U.S. patent application number 11/303903 was filed with the patent office on 2007-06-21 for 6-monoacetylmorphine derivatives useful in immunoassay.
This patent application is currently assigned to Roche Diagnostics Operations, Inc.. Invention is credited to Mitali Ghoshal, Richard Terry Root, Gerald F. Sigler.
Application Number | 20070142628 11/303903 |
Document ID | / |
Family ID | 38050990 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070142628 |
Kind Code |
A1 |
Ghoshal; Mitali ; et
al. |
June 21, 2007 |
6-Monoacetylmorphine derivatives useful in immunoassay
Abstract
Analogs of 6-monoacetyl morphine (6-MAM) are described. These
include analogs derivatized at either the C-3 position, the C-6
position, or the nor position of the molecule. These analogs allow
for elaboration with linkers terminated by a functional group such
as an activated ester, the functional groups being useful for
attaching the molecule to other entities such as proteins,
polysaccharides, and reporter groups.
Inventors: |
Ghoshal; Mitali;
(Noblesville, IN) ; Sigler; Gerald F.; (Carmel,
IN) ; Root; Richard Terry; (Fishers, IN) |
Correspondence
Address: |
ROCHE DIAGNOSTICS OPERATIONS INC.
9115 Hague Road
Indianapolis
IN
46250-0457
US
|
Assignee: |
Roche Diagnostics Operations,
Inc.
Indianapolis
IN
|
Family ID: |
38050990 |
Appl. No.: |
11/303903 |
Filed: |
December 16, 2005 |
Current U.S.
Class: |
530/400 ;
530/409; 536/17.4 |
Current CPC
Class: |
C07D 489/02 20130101;
A61K 47/542 20170801; A61K 47/646 20170801; Y10S 530/807 20130101;
C07K 16/44 20130101 |
Class at
Publication: |
530/400 ;
530/409; 536/017.4 |
International
Class: |
C07K 14/80 20060101
C07K014/80; C07K 14/765 20060101 C07K014/765 |
Claims
1. A compound having the structure: ##STR15## where R is a
saturated or unsaturated, substituted or unsubstituted, straight or
branched chain comprising 0-10 carbon or hetero atoms, L is a
linker group comprising 0-2 substituted or unsubstituted aromatic
rings, X is NH or O, and Y is air activated ester.
2. (canceled)
3. (canceled)
4. A compound having the structure: ##STR16## where Q is a carrier
or label.
5. The compound of claim 4 wherein the carrier is selected from the
group consisting of keyhole limpet hemocyanin, bovine
thyroglobulin, bovine serum albumin, and aminodextran.
6. (canceled)
7. (canceled)
8. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention pertains to the field of drug
monitoring, and in particular, to the field of detecting drugs of
abuse in biological samples via immunoassay. More particularly, the
present invention relates to chemical analogs of morphine useful in
development of antibodies that recognize 6-monoacetyl morphine
(6-MAM) and 6-monoacetyl codeine (6-MAC) and in production of
conjugates useful in immunoassay methods for detection of 6-MAM and
6-MAC.
BACKGROUND OF THE INVENTION
[0002] Heroin (diacetylmorphine) is an opioid developed as an
antitussive agent during the late nineteenth century. Heroin abuse
soon became a problem and continues to be so now, more than a
century later. Following ingestion, heroin is quickly metabolized
to 6-monoacetyl morphine (6-MAM) and then to morphine, which in
turn undergoes extensive metabolism. ##STR1##
[0003] Acetylcodeine is a synthetic byproduct present in street
heroin but not in pharmaceutical diacetylmorphine, which is used in
heroin-assisted treatment for opiate dependent drug users.
Acetylcodeine (AC) was investigated as a urinary biomarker for
detection of illicit heroin use. Detection of acetylcodeine could
play an important role in determining if addicts enrolled in heroin
maintenance programs were supplementing their supervised
diacetylmorphine doses with illicit heroin. ##STR2##
[0004] The problem presently unsolved by the prior art is the
unavailability of antibodies which will allow detection of 6-MAM or
6-MAC as markers of heroin abuse without interference from other
opiates such as morphine or codeine which may derive from
legitimate medical prescriptions, e.g., cough syrup, or even diet,
e.g., poppy seeds. The present invention provides chemical analogs
useful in development of antibodies that recognize 6-MAM and 6-MAC
and in production of conjugates useful in immunoassay methods for
detection of 6-MAM and 6-MAC.
SUMMARY OF THE INVENTION
[0005] It is against the above background that the present
invention provides certain unobvious advantages and advancements
over the prior art. In particular, the inventor has recognized a
need for improvements in 6-monoacetylmorphine derivatives useful in
immunoassay.
[0006] Although the present invention is not limited to specific
advantages or functionality, it is noted that the present invention
provides an immunogen for use in production of an antibody that
recognizes 6-monoacetylmorphine and 6-monoacetylcodeine. This
immunogen is derived at the C-3 position of an analog of the
6-monoacetylmorphine molecule. An immunogen structure having a
reduced double bond in the C-ring is also described.
[0007] In accordance with one embodiment of the present invention,
a compound is provided having the structure: ##STR3## where R is a
saturated or unsaturated, substituted or unsubstituted, straight or
branched chain of 0-10 carbon or hetero atoms, L is a linker group
consisting of 0-2 substituted or unsubstituted aromatic rings, and
Y is an activated ester or NH-Z where Z is a carrier or label and X
is NH or O.
[0008] In accordance with another embodiment of the present
invention, a compound is provided having the structure: ##STR4##
where R is CH.sub.2 or C.dbd.O, L is a saturated or unsaturated,
substituted or unsubstituted, straight or branched chain of 0-10
carbon or hetero atoms, and Y is an activated ester or NH-Z where Z
is a carrier or label and X is NH or O.
[0009] In accordance with another embodiment of the present
invention, a compound is provided having the structure: ##STR5##
where Q is a carrier or label.
[0010] In accordance with another embodiment of the present
invention, antibodies are provided which have specificity for
6-monoacetylmorphine and which are produced in response to a
compound having the structure: ##STR6## where R is a saturated or
unsaturated, substituted or unsubstituted, straight or branched
chain of 0-10 carbon or hetero atoms, L is a linker group
consisting of 0-2 substituted or unsubstituted aromatic rings, X is
NH or O, and Y is NH-Z where Z is a carrier.
[0011] In accordance with another embodiment of the present
invention, antibodies are provided which have specificity for
6-monoacetylcodeine and which can be produced in response to a
compound having the structure: ##STR7## where Q is a carrier.
[0012] In accordance with another embodiment of the present
invention, antibodies are provided which have specificity for
6-monoacetylmorphine and 6-monoacetylcodeine and which can be
produced in response to a compound having the structure: ##STR8##
where R is CH.sub.2 or C.dbd.O, L is a saturated or unsaturated,
substituted or unsubstituted, straight or branched chain of 0-10
carbon or hetero atoms, X is NH or O, and Y is NH-Z where Z is a
carrier.
