Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Vaccine and Immune Serum Against Drugs of Abuse
The invention describes a procedure for the manufacturing of a vaccine against
drugs that can generate a dependence, wherein one or more drugs are linked as
a hapten
to a carrier substance in order to get the hapten antigenic, or wherein one or
more drugs
are chemically cross linked as a hapten in order to get an antigenic effect
without a carrier
compounds. The invention describes also a procedure wherein the vaccines,
manufactured
according to a process wherein one or more drugs are linked as a hapten to a
carrier
substance in order to get the hapten antigenic, are used as antigens for the
production of
antibodies against drugs, which may cause a dependence.
Dependence on drugs is often understood as a physiological and psychological
dependence. One or the other component can be dominant, but both components
are
normally present in an addict. Drugs which cause a physical dependence are
e.g. opiates,
barbiturates, alcohol, cigarettes, anxyolytic drugs and some sedatives.
Psychological dependence on the other hand is described as a desire for a
drug, which
temporarily becomes extinct, after ingestion of the drug and when the desired
effect has
taken over. Examples for a drug which can generate a psychological dependence
are opiate
and cigarettes.
A successful therapy implies a liberation of the psychological as well as the
physical
component of dependence. The classical therapy tries to obtain a
detoxification through a
successive diminution or in some cases (methadone) substitution of the drug.
The
psychological component of dependence is treated through a program of
rehabilitation.
The vaccine of this invention allows a new approach to therapy and prevention
for
patients who are addicted to a drug. The continued use of the drug in the
presence of the
antibody after vaccination inactivates the drug and stimulates the production
of new
specific antibodies. The desired drug effect is therefore eliminated and the
vicious circle
between stimulation and application is interrupted.
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The new vaccine broadens the application of vaccines
into the field of drugs. There are the following
fundamental differences between a classical vaccine and the
present vaccine: 1) The vaccine is not directed against an
infectious agent but against a drug. 2) The typical
application of this vaccine is not preventive but
therapeutic. 3) The antigen against which the vaccine is
directed is in most cases not by itself antigenic, but must
be bound to a carrier protein in order to elicit antibodies.
4) The effect of the drug which causes dependence is based
on the interaction of the drug with the receptor. This
interaction is eliminated through binding of the antibody
with the drug.
It is therefore a goal of this invention to describe a
vaccine against drugs of abuse:
1) Which is directed against one or more drugs which cause
dependence and which is linked to a carrier protein in order
to make the drug immunogenic.
2) Which is effective against drugs causing a physical
dependance as well as against drugs causing a psychological
dependence.
3) Which can be applied therapeutically (a drug addict is
vaccinated in order to inactivate the drug) as well as
preventively (in order to protect the fetus of a drug addict
pregnant woman from the direct damage of the drug as well as
a later dependence on the drug).
4) Which can be directed against all kind of drugs: for
example opiates and opiate-like drugs, marihuana, cocaine,
amphetamine, antipsychotic drugs, barbiturates and other
sedatives, psychomimetic drugs, anticholinergic drugs as
well as compounds which contaminate these drugs.
5) Which typically activates the B-cell branch of the
immune system, but which may gain an additional efficiency
through the activation of the T-cell arm of the immune
system or through an allergenic effect.
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Definitions:
Drugs which may cause a dependance: All compounds,
which in the largest sense of the term may cause a physical
or psychological dependence after one or repeated
application. A drug elicits a physical dependency, if a
withdrawal of the drug causes withdrawal symptoms.
Psychologital dependence on the other hand is caused by a
drug to which the addict has gotten used and which causes
with the addict a desire for a specific effect. Drugs which
may cause a dependance are among others: cocaine and cocaine
derivatives(cocaine consumed by drugs addicts contains
frequently among other compounds in addition mannitol,
lactose, nicotine, ephedrine, caffeine, procaine and
amphetamines), barbiturates and other sedatives,
benzodiazepine, methaqualone, glutethimide, chloral hydrate,
methyprylon, paraldehyde and bromides, antipsychotic drugs,
psychomimetic drugs like phenylcyclidine or LSD (lysergic
acid diethylamide), phenylcyclidin and analog compounds,
amphetamine and tryptamine derivatives, psylocybine,
volatile nitrite and anticholinergic drugs.
