U.S. patent application number 09/997580 was filed with the patent office on 2002-05-30 for method to reduce the physiologic effects of drugs on mammals.
Invention is credited to Bohannon, Robert C..
Application Number | 20020064529 09/997580 |
Document ID | / |
Family ID | 26681736 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020064529 |
Kind Code |
A1 |
Bohannon, Robert C. |
May 30, 2002 |
Method to reduce the physiologic effects of drugs on mammals
Abstract
A method to reduce the physiologic effects of drugs in vivo by
inducing specific anti-drug antibodies using drugs conjugated to
carrier molecules so as to reduce a drug's toxicity and its
physiologic effects upon the recipient. This method includes the
treatment and prophylactic prevention of drug abuse, specifically
for cocaine and nicotine, and to help reduce the toxic effects of
drugs, such as anti-neoplastics.
Inventors: |
Bohannon, Robert C.;
(Lakeside, AZ) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
26681736 |
Appl. No.: |
09/997580 |
Filed: |
November 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09997580 |
Nov 28, 2001 |
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09715238 |
Nov 17, 2000 |
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09715238 |
Nov 17, 2000 |
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09112065 |
Jul 7, 1998 |
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09112065 |
Jul 7, 1998 |
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08203002 |
Feb 28, 1994 |
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08203002 |
Feb 28, 1994 |
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08010903 |
Jan 29, 1993 |
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Current U.S.
Class: |
424/184.1 ;
424/193.1; 424/196.11; 424/197.11; 514/54 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 47/61 20170801; Y02A 50/466 20180101; A61K 47/646 20170801;
A61K 2039/6037 20130101; A61P 25/30 20180101; A61K 2039/6081
20130101; A61P 37/02 20180101; A61P 25/26 20180101; A61K 39/0013
20130101 |
Class at
Publication: |
424/184.1 ;
514/54; 424/193.1; 424/196.11; 424/197.11 |
International
Class: |
A61K 039/00; A61K
031/715; A61K 031/739 |
Claims
What is claimed is:
1. A method for suppressing or reducing in mammals the
physiological effects caused by a drug comprising: administering to
a mammal, prior to the administration of the drug, a
drug-conjugated immunogen which consists of the drug conjugated to
a carrier molecule; and inducing in the recipient the production of
anti-drug antibodies wherein said drug is selected from the group
consisting of cocaine, cocaine-derivatives, nicotine and
anti-neoplastic compounds.
2. The method of claim 1 wherein the drug is cocaine or a
cocaine-derivative.
3. The method of claim 2, wherein the cocaine derivative is
benzoylecgonine.
4. The method of claim 1, wherein the carrier molecule is selected
from the group consisting of albumin, polysaccharide, and
lipopolysaccharide.
5. The method of claim 4, wherein the polysaccharide is mannan.
6. The method of claim 1, wherein the carrier molecule is selected
from the group consisting of Diphtheria, Tetanus, Pertussis,
poliovirus, Rubella, Mumps, Measles, Hepatitis, Haemophilus,
smallpox and varicella-zoster vaccines, or components thereof.
7. The method of claim 4, wherein the drug is cocaine or a
cocaine-derivative.
8. The method of claim 7, wherein the polysaccharide is mannan.
9. A method for reducing the toxicity of a drug in mammals
comprising: administering to a mammal, prior to the administration
of the drug, a drug-conjugated immunogen which consists of the drug
conjugated to a carrier molecule; and inducing in the recipient the
production of anti-drug antibodies wherein said drug is selected
from the group consisting of cocaine, a cocaine-derivative and
anti-neoplastic compounds.
10. The method of claim 9, wherein the drug is cocaine or a
cocaine-derivative.
11. The method of claim 8, wherein the cocaine derivative is
benzoylecgonine.
12. The method of claim 9, wherein the conjugate is administered to
the subject by injection, inhalation or orally.
13. The method of claim 12, wherein the conjugate is administered
by subcutaneous injection.
