THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS. NEW PERSPECTIVES IN THE COMMON EUROPE

Pharmacology is only just a small part of the science and scientists are a small fraction of each country. However, the example of joint activities of Polish and German pharmacologists shows that making new human relationships is possible.

Jerzy Maj n

Official Symposium of the Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie and of the Polish Pharmacological Society n

Białowieża, September 18–21, 2003 ENDOGENOUS 1,2,3,4-TETRAHYDROISOQUINOLINE DERIVATIVES POSSESS ANTIDOPAMINERGIC AND NEUROPROTECTIVE ACTIVITY IN RAT BRAIN

Lucyna Antkiewicz-Michaluk, Jerzy Michaluk, Irena Romañska, Andrzej Bojarski, Jerzy Vetulani Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

1,2,3,4-Tetrahydroisoquinoline derivatives (TIQs) on both pathways of DA catabolism, inhibition of are present both in man and animal brain, and may MAOB-dependent N-oxidation, and acceleration at play important physiological role. However, the least twice the COMT-dependent O-methylation. earlier studies have suggested neurotoxic effect of Such an effect on DA catabolism may reduce the TIQs, resulting from their similarity to 1-methyl- generation of free radicals accompanying this pro- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and cess. We also investigated whether 1MeTIQ exerts ability to form tetrahydroisoquinolinium ions, ana- a protective action against the degeneration of do- logous to 1-methyl-4-phenylpyridinium ion (MPP+), paminergic terminals induced by neurotoxic herbi- and in fact experimental parkinsonism was induced cide – rotenone (7 days, 10 mg/kg sc) in rat brain. by TIQs (TIQ, salsolinol, 1-BnTIQ) in rodents and We have found that repeated but not single admini- marmosets [1, 2]. Another TIQ derivative – 1-me- stration of rotenone causing abnormalities in ge- thyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) – neral behavior of rats, produced considerable mor- was regarded as a neuroprotectant antagonizing the tality, and dramatic increases in DA metabolism, parkinsonism-like behavioral abnormalities in mice which may be ascribed to an increase in the oxida- [3]. In our study, we investigated the acute and tive pathways (strong increase in DOPAC level), chronic effect of TIQs in the behavioral and bio- and 1MeTIQ completely antagonized these effects. chemical experiments. In summary, we have demonstrated that TIQs The results have shown that all investigated tet- are the ideal candidates for natural regulators of rahydroisoquinoline compounds: TIQ, salsolinol, DA system, that would prevent its hyperactivity, 1BnTIQ and 1-MeTIQ administered to rodents and additionally the results have shown neuropro- (25–100 mg/kg ip) antagonized effectively in dose- tective activity of 1MeTIQ against rotenone-indu- related manner the behavioral action (locomotor ced neurodegeneration. hyperactivity) induced by dopamine (DA) agonists: apomorphine and amphetamine (0.25 and 0.5 mg/kg REFERENCES sc). Biochemical experiments have shown the dif- 1. ferences in effects of two tetrahydroisoquinolines: Antkiewicz-Michaluk L., Michaluk J., Mokrosz M., Romañska I., Lorenc-Koci E., Ohta S., Vetulani J.: 1MeTIQ and 1BnTIQ on DA metabolism and ca- Different action on dopamine catabolic pathways of tabolism which would explain their neuroprotec- two endogenous 1,2,3,4-tetrahydroisoquinolines with tive and neurotoxic ability. 1MeTIQ did not change similar antidopaminergic properties. J. Neurochem., the DA concentration either after acute or chronic 2001, 78, 100–108. treatment in any investigated structures (substantia 2. Nagatsu T., Yoshida M.: An endogenous substance of nigra, striatum, nucleus accumbens). In contrast, the brain, tetrahydroisoquinoline, produces parkinson- ism in primates with decreased dopamine, tyrosine hy- 1BnTIQ produced a dramatic fall of DA level in droxylase and biopterin in the nigrostriatal region. the striatum and nucleus accumbens with the in- Neurosci. Lett., 1988, 87, 178–182. crease in homovanillic acid level (HVA), which in- 3. Tasaki Y., Makino Y., Ohta S., Hirobe M.: 1-Methyl- dicates a massive release of DA from the nerve 1,2,3,4-tetrahydroisoquinoline, decreasing in 1-methyl- endings. This effect may be the cause of neurotoxic 4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse, pre- properties of 1BnTIQ. The neuroprotective action vents parkinsonism-like behavior abnormalities. J. Neu- of 1MeTIQ may be explained by its different action rochem., 1991, 57, 1940–1943.

