KR100538388B1 - Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same - Google Patents

Abstract

The present invention relates to an incomplete antigen-carrier conjugate capable of inducing anti-hapten antibody in vivo and a method of preparing the conjugate and the therapeutic composition. If the incomplete antigen (hapten) is an addictive drug, the therapeutic composition of the present invention containing an antiincomplete antigen (hapten) antibody is particularly useful for the treatment of drug addiction. In addition, the therapeutic composition can be used in combination with other existing drugs. The present invention has also been described with respect to passive immunization using antibodies that are enhanced against incomplete antigen-carrier conjugates.

(constitutional formula)

Description

Incomplete antigen-carrier conjugates used in the treatment of substance abuse and a method for preparing the conjugates {HAPTEN-CARRIER CONJUGATES FOR USE IN DRUG-ABUSE THERAPY AND METHODS FOR PREPARATION OF SAME}

FIELD OF THE INVENTION The present invention relates to the treatment of drug abuse, and more particularly, by using an antibody against a drug / hapten-carrier and / or an antibody against a drug / hapten-carrier that induces an antibody response. A method for treating drug abuse.

Drug use and abuse around the world are spreading like epidemics, especially in the United States. The problem of legal and illegal drug abuse is becoming a serious social problem across all classes of society, and medical expenses and social burdens are increasing accordingly. Some drug users are extremely high risk groups and are involved in poverty and illegal social activities. In addition, other drug users may classify drugs for their filter line, which may include: 1) the nature of the drug causing addiction, 2) the tendency to be a habitual drug user, or 3) the personal situation, hardship, environment, and acquisition. Since it is easy to do, the risk of drug addiction can be said to be large. Innovative and innovative treatment programs must be developed to adequately treat drug abuse, including multiple drug abuse.

In particular, examples of three drugs that are problematic for addiction include cocaine, heroin and nicotine. Cocaine is an alkaloid drug extracted from the leaves of coca plants (Erythoxylon coca). In the United States, there are more than 5 million habitual cocaine users, at least 600,000 of whom are classified as serious addicts (see Miller et al. (1989) NY State J. Med. Pp.390-395; and Carrol et al. (1994) Pharm. News. 1: 11-16). Many of the addicts among the cocaine users are actively seeking appropriate treatments. For example, in 1990, 380,000 cocaine users wanted to be treated for drug addiction, and the number is increasing every year. At that time, it is estimated that 100,000 cocaine users are admitted to emergency hospitals every year. The cumulative effects of violent crimes involving cocaine, a decline in personal productivity, illness and death are becoming international issues.

Since there is no effective treatment for the treatment of cocaine addiction, the development of a new approach to cope with this is desired. A side effect of no successful treatment is the type of cocaine addiction that is changing over time. Carol et al., In the article "Chemical Approach to Treat Cocaine Poisoning in 1994," used hydrochloric acid (coke, snow and blow) and cocaine free base as intravenous and nasal administrations as usual routes of administration since the mid-1980s. Inhalation has been reported to increase the feeling of euphoria and psychomotor activity for 30 to 60 minutes (Carroll et al. (1994) Pharm. News, Vol. 1, No. 2). Cocaine, unlike other indulgence drugs, can induce excitement over the user for hours. This behavior of cocaine users causes addiction and in some cases addiction (Carroll et al. Pharm. News, supra.).

Therapies to cope with drug abuse are extremely limited and no long-term effective treatments for cocaine poisoning have been developed. Cocaine addiction is treated by counseling while administering drugs that act as antagonists to opiate receptors or drugs that reduce cravings due to drug addiction. Detoxification is an approach to treating cocaine addiction. Temporary palliative therapy is desirable for the subsequent physical, social, and psychological improvements, as well as for continuous drug abuse, gradual acceleration, and associated medical costs or personal relationships (Wilson et al. In Harrison's Principle of Internal Medicine Vol. 2, 12th Ed., McGraw-Hill (1991) pp. 2157-8). More specifically, pharmacological approaches to treating cocaine addiction include antidepressants such as desipramine or fluoxetine, which can treat the psychological aspects of withdrawal symptoms. In general, they are not directly involved in the physical properties of cocaine (Kleber (1995) Clinical Neuropharmacology 18: 96-109). In addition, the effectiveness of these antidepressants varies widely (Brooke et al. (1992) Drug Alcohol Depend. 31: 37-43). Clinical trials have shown that deciflavin reduced self-administration (Tella (1994) College on Problem of Drug Dependence Meeting Abstract; Mello et al. (1990) J. Pharmacol. Exp. Ther. 254: 926-939; and Kleven et al. (1990) Behavl. Pharmacol. 1: 365-373), the rate of withdrawal withdrawal following treatment did not exceed 70% (Kosten (1993) Problems of Drug Dependence, NIDA Res. Monogr. 85). In addition, there is an example of using a drug that enhances sympathetic excitability, such as bromocriptine, but its use is limited due to toxicity (Taylor et al. (1990) West J. Med. 152 : 573-577). In order to replace opioid poisoning, a new drug such as buprenorphine has been used to replace methadone, but cross-inteference of the sympathetic nervous system is limited, but practical clinical applications are limited. et al. (1991) NIDA Research Monograph, 105: 587-588). Vprenorphine has been reported to reduce cocaine self-administration (Carroll et al. (1991) Psychopharmacology 106: 439-446; Mello et al. (1989) Science 245: 859-862; and Mello et al. 1990) J. Pharmacol.Exp. Ther. 254: 926-939). However, the rate of reduction of cocaine withdrawal following treatment did not exceed 50% (Gastfried et al. (1994) College on Problems of Drug Dependence Meeting Abstracts; and Schottenfeld et al. (1993) Problems on Drug Dependence, NIDA, 10 Res) Monogr. 311).

Existing therapies used to treat cocaine addiction have four limiting factors, causing high recidivism. First, although perhaps very basic, the neurochemical trends of cocaine abuse and addiction are complex (Carroll et al. (1994) supra .). As a result, neuropharmacological approaches by single action, such as dopamine uptake inhibition, are not considered to sufficiently overcome the addiction of drugs. Second, existing drugs used in the treatment of cocaine addiction have significant side effects and limit their use. Third, patient compliance with drug therapy is a problem. Existing therapies require frequent visits to medical workers and self-administration of drugs to treat addiction in patients with drug addiction. The majority of these drugs suppress euphoria following the administration of cocaine, resulting in a strong rejection of drug treatment (Carroll, et al. (1994) supra; Kosten et al. (1993) Problems of Drug Dependence, NIDA Res. Monogr. 132: 311). Fourth, due to the complex chemical properties associated with pharmacological therapy, it is inconsistent with existing therapies in use or in clinical practice. Finally, most pharmacotherapy-based clinical trials were conducted on outpatients with low drug compliance, and therefore were not associated with highly compliant outpatient or other psychosocial treatment deemed necessary for successful treatment of cocaine dependent patients. It is not.

Heroin is an opiate drug extracted from poppy. Heroin is the most addictive opiate drug in the United States, making illegal purchases easy on the market. The quality of heroin used by addicts is high at 45-80% in purity and the physical dependence is much higher than in the past. More powerful heroin is administered through tobacco or inhalation, which is favored by users who avoid intravenous injection, a common dosage form. The death rate of heroin addicts is high. Premature death can be caused by a variety of situations, such as severe bacterial infections and human immune virus (HIV) infections caused by the use of injection devices. There is no accurate record of a number of heroin addicts in the United States, but it is estimated that 750,000-1,000,000 people will be estimated from overdose of the estimated deaths, arrested addicts and those applying for treatment.

Injecting the heroin solution quickly produces various sensations of excitement, flavor and extremes (as can be compared to organism). Heroin is rapidly hydrolyzed to 6-monoacetyl morphine (hereinafter "6-MAM") which is then converted to morphine. Heroin and 6-MAM are highly lipid soluble and cross the blood brain barrier faster than morphine. The primary euphoria that occurs when heroin is administered lasts from 45 seconds to several minutes, depending on the dose, calming occurs over 3-5 hours. When this condition is terminated, the withdrawal symptom causes heroin addicts to become nervous and aggressive, and to become "pathological."

In general, existing therapies have attempted pharmacological interventions to treat withdrawal symptoms or the syndrome. Typically, detox therapy involves long-lived opiates such as methadone. Another approach is to use antihypertensives such as clonidine, which can alleviate many of the side effects of withdrawal symptoms, but not alleviate characteristic symptoms such as craving for drugs and systemic pain. Like most addictions, there is a high degree of habituality. Long-term treatment for heroin addicts and stabilization following methadone administration are known to be the most successful treatments to date, and attention should be paid to thorough monitoring and medication status for drug addicts.

Nicotine (1-methyl-2- (3-pyridyl) pyrrolidine) is an alkaloid drug extracted from tobacco leaves. Nicotine is widely used throughout the world and is manufactured and marketed in many forms, such as cigarettes, cigars, pipe tobacco and smokeless (chewing) tobacco. The addiction to nicotine and the dangers of tobacco have long been known (Slade et al. (1995) JAMA 274 (3): 225-233), but cigarettes are still popular among smokers. It is estimated that 51 million Americans smoke, and 420,000 Americans die each year from tobacco-related diseases, according to statistics from the Center for Disease Control and Prevention.

The most popular nicotine delivery system is cigarettes. Cigarettes contain 6-10 mg of nicotine, of which smokers typically absorb 1-3 mg. A pack of smokers every day absorbs 20-40 mg of nicotine per day and reaches a concentration of 25-50 ng per milliliter of plasma. The plasma half-life of nicotine is about 2 hours, and the half-life of cotinine, the major metabolite, is 19 hours (Henningfield (1995) The New England Journal of Medicine 333 (18): 1196-1203).

Nicotine is legally widely used, so unlike cocaine and heroin, the pressure due to its use is relatively low. Many habitual smokers have expressed their intention to quit smoking, and in practice, only 2-3% of them succeed in quitting each year (Henningfield (1995) supra.). The high degree of habituality among smokers who want to quit smoking has a strong effect of nicotine dependence (O'Brien et al. (1996) Lancet 347: 237-240).

Nicotine addiction is a chronic and recurrent disease. Nicotine targets the emsolimbic reward system and ultimately results in psychological dependence. According to clinical results, nicotine binds to alpha-nicotinacetylcholine receptors in the central and peripheral nervous system to enhance the release of dopamine. Increased nicotinacetylcholine receptors in the brain increase psychological dependence on nicotine (Balfour (1994) Addiction 89: 1419-1423). Nicotine's psychological effects strongly increase psychological addiction. Nicotine users have a strong incentive to continue smoking because they increase their cognition and feel better, but have negative effects (ie withdrawal symptoms) when refraining from it.

Given the lack of effective therapies for nicotine dependence and the rare success rate for those who want to quit smoking, the development of new therapies is desired. Two of the most popular therapies at present are the nicotine Polacrerex (“nicotine gum”) and the transdermal absorption system (“nicotine patch”). Nicotine is administered to reduce the blood levels of nicotine gradually, reducing the withdrawal symptoms and making the smoker no longer dependent on cigarettes (e.g. maintaining desirable emotional state and concentration). (Henningfield (1995) supra.) However, it is pointed out that the method of administration does not have a low nicotine penetration and coping habits for non-motivated non-smokers. It has side effects such as oral irritation, pain in the jaw muscles, dyspepsia, nausea, hiccups and paresthesia. Side effects reported when applying patch preparations include skin reactions (itch and swelling), insomnia, gastrointestinal disorders, lethargy, neurosis, dizziness and sweating (Haxby (1995) Am. J. Health-Syst. Pharm. 52: 265-281).

Laboratory diagnostics and therapies for the treatment of drug addiction have been published in the literature but have not yet been carried out. For example, methods for treating drug addiction through vaccination have already been proposed in principle. Bonez et al. Immunized rhesus monkeys with morphine and observed the change by self-administering heroin (Bonese et al. (1974) Nature 252: 708-710). Basgastra et al. Experimented to suppress drug addiction by inoculating cocaine-KLH (Immunopharmacol. (1992) 23: 173-179), but no clear conclusions were reached, and the method used by Basgastra et al. Still remains controversial. (Gallacher (1994) Immunopharm. 27: 79-81). It is clear that if one conjugate is considered effective as a pharmacotherapy, it should be able to enhance antibodies that can recognize free cocaine, heroin or nicotine circulating in vivo. Kerney (WO 92/03163) mentioned an immune serum for one vaccine and drug. The vaccine consists of incomplete antigens (hapten) bound to the carrier protein producing the antibody. He also mentions the use of such antibodies in connection with the production of antibodies to the drug and the translation of the drug users. Nature 378: 727-730 (1995) reported on the synthesis of cocaine-KLH vaccines to induce anticocaine antibodies that block the motility effects of drugs in rats. A vaccine has been reported consisting of a carrier protein bound to one incomplete antigen (hapten) from the desipramine / imipramine drug group and another incomplete antigen (hapten) from the nortriptrin / amitriptrin drug group ( See, US Pat. No. 5,256,409. Liu et al. Reported the use of incomplete antigen-polymer solid support complexes to induce an immune response (US Pat. No. 5,283,066).

Manual administration of monoclonal antibodies to treat drug abuse has already been reported (Killian et al. (1978) Pharmacol. Biochem. Behavior 9: 347-352; Pentel et al. (1991) Drug Met Dispositions 19: 24-28). The method is to passively administer to the animal a preformed antibody for a particular drug. The above data suggest the feasibility of immunological approaches in the treatment of drug addiction, but there are various problems in the method of passive immunization of the human body over a long period of time. First, if the antibody used for passive therapy is a source other than the human body or a monoclonal antibody, the related agent may be recognized as a foreign protein in the patient's body and the immune response to the foreign antibody can be rapidly achieved. This immune response can neutralize passively administered antibodies and block their effectiveness as well as shorten the protection time of the antibodies. It can also be pointed out that re-administration of the same antibody could potentially lead to hypersensitivity. The problems can be solved by producing the immunoglobulin from human donors immunized with the vaccine. This approach is described in detail in the following examples. Secondly, passively administered antibodies are removed relatively quickly during blood circulation. The half-life of certain antibodies in vivo is 2.5-23 days depending on the isotope. Thus, the effectiveness of passive administration of the antibody may last for a shorter period of time than that induced by immunity.

