An assessment of the mechanistic differences between two integrin α4β1 inhibitors, the monoclonal antibody TA-2 and the small molecule BIO5192, in rat experimental autoimmune encephalomyelitis

Article abstract of DOI:10.1124/jpet.102.047332

Integrin α₄β₁ plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of α₄β₁ inhibitors, the anti-rat α₄ monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-{[1(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino} 4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}amino)- pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (K_D of <10 pM) inhibitor of α₄β₁. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in α₄ integrin expression on the cell surface, which resulted from internalization of α₄ integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface α₄β₁. Our results with BIO5192 indicate that α₄β₇ does not play a role in this model and that blockade of α₄β₁/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to α₄β₁ from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of α₄β₁ integrin, will be a valuable reagent for assessing α₄β₁ biology and may provide a new therapeutic for treatment of human inflammatory diseases.

Full text of DOI:10.1124/jpet.102.047332

0022-3565/03/3053-1150–1162$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 305:1150–1162, 2003
Vol. 305, No. 3
47332/1066457
Printed in U.S.A.
An Assessment of the Mechanistic Differences Between Two
Integrin ␣4␤1 Inhibitors, the Monoclonal Antibody TA-2 and the
Small Molecule BIO5192, in Rat Experimental Autoimmune
Encephalomyelitis
D. R. LEONE, K. GIZA, A. GILL, B. M. DOLINSKI, W. YANG, S. PERPER, D. M. SCOTT, W.-C. LEE, M. CORNEBISE,
K. WORTHAM, C. NICKERSON-NUTTER,1 L. L. CHEN, D. LEPAGE, J. C. SPELL,2 E. T. WHALLEY, R. C. PETTER,
S. P. ADAMS,3 R. R. LOBB, and R. B. PEPINSKY
Biogen, Inc., Cambridge, Massachusetts
Received December 6, 2002; accepted March 5, 2003
ABSTRACT
Integrin ␣4␤1 plays an important role in inflammatory processes by Both inhibitors induced leukocytosis, an effect that was used as a
regulating the migration of lymphocytes into inflamed tissues. pharmacodynamic marker of activity, and both were efficacious in
Here we evaluated the biochemical, pharmacological, and phar- the EAE model. Treatment with TA-2 caused a decrease in ␣4
macodynamic properties and efficacy in experimental autoim- integrin expression on the cell surface, which resulted from inter-
mune encephalomyelitis (EAE), a model of multiple sclerosis, of nalization of ␣4 integrin/TA-2 complexes. In contrast, BIO5192 did
two types of ␣4␤1 inhibitors, the anti-rat ␣4 monoclonal antibody not modulate cell surface ␣4␤1. Our results with BIO5192 indicate
TA-2 and the small molecule inhibitor BIO5192 [2(S)-{[1- that ␣4␤7 does not play a role in this model and that blockade of
(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}- ␣4␤1/ligand interactions without down-modulation is sufficient for
4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}- efficacy in rat EAE. BIO5192 is highly selective and binds with high
amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively affinity to ␣4␤1 from four of four species tested. These studies
studied in rats and provides a benchmark for assessing function. demonstrate that BIO5192, a novel, potent, and selective inhibitor
BIO5192 is a highly selective and potent (KD of Ͻ10 pM) inhibitor of ␣4␤1 integrin, will be a valuable reagent for assessing ␣4␤1
of ␣4␤1. Dosing regimens were identified for both inhibitors, which biology and may provide a new therapeutic for treatment of hu-
provided full receptor occupancy during the duration of the study. man inflammatory diseases.
Integrins are a large family of cell surface receptors that costimulatory signal supporting cell activation (Clark and
mediate cell/cell and cell/matrix interactions and signal trans-
duction. They exist as noncovalent ␣␤ heterodimers of different
combinations of ␣ and ␤ chains and share extensive structural
homology. The leukocyte integrin ␣4␤1 regulates normal lym-
phocyte trafficking (Lobb and Hemler, 1994) and provides a key
Brugge, 1995). During inflammatory responses, it regulates
lymphocyte migration into the damaged tissues and thus has
been recognized as an attractive therapeutic target. In vivo
studies using blocking monoclonal antibodies (Lobb and Hem-
ler, 1994) and inhibitory peptides (Molossi et al., 1995) have
verified the critical role of ␣4␤1 integrins in leukocyte-mediated
inflammation. Numerous EAE models of multiple sclerosis
have been designed to recapitulate important aspects of the
disease and are responsive to ␣4 inhibitors (Yednock et al.,
1992). Recent positive phase II data using the anti-␣4 antibody
1 Current address: Wyeth, Cambridge, MA.
2 Current address: Bayer Corporation, West Haven, CT.
3 Current address: Neogenesis, Cambridge, MA.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.047332.
ABBREVIATIONS: EAE, experimental autoimmune encephalomyelitis; mAb, monoclonal antibody; LIBS, ligand-induced binding site;
VCAM-1, vascular cell adhesion molecule-1; CS1, connecting segment 1; BIO1211, 4-((NЈ-2-methylphenyl)ureido)-phenylacetyl-leucine-
aspartic acid-valine-proline; BIO7662, 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-{2-[4-(3-o-tolyl-ure-
ido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; PBS,
phosphate-buffered saline; TBS, TRIS-buffered saline; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; BOC, t-butoxy-
carbonyl; DMF, dimethyl formamide; HATU, O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate; PBL, periph-
eral blood lymphocyte; PEG, polyethylene glycol; PE, phycoerythrin; NHS, N-hydroxysuccinimide; PK, pharmacokinetics; PD, pharmaco-
dynamics; AUC, area under the curve; ELISA, enzyme-linked immunosorbent assay; MES, 2-(N-morpholino)ethanesulfonic acid; MBP,
myelin basic protein; TNF, tumor necrosis factor; MAdCAM, mucosal addressin cell adhesion molecule; MS, mass spectroscopy.
1150