ISSN 1230-6002 479 Abstracts of Symposium

EFFECTS OF FELBAMATE AND ITS COMBINATIONS WITH CONVENTIONAL ANTIEPILEPTIC DRUGS IN AMYGDALA-KINDLED RATS

Kinga K. Borowicz, Stanis³aw J. Czuczwar Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland

Felbamate modified behavioral and electro- doses of valproate, phenobarbital, diphenylhydan- graphic correlates of kindled seizures in rats. In de- toin or clonazepam did not express any protective tail, when applied at the dose of 5 or 10 mg/kg, action. The concomitant treatment of felbamate felbamate significantly increased afterdischarge with conventional antiepileptics did not induce threshold, shortened seizure and afterdischarge du- any significant motor impairment in the chimney rations, but remained without effect on seizure se- test or memory deficit in the passive avoidance verity. Furthermore, the combination of felbamate task. A pharmacokinetic interaction between felba- (2.5 mg/kg) with carbamazepine (7.5 mg/kg; both mate and carbamazepine can be excluded, at least drugs at their subeffective doses), reduced the sei- at the level of plasma and brain concentrations. The zure severity and afterdischarge duration of kindled antiseizure efficacy of felbamate/carbamazepine seizures. In respect of afterdischarge duration, the combination evaluated in the present study may antiseizure potency of the felbamate/carbamaze- justify its usage as an add-on therapy, being worth pine combination was comparable to that of carba- considering particularly in cases of multiple drug- mazepine alone administered at 10 mg/kg. Neither resistant partial epilepsy. carbamazepine (7.5 and 10 mg/kg) nor felbamate (2.5–10 mg/kg) affected seizure severity, whereas REFERENCES combined treatment of felbamate (2.5 mg/kg) with 1. Borowicz K.K., Stasiuk G., Teter J., Kleinrok Z., carbamazepine (7.5 mg/kg) led to significant re- G¹sior M., Czuczwar S.J.: Low propensity of conven- duction of seizure severity from 5th to 3rd stage of tional antiepileptic drugs for interaction with felba- Racine’s scale. Among conventional antiepileptic mate against maximal electroshock-induced seizures in mice. J. Neural Transm., 2000, 107, 733–743. drugs tested in this study, only valproate (100 2. Ebert U., Reissmüller E., Löscher W.: The new antie- mg/kg) and clonazepam (0.1 mg/kg) exerted simi- pileptic drugs lamotrigine and felbamate are effective lar action on seizure severity. However, the combi- in phenytoin-resistant kindled rats. Neuropharmacolo- nations of felbamate (2.5 mg/kg) with subeffective gy, 2000, 39, 1893–1903.

EFFECT OF CYCLOOXYGENASE AND NITRIC OXIDE SYNTHASE INHIBITORS ON ANTINOCICEPTIVE ACTION OF ACETAMINOPHEN

Magdalena Bujalska Department of Pharmacodynamics, Medical University, Krakowskie Przedmieœcie 26/28, PL 00-927 Warszawa, Poland

Acetaminophen (ACETA), an active metabolite from those of aspirin (ASA), this drug has widely of phenacetin, is a commonly used antinocicep- replaced ASA and other salicylates in the treatment tive/antypyretic drug. Although its antinociceptive of mild to moderate pain in conditions not associ- and antipyretic activities do not differ markedly ated with inflammatory processes such as head-

