- Email: [email protected]
SC9 “animal welfare” in the cortical stimulation model for epilepsy research in the rat
Short Communications / European Journal o f Pharmaceutical Sciences 2 (1994) 8 9 - 9 7
SC9 "i~IIM/U. WELFARE' IN THE CORTICAL STIMULM1ON MODELFOR EPILEPSY RESEARCH IN THE RAT O.E. Delia Paschoa,K.B. Postel, R.A.Voskuyl, M.R. Kruk, M. Danhot Divisionof Pharmacology,J.e~n/Amster{JsmCenter forDragResearch,2300RA~ n , Netherlands
The development of new, more accurate techniques has been a claim in epilepsy research. On the other hand, concern over ethical aspects of the animal experimentation has increased and attention has been focused on ways of minimizing the use of laboratory animals, so that reduction of the number of animals, replacement and methodological refinement ought to be considered. Recently reported, the direct cortical stimulation model (CSM) is an approach which seems to have allowed a move forward in beth directions [1]. Briefly, it supplies information on the ability of an antiepileptic drug (AED) to supress the onset and/or propagation of convulsive activity on the basis of stimulation with a ramp-shaped pulse train. A pulse of linearly increasing amplitude yields two thresholds, one for localized (TLS) and another for generalized (TGS) seizure activity within the same animal. Moreover, this approach offers the possibility of thorough kinetic disposition study in an individual animal. Aim of the present investigation was to assess animal welfare in the CSM in comparison to the actually used eleetrocoovulsive shock (ECS) and kindling models. Therefore, the intensity and severity of seizure activity and the interictal behavioural patterns were evaluated in the rat. 35 female Wistar rats were randoruly divided into five groups. Prior to the actual experiments, stimulation electrodes were chronically implanted in all groups. Seizures were elicited by electrical stimulation accordingly, as previously reported for each model. The control group was treated with a placebo stimulation. For each group, scores were based upon a 30-min recording of the interictal bebaviour and seizure activity. The behavioural sequences and patterns, during stimulation, seizure activity and in the periods in between were encoded off-line. The ethogram consisted of basic postural (sit, walk, lean, upright, floor, oral, care, rest and immobility) and ictal items (eye closure, jerk, paws, gasp, chew, jump and fall). Seizure pattern were described by a frame-by-frame analysis. Behavioural components were analysed by frequency, duration, and survival functions, using the video time-frame encoder/decoder CAMERA. Our results show that the repeated stimulation in individual animals in the CSM seems not to induce considerable alteration in animal behaviour or seizure intensity as compared to the isolated stimulation. The marked changes observed in basic postaral components in the ECS and kindling models are significantly reduced in the CSM. Furthermore, seizure severity was shown to be clearly diminished by the stimulation technique used in the CSM. In summary, it can be concluded that besides the reduction of variability factors, the cortical stimulation model for AED studies represents a refinement in both qualitative and quantitative aspects of animal experimentation. [1] Hoogerkamp et al: J Pharmacol Exp Ther, in press.