14. The method of claim 9, wherein the carrier molecule is selected
from the group consisting of albumin, polysaccharide,
lipopolysaccharide or Diphtheria, Tetanus, Pertussis, poliovirus,
Rubella, Mumps, Measles, Hepatitis, Haemophilus, smallpox or
varicella-zoster vaccine, or components thereof.
15. The method of claim 14, wherein the carrier molecule is either
mannan or Salmonella typhosa lipopolysaccharide.
16. A method for the treatment of drug abuse comprising the steps
of: administering to a recipient a controlled substance-carrier
conjugated immunogen; inducing in the recipient the production of
anti-controlled substance antibodies; and reducing or eliminating:
(i) the physiological effect and/or (ii) toxicity of a subsequent
intake of controlled substance in the recipient.
17. The method of claim 16 wherein the controlled substance-carrier
conjugate is a cocaine derivative conjugated to a carrier
molecule.
18. The method of claim 17 wherein the cocaine derivative is
benzoylecgonine.
19. The method of claim 16 wherein said therapeutically effective
dose is a dose sufficient to generate in the recipient antibodies
against a controlled substance.
20. The method of claim 16, wherein the carrier molecule is
selected from the group consisting of albumin, polysaccharide,
lipopolysaccharide or Diphtheria, Tetanus, Pertussis, poliovirus,
Rubella, Mumps, Measles, Hepatitis, Haemophilus, smallpox or
varicella-zoster vaccine, or components thereof.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method for reducing the
physiologic effects of drugs on mammals in vivo. Specifically, the
method of this invention consists of inducing specific anti-drug
antibodies in mammals with immunogens consisting of a drug
conjugated to a carrier molecule. More particularly, the method of
this invention consists of generating antibodies towards addictive
substances--particularly cocaine and nicotine--such that the
physiologic effects of such addictive substances are reduced. As a
result, toxicity to such addictive substances is dramatically
lowered in those mammals which have been treated with the
immunogens prior to introduction of the addictive substance. In
addition, mammals can be afforded prophylactic protection from such
addictive substances by the administration of the drug-conjugated
immunogen prior to introduction of the addictive substance.
BACKGROUND OF THE INVENTION
[0002] The National Institute of Drug Abuse National Household
Survey on drug abuse estimated that in 1991 there were
approximately 12 million persons in the United States that abused
drugs. This included approximately 4.5 million occasional abusers
of cocaine and greater than 800,000 habitual users of cocaine. The
United States government is projected to spend an estimated $12
billion in 1993 on federal drug control programs with an estimated
$2.7 billion allocated to help defray the cost of drug treatment
programs. Typical methods of drug treatment therapy include
psychological counseling. Heroin addiction is sometimes treated
with methadone therapy to permit gradual withdrawal, however, the
replacement drug methadone is itself addictive.
[0003] It would be highly desirable to develop a method to
neutralize or minimize the effects of controlled substances or
addictive drugs thereby rendering continued abuse of addictive
substances unproductive due to the reduced physiologic effect of
the drug on the user. Additionally, a physiologic reduction of the
action of the drug, or reduction of the rate in which the drug
effects the mammalian subject, would help contribute to reduce the
toxicity of the drug. Such a method would be highly desirable in
the treatment of habitual substance abusers, in the prophylactic
prevention of abuse, to reduce toxicity of drugs, and may provide
an additional method to slowly release toxic anti-neoplastic
drugs.
SUMMARY OF THE INVENTION
[0004] The present invention consists of the administration of an
immunogen consisting of a drug conjugated to a carrier molecule.
The immunogen induces in the recipient the production of antibodies
to the drug, as well as to the carrier molecule in most cases. Such
antibodies, in turn, sequester a subsequently administered drug
thereby reducing the drug's physiologic effects in the recipient.
To demonstrate the method of this invention, an anti-cocaine
immunogen was synthesized that markedly demonstrated a reduction in
the physiologic effects and toxicity of cocaine in laboratory
animals. A "reduction in the physiologic effect of a drug" is
defined herein as that which can be readily observed and measured,
such as heart rate, breathing rate, auditory stimulus, eye
dilation, irritability, agitation, and pain reflex, in the case of
cocaine.
