Note: Descriptions are shown in the official language in which they were submitted.
2298P/0832A 1240669
- 1 - 16777IB
TITLE OF THE INVENTION
CONJUGATES OF LEUKOTRIENES WITH PROTEINS
RELATIONSHIP TO THE PRIOR _
The concept of using conjugates of
leukotrienes in a radioimmunoassay was described, by
L. Levine, R. A. Morgan, R. A. Lewis, K. F. Austin,
D. A. Clark, A. Marfat, and E. J. Corey, Proceeding
of the National Academy of Sciences, U.S.A., Vol. 78,
No. 12 7692 (1981). This method uses direct coupling
through an activated acid derivative to the protein.
This method is much less effective than the presen~
invention.
Bifunctional cross-linking reagents useful
~n preparation of protein-hapten conjugates have also
been prepared, see Kitagawa, J. Biochem. 79, 233-236;
and Kitagawa, Chem. Pharm. Bull. 29(4), 1130-1135;
1240669
2298P/0832A - 2 - 16777IB
describing maleimido-succinimide derivatives. The
present invention relates to conjugates of
leukotrienes C4, B4, D4 or E4 (preferably
C4 and B4) with a protein selected from hemo-
cyanine from giant keyhole limpets (KLH~, bovineserum albumin (BSA), human serum albumin, tetanus
antigen, diphtheriae toxoid, or CRM 197 (a diphtheriae
toxoid produced by a mutant of Corvnebacterium
diphtheriae), through the coupling agents
1,5-difluoro-2,4-dinitrobenzene or 6-N-maleimido-
alkanoic acid chloride, preferably 6-N-maleimido-
hexanoic acid chloride, wherein the alkanoic moiety
has 2 to 8 carbon atoms. The conjugates are useful
in a sensitive and specific immunoassay and are also
useful immunotherapeutic agents in the treatment of
various allergic and chronic inflammatory diseases of
the skin, lung and airways, including asthma,
allergic rhinitis, rheumatoid arthritis, and skin
diseases such as psoriasis and eczema. The present
invention also relates to useful reagents for
preparing such conjugates.
Leukotriene C4 (LTC4) has the following
structure:
25~ COOH
S ~ COOH
~/I\NH
~/ \~\COOH LTC4_
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2298P/0832A - 3 - 16777IB
Leukotriene B4 (LTB4) has the following
structure:
HO ~ CO2H
H~ ~ OH LTB4_
Leukotriene D4 (LTD4) has the following structure:
S/~N~\CO 2H
H2
~ CO2H
LTD4_
Leukotriene E4 (LTE4) has the following structure:
~C02H
NH 2
~02H
LTE4_
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2298P/0832A - 4 - 16777IB
The present invention also relates to the
following compounds which are useful in preparing the
conjugates (especially the conjugates of LTB4):
1)
H ~\CONH (CH2 ) nNH2
wherein n is 0 to 10, preferably 0 or 2 to 10,
more preferably 0 or 3.
2)
HO~
~\CONH (C32) nNH~Fo2
wherein n is 0 to 10, preferably 0 or 2 to 10, more
preferably 0 or 3.
3)
HO~
~\coN~(cH2)nN3
1~Z4066'~
2298P/0832A - 5 - 16777IB
wherein n is 0 to 10, preferably
0 or 2 to 10, more preferably 0 or 3.
In the above three compounds, the compounds
where n is 1 are likely to be less stable than the
other compounds having the same generic formula.
The preparation of the conjugates of the
present invention may be illustrated by reference to
conjugates of LTC4 and LTB4.
For LTC4, coupling procedures were
selected so that the reactions took place on the free
amino group of the glutamyl residue, thus retaining
the most important parts of the LTC4 molecule
unchanged.
The general conjugation procedure utilized a
stepwise method with well characterized
intermediates. The strong W absorption of the
triene chromophore in LTC4 ( = 40,000 at 280 nm)
was used as a probe for determining coupling
efficiencies and for monitoring the state of the
LTC4 molecules throughout the procedures.
Coupling ratios in the ranges of S to 15
equivalents of LTC4 per 100,000 daltons of protein
were desired.
Conjugates of LTC4
Conjugations using 1,5-difluoro-2,4-
dinitrobenzene as coupling agent:
The reagent, 1,S-difluoro-2,4-dinitrobenzene
(DFDNB) reacts quite specifically with amino
functions, allowing clean stepwise replacement of the
two fluorine atoms (the second fluorine being
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2298P/0832A - 6 - 16777IB
replaced at a much slower rate). In addition, the
strong and characteristic W absorptions of the
reagent, and its mono- and diamino substituted
derivatives allows one to follow the course of the
coupling procedure and to quantitate the final
adducts by W spectroscopy.
LTC4 was found to react essentially
quantitatively with excess DFDNB in pH 7.2 buffered
aqueous methanol within 30 minutes. The
intermediates thus formed cou d be characterized by
HPLC analysis, by the appearance of a strong W band
at 345 nm characteristic of 1-amino-5-fluoro-2,4-
dinitrobenzenes. After removal of methanol from the
reaction the excess DFDNB could be removed by ether
extraction. The intermediates could be further
purified by HPLC but this was found to not offer any
advantage and, in general, the crude reaction mixture
was then allowed to react with protein in pH 8.5
buffer for two days in the dark. Final separation of
the conjugates from unreacted LTC4 or reagents was
achieved by filtration on Sephadex G-50. The derived
coupled products now showed W absorptions at 342 and
420 nm characteristic of 1,5-diaminodinitrobenzenes
as well as the characteristic absorptions of the
triene system at 271, 282, and 291 nm in the case of
the LTC4 conjugates. In this manner, S-p-chloro-
phenacylglutathione when reacted in 10:1 molar ratio
with BSA gave a conjugate with about 6 moles of
hapten per mole of BSA.
