- Email: [email protected]
FC18 central serotonergic and dopaminergic function studied by positron emission tomography (PET)
Free Communications / European Journal o f Pharmaceutical Sciences 2 (1994) 9 9 - 1 1 6
105
FC17 NON-INVASIVEIN VIVO STUDIESOF PHARMACOKINETICSAND METABOLICPARAMETERS USING EPR SPECTROSCOPY K. M~iderl,2,HM. Swarlz2,R. St(~sser3,H.-H. Borchertl 1 Instituteof Pharmacyand3 InstituteofChemistry,Humboldt-Universlty,Redin,Germany 2 DartmouthMedicalSchool,Hanover,NH,03755-3863,USA
FC18 CENTRALSEROTONERGICAND DOPAMINERGICFUNCTIONSTUDIEDBY POSITRON EMISSIONTOMOGRAPHY(PE1) P.Hartvig,J. Tedroff,P. Bjuding,K.J.Lindner,B. L.dtngstr6m ~ a UniversityPETCentre,University~ e l , 751 85 Uppcals,Sweden
The dcveloplnent of EPR equipment designed specifically for in vivo studies makes it possible to detect and follow paramagnetic species selectively' in IMng animals. Because free radicals and other paramagnctic materials arc present in low concentrations under physiological conditions, paramagnetic reporter molecules such as sterically stabilized nitroxide radicals can be introduced and studied selectively. They are available in a wide range of properlies concerning their hydrophilicity, size. stability in biological systems etc. The detected EPR spectra yield i~fformation on a number of processes that usually, are difficult In measure in vivo including pH, microviscosity, micropolarity, redox processes, and the concentration of oxygen. The use of pH- sensilive imidazole derived nitroxides will~ different pKa values permits the localized measurement of proton activity. An important example of the usefulness of this method is cmmected ~ith the in vivo study' of liberation processes inside of drug delivery systems. Although drug deliver)' systems with sustained release characteristics arc commonly used, the nlechanisnls of this release, cspcciall_v under in vivo conditions, arc poorly' understood Using a home made EPR spectrometer operating at 1.1 GHz ~c used this noninvasive mcllmd to investigate the release mechanisms of polymers subcutaneously implanted in the backs of mice The viscosib', polarity', and proton activity inside the polymer depends on the general structure of the polymer, the molecular weight, incorporated drugs, and the time after implantation. The experimental results indicate that in the case of polymers made up of polylactic and polyglycolic acid, compartments ~ith high polarity and low viscosity are formed inside the polymers. The proton activity increases with time. Therefore we have direct c~idencc in vivo that in these polymers bulk hydrolysis is a major mechanism of release. Other examples of the pharmaceutical use of in vivo EPR performed include the noninvasive characterization of drug deliver':, systems and their interactions with biological matrices, co~ering sc~crat routes of administrations (per os. iv. sc injection, ip. sc implantation) and the stud~, of drug derived radical species. Also the localized measurement of oxygca and oxygen gradients is possible b~ EPR using oxygcn-sensiti~c paramaguctic materials such as carbon chars or single cr) slals of lithimn phthaloc3 alfine K. M~ider gratefully' ackno~lcdgcs his support by Dcutschcr Akadcmischcr Austauschdienst (DAAD)
Neumtransmitter deficiency characterizes Parkinson's and Aizheimer diseases and is the working hypothesis for symptoms in a number of other psychiatnc and naurologic diseases. Efforts to increase neumtransmitter synthesis has, however, been met by varying success. It is generally agreed that the synthesis rate for the neurotransmitters dopamine and serotonin are governed by the activity of hydroxylase transferring tryptophan and tyrosine to 5-hydroxitryptophan, 5-HTP and LDOPA, respectively and that the following enzymatic step, the decarboxylation forming serotonin and dopamine can not be regulated by neuronal activity. By means of positron emission tomography, PET and using 5-HTP and L-DOPA radiolabeited in a metabolic stable position with 11C, a regional up-regulation of synthesis has been shown in patients with major depression, schizophrenia and advanced Parkinson's disease. Brain decarboxylase activity was also increased in the monkey following acute treatment with the co-factor pyridoxine, vitamine B6 for the decarboxylass. The decarboxylase enzyme was possible to saturate with the precursor amino acid and more efficiently with 5-HTP than with L-DOPA. This difference may explain the success in therapy with high doses of L-DOPA, but less using the precursors tryptophane and 5-HTP for a desired enhancement of serotonin synthesis. The co-factor for the hydroxylase enzyme, 6R-erythro5,6,7,8-tetrahydrobiopterin also increased decarboxylase activity. Furthermore, infuson of tyrosine somewhat increased, while L-DOPA infusion dramatically enhanced striatal synthesis rate of dopamine in the monkey. The decarboxylass activity was further increased by infusion of 6R-erythro-5,6,7,8-tetrahydrobiopterin. This modulation of decarboxylase activity might be explained by the recently descdbed neuronal L-DOPA receptor which on L-DOPA activation may enhance the dopamine synthesis rate. This activation of receptors may also explain the up-regulation of decarboxylase activity induced by 6R-erythro-5,6,7,8tetrahydrobiopterin as a result of an increased formation of neuronal L-DOPA. The regulation of decarboxylass may have clinical implications in the development of new pharmacological principles for treatment of diseases characterized by neumtransmitter deficiency. Furthermore, the L-DOPA controlled activity of the decarboxylase may also explain mechanisms responsible for fluctuating symptoms in patients with advanced Parkinson's disease and supports the clinical success of continuous L-DOPA therapy for control of fluctuating Parldnson disease symptoms.