Another immunological approach for the treatment of drug addiction is the use of catalytic antibodies that can promote the hydrolysis of cocaine molecules in the patient (Landry et al. (1993) Science 259: 1899-1901). Catalytic antibodies are produced by immunizing an experimental animal with analogs to the transient state of cocaine linked to a carrier protein. Then, a monoclonal antibody having desirable activity is selected. This approach appears to be theoretically valid but has some serious problems. In other words, since the catalytic antibody must be passively administered, all side effects of the passive antibody therapy should be taken. Active immunity to produce catalytic antibodies is not justified because for antibodies that are augmented against analogs in the transient state, catalytic activity is rare and its activity cannot be detected in polyclonal agents. In addition, the general esterase-like activity of the catalytic antibody and the ability to inhibit the active immune response in genetically diverse individuals are potentially toxic molecules, especially when the antibody is produced in the human body.

Yugawa et al. Have proposed the use of cocaine-protein conjugates containing cocaine derivatives as a method for enhancing antibodies for the detection of cocaine or derivatives thereof in blood samples (European Patent 0 613 899 A2). In addition, the Ciba patent has disclosed a conjugate for enhancing cocaine antibodies according to immunoassay, and mentions the conjugate to BSA using benzoyl ecgonine and diazonium derived from cocaine. In addition, the patent discloses conjugates using para-imino ester derivatives of cocaine and norcocaine to conjugate carriers (US Pat. Nos. 3,888,866, 4,123,431 and 4,197,236). Biocite refers to cocaine conjugates that utilize the phenyl ring paraposition of various cocaine derivatives to introduce amide bonds to enhance the stability of hydrolysis (WO 93/12111). The use of protein conjugates of endogenous substances and drugs for the treatment of related diseases is mentioned, which may be used in immunoassays, immunodialysis and immunosorbents, as well as in psychoactive incomplete antigens (hapten). Dependence (US Pat. Nos. 4,620,977, 4,813,924, 4,834,973 and 5,037,645).

Breck et al. Describe the use of cotinine 4'-carboxylic acid conjugates covalently bound to poly-L-lysine to enhance antibodies to nicotine metabolite, cotinine, for use in measuring the presence of cotinine in physiological fluids. (1987 Journal of Immunological Methods 96: 239-246). Abad et al. Also described the use of 3 ′-(hydroxymethyl) nicotine hemisuccinate in which BSA was conjugated to nicotine antibodies for enzyme-linked immunosorbent assay (ELISA), which measures nicotine in smoke extracts of cigarettes. It is mentioned. However, no literature indicates or suggests that nicotine-carrier conjugates have been used for the treatment of nicotine addiction (1993 Anal. Chem. 65 (22): 3227-3231).

As mentioned above, effective therapies have not been developed for drug addiction, particularly drug addiction such as cocaine, heroin and nicotine. Accordingly, there is an urgent need for the development of long-term therapies for drug addiction, particularly cocaine and nicotine addiction, in which the drug addict does not rely entirely on medication or self-administration.

[Summary of invention]

Accordingly, an object of the present invention to improve the problems of the prior art is to provide a method for treating drug abuse. The present invention is directed to a therapeutic composition, in particular an incomplete antigen-carrier conjugate, which is inoculated to an addict who has been continuously exposed to the drug to induce an immune response in the form of an anticomplete antibody in the human body. The pharmacological effect is not lost but the effect is reduced. The present invention also provides a medicament for treating drug abuse, in particular cocaine, heroin and nicotine poisoning by inoculating a subject with a drug / hapten-carrier conjugate, and more particularly cocaine-protein, heroin- To provide a protein or nicotine-protein conjugate. The therapeutic composition of the present invention consists of a carrier containing at least one incomplete antigen (hapten) and at least one T cell epitope, the anti-incomplete antigen during the conjugation process to form an incomplete antigen-carrier conjugate (hapten) can stimulate the production of antibodies. The incomplete antigen (hapten) can be a drug or drug derivative, in particular cocaine, heroin or nicotine. When a therapeutic composition of the present invention containing the drug / hapten-carrier conjugate is administered to a drug addict, anti-hapten antibodies specific for the drug are induced. Therapeutic immune pharmacotherapy induces and maintains significantly high titers of anti-incomplete antigenic antibodies, resulting in a sufficient amount of drug in the body following a series of exposures during the lifetime of the therapeutic agent. Can be neutralized to reduce but not lose the pharmacological effects of the drug. It is also an object of the present invention to provide a novel invention for producing the conjugate. In addition, the present invention provides a method of passive immunity, thereby treating a subject with an antibody produced by inoculating a donor with an incomplete antigen-carrier conjugate of the present invention.

The above objects and other features, characteristics, and advantages of the present invention can be more clearly understood and understood in the following drawings, detailed description of the invention, and claims.

1A shows a large number of possible, randomly marked “branches” identified to facilitate understanding of suitable compounds and conjugates for use in the present invention;

FIG. 1B shows a large number of possible, randomly marked “branched” materials identified to facilitate understanding of the appropriate compounds and conjugates used in the present invention, wherein Q ′ in FIG. Modified T-cell epitopes containing carriers such as protein carriers;

2 shows the structure of five reagents usefully used in the present invention;

3 shows four drug structures suitable for inclusion and administration in accordance with the spirit of the present invention;

4A shows a gel showing comparative molecular weights of native choleratoxin-B (CTB, monomers and pentamers) and recombinant choleratoxin-B (rCTB, monomers);

4B shows a gel showing the stability of CTB pentamer within the range of pH 3-9;

FIG. 4C is a diagram of Western Blot gel shown in the 35 peak fractions of rCTB # 32 and # 32 which become CTB pentamers by expression of cell traits; FIG.

FIG. 5A is a graph showing enzyme-linked immunosorbent assay (ELISA), showing that anti-CTB antibodies bind to ganglioside GM1 on plates of enzyme-linked immunosorbent assay by detecting rCTB function; FIG.

5B is a scan showing flow cytometry binding quantitative analysis, showing that rCTB is bound to eukaryotic cells expressing ganglioside GM1;

6A is a schematic of nicotine structural formula;

Figure 6b is a figure showing a variety of sites when producing a nicotine conjugate according to the present invention. The various sites are randomly assigned to facilitate assignment of compounds and conjugates of the invention and do not refer to specific sites. In addition, the various sites are mentioned in FIG. 7;

Figure 7 shows the "branched" at various leaving sites of nicotine molecules for nicotine conjugates and intermediates of the present invention. The nicotine conjugate of the invention indicates that Q is a T-cell epitope containing a carrier;

8 shows nicotine metabolites useful in making some conjugates of the invention;

9A-B show the results of mouse sera tested according to the enzyme-linked immunosorbent assay (ELISA) for antibodies bound to conjugates of PS-55 and hen egg lysozyme protein (HEL). In FIG. 9A, the mouse is immunized with nicotine-BSA conjugate, and in FIG. 9B, the mouse is immunized with nicotine-CTB conjugate;

FIG. 10 shows the results of testing antinicotine antisera in a competitive enzyme-linked immunosorbent assay for the specificity of antisera to free nicotine upon addition of various concentrations of free nicotine and nicotine metabolites and cortitin and nornicotine. The antiserum was prepared by injecting nicotine conjugate PS-55 BSA into mice. Since no presence of nicotine metabolites has been identified, it has been shown that antinicotine antibodies are specific to nicotine in the antiserum. Anesthetizing lidocaine was used as a negative control and there was no competition for binding of the antibody. In addition, nicotine conjugate PS-55 HEL was used as a positive control, and the binding capacity of PS-55 HEL to the antibody was shown to be inhibited by free nicotine;

11A is a schematic diagram of a heroin structural formula;

Figure 11b is a diagram showing various sites in the preparation of the heroin conjugate according to the present invention. The various sites are randomly assigned to facilitate assignment of compounds and conjugates of the invention and do not necessarily refer to sites. In addition, these various sites are mentioned in FIG. 12;

Figure 12 shows the "branched" at various leaving sites of the heroin molecule for the heroin conjugates and intermediates of the present invention. The heroin conjugate of the present invention indicates that Q is a T-cell epitope containing a carrier.

The patents and academic documents referred to in the present invention are already known to those skilled in the art. In addition, published US patents, PCT publications, and other publications cited in this patent are referred to below by reference.

The present invention is characterized by providing a drug for drug abuse by injecting drug addicts with incomplete antigen-carrier conjugates, more specifically, heroin-protein conjugates or nicotine-protein conjugates. The therapeutic composition of the present invention consists of a carrier containing at least one incomplete antigen (hapten) and at least one T-cell epitope, and anti-incomplete during the conjugation process to form an incomplete antigen-carrier conjugate It can stimulate the production of antigen antibodies. As used herein, the term "T-cell epitope" refers to a basic element or a very small unit recognized by a T-cell receptor. The epitope is composed of amino acids essential for receptor recognition. Amino acid sequences that mimic the amino acids of the T-cell epitopes and modify allergic responses to protein allergens are within the scope of the present invention. "Peptidomimetic" can be defined as the chemical structure derived from bioactive peptides that mimic natural molecules. The incomplete antigen (hapten) can be a drug or drug derivative, in particular cocaine, heroin or nicotine.

When a therapeutic composition of the present invention containing the incomplete antigen (hapten) / drug (or a derivative thereof) is administered to a drug addict, an anti-haptic antibody specific for the drug is induced. Therapeutic immune pharmacotherapy induces and maintains significantly high titers of anti-incomplete antigenic antibodies, resulting in a sufficient amount of drug in the body following a series of exposures during the lifetime of the therapeutic agent. Can be neutralized to reduce but not lose the pharmacological effects of the drug. For example, if the therapeutic composition is a heroin-carrier conjugate, an anti-heroin antibody response upon application thereof may be induced to reduce or neutralize the amount of heroin in the bloodstream or mucosal tissues of the subject, thereby reducing psychological addiction to the drug. Will be blocked.

According to the present invention, since the amount of drug abused is delayed or reduced in reaching the central nervous system, the drug addict may experience a decrease in satisfaction or no feeling of using heroin. When administering nicotine-carrier conjugates, anti-nicotine antibodies are induced according to the above mechanism of action, thereby reducing satisfaction or no feeling at all in using nicotine. It can be said that there are no side effects when administering the medicament of the present invention. For example, instant drug abuse is small, monovalent and incapable of crosslinking with antibodies. Thus, the formation of immunocomplexes and their associated pathology do not occur even upon exposure to abused drugs. It is anticipated that the agents of the present invention may be compatible with current and future pharmacological therapies. In addition, an effective neutralization reaction lasts a long time. For example, neutralizing antibody responses to lesions are known to last for years. Therefore, the high leisure anti-hapten antibody, which is derived from the use of the therapeutic composition of the present invention, can have a long duration of action and remain effective for one year. The therapeutic composition of the present invention, along with the reduction of the medication, the action time lasts for a long time, thereby reducing the habituality that has been pointed out as a problem in the conventional therapy.

In addition, the method of inoculating a therapeutic composition of the present invention in connection with heroin poisoning can be combined with other drugs currently in use or in clinical trials. Indeed, combination therapy at an early clinical stage is desirable, which is necessary to obtain optimal antibody titers.

Similarly, the method of inoculating a therapeutic composition of the present invention in connection with heroin poisoning can be combined with other drugs that minimize the withdrawal symptoms of nicotine. For example, the nicotine-carrier conjugates of the present invention may be used in combination with chloridine, buspyrone and antidepressants, or sedatives. In the case of a vaccine prepared according to the above method, it may be used in combination with existing replacement drugs, ie, gum and patch. Since anti-nicotine antibodies take several weeks to produce, there must be some level of control over drug cravings when using nicotine replacement drugs.

Terms defined in the present invention are intended to help understanding. "Incomplete antigen" as used in the present invention is a low molecular weight organic compound that specifically reacts with antibodies and cannot induce an immune response by itself, but forms a T-cell epitope that forms an incomplete antigen-carrier conjugate. Immunity is produced upon attachment to the carrier containing. In addition, the incomplete antigen is characterized by determining the specificity of the incomplete antigen-carrier conjugate, that is, it can react with the antibody specific to the incomplete antigen in the free state. In the case of non-immunized drug subjects, no antibody against the incomplete antigen is produced. The therapeutic composition of the present invention is inoculated to drug addicts who are addicted to the drug and wish to treat it. According to the present invention, the term incomplete antigen includes a drug, an analog or a derivative of a drug having a partial component of the drug in the concept of a more specific drug / incomplete antigen. Therapeutic compositions or therapeutic antihaptic vaccines will produce "desirable and measurable results" upon first administration. A desirable and measurable result at the first dose is the production of high titer antihapty antibodies. Titer is defined as the serum dilution required for half maximum antibody detection by ELISA. However, choosing an appropriate dose for a drug addict can result in a consistently desirable therapeutic effect. The "desirable therapeutic effect" is that a sufficient fraction of the abused free drug is neutralized, which is dependent on the production of anti-haptic antibodies specific for the drug upon continued exposure of the drug of the present invention to the drug. It can reduce or eliminate the pharmacological effect of the drug within an acceptable time. The time required to elicit sufficient antibody response to a particular drug, and the therapeutically acceptable time for how long the antibody response can be maintained, assesses the characteristics of the immunogenicity to the subject, the drug or dosage form being neutralized By one of ordinary skill in the art. One of ordinary skill in the art can use the above information and other vaccination-related clinical protocols to know that the duration of immunity or antibody response following antibody production can last for months to one year.

In addition, "passive immunity" refers to intact anti-hapten antibody, polyclonal antibody or monoclonal antibody fragments (Fab, Fv, (Fab ') 2 or prepared using the novel conjugates of the invention or Fab ') is defined as administering or exposing. As mentioned above, passive immunization of the anti-heroin or anti-nicotine antibody of the present invention to a human body as it is may be less useful than active immunity. In particular, passive immunization may be the first combination or adjuvant drug therapy (e.g., the initial period of administration of the vaccine or a period prior to the production of antibodies in-house), or an urgent situation to prevent death (e.g., drugs In case of overdose). Passive immunotherapy is desirable only in certain situations, i.e., when a patient is in need of immunocompromise or prompt treatment.