480 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS ache, toothache and dysmenorrhoea. A lack of anti- hibitor preferentially acting on COX-1, as well as inflammatory activity can be attributed to the fact nimesulide (NIM) and celecoxib (CECOX), prefer- that ACETA does not influence or does not sub- ential and selective inhibitor of COX-2 respec- stantially inhibit the activity of cyclooxygenases tively, administered, icv or peripherally almost (COXs) in peripheral tissues [1, 2]. completely blocked the antinociceptive effect of In spite of the numerous investigations, the ACETA in Randall-Selitto method. On the other mechanism of ACETA action is still poorly defined hand, pretreatment with NSAIDs it initially in- and has been not satisfactorily explained. creased and then attenuated the ACETA antinoci- It was, therefore, of interest to investigate the ception. Yohimbine did not modify antinociceptive effects of COX and nitric oxide synthase (NOS) in- action of ACETA when administered alone. How- hibitors on the antinociceptive activity of ACETA. ever, yohimbine decreased the nociceptive threshold Effect of yohimbine, an a2-adrenergic receptor an- increased by co-administration of IND and ACETA, tagonist, was also checked. NIM and ACETA as well as CECOX and ACETA Intrathecal (it), intracerebroventricular (icv)or in Randall-Selitto model. peripheral pretreatment with L-G-nitro-L-arginine The results of this study indicate that both COX (L-NO-Arg), a non-selective inhibitor of NOS ac- and nNOS systems are involved in the antinocicep- tivity, as well as with 7-nitroindazole (7-NI), an se- tive activity of ACETA. lective inhibitor of neuronal NOS, potentiated the antinociceptive activity of subceiling doses of REFERENCES ACETA, but was without effect on the action of su- 1. pramaximal doses in Randall-Selitto test. In con- Day R.O., Graham G.G., Whelton A.: The position of paracetamol in the world of analgesics. Amer. J. Ther., trast, L-NIL, a relatively selective inhibitor of in- 2000, 7, 51–54. ducible NOS, did not influence the action of 2. Prescott L.F.: Paracetamol: past, present, and future. ACETA in this model. Indomethacin (IND), an in- Amer. J. Ther., 2000, 7, 143–147.

DYNAMICS OF mRNA EXPRESSION FOR PRO- AND ANTI-INFLAMMATORY CYTOKINES IN THE STRIATUM OF C57BL/6 MALE MICE FOLLOWING INTRAPERITONEAL ADMINISTRATION OF MPTP

Agnieszka Ciesielska1, Ilona Joniec2, Adam Przyby³kowski2, Gra¿yna Gromadzka1,2, Anna Cz³onkowska1,2, Andrzej Cz³onkowski2 Institute of Psychiatry and Neurology, Second Department of Neurology, Sobieskiego 1/9, PL 02-957 Warszawa, Poland, Department of Experimental and Clinical Pharmacology, Medical University, Krakowskie Przedmieœcie 26/28, PL 00-927 Warszawa, Poland

The inflammatory reaction has been linked with With the method of semi-quantitative RT-PCR we Parkinson’s disease (PD) [1]. The neuroinflamma- evaluated mRNA levels for IL1b, IL6, TNFa, IFNg tion is regulated by numerous signal molecules, in- and IL10 in the striatum of mice 6 h, 1, 3, 7 and 14 cluding cytokines. The increased density of pro- days after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri- inflammatory cytokine-producing glial cells in the dine (MPTP) intoxication. MPTP is a selective neu- substantia nigra of patients with PD was docu- rotoxin of dopaminergic nigrostriatal neurons. mented [2]. This may have several implications for The IL1b mRNA expression rapidly increased the phatophysiology of the disease. Although the as early as at 6 h after MPTP injection and peaked possibility that these cytokines have a neuroprotec- at 6 h to 24 h. The expression of TNFa and IFNg tive effect in these conditions cannot be excluded. mRNA appeared to be biphasic with the first in-