Similarly, LTC4 in 30 fold mol r excess
gave a conjugate with BSA with 9-10 moles LTC4 per
mole BSA, and LTC4 in ca. 30 fold molar excess
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2298P/0832A - 7 - 16777IB
(calculated per 100,000 daltons protein~, gave a
conjugate with KLH with 11-12 equivalents LTC4 per
100,000 daltons KLH.
Conjugation using 6-N-maleimidohexanoic acid
chloride as coupling agent
Since this invention provides a second
LTC4 protein conjugate using a different spacer
group, a number of potential coupling methods were
examined. A direct coupling using a reagent such as
DCC or ECDI (6) was considered but quickly rejected
due to the expectation that a heterogeneous mixture
of adducts would be formed. Also, preliminary
experiments indicated that the efficiency of such a
coupling would be low. The known agents, toluene
diisocyanate and m-maleimidobenzoyl-N-hydroxy-
succinimide ester were not used due to the
possibility of immunological cross reactivity with
respect to the spacer units between the two
conjugates.
The coupling agent 6-N-maleimidohexanoic
acid chloride provides rapid, selective
functionalization of the glutamyl amino group of
LTC4, as well as high coupling efficiency.
The agent chosen was 6-N-maleimidohexanoic
acid chloride which was readily prepared from
6-aminohexanoic acid. Other analogous reagents
having from 2-8 carbon atoms in the chain can be
used, e.g., 2-aminoacetic acid up to 8-amino octanoic
acid.
The 6-N-"laleimidohexanoic acid amide of
LTC4 was prepared by reacting a methanolic solution
~Z~Q66~
2298P/0832A - 8 - 16777IB
of LTC4 tripotassium salt with the reagent (1.5
equivalents in dry THF) in the presence of excess
Et3N. HPLC analysis showed essentially complete
conversion to the amide (eluting before LTC4 on
RP-HPLC). A portion of this adduct, isolated from
HPLC, had W characteristics essentially unchanged
from those of LTC4. For subsequent coupling with
thiolated protein (KLH) the crude mixture (in pH 7.2
borate buffer) was used as such.
The thiolated protein used, in this case
derived from KLH, was prepared by reaction with
S-acetylmercaptosuccinic anhydride. As no report of
thiolation of KLH could be found in the literature,
trials were done to determine conditions for
obtaining KLH with about 20 S-acetyl groups per
100,000 daltons protein [thio] content, after
hydrolysis of the acetyl groups, was determined by
Elleman's method. The S-acetylmercaptosuccinyl
derivatized XLH was highly unstable to oxygen until
further reacted with N-ethyl maleimide (NEM).
However, once any free SH groups were thus reacted,
the material could be handled and purified by
Sephadex~G-50 filtration.
Concentration of the resulting purified
protein was accomplished by dialysis against a
packing of anhydrous Sephadex~G-200 resin. Just
prior to coupling with derivatized LTC4, the thiol
groups were liberated by hydrolysis of the rigorously
deoxygenated solution at pH 11.5 followed by
reducti~n of the pH to 7.2.
This mixture was then reacted with the
deoxygenated solution of the 6-N-maleimidohexanoic
~24~669
2298P/0832A - 9 - 16777IB
acid amide of LTC4 in a ratio of 80 equivalents
LTC4 per 100,000 daltons KLH. After stabilization
with NEM and purification by Sephadex G-50, the
protein conjugate showed 7-10 moles LTC4 per
100,000 daltons KLH by W analysis.
The protein solution has proven to be very
stable during several months storage frozen at -78C.
More detailed examples follow. It is noted
that IR spectra were recorded on a Perkin-Elmer 267
Grating Spectrophotometer. PMR spectra were recorded
on a Varian EM-390 spectrometer. UV spectra were
recorded on a Cary 210 spectrophotometer. Spectra
were recorded in water unless otherwise indicated.
Sephadex~G-50 (medium grade) was obtained from
Pharmacia Fine Chemicals.
Bovine Serum Albumin was obtained as
crystalli~ed and lyophilized grade from Sigma
Chemical Co. and Hemocyanin (Keyhole Limpet) was
obtained as lyophilized powder from Calbiochem
Behring Corp. Leukotriene C4 was synthetic material
prepared in our laboratories using known procedures,
Rokach _ al., Tet. Lett., 21, 1485 (1980)._
Preparation of LTB4 conjugates is
illustrated by the following reaction schemes:
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Scheme 1
HO~=\~\C02Et
H~ O~Ph
II
H 2N~NH 2
HO~CON~\NH2
III
2 ~NO2
H.~/\CON~ N~,~F
2 NO2
1 H2N-BSA
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2298P/0832A - 11 - 16777IB
r N ~ 2 ~ ~2
This method makes use of the immediate
synthetic precursor to LTB4, Ethyl 5(S)benzoyloxy-
12 (R) -hydroxy-6 , 14 (z) -8 ,10 (E)-eicosatetraenoate
(II). We reasoned that reaction of II with a
volatile diamine such as 1,3-diaminopropane would at
the same time remove the benzoate protecting group
and convert the ethyl ester to the ~-aminopropyl-
lS amide, all under mild weakly basic conditions. The
solvents could then be removed under vacuum leaving
only a mixture of the product (III) and N-~-amino-
propylbenzamide. In model studies, using ethyl
5-(4-octylphenyl)-~-benzoyloxypentanoate this
reaction was found to be extremely sluggish, even in
neat 1,3-diaminopropane. However, when a catalytic
amount of 2-hydroxypyridine was added to the reaction
mixture, the diester was smoothly converted to the
desired aminoamide. When applied to the protected
LTB4 (II) a similar smooth conversion to III was
effected. III could be reacted directly in the next
step, after removal of the volatile components. The
aminoamide (III) was reacted with excess 1,5-difluoro-
2,4-dinitrobenzene in the presence of triethylamine
to provide the dduct IV in high yield. This product
was purified by reverse phase HPLC and was fully
characterized by W and PMR spectroscopy. Finally,
~2406~i9
2298P/0832A - 12 - 16777IB
IV reacted smoothly with bovine serum albumin (BSA)
(mole ratio-12:1) in a mixture of dimethylformamide
and pH 8.5 borate buffer to provide the conjugate V
which was purified by chromatography on Sephadex
G-50. W spectral analysis indicated that the triene
chromophore was unchanged and allowed the estimation
that 5.5-8.3 moles of LTB4 were coupled per mole of
BSA. (45-70~ coupling efficiency).
