[Evaluation of the prolonged immunotropic effect of the potential antiparkinson agent himantane]. / Otsenka prolongirovannogo immunotropnogo éffekta gimantana--potsentsial'nogo protivoparkinsonicheskogo sredstva.

A prolonged immunotropic effect of himantane, a new potential antiparkinsonian drug with a multicomponent (including dopaminopositive) mechanism of action, upon the functional activity of splenic B-cells in mice was studied in comparison to the effect of a typical neuroleptic drug haloperidol. A single administration of himantane (10 mg/kg) stimulated the B-cell activity over a period of 21 days, while haloperidol (0.25 mg/kg) suppressed this activity for 14 days after administration. The results of experiments on the adjuvant arthritis (paw edema) model showed that a single administration of himantane in the same dose under these pathological conditions does not influence the immune response (B-cell activity) for 14 days and increases the number of antibody-forming cells only 21 day after injection. Himantane enhanced the model arthritis manifestations in the early stage and reduced them in the late stage. It was established that the pronounced effect of himantane on the activity of immunocompetent cells is probably related to the drug action upon the central mechanisms of immunoregulation (which is consistent with a prolonged effect observed upon a single administration). This immunotropic activity indicates that the drug may affect immunological mechanisms involved in the pathogenesis of Parkinson's disease.

Anti-idiotypes against an anti-haloperidol antibody bind to sigma receptors.

Anti-idiotypic monoclonal antibodies that interact with the binding site of sigma receptors were generated. First, BALB/c mice were immunized with a haloperidol-bovine serum albumin conjugate, and monoclonal anti-haloperidol antibodies that recognize the piperidinyl moiety of haloperidol molecule were obtained. Second, for generation of anti-idiotypic antibodies, BALB/c mice were immunized with the anti-haloperidol monoclonal antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antisera and three hybridomas secreting anti-idiotypic monoclonal antibodies were obtained. All of them were shown to inhibit [3H]haloperidol binding to the anti-haloperidol antibodies. The anti-idiotypes were potent in displacing the binding of [3H]haloperidol to rat brain sigma receptors. Furthermore, they significantly immunoprecipitated the sigma receptors from a detergent-solubilized preparation. These findings demonstrate the generation of anti-idiotypic monoclonal antibodies specifically interacting with membrane-bound and solubilized sigma receptors.

Binding of the neuroleptic drug haloperidol to a monoclonal antibody: refinement of the binding site molecular model using canonical structures.

The ligand binding site of a monoclonal antibody (185), which binds the neuroleptic drug haloperidol, has been modelled using canonical structures and energy minimization techniques. This refined modelling protocol has allowed us to predict the variable region loop conformations. Three key residues, H:50(W), H:100a(D) and L:96(Y) appear to create the basis of the electrostatic, pi-pi stacking interactions and hydrogen bonding required for the high affinity binding site characteristics present in this antibody. The use of computer-aided graphics techniques and appropriate three-dimensional modelling permits inspection of the predicted molecular recognition features of the ligand binding site.

[Antibodies to neuroleptics and their role in the mechanisms of the development of drug resistance in psychopharmacotherapy of patients with paranoid schizophrenia]. / Antitela k neiroleptikam i ikh rol' v mekhanizmakh formirovaniia terapevticheskoi rezistentnosti pri psikhofarmakoterapii bol'nykh paranoidnoi shizofreniei.

A study was made of the content of drug antibodies (DAT) to chlorpromazine, triphthazine and haloperidol in the blood sera of 112 patients with paranoid schizophrenia (acute period, curable exacerbation associated with the chronic course, and a group of therapeutically resistant subjects). Antibodies to neuroleptics were demonstrable in all the patients' groups. However, the rate of positive reactions and the content of DAT in the therapeutically resistant patients differed significantly from the characteristics in "acute" patients and in the group of "nonresistant" ones. The maximum "antigenicity" was shown by chlorpromazine, the minimum by haloperidol. The appearance and the content of DAT agree well with the duration of the drug intake, especially if the treatment is repeated. In acute conditions, the content of DAT was found to depend on the increment of the daily dose (with the exception of haloperidol). In chronic conditions and repeated treatments, the rise of the content of DAT was feasible with the low daily doses, particularly to chlorpromazine.