[0013] These and other features and advantages of the present
invention will be more fully understood from the following detailed
description of the invention taken together with the accompanying
claims. It is noted that the scope of the claims is defined by the
recitations therein and not by the specific discussion of features
and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The following detailed description of the embodiments of the
present invention can be best understood when read in conjunction
with the following drawings in which:
[0015] FIG. 1 is a schematic representation of the synthesis of
6-acetamido codeine intermediate,
[0016] FIG. 2 is a schematic representation of the synthesis of
6-MAM analog immunogen out of 3-position,
[0017] FIG. 3 is a schematic representation of the synthesis of
6-MAM screening conjugate out of 3-position,
[0018] FIG. 4 is a schematic representation of the synthesis of
6-MAM 3-O-acetyl-N-position alkylated conjugate as an
immunogen,
[0019] FIG. 5 is a schematic representation of the synthesis of
6-MAM-3-N-acetamido-N-position N-alkylated conjugate as an
immunogen,
[0020] FIG. 6 is a schematic representation of the synthesis of
6-MAM analog-N-position N-acylated immunogen,
[0021] FIG. 7 is a schematic representation of the synthesis of
6-MAC-analog N-position immunogen, and
[0022] FIG. 8 is an inhibition curve generated using monoclonal
antibody MAM 9.3 and varying concentrations of free 6-MAM.
DETAILED DESCRIPTION OF THE INVENTION
[0023] It is noted that terms like "preferably", "commonly", and
"typically" are not utilized herein to limit the scope of the
claimed invention or to imply that certain features are critical,
essential, or even important to the structure or function of the
claimed invention. Rather, these terms are merely intended to
highlight alternative or additional features that may or may not be
utilized in a particular embodiment of the present invention.
[0024] For the purposes of describing and defining the present
invention, it is noted that the term "substantially" is utilized
herein to represent the inherent degree of uncertainty that may be
attributed to any quantitative comparison, value, measurement, or
other representation. The term "substantially" is also utilized
herein to represent the degree by which a quantitative
representation may vary from a stated reference without resulting
in a change in the basic function of the subject matter at
issue.
[0025] As used herein, the term "analyte" refers to a substance, or
group of substances, whose presence or amount thereof is to be
determined. As used herein, the term analyte subsumes the term
"antigen", which refers to any compound that can bind to an
antibody.
[0026] The term "antibody" means a specific binding partner of the
analyte and is any substance, or group of substances, which has a
specific binding affinity for the analyte to the essential
exclusion of other unrelated substances. The term includes
polyclonal antibodies, monoclonal antibodies, and antibody
fragments.
[0027] The term "hapten" refers to a partial or incomplete antigen.
Haptens are protein-free substances, mostly low molecular weight
substances, which are not capable of stimulating antibody
formation, but which do react with antibodies. The latter are
formed by coupling a hapten to a high molecular weight carrier and
injecting this coupled product into humans or animals. Examples of
haptens include 6-monoacetylmorphine and 6-monoacetylcodeine.
[0028] The term "activated hapten" refers to a hapten that has been
provided with an available reaction site, for example, by the
attachment of a linking group carrying a reactive moiety, that can
be used to connect the hapten to a carrier, immunogen, label,
tracer, or other moiety.
[0029] The term "linker" refers to a chemical moiety that connects
a hapten to a carrier, immunogen, label, tracer, or another linker.
Linkers may be straight or branched, saturated or unsaturated
carbon chains. They may also include one or more heteroatoms within
the chain or at termini of the chains. By heteroatoms is meant
atoms other than carbon which are chosen from the group consisting
of oxygen, nitrogen, and sulfur. The use of a linker may or may not
be advantageous or needed, depending on the specific hapten and
carrier pairs.
[0030] A "carrier", as the term is used herein, is an immunogenic
substance, commonly a protein, which can join with a hapten,
thereby enabling the hapten to stimulate an immune response, or a
substance that can form a conjugate useful in immunoassay, e.g.,
aminodextran and bovine serum albumin (BSA). Carrier substances
include proteins, glycoproteins, complex polysaccharides, and
nucleic acids that are recognized as foreign and thereby elicit an
immunologic response from the host. Poly(amino acids) useful as
carriers include keyhole limpet hemocyanin, bovine thyroglobulin,
and bovine serum albumin.
[0031] The terms "immunogen" and "immunogenic" as used herein refer
to substances capable of producing or generating an immune response
in an organism.
[0032] The term "derivative" refers to a chemical compound or
molecule made from a parent compound by one or more chemical
reactions.
[0033] The term "conjugate" refers to any substance formed from the
joining together of two parts. Representative conjugates in
accordance with the present invention include those formed by the
joining together of a small molecule and a large molecule, such as
a protein. The term conjugate subsumes the term immunogen.
[0034] As used herein, a detector molecule, label, or tracer is an
identifying tag which, when attached to a carrier substance or
molecule, can be used to detect an analyte. A label may be attached
to its carrier substance directly or indirectly by means of a
linking or bridging moiety. Examples of labels include enzymes such
as .beta.-galactosidase and peroxidase, fluorescent compounds such
as rhodamine and fluorescein isothiocyanate (FITC), luminescent
compounds such as dioxetanes and luciferin, and radioactive
isotopes such as .sup.125I.
[0035] The term active ester within the sense of the present
invention encompasses activated ester groups which can react with
nucleophiles such as, but not limited to, free amino groups of
peptides, polyaminoacids, polysaccharides, or labels under such
conditions that no interfering side reactions with other reactive
groups of the nucleophile-carrying substance can usefully
occur.
[0036] An object of the present invention is to provide a compound
having the structure: ##STR9## where R is a saturated or
unsaturated, substituted or unsubstituted, straight or branched
chain of 0-10 carbon or hetero atoms, L is a linker group
consisting of 0-2 substituted or unsubstituted aromatic rings, and
Y is an activated ester or NH-Z where Z is a carrier or label and X
is NH or O.
[0037] Another object of the present invention is to provide a
compound having the structure: ##STR10## where R is CH.sub.2 or
C.dbd.O, L is a saturated or unsaturated, substituted or
unsubstituted, straight or branched chain of 0-10 carbon or hetero
atoms, and Y is an activated ester or NH-Z where Z is a carrier or
label and X is NH or O.
[0038] Yet another object of the present invention is to provide a
compound having the structure: ##STR11## where Q is a carrier or
label.
[0039] A further object of the present invention is to provide
antibodies which have specificity for 6-monoacetylmorphine and
which are produced in response to a compound having the structure:
##STR12## where R is a saturated or unsaturated, substituted or
unsubstituted, straight or branched chain of 0-10 carbon or hetero
atoms, L is a linker group consisting of 0-2 substituted or
unsubstituted aromatic rings, X is NH or O, and Y is NH-Z where Z
is a carrier.