In addition, the drug-complex of the present invention, including the therapeutic composition of the present invention, can be used as a prophylactic agent. That is, the drug-complex or composition thereof may be administered to a mammal to produce anti-haptic antibodies prior to exposure to the human body. The resulting anti-haptic antibodies may be present in mammals and combined with drugs introduced upon administration of the conjugates or compositions thereof to minimize or prevent the possibility of drug addiction.

Therapeutic compositions of the present invention, and more particularly for therapeutic antihapta vaccines, at least one drug / incomplete that can induce the production of sufficiently high titers of antibodies specific for drug / hapten antigens. A composition containing an antigen-carrier conjugate, wherein upon exposure to a drug / hapten, the antibody can reduce the addiction of the drug. The expected immune response to the incomplete antigen-carrier conjugate is the formation of anti-haptic and anti-carrier antibodies. When a sufficient amount of anti-haptic specific antibody is introduced, it is a therapeutically feasible step to initiate a neutralizing attack on the introduced drug after inoculation. Therapeutic doses of the invention confer sufficient production time for the initial inoculation and after activation. In addition, optimal antihap vaccines contain at least one drug / hapten carrier that suitably combines the drug as a hapten and a carrier. It can bring about optimal treatment effect. That is, the antibody may remain at a sufficiently high titer in vivo and may neutralize it even when the human body is exposed to a certain drug for several months. More specifically, since the titer of the antibody is high enough, it may bring about an effective response upon exposure to the drug for about 2 months to 1 year or more, and usually at least 3 months, depending on the individual. The optimal composition may contain incomplete antigen-carrier conjugates, excipients and optionally adjuvants.

The present invention defines an incomplete antigen (hapten) -carrier conjugate in the treatment of heroin poisoning, wherein the incomplete antigen (hapten) is a heroin or a heroin derivative that can immunize mammals, especially humans, and is a free drug Induces antiheroin antibodies that can bind to and inhibits intoxicant drug behavior by preventing the drug from entering the reward system in the brain. As discussed for cocaine and nicotine, antiheroin antibodies appear to reduce the pharmacological effects of the drug by limiting the distribution of heroin across the blood-brain barrier.

The present invention defines an incomplete antigen (hapten) -carrier conjugate when treating nicotine poisoning, wherein the incomplete antigen (hapten) is capable of immunizing mammals, especially humans, as nicotine or nicotine derivatives, and is a free drug. Induces antinicotine antibodies that can bind to and inhibits addiction drug activity (eg, smoking) by preventing the drug from entering the reward system in the brain. Nicotine is known to increase dopamine release by binding to the alpha-subunit of the nicotine acetylcholine receptor in the brain. In addition, the increased nicotine acetylcholine receptor in the brain seems to promote the psychological dependence of nicotine. As mentioned above, antinicotine antibodies appear to reduce the pharmacological effects of the drug by limiting the distribution of nicotine across the blood-brain barrier.

For example, there are some standardization steps for nicotine transport. That is, each cigarette contains 9 mg of nicotine on average, of which 1-3 mg of nicotine is effectively dispersed during smoking. In addition, the highest blood concentration of nicotine is 25-50 ng / ml, which is significantly lower than cocaine (0.3-1 ug / ml). Thus, an appropriately high affinity antibody provides an opportunity for ideal involvement.

The first inoculation of a therapeutically hapten-carrier conjugate composition of the invention produces high titers of hapten-specific antibodies in vivo. Examining the plasma of subjects who have been periodically inoculated is effective to determine the effective dose for each individual. The titer value is kept rising through periodic activation. The composition of the present invention can be administered in parallel with rehabilitation training of the existing drug, including counseling. In addition, the therapeutic composition of the present invention may be administered in a single dose or simultaneously or continuously with several drugs and may be co-administered with other drugs. For example, the therapeutic incomplete antigen-carrier conjugate composition of the present invention and its preparation method can be used in combination with existing pharmacological approaches and the "short-term" passive immunity method discussed above to enhance the overall therapeutic effect. do.

Therapeutic incomplete antigen-carrier conjugate compositions of the invention are prepared by coupling one or more incomplete antigen molecules to a T-cell epitope containing a carrier, wherein the incomplete antigen (hapten) -prepared above is prepared. Carrier conjugates can stimulate T cells (immunogenesis) and thus stimulate the increase of T cells and the characteristic release of regulators, thereby activating the relevant B cells to stimulate specific antibody production. The antibody of interest is an antibody specific for the incomplete antigen (hapten) portion of the incomplete antigen (carrier) -carrier complex (also called the incomplete antigen-carrier complex). A composition containing a conjugate complex for the same drug (crossimmune) or multidrug (mixed immunity) is disclosed. Mixtures of the conjugates in the multi-drug are particularly effective in the treatment of addiction by the multi-drug.

In selecting a drug suitable for the conjugate according to the present invention, those skilled in the art can select a drug capable of inducing high antibody titers. However, if the selected molecule is similar to a molecule that is endogenous to the drug addict, an increase in the antibody to the molecule may cause cross-reaction with many molecules in the body, which may have a detrimental effect. Therefore, drugs selected as incomplete antigens (drugs / haptens) are sufficiently heterogeneous and of sufficient size to prevent the induction of antibodies to molecules commonly found in the human body. Antibodies elevated for therapeutic compositions have high specificity and are present in sufficient amounts to neutralize drugs in the bloodstream, mucosa or both. Drugs suitable for the therapeutic composition without departing from the scope of the present invention are as follows.

Hallucinogenic agents (eg, mescarin and LSD);

Marijuana (eg, THC);

Stimulants (eg, amphetamines, cocaine, penmetrazine, methylphenidate);

nicotine;

Depressants (eg, bibarbitrates; bromides, chloral hydrates, etc.), metacelons, barbitrates, diazepam, flulazepam, phencycline and fluorocetin;

Opiates and derivatives thereof (eg, heroin, methadone, morphine, meperidine, codeine, pentazosin and propoxyphene; and

"Designated drugs" such as "psychedelic".

3 shows the structure for four drugs suitable for inclusion in accordance with the present invention.

The carrier of the present invention is a molecule containing at least one T-cell epitope capable of stimulating a T cell of a subject, thereby stimulating B cells to sustain antibodies against the entire conjugate part, including the incomplete antigen part. Keeps the creation Therefore, the selection of a carrier that produces immunity is expected to have a strong immune response to the vaccine in a patient population. As with incomplete antigens, the carrier must be sufficiently heterogeneous to induce a strong immune response to the vaccine. A conservative or non-essential approach to drugs is to avoid carrier-induced inhibition of epitopes using carriers that have not been exposed to most patients. However, carrier-induced epitope inhibition occurs, but the inhibition is controlled by CTB (Stok et al. (1994) Vaccine 12: 521-526). -1418) and other amendments to the protocol (Etlinger et al. (1990) Science 249: 423-425). Vaccines using carrier proteins in patients already in an immune state are currently commercially available. In addition, carriers containing large amounts of lysine are particularly suitable for conjugation according to the process of the invention. There are a significant number of carriers available, for example:

Bacterial toxins or toxins such as cholera toxin B- (CTB), diphtheria toxin, tetanus toxin, pertussis toxin and fibrous hemagglutinin, Shiga toxin, Pseudomonas exotoxin;

Lectins such as lysine B subunit, abrin and sweet kidney bean lectin;

Subviruses such as retroviral nuclear protein (retro NP), rabies RNP, plant virus (eg, TMV, photosensitive and cauliflower mosaic virus), vesicular stomatitis virus-nucleocapsid (VSA) -N), poxvirus vectors and Semiki forest virus vectors; Prodrugs such as polyantibody peptides (MAPs), centrosomes;

Yeast virus positive particles (VLPs);

Malaria protein antigen;

And protein and peptide modifiers, derivatives, or analogs thereof.

In order to evaluate the characteristics of suitable carriers, the first experiment was performed using bovine serum albumin as a protein carrier. Bovine serum albumin was considered to be an ideal carrier because of its low cost and high concentration of lysine in the inclusion. However, the bovine serum albumin has been shown to be inadequate upon inoculation in humans because the production of anti-BSA antibodies has the potential to cause side effects. Therefore, the above-mentioned criteria were applied to a large number of candidate carriers according to the experimental results. Accordingly, examples of the above-mentioned carriers are suitable for the practice of the present invention.

Carriers for preferred embodiments include proteins or peptides (eg, polyantibody peptides (MAP)) or short chain peptides. An ideal carrier is a protein or peptide that is not commonly used for vaccination in countries where drug addiction is being treated, thereby avoiding the possibility of "carrier-induced epitope inhibition." For example, in the United States, standard pediatric immunity includes diphtheria and tetanus, but tetanus toxin and diphtheria toxin are undesirable as appropriate toxins unless chemical modifications have been previously made. In addition, the carrier protein should not have a tolerant to the human body. The human body excludes human serum albumin, which is not chemically modified. In addition, most food proteins must be carefully examined before being used as a carrier. In the human body, bovine serum albumin is not a suitable carrier because most humans consume beef from their daily diet. It is also very beneficial when the carrier has inherited immunity or support. Careful balance must be made between the requirements for the immunization of the carrier and the requirements for maximizing anti-hapten antibodies. In addition, preferred carriers should induce a systemic response and a response at the site of exposure, which is particularly required in the case of heroin, cocaine and nicotine, which are commonly administered through the mucosa of drug addicts. Response rate is especially important in patients with cocaine or heroin aspiration. Thus, in the case of heroin, cocaine and nicotine, preferred carriers not only induce systemic responses but also induce previously existing mucosal antibody responses. The mucosal reaction occurs rapidly and rapidly enough that the antibody reaction with heroin, cocaine and nicotine causes sufficient adverse effects on the drug before the drug is circulated in the blood.

An example of one preferred carrier is Colesotoxin B (CTB), which is a highly immunogenic protein capable of strongly stimulating systemic and mucosal antibody responses (Lycke (1992) J. Immunol. 150 : 4810-4821; Holmgren et al. (1994) Am. J. Trop. Med. Hyg. 50 "42-54; Shilbart et al. (1988) J. Immun. Meth. 109: 103-112; Katz et al (1993) Infection Immun. 61: 1964-1971) This combined IgA and IgG anti-incomplete antigen response (hapten) blocks nicotine absorbed through the mouth or lungs by blocking heroin or cocaine administered by nasal or inhalation routes. In addition, CTB has immigration safety in clinical trials for cholera vaccines (Holmgren et al., Supra; Jerborn et al. (1994) Vaccine 12: 1078-1082; "The Jordan Report , Accelerated Development of Vaccines' 1993., NIAID, 1993).

Other useful carriers include those that enhance mucosal reactions. Specific examples include LTB family of bacterial toxins, retroviral nucleoprotein (Retro NP), rabies riboprotein (rabies RNP), vesicular stomatitis virus-nucleo Capsid proteins (VSV-N), recombinant smallpox virus subunits, and the like.

In another embodiment, various protein derivatives, peptide fragments or analogs of allergens are used as carriers. The reason for selecting these carriers is that they induce a response of T cells that can induce B cells of anti-hapten antibodies. Examples of methods for making allergen proteins and peptides and sequences thereof are disclosed in patent (WO) 95/27786, published October 19, 1995. Particularly suitable allergens as carriers are Cryptomeria japonica, more specifically recombinant Cry j 1, the sequence of which is disclosed with nocturnal modifications. Cryptomeria japonica does not exist outside of Japan. Thus, Cry j 1 allergens generally meet the criteria of a suitable carrier, which is a carrier to which the subject has not been exposed to humans.

The preparation method and composition according to the present invention, more specifically, the use technique is described in the following Examples and those skilled in the art in connection with the selected carrier / selected drug / incomplete antigen (hapten) incomplete antigen of the present invention (hapten) -carrier conjugates can be prepared.

In one embodiment of the invention, the antibody induced by the therapeutic composition acts at a time when the drug is transferred from the lungs through the heart to the brain over a period of time. The ability to induce such an antibody reaction is possible by careful selection of carrier molecules.

Generate recombinant B subunit of cholera toxin

Cholera toxin is an enterotoxin produced by Vibrio cholera and consists of five identified B subunits. Each subunit has a molecular weight of 11.6 KDa (103 amino acids) and one A subunit with a molecular weight of 27.2 KDa (230 amino acids) (Finkelstein (1988) (immunochem. Mol. Gen. Anal. Bac. Path. 85). CTB, a binding subunit, binds ganglioside GM 1 at the surface of cells (Sixma et al. (1991) Nature 351: 371-375; Orlandi et al. (1993) J. Biol. Chem. 268: CTA enters cells as an enzymatic subunit, catalyzes ADP-ribosylation of G protein and essentially activates adenylate cyclase (Finkelstein (1988) Immunochem. Mol. Gen. Anal.Bac.Path.pp. 85-102) The cholera toxin is not toxic if the A subunit is not present.

Other publications include methods for the preparation of highly recombinant expression in CTB pentamers (L'hoir et al. (1990) Gene 89: 47-52; Slos et al. (1994) Protin Exp. Purif. 5: 518-526). Natural CTB can be commercialized, but it is difficult to rule out contamination with CTA. Thus, recombinant CTB is expressed in E. coli and thus manifests its characterization. A choleragenoid construct was purchased from the American Type Culture Collection (according to US Pat. No. 4,666,837). Recombinant CTB was cloned in the original vector (pRIT10810) and expressed in the plasmid of the N-terminal sequence containing His5 label (pET11d, Novagen), which was expressed in Escherichia coli and the concentration was 25 mg / liter of the culture. The protein was purified on a Ni2 + column using standard techniques and it was SDS-PAGE (see Figures 4a, 4b and 4c). The recombinant CTB was larger than the native CTB monomer due to the extended N-terminus as a monomer at the time of quantification.