93
SC10 DESIGN OF NON-'rHIOURrcAH3.RECEPTOR HISTMIINE/~rrAGOI~S'I'S C.R. Ganellinl, A. Fkyeratl, S.K. Hosseinil, W. Tertiukl, M. Garbarg2,X. Ligneau2, J.-C. Schwadz2 1 Gepat,mentof Chemistry,UniversityCollegeLondon,LondonWClH 0AJ, U.K• 2 Unit6109de Neurobiofogleel Pharmacofogle,CentrePaulBc,ocade I'INSERM,75014Paris,France
Thioperamide was the first potent and specific antagonist used for characterisation of histamine H 3 receptors (Arrang et al, Nature, 1987, 327, 117). However, it possesses a thiourea group and since such moieties have been associated with toxicity of H2-antagonists (eg. metiamide) it seemed worthwhile to find alternative structures. There are strong similarities in the structures of some H 2 and H 3 antagonists (eg. burimamide) and it seemed worthwhile exploring whether "urea equivalent" groups of H 2 antagonists could be used for H 3 antagonists. In particular, and to facilitate brain penetration, the thiourea group was replaced by the less polar (and weaker hydrogen bonding) group NH - Het as was used in the design of the H2-antagonist, zolantidine (Young et al, J. Med Chem, 1988, 3 1 , 6 5 6 ) ; where Het is an aromatic nitrogen heterocycle. A series of compounds of structure I (X = NH, O,S; n = 2,3) has been synthesised and tested for antagonism of histamine at H 3 receptors on rat cerebral cortex slices or synaptosomes. It has also been found that Het can be replaced by substituted phenyl (structure II). Many of the compounds are potent H 3 antagonists e.g. UCL 1344, n = 2, R = pPr has Ki=19+9nM and UCL 1390, n = 3, R = pCN has K i = 12+3nM. (Schwartz et al, Fr. Pat 2, 686, 084, July 1993). The structure-activity studies leading to these compounds will be presented.
/ ~ HNvN
(CH2)"X-Het I
/ ~ HNvN
(CH2)"O-
-
~
R
11
We thank Dr. J.M. Lecomte, Bioprojet, Paris for financial support to AF, SKH and XL.
SCll GSAR OF Y1-RECEPTOR ANTAGONISTIC IMIDAZOLYLPROPYLGUANIDINESDERIVED FROM ARPROMIDINE AND MEPYRAMINE S. Dove1, MC. Michel2, S. Knieps3, A. Buschauerl 1 Jnstiluteol Pharmacy,Universityof Regensburg,93040Regensburg,Germany 2 Departmentof Medicine,Universityof Essen,45122Essen,Germany 3 Instiluteof Pharmacy,FreeUniversityol Berlin,14195Bedin,Germany
SC12 G-PROTEIN ACTIVATION VIA NUCLEOSIDE DIPHOSPHATE KINASE J. Klinker,A. Hagel0ken,B. N0mberg, R. Seifert Inetitutl(JrPharmakologie,FreieUniversitAtBerlin,14195Bedin,Germany
TO investigate the physiological role of neuropeptide Y (NPY) and to characterize re-
The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX}-sensitive G-proteins in a receptor-independent manner, presumably by mimicry of receptor activation. MP also activates nucleoside diphosphate kinase (NDPK), and NDPKmediated GTP formation is known to be involved in G-protein activation. It is, however, unknown of whether a functional link exists between MP-induced activations of NDPK and of G-proteins. Therefore, we studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cell membranes and a reconstituted system and on NDPK-catalyzed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an ECr. of I-2 pM and a maximum at 10 i~M. Unlike tile effects of the f~rmyl peptide receptor agonist, N-formyl-Lmethionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only p a r t i a l l y PTX-sensitive. The alkylating substance, N-ethylmaleinimide, inhibited MP-stimulated GTP hydrolysis to a greater extent than the one stimulated by fMetLeu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalyzed ADP~ribosylation of, G~-protein (~-subunits in, HL-60 membranes. By coutrast to fMet-Leu-Phe, MP did not or only weakly stimulate binding of guanosine 5'-O-[3-thio]triphosphate to Gi-protein (~subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted bovine brain Gi/Go-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [gH]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (i) MP activates G.-proteins in HL-60 membranes but ( i i ) the venom does not slmply mlmlc receptor activatlon, ( i l l ) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (iv) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective. (v) NDPK is a novel target for the design of drugs which interfere with G-protein-mediated signal transduction processes at a post-receptor level.