The aminoamide III could also be prepared by
direct reaction of LTB4 -lactone with
1,3-diaminopropane at room temperature. This
provided III in quantitative yield free of side
products.
124()~;~;9
2298P/0832A - 13 - 16777IB
Scheme 2
~K 1~=~ \COGH
H ~ CH 6 2 H' ~
10 R.T. ~ ~ HO~=~( 2
H~ ~ H O
VI VII
0
20~
~eaH, Et3N VIII
~ ~W~ W
~24~6g
2298P/0832A - 14 - 16777IB
Another type of LTB4 conjugate
could be ~repared as illustrated in Scheme 2.
Lact one VI reacted cleanly with hydrazine to
provide the hydrazide (VII) in quantitative yield.
VII was reacted further with 6-N-maleimidohexanoic
acid chloride to give the diacyl hydrazide (VIII).
This material could be purified by reverse phase HPLC
to remove the excess reagent byproducts. However,
attempts to concentrate the product in order to
obtain a PMR spectrum led to partial decomposition
apparently due to hydration or methanolysis of the
maleimide system. It was found however, that the
crude reaction product could be used in the
subsequent coupling reaction. VIII was reacted with
thiolated KLH in a ratio of 50 moles of VIII per
100,000 daltons KLH, to provide the desired conjugate
IX which was purified by filtration through Sephadex~
~-50. UV analysis indicated that 12 equivalents of
LTB4 were bound per 100,000 daltons of KLH.
EXAMPLE 1
Conjugates of LTC4 Using 1,5-Difluoro-2,4-
dinitrobenzene as Coupling Agent
A. Conjugation of S-p-Chlorophenacylgluta-
thione and 80vine Serum Albumin (BSA)
1,5-Difluoro-2,4-dinitrobenzene (120 mg,
0.59 mmol) in methanol (6 ml) was added to a solution
of S-p-chlorophenacylglutathione (88 mg, 0.19 mmol)
in 9 mL of phosphate buffer (pH 7.2, O.lN). After
stirring 12 hours at ro~ temperature the methanol
was removed in vacuo and the resulting aqueous
solution was washed with ether. The aqueous layer
~If
, ~' ;,
1~4~)6~9
2298P/0832A - 15 - 16777IB
was chromatographed on C-18 Silica Gel (eluting with
methanol:water (1:1) to provide the pure adduct
intermediate (105 mg). UV: ~max (~) 260 (24,000),
347 nm (19,000). PMR (D2O): ~ 8.62 (lH, d, J ~ 7.5
Hz), 7.6 (2H, d, J = 9Hz, A of AB), 7.1 (2H, d, J =
9Hz, B of AB), 6.7 (lH, d, J = 15Hz), 3.9 (2H, s,
phenacyl CH2).
The adduct (1.05 mg, 1.63 X 10 6 mol) in
water (0.1 mL) was added to a solution of BSA (10 mg,
1.49 X 10 7 mGl) in borate buffer (pH 8.5, 0.2 N, 1
mL). After standing in the dark at room temperature
for 71 hours the solution was centrifuged and
filtered on Sephadex~G-50 (1.5 X 75 cm) eluting with
water. Fractions (10.5 mL) eluting after the void
volume (55 mL) contained protein and were analyzed by
UV. A sample of this solution diluted 5 times had a
W spectrum (in H2O) ~max (Absorbance) 342 (0.3S9),
425 nm (0.133). Assuming 8 mg of protein were
recovered and assuming for the 1,5-diamino-2,4-
dinitrobenzene chromophor of ca. 27,000 (3) at 342 nm
the W indicated 6 moles of S-~-chlorophenacyl-
glutathione were conjugated per mole of BSA.
B. Conjugation of Leukotriene C4 and
Bovine Serum Albumin:
Leukotriene C4 (tripotassium salt) (2.5 mg) was
dissolved in 1 mL of phosphate buffer (pH 7.2, 0.1
N). 1,5-Difluoro-2,4-dinitrobenzene (1 mg) in
methanol (0.6 mL) was added and the mixture was left
3( min. at room temperature. The methanol was
removed under a stream of N2 and then ln vacuo to
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2298P/0832A - 16 - 16777IB
rémove final traces followed by extraction with ether
(3 x 2 mL) to remove unreacted reagent. The last
traces of ether were removed under N2 and in
vacuo. To this mixture was added bovine serum
albumin (BSA) (10 mg) in borate buffer (0.2M, pH 8.5,
1 mL), and the mixture was left to stand at room
temperature in the dark for two days. The reaction
mixture was filtered on a column of Sephadex G-50
(1.5 x 75 cm) eluting with water and the yellow
protein eluting in 18 mL, after the void volume, of
ca. 55 mL, was collected. At about the 140 mL dead
volume a peak considered to contain unreacted LTC4
eluted. Direct W analysis on the protein fractions
tcombined) gave a spectrum ~max (A) 271 (sh), 282
15 (3.57), 291, 342 (1.835) and 420 nm (0.91). Assuming
about 9 mg of protein were recovered, and assuming
for the l,5-diamino 2,4-dinitrobenzene of about
27,000 at 340 nm and for LTC4 at 280 nm of
40,000, calculations based on the 282 nm absorption
about 10.0 mole of LTC4 per mole BSA while
calculations based on the absorption of 342 nm
indicated 9.1 moles LTC4 per mole BSA.
