Developing Translational Vaccines against Heroin and Fentanyl through Investigation of Adjuvants and Stability.

The nearly insurmountable adversity that accompanies opioid use disorder (OUD) creates life-altering complications for opioid users. To worsen matters, existing small-molecule drugs continue to inadequately address OUD due to their engagement of the opioid receptor, which can leave the user to deal with side effects and financial hardships from their repeated use. An alternative therapeutic approach utilizes endogenously generated antibodies through active vaccination to reduce the effect of opioids without modulating the opioid receptor. Here, we explore different adjuvants and storage conditions to improve opioid vaccine efficacy and shelf life. Our results revealed that inulin-based formulations (Advax) containing a CpG oligodeoxynucleotide (ODN) acted as effective adjuvants when combined with a heroin conjugate: immunized mice showed excellent recovery from heroin-induced antinociception accompanied by high titer, high opioid affinity serum antibodies similar to the immunopotentiating properties of traditional alum-based adjuvants. Moreover, nonhuman primates vaccinated with a heroin/fentanyl combination vaccine demonstrated potent antibody responses against opioids when formulated with both inulin and alum adjuvants. Finally, storing a freeze-dried opioid vaccine formulation maintained efficacy for up 1 year at room temperature. The results from our studies represent an advance toward a clinically feasible opioid vaccine.

Enhancement of a Heroin Vaccine through Hapten Deuteration.

The United States is in the midst of an unprecedented epidemic of opioid substance use disorder, and while pharmacotherapies including opioid agonists and antagonists have shown success, they can be inadequate and frequently result in high recidivism. With these challenges facing opioid use disorder treatments immunopharmacotherapy is being explored as an alternative therapy option and is based upon antibody-opioid sequestering to block brain entry. Development of a heroin vaccine has become a major research focal point; however, producing an efficient vaccine against heroin has been particularly challenging because of the need to generate not only a potent immune response but one against heroin and its multiple psychoactive molecules. In this study, we explored the consequence of regioselective deuteration of a heroin hapten and its impact upon the immune response against heroin and its psychoactive metabolites. Deuterium (HdAc) and cognate protium heroin (HAc) haptens were compared head to head in an inclusive vaccine study. Strikingly the HdAc vaccine granted greater efficacy in blunting heroin analgesia in murine behavioral models compared to the HAc vaccine. Binding studies confirmed that the HdAc vaccine elicited both greater quantities and equivalent or higher affinity antibodies toward heroin and 6-AM. Blood-brain biodistribution experiments corroborated these affinity tests. These findings suggest that regioselective hapten deuteration could be useful for the resurrection of previous drug of abuse vaccines that have met limited success in the past.

Sulfonate-isosteric replacement examined within heroin-hapten vaccine design.

Heroin overdose and addiction remain significant health and economic burdens in the world today costing billions of dollars annually. Moreover, only limited pharmacotherapeutic options are available for treatment of heroin addiction. In our efforts to combat the public health threat posed by heroin addiction, we have developed vaccines against heroin. To expand upon our existing heroin-vaccine arsenal, we synthesized new aryl and alkyl sulfonate ester haptens; namely aryl-mono-sulfonate (HMsAc) and Aryl/alkyl-di-sulfonate (H(Ds)2) as carboxyl-isosteres of heroin then compared them to our model heroin-hapten (HAc) through vaccination studies. Heroin haptens were conjugated to the carrier protein CRM197 and the resulting CRM-immunoconjugates were used to vaccinate Swiss Webster mice following an established immunization protocol. Binding studies revealed that the highest affinity anti-heroin antibodies were generated by the HMsAc vaccine followed by the HAc and H(Ds)2 vaccines, respectively (HMsAc > HAc≫HDs2). However, neither the HMsAc nor H(Ds)2 vaccines were able to generate high affinity antibodies to the psychoactive metabolite 6-acetyl morphine (6-AM), in comparison to the HAc vaccine. Blood brain bio-distribution studies supported these binding results with vaccine efficiency following the trend HAc > HMsAc â‰« H(Ds)2 The work described herein provides insight into the use of hapten-isosteric replacement in vaccine drug design.

Synthesis of a deuterated 6-AmHap internal standard for the determination of hapten density in a heroin vaccine drug product.