ISSN 1230-6002 481 Abstracts of Symposium crease noticed at 6–24 h and the second on the 7th influence on nigrostriatal system. Results of such day after MPTP intoxication. The moderate in- studies could be useful in planning of therapeutic crease in the level of IL6 mRNA was observed strategies based on pharmacological modification within 1–3 days following MPTP intoxication and of the cytokine synthesis aimed to diminish neuro- the peak of mRNA level for IL6 was observed on degeneration and enhance neuroregeneration. the 7th day of experiment. IL10 mRNA showed also phasic expression pattern. Two peaks of IL10 REFERENCES mRNA were seen, immediately (6 h) and on the 3rd 1. day after MPTP injection. Cz³onkowska A., Kohutnicka M., Kurkowska-Jastrzêb- ska I., Cz³onkowski A.: Microglial reaction in MPTP Those findings suggest that cytokine network is (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced involved in the development of immune changes ac- Parkinson’s disease mice model. Neurodegeneration, companying the nigrostriatal system degeneration. 1996, 5, 137–143. The role of subsequent cytokine treatment following 2. Hirsch E.C., Hunot S., Damier P., Faucheux B.: Glial MPTP intoxication should be established taking into cells and inflammation in Parkinson’s disease: a role in consideration their potential harmful or regenerative neurodegeneration? Ann. Neurol., 1998, 44, 115–120.

GLUTAMATE RECEPTOR ANTAGONISTS FOR TREATMENT OF DRUG-RESISTANT EPILEPSY?

Stanis³aw J. Czuczwar1,2 Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland, Isotope Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland

Excitatory amino acids, glutamate or aspartate, botropic glutamate receptor ligands are now inten- are involved in the generation of seizure activity in sively tested for their interactions with antiepileptic animal studies. Some cases of epilepsy are also as- drugs and the initial data indicate that their combina- sociated with a significant increase in plasma exci- tions with benzodiazepines and valproate are quite tatory amino acids. This may indicate that gluta- promising, as undesired effects are completely absent. mate and/or other excitatory amino acids may be Recent data indicate that talampanel very po- responsible for the occurrence of epileptic activity tently enhances the protective effects of benzodi- in humans. Around 20–30% of epileptic patients azepines against amygdala-kindled seizures in rats are refractory to the treatment with available antie- (a model of complex partial seizures [1, 2]). pileptic drugs. Therefore, a continuous search for new treatment strategies is carried out [3]. REFERENCES Available evidence suggests that especially anta- 1. gonists of AMPA/KA receptors potentiated the pro- Borowicz K.K., Kleinrok Z., Czuczwar S.J.: The AMPA/kainate receptor antagonist, LY 300164, in- tective activity of conventional antiepileptic drugs creases the anticonvulsant effects of diazepam. Nau- against maximal electroshock-induced convulsions in nyn-Schmied. Arch. Pharmacol., 2000, 361, 629–635. mice (a model of generalized tonic-clonic seizures), 2. Borowicz K.K., £uszczki J., Szadkowski M., Kleinrok with adverse effects being minimal or absent. Espe- Z., Czuczwar S.J.: Influence of LY 300164, an antago- cially, talampanel (LY 300164) is a promising drug in nist of AMPA/kainate receptors, on the anticonvulsant this respect. Interestingly, in most cases high-affinity activity of clonazepam. Eur. J. Pharmacol., 1999, 380, 67–72. NMDA receptor antagonists enhanced the anticon- 3. Deckers C.L.P., Czuczwar S.J., Hekster Y.A., Keyser vulsant potential of conventional antiepileptic drugs, A., Kubova H., Meinardi H., Patsalos P.N., Renier however, the adverse effects were evidently present. W.O., Van Rijn C.M.: Selection of antiepileptic drug Low-affinity NMDA receptor antagonists (remacemi- polytherapy based on mechanism of action: the evi- de) possess much better profile of activity. Meta- dence reviewed. Epilepsia, 2000, 41, 1364–1374.