ceptor subtypes, selective antagonists are required. They could also be therapeutically useful, e.g., Y1 antagonists as antihypertensives. However, potent and selective nonpeptide antagonists of NPY are not described so far. We found the H 2 agonist arprumidine (I) and a number of its analogs being weak competitive antagonists at Y1 receptors, indicated by their inhibitoq/ effect on the NPY-stimulated mobilization of intracellular Ca 2. in human erythroleukemia (HEL) cells t2` The pA2 values of some compounds like II, the 3,4-di-CI derivative of arpromidine, are greater than 6. H ,K./~.N
H
N~'
y~I
NH
I: arpremidine, R = 4-F I1: Bu-E-64, R = 3,4.-di-Cl
II1: common structure X, Y = H, unsubstituted or substituted alkyl, awl, haterearyl or awlalkyl; B = CH, N; n = 1 , 2 . 3 , 4
Quantitative structure-activity relationships (QSAR) of NPY antagonism within a series of about 50 imidazol-4-yl-propylguanidines (common structure III) were investigated by fragment regression analysis. Results show that there is an optimal number of four C-atoms, i.e. probably optima/distance, between the guanidine site and the pyndine N of arpromidine derivatives or an amine N (B = N) of other analogs. A benzyl replacing the phenyl of arpromidine leads to markedly higher activity. NPY antagonism significantly correlates with the hydrophobicity of substituents in the para position of phenyl or benzyl, indicating binding at hydrop~obic amino acids of the YI receptor. Using the molecular modelling software SYBYL (Tripos Ass.), nor-F-arpromidine was fitted by template forcing to NPY conformations dedved from the crystal structure of the strongly homologous avian pancreatic polypeptide (APP). One possible explanation for the affinity of arpremidine analogs to Y1 receptors is that the imidazole group replaces the Arg-35 guanidine, the guanidino group the An3-33 guanidine, and the pyddine N the Tyr-1 hydroxyl of NPY. Yl selectivity might be attributed to the occupation of a Tyr-1 site• The phenyl of aqxomidine analogs can fit to hydrophobic domains of NPY. This fit is improved by hydrophoblc para-substituents and exchange of pheny! against benzyl, corresponding to QSAR results. 1 A. Buschauer, J. Meal. Chem, 32 (1989) 1963 - 1970. 2 M. C. Michel, H. J. Motulsky, Ann, NY Aced, Sci. 611 (1990) 392 - 394.
SC9 "i~IIM/U. WELFARE' IN THE CORTICAL STIMULM1ON MODELFOR EPILEPSY RESEARCH IN THE RAT O.E. Delia Paschoa,K.B. Postel, R.A.Voskuyl, M.R. Kruk, M. Danhot Divisionof Pharmacology,J.e~n/Amster{JsmCenter forDragResearch,2300RA~ n , Netherlands
The development of new, more accurate techniques has been a claim in epilepsy research. On the other hand, concern over ethical aspects of the animal experimentation has increased and attention has been focused on ways of minimizing the use of laboratory animals, so that reduction of the number of animals, replacement and methodological refinement ought to be considered. Recently reported, the direct cortical stimulation model (CSM) is an approach which seems to have allowed a move forward in beth directions [1]. Briefly, it supplies information on the ability of an antiepileptic drug (AED) to supress the onset and/or propagation of convulsive activity on the basis of stimulation with a ramp-shaped pulse train. A pulse of linearly increasing amplitude yields two thresholds, one for localized (TLS) and another for generalized (TGS) seizure activity within the same animal. Moreover, this approach offers the possibility of thorough kinetic disposition study in an individual animal. Aim of the present investigation was to assess animal welfare in the CSM in comparison to the actually used eleetrocoovulsive shock (ECS) and kindling models. Therefore, the intensity and severity of seizure activity and the interictal behavioural patterns were evaluated in the rat. 35 female Wistar rats were randoruly divided into five groups. Prior to the actual experiments, stimulation electrodes were chronically implanted in all groups. Seizures were elicited by electrical stimulation accordingly, as previously reported for each model. The control group was treated with a placebo stimulation. For each group, scores were based upon a 30-min recording of the interictal bebaviour and seizure activity. The behavioural sequences and patterns, during stimulation, seizure activity and in the periods in between were encoded off-line. The ethogram consisted of basic postural (sit, walk, lean, upright, floor, oral, care, rest and immobility) and ictal items (eye closure, jerk, paws, gasp, chew, jump and fall). Seizure pattern were described by a frame-by-frame analysis. Behavioural components were analysed by frequency, duration, and survival functions, using the video time-frame encoder/decoder CAMERA. Our results show that the repeated stimulation in individual animals in the CSM seems not to induce considerable alteration in animal behaviour or seizure intensity as compared to the isolated stimulation. The marked changes observed in basic postaral components in the ECS and kindling models are significantly reduced in the CSM. Furthermore, seizure severity was shown to be clearly diminished by the stimulation technique used in the CSM. In summary, it can be concluded that besides the reduction of variability factors, the cortical stimulation model for AED studies represents a refinement in both qualitative and quantitative aspects of animal experimentation. [1] Hoogerkamp et al: J Pharmacol Exp Ther, in press.