Components of drugs: drugs are not used all the time in
a chemically pure form and are often mixed with other
compounds. It may under certain conditions be an advantage
to direct the vaccine against more than one compound in
order to obtain a better protective effect.
Vaccine: a preparation which contains an immunogen
being able to activate the B-cell arm and eventually also
the T-cell arm of the immune system. The immunogen of the
vaccine of this invention is typically a drug, which is
bound as a hapten to the carrier substance. The binding
between hapten and carrier can be covalent or ionic or be
based on van der Waal forces or hydrogen bridges. The
linkage may also contain one or more atoms forming a bridge.
A drug can also be rendered immunogenic through a simple
chemical crosslinking(for example through glutaraldehyde) in
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order to gain a molecular weight in excess of 5000 Dalton.
It is in these cases not necessary to use a carrier
compound.
The manufacturing process of the vaccine contains
typically two steps: 1) Conjugation: the drug is linked to
the carrier compound 2) Purification: the hapten-carrier
conjugate is purified from products of the conjugation
procedure and dissolved into a physiological solution. The
conjugation can be performed in an organic solvent or in a
aqueous environment. Substances, which are frequently used
for coupling are: carbodiimides, imidoesters, N-
hydroxysuccinimide ester or their water-soluble sulfo-
derivatives, maleimide-derivatives and phenyl azides.
The purification is usually done by dialysis or with
the help of gel- or ion chromatography. The hapten-carrier
conjugate has mostly a molecular weight exceeding 100,000
.Dalton and can therefore easily be purified from an excess
of coupling substance, which typically has a molecular
weight under 500 Dalton. Preferred methods of separation are
extensive dialysis with dialysis tubing in an aqueous
solution, for example through multiple exchanges of
Phosphate Buffered Saline (PBS) or chromatography using a
Sephadex* G 25 column (Pharmacia LKB Biotechnology, Bromma,
Sweden). Sterile filtration with a filter having a pore size
of 0.2 micrometer and the removal of pyrogenic material are
the final steps of the purification.
Vaccine for preventive purposes: the person to be
vaccinated is not yet drug dependant. A candidate suitable
for a preventive vaccination may for example be a baby,
which obtains the drug through the mother's milk.
Vaccine for therapeutic purposes: The person to be
vaccinated is already drug addicted or has started to,use
the drug. A person suitable for the vaccine is for example a
morphine addict, who determines to get detoxified.
trade-mark
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Immunization against drugs: with patients who suffer
from symptoms of intoxication through drugs, there is no
time to elicit with the help of a vaccine antibodies against
the drug. It is possible to help in such cases through
direct application of specific antibodies of human or animal
origin. The specific antibody links in a short period of
time after application to the drug and the immune complexes
are eliminated through the reticulo- endothelial system.
This leads to a removal of toxic compounds from the body.
It is possible to use a mixture of antibodies against
different drugs, because in lots of cases it is not clear
with which drug the patient got intoxicated. The antibodies
used may be monoclonal or polyclonal antibodies. Monoclonal
antibodies are generated in vitro through fusion with a B-
cell line containing the genetic material for the specific
immunoglobulin. The hybrid cell line so generated secretes
only antibodies of the desired specificity. There is also
the possibility to obtain monoclonal antibodies through
transformation of a B cell with the help of a Epstein-Barr
virus infection.
It is possible to give the antibodies orally, because
patients showing symptoms of an overdose have often not yet
absorbed entirely the drug. The antibodies have to be
applied in this case in a capsule protecting them from
digestion in the stomach and small gut.
Antibodies: This is a class of plasma proteins, which
are produced by the B-cells of the immune system after
stimulation through an antigen. Human antibodies are
immunoglobulins of the Ig G, M, A, E or D class.
Antigen: This is a compound with the ability to elicit
a response of the immune system. Antigens are typically
proteins, but they can also contain sugars or lipid
moieties. Antigens typically have a molecular weight of
over 10,000 Dalton.