The pentameric recombinant CTB was prepared in two forms with or without histidine labeling using cDNA modified by PCR to include the Pel b leader sequence. The histidine label was removed by inserting a C-terminal stop code. Both materials were expressed in E. coli from the pET22b vector (Novagen). Histidine labeled proteins were purified on Ni2 + affinity chromatography as described above (13 mg / L). Unlabeled recombinant CTB was purified on ganglioside GM1 column affinity chromatography as mentioned above (Tayot et al. (1981) Eur. J. Biochem. 113: 249-258).

A pentameric recombinant CTB may be preferred upon binding to ganglioside GM1. The pentamer is stable to SDS unless the samples are boiled and the pentamer polymerization can be evaluated on SDS-PAGE. 4A shows that native CTB is a pentamer and can be quickly distinguished from denatured monomer CTB. The structure of the pentamer is maintained at pH 4-9 (see Figure 4b) and under these conditions promotes various conjugation chemistries. Initially expressed recombinant CTB is a monomer. One way to obtain pentameric CTBs is to control the appropriate overlap of pentameric CTBs upon expression. It has been found that expression of the cytoplasm produces higher levels of monomeric CTB. One skilled in the art recognizes how to overlap a CTB of a monomer with a CT of a pentameric (for example, L'hoir et al. (1190) Gene 89: 47-52). An alternative to overlapping CTBs of monomers to obtain pentameric CTBs is the expression of cell morphology, which produced a pentameric recombinant CTB capable of binding GM1-gangliosides by enzyme-linked immunosorbent assay. 5a and 5b point to data supporting the above contents. Those skilled in the art can express cell traits according to the leader (Slos et al., Supra: Sandex et al. (1989) Proc. Nat'l. Acad. Sci) as a method for obtaining a pentameric recombinant CTB. 86-481-485; Lebens et al. (1993) BioTechnol. 11: 1574-1578) or post-translational overlap (L'hoir et al., Supra; Jobling et al. (1991) Mol. Microbiol. 5: 1755-1767).

Another useful carrier is cholera toxin, which provides an improved mucosal response to CTB. Enzymatically active A subunit adjuvants have been reported to enhance activity (Liang et al. (1988) J. Immunol. 141: 1495-1501; Wilson et al. (1993) Vaccine 11: 113-118; Snider et al. (1994) J. Immunol. 153: 647).

One embodiment in which the conjugates of the present invention can be prepared are chemically modified incomplete antigens (hapten) so that they can be conjugated or bound to the carrier, and also maintain sufficient structure to maintain them in the free state of the incomplete antigens (hapten) (For example, free heroin or free nicotine). Each individual to be vaccinated should have an antibody that recognizes the free hapten (heroin or nicotine). Experimental examples by radioimmunoassay and competitive enzyme-linked immunosorbent assay were described in detail in the following examples and the antibody titers against free incomplete antigens (hapten) can be measured by these experiments. The antibody of interest is an incomplete antigen-specific antibody and in some embodiments is a heroin- or nicotine-specific antibody. Recognize that various principles or methods used to describe preferred embodiments may range from the scope of the present invention to a wide range of incomplete antigen-carrier conjugates useful for the treatment of various drug addiction and toxic reactions. shall.

Conjugate

The preparation of the novel nicotine-carrier conjugates according to the present invention is derived from nicotine and nicotine metabolites. FIG. 8 shows nicotine and its metabolites. The preparation of the novel heroin conjugate according to the invention is derived from heroin and its metabolites. 11a and 11b show heroin and its various sites for the production of heroin conjugates.

Looking at the length and nature of the incomplete antigen (hapten) -carrier binding, in the case of the hapte has been deviated by a sufficient distance from the carrier region, the initially increased antibody to the incomplete antigen (hapten) is incomplete optimal (hapten) ) To be recognized. The length of the bond is optimized by varying the number of —CH 2 — groups, which are strategically placed within the “branch phase” selected from the group consisting of and are shown in FIGS. 1a and 1b of the present invention.

CJO Q

CJ1 (CH ₂ ) nQ

CJ1.1 CO ₂ Q

CJ1.2 COQ

CJ2 OCO (CH ₂ ) nQ

CJ2.1 OCOCH = Q

CJ2.2 OCOCH (O) CH ₂

CJ2.3 OCO (CH ₂ ) nCH (O) CH ₂

CJ3 CO (CH ₂ ) nCOQ

CJ3.1 CO (CH ₂ ) n CNQ

CJ4 OCO (CH ₂ ) nCOQ

CJ4.1 OCO (CH ₂ ) n CNQ

CJ5 CH ₂ OCO (CH ₂ ) nCOQ

CJ5.1 CH ₂ OCO (CH ₂ ) n CNQ

CJ6 CONH (CH ₂ ) nQ

CJ7 Y (CH ₂ ) nQ

CJ7.1 CH ₂ Y (CH ₂ ) nQ

CJ8 OCOCH (OH) CH ₂ O

CJ8.1 OCO (CH ₂ ) nCH (OH) CH ₂ O

CJ9 OCOC ₆ H ₅

CJ10 shown in Figure 2b

CJ11 YCO (CH ₂ ) nCOQ

On the branch, n is preferably selected from the range of about 1-20 as an integer, more preferably in the range of about 2-8, with a very preferred range of 3-5;

Y is preferably selected from the group consisting of S, O and NH;

It is preferable to select Q from the group comprised below.

(1) -H

(2) -OH

(3) -CH ₂

(4) -CH ₃

(4a) -OCH ₃

(5) -COOH

(6) halogen

(7) protein or peptide carriers

(8) chemically modified protein or peptide carriers

(9) activated esters such as 2-nitro-5-sulfophenyl ester and N-oxyrotynimimidyl ester

(10) mixed anhydrides, acyl halides, acyl azides, N-maleimides, imino esters, isocyanates, isothionates; or

(11) another "branch" identified by a "CJ" reference number

T-cell epitopes containing carriers (eg protein or peptide carriers) are chemically modified by methods conventional to those skilled in the art to promote inclusion to incomplete antigens (eg Thiolation). For example, 2-iminothiolane (Traut's reagent) or succinate reaction. If Q = H for (CH ₂ ) Qi, this can be simply defined as (CH ₃ ), methyl or Me, but it is shown that the subject is identified as "branched" as shown in Figures 1a and 1b. Can be.

In addition, the abbreviated chemicals that are commercially available and purchased in the present invention are as follows.

BSA = bovine serum albumin

DCC = dicyclohexylcarbodiimide

15DMF = N, N-dimethylformamide

EDC (or EDAC) = N-ethyl-N '-(3- (dimethylamino) propyl) carbodiimide hydrochloride

EDTA = ethylenediamine tetraacetic acid, disodium salt HATU = 0- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate

NMM = N-methylmorpholine

HBTh = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate

TNTU = 2- (5-norbornene-2,3-dicarboximido) -1,1,3,3-tetramethyluronium hexafluorophosphate

PyBroP (R) = bromo-tris-pyrrolidino-phosphonium hexafluorophosphate

HOBt = N-hydroxybenzotriazole

In addition, the types of compounds according to the nomenclature of IUPAC are as follows.

Nicotine: 1-methyl-2- (3-pyridyl) pyrrolidine

Cotinine: N-methyl-2- (3-pyridyl) -5-pyrrolidine

reaction

As one specific example, the precursor of conjugate PS54 was synthesized by acylating racemic nornicotine with succinic anhydride dissolved in methylene chloride in the presence of 2 equivalents of diisopropylethylamine. The reaction product was then coupled to the lysine residue of the carrier protein by HATU to give conjugate PS54 (see Example 1, Method B).

In another embodiment, the precursors of conjugates PS-55, PS56, PS-57 and PS-58 are each converted to pyridine nitrogen of (S)-(-)-nicotine dissolved in anhydrous methanol in ethyl 3-bromobutylate. It was synthesized by selective alkylation with, 5-bromovaleric acid, 6-bromohexanoic acid or 8-bromooctanoic acid (see Example 2, Method A, B, C and D). The reaction product was then conjugated to the carrier protein by HATU to obtain conjugates PS-55, PS56, PS-57 and PS-58 (see Example 3, Method A).

Conjugate Glycoprotein  Incomplete antigen / monomer        Conjugation Method PS-54     BSA     19.5 Example 1, Method B PS-54     HEL     3.2 Example 1, Method B PS-55     BSA     33.2 Example 3, method A PS-55     HEL     1.09 Example 3, method A PS-56     BSA     27 Example 3, method A PS-56     HEL     2.2 Example 3, method A PS-57     BSA     81 Example 3, method A PS-57     HEL      8 Example 3, method A PS-58 BSA     66.8 Example 3, method A PS-58 HEL     7.4 Example 3, method A

1 is a non-limiting list of conjugates. Example 3, Method A Other conjugates are prepared with one or more incomplete antigens coupled to a carrier containing a T-cell epitope. It is preferred that 1-100 incomplete antigens are coupled to a carrier containing a T-cell epitope. It is also preferred that 1 to 70 incomplete antigens are coupled to a carrier containing T-cell epitopes.

Methods of synthesizing PS-55, PS56, PS-57 and PS-58 compounds are described in the Examples below. Those skilled in the art can synthesize the compounds according to known methods such as the use of activators.

There are a variety of compounds that have been developed to facilitate cross-linking of proteins / peptides or to facilitate the conjugation of proteins to molecules derived (eg, incomplete antigens, etc.). Examples of the specific compounds known to those skilled in the art include active esters derived from carboxylic acids (activated compounds), mixed anhydrides, acyl halides, acyl azides, alkyl halides, N-maleimide Drew, imino ester, isocyanate, isocyanate, etc. are mentioned. These compounds can form covalent bonds with reactors of protein molecules. Depending on the active group, the reactor is either an amino group of lysine residues in a protein molecule or a thiol group in a carrier protein, or a amide, amine, thioester, amidineurea or thiourea upon reaction as a chemically modified carrier protein molecule. Those skilled in the art can specifically identify appropriate activators in general reference books such as, for example, Chemistry of Protein Conjugation and Cross-linking (Wong (1991) CRC Press, Inc., Boca Raton, FL). For conjugation, it is ideal to carry out on amino groups with lysine side chains. Most reagents react preferentially with lysine. A particularly suitable carrier is CTB, which in its natural form has nine lysine residues per monomer. In order to determine whether the conjugated pentameric CTB maintains its structure and activity, it can be evaluated by GM1 ganglioside binding.

The inventors have expressed and purified recombinant CTB prepared in a large scale fermentation medium when appropriate. The process of expressing and purifying the recombinant protein has been reported in existing methods (USSN 07 / 807,529). For example, CTB can be purified on affinity chromatography (Tayot et al. (1981) Eur. J. Biochem. 113: 249-258) and conjugated to heroin or nicotine derivatives. The conjugate can then be purified once more. The purified CTB and the resulting conjugates were analyzed to confirm that the purity and the pentameric structure of CTB were maintained. Examples of the assay include SDS-PAGE, native PAGE, gel filtration chromatography, Western blotting, direct and GM1-capture enzyme-linked immunosorbent assays, and biotinylated CTB and competitive enzyme-linked immunosorbent assays. have. Levels following incomplete antigenization can be determined by analyzing the increase in ultraviolet absorption resulting from mass spectrometry, reverse phase HPLC and the presence of incomplete antigens. The solubility and stability of the conjugates are optimized by preparation according to large scale preparation. Details of the assay are described in the Examples below.

The pentameric structure of the CTB according to the present invention is a preferred carrier and GM1 binding is an effective quantitative method for determining the presence of the pentameric form of CTB, but the present invention is not limited to the use according to the pentameric form of CTB. The content within the scope of the present invention includes other forms of CTB (e.g., monomers, dimers, etc.) that can be derived from use in the present invention, as well as other T-cell epitope carriers. If carriers other than the pentameric form of CTB are used, one of ordinary skill in the art can determine the presence and activity of the required carrier using appropriate assays (e.g., if a pentameric form of CTB is present Use GM1 binding to determine that).

Replace the approach with another in order to vary the value of the incomplete antigenization. In one embodiment the carrier is incomplete antigenized with multivalent heroin or nicotine material. This concept is derived from the polyantibody peptide (MAP) (Lu et al. Mol. Immunol., 28: 623-630 (1991)). Under this system, multiple branched lysine residues can be used to maximize the density and valency of the incomplete antigen. According to the concept of this approach, the immune response is enhanced when there are a large number of incomplete antigens (hapten) that bind to the same peptide or protein molecule. Therefore, multivalent incomplete antigens (hapten) which should be bound only at one or two sites on the carrier pentomer CTB were prepared as described in the present invention. The nucleus of the polyantibody incomplete antigen (hapten) is a branched polylysine nucleus proposed by Tam (Lu et al., Supra). Chemically reactive handles are conserved by inclusion of protected cysteine residues. After all possible amino group heroin or nicotine incomplete antigenization (hapten) has been made, the cysteine sulfhydryl group is unmasked to allow the protein to be coupled to various difunctional sulfhydryl groups / amino specific chemical linkages. (Yoshitake et al. (1979) Eur. J. biochem. 101: 395-399). Numerous dendrimeric structures have been used as nuclei.

Reinforcement

Adjuvants that do not hide the effect of the carrier are considered useful in vaccines for the treatment of heroin and nicotine addiction of the present invention (see Edelman (1980) Rev. Infect. Dis. 2: 370-373). Initial experiments conducted by the inventors focused on validating the therapeutic vaccine against cocaine poisoning using the potent adjuvant CFA. However, CFA is undesirable for the human body. Useful adjuvant currently approved for human use is aluminum hydroxide (Spectrum Chem. Mtg. Corp., New Brunswick, NJ). Or aluminum with aluminum phosphate (Spectrum). Typically the vaccine is adsorbed on aluminum and therefore exhibits very limited solubility. Preliminary data from rats suggest that aluminum can induce potent anticocaine antibody responses and that MF59 (Chiron, Emeryville, CA) or RIBI adjuvant is also suitable.

If CTB is used as a carrier protein, a potent adjuvant is not necessary when effective immunity is shown. As shown in the examples described below, high titer antinicotine antibody responses are induced by using alumininium as adjuvants or by using CTB-nicotine conjugates without the addition of adjuvants. Even in the case of carriers other than CTB, those skilled in the art can determine the appropriate adjuvant if necessary.