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1151
natalizumab in patients with multiple sclerosis have validated of action of the two inhibitors has provided a means for better
understanding the role of ␣4␤1 in EAE.
␣4␤1 as an important clinical target (Miller et al., 2003).
The anti-rat ␣4-chain monoclonal antibody, TA-2, has been
used extensively to study the role of ␣4 integrins in models of
inflammatory disease. TA-2 blocked ␣4 integrin-mediated
lymphocyte migration to inflammatory sites and homing to
lymphoid tissues (Issekutz, 1991; Issekutz and Wykretowicz,
1991). In studies of lung inflammation in the Brown Norway
rat, TA-2 treatment blocked ␣4-expressing eosinophil and
neutrophil migration into the pleural cavity and decreased
late airway responses in ovalbumin-sensitized and chal-
lenged rats, suggesting a use in the treatment of asthma
(Taylor et al., 1997; Hojo et al., 1998; Schneider et al., 1999;
Ramos-Barbon et al., 2001). TA-2 was also used in Lewis rats
to probe for ␣4/VCAM-1 (vascular cell adhesion molecule-1)
interactions in experimental autoimmune neuritis, a model
of Guillain-Barre´ syndrome, where it reduced disease sever-
ity and inflammation through a possible block of cell trans-
migration to the damaged nerve (Enders et al., 1998). A more
recent study using TA-2 in this model suggested that at least
part of the effect of the antibody was the result of induction
of apoptosis of P2-specific T-cells (Leussink et al., 2002).
Other studies demonstrated that TA-2 blocked blood poly-
morphonuclear cell migration to the inflamed joint even after
joint inflammation had developed, proving that an anti-␣4
regimen is an effective arthritis treatment (Issekutz et al.,
1996). It is not clear whether the mechanism of action of TA-2
is solely a consequence of its blocking ␣4/ligand interactions
or other mechanisms or both. Although TA-2 is limited to
studies in rats, other anti-␣4 mAbs have been used to further
extend the list of potential uses for an inhibitor of ␣4␤1 (Lobb
and Hemler, 1994). Because anti-␣4 antibodies block both
␣4␤1 and ␣4␤7, it is not possible to rule out involvement of
␣4␤7 in these studies.
Materials and Methods
Cell Lines. The ␣4␤1-expressing human T-cell line, Jurkat (a gift
from S. Burakoff, Dana Farber Cancer Institute, Boston, MA) and
the ␣4␤7-expressing human B-cell line, JY, and the ␣4-expressing rat
cell line, RBL.1 (American Type Tissue Collection, Manassas, VA)
were maintained in culture at 37°C in RPMI-1640 medium and
Earle’s MEM, 1 mM sodium pyruvate, respectively, supplemented
with 10% fetal bovine serum (FBS). K562 cell lines transfected with
either the human ␣9 integrin chain (Diane Leone, Biogen, Inc.) or
with the human ␣2 integrin chain (a gift from M. Hemler, Dana
Farber Cancer Institute) were maintained in RPMI-1640 medium
supplemented with 10% FBS and 1 mg/ml G418. An enriched popu-
lation of the ␣9-K562 cells that exhibited high levels of ␣9␤1 expres-
sion was obtained by fluorescence-activated cell sorting (FACS), and
this subclone was used for all subsequent work. All cells were peri-
odically monitored for high integrin surface expression by FACS
analysis (data not shown).
Synthesis of ␣4␤1 Small Molecule Inhibitors. BIO5192 was
synthesized as previously described (D. M. Scott, manuscript in
preparation). BIO8139, an amine-containing derivative of BIO5192
that was used for conjugation and the development of assays for
assessing ␣4␤1 function, was prepared as follows (see Scheme below).
SOCl2 (14.6 ml, 200 mmol) was added dropwise over a period of 15
min to a suspension of 8.4 g (33.3 mmol) N␣-CBZ-L-2,4-diaminobu-
tyric acid in 200 ml of methanol at 0°C and stirred overnight at 23°C.
The solution was concentrated and redissolved in methanol 3 times,
then dissolved in CH2Cl2, concentrated, and placed under high vac-
uum to give 10.33 g of the methyl ester. The crude ester was dis-
solved in 200 ml of CH2Cl2 and 16.2 ml (116.6 mmol) of triethylamine
was added followed by 8.96 g (41 mmol) di-tert-butyl dicarbonate.
After stirring at 23°C for 4 h, the solution was washed once with 1 N
HCl, once with saturated NaHCO3 solution, and once with saturated
NaCl, then dried (Na2SO4), filtered, and concentrated to give a
yellow syrup. Purification by flash column chromatography gave
9.6 g (26.3 mmol, 79%) of t-butoxycarbonyl (BOC) protected amine as
a colorless syrup. Then 250 mg of 10% Pd/C was added to the solution
of the BOC-protected amine in 100 ml of methanol and was stirred
overnight under 60 psi of H2. The mixture was filtered through a
plug of Celite and concentrated. 6.0 g (26 mmol, 78% yield) of com-
pound 1 (N␥-Boc-L-2,4-diaminobutyric acid methyl ester) as a color-
less oil was recovered, which was used without further purification.
MS: m/z 232 (M ϩ Hϩ).
␣4␤1 mediates cell adhesion by binding to either of two
protein ligands, VCAM-1, or the alternatively spliced con-
necting segment 1 (CS1)-containing fibronectin variant (Os-
born et al., 1989; Wayner et al., 1989). More recently other
potential ligands have been identified (Bayless et al., 1998;
Takahashi et al., 2000); however, the biological significance
of these interactions is less clear. The key residues in
VCAM-1 (QIDSP) and CS1 (EILDVP) necessary for their
interactions with ␣4␤1 have been defined by molecular and
Triethylamine (70 ml, 0.5 mol) was added to 25 g (0.15 mol) of
L-proline methyl ester hydrochloride in 500 ml of CH2Cl2. The resulting
biochemical techniques (Wayner and Kovach, 1992; Wang et white precipitate was removed by filtration, and the filtrate was cooled
to 0°C. 20 ml (0.15 mol) of benzenesulfonyl chloride in 50 ml of CH2Cl2
was added to the filtrate dropwise over a period of 15 min and stirred
overnight at 23°C. The solution was washed with 1 N HCl, 1 NaOH, and
saturated NaCl, then dried (MgSO4), filtered, and concentrated to give
a pale yellow solid. This material was recrystallized 3 times from ethyl
acetate/hexane to give 38.2 g (0.142 mol, 95%) of N-(benzenesulfonyl)-
proline methyl ester (thin layer chromatography versus 2:1 hexane/
ethyl acetate, Rf ϭ 0.35). The methyl ester was dissolved in 500 ml of
methanol. 140 ml (0.28 mol) of freshly-prepared 2 M aqueous LiOH was
added to the solution and stirred at 23°C overnight. The methanol was
removed using a rotary evaporator. The residue was dissolved in 200 ml
of CH2Cl2 acidified with 1 N HCl. The phases were separated and the
aqueous layer was extracted again with CH2Cl2. The organic phases
were combined, washed with saturated NaCl, dried (MgSO4), and con-
centrated to provide a white solid. The solid was recrystallized twice
from ethyl acetate/hexane to give 34.7 g (0.136 mol, 96%) of compound
2 (N-benzenesulfonylproline). MS: m/z ϭ 256.2 (M ϩ Hϩ).
al., 1995). Many groups have used these sequences as start-
ing points to develop small molecule inhibitors that can block
the interaction between ␣4␤1 and its ligands (Abraham, 1997;
Lin et al., 1999; Kudlacz et al., 2002; van der Laan et al.,
2002). In studying the selectivity of different classes of ␣4␤1
inhibitors, we identified the small molecule inhibitor
BIO5192 [2(S)-{[1-(3,5-dichlorobenzenesulfonyl)-pyrrolidine-
2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-
tolyl-ureido)-phenyl]-acetyl}-amino)pentanoylamino]-butyric
acid] (D. M. Scott, manuscript in preparation). BIO5192 was
of special interest because of its high affinity for ␣4␤1 under
all states of activation, high selectivity for ␣4␤1, and slow
dissociation rate from the bound complex. Here we compared
and contrasted the biochemical, pharmacokinetic, and phar-
macodynamic properties of BIO5192 and mAb TA-2, and
demonstrate that both inhibitors effectively block the disease
To a solution of 62 mg (0.27 mmol) of compound 1 and 77 mg (0.30
progression of EAE in rats. The clear distinction in the modes mmol) of compound 2 in 3 ml of dimethyl formamide (DMF) was

1152
Leone et al.
Scheme 1.
added 137 mg (0.36 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ- low oil, which was used without further purification. MS: m/z 746 (M
tetramethyluronium hexafluorophosphate (HATU), followed by 175 ϩ Hϩ), 646 (M Ϫ BOC ϩ Hϩ); 100 mg (0.13 mmol) of compound 4 was
␮l (1.0 mmol) of diisopropylethylamine to give a yellow solution. dissolved in 3 ml of CH2Cl2; a 1 ml aliquot of trifluoroacetic acid was
After stirring at 23°C for 2 h, the solution was diluted with ethyl
added with stirring. After 2 h, the solution was redissolved in CH2Cl2
acetate, washed with 1 N HCl, then with 1 N NaOH, followed by a and concentrated three times, then dissolved in 3 ml of DMF; 43 mg
wash with saturated NaCl, and dried using Na2SO4. The filtered (0.15 mmol) of o-methyl-tolyl-ureido-phenyl-acetic acid (PUPA)-OH,
solution was concentrated by vacuum to give 128 mg of compound 3 64 mg (0.17 mmol) of HATU, and 175 ␮l (1.0 mmol) of diisopropyl-
[2(S)-{[1-benzenesulfonyl-pyrrolidine-2(S)-carbonyl]-amino}-4-tert- ethylamine were added to give a yellow solution. After stirring at
utoxycarbonylamino-butyric acid methyl ester] as a yellow oil, which 23°C for 4 h, the solution was diluted with ethyl acetate and pro-
was used without further purification. MS: m/z 470 (M ϩ Hϩ), 370 (M cessed as described above for compound 4 to give a yellow foamy
– BOC ϩ Hϩ); 75 mg (0.16 mmol) of compound 3 was dissolved in 3 solid. Chromatography in (2:1 CH2Cl2/acetonitrile, then 1:1 CH2Cl2/
ml of CH2Cl2, and 1 ml of trifluoroacetic acid was added. After acetonitrile, then 5% methanol in CH2Cl2 versus SiO2) was per-
stirring for 2 h at 23°C, the solution was concentrated in CH2Cl2
times, and then dissolved in 2 ml of DMF; 56 mg (0.14 mmol) of
3
formed to give 83 mg (0.09 mmol, 65%) of compound 6 [2(S)-[(1-
benzenesulfonyl-pyrrolidine-2(S)-carbonyl)-amino]-4-[6-benzyloxy-
amino acid 5 (Boc-N-Me-Lys(Z)-OH.DCHA (catalog no. A-3690; carbonylamino-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-
Bachem Bioscience, Inc., King of Prussia, PA), 64 mg (0.17 mmol) of amino)-hexanoylamino]-butyric acid methyl ester] (Rf ϭ 0.17 in 1:1
HATU, and 175 ␮l (1.0 mmol) of diisopropylethylamine were added CH2Cl2/acetonitrile). MS: m/z 912 (M ϩ Hϩ); 83 mg (0.09 mmol) of com-
to give a yellow solution. This mixture was stirred for 4 h at 23°C, pound 6 was hydrogenated as above for compound 1. The resulting solid
then diluted with ethyl acetate, washed with 1 N HCl, 1 N NaOH, was dissolved in 2 ml of DMF, and then 21 mg (0.09 mmol) of 6-(BOC-
then with saturated NaCl, and dried (Na2SO4). The filtered solution amino)caproic acid, 41 mg (0.11 mmol) of HATU, and 175 ␮l (1.0 mmol) of
was concentrated by vacuum to give 100 mg (0.13 mmol, 96%) of
diisopropylethylamine were added. After stirring for 2 h at 23°C, the
compound 4 [2(S)-{[1-benzenesulfonyl-pyrrolidine-2(S)-carbonyl]- solution was diluted with ethyl acetate and processed as for compound 4;
amino}-4-[6-benzyloxycarbonylamino-2(S)-(tert-utoxycarbonyl- 52 mg (0.05 mmol, 60%) of compound 6a [2(S)-[(1-benzenesulfonyl-
methyl- amino)-hexanoylamino]-butyric acid methyl ester] as a yel- pyrrolidine-2(S)-carbonyl)-amino]-4-[6-(6- tert-utoxycarbonylamino-