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Haptens: These are small molecules, which are not able to
elicit an immune answer only by themselves. A hapten has to be
linked to a carrier in order to become antigenic. The immune answer
to a hapten-carrier can be directed against the hapten, the carrier
or both compounds. The hapten of the present invention is normally
a drug, a metabolite of a drug or a component of a drug as for
example a component of cigarette smoke.
Some drugs are rapidly metabolized after absorption by the
body. Morphine for example is rapidly transformed into morphine-3--
glucuronate and morphine-6-glucuronate. It may therefore in certain
cases be an advantage, if metabolites are used as a hapten in place
of the original drug.
Carrier substance: The problem of eliciting antibodies against
a small molecule (hapten) is resolved by linking the small molecule
to a carrier. This linkage makes the hapten immunogenic. This
means antibodies are generated after injection into the body. The
binding of the hapten to the carrier protein is often covalent,
but, it can be ionic or be effected through a chemical component
bridging the hapten and the carrier. The carrier is typically a
protein, but it can also contain sugar and fat in mono- or polymer
form. Under certain conditions it is possible to crosslink the
hapten and no carrier substance is needed in order to make it
immunogenic.
Antigen/antibody interaction: The interaction of a specific
antibody with the surface of the corresponding antigen (hapten plus
carrier compound) is reversible and the complex can dissociate
depending on the force of binding. There is an interaction due to
the fact that the two partners have on their surface a
complementary shape of their electron clouds (similar as a key with
the lock). This interaction is based on hydrogen bridges, the van
der Waals force, electrostatic force and hydrophobic interaction.
Adjuvant: This is a compound or mixture of compounds which are
added to the vaccine in order to enhance the
efficiency of the antibody production or to help generate a
specific class of antibodies as for example IgM immunoglobulins
or antibodies able to bind complement. Substances, which can be
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used as adjuvant are for example mineral oils, derivatives of
aluminium or compounds of mycobacteria. The vaccines of this
invention can be used with or without adjuvant.
Different ways to apply the vaccine: The vaccine can be
injected intravenously, intramuscularly, subcutaneously or
intradermaly. It can also be given orally (e.g. in a
capsule, protected against digestion in the stomach and
small bowel). It is in certain cases also possible to give
the vaccine as an aerosol or onto the skin for an absorption
through the skin. The vaccine can be given one time or more
than one time.
In order to improve the understanding of the invention
the following examples are given.
Example 1
This example shows with the help of a mouse model how
antibodies are generated and how the drug is eliminated from
the circulation after vaccination against morphine
derivatives or barbiturates.
Conjugation of the hapten to the carrier:
The method of Wainer is used in order to synthesize
morphine 6-hemisuccinate (Wainer B.B. et al., 1972, Science
176, 1143-45, Wainer B.B. et al., 1972, Science 178, 647-8)
and to bind it to Bovine Serum Albumin (BSA). Another
preparation is made by transforming morphine into 3-0-
carboxy-methylmorphine by reaction of the free base of
sodium-beta-chlorazetate in absolute alcohol. Carboxymethyl-
morphine (8 mg) is dissolved in 2 ml of distiled water
containing 10 mg BSA and 8 mg of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide are added after adjusting
the pH of the solution to 6. After incubation at room
temperature, the mixture is extensively dialysed against
Phosphate Buffered Saline (PBS).
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The following method is used in order to obtain a
barbiturate-Keyhole Lympet Hemocyanin (KLH, Sigma, St.
Louis, USA) conjugate for the vaccine:
5-allyl-5-(1-carboxyisopropyl)barbituric acid is
transformed into 5-allyl-5(1--p-
nitrophenyloxycarbonylisopropyl)barbituric acid through
reaction of 10 mg of the free base with p-nitrophenol (12mg)
in a N,-N-Dimethylformamide solution during 24 hours at 4 C.