93
SC10 DESIGN OF NON-'rHIOURrcAH3.RECEPTOR HISTMIINE/~rrAGOI~S'I'S C.R. Ganellinl, A. Fkyeratl, S.K. Hosseinil, W. Tertiukl, M. Garbarg2,X. Ligneau2, J.-C. Schwadz2 1 Gepat,mentof Chemistry,UniversityCollegeLondon,LondonWClH 0AJ, U.K• 2 Unit6109de Neurobiofogleel Pharmacofogle,CentrePaulBc,ocade I'INSERM,75014Paris,France
Thioperamide was the first potent and specific antagonist used for characterisation of histamine H 3 receptors (Arrang et al, Nature, 1987, 327, 117). However, it possesses a thiourea group and since such moieties have been associated with toxicity of H2-antagonists (eg. metiamide) it seemed worthwhile to find alternative structures. There are strong similarities in the structures of some H 2 and H 3 antagonists (eg. burimamide) and it seemed worthwhile exploring whether "urea equivalent" groups of H 2 antagonists could be used for H 3 antagonists. In particular, and to facilitate brain penetration, the thiourea group was replaced by the less polar (and weaker hydrogen bonding) group NH - Het as was used in the design of the H2-antagonist, zolantidine (Young et al, J. Med Chem, 1988, 3 1 , 6 5 6 ) ; where Het is an aromatic nitrogen heterocycle. A series of compounds of structure I (X = NH, O,S; n = 2,3) has been synthesised and tested for antagonism of histamine at H 3 receptors on rat cerebral cortex slices or synaptosomes. It has also been found that Het can be replaced by substituted phenyl (structure II). Many of the compounds are potent H 3 antagonists e.g. UCL 1344, n = 2, R = pPr has Ki=19+9nM and UCL 1390, n = 3, R = pCN has K i = 12+3nM. (Schwartz et al, Fr. Pat 2, 686, 084, July 1993). The structure-activity studies leading to these compounds will be presented.
/ ~ HNvN
(CH2)"X-Het I
/ ~ HNvN
(CH2)"O-
-
~
R
11
We thank Dr. J.M. Lecomte, Bioprojet, Paris for financial support to AF, SKH and XL.