C. Conjugation of Leukotriene C4 and
Hemocyanin from Giant Keyhole Limpets
(KLH):
Leukotriene C4 (tripotassium salt) (2.1
mg), and 1,5-difluoro-2,4-dinitrobenzene (8 mg) were
reacted as in reaction A. To the resultant adduct
30 was added KLH (15 mg) in borate buffer (pH 3.5, 0.2M,
0.83 mL) and the mixture was allowed to stand at room
temperature 60 hours. At this time a precipitate of
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2298P/0832A - 17 - 16777IB
denatured KLH had formed which was removed by
centrifugation (6 mg, dry weight). The supernatant
was filtered on Sephadex G-50 as before yielding a
yellow protein fraction eluting in 17 mL following
the void volume which by W analysis indicated 11-12
equivalents of LTC4 per 100,000 daltons of KLH.
D. Conjugation of 2,4(E),6,9(7)-Penta-
decatetraen-l-ol with BSA
A solution of DFDN~ t2.04 9, 10 mmol) in
dioxane (20 mL) was added to L-proline (0.58 g, 5
mmol) in phosphate buffer tpH 7.5, 0.1 N, 5 mL) and
the mixture was stirred 2 hours at room temperature.
The mixture was reduced to dryness and the residue
was chromatographed on silica gel (eluting with
chloroform:methanol (9:1) to yield N-2,4-dinitro-
5-fluorophenylproline as a foam (1.1 g).
PMR (CDC13): ~9.43 (lH, broad, exchanged
by D2O, COOH), 8.55 (lH, d, JH F = 7.5 Hz, H-3 of
phenyl), 6.62 (lH, d, JH F = 15 Hz, H-6 of phenyl),
4.5 (lH, broad t, J = 6Hz, proline methyne), 3.7-3.1
t2H, m), 2.7-1.9 ppm (4H, m).
To a mixture of 2,4(E),6,9(Z)-pentadeca-
tetraen-1-ol (123 mg, 0.56 mmol) and the proline
derivative above (170 mg, 0.57 mmol) in methylene
chloride, at -10 C, were added successively,
l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide
methyl-~-toluenesulphonate (266 mg, 0.63 mmol) and
pyrrolidinopyridine (9 mg, 0.06 mmol). The solution
was stirred under N2 at room tem; !rature for 7
hours. The mixture was filtered and the filtrate was
washed with water, 5% NaHCO3, brine and dried over
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2298P/0832A - 18 - 16777IB
Na2SO4. The residue after concentration was
chromatographed on silica gel [eluting with
chloroform:ethano] (99.25:0.75)] to yield the pure
adduct as an oil.
PMR (CDC13): ~ 8.57 ~lH, d, J = 7.5 Hz),
6.55 (lH, d, J = 15 Hz), 6.7-5.2 ~8H, m, olefinic),
4.65 (2H, d, J = 6Hz, -COOCH2-), 4.47 (lH, t, J =
6Hz , proline methyne), 3 . 4 5 (2H, m), 2 . 95 (2E, m)
2.7-1.8 (6H, m), l.S-1.2 (6H, m), 0.88 ~3H, t).
W (dioxane: ~max (~) 275 (48,700), 347nm (18,450).
Anal- calcd for C26H32N36F C, 62-26; H~ 6-43;
N, 8.38; F, 3.7g. Found: C, 61.88; H, 6.72 N,
8.48; F, 3.47.
A suspension of the add~ct (5 mg, 1 X 10 5
mol) and BSA (10 mg, 1.5 X 10 7 mol) in dioxane (1
mL) and borate buffer (pH 8.5, 0.2M, 3 mL) was slowly
stirred at room temperature for 4 days in the dark.
The mixture was centrifuged and the suspernatant was
filtered on Sephadex~G-50 (1.5 X 75 cm), eluting with
water. The protein fraction eluting in 7 ml after
the void volume analyzed by W for approximately 4
moles hapten per mole of BSA.
EXAMPLE 2
Conjugates of LTC4 Using 6-N-Maleimidohexanoic
Acid Chloride as Couplinq Agent
A. Preparation of 6-N-Maleimidohexanoic
Acid Chloride
6-Aminohexanoic acid (2 g, 0.02 mol) and
maleic anh;dride (2 g, 0.02 mol) were refluxed
together in xylene (20 mL) under a Dean-Stark water
separator such that the internal temperature reached
~Z~V669
2298P/0832A - 19 - 16777IB
ca. 165C. The mixture was cooled, diluted with
chloroform-methanol and washed with lN hydrochloric
acid. The orqanic layers were washed with water,
dried, and reduced to dryness to yield a residue (1
g) which after chromatography on silica gel (eluting
with 5% methanol-chloroform) provided pure
6-N-maleimidohexanoic acid, m.p. 84-85C.
IR(KBr): 3300-2500 (COOH), 1700 cm 1 (maleimide and
COOH). PMR (CDC13): ~ 11.10 (lH, s, exchanged by
D2O, COOH), 6.72 (2H, s, maleimide CH), 3.53 (2H,
t, J = 7Hz), 2.34 (2H, t, J = 7Hz), 1.6 ppm (6~, m).
Mass spectrum: m/e 211 (M ).
Anal. Calcd for CloH13NO4: C, 56.87; H, 6.20;
N, 6.63. Found: C, 56.87; H, 6.24; N, 6.62.
6-_-Maleimidohexanoic acid (50 mg, 0.23
mmol) and a,a-dichloromethyl methyl ether (150 ~1,
1.5 mmol) were refluxed together in anhydrous
dichloromethane (1 mL) overnight. The mixture was
reduced to dryness and the resultant highly
hygroscopic solid (6-N-maleimidohexanoic acid
chloride (54 mg) was used, freshly prepared, in the
coupling reactions.
IR (film): 1795 (COCl), 1700 cm 1 (maleimide).
PMR (CDC13): ~ 6.60 (2H, s, maleimide CH), 3.53
(2H, t, J = 7Hz), 2.90 (2H, t, J = 7Hz), 1.6 ppm (6H,
m).