A deuterated hapten was designed and synthesized that will be essential for a future study of residual hapten and stability of a hapten-protein conjugate. This hapten, 6-AmHap, was chosen for a heroin vaccine that is now slated for a Phase 1 clinical trial. A maleimide-thiol bioconjugation strategy was successfully applied to our heroin vaccine to connect the hapten 6-AmHap with an immunogenic carrier protein (tetanus toxoid, TT) through a trityl-protected 3-mercaptopropanamide linker. The antibodies induced by the vaccine have been found to have activity against several opioids, including heroin and its metabolites, and, importantly, leave alternate pain treatment medications such as methadone untouched. To the best of our knowledge, no other hapten for a heroin vaccine has been deuterated, yet this tool may prove to be of great importance in the study of residual hapten during product release and the long-term stability program of a hapten-protein conjugate as part of FDA regulatory requirements. Hydrocodone was the starting material for the synthesis of the deuterated 6-AmHap, with a stable amide at C6 and a 3-mercaptopropanamide linker attached at C3. The desired deuterated product was prepared as the disulfide, 3,3'-disulfanediylbis(N-((7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl)-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)propanamide), that could be easily reduced to form the needed hapten, N-((4aR,7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl]-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)-3-mercaptopropanamide.

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin.

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine's performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin's psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.

Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins.

Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)4] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)4-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development.

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Dynamic vaccine blocks relapse to compulsive intake of heroin.

Heroin addiction, a chronic relapsing disorder characterized by excessive drug taking and seeking, requires constant psychotherapeutic and pharmacotherapeutic interventions to minimize the potential for further abuse. Vaccine strategies against many drugs of abuse are being developed that generate antibodies that bind drug in the bloodstream, preventing entry into the brain and nullifying psychoactivity. However, this strategy is complicated by heroin's rapid metabolism to 6-acetylmorphine and morphine. We recently developed a "dynamic" vaccine that creates antibodies against heroin and its psychoactive metabolites by presenting multihaptenic structures to the immune system that match heroin's metabolism. The current study presents evidence of effective and continuous sequestration of brain-permeable constituents of heroin in the bloodstream following vaccination. The result is efficient blockade of heroin activity in treated rats, preventing various features of drugs of abuse: heroin reward, drug-induced reinstatement of drug seeking, and reescalation of compulsive heroin self-administration following abstinence in dependent rats. The dynamic vaccine shows the capability to significantly devalue the reinforcing and motivating properties of heroin, even in subjects with a history of dependence. In addition, targeting a less brain-permeable downstream metabolite, morphine, is insufficient to prevent heroin-induced activity in these models, suggesting that heroin and 6-acetylmorphine are critical players in heroin's psychoactivity. Because the heroin vaccine does not target opioid receptors or common opioid pharmacotherapeutics, it can be used in conjunction with available treatment options. Thus, our vaccine represents a promising adjunct therapy for heroin addiction, providing continuous heroin antagonism, requiring minimal medical monitoring and patient compliance.

Endophthalmitis related to lemon allergy in a heroin addict.

BACKGROUND: Heroin and its contaminants may be an important source of allergens in young people. We present a case of severe endophthalmitis in a patient that also suffered from anaphylactoid symptoms (hypotension, urticaria, glottic oedema) whenever he ingested lemon. METHODS: Prick tests with a battery of 42 aeroallergens including fruits and citrus fruits (orange, mandarin, grapefruit and lemon) and specific IgE to these allergens were carried out. Immunodetection was performed using the patient's serum and the following allergens: lemon, Candida, Aspergillus, Penicillium and Alternaria recombinant Alt 1 (Laboratories Diater). RESULTS: Skin tests were negative for Candida, Penicillium, Aspergillus and Cladosporium (ALK-Spain) as were specific IgE antibodies for CAP (Thermofisher, Sweden) and positive only for lemon and, doubtfully, to Candida. Specific IgE tests to pollen, arthropods, fungi, dander and foods were positive only for lemon (0.49kU/L). Serological study of fungi ruled out fungal infection at that time. The immunodetection showed that the patient's serum recognised a protein of approximately 25kDa of lemon peel, one of approximately 12-13kDa of Penicillium, and perfectly recognised Alt a 1. CONCLUSIONS: Lemon surface can be contaminated by Candida and other fungi. In heroin addicts with positive skin tests for lemon, the possibility of these serious complications should be taken into account.

Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 µg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation method, and the methods for the accurate quantification of hapten density are described.

Injection route and TLR9 agonist addition significantly impact heroin vaccine efficacy.

Active immunization is an effective means of blocking the pharmacodynamic effects of drugs and holds promise as a treatment for heroin addiction. Previously, we demonstrated the efficacy of our first-generation vaccine in blocking heroin self-administration in rats, however, many vaccine components can be modified to further improve performance. Herein we examine the effects of varying heroin vaccine injection route and adjuvant formulation. Mice immunized via subcutaneous (sc) injection exhibited inferior anti-heroin titers compared to intraperitoneal (ip) and sc/ip coadministration injection routes. Addition of TLR9 agonist cytosine-guanine oligodeoxynucleotide 1826 (CpG ODN 1826) to the original alum adjuvant elicited superior antibody titers and opioid affinities compared to alum alone. To thoroughly assess vaccine efficacy, full dose-response curves were generated for heroin-induced analgesia in both hot plate and tail immersion tests. Mice treated with CpG ODN 1826 exhibited greatly shifted dose-response curves (10-13-fold vs unvaccinated controls) while non-CpG ODN vaccine groups did not exhibit the same robust effect (2-7-fold shift for ip and combo, 2-3-fold shift for sc). Our results suggest that CpG ODN 1826 is a highly potent adjuvant, and injection routes should be considered for development of small molecule-protein conjugate vaccines. Lastly, this study has established a new standard for assessing drugs of abuse vaccines, wherein a full dose-response curve should be performed in an appropriate behavioral task.

Synthesis and immunological effects of heroin vaccines.

Three haptens have been synthesized with linkers for attachment to carrier macromolecules at either the piperidino-nitrogen or via an introduced 3-amino group. Two of the haptens, with a 2-oxopropyl functionality at either C6, or at both the C3 and C6 positions on the 4,5-epoxymorphinan framework, as well as the third hapten (DiAmHap) with diamido moieties at both the C3 and C6 positions, should be much more stable in solution, or in vivo in a vaccine, than a hapten with an ester in one of those positions, as found in many heroin-based haptens. A "classical" opioid synthetic scheme enabled the formation of a 3-amino-4,5-epoxymorphinan which could not be obtained using palladium chemistry. Our vaccines are aimed at the reduction of the abuse of heroin and, as well, at the reduction of the effects of its predominant metabolites, 6-acetylmorphine and morphine. One of the haptens, DiAmHap, has given interesting results in a heroin vaccine and is clearly more suited for the purpose than the other two haptens.

Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines.

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman's test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3-5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.

Selective effects of a morphine conjugate vaccine on heroin and metabolite distribution and heroin-induced behaviors in rats.

Morphine conjugate vaccines have effectively reduced behavioral effects of heroin in rodents and primates. To better understand how these effects are mediated, heroin and metabolite distribution studies were performed in rats in the presence and absence of vaccination. In non-vaccinated rats 6-monoacetylmorphine (6-MAM) was the predominant opioid in plasma and brain as early as 1 minute after i.v. administration of heroin and for up to 14 minutes. Vaccination with morphine conjugated to keyhole limpet hemocyanin (M-KLH) elicited high titers and concentrations of antibodies with high affinity for heroin, 6-MAM, and morphine. Four minutes after heroin administration vaccinated rats showed substantial retention of all three opioids in plasma compared to controls and reduced 6-MAM and morphine, but not heroin, distribution to brain. Administration of 6-MAM rather than heroin in M-KLH vaccinated rats showed a similar drug distribution pattern. Vaccination reduced heroin-induced analgesia and blocked heroin-induced locomotor activity throughout 2 weeks of repeated testing. Higher serum opioid-specific antibody concentrations were associated with higher plasma opioid concentrations, lower brain 6-MAM and morphine concentrations, and lower heroin-induced locomotor activity. Serum antibody concentrations over 0.2 mg/ml were associated with substantial effects on these measures. These data support a critical role for 6-MAM in mediating the early effects of i.v. heroin and suggest that reducing 6-MAM concentration in brain is essential to the efficacy of morphine conjugate vaccines.