FC19 ACTIVATIONOF G-PROTEINSIN PHYSIOLOGICALAND RECONSTITUTEDMEMBRANOUS SYSTEMS B. Nfimbergl, C. Harteneckl,D. Leopoldtl,R. Harhammerl,R. Seifertl, A. Hagel0kenl, H. Deted2, W. Schunack2,R. Hoppe3,E. NQmberg3 1 InstitutforPharmakologie,FreieUniversit8tRedin,14195Redin,Germany 2 InstitutffirPharmazie,FreieUniversitStBedin,14195Bedin,Germany 3 LehrstuhlforPharmazeutischeTechnologie,UniversitAtEdangen-N0mberg,91054Edangen,Germany
FC20 RIBOSEMODIFIEDADENOSINEANALOGUESAS POTENTIALPARTIALAGONISTSFOR THE ADENOSINERECEPTOR E.M.vanderWenden,J.K.K,~nzel,R.A.A.Math6t,A.F.IJzerman,M.Danhof,W. Soudijn DivisionofMedicalChemistryandPharmacdtogy,Leiden/AmsterdamCenterforDrugResearch,2300RALeiden, Netherlands
Heterotrimeric regulatory G-proteins modulate cellular effectors following activation by ligand-bound receptors. Additionally, direct stimulation of pertussis toxin-(PT) sensitive G-proteins by peptides and amphiphilic drugs has been demonstrated. Hence, it appears feasible to influence cellular functions by drug targeting o f signal transduction components downstream of the receptor, i.e. G-proteins. We compared the effects of mastoparan peptides and histamine derivatives known to be direct stimulators of G-proteins in different physiological and G-protein reconstituted membranous systems. The aim o f the study was to establish models designed to detect and analyze compounds interfering specifically with G-proteins. GTPase activity o f and GTPTS-binding to proteins were analyzed in (i) membranes of the human leukemia cell line HL-60 which physiologically expresses a broad variety of different signal transduction systems including seven-transmembranous receptors and PT-sensitive Gi-proteins, (it) different liposomes loaded with distinct mammalian G-proteins o f the G~-subfamily purified from mammalian brains or blood cells, and (iii) membranes of the eukaryotic Baculovirus/Spodoptera frugiperda (Sf9) expression system overexpressing different mammalian Gi-proteins. Whereas HL-60 membranes resembled the physiological situation very closely compared to all systems tested they exhibited the lowest level of G-protein specificity of either assay, i.e. GTPase or GTPyS-binding, due to the presence of various interfering proteins, e.g. receptors, different GTP-binding proteins and GTPases or kinases. On the other hand, artificial lipid membranous systems reconstituted with distinct purified G-proteins obviously favored specificity and allowed detection of potential new G-protein-activating compounds and analysis of the molecular mechanism of their action. Using this system, we were able to compare the mode of action and the G-protein specificity of different mastoparans and synthetic drugs. However, clear disadvantages were the time and cost consuming preparation of G-proteins and preparation of lipid vesicles. Interestingly, GTPase-activity of membranes of uninfected Sf9-cells was not stimulated by the peptides and drugs tested. In contrast, GTPase activity of Sf9 membranes expressing mammalian Ga~2- or Gcti2132yz-proteins was enhanced by all substances tested. From these experiments we suggest that the Baculovirus/Sf9 expression system may be an elegant screening model for detection of receptor-independent G-protein modulators. Supported by Deutsche Forschungsgemeinschafi (SFB 366)
Agonists for the adenosine receptor reduce blood pressure, but a hypotension can be the result. Partial agonists, however, should decrease the blood pressure to a lesser extent titan full agonists. It had been shown in the literature that an intact ribose moiety is necessary for full activity on the adenosine receptor. Thus, we synthesised and tested several 2"
7--7
105
FC17 NON-INVASIVEIN VIVO STUDIESOF PHARMACOKINETICSAND METABOLICPARAMETERS USING EPR SPECTROSCOPY K. M~iderl,2,HM. Swarlz2,R. St(~sser3,H.-H. Borchertl 1 Instituteof Pharmacyand3 InstituteofChemistry,Humboldt-Universlty,Redin,Germany 2 DartmouthMedicalSchool,Hanover,NH,03755-3863,USA
FC18 CENTRALSEROTONERGICAND DOPAMINERGICFUNCTIONSTUDIEDBY POSITRON EMISSIONTOMOGRAPHY(PE1) P.Hartvig,J. Tedroff,P. Bjuding,K.J.Lindner,B. L.dtngstr6m ~ a UniversityPETCentre,University~ e l , 751 85 Uppcals,Sweden