[0040] A further object of the present invention is to provide
antibodies which have specificity for 6-monoacetylcodeine and which
can be produced in response to a compound having the structure:
##STR13## where Q is a carrier.
[0041] Yet a further object of the present invention is to provide
antibodies which have specificity for 6-monoacetylmorphine and
6-monoacetylcodeine and which can be produced in response to a
compound having the structure: ##STR14## where R is CH.sub.2 or
C.dbd.O, L is a saturated or unsaturated, substituted or
unsubstituted, straight or branched chain of 0-10 carbon or hetero
atoms, X is NH or O, and Y is NH-Z where Z is a carrier.
[0042] The synthetic schemes for immunogens and screening
conjugates of monoacetyl morphine and monoacetylcodeine are
illustrated in FIG. 1 through FIG. 7.
[0043] Codeine is reduced to dihydrocodeine (see FIG. 1) using
hydrogen gas, preferably palladium on charcoal as catalyst under
pressure. Hydrogenation reactions are well known in the art and can
be performed using many different catalysts such as Raney Ni,
palladium hydroxide on charcoal, Adam's catalyst, etc in solvents
such as methanol, ethanol, or ethyl acetate. The secondary hydroxyl
group of dihydrocodeine is oxidized back to carbonyl group. Many
literature references are known for conversion of a secondary
hydroxyl group to carbonyl functionality, e.g., oxalyl
chloride/DMSO, pyridinium chlorochromate and chromium
oxide/pyridine, most preferably benzophenone and potassium
t-butoxide under reflux conditions to give compound 3. The latter
intermediate then undergoes reductive amination to give
aminodihydrocodeine (4) in the presence of ammonium acetate and a
reducing agent, sodium cyanoborohydride, preferably at a pH of 6 to
7, thus providing a mixture of two diastereomers (compound 4).
Acetylation reaction of the amino group of compound 4 can be
performed using acetic anhydride and pyridine at a temperature
ranging from room temperature to reflux conditions. This reaction
is performed using acetic anhydride as the acetylating agent and a
base using triethylamine in the presence of
4-dimethylaminopyridine. The acetylated product is obtained as a
mixture of diastereomers and can be separated by either column
chromatography or preparative HPLC. Chromatographic techniques to
separate diastereomers are well known in the art. The desired
isomer (compound 5b) is demethylated to give the phenolic compound
6 (FIG. 2). A variety of demethylation reactions are known in the
art, for example, trimethylsilyl iodide, sodium thioethoxide,
potassium thiophenoxide, sodium cyanide in DMSO, aluminium
tribromide in ethane thiol, aluminium chloride/dimethyl sulfide,
and hydrobromic acid boron tribromide (Greene, T. and Wuts, P.,
"Protective groups in organic synthesis", 2.sup.nd edition, Wiley
Intersciences, 1991) at a temperature ranging from 0.degree. to
room temperature. Preferably the demethylation of the compound 5b
is done in the presence of boron tribromide in dichloromethane at
room temperature. The phenolic hydroxyl group of compound 5b is
then extended with a protected carboxyl terminated linker through
an ether linkage (FIG. 2). The phenolic hydroxyl group can also be
converted to a carboxyl leashed linker by an ester or urethane
linkage.
[0044] The t-butylester group of compound 7 is deprotected to a
carboxylic acid functionality followed by conversion to an
N-hydroxysuccinimide ester (compound 9). The activation of a
carboxyl group can be accomplished by an activation step using a
carbodiimide such as dicyclohexylcarbodiimide,
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDC), or N,N,N',N'-O-succinimidyl-tetramethyluronium
tetrafluoroborate. Conjugation to a protein provides an immunogen
(compound 10) and screening conjugates.
[0045] The extension of the phenolic group can also be done using
an alkylating reagent or acylating reagent with protected amino
group (FIG. 3). An example of an amino protecting group is a
phthalimido group, which can be cleaved under methyl amine or
hydrazine. The amino group of the resultant amino leashed product
is amenable to acylation reactions with a wide variety of carboxyl
activated linker extensions or labels that are well known to those
skilled in the art.
[0046] Linker extension is often performed to generate a terminal
activating group. Linker extension using a homobifunctional linker
such as N-hydroxysuccinimide ester of a biscarboxylic acid such as
terephthalic acid or 4,4-bipenyl dicarboxylic acid (FIG. 3) can be
used to generate an activated ester (compound 15) in a single step
by reaction with the aforementioned amino group (compound 14). For
a good treatise on the subject of linkers, the reader is referred
to Hermanson, Greg T., "Bioconjugate Techniques", Academic Press
Inc., 1996.
[0047] The 6-hydroxyl group of compound 15 is acetylated.
Acetylation reaction can be done preferably using pyridine and
acetic anhydride. The resulting active ester is conjugated to
protein for use as a screening conjugate.
[0048] The immunogen leashed out of the nitrogen can use
dihydronormorphine (compound 18) as a starting material. Protection
of amino group in the presence of free phenolic hydroxyl group is
known in the art. These reactions can be done by a proper choice of
protecting group and reaction conditions. An example of an
N-protecting group removed under mild basic conditions is
fluoromethyloxycarbonyl (FMOC). An example of an N-protecting group
easily removed with acid is t-butoxycarbonyl (BOC). An example of
an N-protecting group removed under neutral hydrogenation
conditions is a carbobenzyloxy group (CBz group). In this
embodiment, the preferred protection of the cyclic secondary amine
of dihydronormorphine is use of a carbobenzyloxy group in this
sequence (FIG. 4). The phenolic group of CBz protected derivative
(compound 19) can be protected as a benzyl group. The secondary
hydroxyl group is then converted to an acetyl group (compound 21).
One of the preferred acetylation reactions of the hydroxyl group is
use of pyridine and acetic anhydride under reflux conditions.
Deprotection of N-CBz and benzyl groups can be achieved in one step
using hydrogen and palladium on a charcoal catalyst to give
compound 22. The free secondary amine is extended to a carboxyl
group though a succinylation reaction or a reductive amination
reaction with succinic semialdehyde and sodium cyanoborohydride at
a pH of 6-7. The compound 22 is then converted to an active ester
(compound 23). The activation of a carboxyl group is accomplished
by an activation step using a carbodiimide such as
dicyclohexylcarbodiimide (DCC), EDC, or
N,N,N',N'-O-succinimidyl-tetramethyluronium tetrafluoroborate.
Conjugation to protein provides immunogen and screening
conjugates.
