.6 0.00100 50.5 o 17 0.00043 61.8 0.0700045.3 0.0100055.4 0.00500 50.1 0.00130 50.4 ~, 'o O r 2 3 4 5 ~; 7 # ~ ~û
% ~'68 Fuffer c~ P~n . Buffe~ ~ Fh~ ~y~ %PS~n ~Cjs .. ~Pg~0 ~Cj6.
+~6W ~PS80 +~h$ +0;1%F~X . +Y~}
ImN/m) ~mN/m) ImNlm~ . . .. +P~8~. ImNtm) ~ ~nNlm) IX n (NO~7 fi2.4 0.10000 45.4 O.OS()00 53.6 o olnoo ~8.6 0.00170 49.~
19 0.00050 63.1 0.10000 53.7 0 05000 45.6 0.00200 48.8 0.00060 61.6 0.10000 45.0 0.00500 47.7 21 0.00070 62.5 0.01000 46.7 oo S 22 0.00080 62.0 0.05000 45.4 23 0.00100 61.7 0.10000 45.0 24 0.00300 61.2 0.00500 59.3 26 0.00700 58.9 0 27 0.01000 58.4 28 0.03000 56.6 29 0.07000 56.1 E

71VO 94/17819 2 1 5 S O 0 5 ~usg4~0~9 F..~ ,lc 6 Protein samples were analyzed by Dirrt;lC~Iial SC~nning Calorimetry (DSC) to study the unfolding (or den~lu.~.lion) of the protein. The starting m~tt~ri~l~ for DSC analysis were identi~l to those used in the surface S tension measurement. A series of rBPI2l~cys solutions was pl~cd with varying concentrations of surf~ct~ntc, poloxamer 188, polysoll,ate 80 or combinations ofboth, and diluted with buffer (20 mM sodium citrate, 150 mM sodium chloride, pHS.0) to give a final rBPI2l~cys concentration of 1 mg/mL. A series of buffer solutio~s was also ~lcpalcd with surf~t~nt~ at the sadme concentrations as in the 10 BPl2,~cys solutions to serve as blanks for DSC. Each solution was filtered and placed into a 2 mL sterile plastic vial. The samples were packed into a 4~C coldbox until subjected to DSC Analysis.
The behavior of rBPI2l~\cys was evaluated as the tell-peldlulc of the solution was gradually increased from ambient telllpclalure to about 90~C, at a 15 rate of 1 ~C per minute. As the leln~e.~lurc is increased two events occur. The first event is an unfolding reaction, which is endollle~lllic, and is illustrated by an upward peak in the scans. The second event is precipitation, which is exothermic, and is depicted by a downward peak in the scans. In the scans depicted in Figs. 5, 6 and 8-10, each scan is offset to f~ilit~tP analysis of data.
20 In the rBPI2l~\cys solution not con~ -g s~lrf~ct~nt~ (Figure 5, Scan 1) the unfolding of the protein at 65~C was followed immediately by the second event, precipitation of the protein at 66 to 67~C.
With low poloxamer 188 (PLURONIC~ F68) concentrations ranging between 0.001% to 0.01%, the unfolding and precipitation events are 25 similar to the rBPI2l~cys solution without sl-rfact~ntc (Figure 5, Scans 2 to 5), i.e.
as BPI2l~cys unfolds, precipitation takes place immediately. With poloxarner 188 concentrations excee~ling 0.05%, the unfolding of rBPI2l1\cys still occurs at 65~C, but precipitation does not occur until the telllpelalulc reaches 85~C (Figure - 5, Scan 6). Figure 6 shows that at poloxamer 188 concentrations between 0.01%
30 and 0.05 %, there is a gradual transition of delayed precipitation of unfolded BPI.
These results suggest that at poloxamer 188 concentrations higher than 0.01 ~, WO 94/17819 PCT/US94/0l239 21~5~0~