Excipients and Supplements

Therapeutic compositions optionally comprise one or more pharmaceutically acceptable auxiliaries such as sterile water and saline solutions (e.g. physiological saline, sodium phosphate, sodium chloride, alcohols, gum gum, vegetable oil, benzyl alcohol, polyethyleneethylene glycol, Gelatin, mannitol, carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters, hydroxymethyl cellulose and buffers) and the like. Other suitable excipients can be used by those skilled in the art. Therapeutic compositions optionally contain at least one adjuvant, such as dispersion media, coatings such as lipids and liposomes, surfactants such as wetting agents and emulsifiers, preservatives such as antibiotics and antifungal agents, stabilizers and those skilled in the art And other known agents. In addition, the therapeutic compositions of the present invention may add another adjuvant, adjuvant and pharmaceutically acceptable inert excipients to enhance the therapeutic properties of the drug and to develop other routes of administration.

The highly purified incomplete antigen-carrier conjugate prepared above can be formulated into a therapeutic composition of the present invention suitable for human administration. When the therapeutic composition of the present invention is administered in the form of an injection (ie, subcutaneous injection), the highly purified incomplete antigen-carrier conjugate is an aqueous solution at a pharmaceutically acceptable pH (ie, pH 4-9). It is preferable to dissolve in the composition, so that the composition becomes liquid and easy to administer. However, the highly purified hapten-carrier conjugate can be prepared as a suspension in aqueous solution and administered therein, and the suspension is included within the scope of the present invention. In addition, the therapeutic compositions of the invention optionally include pharmaceutically acceptable excipients, adjuvant and adjuvant or adjuvant active compound. Ingredients optionally added depending on the route of administration may result in the desired physical properties of the therapeutic composition, eg, proper fluidity, undesirable microbial activity, improved bioavailability or absorption over time.

Therapeutic compositions of the present invention should always be sterile and stable at the time of manufacture, storage, distribution and use to prevent contamination of microorganisms such as bacteria and fungi. Therapeutic compositions of the present invention may be prepared in the form of lyophilized powders by formulating with conjugates and pharmaceutically acceptable excipients in a preferred dosage form at the time of manufacture to maintain their integrity, and then at the time of use Add excipients or auxiliaries, for example sterile water again. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred means of the preparation is vacuum drying, lyophilization or spin drying, which can provide a powder containing the active ingredient from the previous sterile filtrate solution and the other preferred auxiliary components. There is this.

The active compounds of the present invention are processed according to conventional galenical pharmacy to produce therapeutic compositions for administration to patients, the subjects of which are mammals, including humans. Preferred routes of administration for the therapeutic compositions of the invention are intranasal, intratracheal, oral, transdermal and parenteral administration. In particular, a suitable co-administration is to administer the injection initially with intranasal administration.

Particularly suitable forms for parenteral administration include suspensions, emulsions or implants and suppositories in injections, sterile solutions, in particular oily or liquid solutions. Ampoules are a convenient unit dose. The most suitable dosage form for application to the intestine may include enteric agents as tablets, dragees, solutions, suspensions, drops, suppositories or capsules. Syrups, elixirs and the like can be applied when sweetening agents are used.

Sustained release compositions can be formulated in liposome-like form and protect the active compound (conjugate) by using different dosage forms of the coating, such as microcapsules, multi-coatings, etc. In addition, the therapeutic composition of the present invention can be applied after lyophilization to inject it.

For topical application it is used in a non-spraying form, viscous to semisolid or solid form, containing a carrier suitable for this purpose and having a higher viscosity than water. Examples of suitable dosage forms include solutions, suspensions, emulsions, creams, ointments and the like, if desired sterilized or used in admixture with adjuvants. Suitable forms of topical application are sprayed aerosols which are packaged in a mixture equipped with a suitable excipient or adjuvant in a compression vessel or a mixture equipped with a pressure volatile, usually gaseous propeller.

The molecular weight of the antibody cultured by the method and composition of the present invention is 150KDa to 1000KDa. When the antibody is exposed to free heroin or nicotine after inoculation with the appropriate conjugate to the therapeutic component, such heroin and nicotine are targeted for heroin specific or nicotine specific antibodies. There is a need for antibodies that recognize drugs in vivo without altering the form and structure of the drug. In the present invention, without any limitation, once the inoculated individual drug is exposed to heroin or nicotine, such anti-drug antibodies will block the action of heroin and nicotine. There are at least three mechanisms of action for this blocking action. The first is that antibodies cannot cross the Blood-Brain Barrier. Therefore, when heroin or nicotine is bound to an anti-heroin or anti-nicotine antibody, it cannot pass through the BBB and thus cannot act as an opioid receptor and as a dopamine transporter, respectively. The second is not limited to any particular hypothesis, and these antibodies simply block the binding of the drug to the receptor by steric blockade. This mechanism of action is expected to block the astral nervous system action (eg, cardiotoxicity) of the drug or to exert antibody action against other drugs that do not act on the central nervous system. Third, both heroin and nicotine have relatively short half-lives in vivo because they inactivate metabolites by enzymatic and non-enzymatic degradation. Heroin and nicotine, in particular, are very small molecules of drug and therefore do not bind to antibodies or form physiologically important immune complexes with drugs.

This invention can be used in practice by specifying mucosal applications. For example, microspherical polymers are used to elicit mucosal immune responses. These small biodegradable microspheres are encapsulated to block conjugates and facilitate induction by mucosal immune systems. Most have been widely used as oral immunization agents, but have been reported to be effective as intranasal immunization agents (Walker (1994) Vaccine 12; 387-399). Inert polymers such as poly (lactide-co-glycolide) (PLG) 1-10 μm in diameter are particularly useful in this respect (Holmgren et al. (1994) Am. J. Trop. Med. Hyg. 50; 42; -54: Sevra (1994) Science 265: 1522-1524).

In addition to the preferred conjugates, cross-immunization with other conjugates minimizes antibody cross-activity. Mice were first inoculated with one conjugate to 14 days after the other conjugate was inoculated in the same manner. Only antibody-secreting B cells that recognized both heroin and nicotine conjugates were most stimulated. The two conjugates have increased recognition specificity because of different sites of contact with the drug molecule. Specificity caused in serum is confirmed by ELISA method.

Therapeutic compositions comprising one or more conjugates promote polyclonal antibodies that produce a continuous immune response.

Volume

Neutralizing antibody responses to pathogens are known continuously and may exhibit anti-heroin and anti-nicotine antibody responses that persist for at least one year. In view of what is obtained with conventional vaccines, the concentration of specific antibodies required to neutralize the plasma concentrations of heroin and nicotine can be reached. Although not shown, pharmacokinetic results in mice showed that the concentration of neutralizing antibodies was reached physiologically. Finally, the ability of the mother's antibody to penetrate the placenta of the mother, who is addicted to heroin or smoker, is blocked in the fetus, so the therapeutic effect of heroin and nicotine can be expected. The optimal treatment that is effective for many people depends on the technology used by understanding the various factors that determine the appropriate therapeutic dose. Furthermore, antibody reactions can be monitored by specific ELISA methods of specimens and other antibodies.

Due to genetic diversity in clearance, interaction with other drugs, excretion and distribution, disease-related diversity and combination with other factors result in varying vaccine responses in patients administered the same dose. Various clinical measures assessed the desired range of drug and did not monitor treatment response with chemical measures. The excretion rate, accumulation of half-life, and homeostasis of plasma concentrations are very difficult to predict and are used to determine the appropriate dose by measuring anti-drug-drug antibody production. Each of the conjugates / carriers / immunoadjuvant of the present invention is to assess the ability to produce an antibody response that binds to heroin and nicotine in the blood as much as possible.

Details of the effects of carriers and adjuvant inducing antibody responses are given in the examples. In certain cases, the appropriate amount of active compound will vary depending upon the particular conjugate employed, the composition prescribed and the particular site and organ. For example, therapeutic compositions comprising suitable carriers are administered parenterally parenterally. As discussed in detail herein, it is very effective for the generation of systemic IgG and local IgA that are immunized with suitable incomplete antigens and carrier complexes.

The murine experimental animals set up in this example have shown antibody titer, free nicotine recognition ability, binding ability with nicotine, affinity with nicotine, antibody specific reaction, antibody isotope, antibody tissue location, and physiological effects of nicotine after administration of nicotine. It is used to measure other characteristics of the antibody response, including.

Antibody titers

After inoculation, the conjugates were first screened to produce high titer antibody responses. Antibody titers were determined using the ELISA method which is well known in this area. For example, plates were coated with nicotine-HEL conjugates and then incubated with extensively washed and variously diluted test serums. The plate was washed again and the enzyme attached anti-mouse IgG2 antibody developed. The titer was taken as the inverse of the diluted serum showing 50% of the maximum response.

Antibody titers are determined by the concentration of the antibody and the affinity of the antibody. Both the concentration and affinity of the antibody were considered to establish the titer of the antibody required.

Antibody affinity reflects the amount of antibody-drug complex that is in equilibrium with a drug that is not bound to an antibody that is not bound. therefore :

Keq = [Ab + drug complex] / [Ab] × [drug]

[Ab] = molecular concentration not bound to the antibody binding site

[drug] = unbound drug concentration

[Ab + drug] = concentration of antibody-drug complex

Antibody Response Specificity

In order to obtain the maximum effect of blocking the action of addictive drugs, the antibodies raised should have minimal affinity with the drug's inactive metabolites. Pharmacologic binding of the drug with the inactive metabolite of the drug reduces the efficacy of the vaccine. Specificity of antisera for metabolites is determined according to ELISA and radioisotope labeled immunoassays. Furthermore, the effectiveness of the vaccine is increased only by the binding of the pharmacologically active metabolite with the drug derivative and the antibody.

In addition, interactions with antibodies from other drugs used in the treatment of addiction and other medications should be minimized. In particular, interactions between heroin and drugs prescribed to many drug abusers should be avoided. Buprenorphine, desipramine, naloxone, haloperidol, chlorproazine, mazindol, bromocriptine and related drugs are used in combination therapy.

Heroin LD ₅₀  Effect

More effective conjugates and immunization protocols, including high titer specific antibody responses, evaluate the ability of heroin to match LD ₅₀ . In these experiments, heroin at a dose that caused LD ₅₀ in heroin-immune and regulatory carrier-immune mice was IV. Ten mice were used and analyzed using the Cochran-Mantel-Haenzel Chi-squared method.

A drug failure model over time was also used for the analysis of death time by heroin. With this model, the effective rate of both the dose of heroin leading to death (a) and the time to death (b) were measured to increase anti-heroin antibodies. This is a quick and accurate test to determine the in vivo effectiveness of an antibody.

Observing physiological effects on humans

Persons in need of medical treatment for heroin abuse are unresponsive to shallow breathing, narrowing of the pupils, tachycardia, hypothermia and foreign stimuli. Overdose leads to cyanosis and death. The heroin blood concentrations at the time of active immunization with the vaccine and when the antibody was administered and all these factors were evaluated and characterized.

Without limiting the invention, details regarding nicotine and heroin and certain specifics have been presented in the compositions and methods of the invention.

The following examples show nicotine and antinicotine antibodies. However, these examples may also apply to heroin. For example, mice may be injected with tritium-labeled nicotine and killed at various times to track the redistribution of heroin (minimal amount in the brain). The effect of anti-heroin antibodies on heroin metabolism and excretion can be analyzed by plasma TLC and HPLC of 3H-heroin injected mice.

The examples and specific details set forth herein are understood for the purpose of practical examples only, and various modifications can be suggested to a person skilled in the art. Accordingly, many of the following examples provide guidance as a practical example of the invention.

Example 1

Nicotine conjugate preparation

Method A Triethylamine (75 mmol) and tin anhydride (100 mmol) were added to a solution of nornicotine 50 mmol methylene chloride. This solution was heated to reflux for 18 hours. The reaction mixture was washed with 10% hydrochloric acid, saturated sodium bicarbonate solution, brine and water. After drying over magnesium sulfide, the solvent was removed under reduced pressure, and the residue was purified using silica gel fresh chromatography to obtain the desired material.

Method B Nornicotin oxide was used to synthesize nicotine conjugate PS-54 as shown in FIG. DMF (0.1 ml), diisopropylethylamine (10 um) was added nornicotin oxide (5 um) and HATU (5.5 um). After 10 minutes, the pale yellow solution was added to a HEL or BSA (500 ug) solution of 0.1 M sodium borate buffer at pH 8.8 (0.9 ml) and the mixture was stirred at ambient temperature for 18 hours. The conjugate solution was adjusted to pH 7.0 with 0.1 M hydrochloric acid and purified by dialysis against PBS. Dialysis was filtered with 0.2um filter paper and the concentration of incomplete antigen was analyzed by mass and UV absorbance.

Induced nicotine-specific antibody response

In order to induce antibody-specific reactions with incomplete antigens such as small molecules or nicotine, it is necessary to link with T cell-epitope-containing carriers such as protein carriers. Carriers are recognized by T cells necessary for induction and maintenance of antibody production by nicotine-specific B cells. For example, BSA is used as the carrier. Structurally distinct nicotine-BSA conjugates are made by linking several other linkers and nicotine molecules through different moieties (FIG. 6B). Another conjugate was used to test the altered nicotine molecules. Nicotine molecules induce a variety of antibodies that recognize free incomplete antigen (nicotine) or a carrier or a conjugate used next (not recognized as nicotine).

Four Balb / c female mice 2-3 months old were immunized with intraperitoneal injection of 50 ug of nicotine-BSA conjugate, PS-55-BSA and Freund's immune adjuvant. Mice under this study have good reproducibility to antigens. PS-55-BSA was inoculated on day 21 and lethal on day 35. Serum was shown in Figure 9a by testing the binding between the PS-55 and the hen egg lysozyme protein (HEL) conjugate and the antibody by ELISA. These results indicate that nicotine-BSA conjugates can induce a strong antibody response.

Ten Balb / cbyj female mice, 2-3 months old, were immunized with anti-nicotine antiserum. In this experiment, CTB was used as a carrier. Mice were immunized with 10 ug of alhydrogel PS-55-CTB and immunized three times in the same manner. Mice were killed and serum isolated. Anti-nicotine antibodies were measured by direct nicotine ELISA with the serum of each experimental animal, and whether an antibody capable of recognizing free nicotine was induced.