[0049] In a different embodiment, N-leashed 6-acetamido 6-MAM
protein conjugate (FIG. 5 and FIG. 6) can also be made. The
phenolic hydroxyl group of compound 6 can be protected as a benzyl
group. N-demethylation of cyclic secondary amine is well known in
the literature and can be achieved under various reaction
conditions, such as 1-chloroethyl chloroformate followed by
methanol, cyanogens bromide followed by Zn dust, or with vinyl
chloroformate followed by treatment with hydrobromic acid. The
preferred reaction used for N-demethylation of compound 26 is
treatment of 1-chloroethyl chloroformate followed by methanol to
give compound 28 (FIG. 5). Deprotection of the benzyl group is done
under hydrogenation conditions using 10% palladium hydroxide on
charcoal. Extension at the nitrogen position to a carboxyl terminal
group by reductive amination (FIG. 5) followed by a carboxyl group
activation procedure can be done as described earlier.
[0050] Extension at the nor position to a carboxy terminal group
can also be achieved by succinylation of compound 28 to compound 33
(FIG. 6). Deprotection of the benzyl group followed by activation
of the carboxylic group functionality can be achieved as described
earlier.
[0051] FIG. 7 describes the preparation of a monoacetylcodeine
immunogen. This can be used as a booster antibody for preparation
of a 6-MAM antibody cross-reactive with 6-monoacetyl codeine.
During the N-demethylation of compound 5b using 1-chloroethyl
chloroformate in 1,2-dichloroethane, compound 38 was formed in one
step. It was anticipated that the intermediate
N-(1-chloroethoxy)carbonyl derivative (37) underwent hydrolysis
under the reaction conditions to give compound 38.
[0052] Extension at the nor position to a carboxyl terminal
functionality has been discussed earlier. Activation is followed by
conjugation to a protein for preparation of the immunogen (compound
41).
[0053] In order that the invention may be more readily understood,
reference is made to the following examples, which are intended to
illustrate the invention but not limit the scope thereof.
SPECIFIC EMBODIMENTS
[0054] In the examples that follow, boldface numbers refer to the
corresponding structure in the drawings.
Example 1
Preparation of Dihydrocodeine (2)
[0055] To 1.2 g (4.0 mmol) of codeine in 40 mL of methanol was
added 100 mg of 10% Pd-C. The resulting reaction mixture was
hydrogenated under pressure of 50 psi. The solution was filtered
through CELITE (Celite Corporation), and the filtrate was
concentrated to give 1.12 g (3.7 mmol, 93%) of dihydrocodeinone as
a white solid. ES (+) m/z 301.
Example 2
Preparation of Dihydrocodeinone (3)
[0056] To a mixture of 1.25 g (11.1 mmol) of potassium-t-butoxide
in 50 mL of benzene were added 6.78 g (37 mmol) of benzophenone and
1.12 g (3.7 mmol) of dihydrocodeine, which was allowed to reflux
for 2 hours. The reaction mixture turned yellow. The reaction
mixture was allowed to cool to room temperature, and 50 mL of 2N
HCl was added. The resulting reaction mixture was allowed to stir
for 10 minutes. The organic layer was separated and extracted with
3.times.40 mL of 2N HCl. The organic layer was discarded, and the
aqueous layer was again extracted with 3.times.20 mL of
dichloromethane. The aqueous layer was basified with aqueous
potassium hydroxide solution to pH 12 and extracted with 5.times.60
mL of dichloromethane. The organic layers were combined and dried
over MgSO.sub.4 and filtered. The filtrate was concentrated to give
691 mg (2.3 mmol, 55%) of 3 as a yellow solid.
Example 3
Preparation of amino dihydrocodeine (4)
[0057] To 3.46 g (11.5 mmol) of compound 3 in 70 ml of methanol was
added 8.92 g (115 mmol) of ammonium acetate, which was and allowed
to stir at room temperature for 15 minutes, at which time a clear
solution was obtained. To the reaction mixture a solution of 727 mg
(11.5 mmol) of sodium cyanoborohydride was added, and the pH of the
reaction mixture was adjusted to pH 6-7 by addition of conc. HCl.
The reaction mixture was allowed to stir at room temperature for 18
hours and concentrated under reduced pressure to give yellow oil.
To this yellow oil 250 mL of water was added, and the pH was
adjusted again to 1 with 6N HCl. The resulting aqueous reaction
mixture was extracted with 2.times.250 mL of dichloromethane. The
pH of the aqueous part was readjusted to 1 using 6N HCl and
extracted with 3.times.250 mL of dichloromethane. The organic
layers were combined, dried (MgSO.sub.4), and concentrated to give
3.2 g (10.6 mmol, 92%) of 4 as a thick yellow oil. This product was
used in the next step without purification.
[0058] HR-ES (+); calculated for C.sub.18H.sub.24N.sub.2O.sub.2,
M+H 301.1911; observed 301.1913.
Example 4
Preparation of acetamido dihydrocodeine derivative (5b)
[0059] To 3.2 g (10.6 mmol) of amino dihydrocodeine 4 in 125 mL of
dichloromethane were added 87 mg (0.71 mmol) of
4-dimethylaminopyridine and 9 mL of triethylamine. The reaction
mixture was allowed to cool to 0.degree. C., and 7.1 mL (74 mmol)
of acetic anhydride was added. The reaction mixture was allowed to
warm up to room temperature and allowed to stir for 18 h. The
reaction mixture was concentrated under reduced pressure, and a
yellow oil was obtained. To this yellow oil 125 mL of water was
added, and the product was extracted with 4.times.150 mL of
dichloromethane. The pH of the aqueous solution was adjusted to 10,
and the solution was re-extracted with chloroform. All the organic
layers were combined, dried (MgSO.sub.4), and concentrated to give
a colorless crude oil. LC-MS analysis indicated formation of two
diastereomers. One half of the crude product was purified by
RP-HPLC (C-18 column) using a gradient run with acetonitrile-water
containing 0.1% trifluoroacetic acid. Fractions containing the
desired isomer were combined and concentrated, followed by
lyophilization to give 614 mg of product 5b as an off-white powder.
HR-ES (+) calculated for C.sub.20H.sub.26N.sub.2O.sub.3, M+H
343.2016; observed 343.2022.
Example 5
Preparation of Acetamido Dihydromorphine Derivative (6)
[0060] To 644 mg (1.8 mmol) of dihydrocodeine derivative (5b) was
added 12 mL dichloromethane. This solution was added to a
magnetically stirred solution of 11.3 mL (11.2 mmol) of 1M
BBr.sub.3 in 20 mL of dichloromethane at room temperature. The
reaction mixture was allowed to stir at room temperature for 1 h
and was poured into 60 mL of conc NH.sub.4OH solution and 30 g of
ice. The reaction mixture was allowed to stir for 1 h and extracted
with 5.times.100 mL of 80% ethanol in dichloromethane. The organic
layers were combined, dried, and concentrated. The residue was
purified by preparative RP-HPLC using using a gradient run with
acetonitrile-water containing 0.1% trifluoroacetic acid. Fractions
containing the desired product were combined and concentrated
followed by lyophilization to give 480 mg (78%, 1.46 mmol) of 6 as
an off-white solid. LR-ES (+) M+H 329.