482 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

MEMANTINE AS A NOVEL TREATMENT FOR ALZHEIMER’S DISEASE – PRECLINICAL PERSPECTIVE

Wojciech Danysz, Chris G. Parsons Merz Pharmaceuticals, D 60318 Frankfurt am Main, Germany; e-mail: [email protected]

The real therapeutic challenge is not only to al- therapeutic range. However, since it is well known leviate the symptoms, but also to interfere with that NMDA receptors are involved in the synaptic pathomechanism of the disease. There are many in- plasticity, one could expect negative effects of me- dications that glutamate might be involved in the mantine on learning, which would be contraindica- pathomechanism of Alzheimer’s disease [1]. Hen- tion for the treatment of dementia. Surprisingly, at ce, NMDA receptor antagonists should be able to the neuroprotective doses positive effects on learn- slow down the progression of neurodegeneration. ing were observed similarly to clinical studies [3]. The moderate affinity NMDA receptor antagonist, This has been shown for instance in the radial maze memantine, has been used for many years in Ger- test in rats after entorhinal cortex lesion. Moreover, many and reached recently approval in the EU for to mimic conditions of glutamatergic system over- the treatment of patients with moderately severe to activation – a condition believed to take place in severe Alzheimer’s disease. Memantine attenuates Alzheimer’s disease – we injected NMDA in rats NMDA receptor-mediated responses with an IC50 and observed a deficit in passive avoidance learn- of 1–3 mM and similar plasma levels (0.4–1.2 mM) ing that was attenuated by memantine. Similarly, are usually observed at effective doses both in pa- memantine (1 mM) was able in vitro to restore LTP tients and in experimental animals [2]. Hence, at in hippocampal slices impaired by the presence of therapeutic doses memantine blocks NMDA recep- NMDA in the buffer. Decreasing concentrations of 2+ tors in the CNS in humans which should be suffi- Mg also resulted in inhibition of LTP which was cient for neuroprotective activity. In fact, in many again restored by memantine. The hypothesis uni- animal models memantine has been shown to pro- fying neuroprotective and plasticity improving ef- vide neuroprotection. Memantine prevented the le- fects of memantine will be presented. sions of the NBM in rats induced by a direct injec- tion of NMDA with ED50 = 2.8 mg/kg. Infused sc REFERENCES for two weeks, it also attenuated the biochemical 1. and behavioral deterioration produced by infusion Danysz W., Parsons C.G., Möbius H.J., Stöffler A., Quack G.: Neuroprotective and symptomatological of NMDA receptor agonist, quinolinic acid, into action of memantine relevant for Alzheimer’s disease the lateral ventricle. Similarly, its sc infusion (15 – an unified glutamatergic hypothesis on the mecha- mg/kg/day) prevented pathological alteration in the nism of action. Neurotoxicity Res., 2000, 2, 85–97. hippocampus produced by a direct injection of 2. Parsons C.G., Danysz W., Quack G.: Memantine is a b-amyloid. Also, the loss of cholinergic neurons in clinically well tolerated N-methyl-D-aspartate (NMDA) the NBM produced by chronic inflammation in- receptor antagonist – a review of preclinical data. duced by LPS was decreased by memantine co- Neuropharmacology, 1999, 38, 735–767. 3. Reisberg B., Doody R., Stoffler A., Schmitt F., Ferris administration. Thus, memantine is neuroprotec- S., Mobius H.J.: Memantine in moderate-to-severe tive in animal models of neurodegenerative dis- Alzheimer’s disease. N. Engl. J. Med., 2003, 348, eases at doses producing plasma levels within the 1333–1341.

ISSN 1230-6002 483 Abstracts of Symposium

REGULATION OF GENE EXPRESSION IN HUVEC AND HUMAN UMBILICAL CELL PROGENITORS BY GROWTH FACTORS AND PPAR g ACTIVATOR

Aldona Dembiñska-Kieæ, Anna Polus, Joanna Grzybowska, Ewa Pi¹tkowska Department of Clinical Biochemistry, Collegium Medicum, Jagiellonian University, Kopernika 15a, PL 31-501 Kraków, Poland; e-mail: [email protected]