The dcveloplnent of EPR equipment designed specifically for in vivo studies makes it possible to detect and follow paramagnetic species selectively' in IMng animals. Because free radicals and other paramagnctic materials arc present in low concentrations under physiological conditions, paramagnetic reporter molecules such as sterically stabilized nitroxide radicals can be introduced and studied selectively. They are available in a wide range of properlies concerning their hydrophilicity, size. stability in biological systems etc. The detected EPR spectra yield i~fformation on a number of processes that usually, are difficult In measure in vivo including pH, microviscosity, micropolarity, redox processes, and the concentration of oxygen. The use of pH- sensilive imidazole derived nitroxides will~ different pKa values permits the localized measurement of proton activity. An important example of the usefulness of this method is cmmected ~ith the in vivo study' of liberation processes inside of drug delivery systems. Although drug deliver)' systems with sustained release characteristics arc commonly used, the nlechanisnls of this release, cspcciall_v under in vivo conditions, arc poorly' understood Using a home made EPR spectrometer operating at 1.1 GHz ~c used this noninvasive mcllmd to investigate the release mechanisms of polymers subcutaneously implanted in the backs of mice The viscosib', polarity', and proton activity inside the polymer depends on the general structure of the polymer, the molecular weight, incorporated drugs, and the time after implantation. The experimental results indicate that in the case of polymers made up of polylactic and polyglycolic acid, compartments ~ith high polarity and low viscosity are formed inside the polymers. The proton activity increases with time. Therefore we have direct c~idencc in vivo that in these polymers bulk hydrolysis is a major mechanism of release. Other examples of the pharmaceutical use of in vivo EPR performed include the noninvasive characterization of drug deliver':, systems and their interactions with biological matrices, co~ering sc~crat routes of administrations (per os. iv. sc injection, ip. sc implantation) and the stud~, of drug derived radical species. Also the localized measurement of oxygca and oxygen gradients is possible b~ EPR using oxygcn-sensiti~c paramaguctic materials such as carbon chars or single cr) slals of lithimn phthaloc3 alfine K. M~ider gratefully' ackno~lcdgcs his support by Dcutschcr Akadcmischcr Austauschdienst (DAAD)
Neumtransmitter deficiency characterizes Parkinson's and Aizheimer diseases and is the working hypothesis for symptoms in a number of other psychiatnc and naurologic diseases. Efforts to increase neumtransmitter synthesis has, however, been met by varying success. It is generally agreed that the synthesis rate for the neurotransmitters dopamine and serotonin are governed by the activity of hydroxylase transferring tryptophan and tyrosine to 5-hydroxitryptophan, 5-HTP and LDOPA, respectively and that the following enzymatic step, the decarboxylation forming serotonin and dopamine can not be regulated by neuronal activity. By means of positron emission tomography, PET and using 5-HTP and L-DOPA radiolabeited in a metabolic stable position with 11C, a regional up-regulation of synthesis has been shown in patients with major depression, schizophrenia and advanced Parkinson's disease. Brain decarboxylase activity was also increased in the monkey following acute treatment with the co-factor pyridoxine, vitamine B6 for the decarboxylass. The decarboxylase enzyme was possible to saturate with the precursor amino acid and more efficiently with 5-HTP than with L-DOPA. This difference may explain the success in therapy with high doses of L-DOPA, but less using the precursors tryptophane and 5-HTP for a desired enhancement of serotonin synthesis. The co-factor for the hydroxylase enzyme, 6R-erythro5,6,7,8-tetrahydrobiopterin also increased decarboxylase activity. Furthermore, infuson of tyrosine somewhat increased, while L-DOPA infusion dramatically enhanced striatal synthesis rate of dopamine in the monkey. The decarboxylass activity was further increased by infusion of 6R-erythro-5,6,7,8-tetrahydrobiopterin. This modulation of decarboxylase activity might be explained by the recently descdbed neuronal L-DOPA receptor which on L-DOPA activation may enhance the dopamine synthesis rate. This activation of receptors may also explain the up-regulation of decarboxylase activity induced by 6R-erythro-5,6,7,8tetrahydrobiopterin as a result of an increased formation of neuronal L-DOPA. The regulation of decarboxylass may have clinical implications in the development of new pharmacological principles for treatment of diseases characterized by neumtransmitter deficiency. Furthermore, the L-DOPA controlled activity of the decarboxylase may also explain mechanisms responsible for fluctuating symptoms in patients with advanced Parkinson's disease and supports the clinical success of continuous L-DOPA therapy for control of fluctuating Parldnson disease symptoms.