Example 6
Preparation of Acetamido Dihydromorphine Butyric t-butyl Ester
Derivative (7)
[0061] To 50 mg (0.15 mmol) of acetamido dihydromorphine derivative
(6) was added 6 mL of anhydrous acetone, 0.5 mL of anhydrous
dimethylformamide, 150 mg (1.08 mmol) of potassium carbonate, and
140 .mu.L (0.78 mmol) of t-butylbromobutyrate. The reaction mixture
was allowed to reflux under argon atmosphere for 18 h, cooled to
room temperature and filtered. The filtrate was concentrated under
reduced pressure, redissolved in 60 mL of chloroform, and 25 mL of
water was added. The organic part was separated, and the aqueous
part was extracted with 3.times.50 mL of chloroform. All organic
layers were combined, dried (MgSO.sub.4), and concentrated. The
residue was purified by silica gel flash column chromatography
using 10% methanol in chloroform to give 35 mg (48%, 0.074 mmol) of
7 as a thick colorless oil. LR-MS-ES (+): M+H 471.
Example 7
Preparation of Acetamido Dihydromorphine Butyric Acid Derivative
(8)
[0062] To 35 mg (0.074 mmol) of acetamido dihydromorphine butyric
acid derivative (7) was added 3 mL of dichloromethane and 1 mL of
trifluoroacetic acid. The reaction mixture was allowed to stir at
room temperature for 30 minutes and concentrated. To the residue 10
mL of dichloromethane was added and concentrated. The residue was
purified by preparative RP-HPLC using using a gradient run with
acetonitrile-water containing 0.1% trifluoroacetic acid. Fractions
containing the desired product were combined and concentrated
followed by lyophilization to give 19 mg (0.045 mmol, 63%) of
desired product 8 as white powder. HR-ES (+): Calculated for
C.sub.23H.sub.30N.sub.2O.sub.5; M+H 415.2228; observed
415.2227.
Example 8
Preparation of Acetamido Dihydromorphine Butyric Acid NHS Ester
(9)
[0063] To 17 mg (0.041 mmol) of acetamido dihydromorphine butyric
acid (8) was added 3 mL of freshly distilled THF and the mixture
was cooled to 0.degree. C. To the reaction mixture was added 21.4
.mu.L (0.12 mmol) of N,N-diisopropylethylamine followed by 37 mg
(0.12 mmol) of O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate. The reaction mixture was allowed to warm up to
room temperature and allowed to stir at room temperature 18 h. The
reaction mixture was concentrated and the residue was purified by
preparative RP-HPLC using a gradient run with acetonitrile-water
containing 0.1% trifluoroacetic acid. Fractions containing the
desired product were combined and concentrated followed by
lyophilization to give 13 mg (0.02 mmol, 62%) of 9 as white solid.
LR-MS-ES (+): M+H 512.
Example 9
Preparation of 6-MAM KLH Conjugate (10)
[0064] A solution of 37 mg of keyhole limpet hemocyanin (KLH) in
1.2 ml of 50 mM potassium phosphate (pH 7.5) was cooled in an ice
bath. To the reaction mixture 1.5 mL of DMSO was added dropwise,
and the reaction temperature was maintained below room temperature.
Then a solution of 8 mg (0.015 mmol) of 9 in 1 mL of DMF was added
to the protein solution dropwise. The mixture was allowed to warm
up to room temperature and was allowed to stir at room temperature
18 h. The resulting conjugate was placed in a dialysis tube (10,000
MW cut-off) and was dialyzed in 1 L of 70% DMSO in 50 mM potassium
phosphate (pH 7.5, at least 3 hour), 1 L of 50% DMSO in 50 mM
potassium phosphate (at least 3 hours), 1 L of 30% DMSO in 50 mM
potassium phosphate (at least 3 h), 1 L of 10% DMSO in 50 mM
potassium phosphate (at least 3 h) at room temperature followed by
50 mM potassium phosphate buffer (6 times, at least 6 h each at
4.degree. C.). The protein concentration was determined to be 2.2
mg/mL using BioRad Coomassie blue protein assay (Bradford, M.,
Anal. Biochem. 72, 248, 1976). The extent of available lysine
modification was determined to be 46% by the TNBS method (Habeeb
AFSA, Anal. Biochem. 14, 328-34, 1988).
Example 10
Preparation of 1,1'-biphenyl-4,4'-dicarbonyl chloride (11)
[0065] A mixture of 10.0 g (0.041 mol) of 4,4'-biphenyldicarboxylic
acid in 200 mL of anhydrous THF (distilled over Na and
benzophenone) under argon was treated with 25.0 mL (0.286 mol) of
oxalyl chloride followed by 0.1 mL of anhydrous DMF. The reaction
was then concentrated at reduced pressure to a yellow oil. This was
stripped down 5 times with anhydrous THF to drive off any residual
oxalyl chloride to yield 11.5 g of the desired product 11 as a
yellow solid. This was used in the next step without purification.
MS: m/e 278 (M.sup.+).
Example 11
Preparation of 1,1'-biphenyl4,4'-di-N-hydroxysuccinimide ester
(12)
[0066] A solution of 11.5 g (0.041 mol) of 11 in 500 mL of
anhydrous dichloromethane under argon was treated with 25.0 g
(0.217 mol) of N-hydroxysuccinimide followed by 25 mL of
triethylamine and stirred at room temperature overnight. The
resulting precipitate was collected by suction filtration and
washed with dichloromethane to yield 10.7 g of product as a white
solid. The filtrate was concentrated at reduced pressure, and the
residue was triturated with dichloromethane to yield 3.84 g of a
second crop as a white solid for a combined yield of 14.5 g (81%)
of the desired product 12. MS m/e 437 (M+H).
Example 12
Preparation of 3-O-phthalimidopropyl morphine (13)
[0067] 1.5 g (37.5 mmol) of NaH (60% dispersion in mineral oil) was
rinsed with 2.times.25 mL of hexanes to remove the oil. To the
reaction mixture was added 140 mL of anhydrous DMF followed by 10.1
g (35 mmol) of morphine. This mixture was allowed to stir at room
temperature for 30 minutes under nitrogen atmosphere, and 14.5 g
(53 mmol) of N-(3-bromopropyl) phthalimide was added. The reaction
mixture was allowed to stir 18 h at room temperature. The reaction
was monitored by thin layer chromatography (silica, 7:2:1:1,
MeOH:EtAc:NH.sub.4OH:H.sub.2O, I.sub.2 visualization), which showed
that the reaction was incomplete. To the reaction mixture 0.15 g
(3.75 mmol) of NaH (60% in oil) was added, and the mixture was
allowed to stir for an additional 1.5 h. The mixture was poured
into 350 mL of ice-water and extracted with 250 mL of ethyl
acetate. The organic layer was separated, and the aqueous layer was
extracted with 2.times.250 mL of ethyl acetate. The organic layers
were combined and washed with 2.times.150 mL of water, 150 mL of 1N
NaOH and 2.times.150 mL of water. The organic layer was dried
(Na.sub.2SO.sub.4) and concentrated. The residue was crystallized
with diethyl ether to give 12.9 g (27.3 mmol, 77%) of the desired
product 13 as a white solid.