Use of antisera in the isolation of human specific conjugates of haloperidol.

1. Three conjugated metabolites of haloperidol were isolated from urine of patients on haloperidol and purified by h.p.l.c. with immunological detection, using three types of anti-haloperidol antisera. 2. Structures of the metabolites were: a sulphate conjugate of the 2-hydroxylated 4-fluorophenyl ring of reduced haloperidol (MH-1), a glucuronide conjugate at the same position as MH-1 (MH-2), and a glucuronide conjugate of the hydroxy group of haloperidol (MH-3). 3. MH-3 was the main urinary metabolite in volunteers receiving haloperidol, who excreted 18% of the dose in the 24 h urine as MH-3, while other conjugates were less than 1%. MH-3 could not be hydrolysed with beta-glucuronidase, due to steric hindrance. 4. Immunological detection of conjugated metabolites is very useful in metabolic studies in humans because of its sensitivity and specificity.

Production and characterization of monoclonal idiotypes and anti-idiotypes for small ligands.

The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availability of labeled antigens. Use of the intrasplenic injection technique for the final booster immunization prior to the fusion protocol has enabled us to achieve more consistent results than the usual intravenous or intraperitoneal injection routes. Isoelectric focusing of tissue culture supernatant from monoclonal antibody-secreting clones can easily identify possible duplicate clones, and thereby reduces the amount of labor required for extensive characterization of a large number of clones. We have found that these techniques have enabled us to identify "sister clones" or redundancies in our collection of antiligand and anti-idiotype antibodies rapidly and accurately. These various techniques have allowed us to save much time, labor, and money in the search for specific antibodies with desired characteristics.

Antibodies against haloperidol specific to the butyrophenone moiety.

Polyclonal and monoclonal antibodies to the D2-dopamine receptor antagonist haloperidol were produced by immunization with haloperidol conjugated to bovine serum albumin either through the tertiary hydroxy group of the piperidine ring (halo(OH)-BSA), or through the keto group of the butyrophenone (halo(CO)-BSA). Polyclonal rabbit antisera raised against halo(OH)-BSA exhibited high affinity for [3H]haloperidol. A fraction of these antibodies also bound [3H]spiperone with high affinity. Inhibition of the [3H]spiperone binding by various butyrophenone derivatives displayed a specificity similar to that observed for the inhibition of [3H]spiperone binding to the D2-dopamine receptor. Both monoclonal antibodies raised against halo(CO)-BSA(AG-58) and against halo(OH)-BSA(AC-91) exhibited high binding affinities to haloperidol. Monoclonal antibody AG-58 cross-reacted primarily with butyrophenone derivatives that are closely related to haloperidol in their substitutions on the piperidine ring. On the other hand, monoclonal antibody AC-91 cross-reacted with a wide range of butyrophenones with binding specificities resembling those of the dopamine receptor.

Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor.

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [3H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D2-dopamine receptors.

Haloperidol binding to monoclonal antibodies: conformational analysis and relationships to D-2 receptor binding.

A library of 22 monoclonal antibodies to the D-2 dopaminergic receptor antagonist haloperidol has been developed by immunizing BALB/c mice with two conformationally distinct immunogens. The two immunogens were prepared by coupling haloperidol to bovine serum albumin through the tertiary alcohol at the 4-position of the piperidine ring with a succinic acid linkage and by coupling to bovine serum albumin through the ketone group of the butyrophenone with a carboxymethyl oxime linkage. Seventeen monoclonal antibodies displayed specific, saturable, high affinity binding of [3H]haloperidol which could be inhibited by a variety of neuroleptic drugs. Three monoclonal antibodies raised against the succinic acid conjugate and two monoclonal antibodies raised against the oxime conjugate were selected for detailed analysis of the molecular characteristics of binding specificity and for relationships to bovine striatal D-2 dopaminergic receptor binding. The monoclonal antibody with highest affinity for haloperidol, 185(2)-1 (raised against the succinic acid conjugate and herein referred to as mAb A), had a Kd of 3.3 (+/- 0.06) nM and primarily recognized chemical determinants in the butyrophenone ring (ring 1) and side chain. Inhibition of [3H]haloperidol binding to mAb A by 16 unlabeled haloperidol analogs displayed a good correlation [r = 0.82, n = 16, m = 1.06(+/- 0.38)] with D-2 receptor binding affinity, suggesting that the parts of the D-2 receptor combining site which recognize butyrophenone antagonists may have molecular characteristics which are similar to those of the monoclonal antibody. Other dopaminergic ligands such as dopamine and the D-1 antagonist SCH-23390 were not recognized by monoclonal antibodies raised against the succinic acid conjugate. Monoclonal antibodies raised against the oxime conjugate such as 258(2)-1 (herein referred to as mAb D) primarily recognized chemical determinants in ring 2 and the tertiary amino group of the piperidine ring. Although the D-2 receptor and mAb D both prefer electron-withdrawing substituents in the para position of ring 2, the antibody was more sensitive than the receptor to changes and displayed affinities that were much lower for substituents attached to the p-chlorophenyl ring (ring 2), which were electron donating. In addition, dopamine was able to completely displace 4 nM [3H]haloperidol from mAb D at a concentration of 6 mM.