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1153
hexanoylamino)-2(S)-(methyl-{2-[4-(3-o- tolyl-ureido)-phenyl]-acetyl}- added to wells either in Mg2ϩ-TBS or with serial dilutions of
amino)-hexanoylamino]-butyric acid methyl ester] was recovered as a
BIO5192 in Mg2ϩ-TBS. The plate was incubated for 30 min at 37°C;
yellow solid that was used without further purification. MS: m/z 991 (M ϩ then the plates were washed three times with Mg2ϩ-TBS to remove
Hϩ), 891 (M Ϫ BOC ϩ Hϩ).
unbound cells. The fluorescent cells that bound to mAb 9EG7 were
To 52 mg (0.05 mmol) of compound 6a in 3 ml of methanol, 265 ␮l analyzed by a Cytofluor fluorescence plate reader. Data reductions
(0.53 mmol) of 2 N LiOH was added while stirring. After 2 h, the were done using Microsoft Excel 98.
solution was redissolved in acetone and concentrated. The residue
Isolation of Peripheral Blood Lymphocytes (PBLs). Rat ve-
was dissolved in 2 ml CH2Cl2, and 2 ml of trifluoroacetic acid was nous blood was collected in sodium citrate and centrifuged at 200g
added. The sample was stirred for 90 min at 23°C and then concen- for 5 min. The platelet-rich plasma was discarded. The cell pellet was
trated. The residue was dissolved in minimal methanol and purified diluted 1:1 with Dulbecco’s PBS without Ca2ϩ and Mg2ϩ (Sigma-
by reverse-phase high pressure liquid chromatography (5 to 95% Aldrich), layered onto Ficoll-Hypaque solution (Pharmacia, Peapack,
acetonitrile in water with 0.1% trifluoroacetic acid) to give 26 mg NJ) and subjected to centrifugation at 900g for 20 min. The mono-
(0.026 mmol, 50%) of compound 6b [4-[6-(6-amino-hexanoylamino)- nuclear cell layer at the plasma-Ficoll interface was collected. The
2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-hex- cells were washed twice with RPMI 1640 medium containing 10%
anoylamino]-2(S)-[(1-benzenesulfonyl-pyrrolidine-2(S)-carbonyl)-- FBS (RPMI/10) with each step followed by centrifugation. The
amino]-butyric acid] (BIO8139) as a white solid. MS: m/z 878 (M ϩ Hϩ). washed PBLs were resuspended in TBS-Mn.
Cell Adhesion Assays. The ability of BIO5192 to block ␣2␤1/
Isolation of Splenocytes. Spleens were dissected from 240-g
collagen I and ␣9␤1/VCAM-1 interactions were evaluated in cell Lewis rats and gently forced through a 100-␮m mesh screen. Con-
adhesion assays. Collagen I and VCAM-1 were immobilized onto a nective tissue was excluded from the cell suspension by the screen,
96-well Corning Easy Wash plate (catalog no. 25801; Corning Inc., and the resulting splenocytes were suspended in RPMI/10 medium.
Corning, NY). Human integrin-expressing cell lines (2 ϫ 105/well) The cells were washed twice in RPMI/10 with each wash followed by
were labeled with a fluorescent compound, 2 ␮M 2Ј,7Ј-bis-(2-carboxy- a centrifugation step. Residual red blood cells in the splenocyte
ethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (catalog no. suspension were excluded from the cell count. Splenocytes (1.8 ϫ
B1150; Molecular Probes, Eugene, OR), and added plus or minus 109) were obtained from a single spleen.
BIO5192. After 30 min, the plates were washed, and bound cells
BIO8139-Biotin Occupancy FACS Assay. Splenocytes were
were quantified in a Cytofluor fluorescence plate reader. The assay isolated from control rats or from rats treated with BIO5192 (30
buffer was TRIS-buffered saline (TBS: 24 mM TRIS, 137 mM NaCl, mg/kg, s.c.) or TA-2 (2.5 mg/kg, i.v.), as described above. Control rat
2.7 mM KCl, 2 mM glucose, 0.1% BSA, pH 7.4), containing 1 mM
splenocytes were incubated at 23°C for 15 min with a titration of
MnCl2. The specificity of binding was controlled for using integrin- either BIO5192 or TA-2 and washed twice with TBS-Mn with a
specific neutralizing monoclonals. The antibodies were run at 10 centrifugation step between each wash. The samples were then in-
␮g/ml on each day of assay and were as follows: anti-␣4␤1, mAb cubated with 20 nM BIO8139-biotin at 23°C for 15 min, washed
HP1/2 (Biogen), anti-␣2␤1, mAb 26G8 (Biogen) and ␣9␤1-mAb Y9A2 twice with TBS-Mn, treated with a 1:500 dilution of streptavidin-PE
(Chemicon).
(catalog no. 016-110-084; Jackson ImmunoResearch Laboratories,
Expression and Purification of VCAM-Ig and Direct Bind- Inc., West Grove, PA) for 15 min at 23°C, and again washed twice.
ing Assay Protocol. Details for the construction of the VCAM-Ig The samples were resuspended in 200 ␮l of assay buffer and ana-
animal cell expression vector, the generation of a Chinese hamster
lyzed by FACS. BIO8139-biotin was generated by reacting BIO8139
ovary cell line expressing VCAM-Ig, conjugation of the VCAM-Ig to with biotin-PEG-CO2-NHS, mol.wt. 3.4 kDa (Shearwater Polymers
alkaline phosphatase, and the development of a direct binding assay Inc., Huntsville, AL) and separating the modified BIO8139 from
for characterizing ␣4␤1 and ␣4␤7 binding to VCAM-Ig were as previ- unmodified by size exclusion-HPLC on a Superdex peptide column
ously described (Lobb et al., 1995).
(Amersham Biosciences, Inc., Piscataway, NJ). Where indicated in
The ␣IIb␤3-Fibrinogen Platelet Aggregation Assay. Whole the text, this assay was replaced with a simpler version where
blood (50 ml) was collected into 10-ml vacutainer tubes containing 1 BIO8139 was directly conjugated to the PE.
ml of 3.8% sodium citrate. The citrate-treated blood was centrifuged
Monitoring ␣4␤1 Levels on PBLs and Splenocytes by FACS
for 5 min at 200g and the platelet-rich plasma was collected. Plate- Analysis. Female Lewis rats were injected with a single dose of TA-2
let-poor plasma was prepared by centrifuging the remaining blood (2.5 mg/kg, i.v. in PBS) or with PBS at 0 h, or two doses of BIO5192
specimen at 1500g for 15 min. The platelet count in the platelet-rich (30 mg/kg, s.c. in TRIS/lactose) or vehicle at 0 and 24 h. The animals
plasma was adjusted to 2 ϫ 108 platelets/ml using the platelet-poor were sacrificed at 48 h. PBLs and splenocytes were isolated as
plasma. A Biodata 4-channel platelet aggregation profiler (PAP-4; described. BIO8139-PE (10 nM) or TA-2 (10 ␮g/ml) followed by goat
Biodata Corp., Hatboro, PA) was blanked using a cuvette containing anti-mouse-PE, were added to the test samples and the cells were
only platelet-poor plasma. Compounds in TBS containing 1 mM analyzed by FACS for the presence of cell surface ␣4␤1 integrin. In a
MnCl2 (TBS-Mn) were tested at 100 ␮M concentration. 350 ␮l of separate experiment, samples were examined by FACS to determine
platelet-rich plasma plus 100 ␮l of compound were added to cuvettes the specific cell types present. The cells were analyzed using rat
containing stir bars and placed into the aggregometer. To start markers for T-cells, CD3 (catalog no. 22014D; BD Biosciences
aggregation, 50 ␮l of freshly made ADP at 2 ϫ 10Ϫ4 M was added to Pharmingen), and B-cells, CD45R (catalog no. 22164D; BD Bio-
each cuvette. A TBS-Mn control was run with each set of test sam- sciences Pharmingen), and their isotype-matched controls, IgG2b
ples. A 4-min aggregation tracing was generated for each sample and (catalog no. 03044C; BD Biosciences Pharmingen), and IgG3 (catalog
percent aggregation was calculated by the profiler. A positive con-
trol, 100 ␮M of the RGD-containing peptide GRGDSP (Sigma-Al-
drich, St. Louis, MO), was run on each day.
no. 03064C; BD Biosciences Pharmingen).
PE-Labeled BIO8139 was prepared as follows: 200 ␮l of 2.5 mM
BIO8139, 5 mM succinimidyl-4-[N-maleimidomethyl]-cyclohexane-
LIBS Assay. LIBS induction by BIO5192 was assessed in vitro by 1-carboxylate sulfo-SMCC (Pierce Chemical, Rockford, IL), 100 mM
an adhesion assay. Corning Easy Wash 96-well plates were coated HEPES, pH 7.5, in 90% dimethyl sulfoxide/10% water was incubated
overnight at 4°C with the LIBS-recognizing mAb 9EG7 (catalog no.
at 23°C for 4 h. Ethanolamine was added to 20 mM final to quench
09351D; BD Biosciences Pharmingen, San Diego, CA) (10 ␮g/ml) in any further reaction and the BIO8139-SMCC conjugate was stored
phosphate-buffered saline (PBS). The next day, plates were washed at Ϫ70°C for subsequent use. R-Phycoerythrin pyridylsulfide deriv-
with PBS and blocked with 1% BSA in PBS for 1 h at 37°C. Jurkat ative (2 mg/ml, 1.8 pyridylsulfide residues/PE) was obtained from
cells (1 ϫ 105/well) were labeled with Calcein (Molecular Probes, Molecular Probes. Three milliliters of the PE derivative was incu-
Eugene, OR) at 37°C for 20 min in TBS, containing 2 mM MgCl2 bated with 10 mM DTT for 30 min at 23°C and desalted on a 35-ml
(Mg2ϩ-TBS) and washed twice. Calcein-labeled Jurkat cells were G25M column in 5 mM MES, pH 5.5, and 100 mM NaCl. The PE

