Search

The possibility that immunological mechanisms are involved in the development of tolerance to the analgesic effect of morphine was investigated, focusing on several immunological parameters. The data indicate that transfer of spleen cells and bone marrow cells obtained from morphine-tolerant donors into nontolerant syngeneic recipient mice did not result in the transfer of tolerance to the analgesic activity of morphine. Furthermore, the data indicate that the thymus, which is essential for the immunological response to many antigens, was not involved in the induction of tolerance to the opiate since tolerance was induced in thymectomized mice as well as in genetically athymic mice. The results suggest that immunological factors do not seem to play a crucial role in the phenomenon of tolerance to the analgesic activity of morphine.

Antiserum against morphine was produced in rabbits immunized with morphine hapten conjugated to bovine serum albumin. The carrier protein was conjugated to the nitrogen atom of the opiate alkaloid in order to make the phenolic hydroxy group on C3 and the alcoholic group on C6 as determinant groups. The antibody does not recognize codeine or the major metabolite of morphine, 3-O-monoglucuronide. This antibody was used in conjunction with an antibody prepared against 3-O-carboxymethylmorphine to develop a radioimmunoassay which can measure codeine in the presence of morphine. The assay was used to follow both the plasma and brain levels of codeine and its biotransformation to morphine. Codeine when administered at a dose of 5 mg/kg i.v. showed a biphasic plasma decay curve the first phase of which had a +1/2 of 26 min. Peak concentrations of morphine were detected in the plasma following that dose of codeine at 0.5 h. 30 min after the injection of 20 mg/kg i.p. codeine, the brain levels of morphine were only 2% that of codeine. Thereafter, the brain levels of morphine slowly declined.

Morphine was demonstrated to exacerbate infections. Experiments were performed to evaluate variations of phagocytic physiology during morphine treatment. In mice, morphine drastically reduced reticuloendothelial system activity, phagocyte count, phagocytic index, killing properties, and superoxide anion production in polymorphonuclear leukocytes and macrophages. Similar effects on alveolar macrophage count, phagocytosis, and killing were found in rabbits, a result which suggested a lack of species specificity. Additional experiments demonstrated that morphine (1) induces a reduction of lymphoid organ weight, (2) impairs the ability to eradicate infections and (3) is counteracted in its depressing activity on phagocytic physiology by small amounts of Corynebacterium parvum. The results suggest that there is a close relationship between the fact that morphine exacerbates infections and the fact that morphine depresses phagocytic functions; therefore, the negative effect of morphine on phagocytosis is at least one of the reasons for its negative effect on the development of infections.

IgE antibodies that reacted with morphine and codeine were detected in the serum of a subject who experienced a life-threatening anaphylactic reaction following the administration of Omnopon-Scopolamine (papaveretum-hyoscine). Hapten inhibition studies with morphine and a number of structurally-related analogues revealed that morphine and codeine were the most potent inhibitors of IgE binding to a morphine-solid phase. Nalorphine, meperidine, and methadone were also good inhibitors of IgE binding, but naltrexone, buprenorphine, and fentanyl proved to be poor inhibitors. From a detailed examination of structure-activity relationships, the authors conclude that the important structural features of the morphine allergenic (that is, IgE binding) determinant comprises the cyclohexenyl ring with a hydroxyl group at C-6 and, most important of all, a methyl substituent attached to the N atom. The authors' findings suggest that morphine analogues administered to such a patient may provoke clinical anaphylaxis. Hyoscine reacted weakly with IgE antibodies in the subject's serum, but this was thought to be due to weak cross-reaction between this compound and morphine.

beta-Endorphin was isolated from 200 rat pituitaries by means of high-performance column chromatography, using sensitive fluorometric methods and a radioreceptor assay for opioid activity. The beta-endorphin was characterized as to molecular weight, amino acid composition, and mapping of tryptic peptides by a new microtechnique. It was found that rat beta-endorphin is identical to camel and sheep beta-endorphin. Furthermore, alpha-endorphin, Met-enkephalin, a nonpeptide morphine-like compound (MLC), and an additional unidentified opioid compound were detected in the extract of rat pituitary.

Antimorphine antibodies produced in Rhesus monkeys immunized with morphine-6-hemisuccinate-BSA were passively administered to recipient monkeys trained to self-administer heroin and cocaine. Following antibody administration, changes in heroin self-administration behavior were observed which were similar to those achieved with low doses of naloxone. Both manipulations increased heroin self-administration without affecting cocaine responding.

This report describes a new technique, immunofluorescence, for the detection and possible characterization of drug content in postmortem tissues. By using antisera generated against a drug-protein conjugate, the stabilization of tissue-sequestered drug is accomplished by incubation of fresh frozen sections of tissue with dilute solutions of rabbit anti-drug antibodies. Secondary incubation with a fluorescence-labeled anti-rabbit immunoglobulin labels these points of sequestration. Tissue sections so stained are examined by fluorescence microscopy. In studies with rats given graded doses of morphine sulfate, there were discernible differences in tissue binding of morphine in brain sections from animals treated "therapeutically," fatally, and chronically. Extension of these studies to human autopsy material is anticipated and potential problems are discussed. This technique offers the forensic toxicologist the potential for evaluating the drug content of tissues in situ.

The biologic impact and clinical toxicity of Corynebacterium parvum administered at a dose of 5 mg/M2 by intravenous or subcutaneous routes were evaluated in 18 children receiving combination chemotherapy for maintenance of acute lymphoblastic leukemia (ALL) in remission. Several nonspecific immunologic and hematologic parameters were evaluated. Patients were also monitored for changes in cutaneous sensitivity to histamine. No changes in any parameter were observed in patients after only one course of C parvum injection. However, after 6-10 courses, glass-adherent peripheral blood leukocytes of C parvum-treated patients augmented the response of PHA-stimulated autologous lymphocytes. In all nine patients studied who received C parvum injection subcutaneously for at least six months, there were significant increases in the mean bone marrow myelocyte-erythrocyte (ME) volumes compared to pretreatment values. These results suggest that periodic evaluations are desirable in patients receiving repeated administration of C parvum, since changes in immunologic and hematologic responses may be demonstrable only after several injections. In contrast to the reported experience in adults, subcutaneous C parvum administration children was not well tolerated, whereas intravenous infusion was generally well tolerated.

A general immunochemical method for the assay of haptens and proteins has been devised and applied to morphine, a morphine-albumin conjugate, and human immunoglobulin G. A fluorescein-labeled antigen and a quencher-labeled antibody are employed. By use of fluorescein and rhodamine as the fluorescer and quencher, respectively, dipole-dipole-coupled excitation energy transfer can occur within the antigen-antibody complex. The resulting quenching of fluorescence can be inhibited by competitive binding with unlabeled antigen, Alternatively, separate antibody samples can be labeled with fluorescein and rhodamine, respectively. Unlabeled antigen causes aggregation of the separately labeled components with resultant quenching. Using the latter method, experiments suggest that up to about 20 anti-morphine antibody binding sites will associate with morphine-albumin conjugates. When an excess of the conjugate is present the antibodies appear to assemble in clumps on the protein surface. Mathematical analysis of the quenching of fluorescein-labeled morphine by rhodamine-labeled anti-morphine gives an approximate fit to the quenching data, but the calculations are very dependent on the assumptions used.

1 Fragments of the N terminal part of adrenocorticotrophic hormone (ACTH) inhibited the electrically evoked contractions of the mouse vas deferens. This inhibition could be antagonized by naloxone. 2 The same fragments displaced radiolabelled morphine from morphine antiserum. 3 Structure-activity relationship studies showed that in both assay systems the active core is located within the sequence ACTH 7--10. 4 It is postulated that the Trp9 residue and the peptide bond between Trp9 and Gly10 are particularly important for interaction of ACTH fragments with morphine receptors.

We describe nonpeptide opioids found in extracts of beef hypothalamus and adrenal, which are recognized by antisera raised against morphine. Four have been purified to homogeneity. One is morphine. The structures of the other three have not been determined yet. None of them are derived from morphine or normorphine after extraction from the tissues. It is not known whether the opiates described here are of endogenous or exogenous origin.

The suppression of skin test reactivity by single doses of six antihistamines was measured before and after a period of daily antihistamine ingestion in 18 subjects. Single doses of hydroxyzine, 50 mg; chlorpheniramine, 16 mg; and promethazine, 50 mg; induced significant suppression of skin test reactivity at 2 hr, whereas the suppression produced by tripelennamine, 100 mg; diphenhydramine, 50 mg; and cyproheptadine, 16 mg; did not differ significantly from that produced by placebo. After 3 wk of treatment with hydroxyzine, 75 mg per day, the suppressive effect of hydroxyzine as well as the five clinically unrelated antihistamines was significantly reduced. Although the response to chlorpheniramine was also reduced after chronic treatment with chlorpheniramine, 24 mg per day, the difference was not statistically significant. We conclude that antihistamines in the doses used differ greatly in their suppressive effect on skin test reactivity. The antihistamine producing the most skin test suppression, hydroxyzine, when it was taken daily for 3 wk, caused the development of partial tolerance not only to its own effect but to those of clinically unrelated antihistamines.

The role of the immune system in the development of physical dependence and tolerance to morphine was studied in mice in which the immune response was either stimulated or suppressed. Immunization of mice against morphine increased the blood and brain levels of morphine as compared to controls. However, the development of physical dependence and tolerance was decreased. The chronic responses to morphine were also decreased by nonspecific immunosuppression (vincristine-cyclophosphamide treatment and gamma-irradiation exposure) and specific immunosuppression (antithymocyte and antilymphocyte sera treatment). Immunosuppressive treatments did not alter the rate of morphine absorption from the subcutaneous depot used to induce chronic exposure to the drug. However, the blood and brain levels of morphine were higher than control after 72 hours of morphine pellet implantation. It is apparent that manipulation of the immune system can alter the physical dependence and tolerance development to morphine.

Two radioimmunoassays (RIA-A and RIA-B) were used to estimate morphine equivalents (ME) in rabbit serum after i.v. administration of morphine or morphine-3-glucuronide (M-3-G). RIA-A and RIA-B differed only with respect to the particular pool of antibodies used, and both were elicited from rabbits immunized with a 2-diazomorphine-protein conjugate. In vitro inhibition studies indicated the molar ratio of M-3-G/morphine which inhibited 3H-morphine-antimorphine binding by 50% (150) was 90 and 14 for RIA-A and RIA-B, respectively. RIA-B 150 values were dependent on the final protein concentration of the incubation mixture. RIA-A and RIA-B estimates of the ME per milliliter were in agreement in serum samples obtained from rabbits after i.v. morphine 0.01 mg/kg; however, after higher doses only samples obtained in the first minutes agreed and at later times RIA-B estimates exceeded RIA-A by increasing amounts. After i.v. M-3-G (1.0 mg/kg) RIA-B estimated 20 to 25 times more ME per milliliter than RIA-A. After rapid i.v. administration of morphine (1 or 5 mg/kg) the disappearance curves obtained with RIA-B were multiphasic and contained a prolonged terminal phase with an estimated half-life of 24 hours. The higher estimates obtained with RIA-B are best explained by cross-reactions with M-3-G or other morphine metabolites. With RIA-A the mean serum morphine clearance (86 ml/min/kg) and half-life of the elimination phase (74 minutes were independent of dosage. The least complicated pharmacokinetic model which describes the disposition of morphine in the rabbit is the open two-compartment model.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.

Polyclonal, anti-idiotypic, rabbit antibodies have been raised against four murine monoclonal anti-morphine Fab fragments. The antibody preparations, after affinity purification, have been shown to contain an anti-paratypic fraction which reversibly inhibits morphine binding to the anti-morphine antibodies and to cellular opiate receptors. Using some of the unique properties of this system, for the first time cross-reactivities of anti-paratypic antibodies with the monoclonal anti-morphine IgGs have been examined including competition for the same binding sites by a classical opiate agonist/antagonist pair.

Rabbit antibodies have been raised against murine monoclonal anti-morphine Fab fragments. Following affinity purification, these antibodies competitively inhibit morphine binding to anti-morphine monoclonal antibodies, yet do not recognize normal mouse IgGs, suggesting that the antibodies produced are anti-idiotypic with respect to the anti-morphine IgG. More importantly, the purified antibodies competitively inhibit binding of morphine, naloxone, and D-ala-2-D-leu-5-enkephalin to rat brain opiate receptors.