A simple 'free 3H-ligand' assay for rapid screening of hybridoma monoclonal antibodies against neurotransmitter analogs and anti-idiotype antibodies.

Effective, rapid screening of hybridoma supernatants for monoclonal antibodies against the dopaminergic antagonists pimozide and haloperidol, and the serotonergic antagonist ketanserin was performed using a 'free 3H-ligand' assay. Anti-mouse Ig-coated microtiter plates were incubated with hybridoma supernatants prior to incubation with excess 3H-ligand. After removal of free 3H-ligand, bound 3H-ligand was eluted with acid for liquid scintillation counting. With minor modification, the assay can be used to screen hybridomas for anti-anti-ligand (anti-idiotypic) antibodies.

Preparation and characterization of antisera and monoclonal antibodies to haloperidol.

For the first time a library, of monoclonal antibodies (MoAbs) to the butyrophenone haloperidol (D-2 antagonist) has been prepared. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was used to develop two polyclonal antisera and twelve MoAbs specific for the hapten. Our library of MoAbs can be grouped into three classes; 1) high affinity and specificity for free 3H-haloperidol, 2) moderate affinity with significant cross-reactivity to other butyrophenone ligands, and 3) a group which binds poorly to free 3H-haloperidol but instead recognizes the ligand only when it is coupled to carrier protein. Clone (189(2)-6) was found to have the highest equilibrium binding affinity (Kd = 4 nM) and is far more specific than the currently available antisera to haloperidol. This MoAb has significantly lower affinity for all of the common metabolites of haloperidol. This capability makes 189(2)-6 a candidate for further development with regard to use in clinical radioimmuno-assays of therapeutic drug levels. In addition, one of the anti-haloperidol Moabs (190(2)-6) binds more tightly to spiperone than to haloperidol and displays a qualitative correlation in the rank order of neuroleptic binding affinity for a limited series of analogs when compared to membrane bound D-2 receptor binding.

Antisera raised against the drug haloperidol.

Antisera against haloperidol coupled to albumin have been raised in two rabbits. Both antisera bind haloperidol with high affinity but differ in their selectivity for binding other substances. A sub-population of anti-haloperidol antibodies has been partially purified from one antiserum by affinity chromatography. This sub-population resembles the D2 dopamine receptor in its ability to bind some D2 receptor selective antagonists.

Preparation and characterization of antisera and monoclonal antibodies to serotonergic and dopaminergic ligands.

In an attempt to produce polyclonal antisera and monoclonal antibodies to serotonin, SKF 38393 (D-1 agonist), dopamine, and haloperidol (D-2 antagonist) several procedures for the preparation of immunogenic ligand-protein carrier conjugates were investigated. The Mannich reaction utilizing formaldehyde as the chemical linker was used to prepare serotonin-protein conjugates; antibodies raised to this conjugate reacted specifically to the conjugated serotonin moiety but did not react to native serotonin. Chemical conjugations involving dimethylpimelylimidate or N-carboxymethyl derivatives for the coupling of serotonin, dopamine and SKF 38393 to carrier proteins produced antibodies primarily directed against the 'chemical coupling arm' and very little antibody activity against the ligand itself could be detected. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was capable of eliciting both polyclonal and monoclonal antibodies specific for the hapten. The pitfalls of the various chemical conjugation procedures and the difficulties of producing antibodies to free ligands are discussed.