[0005] The present invention describes a method for preventing or
treating drug abuse by administering drug-conjugated immunogen that
induces in the recipient antibodies, which specifically recognize
and neutralize a targeted controlled substance. Such antibodies are
then available in the treated recipient to reduce or eliminate the
effect of a subsequent intake of the drug including reduction of
the physiologic effects of subsequent drug use and reduction of the
toxicity of the drug. The invention therefore is of great
assistance in drug treatment programs.
[0006] The method of this invention may also be employed to reduce
anti-neoplastic drug toxicity and to provide for a method of slow
in vivo release of drugs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is an immunoblot showing reactivity of
cocaine-conjugated immunogens with antisera of an animal treated
with the anti-cocaine immunogen.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0008] The drug-conjugated immunogen of the present invention is a
substance which induces in a mammal the production of antibodies
which minimize or neutralize the effect of a targeted controlled
substance or drug. Antibodies against addictive substances, drugs,
or anti-neoplastic compounds also reduce the toxic effects of the
respective drug by keeping the blood level of unbound drugs in the
bloodstream at a relatively low level while the drug is cleared, by
enzymes, the reticulo-endothelial system, the liver, secreted via
the kidneys, or other methods used in mammals to dispose or clear
toxic substance. The ability of the drug-conjugated immunogen to
reduce such toxic effects thereby minimizes the likelihood of a
drug overdose. Toxicity resulting in death, seizure and tissue
damage is greatly diminished thereby.
[0009] A controlled substance or drug is defined-herein to include
those substances known to be subject to abuse, e.g., cocaine,
morphine (opium), heroin, and tetrahydrocannabinol (marijuana).
Additional substances included in the invention are those which are
habit forming such as various pain relievers, stimulants,
anti-depressant drugs and nicotine. These substances, as well as
other drugs which are potential candidates for abuse or that are
toxic, are contemplated as targets for the drug-conjugated
immunogen and method of the present invention, and are thus to be
included in the definition of "controlled substances" or "drugs"
for purposes of the present invention.
[0010] Anti-cocaine immunogens and a method to treat cocaine abuse
are exemplified herein, but it is understood that the immunogen and
method of the present invention may similarly be applied to other
controlled substances or drugs. The method of the invention reduces
the toxicity of drugs--due to antibody sequestering of the target
drug induced by the drug-conjugated immunogen--and thus provides a
method of releasing toxic drugs, such as anti-neoplastic compounds,
into the bloodstream of the host mammal, thereby slowly reducing
the side-effects of toxic compounds or drugs.
[0011] Cocaine, most controlled substances, and anti-neoplastic
drugs are compounds having low molecular weights and do not tend to
elicit an effective immune response when injected into mammals.
However, cocaine, controlled substances, and anti-neoplastic
compounds may be conjugated to carrier molecules. The resulting
conjugate is of sufficient size and thus becomes immunogenic so as
to generate an immune response, thereby producing antibodies
against the drug of the conjugate.
[0012] Carrier molecules suitable for conjugation with controlled
substances and drugs that would generate an immunogen include, for
example, sheep albumin, polysaccharide such as mannan, and various
lipopolysaccharides such as those derived from Salmonella typhosa.
Many conventional carriers known to those skilled in the art may
also be used in this invention, including those approved by
regulated governmental agencies for use in humans, such as, for
example: Diphtheria, Tetanus, and Pertussis vaccines or components
thereof; poliovirus vaccines and components thereof; Rubella,
Mumps, and Measles vaccines or components thereof; Hepatitis
vaccines (A,B,C, and delta) and components thereof; Haemophilus (A
and B) vaccines and components thereof; vaccinia and smallpox
vaccines and components thereof; varicella-zoster vaccines and
components thereof, as well as synthetic multiple antigenic
peptides (MAPs) and derivatives thereof.
[0013] The conjugation method used will depend upon the chemistry
of coupling a particular drug to a particular carrier. Suitable
agents and methods for conjugating a variety of compounds are
commercially available from Pierce Chemical Co. (Rockford, Ill.),
as described, for example in the Pierce Chemical Co. catalog. Known
methods for conjugating two or more compounds via specific reactive
groups may be applied to the preparation of the drug-carrier
molecule conjugates of the present invention. In general, a
reactive site on a first compound is linked to a reactive site on a
second compound, using a coupling agent or catalyst.