B. Reaction of 6-N-Maleimidohexanoic Acid
Chloride with Leukotriene C4
LTC4 tripot~ssium salt (5 mg) was
dissolved in anhydrous methanol (1 mL) and
triethylamine (80 ~L) under nitrogen and the acid
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2298P/0832A - 20 - 16777IB
chloride (25 ~L of a solution of 10 mg acid chloride
in 100 ~L anhydrous THF) was added. The reaction was
stirred at room temperature and was followed by HPLC
(Whatman Partisil M9 10/25 ODS, eluting with
MeOH:H2O:HOAc; 70:30:0.01, 4 mL/min). The adduct
eluted at 4.8 min. and LTC4 eluted at 6.6 min.
After 10 and 30 min. about 15% of unreacted LTC4
remained. More of the acid chloride solution (5 ~L)
was added and after a further lO min. 5% unreacted
LTC4 remained. The reaction mixture was
concentrated to 0.2 mL under a stream of N2,
diluted with borate buffer (pH 7.2, 0.1M, 0.5 mL) and
the residual methanol was removed in vacuo. This
solution had W spectrum essentially unchanged from
LTC4 itself, and was used as such in reaction with
thiolated KLH (see following).
C. Reaction of KLH with S-Acetylmercapto-
succinic Anhvdride
KLH (60 mg) was dissolved in borate buffer
(0.2M, pH 8, 1.5 mL) and centrifuged to remove
denatured protein. The resultant solution analyzed
for 24.6 mg/mL by W [E278(mg/mL) = 1.36]. The
solution was deoxygenated (by three purges
alternating high vacuum and pure N2 flush) then
treated under N2 with S-acetylmercaptosuccinic
anhydride (45 mg added in 5 mg portions over one
hour). The pH was maintained at 8 by addition of lN
NaOH (total 400 ~L). After s-anding one hour more,
N-ethylmaleimide (20 mg in 0. mL MeOH) was added to
bind any free thiol groups and stabilize the solution
to air. After standing 1.5 hours more the solution
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2298P/0832A - 21 - 16777IB
was centrifuged and applied to a column Sephadex G-50
(1.5 x 75 cm) eluting with 0.1N saline buffered with
0.01N pH 6.2 phosphate buffer. Two fractions (7 mL)
eluting after the void volume contained the bulk of
the protein (2.4 mg/mL). An aliquot analyzed for
thiol content, after hydrolysis at pH 11.5 for one
hour, indicated 18 thiol groups per 100,000 daltons
protein.
D. Coupling of 6-N-Maleimidohexanoic Acid
Amide of LTC4 and Thiolated KLH
A solution of S-acetylmercaptosuccinate
derivative of KLH (from reaction C) (10.8 mg, in 4.5
mL 0.1N saline, buffered to pH 6.2 with 0.01N
phosphate) was rigorously deoxygenated and then the
pH was raised to 11.5 with lN NaOH (150 ~L) under
N2 and the mixture was left at room temperature for
one hour. The pH was then reduced to 7.2 by addition
of deoxygenated lN HCl (150 ~L) and the solution of
the 6-N-maleimidohexanoic acid amide derivative of
LTC4 from reaction B was added. After standing 2
hours at room temperature, _-ethylmaleimide (1 mg in
10 ~L methanol) was added and the mix~ure was left
one hour more at room temperature. This solution was
25 applied to a Sephadex~G-50 column (1.5 x 7.5 cm)
eluting with 0.lN saline buffered to pH 6 with 0.01N
phosphate. The protein fraction eluted with 85% in
11 mL after the void volume. Unreacted reagents
eluted at the dead volume (150 mL). The protein
solution was adjustad to pH 7.2 with lN NaOH for
storage.
~r
lZ4066~
2298P/0832A - 22 - 16777I8
Analysis of the protein solution by UV
indicated 7-10 equivalents of LTC4 were coupled per
100,000 daltons protein.
The conjugates of LTC4 with the proteins
BSA and KLH have been used to raise antibodies with
rabbits, at a dose of 200 ~g/rabbit, approximately
weighing 1 kg; the antibodies specifically recognize
Leukotrienes C4, D4, and E4. A detailed
description of the antibody production, specificity,
and the use of these conjugates in an immunoassay for
the leukotrienes follows.
In addition to LTC4 and the specific
proteins used, it will be appreciated that other
leukotrienes, such as LTD4 and LTE4 can be
conjugated with other antigenic proteins such as
tetanus antigen, human serum albumin (HSA), as well
as diphtheriae toxide, tetanus antigen, and CRM 197
(from coryne bacterium diphtheriae) and other similar
antigenic materials.
EXAMPLE 3
Immunization Using LTC4 Conjugates
The following is the immunization regime
used employing KLH-maleimido-LTC4 as the immunogen.
Three 4 month old New Zealand White rabbits
each received sub-cutaneous injections at multiple
sites of 200 ~g KLH-LTC4 in complete Freunds
adjuvant followed in three weeks by sub-cutaneous
injections at multiple sites with 100 ~g LKH-LTC4
in incomplete Freunds adjuvank . The rabbits were
bled 10 days after the second injection and every
three weeks thereafter. When a significant decline
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in the level of antibody was observed, the animals
were boosted with 200 ~g KLH-LTC4 in incomplete
Freunds adjuvant and the animals bled again on the
same schedule.
The antigen BSA-DNP-LTC4 was employed in a
solid-phase-immuno-radioassay (SPIRA) in order to be
used for the dectection of leukotrienes.
Polyvinyl chloride - 96 well microtiter
plates (Dynatech Laboratories) were coated with
antigen (BSA-DNP-LTC4) by incubating 100 ~1
aliquotes of the antigen at 0.1 mg protein/ml in PBS
for 18 h at 4C. The wells were washed three times
with 200 ~1 PBS and then unreacted sites in the wells
were blocked by incubating a 200 ~l aliqout of 10%
horse serum in PBS in the wells for 2 h at 22C. The
wells were then washed three times with 200 ~l of
PBS-1.5 H.S.(1.5% horse serum in PBS). One hundred
(100)~1 of a reaction mixture containing a dilution
- of the immune or pre-immune rabbit serum was added to
the wells and the plates incubated for 4 h at 22C.