FC19 ACTIVATIONOF G-PROTEINSIN PHYSIOLOGICALAND RECONSTITUTEDMEMBRANOUS SYSTEMS B. Nfimbergl, C. Harteneckl,D. Leopoldtl,R. Harhammerl,R. Seifertl, A. Hagel0kenl, H. Deted2, W. Schunack2,R. Hoppe3,E. NQmberg3 1 InstitutforPharmakologie,FreieUniversit8tRedin,14195Redin,Germany 2 InstitutffirPharmazie,FreieUniversitStBedin,14195Bedin,Germany 3 LehrstuhlforPharmazeutischeTechnologie,UniversitAtEdangen-N0mberg,91054Edangen,Germany
FC20 RIBOSEMODIFIEDADENOSINEANALOGUESAS POTENTIALPARTIALAGONISTSFOR THE ADENOSINERECEPTOR E.M.vanderWenden,J.K.K,~nzel,R.A.A.Math6t,A.F.IJzerman,M.Danhof,W. Soudijn DivisionofMedicalChemistryandPharmacdtogy,Leiden/AmsterdamCenterforDrugResearch,2300RALeiden, Netherlands
Heterotrimeric regulatory G-proteins modulate cellular effectors following activation by ligand-bound receptors. Additionally, direct stimulation of pertussis toxin-(PT) sensitive G-proteins by peptides and amphiphilic drugs has been demonstrated. Hence, it appears feasible to influence cellular functions by drug targeting o f signal transduction components downstream of the receptor, i.e. G-proteins. We compared the effects of mastoparan peptides and histamine derivatives known to be direct stimulators of G-proteins in different physiological and G-protein reconstituted membranous systems. The aim o f the study was to establish models designed to detect and analyze compounds interfering specifically with G-proteins. GTPase activity o f and GTPTS-binding to proteins were analyzed in (i) membranes of the human leukemia cell line HL-60 which physiologically expresses a broad variety of different signal transduction systems including seven-transmembranous receptors and PT-sensitive Gi-proteins, (it) different liposomes loaded with distinct mammalian G-proteins o f the G~-subfamily purified from mammalian brains or blood cells, and (iii) membranes of the eukaryotic Baculovirus/Spodoptera frugiperda (Sf9) expression system overexpressing different mammalian Gi-proteins. Whereas HL-60 membranes resembled the physiological situation very closely compared to all systems tested they exhibited the lowest level of G-protein specificity of either assay, i.e. GTPase or GTPyS-binding, due to the presence of various interfering proteins, e.g. receptors, different GTP-binding proteins and GTPases or kinases. On the other hand, artificial lipid membranous systems reconstituted with distinct purified G-proteins obviously favored specificity and allowed detection of potential new G-protein-activating compounds and analysis of the molecular mechanism of their action. Using this system, we were able to compare the mode of action and the G-protein specificity of different mastoparans and synthetic drugs. However, clear disadvantages were the time and cost consuming preparation of G-proteins and preparation of lipid vesicles. Interestingly, GTPase-activity of membranes of uninfected Sf9-cells was not stimulated by the peptides and drugs tested. In contrast, GTPase activity of Sf9 membranes expressing mammalian Ga~2- or Gcti2132yz-proteins was enhanced by all substances tested. From these experiments we suggest that the Baculovirus/Sf9 expression system may be an elegant screening model for detection of receptor-independent G-protein modulators. Supported by Deutsche Forschungsgemeinschafi (SFB 366)
Agonists for the adenosine receptor reduce blood pressure, but a hypotension can be the result. Partial agonists, however, should decrease the blood pressure to a lesser extent titan full agonists. It had been shown in the literature that an intact ribose moiety is necessary for full activity on the adenosine receptor. Thus, we synthesised and tested several 2"
7--7