[0014] Coupling agents include EDC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCL, (Pierce
Chemical Company, Catalog No. 22980), glutaraldehyde and other
similar agents. (See, for example, Axen et al., Nature,
214:1302-1987; and Okawa et al., Journal of Immunolocrical Methods,
149:127, 1992).
[0015] The compound conjugated to the carrier may be a natural or
synthetic drug, controlled substance, anti-neoplastic compound, or
a derivative thereof. For example, the cocaine derivative
benzoylecgonine was used to form a drug-carrier conjugate which
induces the production of antibodies against cocaine in a
recipient.
[0016] The drug-carrier conjugate may contain a drug to carrier
ratio of 1:1 or greater, e.g., 75:1, depending upon available
conjugation sites on the carrier molecule. In some cases, a
polymeric carrier may be used which may carry a large number of
drug molecules, e.g., greater than 100:1 drug:carrier.
[0017] The drug-conjugated immunogens of the present invention are
immunogenic. A particular drug-conjugated immunogen may be tested
for immunogenicity, for example, by immunization of test animals
and analysis of the resulting antisera. A drug-conjugated immunogen
may be modified to make the composition more immunogenic. Such
modifications includes conjugation to lipopolysaccharides,
synthetic peptides, or to molecules which stimulate the immune
system.
[0018] The drug-carrier conjugate immunogens of the present
invention are administered to a recipient by known, conventional
methods, e.g., injection, inhalation, or by oral delivery,
depending upon the physiological characteristics of the drug and
carrier compounds. Preferably, the agent is administered by
subcutaneous injection. The conjugate is prepared in a
pharmaceutically acceptable carrier, for example, in an aqueous
medium such as water, buffer, saline, glycine, or an oil based
carrier, as appropriate for the specific drug-carrier conjugate and
the desired mode of delivery.
[0019] A therapeutically effective dose of the drug-conjugated
immunogen is administered to a recipient mammal, e.g., human,
rabbit, monkey or mouse. A therapeutically effective dose of the
drug-conjugated immunogen is one that induces in the recipient the
production of antibodies to the desired drug or controlled
substance, which antibodies are effective to reduce or eliminate a
response to a subsequent challenge or intake of the controlled
substance, anti-neoplastic agent, or drug. It is understood that a
single administration or multiple administrations of the
drug-conjugated immunogen may be required to induce adequate
antibody titres, depending upon each recipient's immune competence.
In general, one to four injections will be used.
[0020] Successful immunization of the recipient may be monitored,
for example, by analyzing the ability of a recipient's anti-serum
to bind the controlled substance or drug of choice. Immunoassay
methods such as ELISA and immunoblot may be used for such analysis.
Immunized mammals, when presented with a challenge dose of
controlled substance or drug, exhibit diminished effects or no
effect of the controlled substance or drug.
[0021] The method of the present invention may be used to immunize
mammals, including rabbits and humans, against controlled
substances and other drugs as a method of preventing or treating
drug abuse or to reduce a drug's toxic effects. Immunization
against such drugs as cocaine, heroin, opium, morphine, marijuana,
nicotine, and anti-neoplastic compounds should reduce or nullify
the physiologic effects of these drugs in the recipient and thereby
help to reduce the drug's toxicity.
[0022] The invention may be better understood by reference to the
following examples:
EXAMPLE 1
PREPARATION OF A BENZOYLECGONINE-CONJUGATED IMMUNOGEN
[0023] To form a conjugate between benzoylecgonine, a cocaine
derivative, and sheep albumin, 5 mg of sheep albumin was conjugated
to 1 mg of benzoylecgonine using 100 mg of EDC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl, Pierce
Chemical Company, Catalog No. 22980) in 1 ml of 0.1 M MES buffer
(2-(N-morpholino)ethanesulfonic acid) at pH 4.5 for four hours at
50.degree. C. The mixture was dialyzed against water for two days
at room temperature using a 1,000 m.w. cutoff dialysis tubing.