The 100 ~l reaction employed for the titration of
rabbit serum consisted of 50 ~l of dilutions of the
sera in PBS-l. 5 H.S. and 50 ~l of PBS-l. 5 H.S. For
competition analysis this reaction mixture consisted
of 50 ~l of a dilution of immune se~um in PBS-1.5
H.S. which contained a limiting amount of leukotriene
specific antibody and 50 ~1 of PBS-1.5. H.S~
containing various concentrations of leukotrienes or
chemically related compounds. This lon ~1 reaction
mixture was preincubated l h at 22C I fore it was
added to the well of the microtiter plate.
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The wells of the microtiter plate were then
washed three times with 200 ~1 PBS-1.5 H.S. and then
100 ~1 of 125I-labeled rabbit anti-mouse
[F(ab)2 fragments of rabbit anti-mouse IgG (H +
L)] in PBS containing 10% horse serum was added to
the wells and the plates incubated 4 h at 22C.
Approximately 2 X 104 cpm of the iodinated reagent
was added to each well. After the incubation period,
the wells were washed five times with 200 ~1 PBS-1.5
H.S. and once with 200 ~1 PBS. The wells were then
cut from the plate and the radioactivity in each well
was determined in a gamma counter.
The advantage of this assay is that,
although the rabbits were immunized with
KLH-maleimido-LTC4, therefore antibodies are
present in these animals against KLH, against the
maleimido linker and against the hapten-LTC4,
however, antibodies directed against the KLH and the
maleimido linker do not cross react or bind to the
BSA or DNP linker of the material coated onto the the
surface of the wells. Therefore the only antibodies
that bind to the material coated on the wells
(LTC4-DNP-BSA) are directed against the LTC4.
These rabbit LTC4 antibodies bind to the
LTC4 portion of the conjugate and they in turn are
detected by adding a second species of antibody
(125I-labeled goat anti rabbit antibodies). There
antibodies are radio-labeled with iodine and will
bind to the rabbit antibodies which in turn are bound
to LTC4. The ne~ result is, the more antibodies
directed against LTC4, the more radioactivity
associated with the well.
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2298P/0832A - 25 - 16777IB
In order to determine if free LTC4 is in a
biological sample, an aliquot is added to the plastic
well. Some of this free LTC4 will bind to the
rabbit anti LTC4 displacing if from the antigen
coating which is bound to the surface. This results
in a decrease in the number of counts (125I) bound
to the surface of the well and by comparing this
decrease to the decrease in a standard curve where
known amounts of free LTC4 are added, the amount of
LTC4 in the sample can be determined.
The other compounds described in Examples 1
and 2 can be similarly used in an assay system, as
reagents.
The antisera produced in rabbits by
immunization with these conjugates can also be used
in conjunction with radio-labelled leukotrienes C4,
D4, or E4 as the basis of a radioimmunoassay for
Leukotrienes C4, D4, and E4.
These con~ugates are useful as chemical
immunotherapeutic agents in the treatment various
allergic and chronic inflammatory diseases of the
skin, lung, and airways, including asthma, allergic
rhinitis, rheumatoid arthritis, and skin diseases
such as psoriasis and eczema.
LTC4 ANTIBODY ASSAY
In a standard Guinea Pig Ileum assay, 4
tissues were set up in 10 ml baths of Kreb's buffer
with atropine and pyrilamine both at 10 6M.
Standard contractions were observed using 10
~1 of 2.7 x 10 6 M LTC4 solution in a 10 ml bath,
for a final concentration of 2.7 x 10 M LTC4.
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2298P/0832A - 26 - 16777IB
The standard response tension was 1.1-2.0
grams.
20 ~1 of stock LTC4 solution was mixed
with a varying amount (10 ~1, 40 ~1, 100 ~1 and 400
~1) of anti-LTC4 serum (rabbit).
(1 ml of serum contained 7.9 x 10 M of
specific anti-LTC4 antibody).
The serum was incubated on ice (in the dark)
for 1/2 hour before use.
Control samples were run using similar
amounts of normal rabbit serum.
The mixed samples (15 ~1, 30 ~1, 60 ~1 and
210 ~1 respecitvely) were added to baths and the
response recorded.
RESULTS
Volume of Antibody Serum % of Control Response
205 ~1 100.0
20 ~1 72.7
50 ~1 92.3
200 ~1 64.7
Volume of Normal Serum
5 ~1 108.6
20 ~1 85.7
50 ~1 102.6
30200 ~1 114.3
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From the above results, clearly the
anti-LTC4 serum diminished the effect of LTC4 in
each sample. Thus the conjugates can be used to
raise antibodies to LTC4, LTD4, LTE4, in humans
(in a manner similar to that employed herein in
rabbits). The resulting circulating levels of
antibodies would serve to diminish plasma levels of
LTC4, and LTD4 and LTE4 released during an
asthmatic anaphylactic response and thus serve to
alleviate the symptoms. Since the antibodies would
be present during long periods of time, this would
represent a long term asthma therapy.
EXAMPLE 4 - Conju~ates of LTB4
Materials and Conditions
PMR spectra were recorded on a Varian EM-390
or Bruker WM-400 spectrometer. W spectra were
recorded on a Cary 210 spectrophotometer. Optical
rotations were measured using a Perkin Elmer Model
241 Polarimeter. Sephadex G-50 (medium grade) was
obtained from Pharmacia Fine Chemicals.
Bovine serum albumin was obtained as
crystallized and lyophilized grade from Sigma
Chemical Co. and Hemocyanin (Keyhole Limpet) was
obtained as lyophilized powder from Calbiochem
Behring Corp.