A morphine/heroin vaccine with new hapten design attenuates behavioral effects in rats.

Heroin use has seriously threatened public heath in many countries, but the existing therapies continue to have many limitations. Recently, immunotherapy has shown efficacy in some clinical studies, including vaccines against nicotine and cocaine, but no opioid vaccines have been introduced in clinical studies. The development of a novel opioid antigen designed specifically for the prevention of heroin addiction is necessary. A morphine-keyhole limpet hemocyanin conjugate was prepared and administered subcutaneously in rats. Antibody titers in plasma were measured using an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was used to assess the selectivity of the antibodies. Dopamine concentrations in the nucleus accumbens in rats after vaccine administration were determined by high-performance liquid chromatography with electrochemical detection. The effects of the vaccine on the heroin-primed restatement of self-administration and locomotor sensitization were evaluated. A novel hapten, 6-glutarylmorphine, was produced, and the vaccine generated a high antibody titer response. This vaccine displayed specificity for both morphine and heroin, but the anti-morphine antibodies could not recognize dissimilar therapeutic opioid compounds, such as buprenorphine, methadone, naloxone, naltrexone, codeine, and nalorphine. The morphine antibody significantly decreased morphine-induced locomotor activity in rats after immunization. Importantly, rats immunized with this vaccine did not exhibit heroin-primed reinstatement of heroin seeking when antibody levels were sufficiently high. The vaccine reduced dopamine levels in the nucleus accumbens after morphine administration, which is consistent with its behavioral effects. These results suggest that immunization with a novel vaccine is an effective means of inducing a morphine-specific antibody response that is able to attenuate the behavioral and psychoactive effects of heroin.

The morphine/heroin vaccine decreased the heroin-induced antinociceptive and reinforcing effects in three inbred strains mouse.

Clinical trials have indicated that a vaccine must be immunogenic in genetically diverse human populations and that immunogenicity and protective efficacy in animal models are two key indices required for the approval of a new vaccine. Additionally, the immune response (immunogenicity) and immunoprotection are dependent on the mouse strain. Therefore, the objective of the present study was to determine the immune response (immunogenicity) and the protective efficacy (behavioral response) in three inbred mouse strains immunized with the M6TT vaccine. Female BALB/c, C57Bl/6, and DBA/2 inbred mice were immunized with the M6-TT vaccine. A solid-phase antibody-capture ELISA was used to monitor antibody titer responses after each booster dose in vaccinated animals. The study used tail-flick testing to evaluate the antinociceptive effects induced by heroin. Additionally, heroin-induced locomotor activity and place preference were evaluated. The M6-TT vaccine was able to generate a specific antibody titer in the three inbred mouse strains evaluated. The antibodies reduced the antinociceptive effect of different doses of heroin. In addition, they decreased the heroin-induced locomotor activity and place preference. These findings suggest that the M6-TT vaccine generates a powerful immunogenic response capable of reducing the antinociceptive and reinforcing effects of heroin in different inbred mouse strains, which supports its possible future use in clinical trials in genetically diverse human populations.

Neuronal adaptations, neuroendocrine and immune correlates of heroin self-administration.

Opioid receptor-mediated action in the central nervous system (CNS) has been consistently shown to trigger changes in the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) and suppress a variety of parameters of immune function in investigator-delivered paradigms. Overwhelming evidence supports the concept that the CNS undergoes numerous and complex neuronal adaptive changes in addicts, and in animal models of heroin addiction as a result of the training of drug stimuli to serve as reinforcers, altering the function of individual neurons and the larger neural circuits within which the neurons operate. Taken together, these advances suggest that since plastic neuronal changes occur in drug addiction and related animal model paradigms, profiles of neuroendocrine and immune function would differ in a rat model of heroin self-administration compared to passive infusion of drug. Self-administration of heroin induces neuronal circuitry adaptations in specific brain regions that may be related to alterations in neuroendocrine and T lymphocyte function also observed. Animals self-administering (SA) heroin exhibit increased mu-opioid receptor agonist ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO))-stimulated guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding in the anterior hypothalamus (50% and 33%) and rostral medial thalamus (33% and 36%) compared with control animals receiving identical non-contingent injections of yoked-heroin (YH) or yoked-saline (YS), respectively. No changes in agonist-stimulated G-protein sensitization were observed in 14 other brain regions studied. No changes in mu-opioid receptor density, ((3)H-DAMGO binding) were seen in all brain regions examined. The neuronal changes in SA animals were correlated with elevated adrenocorticotrophic hormone (ACTH) (64% and 104%) and glucocorticoid production (198% and 79%) compared with YH and YS groups, respectively. Neuroendocrine adaptive changes in SA animals were associated with thymic hypoplasia. Splenic T lymphocytes from animals that had self-administered heroin showed a profoundly reduced ability to proliferate in response to concanavalin A (50% and 48% compared with YH and YS controls, respectively; Fig. 1A), or a monoclonal antibody (R73) to the CD3/T-cell receptor complex (anti-TCR) plus IL-2 (55% and 59% compared with YH and YS controls, respectively; Fig. 1B). Self-administration of heroin selectively alters T lymphocyte function, as no effects on natural killer cell activity or macrophage functions were observed. These findings may have relevance to the acquisition and documented increased incidence of infectious diseases, including HIV, in heroin addicts, due to a pre-existing T-cell immunodeficient state.