Serum from each experimental animal was tested by anti-nicotine direct ELISA to measure antibodies in the following manner. The Immulon 2 96 well ELISA plate was coated with 10 μg / ml of secondary conjugated nicotine, HEL, PS-55-HEL, and 50 μl of diluted 1 × PBS at 4 ° C. overnight. Plates were blocked with PBS 0.5% gelatin at room temperature for 1 hour. Plates were washed three times with 1 × PBS containing 0.05% Tween20 (PBS-T). Serum samples were placed in 1/300 diluted plates coated with PS-55-HEL. Serum was diluted three-fold and incubated with plate blocking at room temperature for 2 hours. Plates were washed three times with PBS-T. Biotinylated goat anti-mouse IgG was diluted 1/10000 with PBS-T and 100ul was added to each well and incubated at room temperature for 1 hour. Streptavidin-HRP was diluted 1/10000 and added to the wells for 30 minutes and washed with PBS-T. Plates were washed three times with PBS-T and TMB Substrate was added to each well. The reaction was stopped after 5 minutes with 1M phosphoric acid. Plates were analyzed using an ELISA recorder at 450 nm. Serums that produced anti-nicotine antibodies by direct ELISA were tested by ELISA competition.

Free Nicotine Recognition

Competitive enzyme-linked immunosorbent assays were performed to determine whether the induced antibodies could recognize free nicotine molecules. In this assay free nicotine begins to compete with PS-55 HEL coated on a plate of enzyme-linked immunosorbent assay for binding to antibodies in serum. When an antibody having high affinity for nicotine is mostly bound to PS-55 HEL, nicotine at a low concentration can effectively inhibit the antibody binding.

As mentioned above, when three of four mice were injected with PS-55 HEL, the antibody binding to PS-55 HEL was inhibited by free nicotine (without relevant data). It should be noted that the presence of antibodies specific to the conjugate does not interfere with the action of the antinicotine antibodies. This suggests that the antibodies present in each serum recognize free nicotine. In addition, a competitive enzyme-linked immunosorbent assay was also performed on cotinine, a major metabolite of nicotine. RIA was used to measure specific binding to [3 H] -nicotine to demonstrate whether the induced antibody can recognize free nicotine molecules. From this experiment, the immunoserum is incubated with [3H] -nicotine and protein-G-conjugated Sepharose beads (Gammabind-G Sepharose, Pharmacia) to bind to IgG in serum samples. [3H] -nicotine bound to the antibody was separated by centrifugation of the beads and examined by scintillation counting of the beads. Free [3H] -nicotine was significantly bound to three of four mice (without relevant data). The serum was prenatally cultured with 50-fold excess unlabeled nicotine to completely inhibit the binding of [3H] -nicotine to the antibody. The data demonstrate that the synthesized nicotine-carrier conjugates induce nicotine specific antibody responses that can prevent nicotine from being distributed to the brain in vivo.

For the ten second groups immunized with PS-55-CTB, binding of the antibody to PS-55 HEL was inhibited by free nicotine (FIG. 10). The diluted serum used showed a 50% point titer. 96 well Immunon 2 enzyme-linked immunosorbent assay plates were applied overnight to 50 μl of 10 μg / ml PS-N-3.2 HEL dissolved in 1 × PBS at 4 ° C. The plates were blocked with 0.5% gelatin dissolved in PBS for 1 hour at room temperature and washed three times with 1 × PBS containing 0.05% Tween-20 (PBS-T). The plates were incubated with antiserum at room temperature for 2 hours under respective concentrations of nicotine metabolites, drugs and related compounds as well as free nicotine. The plates were then washed three times with PBS-T. Biotinylated goat antimouse IgG (lot #: J194-N855B or C) was diluted to 1 / 10,000 using PBS-T, and then 100 μl solution was added to each well and washed at room temperature for 1 hour. Streptavidin-HRP was diluted to 1 / 10,000 and washed with PBS-T and then added to each well. The plates were washed three times with PBS-T and then expressed by adding TMB substrate to each well. After addition of 1 M phosphoric acid, the reaction was stopped. Then, using the enzyme-linked immunosorbent assay, the O.D. The plate was measured at 450 nm. Competitive curves for each test serum are shown in FIG. The increase in competition was shown on the X-axis and the absorbance at 450 nm on the Y-axis.

As shown in FIG. 10, there was little recognition of the antibody with respect to metabolites of nicotine such as cotinine or norcotinine. Lidocaine anesthesia was used as a negative control, and thus could not compete for binding to the antibody. In addition, since the conjugate itself was used as a competitor, it was able to compete with the antibody.

In order to block the activity of nicotine as effectively as possible, the induced antibody should have minimal affinity for nicotine metabolites. Binding of the antibody to the metabolite more stably reduces the titer of the vaccine. Only nicotine was able to inhibit the binding of the antibody to the conjugate at the time of antisera screening. In this experiment little recognition of the antibodies to the products (cotinine and norcotinine) and anesthetics (lidocaine) was observed. Thus, the nicotine-CTB conjugate induces an antibody that recognizes nicotine, but the antibody does not recognize metabolites. In addition, the nicotine-CTB conjugate does not recognize a compound having a related structure and a drug currently used to treat nicotine poisoning.

Specificity of Antibodies Specific to Nicotine

In order to investigate the specificity of anti-nicotinic antibodies induced by nicotine vaccines, sera from mice immunized with nicotine-CTB conjugates were subjected to a competitive enzyme-linked immunosorbent assay. Different concentrations of nicotine metabolites and related molecules were tested. If the antibody has a high affinity for the metabolite, it can effectively compete for the assay at low concentrations. Relative reactivity was expressed as LD50, which indicates the concentration of inhibitor that reduced the signal of the competitive enzyme-linked immunosorbent assay by 50%. Reactivity to the following metabolites was tested: Nacotin glucuronide, cotinine, cotinine glucuronide, trans-3'-hydroxycotinine, trans-3'-hydroxycotinine glucuronide, nicotine 1 '-N-oxide, cotinine N-oxide and norcotinine.

Effect of Nicotine-Specific Antibodies on Inhibiting Nicotine Distribution

In vivo Inhibition of Nicotine Distribution to the Brain

To assess the changes in nicotine tissue distribution caused by nicotine specific antibodies, the distribution of 3H-nicotine was examined in mice immunized with nicotine-CTB and compared with natural control mice (non-immunized). Immune mice and control immune mice were injected intravenously with 0.08 mg / kg 3H-nicotine and killed 10 minutes after injection. Blood was collected and placed in a tube containing EDTA to prevent coagulation. Brain and plasma samples were placed in scintillation vials containing cell homogenizers. Digestion of the sample occurred over 3 days at room temperature. The sample was decolorized and a scintillation cocktail was added to each sample. Glacial acetic acid was added to make the sample clear. Samples were measured on a scintillation counter and then the data was converted to ng / g or ng / ml of cells. In the brain tissues of mice immunized with nicotine-CTB after 3H-nicotine injection, nicotine concentrations were significantly lower than those of natural control mice.

Example 2

Method A: N'-butyric acid additive of (S) -nicotine

To a (S) -nicotine (0.031 moles) solution in anhydrous methanol (50 mL) was added ethyl-4-bromobutyrate (0.0341 moles) dropwise over 10 minutes under ice-water temperature and argon. The orange solution thus prepared was heated up to room temperature and stirred for 18 hours. The solvent was then removed under reduced pressure to give a brown residue which was precipitated with hexane to give a pure sample for analysis of the desired ester.

Then, the ester (36 mg) was dissolved in methanol (3 mL) and sodium hydroxide solution (5 mL) and stirred at room temperature for 18 hours. Then, the solvent was removed under reduced pressure, and the residue was dissolved in 10% hydrochloric acid, and then extracted with ethyl acetate. Then dried (MgSO4) and the solvent was removed under reduced pressure to give the desired compound.

Method B: N'- valeric acid additive of (S) -nicotine

To a (S) -nicotine (0.031 moles) solution in anhydrous methanol (50 mL) was added dropwise 1-bromovaleric acid (0.0341 moles) over 10 minutes under ice-water temperature and argon. The orange solution thus prepared was heated up to room temperature and stirred for 18 hours. The solvent was then removed under reduced pressure to give a brown residue which was precipitated with hexane to give a pure sample for analysis of the desired ester.

Method C: N'-hexanoic acid additive of (S) -nicotine

To the (S) -nicotine (0.031 moles) solution in anhydrous methanol (50 mL) was added dropwise 1-bromohexanoic acid (0.0341 moles) over 10 minutes under ice-water temperature and argon. The orange solution thus prepared was heated up to room temperature and stirred for 18 hours. The solvent was then removed under reduced pressure to give a brown residue which was precipitated with hexane to give a pure sample for analysis of the desired ester.

Method D: N'-octanoic acid additive of (S) -nicotine

To a solution of (S) -nicotine (0.031 moles) in anhydrous methanol (50 mL) was added dropwise 1-bromooctanoic acid (0.0341 moles) over 10 minutes under ice-water temperature and argon. The orange solution thus prepared was heated up to room temperature and stirred for 18 hours. The solvent was then removed under reduced pressure to give a brown residue which was precipitated with hexane to give a pure sample for analysis of the desired ester.

Example 3

Method A: General Preparation of PS-55, PS-56, PS-57, and PS-58

To a suitable N'-alkanoic acid analog (6.27 × 10-5 moles) solution of nicotine obtained in Example 1 above in DMF (1.6 mL), DIEA (1.25 × 10-4 moles) and HATU (7.53 × 10-5) moles) were added. After 10 minutes at room temperature, a pale yellow solution was added to HEL or BSA (16.5 mg) in 0.1 M sodium bicarbonate (pH 8.3, 14.4 mL) and stirred for 18 h. The conjugation solution was then purified by dialysis overnight at 4 ° C. with PBS solution. The conjugate was then analyzed using a laser desorption mass spectrum analyzer to determine the number of hapten.

Method B: Preparation of PS-55

DIEA (54 mL, 3.10 × 10-4 moles) in a solution of the N'-butyric acid additive (39 mg, 1.56 × 10-4 moles) of nicotine obtained in Method A of Example 2 above in DMF (0.4 mL) And HATU (71 mg, 1.87 × 10-4 moles). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. The conjugation solution was then purified by dialysis overnight at 4 ° C. with PBS solution. The conjugate was then analyzed using a laser desorption mass spectrum analyzer to determine the number of hapten.

Method C: Preparation of PS-56

DIEA (2 mL) and HATU (2 mg) were added to the N'-valeric acid adduct (2 mg) solution of nicotine obtained in Method B of Example 2 above in DMF (0.4 mL). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. The conjugation solution was then purified by dialysis overnight at 4 ° C. with PBS solution. The conjugate was then analyzed using a laser desorption mass spectrum analyzer to determine the number of hapten.

Method D: Preparation of PS-57

To the N'-hexanoic acid adduct (2 mg) solution of nicotine obtained in Method C of Example 2 above in DMF (0.4 mL) was added DIEA (2 mL) and HATU (2 mg). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. The conjugation solution was then purified by dialysis overnight at 4 ° C. with PBS solution. The conjugate was then analyzed using a laser desorption mass spectrum analyzer to determine the number of hapten.

Method E: Preparation of PS-58

DIEA (2 mL) and HATU (2 mg) were added to the N'-octanoic acid adduct (2 mg) solution of nicotine obtained in Method D of Example 2 above in DMF (0.4 mL). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. The conjugation solution was then purified by dialysis overnight at 4 ° C. with PBS solution. The conjugate was then analyzed using a laser desorption mass spectrum analyzer to determine the number of hapten.

Method F: Preparation of PS-60

Iii) preparation of N-pyrrolidone additives

To a solution of nornicotine (10 mmol) in methanol was added dropwise ethyl-5-bromovaleric (10 mmol). After consumption of the starting material identified by TLC, the solvent was removed under reduced pressure and the residue was purified using silica gel flash chromatography to prepare the desired N-pyrrolidone additive.

Ii) preparation of the conjugate

The ester was dissolved in 5% aqueous methanol solution and sodium hydroxide (15 mmol) was added. After consumption of the starting material identified by TLC, the pH was adjusted to 2 by carefully adding 1M hydrochloric acid solution, and extracted by adding ethyl acetate. The desired free acid was then prepared by drying (MgSO4), removing the solvent under reduced pressure and then purifying the residue using silica gel flash chromatography.

To the solution of the free acid (1.55 × 10-4 mole) in DMF (0.4 mL) was added DIEA (3.1 × 10-4 mole) and HATU (1.86 × 10-4 mole). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. Subsequently, the mixed solution was allowed to stand at room temperature for 1 hour, neutralized, and then dialyzed overnight at 4 ° C. with PBS solution to obtain PS-60.

Method G: Preparation of PS-51

Viii) preparation of methyl 6-methylnicotinate

To a reflux solution of concentrated sulfuric acid (25 mL) in methanol (250 mL) was added 6-methylnicotinate (0.375 mol) and stirred under reflux for 3 h. Methanol (250 mL) was then added and the mixture was heated to reflux for 18 h. The reaction mixture was then cooled and concentrated under reduced pressure to make a slurry, which was then added to a solution of sodium bicarbonate (80 g) in water (450 mL). The mixture was then concentrated under reduced pressure to remove methanol. The blurry result was extracted with methylene chromide, then dried (MgSO 4), filtered and concentrated under reduced pressure. The resultant was then distilled off under reduced pressure to purify the desired ester.

Ii) Preparation of 6-methylmyosmine

To a solution of diisopropylamine (0.33 mol) in diethyl ether (500 mL) was added n-butyllithium (0.247 mol of 1.6M solution in hexanes) at -70 ° C under argon. N- (trimethylsilyl) pyrrolidone (0.265 mol) was then added to the prepared lithium diisopropylamine (LDA) and stirred at −70 ° C. for 15 minutes. To the solution was added methyl 6-methylnicotinate (0.165 mol) in diethyl ether (25 mL) and warmed to room temperature overnight. The mixture was then cooled in an ice-bath, water (33 mL) was added and then the ether layer was removed. Additional ether was added, followed by two ether layer removal procedures. Concentrated hydrochloric acid was added to the aqueous layer, and the resulting solution was refluxed overnight. The acidic solution was then washed with diethyl ether, concentrated under reduced pressure, cooled in an ice-bath and basified with 50% potassium hydroxide. The aqueous layer was extracted with diethyl ether (3 × 150 mL), dried (Na 2 SO 4), concentrated under reduced pressure and distilled under reduced pressure to afford the desired 6-methylmymine.