1. Conjugation of LTB4 with Bovine Serum Albumin
A. 5(S),12(R)-dihydroxy-6,14(Z)-8,10(E)-eicosatetra-
z- 30 eonic acid ~-lactone (LTB4 -lactone) (VI):
5(S),12(R)-dihydroxy-6,14(Z)-8,10(E)-eico
satetraenoate (12 mg) was stirred under nitrogen in
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2298P/0832A - 28 - 16777IL
methanol (1.5 mL) and water (0.4 mL) with postassium
carbonate (22 mg) for 2.5 days at ambient
temperature. Most of the methanol was removed under
a stream of nitrogen (to leave about 0.4 mL volume)
and the mixture was diluted with 0.lN pH 6.2
phosphate buffer (2.5 mL). The mixture was extracted
with ether (5 x 2 mL) and the combined ether extracts
were dried (Na2SO4) and reduced to dryness. uv
analysis of the resulting oil indicated that 8 mg of
LTB4 free acid was thus obtained. The oil was
dissolved in anhydrous ether (5 mL) and treated with
dicyclohexylcarbodiimide (DCC) (20 mg) at 0 under
nitrogen for 24 hours. TLC analysis (ethyl acetate:
hexane 2:3) indicated about 50% conversion of LTB4
to the ~-lactone (Rf LTB4 = 0, Rf LTB4
lactone = 0.6). More DCC (30 mg) was added and after
2 days at 0 TLC indicated essentially complete
conversion to the ~-lactone. The mixture was
concentrated to 1 mL under N2, filtered, reduced to
dryness, taken up in ethyl acetate:hexane (2:3) (1
mL) and chromatographed on silica gel column (10 g)
eluting with the same solvent ot provide the
~-lactone contaminated with a small amount of
dicyclohexylurea. This material was further purified
by HPLC (Waters 10~, ~-porasil; ethylacetate:hexane;
(1:2) 4 mL/min) to provide the pure LTb4 r-lactone
(VI) eluting at 5.7 min (6.3 mg, 77% yield from II).
The lactone crystallized as fine needles from
ether:hexane, mp 50.0-50.5 [a]RDT = +231.0
( 0.3, CHC13) W : ~max (~)(MeOH) 260 (37,200), 270
(~0,000), 280 nm ~39,400). S PMR (400 MHz) (CDC13~:
0.87 (3H, t), 1.2-1.4 (6H, m), 1.65 (2H, m), 1.93
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2298P/0832A - 29 - 16777IB
(2H, m), 2.03 (2H, q, CH2, C-16), 2.32 (2H, m, CH,
C-13), 2.48 ~lH, dt, J=17.5, 7 Hz, one of CH2,
C-2), 2.62 (lH, dt, J=18, 5 Hz, one of CH2, C-2),
4.22 (lH, m, methine, C-12), 5.23 (lH, dt, J=10.5,
2Hz, methine, C-5), 5.35 (lH, dd), 5.45 (lH, t),
5.58 (lH, dd), 5.81 (lH, dd), 6.15 (lH, t), 6.29 (2H,
m), 6.41 (1~, dd).
B. N- (3-aminopropyl) -5- (S) ,12 (R) -dihydroxy-6,14 (Z) -
8,10(E)-eicosatetraenoic acid amide (III):
Method 1. LTB4 ~-lactone (VI) 1.75 mg)
was dissolved in redistilled 1,3-diaminopropane (0.5
mL) and the mixture was left at room temperature for
18 hours. The excess diaminopropane was removed
under high vacuum to give the amide III, quantitative
yield, [a]DR = -2 (C=0.17, CHC13).
W: ~max (~) (MeOH) 259.5 (29,800) 269.5 (46,500),
280 (36,500). ~PMR (400 MHz): 2.03 (2H, q, CH2
C-16), 2.21 (2H, t, -CH2-CONH-), 2.31 ~2H, m,
CH2, C-13), 2.76 (2H, t, -CH2-NH2), 3.33 (2H,
q, -CONH-CH2-), 4.20 (lH, q, methine, C-12), 4.58
(lH, q, methine, C-5), 5.3-5.43 (2H, m), 5.55 (lH,
dd), 5.78 (lH, dd), 6.05 (lH, t), 6.18-6.31 (2H, m),
6.36 (lH, broad NH, amide), 6.47 (lH, dd).
Method 2. Ethyl 5(S)-benzoyloxy-12(R)-
hydroxy-6,14(Z)-8,10(E)-eicosatetraenoate (2.5 mg)
and 2-hydroxypyridine (1.5 mg) were dissolved in
1,3-diaminopropane (0.5 mL) and the mixture was left
at room temperature, under nitrogen, for 3 days. The
excess diaminopropane was removed under high vacuum
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2298P/0832A - 30 - 16777IB
at room temperature to provide crude III which was
used as such in the next reaction (W : ~ max 227,
260, 270, 280, 298 mm).
C. N-(3-[2,4-dinitro-5-fluorophenyl]aminopropyl)-
5(S), 12(R)-dihydroxy-6,14 (Z)-8,10(E)-
eicosatetraenoic acid amide (IV):
The crude amino amide (III) from Step B,
Method 2 (2 mg) in anhydrous methanol (400 ~) and
triethylamine (8 ~1) was treated with 1,5-difluoro-
2,4-dinitrobenzene (4 mg) in methanol 200 ~1) at room
temperature for 15 minutes at which time reverse
phase TLC (RPTLC) (acetonitrile:water, 85:15:)
indicated complete reaction of III (Rf=0.1) and the
appearance of a new yellow product (Rf=0.7). The
mixture was chromatographed on RPHPLC (Waters, 10 ~
~bondapak, C-18, acetonitrile:water, 70:30, 1 mL/min)
to provide the product IV (1.8 mg) []RT =18.9
~C=0.37, MeOH).