1154
Leone et al.
elution peak was collected visually and quantified by absorbance at
Rat EAE Model. Healthy female Lewis rats weighing 150 g were
280 nm; 3.7 ml of 5 ␮M PE was treated with 20 ␮l of BIO8139-SMCC obtained from Harlan (Indianapolis, IN) and housed in ventilated
(10 ␮M final) and 0.3 ml of 0.5 M MES, pH 6.5. The sample was cage racks and allowed food and water ad libitum. At approximately
incubated at 23°C for 2 h, then N-ethylmaleimide (Pierce Chemical) 9 weeks of age, animals were immunized with an emulsion of guinea
was added to 60 ␮M, and the sample incubated further for 20 min. pig myelin basic protein (MBP) peptide in complete Freund’s adju-
The sample was desalted on a 35-ml G25M column in 10 mM vant. MBP peptide sequence of GPMBPYGSLPQKSQRSDENPV
HEPES, pH 7.5, and 150 mM NaCl. The PE-BIO8139 conjugate was (amino acid residues 68–86), 100 ␮g/ml in PBS, was diluted with an
equal volume of incomplete Freund’s adjuvant. Before emulsifica-
tion, ground Mycobacterium tuberculosis was added to 4 mg/ml.
Animals were anesthetized with isoflurane and immunized with a
single footpad injection of 100 ␮l of the emulsion. Animal body
weights, and observations for signs of paralysis starting on day 8
post immunization, were collected daily. Each treatment group con-
tained 13 animals. BIO5192 was administered at 30 mg/kg in TRIS/
lactose, s.c., b.i.d., during days 5 through 14. Untreated rats and
TRIS/lactose vehicle-treated controls were also monitored. For TA-2
treatment, rats were injected i.v. on day 9 and day 13 with TA-2 (2.5
mg/kg). The following grading system was used to quantify disease
severity: 0.5 ϭ half of tail limp; 1.0 ϭ whole tail limp; 1.5 ϭ whole tail
limp with a small amount of gait disruption; 2.0 ϭ hind limb weak-
ness (waddling gait or one dragging leg); 2.5 ϭ hind limb weakness
(one dragging leg with a small amount of loss of motile function in
opposite leg); 3.0 ϭ hind limb paralysis (both legs dragging; some
slight hind limb movement); 3.5 ϭ hind limb paralysis (both legs
dragging with a small amount of forelimb weakness); 4.0 ϭ hind limb
and front limb paralysis (sufficient to prevent movement); 5.0 ϭ
moribund or death. Statistical significance of differences in severity
of disease (peak height) and day of peak disease score (peak day)
were assessed using a one-way analysis of variance followed by
Fisher’s protected least significant difference test for multiple com-
parisons among means. P values of less than 0.05 were taken to be
statistically significant. All procedures using animals were con-
ducted in accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and approved by the Insti-
tutional Animal Care and Use Committee.
Assessing ␣4␤1 Expression by Confocal Microscopy. TA-2
and BIO8139 were conjugated with AlexaFluor594 (Molecular
Probes, Inc.) following the manufacturer’s instructions. The
BIO8139-Alexa594 conjugate was generated by first reacting
BIO8139 with NHS-PEG-maleimide 2.4 K (Shearwater Polymers
Inc.), then treating the BIO8139-PEG with sonic hedgehog (Shh)
protein that contains a single thiol for modification, and finally
reacting the BIO8139-PEG-Shh with Alexa594. RBL.1 cells were
incubated with TA-2-Alexa594 (10 ␮g/ml) or BIO8139-Alexa594 (50
nM) for 30 min at 4°C. The cells (100,000 cells/well) were plated onto
a 96-well flat-bottomed plate in Earle’s minimal essential medium,
10% FBS, 1 mM sodium pyruvate, and further incubated with the
inhibitors at 37°C for either 0 or 48 h. The cells were washed and
fixed in 2% paraformaldehyde at designated time points. Freshly
isolated rat splenocytes were incubated with unlabeled TA-2. Spleno-
cytes were washed and fixed in 2% paraformaldehyde following 0, 4,
24, and 48 h of continual TA-2 treatment at 37°C. Cell suspensions
were permeabilized using the Cytoperm/cytofix kit (BD Bioscience
Pharmingen) and incubated with goat anti-mouse-Alexa594. The
cells were placed onto slides with coverslips and fluorescence detec-
tion of the Alexa594-labeled complex ␣4␤1/inhibitor was analyzed by
confocal laser-scanning microscopy using a Leica TCS SP confocal
microscope equipped with a krypton/argon/helium/neon laser (Leica
Lasertechnik GmbH, Heidelberg, Germany). For Alexa594 staining,
red channel (600–700 nm range) images were collected from the
center of representative cells showing intact cell membranes.
filtered through a 0.2-␮m filter and stored at 4°C. Batches were
analyzed for relative potency by measuring dose-dependent staining
on Jurkat cells by FACS analysis. Conjugates stored at 4°C were
stable for about 2 months.
Assessing PK Properties of BIO5192 and TA-2 in Lewis
Rats. Female Lewis rats, three per route of administration, received
BIO5192 (30 mg/kg, s.c. or 1 mg/kg, i.v.) or TA-2 at 3 mg/kg, i.v. Blood
samples (250 ␮l/bleed) were obtained at specific time points after
administration. For TA-2, blood samples were drawn at 0, 1, 2, 6, 8,
10, and 14 days. Serum samples were analyzed for TA-2 levels by
ELISA. For BIO5192, blood samples were drawn at 0, 2, 6, 24, and
48 h. Serum samples were analyzed for BIO5192 levels using mass
spectrometry. PK parameters were calculated from the mass spec-
trometry data by noncompartmental analysis. PK parameters in-
clude CMAX (maximum serum concentration), tMAX (time to achieve
maximum serum concentration), CL (systemic clearance), Vss (vol-
ume of distribution at steady state), t1/2 (terminal phase half-life),
and bioavailability. Area under the curve (AUC) was calculated
using the trapezoidal rule. Percent bioavailability was calculated
from the following equation: (AUCextravascular/AUCIV) ϫ (DoseIV
/
Doseextravascular) ϫ 100. For analysis of BIO5192 levels in serum by
mass spectrometry, 100-␮l serum samples were extracted into
methyl tert-utyl ether and spiked with an internal standard. Mass
spectroscopy was performed on a Sciex API365 triple quadrupole
mass spectrometer with a TurboIonSpray interface. The assay limit
of quantification was 2 ng/ml.
TA-2 levels in serum were measured using a sandwich ELISA.
Costar 96-well Easy Wash plates (Corning Inc.) were coated over-
night at 4°C with 5 ␮g/ml (50 ␮l/well) of anti-AffiniPure goat-anti
mouse IgG Fc fragment (Jackson ImmunoResearch Laboratories) in
50 mM sodium bicarbonate, pH 9.2. All subsequent manipulations
were performed at room temperature. The plates were blocked for 30
min with 200 ␮l/well 1% BSA (cat. no. A-7030; Sigma-Aldrich) in 10
mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 0.01% thimer-
osal, and washed three times with TBS (50 mM Tris-HCl, pH 7.5, 150
mM NaCl). The wells were then treated for 1 h with serial dilutions
of PK samples or of the appropriate dosing solution standards (50
␮l/well) diluted in blocking buffer plus 1% rat plasma. The plates
were washed three times and incubated for 1 h with 50 ␮l/well of goat
anti-mouse IgG1-alkaline phosphatase (catalog no. 1070-04; South-
ern Biotechnology Associates, Inc., Birmingham, AL) at a 1:2000
dilution in blocking buffer plus 1% rat plasma (100 ␮l/well). The
plates were again washed three times, incubated for 1 h with 5
mg/ml 4-nitrophenyl phosphate in 100 mM glycine, pH 10.5, 1 mM
ZnCl2, 1 mM MgCl2, and read at 405 nm on a Molecular Devices
Thermo Max microplate reader. Each sample was analyzed in dupli-
cate, and three animals were analyzed per time point. TA-2 concen-
trations were calculated by interpolation from a standard curve.
Assessing Lymphocyte Counts and Subtypes following In-
hibitor Treatments. Female Lewis rats were injected with a single
dose of either TA-2 (2.5 mg/kg, i.v. in PBS), or BIO5192 (30 mg/kg,
s.c., in TRIS/lactose) or with their respective vehicles at time 0. At
each time point, 0.3 ml of blood from triplicate animals was drawn
from the jugular vein without anesthesia using indwelling catheters
and collected into Capiject purple-top microtainer tubes containing
EDTA (catalog no. T-MQK; Terumo Medical Corp., Somerset, NJ).
Plasma samples were analyzed for lymphocyte count using an Abbott
CellDyn 3500 cell analyzer (Abbott Diagnostics, Abbott Park, IL).
Blood samples from the TA-2-treated animals were drawn on days 1,
2, 4, 6, 8, 10, and 14 and for the BIO5192-treated animals after 2, 6,
24, and 48 h.
Results
Characterization of the Biochemical Properties of
BIO5192. The small molecule inhibitor BIO5192 was iden-
tified as a potent inhibitor of ␣4␤1 (KD Ͻ 10 pM) from a