Approximately, 5 mg of the benzoylecgonine-albumin conjugated
immunogen (COALB) in a volume of 1.5 ml was recovered and used in
the subsequent studies.
[0024] Similarly, conjugates of benzoylecgonine to mannan or
lipopolysaccharide were formed by dissolving 10 mg of mannan (Sigma
Chemical Co. #M7504) or Salmonella typhosa lipopolysaccharide
(Sigma Chemical Co. #L6386) in 0.1 M sodium carbonate-buffer, pH
10.7, to which 10 mg of cyanobromide (CNBr) was added in a volume
of 1 ml. The mixture was allowed to react for ten minutes at room
temperature. Diaminopropane (50 .mu.l) was added and the mixture
was allowed to sit overnight at room temperature. This process
provided amine groups on the polysaccharide for coupling of
benzoylecgonine. The mixture was dialyzed with water overnight
using a 1000 molecular weight cutoff dialysis membrane to yield
activated polysaccharide. To 5 mg of the activated polysaccharide
in 0.9 ml volume was added 5 mg of benzoylecgonine in 50 .mu.l of
30% ethanol followed by the addition of 50 mg of EDC in 50 .mu.l of
0.5 M MES buffer at pH 4.5. This solution was incubated at
50.degree. C. for a period of four hours then dialyzed against
water for 24 hours using 1000 molecular weight cutoff dialysis
membrane. Approximately 5 mg of each conjugate,
benzoylecgonine-mannan (COMAN) and
benzoylecgonine-lipopolysaccharide (COLPS) in a volume of 1.5 ml
was recovered and used in the subsequent studies.
EXAMPLE 2
CONJUGATION OF BENZOYLECGONINE TO DIPHTHERIA TOXIN
[0025] Similarly benzoylecgonine can be coupled to other carrier
molecules such as Diphtheria Toxin. In this example, 5 mg. of
Diphtheria toxin (Sigma D7544) is dissolved in 0.9 ml of 0.1M MES
(pH 4.5) to which is added 1 mg. of benzoylecgonine dissolved in a
volume of 0.05 ml. 0.1M MES (pH 4.5) and to which 10 mg. EDC in
0.05 ml of 0.1M MES (pH 4.5) is added. This coupling reaction is
performed at 50.degree. C. for four hours then the mixture is
dialyzed against water or PBS using a 1000 molecular weight cut-off
dialysis membrane or purified by passing the mixture over an gel
filtration column. The recovered conjugate can then be used as
drug-conjugated immunogen.
EXAMPLE 3
PREPARATION OF ANTI-NEOPLASTIC-CONJUGATED IMMUNOGENS
[0026] Anti-neoplastic drugs, or other highly toxic drugs, may be
directly coupled to carrier molecules as a method to lower the
toxicity of the drug and to provide controlled release or
degradation of the drug. For example, methotrexate can also be
coupled to albumin, diphtheria toxoid, tetanus toxoid, and modified
polysaccharide as described above. In this example, 5 mg. of a
carrier molecule, which has amine groups for binding, such as
tetanus toxoid, is coupled to 1 mg. of methotrexate in 0.9 ml of
0.1M MES (pH 4.5) to which is added 10 mg. of EDC dissolved in 0.1
ml of 0.1M MES (pH 4.5). The mixture is allowed to couple the
anti-neoplastic, or other toxic substance, to the carrier
molecule--tetanus toxoid in this example--for four hours at
50.degree. C. after which the drug-conjugated immunogen is purified
by dialyzing against water or a buffered solution, such as PBS, or
is purified by column filtration or by High Pressure Liquid
Chromatography. The resulting drug-conjugated immunogen elicits an
immune response in a mammal thereby generating antibodies against
the conjugated agent:methotrexate.