The coupling to KLH (19) (mg) is thereafter done in a
solution containing equal volumes of glycerin and water and
mg dicyclohexylcarbodiimide. After 24 hours incubation at
4 C., the mixture is dialysed with extensive exchange of
Buffer against PBS pH 7.6. The degree of substitution of
the drug at the carrier substance is calculated as an
increase in optical density of the conjugate at 202 nm as
compared to a KLH control solution (molar extinction
coefficient of barbiturate is 19,500).
Immunization: 5 female Balb/C mice are injected
subcutaneously 8 times in intervals of 2 weeks with 40,
microgram per mouse and dose of the morphine-BSA conjugate.
Another group of the mice receives the barbiturate-KLH
conjugate under the same conditions and a control group of
10 female Balb/C mice of the same age is treated with the
buffer of the vaccine preparation without the vaccine.
Bleeding: The blood is taken from the tail vein.
Assay of the drug concentration: The High pressure
Liquid Chromatography (HPLC) method of Joel et al. (1988,
Chromatography, 430: 394-9 is used as a reference method in
order to measure Morphine, morphine -6-glucuronide and
morphine 3-glucuronide. The morphine as well as the
barbiturate are likewise measured with a commercial enzyme
immunoassay (EMIT, Shyva Corp., Palo Alto, USA) in
accordance with the manufacturers instructions (EMIT
operator's manual, Shyva Corporation, Palo Alto, USA). It is
important for the evaluation of the vaccine to differentiate
free drug from antibody-bound drug. The EMIT assay is
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therefore modified as follows: the already diluted serum is
purified from antibodies before the EMIT assay is executed.
The sample is incubated at room temperature for two hours in
a PBS Buffer, pH 7.6 which contains CnBr-activated
Sepharose* 4 B, to which polyclonal rabbit antibodies
against mice light chain immunoglobulins are linked
(Pharmacia LKB Biotechnology, Bromma, Sweden).
Five mice, which have been vaccinated and 5 mice of the
control group are 2 weeks after the last immunization
injected with 0.5 mg per mouse. The serum samples are taken
immediately after injection and 2 hours after injection and
the quantity of the drug in the serum is determined with the
help of the modified EMIT assay. It is still possible to
detect the drug in the samples which were taken immediately
after the injection of the drug, but 2 hours after injection
the drug is no longer detectable. The discrimination in the
test system between positive and negative sample is
determined to be 5 ng/ml as well for the barbiturates as for
morphine. The interpretation of the test results is, that
the antibodies have bound to the drug after a successful
vaccination and the free drug is therefore no longer
detectable.
Example 2:
This experiment demonstrates that the "immunity"
against the drug is still present even after months. Two of
the immunized mice of example I and three mice of the
control group are injected 2 and 4 months after the first
experiment with 0.5 mg morphine. One blood sample is taken
as in the first experiment immediately after injection and 2
hours after injection and the serum is tested for morphine
in the EMIT assay. The serum of the mice, which were prior
immunized with the morphine -BSA conjugate is again negative
and only the samples, which were taken immediately after
injection show a positive result. The group of controls show
in all samples detectable levels of morphine. The conclusion
* trade-mark
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is made that the vaccine is efficient over an extended time
span.
Example 3:
This example serves to demonstrate the protective
effect of specific antibodies, respectively specific immune
serum containing the antibodies.
Two mice each of the control group receive 0.5 mg
morphine mice (A and B) and 0.5 mg phenobarbital (mice C and
D). Mice A and C receive each 0.5 ml of the serum of the
mice which were previously vaccinated with the anti-morphine
vaccine. Two hours after injection of the immune serum the
blood samples are taken and examined for the presence of
drug (modified EMIT assay). The mouse which has received
morphine and anti-morphine serum shows no free morphine in
the assay. The mouse which got morphine and anti-barbiturate
serum, shows free morphine in the serum. An analogous
phenomenon is seen with mice which have received
phenobarbital: no free drug can be detected with the mouse
which received the specific antibodies (mouse D). The mouse
with the anti-morphine serum (mouse C) shows still traces of
the drug in her serum.
This experiment can likewise be done with an anti-
serum consisting of monoclonal antibodies or a mixture
ofmonoclonl antibodies: as an analogous result is expected.