SCll GSAR OF Y1-RECEPTOR ANTAGONISTIC IMIDAZOLYLPROPYLGUANIDINESDERIVED FROM ARPROMIDINE AND MEPYRAMINE S. Dove1, MC. Michel2, S. Knieps3, A. Buschauerl 1 Jnstiluteol Pharmacy,Universityof Regensburg,93040Regensburg,Germany 2 Departmentof Medicine,Universityof Essen,45122Essen,Germany 3 Instiluteof Pharmacy,FreeUniversityol Berlin,14195Bedin,Germany
SC12 G-PROTEIN ACTIVATION VIA NUCLEOSIDE DIPHOSPHATE KINASE J. Klinker,A. Hagel0ken,B. N0mberg, R. Seifert Inetitutl(JrPharmakologie,FreieUniversitAtBerlin,14195Bedin,Germany
TO investigate the physiological role of neuropeptide Y (NPY) and to characterize re-
The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX}-sensitive G-proteins in a receptor-independent manner, presumably by mimicry of receptor activation. MP also activates nucleoside diphosphate kinase (NDPK), and NDPKmediated GTP formation is known to be involved in G-protein activation. It is, however, unknown of whether a functional link exists between MP-induced activations of NDPK and of G-proteins. Therefore, we studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cell membranes and a reconstituted system and on NDPK-catalyzed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an ECr. of I-2 pM and a maximum at 10 i~M. Unlike tile effects of the f~rmyl peptide receptor agonist, N-formyl-Lmethionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only p a r t i a l l y PTX-sensitive. The alkylating substance, N-ethylmaleinimide, inhibited MP-stimulated GTP hydrolysis to a greater extent than the one stimulated by fMetLeu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalyzed ADP~ribosylation of, G~-protein (~-subunits in, HL-60 membranes. By coutrast to fMet-Leu-Phe, MP did not or only weakly stimulate binding of guanosine 5'-O-[3-thio]triphosphate to Gi-protein (~subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted bovine brain Gi/Go-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [gH]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (i) MP activates G.-proteins in HL-60 membranes but ( i i ) the venom does not slmply mlmlc receptor activatlon, ( i l l ) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (iv) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective. (v) NDPK is a novel target for the design of drugs which interfere with G-protein-mediated signal transduction processes at a post-receptor level.
ceptor subtypes, selective antagonists are required. They could also be therapeutically useful, e.g., Y1 antagonists as antihypertensives. However, potent and selective nonpeptide antagonists of NPY are not described so far. We found the H 2 agonist arprumidine (I) and a number of its analogs being weak competitive antagonists at Y1 receptors, indicated by their inhibitoq/ effect on the NPY-stimulated mobilization of intracellular Ca 2. in human erythroleukemia (HEL) cells t2` The pA2 values of some compounds like II, the 3,4-di-CI derivative of arpromidine, are greater than 6. H ,K./~.N
H
N~'
y~I
NH
I: arpremidine, R = 4-F I1: Bu-E-64, R = 3,4.-di-Cl
II1: common structure X, Y = H, unsubstituted or substituted alkyl, awl, haterearyl or awlalkyl; B = CH, N; n = 1 , 2 . 3 , 4
Quantitative structure-activity relationships (QSAR) of NPY antagonism within a series of about 50 imidazol-4-yl-propylguanidines (common structure III) were investigated by fragment regression analysis. Results show that there is an optimal number of four C-atoms, i.e. probably optima/distance, between the guanidine site and the pyndine N of arpromidine derivatives or an amine N (B = N) of other analogs. A benzyl replacing the phenyl of arpromidine leads to markedly higher activity. NPY antagonism significantly correlates with the hydrophobicity of substituents in the para position of phenyl or benzyl, indicating binding at hydrop~obic amino acids of the YI receptor. Using the molecular modelling software SYBYL (Tripos Ass.), nor-F-arpromidine was fitted by template forcing to NPY conformations dedved from the crystal structure of the strongly homologous avian pancreatic polypeptide (APP). One possible explanation for the affinity of arpremidine analogs to Y1 receptors is that the imidazole group replaces the Arg-35 guanidine, the guanidino group the An3-33 guanidine, and the pyddine N the Tyr-1 hydroxyl of NPY. Yl selectivity might be attributed to the occupation of a Tyr-1 site• The phenyl of aqxomidine analogs can fit to hydrophobic domains of NPY. This fit is improved by hydrophoblc para-substituents and exchange of pheny! against benzyl, corresponding to QSAR results. 1 A. Buschauer, J. Meal. Chem, 32 (1989) 1963 - 1970. 2 M. C. Michel, H. J. Motulsky, Ann, NY Aced, Sci. 611 (1990) 392 - 394.