EXAMPLE 4
IMMUNIZATION WITH BENZOYLECGONINE-CONJUGATED IMMUNOGEN
[0027] Two rabbits were immunized with the cocaine-conjugated
immunogen COALB, prepared as described for Example 1. Rabbits were
immunized with COALB by subcutaneous injection of 100 .mu.g of the
benzoylecgonine-conjugated immunogen every two weeks for a period
of two months (four injections). Freund's complete adjuvant was
used in the first injection and Freund's incomplete adjuvant was
utilized for subsequent injections.
[0028] To test for the presence of anti-cocaine antibodies, the
serum of the treated rabbits was analyzed by immunoblot assay for
its ability to bind the benzoylecgonine conjugates produced in
Example 1.
[0029] Approximately 1 .mu.g of each drug-carrier conjugated
immunogen was spotted onto nitrocellulose strips and allowed to air
dry. These strips were blocked for one hour at room temperature
using 1:10 Mega-block I (ONASCO Products, Houston, Tex.).
[0030] Sera obtained from the test rabbits both before and after
immunization with COALB were diluted 1:100 in the blocking solution
and applied to the test strips. After incubating for two hours at
room temperature, the strips were washed four times in a solution
of saline and 0.01% tween-20 and the secondary antibody was added
(1:1000 dilution of goat anti-rabbit antibody conjugated with
alkaline phosphatase, in 1:100 Mega-block I blocking buffer). The
strips were incubated for two hours at room temperature then washed
four times in 0.01% tween-20 solution. Bound antibodies were
detected by reacting the substrate NBT/BCIP (Promega Biotech) with
the bound secondary antibody which resulted in a visually
detectable signal. The results of this immunoblot assay are shown
in FIG. 1, where strip A shows rabbit sera prior to immunization
and strip B shows rabbit sera after four doses of the COALB. The
COMAN (1), COLPS (2) and COALB (3) immunogens each reacted with
antibody present in the rabbit sera after immunization. The
background signal in the pre-immunization COALB (3) spot is
expected due to the nature of the albumin carrier.
[0031] The immunized rabbits were then challenged with an
intravenous dose of cocaine-HCl (Sigma Chemical Co., #C5776) in
sterile saline at 2 mg/kg wt (LD.sub.50 of 12 mg/kg). Control
rabbits challenged with the same dose of cocaine showed marked
grande maul convulsions, involuntary eye movements, rapid and
shallow breathing, were unresponsive to both visual, auditory, and
pain stimuli, had dilated eyes, but recovered after 10-15 minutes.
Clearly, the i.v. administered cocaine caused pronounced
physiologic effects upon the animals and nearly caused the death of
one of the animals due to the drug's toxic effects upon the rabbit.
Two rabbits immunized with the COALB immunogen, when injected with
the same dose of cocaine (2 mg/kg), showed none of the dramatic
effects that the control rabbits had demonstrated. The treated
animals remained responsive, and no physiologic effects of the
cocaine injection were noted, which clearly demonstrated that the
anti-cocaine immunogen, COALB, was effective in suppressing the
physiologic effects of the cocaine challenge as well as apparently
lowering the drugs toxicity as demonstrated by the lack of
physiologic trauma when compared to an un-immunized animal.
EXAMPLE 5
IMMUNIZATION WITH BENZOYLECGONINE-CONJUGATED IMMUNOGEN
[0032] In a similar fashion, anti-benzoylecgonine immunogens
described above in Example 2 (those conjugated to diphtheria or
tetanus toxoid) are used to immunize mammals other than rabbits
(such as monkeys and humans) to suppress the action of cocaine and
its derivatives upon the central nervous system and to increase
tolerance of the mammal to the drug. One hundred .mu.g of the
anti-cocaine immunogen is absorbed to an approved adjuvant aluminum
hydroxide and is injected s.c. Similarly, anti-neoplastic or
anti-toxic conjugates could be used as drug-conjugated immunogens
in this example.