Vaccines against morphine/heroin and its use as effective medication for preventing relapse to opiate addictive behaviors.

Current pharmacotherapies for treating morphine/heroin dependence are designed to substitute or block addiction by targeting the drug itself rather than the brain. The heroin addict is still being exposed to addictive opiates, and consequently may develop tolerance to and experience withdrawal and drug's toxic effects from the treatment with high incidence of relapse to addictive drug consumption. As for other drugs of abuse, an alternative approach for morphine/heroin addiction is an antibody-based antagonism of heroin's brain entry. This review summarizes the literature examining important aspects of neurobiological and pharmacological processes involved in opiate dependence. Thereafter, classical pharmacological interventions for opiate dependence treatment and its major clinical limitations are reviewed. Finally, relevant preclinical studies are examined for comparisons in the design, use, immunogenic profile and efficacy of several models of morphine/heroin vaccine as immunologic interventions on the pharmacokinetics and behavioral of morphine/heroin in the rat as animal model.

Group-selective antibodies based fluorescence immunoassay for monitoring opiate drugs.

A novel carboxylic acid derivative of monoacetylmorphine (MAM-COOH) was synthesized and conjugated with bovine serum albumin (BSA) for generating polyclonal antibodies against the target molecule heroin and its major metabolites. The conjugate was characterized by fluorescence spectroscopy, polyacrylamide gel electrophoresis, and mass spectrometry to confirm the extent of haptenization of the carrier protein. A high titer (1:64,0000) of antibody was obtained by using the conjugate with an optimum protein/hapten molar ratio of 1:100. The generated antibody showed good binding affinity with heroin and its metabolites monoacetylmorphine (MAM) and morphine. The relative affinity constant (K (aff)) of the antibody was 3.1 x 10(7) l mol(-1), and the IC(50) values obtained for heroin, MAM, morphine, and codeine were 0.01, 0.013, 0.012, and 0.014 ng ml(-1), respectively. A fluorescence-based competitive inhibition immunoassay procedure was developed for the estimation of heroin and its major metabolites in standard and biofludic samples over a concentration range up to 0.01 ng ml(-1) with good signal reproducibility (p < 0.05). The method can be used as a convenient quantitative tool for the sensitive screening of major metabolites of heroin in biological samples.

Efficacious Vaccine against Heroin Contaminated with Fentanyl.

The sharp increase in overdose deaths involving illicit opioid use has been declared a national crisis in the United States. This growing number of overdose deaths can in part be attributed to the increased frequency of fentanyl contamination in the United States heroin supply. To combat this growing trend, we designed a vaccine containing a mixture of heroin and fentanyl hapten-conjugates as a proof-of-concept immunotherapy targeting a combination of these drugs. Rodents immunized with the admixture vaccine showed drug retention in serum and reduced distribution in the brain after administration of an intravenous bolus of heroin coadministered with fentanyl (10% w/w). Moreover, the admixture vaccine performed as well as or better than individual immunoconjugate vaccines in antinociception behavioral models and recognized six other fentanyl analogues with nanomolar affinity. Taken together, these data highlight the potential of an admixture vaccine against heroin contaminated with fentanyl.