Example 13
Preparation of 3-O-aminopropylmorphine (14)
[0068] To 14 g (29.6 mmol) of 3-O-phthalimidopropyl morphine (13)
was added 188 mL of 2M ethanolic methylamine, and the reaction
mixture was allowed to stir for 3.5 h at room temperature under
nitrogen. The reaction mixture was poured into 650 mL of chloroform
and 425 mL of cold water. The organic layer was separated, and the
aqueous layer was extracted with 3.times.90 mL of chloroform. The
chloroform layers were combined and extracted with 2.times.140 mL
of 1N HCl. The aqueous extract was basified with 140 mL of 2.5 N
NaOH and extracted with 3.times.300mL of chloroform. The organic
layer was dried (Na.sub.2SO.sub.4) and concentrated. The residue
was crystallized from diethyl ether to give 8.9 g (26 mmol, 88%) of
14 as a white solid.
Example 14
Preparation of morphine 3-O-aminopropylbiphenyl
N-hydoxysuccinirnide ester hydrochloride salt (15)
[0069] To a mixture of 6.4 g (14.6 mmol) of biphenyl
di-N-hydroxysuccinimide ester (12) in 400 mL of anhydrous THF was
added a solution of 3.2 g (9.3 mmol) of 3-O-aminopropylmorphine (1
) in 400 mL of anhydrous THF dropwise over a period of 30 minutes.
The reaction mixture was allowed to stir at room temperature for 2
h and filtered. The filtrate was concentrated, and the residue was
dissolved in 250 mL of dichloromethane. This was washed with 150 mL
of saturated aqueous sodium bicarbonate solution, dried
(Na.sub.2SO.sub.4), and concentrated. The residue was purified by
silica gel column chromatography, first by eluting with ethyl
acetate to remove the higher Rf impurities followed by eluting with
anhydrous THF to give the desired product as a free base. All the
fractions containing the desired product (Rf 0.13) were combined
and treated with 12 mL of 1M HCl in ether. The solution was then
concentrated at reduced pressure to give 3.2 g (49%, 4.5 mmol) of
15 as a white solid. MS: m/e 664 (M+H).
Example 15
Preparation of 6-monoacetylmorphine-3-O-aminopropylbiphenyl
N-hydoxysuccinimide ester (16)
[0070] To 50 mg (0.071 mmol) of morphine 3-O-aminopropylbiphenyl
N-hydoxysuccinimide ester hydrochloride salt 15 was added 1 mL of
anhydrous DMF, 7.1 .mu.L of acetic anhydride, and 6.1 .mu.L of
anhydrous pyridine. The reaction mixture was allowed to stir 18 h.
An analytical HPLC was run using RP-HPLC column (C-18) using
acetonitrile and water containing 0.1% trifluoroacetic acid to
monitor the progress of the reaction. The result indicated the
presence of only starting material. To the reaction mixture 14
.mu.L of acetic anhydride and 12 .mu.L of pyridine were added. The
mixture was allowed to stir for 6 h, and HPLC analysis indicated no
reaction. To the reaction mixture 20 .mu.L of acetic anhydride was
added and allowed to stir 18 h. To the reaction mixture 0.5 mL of
pyridine and 40 .mu.L of acetic anhydride were added. The reaction
mixture was allowed to stir at room temperature 18 h, and HPLC
analysis indicated product formation. This was concentrated under
reduced pressure to give a yellow oil and was purified by RP-HPLC
(C-18 column) using a gradient run with acetonitrile-water
containing 0.1% trifluoroacetic acid. Fractions containing the
desired isomer were combined and concentrated followed by
lyophilization to give 32 mg (64%, 0.04 mmol) of desired product 16
as white solid. HR-ES (+): calculated for C40H39N.sub.3O.sub.9, M+H
706.2759; observed 706.2758.
Example 16
Preparation of 6-MAM BSA conjugate (17)
[0071] To 500 mg of bovine serum albumin (BSA) was added 6 mL of 50
mM potassium phosphate, and the solution was allowed to cool to
0.degree. C. To the reaction mixture 6 mL of DMSO were added
dropwise for a period of 5 minutes. A solution of 13 mg (0.018
mmol) 6-MAM NHS ester derivative (6) in 1 mL of anhydrous DMF was
added to the reaction mixture dropwise at 0.degree. C. The mixture
was allowed to warm up to room temperature and was allowed to stir
at room temperature 18 h. The resulting conjugate was placed in a
dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of 70%
DMSO in 50 mM potassium phosphate (pH 7.5, 3 hours), 1 L of 50%
DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 30%
DMSO in 50 mM potassium phosphate (at least 3 h), 1 L of 10% DMSO
in 50 mM potassium phosphate (at least 3 h) at room temperature
followed by 50 mM potassium phosphate buffer (6 times, at least 6 h
each at 4.degree. C.). The protein concentration was determined to
be 15 mg/mL using BioRad Coomassie blue protein assay. The extent
of available lysine modification was determined to be 46% by the
TNBS method.
Example 17
Preparation of N-CBz nordihydromorphine (19)
[0072] To 200 mg (0.73 mmol) of dihydro normorphine (18) is added
12 mL of THF and 8 mL of water. To the reaction mixture 310 mg
(2.92 mmol) of sodium carbonate is added followed by 0.12 mL (0.84
mmol) of benzylchloroformate. The mixture is allowed to stir at
room temperature 18 h and concentrated under reduced pressure. To
the residue 10 mL of water is added, and the pH is adjusted to 2
using 3N HCl. This is extracted with ethyl acetate, dried, and
concentrated. The residue is purified by silica gel column
chromatography to give 19.
Example 18
Preparation of N-CBz-3-O-benzyl-nordihydromorphine (20)
[0073] To 100 mg (0.23 mmol) of compound 19 in 5 mL of anhydrous
DMF is added 33 .mu.l (0.27 mmol) of benzyl bromide, 55 mg (0.39
mmol) of potassium carbonate, and the reaction mixture is heated at
60.degree. C. for 6 h, filtered, and the filtrate is concentrated.
The residue is purified by silica gel column chromatography to give
20.
Example 19
Preparation of 6-acetyl N-CBz-3-O-benzyl-nordihydromorphine
(21)
[0074] To 100 mg (0.20 mmol) of 20 is added 1.5 mL of pyridine and
74 .mu.L (0.78 mmol) of acetic anhydride. The reaction mixture is
allowed to stir and is heated at 90.degree. C. for 3 h. The
resulting reaction mixture is allowed to cool to room temperature
and concentrated. The residue is purified by silica gel column
chromatography to give 21.