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1155
structure-activity relationship analysis of ␣4␤1 inhibitors
(D. M. Scott, manuscript in preparation). The high affinity
for ␣4␤1 results from an extremely slow dissociation rate of
the inhibitor from the integrin/inhibitor complex, with a dis-
sociation half-life of Ͼ12 h for both the unactivated and
activated integrin. The following studies were performed to
further characterize the properties of BIO5192. First, the
selectivity of BIO5192 for ␣4␤1 was evaluated by assessing
the binding of BIO5192 to cells expressing ␣4␤7, ␣9␤1, ␣2␤1,
and ␣IIb␤3 (Table 1). BIO5192 was highly selective for ␣4␤1.
The affinity of BIO5192 for ␣4␤1 was 250- to 1000-fold higher
than for the related integrin, ␣4␤7, which shares many of the
same ligands as ␣4␤1. The inhibitor bound even less tightly to
Fig. 1. Assessing binding of [35S]BIO7662 to cells expressing ␣4␤1 using
kinetic measurements. For assessing binding of BIO5192 to ␣4␤1-express-
ing cells the radiolabeled derivation of BIO5192, [35S]BIO7662, was used
as described previously (Chen et al., 2001). A, for on-rate measurements,
Jurkat cells (1 ϫ 106 cells/ml) in TBS containing 1 mM Ca2ϩ and 1 mM
Mg2ϩ plus varying amounts of rat plasma: 0% (f), 20% (F), 50% (Œ), or
100% (᭜) were treated with 2 nM [35S]BIO7662 for 5, 10, 20, 30, or 60
min. Cells were collected by centrifugation and subjected to scintillation
counting. The data were fitted to an exponential equation by nonlinear
regression. B, for off-rates, cells expressing ␣4␤1: 70Z3 cells (f), RBL.1
cells (F), or Jurkat cells (Œ) at 1 ϫ 106 cells/ml were treated with 5 nM
␣2␤1 and ␣IIB␤3 (Table 1). A low but significant level (KD
ϭ
140 nM) of binding was seen on ␣9␤1 integrin in buffer
containing 1 mM Mn2ϩ. The large discrepancy in the binding
constants calculated from the adhesion assay shown in Table
1 (IC50 ϭ 1.8 nM) and from the kinetic data (KD Ͻ 10 pM)
(Scott, 2002; manuscript in preparation) arise because the
affinity constant of BIO5192 for ␣4␤1 is lower than the con-
centration of ␣4␤1 in the binding assay. Consequently, the
IC50 data reflect the concentration of ␣4␤1 in the adhesion
assay and not the affinity of BIO5192 for ␣4␤1, as discussed
elsewhere (Pepinsky et al., 2002).
Second, the effects of protein binding on the affinity of
BIO5192 for ␣4␤1 were evaluated in vitro by kinetic measure-
ments using a radiolabeled analog of BIO5192, [35S]BIO7662
(Chen et al., 2001). Figure 1A shows association curves for
the binding of [35S]BIO7662 to Jurkat cells (␣4␤1 positive) in
[
35S]BIO7662 for 40 min in TBS containing 1 mM Ca2ϩ and 1 mM Mg2ϩ
.
Unlabeled BIO7662 (500 nM) was added, and the cells were further
incubated for the times indicated in the presence of 1 mM Ca2ϩ and 1 mM
Mg2ϩ buffer. Cells were pelleted at each time point, and cell-associated
[
35S]BIO7662 was measured by scintillation counting. Dissociation data
are represented as a percentage of the maximum total counts bound as a
function of time. The data were fitted to a single exponential for all
curves. kon and koff values were calculated from the curve fits. koff is the
observed rate constant; kon is the observed binding constant divided by
the concentration of BIO7662. Data shown are from a single determina-
tion of the on and off rates.
Third, the ability of BIO5192 to recognize ␣4␤1 from differ-
TBS buffer containing 1 mM Ca2ϩ and 1 mM Mg2ϩ alone, ent species was tested by using kinetic measurements to
and in the same buffer with increasing amounts of plasma. calculate affinity constants. On and off-rate measurements
The association rate for BIO7662 binding to ␣4␤1 decreased for binding and dissociation of [35S]BIO7662 for human, rat,
with increasing plasma concentrations. This effect was and mouse cells were similar. Figure 1B shows the dissocia-
caused by nonspecific binding of [35S]BIO7662 to serum al- tion curves for the binding of [35S]BIO7662 from ␣4␤1 on
bumin and can be mimicked by incubating samples with the human Jurkat, rat RBL.1 and mouse 70Z3 cells in buffer
corresponding concentration of albumin (data not shown). As containing 1 mM Ca2ϩ and 1 mM Mg2ϩ. The ␣4␤1 receptors
the amount of plasma was increased from 20 to 100%, the from the three species all remained 90% saturated with
effective concentration of [35S]BIO7662 diminished, causing
[
35S]BIO7662 at 120 min, demonstrating a slow dissociation
a decline in the association rate for the binding of BIO7662 to rate. Off rates of Յ0.19 ϫ 10Ϫ4 sϪ1 were calculated from the
␣4␤1. Plasma binding had no effect on the dissociation rate dissociation data. Binding in the presence of 1 mM Mn2ϩ
constant for the release of
[
[
35S]BIO7662 from ␣4␤1/ changed the ␣4␤1 integrin to a higher activation state (Chen
35S]BIO7662 complexes (data not shown). Although protein et al., 2001). The dissociation of [35S]BIO7662 from ␣4␤1 in
binding in general reduces the effective concentration of a the presence of 1 mM Mn2ϩ was too slow to allow determi-
compound, for tight binding inhibitors such as BIO5192 a nation of an accurate koff, but extrapolation from the avail-
slow off-rate drives the binding equilibrium toward the occu- able data suggests a half-life for dissociation of at least 16 h,
pied state and therefore in part compensates for the apparent corresponding to a koff Յ 0.11 ϫ 10Ϫ4 sϪ1 (data not shown).
loss in concentration due to protein binding.
Since kon ϭ 1.9 ϫ 106 MϪ1 sϪ1 for the binding of BIO7662 to
the unactivated integrin, the resulting KD that was calcu-
lated from kon and koff is 10 pM or less for rat, mouse, and
human ␣4␤1.
TABLE 1
Integrin selectivity data for BIO5192
The selectivity of BIO5192 toward the five integrins indicated were tested in adhe-
sion formats for ␣2␤1 and ␣9␤1 by direct binding for ␣4␤1 and ␣4␤7, and by platelet
aggregation for ␣IIB␤3, as described under Materials and Methods. For all assay
formats, serial dilutions of each compound (from 1 nM to 100 ␮M) were evaluated
and IC50 values were calculated from the concentration dependence of the inhibition
curves.
Fourth, the ability of BIO5192 binding to induce expres-
sion of LIBS epitopes was evaluated. Ligand binding induces
a cascade of conformational changes within the integrin that
has been studied in detail using mAbs whose epitopes are
either exposed (termed LIBS; also referred to as CLIBS due
to modulation of the epitope by cations) or lost following
ligand binding (Humphries, 1996; Newham et al., 1998).
Monoclonal antibody 9EG7 recognizes and binds to exposed
epitopes on the ␤1 chain of ␣4␤1 that are presented when the
ligand binding site is occupied. BIO5192 induced binding of
9EG7 with an EC50 of approximately 0.3 nM indicating that
BIO5192 is a LIBS inducer. The EC50 for BIO5192 LIBS
Integrin
IC50 (n)
nM
a
␣4␤1
1.8 Ϯ 0.7 (10)
Ͼ500 (1)
␣4␤7
␣9␤1
␣2␤1
138 (2)
1,053 Ϯ 143 (3)
Ͼ10,000 (1)
␣
␤
3
IIb
a Due to the high affinity of BIO5192 for ␣4␤1, its binding constants were also
calculated from kinetic measurements (KD ϭ Ͻ10 pM).

1156
Leone et al.
induction (Fig. 2) and the nanomolar potency in the binding or s.c. administration in female Lewis rats (Fig. 4). BIO5192,
assay were similar.
dosed at 1 mg/kg, i.v. showed a multiexponential disposition
with a rapid initial decline followed by a less steep terminal
phase. The terminal half-life was 1.1 h. Prolonged exposure
was observed following s.c. administration. Dose-dependent
increases in available drug were observed with CMAX occur-
ring from 30 to 60 min in all treatment groups. Half-lives of
1.7, 2.7, and 4.7 h were observed for the 3, 10, and 30 mg/kg,
s.c. doses, respectively. The blood plasma curves show that
the AUC for the s.c. route of administration increased about
2.5-fold from 5,460 h ϫ ng/ml for the 3 mg/kg dose to 14,175
h ϫ ng/ml for the 30 mg/kg dose. Following the 30 mg/kg, s.c.
dosing, blood levels of Ͼ100 ng/ml were maintained for over
24 h, and consequently, once a day dosing at 30 mg/kg was
chosen for all subsequent studies. The increase in the half-
life of BIO5192 at the 30 mg/kg dose relative to the half-life
at lower doses is the result of a depot effect. At this high dose,
dissolution of the BIO5192 results in a slow release of the
compound into the bloodstream.
In Vivo Measurements of the Binding of BIO5192 to
Rat Lymphocytes. To compare the treatment effects of
BIO5192 and TA-2 in a rat EAE model, the PK and PD
properties of the two inhibitors were evaluated to select ap-
propriate dosing regimens. First, the binding of BIO5192 and
TA-2 to ␣4␤1 was measured on freshly isolated rat PBLs.
These titration curves are shown in Fig. 3A. The binding of
BIO5192 and TA-2 on PBLs was dose-dependent and satu-
rable; TA-2 had a 2- to 3-fold higher apparent binding affin-
ity. The binding of BIO5192 to isolated rat splenocytes is
shown in Fig. 3B. The binding is dose-dependent and satu-
rable with an IC50 of 1 nM.
The in vivo time course of receptor occupancy was studied
using rat splenocytes ex vivo to characterize binding. Figure
3C shows the results from a study in which female Lewis rats
were treated with a single dose of BIO5192 at 30 mg/kg, s.c.,
at t ϭ 0 min, and splenocytes were harvested at 24, 48, and
72 h and assayed for receptor occupancy. At 24 h, all ␣4␤1
receptors were occupied by BIO5192. After 48 h, 50% remain
occupied, and at 72 h, less than 40% were occupied. Blood
levels of BIO5192 in these animals as measured by mass
spectroscopy were 180 ng/ml at 24 h but were below limits of
detection (Յ2 ng/ml) at 48 and 72 h. These data demonstrate
that BIO5192 remains bound to the ␣4␤1 receptor even in the
absence of circulating plasma levels of compound and suggest
that the slow off-rate for BIO5192 that was observed in vitro
also occurs in vivo. This hypothesis was supported by leuko-
cytosis data that was routinely used as an in vivo PD marker
for receptor occupancy (see below).
TA-2 was administered i.v to rats at 2.5 mg/kg for deter-
mination of half-life (Fig. 5). Levels of antibody of Ͼ2 ␮g/ml
were observed for 7 days. Based on the data, a t1/2 of 2 days
was calculated.
Comparative Pharmacodynamics of BIO5192 and
TA-2 in Rats. Administration of BIO5192 and TA-2 to rats
produced a lymphocytosis that was sustained as long as
sufficient concentrations of the inhibitors were maintained in
circulation to provide Ͼ90% of ␣4␤1 receptors occupied. Fig-
ure 5A shows lymphocytosis induced in vivo by TA-2 treat-
ment. A 3- to 4-fold increase in lymphocyte levels was ob-
served. The maximal level of induction was reached at the
earliest time point tested, after 24 h. Elevated lymphocyte
levels were maintained for 6 days with the half maximum
occurring on day 7. The elimination half-life of TA-2 is 2 days.
The biological effect of the TA-2 antibody was clearly associ-
ated with its PK. At 7 days, the TA-2 blood level had dropped
to 2 ␮g/ml, and by 8 days there was no detectable TA-2 in the
blood. During the analysis of the lymphocyte levels, circulat-
ing differential white blood cell counts were examined from
three animals on day 1 and day 10 following TA-2 treatment.
Lymphocyte levels increased from a baseline of 6,800 Ϯ 300
to 18,400 Ϯ 400/␮l(mean Ϯ S.E.M.) on day 1 and returned to
baseline on day 10. Basophil levels increased to 50 Ϯ 20/␮l on
day 1 and returned to normal levels of 8 Ϯ 1/␮l by day 10.
Levels of neutrophils (600 Ϯ 40/␮l, day 1; 900 Ϯ 70/␮l, day
10), monocytes (300 Ϯ 50/␮l, day 1; 300 Ϯ 20/␮l, day 10), and
eosinophils (20 Ϯ 2/␮l, day 1; 40 Ϯ 10/␮l, day 10) did not
change in response to TA-2 treatment.
Pharmacokinetics of BIO5192 and TA-2 in Female
Lewis Rats. The pharmacokinetic properties of BIO5192
were evaluated by noncompartmental analysis following i.v.
Figure 5B shows the pharmacodynamic effect of BIO5192.
The lymphocyte count rose about 1.5-fold after 24 h of drug
treatment. Half as many cells were released into the circu-
lation following BIO5192 treatment as when TA-2 was given.
The transient leukocytosis was sustained for 30 h. The ob-
served t1/2 of BIO5192 is approximately 12 h. Although the
PK data would suggest that lymphocytosis should have been
reversed at an earlier time, the receptor occupancy study
described above showed that BIO5192 remains bound to
100% of the ␣4␤1 receptors for 24 h and 50% bound for 48 h,
even in the absence of compound plasma levels. These data
demonstrate that the slow dissociation rate and prolonged
receptor occupancy of BIO5192 is associated with a biological
Fig. 2. Induction of the expression of the 9EG7 LIBS epitope by BIO5192.
The effects of BIO5192 on the expression of the LIBS epitope that can be
detected with the 9EG7 mAb were evaluated on Jurkat cells expressing
␣4␤1 in an adhesion assay. Calcein-labeled Jurkat cells (1 ϫ 105 cells) in
TBS plus 2 mM Mg2ϩ were incubated for 30 min at 37°C with serial
dilutions of BIO5192 or EGTA at the various concentrations indicated.
Costar 96-well Easy Wash plates were coated with 10 ␮g/ml 9EG7 and
blocked with bovine serum albumin. Plates were washed three times and
read on a CytoFluor 4000 multiwell plate reader from Applied Biosystems
(Foster City, CA) (excitation at 485 nm, emission at 530 nm). For ␣4␤1
cells, background binding was 400 mean fluorescence units and maxi-
mum binding was 9000 units. 0% ϭ nonspecific binding to non-9EG7
coated wells. 100% ϭ binding seen in the presence of 14 nM BIO5192. As
a further control for LIBS induction, certain wells were treated with
Mn2ϩ, which itself is a potent inducer of the LIBS epitope. As expected
Mn2ϩ induced expression of the LIBS epitope on Jurkat cells.