20 W : ~ max (MeOH) 260, 270, 280, 335, 380 (sh). PMR
(400 MHz) (acetone-d6: r 3.34 (2H, q, -CONH-CH2-),
3-61 (2H~ m~ -CH2-)~ 3-61 (2H, m, -CH2-NH-Ar)~
3.84 (2H, m, 2-OH), 4.14 (lH, m, methine, C-12) 4.58
(lH, m, methine, C-5), 5.42 (3H, m), 5.78 (lH, dd,
25 J=14, 6Hz, H-ll), 6.00 (lH, t, J=llHz, H-7), 6.21
(lH, dd, J=14, llHz, H-10), 6.30 (lH, dd, J=14, llHz,
H-9), 6.57 (lH, dd, J=14, llHz, H-8), 7.15 (lH, d,
JHF=15Hz), 7.27 (lH, broad, NH, amide), 9.00 (lH,
d, JH, F=8Hz), 9.15 (lH, ~road, MH, amine).
.-
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2298P/0832A - 31 - 16777IB
D. Coupling of compound IV with Bovine Serum
Albumin (BSA)
A solution of compound IV (from step C) (1.5
mg) in dimethylformamide (0.5 mL) was added to a
solution of BSA (15 mg) in 0.2~ pH 8.5 borate buffer
(0.75 mL) and the mixture was allowed to stand in the
dark under nitrogen and at room temperature for 4
days. The mixture was centrifuged and the clear
supernatant was applied to a column of Sephadex G-50
(1.5 x 75 cm) eluting with water. The yellow protein
fraction eluted cleanly in 20 mL, after the void
volume of about 55 mL. At about the 140 mL dead
volume a peak containing unreacted IV and byproducts
eluted. W analysis of the protein fractions gave a
spectrum max 266, (sH), 273, 283, 336, 420 nn.
Assuming 100% recovery of BSA from the column,
calculations based on the peak at 273 nm, correcting
for contributions due to BSA and to the
dinitrobenzene chromophore, indicated that 5.5 moles
of LTB4 were coupled per mole of BSA. The
absorption at 336 nm (assuming for the
1,5-diamino-2,4-dinitrobenzene chromophor of about
27,000) indicated that 8.3 moles of LTB4 were
coupled per mole BSA.
2. Conjugation of LTB4 with Hemocyanin from
Keyhole LimPets (KLH)
LTB4 ~-lactone (VI) (4 mg) was dissolved
in a mixture of THF (1 mL) and 99% hydrazine hydrate
(0.5 mL) and the mixtui~ was stirred vigorously under
nitrogen at room temperature for 0.5 hours. The
mixture was extracted with ether (3 x 2 mL) and the
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2298P/0832A - 32 - 16777IB
combined organic layers were dried (Na2SO4) and
evaporated to dryness under a stream of nitrogen and
then in vacuo to provide the hydrazide VII (4.2 mg).
[]D =8.9 (C=0.28, MeOH). UV ~max (~)=260
(37,000) 269.5 (50,000), 280 (3g,000). PMR (400 MHz)
acetone-d6): ~ 2.1 (2H, t), 2.27 (2H, m), 3.82 (lH,
m, NH2), 3.99 ~lH, broad NH2), 4.14 (lH, m,
methine, c-12), 4.56 (lH, m, methine, C-5), 5.3-5.5
(3H, m), 5.77 (lH, dd, J=14, 6Hz, H-ll), 6.00 (lH, t,
J=llHz), 6.22 (lH, dd, J=14, llHz, H-10), 6.31 (lH,
dd, J=14, llHz, H-9), 6.56 (lH, dd, J=14, 11 Hz,
H-8), 8.22 (lH, broad, -CO-NH-).
B. Reaction of LTB4 hydrazide (VII) with 6-N-
Maleimidohexanoic acid chloride:
LTB4 hydrazine (VII) 2.5 mg, 7 x 10 6
moles), in anhydrous methanol (1 mL) and
triethylamine (20 ~L) was treated with a solution of
6-N-maleimidohexanoic acid chloride (8) (3.3 mg, 1.4
x 10 5 moles) in anhydrous THF (100 ~L) under
nitrogen at room temperature. TLC analysis
(chloroform:methanol, 85:15) indicated complete
conversion to a less polar product. The mixture was
reduced to dryness, and the residue was taken up in
deoxygenated methanol (1.2 mL) and used as such in
the next reaction. The product could be purified if
desired by reverse phase HPLC (Waters 10~, ~-Bondapak
C-18; methanol:water; 75:25, 2mL/min), to give the
pure adduct VIII eluting at 4.5 min. UV ~max (MeOH)
(~): 260 (36,300), 270 (50,000), 280.5 nm (39,400).
On concentration to obtain PMR spectra some
decomposition was noted by TLC. However the spectrum
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2298P/0832A - 33 - 16777IB
(400 MHz) (acetone d6) contained a weak signal at
6.82 ppm indicating that the malemide unit was
present although partially reacted.
C. Coupling of Compound VIII with Thiolated KLH:
S-Acetylmercaptosuccinylated KLH was
prepared as previously described (8). The
derivatized protein SKLHSAc) (10 mg) in 0.~ N Saline
buffered with 0.01 N pH 6.2 phosphate buffer (PBS) (5
mL~ was rigorously deoxygenated; then the pH was
raised ~o 11.5 by addition of 0.lN NaOH. After
standing 1 hour nitrogen at room temperature, the pH
was reduced to 7.2 by addition of 0.lN HCl. The
adduct VIII in methanol (1.2 mL) from reaction B
above, was added and the mixture was stirred slowly
under nitrogen for 18 hours. N-ethylmalemide (5 mg)
in methanol (0.1 mL) was added and the mixture was
stirred 1 hour more. The methanol was removed under
a stream of nitrogen during 1 hour, the mixture was~
centrifuged and the supernatant was filtered on
Sephadex G-50 eluting with pH 6.2 PBS. The protein
eluted with 95% in 19 ml after the void volume and
gave a W spectrum: ~max 264 (sH), 273.5, 283.5 nm.
Assuming 9 mg of protein was recovered from the
column and correcting the absorption at 273.5 nm for
contributions due to coupled per 100,000 daltons KLH.