unfolded rBPI2lAcys can be stabilized and the occurrence of precipitation is delayed. A plot of cle..~ ion and precipitation temperature dependence over the s~-rfact~nt (poloxamer 188) concentration is shown in Figure 7. The effects of poloxamer 188 appear to delay the pr~i~ dlion of rBPI2lAcys to a higher S te~ clalulc but not to stabilize its native structure as the Tm (denaLuldlion temperature) and ~H (energy of ~e~ lion) did not change.
rBPI2l ~\cys formul~t~d with polyso,l,ale 80 at concentrations up to 1 % was likewise analyzed. The isotherms were similar to rBPI2lAcys solution without sl-rf~ct~nt~ (Figure 8: Scans 1 and 8-13, Figure 9: Scans 11, 12).
10 Polysorbate 80 did not m~int~in the rBPI2l Acys in solution at higher tell~pelalul~,s. The stabilization of unfolded rBPI2lAcys is thus unique to poloxamer 188.
The two formulations using combined poloxamer 188 and polysorbate 80, namely 0.1 %F68/0.001 %PS80 and 0.1 %F68/0.002 %PS80, showed the same scan profile as rBPI2l Acys co.. ~ g 0.05 % and 0.1 %
PLURONIC F68, with unfolding at 65~C and precipitation at 85~C (Figure 8:
Scans 14, 15).
In addition to ~let~ g the melting behavior of rBPI2l ~cys, resc~nning was done with rBPI2l~cys formulations co--l;~inillg 0.05% and 0.10%
poloxamer 188 to dele~ e if unfolding is a reversible process. The temperature of the rBPI2lAcys solution was first increased to 75~C (l~lllpelalul~ after delldlu~dlion/unfolding but before p,eci~ildlion), then was cooled down for repeat sc~nnin~. Figure 10 shows that the addition of poloxamer 188 to rBPI2lAcys does not make unfolding reversible. Profiles AS,l and A6,1 show the sc~nning to 75~C, while profile A5,2 and A6,2 are repeat Sc~nnin~ after cooling the system from 75 ~C. If unfolding were a reversible process, 6 and 7 scan profiles would have been obtained.
The experimental results described above demonstrate that poloxamer surfactant alone is capable of stabilizing BPI-related polypeptides in wo g4,l78l9 2 1 S ~i O O S PCT/US94/01239 ~ ,....

solution and delaying the occurrence of precipitation by a mech~ni~m that does not appear to involve modulation of the surface tension of the aqueous solution.- This p~ )elly is unique to poloxamer because other s~ etantc such as polysoll,ale 80 do not affect the precipitation phenomenon and do involve S modulation of the surface tension of the aqueous solution.

Example 7 The rate of rBPI2l ~cys ~leci~i~lion during shipping was simulated in the laboratory by adjustment of the speed of the horizontal shaker. During five - 10 cycles of surface shipping, about 70% of the ul~o~ ted (snrfart~nt free) rBPI2, ~cys precipitated. By varying the speed (rpm) of the flat-bed shaker, shake tests were then co~ ed such that 70 to 90% of ul~o~ lated rBPI2~ ~\cys subjected to the shake test precipitated. No rBPI2, ~cys was precipitated when the unrollllulated product was shaken on a flat bed shaker at 110 rpm or less for 18hours at 4~C. Shaking at 140 rpm (rather than at 150 Ipm as in Examples 1 and 2) most closely ~im~ ted the agitation occurring during five cycles of surface transport. Changes in the flow dynamics of the liquid in the vial are subst~nti~lly different at 140 rpm versus 150 rpm. Compositions including various concentrations of surfactant combinations were screened using the 140 rpm shake 20 condition and the results obtained are set out in Table 4. It was determined that the optimal s-~ ct~nt concentrations for protection from precipitation were 0.2 %
poloxamer 188 with 0.002% polysorbate 80 and 0.15% poloxamer 188 with 0.005% polysorbate 80.

Table 4 Summary of Shake Test at 140 rpm for rl~PI2, ~cys at 4~C
Poloxamer PS80 Visual Concelltratioll by MA7C HPLC (Illg/llll) 188(%) (%) (see note) Before AfterLoss (%) ., 0.005 5 2.14 1.54 28 0.075 0.010 4 2.10 1.56 26 , , 0.020 1 2.14 1.68 21 C~ ~ .
~ 0.002 5 2.24 1.85 17 m rn~ 0.005 4 2.14 1.85 14 ~ ' ~ _~ o.loo o.olo 1 2.10 1.87 1l , c~
c, C 0.020 1 2.13 1.94 9 o m 0.002 3 2.19 1.92 12 ~n 0.005 2 2.08 1.95 6 0.150 0.010 1 2.19 1.94 11 0.020 1 2.06 1.96 5 T~ble 4 Summary of Shake Test at 140 r pm for r I~l'It, ~cys at 4 C
_ . ~
r~, Poloxalller PS80 Visllal Concelllralioll l)y I\/IA7C IlPLC (n~g/nll) 188(%) (%) (see note) Before AlterLoss (%) 0.002 2 2. 19 1 .98 10 0.200 o.005 1 2.19 1.95 11 ~ - 0. () I () I 2 . 22 1 . (~512 w r~i i . ~, ~~ No~e: Tlle scoring for visual ol)servalioll is as tollows:
l. Clear 2. Clear witll few particulales o ~- ~ ril 3. Sliglltly ll~zy ~rr 4. H~y 5. Clo~l(ly WO 94tl7819 PCT/US9410123g '~ _ 2ls~no~

Based on the above data, a p-c;îelr~d formulation for 2 mg/mL
rBPI21 Acys to be stored at 4~C would contain 5 mM citrate, 150 mM NaCl, pH
5 . O, 0.2 % poloxamer 188 and 0.002 % polysoll,ale 80. An ~ltt-rn~tive formulation for 2 mg/ml rBPI2, ~cys to be stored at 4~C would contain 5 mM citrate, 150 mM
NaCl, pH 5.0, 0.15% poloxamer 188 and 0.005% polysorbate 80.
In summary, agglegation/precipitation is one of the major causes of protein instability and can occur when proteins at the air-liquid interface unfold and expose hydrophobic domains. If left unplvlected, proteins self-associate through the interaction of the exposed hydrophobic domains, res--lting in ag~r~alion and/or precipitation. With the use of the surfactants and surfactant combinations of the invention, protein can be stabilized in two ways. First, exposed hydrophobic regions at the air-liquid interface are shielded by poloxamer s~ ct~nt~. Second, additional stabilization can be provided by polysorbate s--rf~ct~nt~ through conventional modulation of the surface activity of the solution.
Numerous mo-lifi-~tions and variations of the above-described invention are expected to occur to those of skill in the art. Accordingly, only such limit~tions as appear in the appended claims should be placed thereon.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a bactericidal/permeability-increasing (BPI) protein or a biologically active fragment, analog or variant thereof, in combination with a polysorbate surfactant and a poloxamer surfactant at solubilizing/stabilizing concentrations.

2. A pharmaceutical composition comprising a biologically active amino-terminal fragment of bactericidal/permeability-increasing (BPI) protein or an analog or variant thereof in combination with a polysorbate surfactant and a poloxamer surfactant at solubilizing/stabilizing concentrations.

3. A pharmaceutical composition according to Claim 1 or 2 wherein the poloxamer surfactant is characterized by an HLB
value greater than 14 and by having a surface tension between 10 and 70 mN/m when measured at room temperature and at a concentration of 0.1%.

4. A pharmaceutical composition according to Claim 1 or 2 wherein the poloxamer surfactant is poloxamer 188.

5. A pharmaceutical composition according to Claim 1 or 2 wherein the polysorbate surfactant is characterized by an HLB of greater than 10 and by having a surface tension between 10 and 70 mN/m as measured at room temperature and at a concentration of 0.1%.

6. A pharmaceutical composition according to Claim 1 or 2 wherein the polysorbate surfactant is polysorbate 80.

7. A pharmaceutical composition according to any one of the preceding claims, wherein the poloxamer surfactant is present at a concentration of from 0.01% to 1% by weight.

8. A pharmaceutical composition according to any one of the preceding claims, wherein the polysorbate surfactant is present at a concentration of from 0.0005% to 1% by weight.

9. A pharmaceutical composition according to any one of the preceding claims, wherein the poloxamer surfactant is present at a concentration of from 0.1% to 0.2% by weight and the polysorbate surfactant is present at a concentration of 0.002% by weight.

10. A pharmaceutical composition comprising a bactericidal/permeability-increasing (BPI) protein or a biologically active fragment, analog or variant thereof, in combination with a poloxamer surfactant at a solubilizing/stabilizing concentration.

11. A pharmaceutical composition comprising a biologically active amino-terminal fragment of bactericidal/permeability-increasing (BPI) protein or an analog or variant thereof in combination with a poloxamer surfactant at a solubilizing/stabilizing concentration.

12. A pharmaceutical composition according to Claim 10 or 11 wherein the poloxamer surfactant is characterized by an HLB value greater than 14 and by having a surface tension between 10 and 70 mN/m when measured at room temperature and at a concentration of 0.1%.

13. A pharmaceutical composition according to Claim 10 or 11 wherein the poloxamer surfactant is poloxamer 188.

14. A pharmaceutical composition according to any of Claims 10 to 13 wherein the poloxamer surfactant is present at a concentration of from 0.1% to 0.2% by weight.

15. A method for solubilization/stabilization of a polypeptide that is a bactericidal/permeability-increasing (BPI) protein or a biologically active fragment, analog or variant thereof in aqueous solution, comprising the step of contacting said polypeptide with a poloxamer surfactant and a polysorbate surfactant at solubilizing/stabilizing concentrations.

16. A method according to Claim 15, wherein the poloxamer surfactant is poloxamer 188 and the polysorbate surfactant is polysorbate 80.

17. A method for solubilization/stabilization of a polypeptide that is a bactericidal/permeability-increasing (BPI) protein or a biologically active fragment, analog or variant thereof in aqueous solution, comprising the step of contacting said polypeptide with poloxamer surfactant at a solubilizing/stabilizing concentration.

18. A method according to Claim 17, wherein the poloxamer surfactant is poloxamer 188.