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1157
Fig. 3. Assessing the occupancy of ␣4␤1 integrin receptors. Binding of BIO5192 and TA-2 to rat PBLs (A) and BIO5192 binding to rat splenocytes (B)
in vitro and BIO5192 splenocyte receptor occupancy following an in vivo administration (C) were investigated. 4.0 ϫ 106 cells/ml of PBLs (A) or
splenocytes (B) were incubated at 23°C for 30 min with the indicated concentrations of BIO5192 (f) in the presence of TBS containing 1 mM Mn2ϩ
or TA-2 (Ⅺ) in the presence of TBS containing 1 mM Ca2ϩ and 1 mM Mg2ϩ. The cells were washed and pelleted by centrifugation. BIO8139-PE (10
nM) was added to the cells treated with the BIO5192 titration, and goat anti-mouse-PE (1:500) was added to the cells treated with the TA-2 titration,
then incubated at 23°C for 30 min. Cells were collected by centrifugation and subjected to FACS analysis. Solid lines represent best fit of the data to
a hyperbolic equation. C, rats were injected once with BIO5192 (30 mg/kg, s.c.) at time 0. At 24, 48, and 72 h post injection of BIO5192, splenocytes
were isolated and 4.0 ϫ 106/ml of splenocytes were incubated with BIO8139-PE (10 nM) for 15 min. Cells were washed and collected by centrifugation
and subjected to FACS analysis. Data are the mean of two animals.
activity that occurs in the absence of compound plasma lev- disease (peak height) and day of peak disease score (peak
els.
day) were assessed using a one-way analysis of variance
TA-2 and BIO5192 Treatments Delay Paralysis Asso- followed by Fisher’s protected least significant difference
ciated with EAE. EAE was induced in female Lewis rats test. In a comparison of the day of peak disease for treated
with MBP peptide. The time course for EAE is shown in Fig. versus controls, P values of Ͻ0.0001 were obtained for
6. The effects of the MBP treatment on the paralytic score BIO5192 treatment versus both vehicle and untreated con-
were monitored from day 8 to day 26. The earliest onset of trols, and similarly, P values of Ͻ0.0001 were obtained for
paralysis in the no treatment and TRIS/lactose vehicle con- TA-2 treatment versus vehicle and untreated controls. Sta-
trol groups occurred on day 9 followed by a peak of disease tistical differences in disease severity (peak height) were also
between days 12 and 13 with a paralytic score of 3. The compared. P values of Ͻ0.0032 were observed for peak height
disease resolved to a baseline paralysis score of 0 by day 18. for BIO5192 versus both the vehicle and the untreated con-
There was a decrease in body weight during the course of the trols, and P values Ͻ0.0011 were obtained for the TA-2-
disease and a return to normal following the resolution of treated group versus the two control groups.
EAE. TA-2 treatment following injections on days 9 and 13
TA-2 Treatment Down-Modulates ␣4␤1 Surface Ex-
(2.5 mg/kg, i.v.) resulted in a 3- to 4-day delay in onset of the pression. As part of the analysis of function, we tested
disease and a decrease in total disease severity. With a half- whether TA-2 or BIO5192 modulated ␣4␤1 levels in vivo. For
life of 2 days, we expected that sufficient blood levels of TA-2 these studies, female Lewis rats were treated with either a
would last 7 days and therefore that dosing on days 9 and 13 single 2.5 mg/kg dose of TA-2 at t ϭ 0 h or with 2 doses of
would be an effective treatment regimen. This result is evi- BIO5192 at 30 mg/kg at t ϭ 0 and t ϭ 24 h. Freshly isolated
dent from the plotted data. Rats treated with BIO5192 (30 rat PBLs and splenocytes were analyzed at t ϭ 48 h for
mg/kg, s.c.) also show a 3-day delay in onset of disease when receptor levels and occupancy. Figure 7 shows the results
dosed b.i.d. (Fig. 6), and a 1- to 2-day shift when dosed q.d. from this analysis. PBLs and splenocytes from the vehicle-
(data not shown) compared with the control groups. The treated control animals showed strong FACS signals when
delay in onset of the disease in the BIO5192 treatment group analyzed by ex vivo treatment with TA-2 and detected with
is consistent with the finding that bound BIO5192 will oc- anti-mouse PE or by direct analysis using a PE-tagged ana-
cupy ␣4␤1 long beyond the point at which the BIO5192 is no log of BIO5192, PE-BIO8139. When animals were treated
longer detected in blood. Once BIO5192 is released, EAE with BIO5192 and analyzed for receptor occupancy using
underwent its normal disease progression. The disease in PE-BIO8139, no binding of the analog was observed. To rule
rats from both the TA-2 and BIO5192 treatment groups out modulation of the receptor, we analyzed the cells from the
reached a paralytic score of 2.0, indicating that both inhibi- BIO5192-treated rats with TA-2 and measured ␣4␤1 levels
tors delayed the onset and decreased the severity of the using a PE-labeled anti-mouse antibody. No change in recep-
disease.
tor number was observed for the BIO5192-treated animals
The statistical significance of differences in severity of versus the untreated control animals, indicating that the loss