Example 20
Preparation of 6-acetyl-nordihydromorphine (22)
[0075] To 100 mg (0.18 mmol) of compound 21 is added 30 ml of
ethanol and 50 mg of 20% Pd(OH).sub.2/C. The reaction mixture is
hydrogenated at 60 psi for 6 h. The reaction mixture is filtered
through CELITE, and the filtrate is concentrated to give compound
22.
Example 21
Preparation of 6-acetyl dihydronormorphine N-butyric acid (23)
[0076] To 100 mg (0.31 mmol) of 6-acetyl dihydronormorphine (22) is
added 25 mL of freshly distilled THF. The reaction mixture is
allowed to stir at room temperature. 266 .mu.l (0.38 mmol) of
succinic semialdehyde (15 wt % solution in water) is added followed
by 24 mg (0.38 mmol) of sodium cyanoborohydride. The pH of the
reaction mixture is adjusted between 6-6.5, and the reaction
mixture is allowed to stir at room temperature for 6 h. The
reaction mixture is concentrated under reduced pressure, and 50 mL
of dichloromethane is added followed by 25 mL of water. The organic
layer is separated, and the aqueous layer is extracted with
2.times.50 mL of dichloromethane. The combined organic layers are
dried and concentrated. The residue is purified by silica gel
column chromatography to give 23.
Example 22
Preparation of 6-acetyl dihydronormorphine N-butyric acid
N-hydroxysuccinimide ester (24)
[0077] To 100 mg (0.24 mmol) of 6-acetyl dihydronormorphine
N-butyric acid (23) is added 30 mL of freshly distilled THF, and
the mixture is cooled to 0.degree. C. To the reaction mixture is
added 0.10 ml (0.57 mmol) of N,N-diisopropylethylamine followed by
171 mg (0.57 mmol) of
O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.
The reaction mixture is allowed to warm up to room temperature and
is allowed to stir at room temperature 18 h. The reaction mixture
is concentrated, and the residue is purified silica gel column
chromatography to give 24.
Example 23
Preparation of 6-acetyl dihydronormorphine N-butyric acid KLH
conjugate (25)
[0078] This conjugate is prepared from compound 24 according to the
procedure described for the conversion of compound 9 to compound
10.
Example 24
Preparation of 6-acetamido-3-O-benzyl dihydromorphine (26)
[0079] To 500 mg (1.52 mmol) of 6 is added 12 mL of anhydrous DMF
followed by 270 .mu.L (2.26 mmol) of benzyl bromide and 734 mg (5.3
mmol) of anhydrous potassium carbonate. The reaction mixture is
allowed to heat at 60.degree. C. for 18 h, then is cooled to room
temperature and filtered. The filtrate is concentrated and purified
by preparative RP-HPLC using using a gradient run with
acetonitrile-water containing 0.1% trifluoroacetic acid. Fractions
containing the desired product are combined and concentrated
followed by lyophilization to give 26.
Example 25
Preparation of 6-acetamido-3-O-benzyl N-[1-chloroethoxy
carbonyl]nordihydromorphine (27)
[0080] To 250 mg (0.59 mmol) of 26 is added 5 mL of
1,2-dichloroethane. The resulting solution is allowed to stir, and
636 .mu.L (5.8 mmol) of 1-chloroethyl chloroformate is added. The
reaction mixture is heated under reflux conditions 18 h, and an
additional 640 .mu.L (5.9 mmol) of 1-chloroethyl chloroformate is
added. The reaction is heated to reflux for 18 h and concentrated.
The residue is used in the next step without purification.
Example 26
Preparation of 6-acetamido-3-O-benzyl nordihydromorphine (28)
[0081] To all of the above [6-acetamido-3-O-benzyl
N-[1-chloroethoxy carbonyl]nordihydromorphine (27)] is added 5 mL
of methanol, and the mixture is allowed to heat to reflux for 6 h.
The reaction mixture is concentrated, and the residue is purified
by preparative RP-HPLC using a gradient run with acetonitrile-water
containing 0.1% trifluoroacetic acid. Fractions containing the
desired product are combined and concentrated followed by
lyophilization to give 28.
Example 27
Preparation of 6-acetamido 3-OH-nordihydromorphine (29)
[0082] To a solution of 100 mg (0.24 mmol) of 28 in ethanol is
added 10% Pd-C, and the mixture is allowed to hydrogenate under 60
psi for 12 h. The reaction mixture is filtered, and the filtrate is
concentrated to give 29.
Example 28
Preparation of 6-acetamido 3-OH-nordihydromorphine N-butyric acid
derivative (30)
[0083] Compound 30 is prepared from compound 29 following the
procedure as described for the preparation of compound 23.
Example 29
Preparation of 6-acetamido 3-OH-nordihydromorphine N-butyric acid
NHS ester derivative (31)
[0084] This active ester is prepared from compound 30 according to
the procedure used to convert the acid 23 to active ester 24.
Example 30
Preparation of 6-acetamido 3-OH-nordihydromorphine N-butyric acid
KLH conjugate (32)
[0085] This conjugate is prepared according to the procedure
described for conjugate 10.
Example 31
Preparation of 6-acetamido nordihydrocodeine (38)
[0086] To 250 mg (0.73 mmol) of 5b was added 5 mL of
1,2-dichloroethane. The resulting solution was allowed to stir, and
636 .mu.L (5.8 mmol) of 1-chloroethyl chloroformate was added. The
reaction mixture was allowed to heat to reflux 18 h, and an
additional 640 .mu.L (5.9 mmol) of 1-chloroethyl chloroformate was
added. The reaction mixture was heated under reflux conditions for
an additional 18 h and concentrated. The residue was purified by
preparative RP-HPLC using a gradient run with acetonitrile-water
containing 0.1% trifluoroacetic acid. Fractions containing the
desired product were combined and concentrated followed by
lyophilization to give 75 mg (0.22 mmol, 31%) of 38 (M+H 329) as a
white solid. [Note: The intermediate 6-acetamido-N-(1-chloroethoxy)
nordihydrocodeine] derivative (37) was anticipated to be formed
under reaction conditions and was hydrolyzed under reaction
conditions].
Example 32
Preparation of 6-acetamido nordihydrocodeine N-butyric acid
(39)
[0087] This compound 39 is prepared from compound 38, following the
procedure as described for the preparation of compound 23.
Example 33
Preparation of 6-acetamido nordihydrocodeine N-butyric acid NHS
ester (40)
[0088] This active ester is prepared from compound 39 according to
the procedure used to convert the acid 23 to active ester 24.
Example 34
Preparation of 6-acetamido-nordihydrocodeine N-butyric acid KLH
conjugate (41)
[0089] This conjugate is prepared from 40 according to the
procedure described for conjugate 10. Having described the
invention in detail and by reference to specific embodiments
thereof, it will be apparent that modifications and variations are
possible without departing from the scope of the invention defined
in the appended claims. More specifically, although some aspects of
the present invention are identified herein as preferred or
particularly advantageous, it is contemplated that the present
invention is not necessarily limited to these preferred aspects of
the invention.