Quantification of reduced haloperidol and haloperidol by radioimmunoassay.

A radioimmunoassay for reduced haloperidol and haloperidol has been developed by using a simple derivatization-separation step prior to assay with an antibody cospecific for both compounds. The detection limit of the assay is less than 25 pg and shows no cross reactivity to other metabolites. The intraassay coefficient of variation for reduced haloperidol and haloperidol were 9.0 and 8.2% respectively and the interassay coefficients of variation were 9.0 and 10.6% respectively at 5-10 ng/ml. As many as 30 patient samples can be analyzed for both compounds in a single day.

[Distribution of functionally differing cells in immunocompetent organs on haloperidol administration]. / Raspredelenie funktsional'no otlichaiushchikhsia kletok v immunokompetentnykh organakh pri vvedenii galoperidola.

Haloperidol blockade of the dopaminergic system entails redistribution of functionally different cells. Suppressors of IgM and IgG response migrate, respectively, from the spleen and lymph nodes to the bone marrow whose cell transfer leads to inhibition of immune response. The data obtained indicate common regularities in cell distribution under blockade of the dopaminergic and activation of serotoninergic systems. Therefore, at the basis of inhibition of immunogenesis there lie monotypic processes of suppressor cell migration whatever the system responsible for it.

Haloperidol pharmacokinetics: a preliminary study in rhesus monkeys using a new radioimmunoassay procedure.

The development, validation, and application of a new radioimmunoassay for haloperidol in biological fluids is described. The antiserum, raised against N-amino-butyl chlorophenyl piperidine bovine albumin conjugate, could not distinguish between haloperidol and its reduced metabolite, but it could discriminate against chlorophenyl piperidine (cross-reaction 2.6%). The fluorophenyl metabolites of haloperidol were not recognized by the antiserum. Haloperidol determinations were made on less than 100 microliter aliquots of human and rhesus monkey plasma or diluted urine without prior extraction of the sample. The radioimmunoassay was applied to the study of the pharmacokinetics of intravenous haloperidol administration to two male rhesus monkeys. Salient features of the results are as follows. As with man, the plasma concentration versus time curve could be resolved into three compartments, but there were differences in the distribution of haloperidol between the compartments. The apparent volume of distribution for the two monkeys examined was 5.87 L kg-1 and 7.37 L kg-1, considerably smaller than in man, a difference almost entirely due to a much smaller tissue compartment. The biological half-life of 15.97 hr and 7.56 hr was similar to man. The mean hepatic extraction ratio was calculated to be 0.032 and 0.056, and the data suggested that hepatic metabolism of haloperidol may be of lesser importance in rhesus monkey than in man. An insignificant proportion (0.01%) of the administered dose was excreted as haloperidol in the urine.

A new radioimmunoassay for haloperidol: direct measurement of serum and striatal concentrations.

A sensitive and specific radioimmunoassay for haloperidol has been developed. Antibodies were elicited in rabbits immunized with haloperidol hemisuccinate coupled to bovine serum albumin. Optimum sensitivity was obtained with a 1:4000 dilution of the antisera, permitting the detection of as little as 2.6 ng/ml of haloperidol. Major metabolites of haloperidol did not cross-react in the assay. The method does not require an extraction procedure and can be performed using as little as a 5-microliter sample. Haloperidol levels in rat serum and striatum were determined with this method, after the i.v. administration of different dosages of the drug. The distribution and elimination of haloperidol was linear over the dose range to have an elimination half-life of approximately 2.6 hr at all three dosage levels.

[Effect of apomorphine and haloperidol on the immune reaction]. / Vliianie apomorfina i galoperidola na immunnuiu reaktsiiu.

It has been established that injection of apomorphine leads to the dose-dependent enhancement of the immune response in CBA mice whereas that of haloperidol suppresses the number of rosette-forming cells. After thymectomy apomorphine does not stimulate rosette-formation. This indicates that the thymus participates in the realization of apomorphine action. Preliminary injection of haloperidol prevents the apomorphine-induced enhancement of the immune response. The results suggest that the enhancement is mediated through the activation of dopamine receptors.