Iii) Preparation of ethyl valerate of 6-methylmyosmine

To a solution of 6-methylmyosmine (10 mmol) in dry toluene was added sodium amide (15 mmol). After 10 minutes, ethyl-5-bromovalericate (20 mmol) was added and the mixture was stirred until the starting material was consumed (checked by TLC). The mixture was then cooled in an ice-bath, terminated with dry ethanol, extracted with ethyl acetate, dried (MgSO 4), filtered and concentrated under reduced pressure. Purification using silica gel flash chromatography gave the desired product.

Viii) Preparation of valeric acid additive of 6-methylnornicotine

To the solution of the additive (10 mmol) obtained in (i) above in methanol, add sodium cyanoborohydride (10 mmol), a small amount of bromocresol green indicator and sufficient 2N hydrochloric acid / methanol to change the color of the solution from blue to yellow. Change and maintained. The solution was then stirred for 7 hours, and concentrated under reduced pressure after addition of 6N hydrochloric acid solution. After basification with sodium bicarbonate solution, diethyl ether extraction and drying (MgSO4) were carried out and purification using distillation under reduced pressure afforded the desired target.

Viii) Preparation of valeric acid additive of 6-methylnicotine

To the nornicotine (10 mmol) solution obtained in (iii) in diethyl ether was added iodomethane (20 mmol). The solution was refluxed under argon using an effective condenser until the starting material was consumed (identified by TLC). The solvent was removed under reduced pressure and the residue was purified using flash chromatography to give a racemic mixture of the desired compounds. Isomers were separated using chiral HPLC to afford the desired (S) -isomer.

Iii) preparation of conjugates

DIEA (3.06 × 10-5 moles) and HATU (1.86 × 10-) in a (S) -isomer (1.53 × 10-5 moles) solution of the valeric acid derivative of 6-methylnicotine obtained in (iii) in DMF 5 moles 2 mg) was added. After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 1.53 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added. After standing at room temperature for 1 hour, it was dialyzed at 4 ° C. with PBS solution, and analyzed using a mass spectrum analyzer to determine the number of hapten.

Method H: Preparation of PS-52

Iii) Preparation of methyl 5-methylnicotinate

To a reflux solution of concentrated sulfuric acid (25 mL) in methanol (250 mL) was added 5-methylnicotinate (0.375 mol) and stirred under reflux for 3 hours. Methanol (250 mL) was then added and the mixture was heated to reflux for 18 h. The reaction mixture was then cooled and concentrated under reduced pressure to make a slurry, which was then added to a solution of sodium bicarbonate (80 g) in water (450 mL). The mixture was then concentrated under reduced pressure to remove methanol. The blurry result was extracted with methylene chromide, then dried (MgSO 4), filtered and concentrated under reduced pressure. The resultant was then distilled off under reduced pressure to purify the desired ester.

Ii) Preparation of 5-methylmyosmine

To a solution of diisopropylamine (0.33 mol) in diethyl ether (500 mL) was added n-butyllithium (0.247 mol of 1.6M solution in hexanes) at -70 ° C under argon. N- (trimethylsilyl) pyrrolidone (0.265 mol) was then added to the prepared lithium diisopropylamine (LDA) and stirred at −70 ° C. for 15 minutes. To the solution was added methyl 6-methylnicotinate (0.165 mol) in diethyl ether (25 mL) and warmed to room temperature overnight. The mixture was then cooled in an ice-bath, water (33 mL) was added and then the ether layer was removed. Additional ether was added, followed by two ether layer removal procedures. Concentrated hydrochloric acid was added to the aqueous layer, and the resulting solution was refluxed overnight. The acidic solution was then washed with diethyl ether, concentrated under reduced pressure, cooled in an ice-bath and basified with 50% potassium hydroxide. The aqueous layer was extracted with diethyl ether (3 × 150 mL), dried (Na 2 SO 4), concentrated under reduced pressure and distilled under reduced pressure to afford the desired 5-methylmyosmine.

Viii) preparation of ethyl valerate additive of 5-methylmyosmine

To a solution of 5-methylmyosmine (10 mmol) in dry toluene was added sodium amide (15 mmol). After 10 minutes, ethyl-5-bromovalericate (20 mmol) was added and the mixture was stirred until the starting material was consumed (checked by TLC). The mixture was then cooled in an ice-bath, terminated with dry ethanol, extracted with ethyl acetate, dried (MgSO 4), filtered and concentrated under reduced pressure. Purification using silica gel flash chromatography gave the desired product.

Iii) Preparation of valeric acid additive of 5-methylnornicotine

To the solution of the additive (10 mmol) obtained in (i) above in methanol, add sodium cyanoborohydride (10 mmol), a small amount of bromocresol green indicator and sufficient 2N hydrochloric acid / methanol to change the color of the solution from blue to yellow. Change and maintained. The solution was then stirred for several hours, and concentrated under reduced pressure after addition of 6N hydrochloric acid solution. After basification with sodium bicarbonate solution, diethyl ether extraction and drying (MgSO4) were carried out and purification using distillation under reduced pressure afforded the desired target.

Iii) Preparation of valeric acid additive of 5-methylnicotine

To the nornicotine (10 mmol) solution obtained in (iii) in diethyl ether was added iodomethane (20 mmol). The solution was refluxed under argon using an effective condenser until the starting material was consumed (identified by TLC). The solvent was removed under reduced pressure and the residue was purified using flash chromatography to give a racemic mixture of the desired compounds. Isomers were separated using chiral HPLC to afford the desired (S) -isomer.

Iii) preparation of conjugates

DIEA (3.06 × 10-5 moles) and HATU (1.86 × 10-) in a (S) -isomer (1.53 × 10-5 moles) solution of the valeric acid derivative of 5-methylnicotine obtained in (iii) in DMF 5 moles 2 mg) was added. After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 1.53 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added. After standing at room temperature for 1 hour, it was dialyzed at 4 ° C. with PBS solution, and analyzed using a mass spectrum analyzer to determine the number of hapten.

Method I: Preparation of PS-53

Viii) preparation of methyl 4-methylnicotinate

To a reflux solution of concentrated sulfuric acid (25 mL) in methanol (250 mL) was added 4-methylnicotinate (0.375 mol) and stirred with reflux for 3 h. Methanol (250 mL) was then added and the mixture was heated to reflux for 18 h. The reaction mixture was then cooled and concentrated under reduced pressure to make a slurry, which was then added to a solution of sodium bicarbonate (80 g) in water (450 mL). The mixture was then concentrated under reduced pressure to remove methanol. The blurry result was extracted with methylene chromide, then dried (MgSO 4), filtered and concentrated under reduced pressure. The resultant was then distilled off under reduced pressure to purify the desired ester.

Ii) Preparation of 4-methylmyosmine

To a solution of diisopropylamine (0.33 mol) in diethyl ether (500 mL) was added n-butyllithium (0.247 mol of 1.6M solution in hexanes) at -70 ° C under argon. N- (trimethylsilyl) pyrrolidone (0.265 mol) was then added to the prepared lithium diisopropylamine (LDA) and stirred at −70 ° C. for 15 minutes. To the solution was added methyl 6-methylnicotinate (0.165 mol) in diethyl ether (25 mL) and warmed to room temperature overnight. The mixture was then cooled in an ice-bath, water (33 mL) was added and then the ether layer was removed. Additional ether was added, followed by two ether layer removal procedures. Concentrated hydrochloric acid was added to the aqueous layer, and the resulting solution was refluxed overnight. The acidic solution was then washed with diethyl ether, concentrated under reduced pressure, cooled in an ice-bath and basified with 50% potassium hydroxide. The aqueous layer was extracted with diethyl ether (3 × 150 mL), dried (Na 2 SO 4), concentrated under reduced pressure and distilled under reduced pressure to afford the desired 4-methylmymine.

Example 4

Preparation of Heroin conjugates

Preparation of PS-61

Iii) production of no heroin

To a solution of heroin (10 mmol) in water was added potassium permanganate (12 mmol) at 0 ° C. After the consumption of starting material (confirmed by TLC), the suspension was warmed to room temperature. Manganese dioxide was removed by filtration and the solvent was removed under reduced pressure to afford the desired compound.

Ii) preparation of conjugate precursors

Ethyl-5-bromovalate (20 mmol) was added dropwise to a solution of noheroin (10 mmol) in THF. The solvent was removed under reduced pressure after the consumption of starting material (confirmed by TLC). The residue was purified on silica gel using flash chromatography to give the desired noheroin ester additive.

Iii) preparation of conjugates

The ester (15 mmol) was dissolved in 5% aqueous methanol solution and sodium hydroxide (15 mmol) was added. After consumption of the starting material identified by TLC, the pH was adjusted to 2 by carefully adding 1M hydrochloric acid solution, and extracted by adding ethyl acetate. The desired free acid was then prepared by drying (MgSO4), removing the solvent under reduced pressure and then purifying the residue using silica gel flash chromatography.

To the solution of the free acid (1.55 × 10-4 mole) in DMF (0.4 mL) was added DIEA (3.1 × 10-4 mole) and HATU (1.86 × 10-4 mole). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.6 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. Subsequently, the mixed solution was allowed to stand at room temperature for 1 hour, neutralized, and then dialyzed overnight at 4 ° C. with PBS solution to obtain PS-61. The conjugates were analyzed using laser desorption mass spectra to determine the number of incomplete antigens.

Example 5

Preparation of PS-62 and PS-63

Iii) preparation of precursors

To a solution of heroin (10 mmol) in THF was added dropwise n-butyllithium (1.5 mmol of 1.6M solution in hexane) at 0 ° C. The mixture was left at 0 [deg.] C. for 2 hours, then ethyl-4-bromobutyrate (22 mmol) in THF was added dropwise over 10 minutes. Then it was heated until the starting material was consumed (identified by TLC) and then cooled to 0 ° C. and then carefully added 10% aqueous hydrochloric acid solution. Then, the aqueous solution layer in the two layers formed was extracted with ethyl acetate. The organic extract was washed with 1M aqueous sodium hydroxide solution, water and brine in that order. Then, dried (MgSO4), the solvent was removed under reduced pressure, and the residue was purified by silica gel flash chromatography to obtain two products, products bound with ortho and meta to an aromatic acetate group.

Ii) preparation of PS-62

An ester of ortho additive (5 mmol) was dissolved in 5% aqueous methanol solution and sodium hydroxide (5 mmol) was added. After consumption of the starting material identified by TLC, the pH was adjusted to 2 by carefully adding 1M hydrochloric acid solution, and extracted by adding ethyl acetate. The desired free acid was then prepared by drying (MgSO4), removing the solvent under reduced pressure and then purifying the residue using silica gel flash chromatography.

To the solution of the free acid (1.55 × 10-4 mole) in DMF (0.4 mL) was added DIEA (3.1 × 10-4 mole) and HATU (1.86 × 10-4 mole). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.5 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. Subsequently, the mixed solution was allowed to stand at room temperature for 1 hour, neutralized, and then dialyzed overnight at 4 ° C. with PBS solution to obtain PS-62. The conjugates were analyzed using laser desorption mass spectra to determine the number of incomplete antigens.

Iii) manufacture of PS-63

The ester of meta additive (5 mmol) was dissolved in 5% aqueous methanol solution and sodium hydroxide (5 mmol) was added. After consumption of the starting material identified by TLC, the pH was adjusted to 2 by carefully adding 1M hydrochloric acid solution, and extracted by adding ethyl acetate. The desired free acid was then prepared by drying (MgSO4), removing the solvent under reduced pressure and then purifying the residue using silica gel flash chromatography.

To the solution of the free acid (1.55 × 10-4 mole) in DMF (0.4 mL) was added DIEA (3.1 × 10-4 mole) and HATU (1.86 × 10-4 mole). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.5 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. Subsequently, the mixed solution was allowed to stand at room temperature for 1 hour, neutralized, and then dialyzed overnight at 4 ° C. with PBS solution to obtain PS-63. The conjugates were analyzed using laser desorption mass spectra to determine the number of incomplete antigens.

Example 6

Manufacture of PS-64

Iii) preparation of acetylated codeine

Triethylamine (12 mmol) was added to a solution of codeine (10 mmol) in methylene chloride, followed by addition of acetic anhydride (12 mmol). After consumption of the starting material identified by TLC, the solvent was removed under reduced pressure and then the residue was purified using silica gel flash chromatography to prepare the desired acetylated compound.

Ii) Demethylation of Acetylated Codeine

Boron tribromide (12 mmol of a 1.0 M solution in methylene chloride) was added dropwise to a solution of acetylated compound (10 mmol) in methylene chloride. After consumption of the starting material identified by TLC, anhydrous methanol was added and concentrated under reduced pressure. The residue was dissolved in methanol, re-concentrated under reduced pressure and purified using silica gel flash chromatography to prepare the desired alcohol.

Viii) Succinylation of demethylated acetylated codeine

Triethylamine (20 mmol) was added to the alcohol (10 mmol) solution in methylene chloride followed by succinic anhydride (20 mmol). Heated under reflux until starting material was consumed (identified by TLC). The solvent was then removed under reduced pressure and the residue was purified using silica gel flash chromatography to prepare the desired hemisuccinate.

Iii) manufacture of PS-64

To the hemisuccinate (1.55 × 10-4 mole) solution in DMF (0.4 mL) was added DIEA (3.1 × 10-4 mole) and HATU (1.86 × 10-4 mole). After 10 minutes at room temperature, the pale yellow solution was added rCTB (2 mg, 3.4 x 10-8 moles [lysine 15.5 x 10-6 moles]) in 4 ml of 0.1 M sodium borate and 0.15 M sodium chloride (pH 8.5). Was added dropwise and stirred for 1 hour. Subsequently, the mixed solution was allowed to stand at room temperature for 1 hour, neutralized, and then dialyzed overnight at 4 ° C. with PBS solution to obtain PS-63. The conjugates were analyzed using laser desorption mass spectra to determine the number of incomplete antigens.