EXAMPLE 6
PREPARATION OF A NICOTINE-CONJUGATED IMMUNOGEN
[0033] Nicotine (3-1-Methyl-2-pyrrolidinyl)pyridine;
1-methyl-2-(3-pyridyl)pyrrolidine;
beta-pyridyl-alpha-N-methyl-pyrrolidin- e) is conjugated to a
carrier molecule (albumin, cationized albumin, amine-linked mannan,
amine-linked polysaccharide, diphtheria toxoid, tetanus toxoid, or
other such molecules) using the PharmaLink Immunogen Kit
commercially available from Pierce Chemical Company, Rockford,
Ill., catalog number 77158 G.
[0034] Following the instructions provided, nicotine-HCl is
dissolved in the conjugation buffer which includes the carrier
compound, SuperCarrier. The coupling agent provided in the Pierce
kit is then added and the mixture is incubated for 2 to 24 hours at
approximately 37-57.degree. C. (The time required is related to the
temperature of incubation.) The resulting conjugate is then
purified from non-conjugated nicotine using a desalting column.
[0035] Alternatively, approximately 1 mg of carrier protein, e.g.
tetanus or diphtheria toxoid, is dissolved in 200 .mu.l of 0.1 M
MES buffer, pH 4.5, 0.15M NaCl, and approximately 1 mg. of
nicotine-HCl in 200 .mu.l of 0.1 M MES buffer, pH 4.5, 0.15 M NaCl
is added. A volume of 50 .mu.l of 37% formaldehyde is added next
and the mixture is allowed to react at 37.degree. C. for
approximately 3 hours. The solution is dialyzed overnight against
water using a 1000 m.w. cutoff membrane.
[0036] The resulting nicotine-carrier conjugated immunogen is then
used to immunize a recipient, following the procedures for Example
2.
EXAMPLE 7
IMMUNIZATION WITH NICOTINE-CONJUGATED IMMUNOGEN
[0037] Humans and monkeys are immunogenized with antinicotine
immunogens wherein the conjugated immunogen is absorbed onto a
pharmaceutically approved adjuvant aluminum hydroxide and injected
i.m. using a dose which causes the generation of relatively high
titre antibodies (100 .mu.g-500 .mu.g). Several injections may be
required before suitable antibody titres are obtained.
[0038] In a similar fashion, toxic compounds, such as
anti-neoplastic agents, are coupled to carrier molecules using the
same method of coupling via a condensation reaction to active
hydrogen molecules. One mg. of a carrier molecule, such as sheep
albumin, is dissolved in 0.2 ml. of 0.1 M MES (pH 4.5) and 0.15 M
NaCl. to which 1 mg. of vinblastine dissolved in 0.2 ml of ethanol
is added followed by the addition of 0.05 ml. of 37% formaldehyde.
The condensation-coupling reaction is allowed to proceed at
37.degree. C. for 3 hours after which the mixture is dialyzed, or
otherwise purified, to remove the salts, buffering agents,
formaldehyde and unbound vinblastine. The immunogen is then used to
generate antibodies towards vinblastine by immunizing a mammal
(mouse, rabbit, monkey, or human) so as to lower the drug's
toxicity during treatment in the same manner as described
above.
EXAMPLE 8
PREPARATION OF MORPHINE-CONJUGATED IMMUNOGEN
[0039] Morphine
(7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol) is conjugated
to albumin, mannan, or lipopolysaccharide using conjugation methods
as described for Examples 1 and 6. Approximately 5 mg of morphine
acetate-trihydrate (Sigma Chemical Co.) is dissolved in 0.5 ml of
0.1 M MES, pH 4.5. To this is added approximately 5 mg of sheep
albumin dissolved in 0.45 ml of 0.1M MES, pH 4.5. EDC, 50 mg in 50
.mu.l of 0.1 M MES, pH 4.5 is added and the mixture reacted for
approximately 4 hours at 50.degree. C. The resultant conjugate is
dialyzed against water overnight at room temperature, changing
water approximately every 2 hours and using a 1000 m.w. cutoff
membrane.
[0040] The resultant morphine-conjugated immunogen is used to
immunize mammals (rabbits, mice, monkeys, or humans) as described
in Example 2. The resulting antibodies to morphine sequester
morphine, and derivatives thereof, thereby suppressing its effects
upon the central nervous system and thereby reducing the toxicity
of the drug.
* * * * *