1158
Leone et al.
Fig. 5. Pharmacodynamic effects of ␣4␤1 inhibitors. A single dose of TA-2
(2.5 mg/kg, i.v. in PBS), BIO5192 (30 mg/kg, s.c. in TRIS/lactose), or the
corresponding vehicle control was administered to Lewis rats at time 0.
Blood was collected into EDTA to form plasma at the time points indi-
cated and analyzed for circulating blood count (CBC) by a CellDyne cell
analyzer. Lymphocyte counts were assessed for each animal from the
CBC. The top panel shows the lymphocyte counts (E) following TA-2
injection relative to PK data for TA-2 (F), which was measured in the
same study. The bottom panel shows the lymphocyte counts (E) following
BIO5192 injection in relation to its PK data (F). Data points are the mean
of three animals Ϯ S.D.
Fig. 4. Pharmacokinetic analysis of BIO5192 in Lewis rats following i.v.
and s.c. administration. Rats were injected with BIO5192 at 1 mg/kg, i.v.
(F) (top panel), or with 3 (F), 10 (f), or 30 (Œ) mg/kg, s.c. (bottom panel).
Whole blood from these animals was collected and treated with heparin
at the time points indicated. BIO5192 was isolated from the resulting
plasma by extraction and BIO5192 levels were assessed by mass spec-
troscopy (see Materials and Methods). Each curve is the mean of data
from three animals. Data are expressed as mean Ϯ S.D.
of signal with PE-BIO8139 was due to the presence of bound
BIO5192. This observation is consistent with the finding that ␣4␤1 levels returned to normal by day 14 when the serum
BIO5192 dissociates slowly from ␣4␤1 and remains bound TA-2 level had fallen below the limits of detection of the
after 24 h. The cells isolated from the TA-2-treated group assay (data not shown). Additionally, we and others (P.
were analyzed for TA-2 binding using anti-mouse PE. With a Kubes, unpublished observations) have observed that a
half-life of 48 h, we had expected to observe full saturation of mouse-specific ␣4␤1 mAb, PS/2, down-modulated ␣4␤1 on
␣4␤1 receptors by TA-2 but instead no bound TA-2 was ob- PBLs and splenocytes isolated from treated mice (data not
served (data not shown). To explain this observation, the shown). Whereas treatment with the PE-BIO8139 probe ver-
cells from this group were treated ex vivo with TA-2 detected ified that there was no free ␣4␤1 on the cells isolated from
with anti-mouse PE or with PE-BIO8139 and directly ana- TA-2-treated animals, the PE label sterically interferes with
lyzed for binding by FACS. In both instances, there was little the binding of BIO8139 to ␣4␤1 that is bound to TA-2. This
or no signal observed indicating that no appreciable ␣4␤1 interference does not occur if the [35S]BIO7662 probe is used.
receptor level was present (Fig. 7). A PK study was conducted Consequently, we also tested binding to ␣4␤1 on cells isolated
separately to examine the ␣4␤1 levels on PBLs and spleno- from rats treated with either TA-2 or BIO5192 using the
cytes following a single injection of TA-2 administered to
[
35S]BIO7662 probe. Specific binding was observed on cells
Lewis rats. Cells were isolated on days 1, 2, 4, 6, 8, 10, and 14 isolated from untreated rats and from the same cells treated
and the presence of ␣4␤1 on the cells was analyzed using TA-2 with TA-2, ex vivo. In contrast, only background binding of
detected with goat anti-mouse PE and compared with TA-2 the probe was observed on cells from animals treated with
serum levels. On days 1, 2, and 4 there was no ␣4␤1 present TA-2 or BIO5192 (data not shown). The loss of [35S]BIO7662
on the lymphocytes. The serum TA-2 concentrations were binding on cells isolated from TA-2-treated animals, but not
between 8 and 25 ␮g/ml on days 4 and 1, respectively. At day on cells from untreated animals that were incubated with
6, when the serum TA-2 level was at 2 ␮g/ml, the ␣4␤1 levels TA-2 in vitro, further verifies that TA-2 treatment down-
began to rise and reached a half-maximal signal by day 8, modulates ␣4␤1 expression. To rule out the selective loss of a
when the serum TA-2 had dropped to Ͻ1 ␮g/ml. Cell surface lymphocyte subtype, PBLs isolated from the TA-2-treated

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1159
trol did not change for T-cells (control: 74%, n ϭ 1; TA-2-
treated: 70 Ϯ 2%, n ϭ 3) but increased for B-cells (control:
12%, n ϭ 1; TA-2-treated: 21 Ϯ 3%, n ϭ 3). Since T- and
B-cells are normally ␣4␤1-positive but are devoid of surface
␣4␤1 following TA-2 treatment, these data provide evidence
that this observed effect is not due to a loss of a lymphocyte
subclass.
Internalization of ␣4␤1 Integrin/TA-2 Complex. To de-
termine whether the ␣4␤1 integrin was being down-modu-
lated and internalized or whether the integrin was shed from
the cell surface following TA-2 treatment, we directly labeled
TA-2 with the fluorescent tag, Alexa594, and used confocal
microscopy to follow the disappearance of ␣4␤1 from the cell
surface. Results from the study are shown in Fig. 8. First, we
isolated splenocytes from an untreated Lewis rat, incubated
them with TA-2 ex vivo for 0, 4, 24, and 48 h and monitored
the arrangement of TA-2/␣4␤1 complexes by confocal micros-
copy. Figure 8A shows the time course of TA-2 binding and
internalization in rat splenocytes. At 0 h, the fluorescently
tagged TA-2 was uniformly bound to the cell surface with
none appearing in the cell cytoplasm. At 4 h, the staining was
less uniform with capping beginning to occur, represented by
the patchy areas of fluorescence on the cell surface. By 24 and
48 h, the cell cytoplasm showed dense areas of internalized
TA-2 with less fluorescence associated with cell surface. Be-
cause of the low density of ␣4␤1 on splenocytes, we were
unable to observe a measurable fluorescent signal when they
were treated with BIO8139-Alexa594. Consequently, we
tested binding of BIO8139-Alexa594 to the RBL.1 rat cell
line, which express a higher density of cell surface integrin.
RBL.1 cells were treated with a saturating dose of BIO8139-
Alexa594 or TA-2-Alexa594 for 30 min at 4°C then incubated
for 0, 4, 24, or 48 h. The observed capping and internalization
of TA-2 is shown (Fig. 8B) compared with BIO8139 (Fig. 8C)
in RBL.1 cells at 0 and 48 h using confocal microscopy. At
time 0, 30 min after the addition of the probes at 4°C, TA-2-
Alexa594 or BIO8139-Alexa594 uniformly saturated the cell
membrane whereas the cell cytoplasm was devoid of fluores-
cence (Fig. 8, B and C, 0 h). At 48 h, capping of the TA-2/␣4␤1
complex was striking on the cell surface (data not shown),
and in most cells, the complex had internalized (Fig. 8B,
48 h). BIO8139 did not internalize and remained on the cell
surface (Fig. 8C, 48 h).
Fig. 6. Evaluation of BIO5192 and TA-2 in rat EAE. Lewis rats were
injected with MBP on day 0 and then monitored daily for progression of
the disease. The treatment groups receiving either no treatment (f), the
TRIS/lactose vehicle (F), TA-2 (2.5 mg/kg, i.v., day 9 and 13) (Ⅺ), or
BIO5192 (30 mg/kg, s.c.) administered b.i.d. during days 5 through 14 (E).
Values are given as the mean Ϯ S.D. for 13 animals.
Discussion
We have investigated the role of ␣4␤1 in rat EAE using two
types of inhibitors, an anti-rat ␣4 monoclonal antibody TA-2,
and the small molecule inhibitor BIO5192. Whereas both
TA-2 and BIO5192 are potent inhibitors of ␣4␤1 and were
efficacious in the EAE model, they are biochemically very
different (see Table 2). The differences in their biochemical
properties provided a means for understanding key features
in the inhibition of ␣4␤1 function that lead to efficacy in EAE.
First, the selectivity of BIO5192 for ␣4␤1 indicates that en-
gagement of ␣4␤1 is a key event in the progression of the
disease. Although TA-2 recognizes ␣4␤1 and ␣4␤7, from the
BIO5192-selectivity data, we can infer that inhibition of ␣4␤7
is not necessary for efficacy in this rat EAE model. Second,
Fig. 7. Modulation of ␣4␤1 integrin by TA-2 but not BIO5192. A single
dose of TA-2 (2.5 mg/kg, i.v. in PBS), BIO5192 (30 mg/kg, s.c. in TRIS/
lactose, QD) or the corresponding vehicle control was administered to
Lewis rats at time 0. After 48 h, PBLs and splenocytes were isolated and
analyzed for ␣4␤1 levels by FACS analysis. 100% represents the binding
seen in vehicle-treated animals. The solid bars represent cells treated
with additional TA-2 (10 ␮g/ml) followed by goat anti-mouse-PE, and the
open bars represent cells treated with BIO8139-PE, for detection of cell
surface ␣4␤1.
and untreated animals were analyzed by FACS using specific although TA-2 treatment down-regulated ␣4␤1 expression,
surface markers for T- and B-cells. At 24 h post TA-2 treat- BIO5192 treatment had no effect on ␣4␤1 expression, indi-
ment, the percentage of total cells versus the untreated con- cating that blockade of ␣4␤1 is sufficient for inhibiting EAE.