Example 7

Differential analysis of CTB functional activity

 Two assays have been developed to investigate CTB functional activity. First, binding of CTB to cells was specified using flow cytometry. Cells were incubated with CTB and then treated with commercially available anti-CTB Gout antiserum and fluorescent isothiocyanate (FITC) -labeled anti-Gout secondary antibody (FIG. 13). Natural CTB pentamers bound to the cells cause large changes in fluorescence intensity. CTB monomers do not have binding capacity in this assay. Second, ELISA assay was used to measure the binding capacity of CTB to ganglioside GM1. ELISA plates were applied with GM1-gangliosides and then incubated with various concentrations of CTB. Binding was analyzed by progress of the reaction with anti-CTB antibodies (control brine), followed by enzyme-labeled secondary antibodies and substrates to be treated. The analytical method is a method capable of quantitatively measuring the binding capacity of CTB pentamers to GM1-gangliosides very accurately. The assay is used to investigate the activity of incompletely antigenized CTB conjugates and recombinants prior to in vivo experiments.

Example 8

Simultaneous treatment with other medicines

In relation to heroin abuse, screening was performed to determine whether treatment with secondary medications would increase the activity of the therapeutic vaccine. Treatment with opiate antagonists such as narroxone and naltrexone, naropin, levalohan, cyclazosine, buprenorphine and pentazosin, is expected to be well coordinated with treatment with heroin conjugates. One or more of the therapeutic agents may inhibit an immune response, thereby inhibiting the induction of a high anti-heroin antibody response. To confirm this possibility, mice were immunized with heroin-carrier conjugates in the presence or absence of therapeutic medication and antibodies were measured at various times. Therapeutic medications found to significantly suppress the immune response are eliminated as a discordant treatment. The screening test can be applied to all medications for which co-treatment is considered.

If no immunosuppression is found, additional screening is performed to determine whether the two approaches are synergistic. After training, immunization and testing, rats are evaluated in two models in the presence of medication. Medication is administered before rats receive other amounts of heroin. An initial experiment with control carrier-immunized mice was conducted to determine the amount of medication that would not completely eliminate the self-inoculation or drug identification system. The data is evaluated to determine whether the action of the therapeutic vaccine can be an addition to co-treatment.

Experimental Example 9

Induction of mucosal reactions

The B subunit of cholera toxin (CTB) is known to induce the activity of complete cholera toxin, ie, the mucosal antibody response, in several systems. Therefore, the carrier must strongly induce an anti-heroin or anti-nicotine IgA antibody response. In addition, oral initiation should strongly induce IgG antibody responses.

An efficient way to trigger a deductive reaction in the respiratory tract is to carry the antigen directly to the site. The antigen is administered with CTB which acts as an adjuvant in brine. Initial experiments were performed only with a carrier to confirm the ability of CTB to trigger by inoculation at the IgA surface of the mucosa. Mice were triggered by this route (oral, nasal or intratracheal) with 50 μg of CTB or heroin-CTB or nicotine-CTB. For oral administration, 250 μg of heroin-CTB or nicotine-CTB conjugate or CTB alone is administered enterally, or directly up using a 23G needle. At 14 days after the trigger, the mice are boosted by the same experimental method. Nasal administration is a simple and common route of triggering. Antigen is applied to lightly anesthetized rat nostril, which is done at a volume of 50 μl per mouse. At 14 days after the trigger, the mice are boosted by the same experimental method. Nasal administration can be easily applied to humans by spraying. Nasal vaccination can be used successfully with live influenza vaccines (Walker et al. (1994) Vaccine 12: 387-399).

Endotracheal immunization carries direct antigens to the bottom of the organ, which enhances immunization in the lungs. Rats are anesthetized with a cocktail of ketamine and xylazine. The rat is then transferred to a device in which the oral cavity is opened and the bronchus is exposed. The bronchus can be seen with a fiber light probe. Transfer 50 μl of solution to the lung using a 23 gauge needle. After 14 days of triggering, the mice are boosted using the same experimental method.

The rats were suffocated with carbon dioxide several hours after boosting (14, 21 or 28 days), then the nasal and intratracheal fluids were collected and IgA examined for inoculated conjugates. Nasal washes are obtained by washing the nasal cavity 4 times with 1 ml PBS as described in Tamura et al. (1989) Vaccine 7: 257-262. Endotracheal fluids are described in Nedrud et al (1987) J. Immunol. Obtained by exposing the bronchus according to the method described in 139: 3484-3492 and inserting 0.5 ml PBS solution into the lungs followed by three washes. After centrifugation to remove cells, samples for antigen-specific IgA are analyzed by ELISA using IgA-specific secondary antibodies. Heroin-specific or nicotine-specific IgG is measured by nasal and lung lavage. As is known, IgG is detected in the lungs and is an important antibody (Cahill et al. (1993) FEBS Microbiol. Lett. 107: 211-216).

The oral immunization route is used to test the ability to produce heroin-specific or nicotine-specific IgA in intestinal lavage fluid and compares it with other routes for the ability to produce serum Ig for heroin or nicotine. Oral administration is particularly preferred for humans because of its ease of administration. Intranasal and intratracheal administrations directly compare the ability to induce IgA responses in pulmonary and nasal fluids. It is desirable to use the best route for subsequent experiments. If both routes are efficient, nasal immunization is preferred because of its ease.

Maximize both systematic IgG and mucosal IgA responses for maximum protection against heroin or nicotine. Thus, mucosal membranes with systematic inoculation of conjugates in alum (or other adjuvants) heroin-CTB or nicotine-CTB conjugates and conjugates are effective in triggering. Three groups are compared. First, the mice were triggered by organ system, followed by 14 days of organ system immunization. Second, mucosa was triggered mucosally, followed by organ immunization for 14 days. Third, the mice were simultaneously triggered by the tracheal system and mucosa and the same boosting was performed 14 days later. Control mice are only triggered mucosally or organally. In each case, the efficiency of boosting was determined by the amount of IgG and IgA.

In the initial measurement of in vivo efficiency of mucosal anti-heroin or anti-nicotine antibodies, changes in pharmacokinetics are measured for each mucosally administered heroin or nicotine.

Example 10

Passive Transport of Immunoglobulins in Mice

Mice are immunized with heroin conjugates using the optimal immunization method described in the Examples. Blood was taken from mice at various times and the amount of anti-heroin antibody was measured using ELISA. Blood was taken by cardiac puncture from mice with antibody amounts of at least about 54,000. Control mice are immunized with carrier protein. Serum obtained from several rats (20 or more) was pooled and IgG was isolated using ammonium sulfate precipitation. Dialysis to remove ammonium sulfate was performed and then the amount of cocaine-specific antibody in the collected immunoglobulins was measured using ELISA. Various amounts of immunoglobulins are administered to natural mice by the i.p or i.v route. After 24 hours, blood was collected from recipient mice and serum was analyzed to determine the amount of cocaine-specific antibodies. Using this method, the amount of antibody to be transferred to determine a certain amount is determined. Immunoglobulins can be given to different groups of mice, and blood can be drawn at various times to determine the clearance of antigen-specific antibodies. Another group of mice is tested with radiolabeled heroin as described in the Examples, and measures the amount of heroin distributed to the brain. Control mice receive IgG from carrier-immunized mice. The experiments show that passively transported immunoglobulins prevent heroin from entering the brain.

Example 11

Passive Transport of Immunoglobulins in Humans

A herd of human donors is immunized with the conjugates of the present invention using the optimized immunization method described in the Examples. At various times, blood is obtained by Bennypuncture from the donor and the amount of anti-incomplete antigen antibody is analyzed using ELISA. Hyperimmune plasmas obtained from different donors are pooled and IgG separated using cold alcohol separation. Precipitated antibodies are treated with buffer, stabilized, stored and normalized to the extent necessary for high immune antibodies for human use. The amount of anti-incomplete antigen antibody is normalized using ELISA and other antibody-using assays.

Purified antibody titers were administered to 20 patients intramuscularly or intravenously without incomplete antigen-CTB vaccine or vaccine. Appropriate doses were determined 24 hours after the administration of hyperimmune antibodies or other effective drugs that inhibit the effects of heroin or nicotine, or by analyzing the serum of the patient by ELISA or other antibody assays.

Passively delivered immunoglobulins inhibit the effects of heroin or nicotine in the patient. The use of human donors, polyclonal antibodies and numerous donors limits the chance of immune response by antibodies delivered to the patient.

Example 12

Manufacture and purification of rCTB

The rCTB of cholera supplied from SBL vaccine AB in 0.22M phosphoric acid, pH 7.3 in 0.9% saline buffer was mixed with 20 mM sodium phosphate pH6.5. The sample was purified by the following solution by Pharmacia SP Sepharose resin cation exchange chromatography. ; Buffer A: 20 mM sodium phosphate at pH 6.5, elution buffer B: 20 mM sodium phosphate at pH 6.5, 1.0 M sodium chloride. The purified fractions were analyzed by Dainichi silver stained SDS-PAGE. Purified samples were filtered through 0.22micron filter paper and sterilized storage at 4 ℃.

Example 13

Method A (analysis) Samples for reverse phase HPLC (RP HPLC) analysis were prepared by the following method. : 100 ul of conjugate CTB-5.200 was precipitated by adding 1.0 ml of ethanol and frozen at -80 ° C. The conjugate was centrifuged at 4 ° C. and 14000 rpm for 20 minutes to remove ethanol and dried the precipitate. The residue was suspended in 25ul of 0.1% trihuloacetic acid (TFA) -20% acetonitrile and the protein concentration was measured by Pierce Micro BCA assay.

The conjugate was analyzed using a C18 reversed phase column (Vydac No. 218TP5215 narrow hole) of 2.1 × 150 mm. ; 5 U, particle rate 200 ul / min; Buffer A-100% water, 0.1% TFA; Buffer B-80% acetonitrile, 0.08% TFA. The concentration was varied from 50% for 50 minutes to 56%, increased to 80% for 60 minutes, and 10 minutes.

Method B (Semi-Preparation) For HPLC on a semi-preparation sample is prepared as follows. Re-suspend 2 vials of CTB-5.200 lysophil with 20% acetonitrile-0.1% TFA, sterile filter and quantify with Pierce Micro BCA. Two injections of 1.24 mg each in semi-preparative RP HPLC using a 10 × 50 mm C18 reversed phase column. ; Particle size 5 U, flow rate 1.8 ml / min; Buffer A-0.1% TFA-water; Buffer B-0.08% TFA-80% Acetonitrile. The concentration was changed as follows. 20% B solution for 10 minutes, 35% B solution for 40 minutes, 55% B solution for 5 minutes, 100% B solution for 5 minutes. Peaks were collected and immediately frozen.

Example 14

Incomplete Antigen vs. Immunity

The ratio of incomplete antigen to carrier protein in the conjugate may vary depending on the ability of the conjugate to promote incomplete antigen specific antibody production. The inclusion reaction changes the production of heroin-CTB conjugates depending on the concentration of some other incomplete antigen. The degree of incomplete antigen is calculated according to the mass absorbance analysis of the conjugate. The conjugates were screened for physiological activity in the immunoassay, and the degree of incomplete antigen was measured by mass absorbance analysis of the conjugates. Complexes made according to other methods using different incomplete antigen ratios are compared to carrier proteins. Incomplete antigen and immunity were compared.

Equivalent way

Those skilled in the art can identify or recognize using common test methods that are equivalent to the specific materials and methods mentioned herein. Such equivalent methods are believed to be present in the rats of the present invention and include them in the claims below.

Claims (54)

An incomplete antigen-carrier conjugate comprising at least one incomplete antigen derived from nicotine and at least one carrier containing a T-cell epitope, wherein the incomplete antigen and the carrier are identified by a CJ reference number consisting of Incomplete antigen-carrier conjugates, characterized in that they are linked by a branch selected from the group of chemical moieties: Where Q 'is a modified protein Wherein n is an integer, Q is a carrier and Y is selected from the group consisting of S, 0 and NH. The incomplete antigen-carrier conjugate according to claim 1, wherein n is 2 to 20. The incomplete antigen-carrier conjugate of claim 1 or 2, wherein the carrier is selected from the group: Proteins or peptides, bacterial toxins or products, subviruses, lectins, allergens and fragments of allergens, malaria protein antigens, artificial multi-antigenic peptides, and variants, analogs and derivatives thereof. The incomplete antigen-carrier conjugate of claim 1 or 2, wherein at least one incomplete antigen is coupled to the carrier. A therapeutic composition for the treatment of drug addiction in humans, comprising an incomplete antigen-carrier conjugate comprising at least one incomplete antigen derived from nicotine and at least one carrier containing a T-cell epitope, wherein the incomplete therapeutic composition A therapeutic composition for the treatment of drug addiction in humans, characterized in that the antigen is connected by a branch selected from the group of chemical moieties identified by the CJ reference number consisting of: Where Q 'is a modified protein Wherein n is an integer, Q is a carrier and Y is selected from the group consisting of S, 0 and NH. The therapeutic composition of claim 5 comprising a pharmaceutically acceptable excipient. 7. The therapeutic composition of claim 5 or 6, which may be administered to the mucosa and comprises copolymer microspheres. 8. The therapeutic composition of claim 7, wherein the copolymer microspheres are poly (lactide-co-glycolide) copolymers. 7. The therapeutic composition of claim 5 or 6, which can be administered topically. The therapeutic composition of claim 9, wherein the composition has a higher kinematic viscosity than water. The therapeutic composition of claim 5 or 6, which may be administered as an injectable sterile solution. The therapeutic composition according to claim 5 or 6, comprising an adjuvant. A method for preparing a conjugate according to claim 1, comprising the step of binding a carrier containing an incomplete antigen and a T-cell epitope by crosslinking with a compound selected from the group: Active esters, mixed anhydrides, acyl azides, acyl halides and imino esters derived from carboxylic acids. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete