Giancarlo Tonon

Effects of bromocriptine on central dopaminergic receptors

Abstract Bromocriptine injected to rats induces an increase of cAMP levels in the striatum in vivo. The time course of this increase is very similar to that of apomorphine. However bromocriptine does not stimulate striatal dopamine-sensitive adenylate cyclase but surprisingly antagonized the activation of this enzyme elicited by dopamine. Possible hypotheses on various sites of action of the drug are discussed.

Alternative BCR/ABL Splice Variants in Philadelphia Chromosome–Positive Leukemias Result in Novel Tumor-Specific Fusion Proteins that May Represent Potential Targets for Immunotherapy Approaches

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor

Poly(ethylene glycol) (PEG) is a widely used polymer employed to increase the circulating half‐life of proteins in blood and to decrease their immunogenicity and antigenicity. PEG attaches to free amines, typically at lysine residues or at the N‐terminal amino acid. This lack of selectivity can present problems when a PEGylated protein therapeutic is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic modification of proteins is one route to overcome this limitation. Bacterial transglutaminases are enzyme candidates for site‐specific modification, but they also have rather broad specificity. The need arises to be able to predict a priori potential PEGylation sites on the protein of interest and, especially, to be able to design mutants where unique PEGylation sites can be introduced when needed. We investigated the feasibility of a computational approach to the problem, using human granulocyte colony‐stimulating factor as a test case. The selected protein is therapeutically relevant and represents a challenging problem, as it contains 17 potential PEGylation sites. Our results show that a combination of computational methods allows the identification of the specific glutamines that are substrates for enzymatic PEGylation by a microbial transglutaminase, and that it is possible to rationally modify the protein and introduce PEG moieties at desired sites, thus allowing the selection of regions that are unlikely to interfere with the biological activity of a therapeutic protein.

LSD and dopamine-sensitive adenylate-cyclase in various rat brain areas.

The mechanism or mechanisms by which LSD induces its hallucinogenic effects are not yet understood 1,~,6,7. Biochemical, physiological and behavioral data suggest that LSD interacts at the level of monoaminergic synapses in brain 9,17. Recently Pieri and Pieri 14 have observed that LSD, as apomorphine, induces circling behavior in rats which had been unilaterally injected in the medial forebrain bundle with 5,6-dihydroxytryptamine or 6-hydroxydopamine. The circling behavior induced by LSD or apomorphine in the lesioned rats was blocked by haloperidol, indicating that LSD, like apomorphine, may stimulate directly dopamine (DA) receptors in the striatum. On the other hand, Uzunov and Weiss 19 have described that D-LSD produces an increase of the concentration of cyclic AMP either when incubated in vitro with slices of rat brain stem or in rat brain stem and cerebrum after in vivo administration. Moreover, these authors showed that the neuroleptic trifluoperazine both in vitro and in vivo inhibits the accumulation of cyclic AMP elicited by D-LSD. However, Uzunov and Weiss have left open the question of how D-LSD increased the concentration of cyclic AMP in brain since none of the psychotomimetic drugs they studied altered the activity of either adenylate cyclase or phosphodiesterase in cerebrum, brain stem, cerebellum or pineal gland homogenates. More recently DA-sensitive adenylate-cyclases have been identified in homogenates of the rat caudate nucleus 10, tuberculum olfactorium 5, nucleus accumbens s and limbic cortex 4. Haloperidol and other antipsychotic neuroleptics antagonize the activation by DA of this enzyme. It was therefore our purpose to investigate whether D-LSD directly and selectively stimulates DA-sensitive adenylate-cyclase in striatum and in other areas of the limbic system which appears to be more strictly linked to perception, feeling and emotional behavior. Male Charles River rats, weighing 120--150 g, were used in our experiments.

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.

A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization

We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH(2)-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8°C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.

Tailored PEG for rh-G-CSF analogue site-specific conjugation.

A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH(2) was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH(2))(11)-Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH(2))(11)-NH(2) was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal (PEG-C(18)-Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by (1)H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal/PEG-NH(2) molar ratio, %]. Both PEG-C(18)-Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to (17)Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C(18)-Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.

INTERACTION AMONG ENKEPHALINERGIC AND DOPAMINERGIC SYSTEMS IN STRIATUM AND LIMBIC FOREBRAIN

ABSTRACT The reciprocal influences of the dopaminergic and enkephalinergic systems in striatum have been studied by measuring ( 3 H)-D-ALA 2 -binding and dopamine (DA) metabolism in various experimental conditions.

Localization of dopamine receptors in the rat cerebral cortex

Only four major dopaminergic neuronal tracts had been described until recently in the mammalian brain: the nigrostriatal tract, the retinal tract, the tuberoinfundibular tract and the mesolimbic tract (Ungerstedt, 1971). Through consideration of the known anatomy and physiology of these tracts, several investigators have concluded that the mesolimbic dopaminergic tract may be the most likely candidate for a relevant role in the schizophrenic process (Matthysse, 1973; Stevens, 1973; Torrey & Peterson, 1974). However, the presence of dopaminergic terminals in some regions of the rat cerebral cortex has been documented more recently (Thierry, Blanc & others, 1973; Thierry & Glowinski, 1973 ; Lindvall & Bjorklund, 1974; Hokfelt, Ljungdahl & others, 1974b; Hokfelt, Fuxe & others, 1974a; Berger, Tassin & others, 1974). Additional observations indicate that the dopamine terminals of the cerebral cortex originate from the same areas (A9 and A10) where the nigrostriatal and mesolimbic terminals have their origin (Lindvall, Bjorklund & others, 1974). Moreover, since the cerebral cortex is most likely associated with some of the major symptoms of schizophrenia, such as disturbances of thinking and symbolic processes (Kety & Matthysse, 1972), the discovery of cortical dopamine terminals gives further support to the hypothesis of a role of dopamine in the pathogenesis of schizophrenia. On the other hand, adenylate cyclases, which are specifically sensitive to dopamine, have also been described in homogenates of bovine superior cervical ganglia (Kebabian & Greengard, 1971), calf and rat retinas (Brown & Makman, 1972), rat caudate nucleus (Kebabian, Petzold & Greengard, 1972), and n. accumbens and tuberculum olfactorium in the mesolimbic system (Horn, Cuello & Miller, 1974). These findings have led to the suggestion that in these areas the dopamine-sensitive adenylate cyclase and the dopamine receptor may be related, and that the physiological effects of dopamine could be mediated by cyclic adenosine monophosphate (cyclic AMP). Hunger & Roberts (1973) have recently reported the presence of a dopamine stimulated adenylate cyclase in a cell-free preparation of rat cerebral cortex. However, no specific dopamine-sensitive adenylate cyclase activity has been described so far in homogenates of discrete areas of the cerebral cortex. We have, therefore, examined areas of the cerebral cortex to establish the existence of a specific dopamine sensitive adenylate cyclase activity. Male Charles River rats, ll(r130 g, were used. Adenylate cyclase activity was measured in homogenates of the striatum and two cortical regions, frontal neocortex and entorhinal cortex, according to Kebabian & others (1972) with some modifications. After 3 min of incubation the reaction was stopped by boiling the samples for 3 min. The cyclic AMP was purified and assayed using the method for cAMP dependent protein kinase described by Kuo & Greengard (1972). Adenylate cyclase activity was measured by adding several concentrations of dopamine and noradrenaline. Section of the various cortical areas was according to Berger & others (1974). Protein was measured according to Lowry, Rosebrough & others (1951).

STEREOSPECIFIC EFFECTS OF (‐)SULPIRIDE ON BRAIN DOPAMINE METABOLISM AND PROLACTIN RELEASE

Abstract— Sulpiride, an unusual neuroleptic, was resolved into its two enantiomers by a newly developed procedure. (‐)Sulpiride is several times more active than (+)sulpiride in increasing rat prolactin secretion and DOPAC concentrations in rat strialum and limbic areas. These results are in agreement with clinical data showing that (‐)sulpiride is the active form of the drug.