1160
Leone et al.
TABLE 2
Comparative properties of BIO5192 and TA-2
Summary of the biochemical, PK, and PD properties of BIO5192 and TA-2 and their
efficacy in rat EAE.
Properties
BIO5192
mAb TA-2
Selectivity
␣4␤1
Human, mouse, rat,
sheep, dog
ϩ
␣4␤1/␣4␤7
Species specificity
Rat
Binds unactivated and
activated ␣4␤1
Binding inhibition by EDTA
LIBS inducer
KD
Valency
PK (i.v.) t1/2
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ͻ10 pM
Monovalent
30 pM
Divalent
48 h
ϩ
1.1 h
ϩ
Ϫ
Ϫ
ϩ
Lymphocytosis
Down modulates ␣4␤1
Clusters ␣4␤1
ϩ
ϩ
ϩ
Efficacy in EAE
maj et al., 1998). The significance is unknown, but may
reflect the contributions of other downstream effects on its
binding. Several groups have reported downstream effects of
blocking the ␣4␤1/VCAM-1 interaction. Leussink et al. (2002)
showed that inhibition of ␣4␤1/VCAM-1 interactions by mAb
TA-2 led to a decrease in cytokine production, including in-
terferon-␥ and TNF␣, and induction of T-cell apoptosis in rat
experimental autoimmunue neuritis. Furthermore, when a
TNF-binding protein was administered in a passive transfer
model of mouse EAE, lower TNF levels caused a decrease in
VCAM-1 expression in the central nervous system and a
down-regulation of ␣4␤1 expression on splenocytes (Selmaj et
al., 1998). In another study, treatment with anti-␣4 mAb
9C10 induced apoptosis of CD4ϩ and CD8ϩ T-cells resulting
in activation of protein kinase C during T-cell development
and following mature T-cell activation (Tchilian et al., 1997).
To determine whether BIO5192 could elicit a signaling re-
sponse, we performed a gene chip analysis on human THP1
cells with and without BIO5192 treatment (A. R. deFoug-
erolles, R. B. Pepinsky, R. R. Lobb, V. E. Koteliansky, data
not shown). As expected BIO5192, as a monomer, was unable
to induce a signal. Although TA-2 treatment may be having
a secondary effect in our studies, the data with BIO5192
indicate that in EAE this is not necessary for efficacy.
TA-2 and BIO5192 treatments both induced a lymphocy-
tosis and were efficacious in the EAE model. To verify the
relationship between lymphocytosis and efficacy, we tested
several other ␣4␤1 inhibitors with distinct biochemical prop-
erties (data not shown). The small molecule ␣4␤1 inhibitor,
(R)-N-[[4-[[(2-methylphenylamino)-carbonyl]amino]pheny-
Fig. 8. Confocal imaging and internalization of the integrin ␣4␤1 receptor/
TA-2 complex. Rat splenocytes were treated with TA-2 for 0, 4, 24, and
48 h (A) or RBL.1 cells treated with either TA-2-Alexa594 (B) or BIO8139-
Alexa594 (C) for 0 and 48 h. Images were taken with a Leitz Plan-
Apochromatic 63ϫ (1.32 numerical aperature, oil immersion) objective
(Leica) for the RBL.1 cells and 100ϫ objective for the splenocytes, with a l]acetyl]-L-prolyl-3-methyl)-␤-alanine, had an IC50 of 25 nM,
2ϫ digital zoom. Each frame represents a single optical section from the
did not elicit a lymphocytosis response, and was inactive in
rat EAE. We also tested an inhibitor compound 3 (Pepinsky
et al., 2002), which induced a very transient lymphocytosis
due to a short serum half-life, which also failed in EAE.
middle section of the cells observed for internalization under all condi-
tions. The fluorescent images were overlayed onto Neumarski images of
the cells. Note: there is no staining in the nucleus.
Other differences in the biochemical properties of TA-2 and Although compound 3 was a low nanomolar inhibitor of the
BIO5192 include metal ion dependencies, valency, and LIBS nonactivated integrin, it failed to maintain receptor occu-
binding. In particular since TA-2 contains two ␣4-binding pancy following a 30 mg/kg s.c. dosing. We infer from these
sites and BIO5192 has only one, the difference in valency is studies that a constant coverage of ␣4␤1 by the inhibitor is a
likely to have lead to the capping phenomenon observed with prerequisite for lymphocytosis and for activity in EAE. Lym-
TA-2 and subsequent events leading to down modulation.
phocytosis is a simple pharmacodynamic marker for blockade
The observation that no cell surface ␣4␤1 remained after of ␣4␤1 function and can be used as a surrogate marker for
treatment with TA-2 was surprising since others have ob- selecting dosing regimens. From our studies, it is unclear if
served only a slight down-regulation (Bretscher, 1992; Sel- the elevated cell number is due to the release of ␣4␤1-positive

Assessing Integrin ␣4␤1 Inhibitors in Rat EAE
1161
leukocytes from VCAM-1 expressed on blood vessel endothe- during clinical trials (Miller et al., 2003) and may be specific
for this mouse model.
lial cells or stroma or a block of trafficking lymphocytes
accompanied by their inability to leave the blood compart-
ment. The speed of the effect following BIO5192 treatment
best supports the displacement hypothesis.
Of the many small molecule inhibitors that have been
developed from the LDV sequence of CS1, BIO5192 is the
most potent and selective ␣4␤1 inhibitor developed to date
(Abraham, 1997; Lin et al., 1999; Kudlacz et al., 2002; van
der Laan et al., 2002). Lin et al. (1999) reported that
BIO1211, KD ϭ 70 pM for Mn2ϩ-activated ␣4␤1, is efficacious
in a sheep asthma model. BIO1211 is only a 20 to 40 nM
inhibitor of nonactivated ␣4␤1 and therefore was not expected
to be effective if the nonactivated ␣4␤1 was important for
function. BIO1211 was inactive in EAE when administered
at 30 mg/kg. A second compound, the CS1 ligand mimic,
phenylacetyl-L-leucyl-L-aspartyl-L-phenylalanyl-D-prolin-
eamide, was efficacious in the sheep asthma model and par-
tially effective in active rat EAE (Abraham, 1997; van der
Laan et al., 2002). A third compound, CP-664511, which is
related to BIO1211, is a 5 nM ␣4␤1 inhibitor in serum and
efficacious in an antigen-induced pulmonary eosinophil infil-
tration model. The extraordinary potency of BIO5192, Ͻ10
pM for activated and unactivated integrin and because it
binds ␣4␤1 from many species, including mouse, rat, sheep,
dog and humans, makes BIO5192 a particularly attractive
compound for investigating ␣4␤1 function. In summary, we
have compared the biochemical, pharmacological, and phar-
macodynamic properties and efficacy in a rat model of EAE,
of two ␣4␤1 inhibitors, mAb TA-2 and BIO5192. TA-2 pro-
vides a benchmark for assessing ␣4␤1 function in rats against
which we tested the highly selective ␣4␤1 inhibitor BIO5192.
Both inhibitors induced a lymphocytosis, a PD marker of
activity, and were efficacious in EAE. Treatment with TA-2
caused a decrease in ␣4␤1 integrin expression on the cell
surface, which resulted from internalization of the ␣4␤1 in-
tegrin/TA-2 complex. In contrast, BIO5192 did not modulate
cell surface ␣4␤1, indicating that blockade of ␣4␤1/ligand in-
teractions is sufficient for function in EAE. The use of potent
selective ␣4␤1 integrin inhibitors therapeutically for treat-
ment of inflammatory diseases may be an important alter-
native to therapy when inhibition is the mechanism neces-
sary to alter disease.
The role of ␣4␤7 in EAE has been controversial. The ␣4␤7/
MAdCAM-1 pathway was shown to contribute to the EAE
response in a nonremitting EAE model induced by myelin
oligodendrocyte glycoprotein 35–55-stimulated T-cells (Kan-
war et al., 2000a,b). Kanwar et al. showed that an anti-
MAdCAM-1 antibody alone or in combination with anti-
VCAM-1 and anti-intercellular adhesion molecule-1 induced
remission of EAE, with combination therapy leading to a
more rapid remission. Conversely, Englehardt et al. (En-
gelhardt et al., 1995, 1998; Laschinger and Engelhardt, 2000)
examined the roles of ␣4␤1 and ␣4␤7 in transfer and active
models of EAE in mice using monoclonal antibodies to ␣4␤1,
␣4␤7, the ␤7-chain and VCAM-1 and demonstrated that ␣4␤7
integrin is not essential for the development of EAE. MAd-
CAM-1 was never detected on blood-brain barrier endothelial
cells during EAE in SJL/J or C57Bl/6 mice. The BIO5192
studies presented here support this latter study demonstrat-
ing that inhibition of ␣4␤7 is not necessary. One explanation
for these conflicting results is that MAdCAM-1 is interacting
with ␣4␤1, and therefore the effect of the anti-MAdCAM-1 is
not on inhibition of ␣4␤7 but was a direct effect on ␣4␤1
function. In support of this hypothesis, Newham et al. (1998)
showed that MAdCAM-1 binds ␣4␤1. Also since ␣4␤7 binds to
osteopontin (Bayless et al., 1998) in the brain, MAdCAM-1
may not be involved. Alternatively, the discrepancy may
represent differences in the EAE model used by Kanwar et
al. (2000a,b) and would require a more thorough evaluation.
Multiple sclerosis is characterized by lymphocytes that
migrate and infiltrate into the central nervous system and
brain affecting demyelination of nerve cells causing the dis-
ease (Miller et al., 2003). Various aspects of multiple sclerosis
have been recapitulated in rodent models of EAE, with dif-
fering effects and efficacy profiles of ␣4␤1 inhibition observed
in rats and mice (Kanwar et al., 2000a,b). Rat EAE is usually
monophasic, and ␣4␤1 inhibition delays the onset of disease.
Our results confirm and extend these original observations.
Mouse EAE is usually relapsing/remitting characterized by
epitope spreading and activated T-cell involvement. En-
gelhardt et al. confirmed in a mouse model of EAE that
Acknowledgments
We thank Chris Ehrenfels for confocal instruction, Sukumori Mo-
han and Akos Szilvasi for performing FACS analysis, Martin Hemler
and S. Burakoff for cell lines, members of the Biogen-Merck Joint
blockade of ␣4/VCAM-1 interaction was involved in inflam- Task Force for helpful discussions, and Shelia Violette for support of
the manuscript.
matory cell recruitment across the blood-brain barrier and
that inhibition of ␣4␤1-dependent adhesion of antigen-spe-
cific T-cells to blood-brain barrier endothelium was impor-
tant for efficacy (Engelhardt et al., 1995, 1998; Laschinger
and Engelhardt, 2000). In another model, PS/2, an anti-
mouse ␣4 antibody, inhibited EAE when administered pro-
phylactically but exacerbated disease when given therapeu-
tically in established disease (Theien et al., 2001). We saw no
evidence of an exacerbation of disease when treatment was
stopped in rats. Furthermore, in human clinical trials in
which the anti-␣4 mAb natalizumab was administered to
patients with relapsing/remitting multiple sclerosis, clinical
benefits were observed while natalizumab maintained ade-
quate serum levels, and disease activity returned to baseline
when the drug was withdrawn. The exacerbation of disease
seen in the relapsing mouse model was not seen in patients
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Address correspondence to: Diane R. Leone, Biogen, Inc., 12 Cambridge
Center, Cambridge, MA 02142. E-mail: diane_leone@Biogen.com