Antimorphine globulin was used to study the mechanism by which naloxone produces a contracture in ileal strips removed from morphine pellet-implanted guinea pigs. Although the antibodies effectively reversed the depressant actions of morphine on electrically stimulated contractions in both naive and tolerant tissues, neither monkey antimorphine (Kd = 1.43 x 10(-7)M) nor goat antimorphine (Kd = 1.36 x 10(-9)M) were able to elicit a naloxone-like contracture. The rate of attenuation of tissue sensitivity to naloxone was measured and no changes were observed after incubation in the presence of monkey antimorphine globulin. High-affinity goat antimorphine globulin, however, was able to accelerate the rate of attenuation of tissue sensitivity to naloxone. The naloxone contracture was accompanied by release of material reactive with morphine antiserum in a radioimmunoassay. The quantities of material were in excess of what could be bound by opiate receptors present in the tissue. Possible mechanisms of naloxone sensitivity observed in the isolated ileum are discussed.

The data on the presence of antibodies to morphine and neurotransmitters (dopamine, norepinephrine, serotonin) in the blood serum of chronic alcoholics are presented. The antibodies were detected by the modification of complement-fixation test. 82% of chronic alcoholics showed significantly elevated levels (P less than 0.01) of antibodies to morphine (5.34 +/- 0.65% of complement fixed) when compared to the control group (2.41 +/- 0.52% of complement fixed), which was an indirect evidence of the presence of endogenous morphine-like compounds in men and their role in the pathogenesis of the disease. A general group of alcoholics could be divided into subgroups with significantly elevated and decreased levels of antibodies to the appropriate neurotransmitter, as compared to the control group, which significantly differed from each other. Similar results were obtained in chronically alcoholized rats.

A nonpeptide morphine-like compound (MLC) which cross reacts with morphine-specific antibodies has been localized with the use of immunocytochemistry. This morphine-like compound is found in neuronal perikarya or processes (or both) in nuclei related to vestibular, cerebellar, and raphe systems.

Long-term effects of morphine administration or immunologic test responses were studied in female rabbits. Implantation of morphine-containing pellets was found to be more effective than injection of morphine sulfate solutions in promoting increased serum binding of 140-morphine. A large part of the increased morphine binding by sera associated with administration of morphine was found in serum fractions containing gamma-globulin and was absent in gamma-globulin-free fractions. These sera showed some degree of specificity for the morphine configuration in tests with other narcotics. They also gave positive immunologic test reactions in passive hemagglutination and radial immunodiffusion tests involving serum albumins conjugated with morphine derivatives. Other evidence for immunologic responsiveness against morphine by morphine-pretreated rabbits was shown by cutaneous hypersensitivity reactions against morphine-carrier conjugates and by a diminution of the serum morphine-binding response in rabbits given an immunosuppressive dose of cyclophosphamide. Failure of naloxone, a morphine antagonist, to alter the serum morphine-binding response suggested that serum levels of the morphine-binding globulin studied here were not direclty related to morphine withdrawal.

Conjugates morphine-protein which are formed in the systems of cytochrome P-450 and horse-radish peroxidase can induce in rabbits synthesis of the specific antibodies to morphine. In the blood of animals immunized with the above mentioned conjugates there are antimorphine-antibodies being constituents on immunochemical complexes with endogenous ligands. Administration to rabbits of the conjugated antigen morphine-microsomes of the rabbit liver causes the development of the reaction of hypersensitivity of delayed type to morphine.

A nonpeptide opioid was found in toad skin and purified to homogeneity by using HPLC with electrochemical detection. A nonpeptide opioid also was detected in bovine brain and adrenals as well as rabbit and rat skin, by reversed-phase HPLC following Sephadex G-15 column chromatography. The material in toad skin was identical to morphine by immunological, pharmacological, and physical chemical criteria.

For constant analgesia after abdominal surgery, 34 patients (Group I) and 14 patients under intensive care (Group II) received epidural morphine infusions at mean hourly rates of 0.23 +/- 0.04 mg morphine in the first hour to 0.01 +/- 0.0 mg in the 50th hour, and 1.55 +/- 0.26 mg in the first hour to 0.20 +/- 0.08 mg in the 50th hour, respectively, after an initial bolus injection of 2 mg morphine. Whereas in Group I metabolized morphine immunoreactivity reached a plateau within 20 minutes which lasted until 8 p.m. on the operation day, in Group II a far higher plateau (p less than 0.001) was attained,--but only after some hours--which lasted until 8 p.m. on the first postoperative day. In the course of the treatment, concentrations of metabolized morphine immunoreactivity in serum and in urine decreased in both groups.