Example 35
Development of monoclonal antibodies to 6-monoacetyl morphine
[0090] Female Balb-c mice, at least 3 months of age, were used for
immunizations. The immunogen contained 100 .mu.g of the 6-MAM KLH
immunogen from Example 9 per mouse emulsified in 50% isotonic
saline and 50% Freund's Adjuvant. Complete Freund's Adjuvant was
used for the initial intraperitoneal (ip) immunization, and
Incomplete Freund's Adjuvant was used for the remaining
immunizations. The mice were reimmunized 30, 84, and 114 days after
the initial immunization. The mouse selected for the fusion was
given a booster immunization identical to immunizations two through
four 509 days after the first immunization. Four days later the
immunized mouse was sacrificed, and its splenocytes were used in a
cell fusion to produce monoclonal antibody secreting
hybridomas.
[0091] The mouse selected for fusion was killed via exsanguinations
and cervical dislocation, and the spleen was aseptically harvested
and ground between two sterile glass slides to release the
lymphocytes. The resulting lymphocyte suspension was fused with the
P3X63Ag8.653 (ATCC cell line number CRL 1580) myeloma line.
[0092] Viable lymphocytes were counted, and 20% of that number of
myeloma cells were added to the tube containing the mouse
lymphocytes. The cells were washed in warm, serum-free Iscove's
Modified Dulbecco's Media (IMDM) by centrifugation, resuspension,
and re-centrifugation. The centrifuge tube containing the resulting
pellet was gently tapped to loosen the pelleted cells. One ml of
warmed PEG/DMSO solution (Sigma Chemicals) was then slowly added to
the cells while gently mixing. The cells were incubated at
37.degree. C. for 1.5 minutes after which pre-warmed serum-free
IMDM was added at the following rates: 1 ml/min, 2 ml/min, 4
ml/min, and 8 ml/min. The tube was filled to 50 ml, capped, and
incubated for 15 minutes at 37.degree. C. The cell suspension was
next centrifuged, the supernatant decanted, and the cells
resuspended in HMT media. HMT media consists of Complete IMDM with
Condimed (77.9% IMDM, 10% FCS, 10% Condimed H1, 1% L-glut, 1%
L-glut-pen/strep, 50 uM 2-mercaptoethanol, 40 .mu.M ethanolamine)
with 50.times. HMT diluted to 1.times.). The cells were resuspended
at a concentration of 2.times.10.sup.5 lymphocytes/ml, and 200
.mu.l were pipetted into each well of 30.5 sterile, covered 96-well
microculture plates. The plates were incubated at 37.degree. C. in
5% CO.sub.2 tissue culture incubator for five days. On day six, 150
.mu.l of supernatant was withdrawn from each well and was replaced
with 150 .mu.l of HT Rescue Media. HT Rescue Media consists of
Complete IMDM with Condimed plus 50.times. HT supplement diluted to
1.times.. The plates were returned to the incubator and inspected
daily for signs of growth. When cell colonies were sufficiently
large, wells were screened for antibody production using an
ELISA.
[0093] 96-well microtiter plates were coated with 50 .mu.l of the
6-MAM-BSA conjugate from Example 16 at 1 .mu.g/ml in 0.1 M
carbonate buffer, pH 9.5 for 1 hour at 37.degree. C. (humidified).
The plates were emptied, and 200 .mu.l of post-coat solution
consisting of pH 7.4 Tris buffer, 1% gelatin hydrolysate, 2%
sucrose, and 0.18% TWEEN 20 were added. The plates were incubated 1
hour at 37.degree. C. (humidified). After the plates were emptied,
200 .mu.l of a 2% sucrose solution in 0.15M Tris, pH 7.4, was
added. The plates were allowed to stand for approximately 5 minutes
at room temperature and then were emptied and air dried overnight
at room temperature. When dried, the plates were packed in zip-lock
bags containing several desiccant pillows, sealed, and stored at
4.degree. C. until use.
[0094] When clones were ready for testing, 20 .mu.l of supernatant
from each well showing cell growth was taken and transferred to
96-well flexible plates. PBST (phosphate buffered saline, pH 7.0,
with 0.2% TWEEN 20) was added to each well to provide a 1:10
dilution of the supernatant sample. Two 6-MAM-BSA coated wells were
used for each culture well tested. One well received 25 .mu.l of
PBST buffer, the other received 25 .mu.l of PBST containing 6-MAM
at a concentration of 800 ng/ml. Twenty-five microliters of the
diluted sample were transferred to each pair of wells to assay
supernatant antibody binding with and without free drug (6-MAM)
present. The plates were incubated in a humid chamber for 1 hour at
37.degree. C., after which they were washed with PBST. The wells
were then filled with 50 .mu.l of properly diluted goat anti-mouse
IgG-HRP conjugate and again incubated for 1 hour. The plates were
washed again, and 50 .mu.l of K-Blue substrate was added. After a
five minute incubation for color to develop, the reaction was
stopped by the addition of 50 .mu.l of 1 N HCl. Color was read via
a microplate reader at 450 nm, and data was transferred to a
computer for analysis. Supernatants that bound the 6-MAM-BSA
conjugate (produced color in the wells) and showed significant
inhibition of binding in the presence of free drug were considered
positives, and the corresponding clones were subcloned. A total of
40 clones were selected for subcloning.
[0095] Subcloning was achieved by stringent cloning via limiting
dilution. Briefly, cells from the original well were counted, and a
dilution was made so that 120 cells were added to 40 mls of
Complete IMDM with 10% Condimed. These cells were then dispensed
into 2 sterile 96-well plates, 200 .mu.l per well. Wells containing
colonies that appeared to have grown from single cells were
screened on 6-MAM coated ELISA plates for binding in the absence of
any free drugs and in the presence of morphine or codeine (each at
800 ng/ml). Clones from selected wells (positive for 6-MAM-BSA)
were then expanded in culture and frozen to create a cell bank.
Example 36
Use of 6-MAM 9.3 Monoclonal Antibody for Quantifying 6-MAM
[0096] 6-MAM 9.3 was the third subclone selected from the 9.sup.th
clone identified in the original fusion plates. Supernatant
collected from 6-MAM 9.3 was titered on a 96-well plate coated with
6-MAM-BSA at 0.1 .mu.g/ml. The dilution for the supernatant which
provided for 90% of maximal OD was 1:270. 6-MAM 9.3 supernatant
diluted 1:270 was then assayed on a 96-well plate coated with 0.1
.mu.g/ml of 6-MAM-BSA with different concentrations of free 6-MAM
added to the well to generate the inhibition curve shown in FIG. 8.
The ED.sub.50 was determined to be 1.22E-08. That parameter which
describes the concentration of the free drug which corresponds to
50% of the binding in the absence of free drug is termed the
ED.sub.50 for that drug.
* * * * *