Affinity chromatography was employed successfully to purify extensively antimorphine antibodies produced in rabbits. Morphine succinylated at position 6 was attached to bovine serum albumin to make it immunogenic and to Sepharose beads for affinity chromatography. A crude gamma-globulin fraction of antimorphine serum, prepared by salt fractionation, was used for purification. After unbound gamma-globulin was washed off the column with phosphate-buffered saline, two populations of antimorphine antibodies were eluted in succession by two low pH buffers. Each of these two purified antibody fractions had an index of heterogeneity of approximately 1. Immunoelectrophoresis indicated that the antibodies were immunoglobulin G and that they formed a single precipitation band with antirabbit serum. The average association constants for morphine of the two purified antibody fractions were similar and ranged from 4.5 to 4.8 X 10(7) LITERS/MOL-1. A 67-fold purification of the antimorphine antibodies could be achieved by a single pass through an affinity column.

The specificity of antimorphine and antimeperidine antisera was measured by competitive displacement of immunizing radiolabeled haptens. Antimorphine antisera demonstrated a high degree of specificity for a conformation of the phenylpiperidine moiety contained within the structures of morphine and its congeners of the morphinan and benzomorphan series. Antimeperidine antisera demonstrated a high degree of specificity for a different conformation of the phenylpiperidine moiety represented within the structures of meperidine and its semisynthetic derivatives. The reactivity of methionine- and leucine-enkephalin, several synthetic enkephalin analogs, and alpha- and beta-endorphin with the antibodies was tested using purified immunoglobulin G in order to avoid serum-induced proteolysis. No significant cross-reactivity of antimorphine antibodies with any of the opioid peptides was detected. All of the opioid peptides tested exhibited weak but immunologically specific cross-reactivity with antimeperidine antibodies. These findings suggest that conformations analogous to the phenylpiperidine moiety in morphine as have been proposed for [Tyr1] in opioid peptides do not appear to be present as measured by immunochemical methods. A conformation with weak stereochemical similarity to the phenylpeperidine moiety in meperidine does appear to be present. The possible homologies between [Phe4] of opioid peptides, meperidine and hydrophobic side chains of certain oripavine derivatives are discussed.

A radioimmunoassay is described for the determination of morphine and morphine-like substances in plasma, serum, biological fluids and tissue homogenates using an antiserum to morphine-6-hemisuccinyl-BSA and 125I-morphine as the labelled tracer. In the B/F separation with ammonium sulphate, calcium sulphate was added to make the precipitate more compact. Some parameters related to the use of this method in direct assay on plasma, serum, other biological fluids and tissue homogenates were evaluated.

Anti-idiotypic antibodies which recognize the opiate receptor were generated in guinea pigs following immunization against purified rabbit anti-morphine antibodies. The anti-idiotypic antibodies produced a concentration-dependent inhibition of [3H]naloxone binding to opiate receptors in a membranous mouse brain preparation. Saturation analysis indicated that the antibodies produced a non-competitive inhibition of naloxone binding. The ability of the antibodies to interact with biological systems was investigated in in vitro systems. In both the isolated guinea pig ileal longitudinal muscle and mouse vas deferens, the antibodies produced a concentration-dependent, opiate agonist-like action. The anti-morphine anti-idiotypic antibodies appear to interact specifically with the opiate receptor and may serve as useful tools in characterization of this receptor system.

Four lines of high affinity monoclonal antibodies directed against morphine have been isolated and affinity purified. Some of their properties, including cross-reactivities to a large set of selected opiate agonists and antagonists are described. Importantly, none of the immunoglobulins cross-react with D-Ala2-D-Leu5-enkephalin.

Displacement of spin-labeled drugs from drug-specific antibodies in human serum can be measured by use of electron spin resonance. This "spin immunoassay" has advantages of speed, simplicity, and small volume requirements over other standard techniques such as gas-liquid, liquid-liquid, or thin-layer chromatography. Unlike radioimmunoassay, there is no need to separate free and bound label and there are no radiation hazards. We present clinical and experimental data showing how this method can be applied to the routine determination of morphine and diphenylhydantoin concentrations in serum.