The Jagged-Notch system is a transmembrane blott methods. The influence of VEGF, bFGF, lep- receptor complex necessary for developmental cell tin and ciglitazone on tubular-like structure forma- differentiation. The fatty acid-induced peroxisome tion was investigated in the 3D assay in matrigel. proliferation-activated receptors (PPARs) and leptin Results. The Notch-4, eNOS, vWF, HoxD3, are the important regulators of the nutrition ingre- HoxB3 gene expression was found in HUVEC but dient-stimulated cell differentiation. The cellular not in UPC. The bFGF and PPARg activator (cigli- CD36 is recognized as the scavenger receptor for tazone), but not VEGF, down-regulated the expres- fatty acid and modified LDL as well as thrombo- sion of Jagged-1 and Notch4 in HUVEC. VEGF spondin (TSP). Since bone marrow and umbilical weekly reduced Jagged-1 expression in UPC. The cord blood CD34+ progenitor cells (UPC) were re- reverse correlation between Jagged-1 and CD36 cently used for experimental activation of angio- gene expression was found. In HUVEC, VEGF genesis in ischemic tissue, the influence of PPARg up-regulated Jagged-1 and down-regulated CD36 agonist and leptin on the expression of Jag- expression, while in UPC, VEGF reduced Jagged-1 ged/Notch, CD36 and participating in proliferation expression with non-significant activation of CD36 (HoxD3) and branching (HoxB3) homeobox genes expression. PPARg activation by ciglitazone in- was compared with the angiogenic activity of creased the CD36 and decreased Jagged-1 expres- VEGF and bFGF in HUVEC and UPC. sion in both cell lines. bFGF did not influence the Methods. Human umbilical cord blood CD34+ CD36 gene expression in the studied cells. All cy- cells and HUVEC were used. VEGF, bFGF, leptin tokines stimulated the tubulogenesis in HUVEC or PPARg activator (ciglitazone) were added for 24 and to the lesser extent in UCP, when ciglitazone h to the cell cultures (70% of confluence) in the inhibited this effect. culture medium. Total RNA was isolated and used Conclusions. The PPAR-g agonists (lipids and for RT-PCR assay to detect mRNA for the CD34, their derivatives) may interfere with the proangio- Jagged-1, Notch-1, Notch-2, Notch-4 and HoxD3, genic activity of VEGF, bFGF and leptin in UPC as HoxB3, PPARg, VEGFR-2 and CD36 gene expres- they do in HUVEC. sion. The Jagged-1, Notch-4 and CD36 gene ex- pression levels were estimated using the real-time Acknowledgment. Investigations have been sponsored PCR method (MJ Research Inc.) with GAPDH as by the EU Comission DLARFID QLRT-2001-00183, SP- the reference gene. Expression of cell surface re- UB-M 156/E-390/SPB/5PR, The State Committee for Sci- ceptors (protein) was detected by FACS or Western entific Research W£ 135/P/L, Warszawa, Poland.

484 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

LOOKING FOR SELECTIVE A1 AND A2A ADENOSINE RECEPTORS LIGANDS AMONG ANNELATED THEOPHYLLINE DERIVATIVES

Anna Drabczyñska1, Christa Müller2, Britta Schumacher2, El¿bieta Pêkala1, Janina Karolak-Wojciechowska3, Katarzyna Kieæ-Kononowicz1 Department of Chemical Technology of Drugs, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, Pharmaceutical Institute Poppelsdorf, University of Bonn, Bonn, Germany, !Institute of General and Ecological Chemistry, Technical University of £ódŸ, £ódŸ, Poland

Adenosine exerts its physiological effects by in- cromolar affinity and selectivity mostly for A2A teracting with four different G protein-coupled re- ARs vs. other AR subtypes ceptors (A1,A2A,A2B,A3) – AR. Recent studies indi- cate widening role for AR in many therapeutic areas: Acknowledgment. This work was supported in part by immunology, cardiovascular system, various CNS- the State Committee for Scientific Research (Grant No. 6 mediated events (sleep, neuroprotection, pain) [2]. P05F 024 21) Warszawa, Poland, and by the Deutsche For- Searching for new selective AR ligands, we schungsgemeinschaft (GRK-677). have synthesized tricyclic configurationally stable derivatives of xanthine with annelated ring contain- REFERENCES ing N or O [1, 3]. 1. To gain insight into the structure-affinity rela- Drabczyñska A., Schumacher B., Müller C.E., Karo- tionships, the lead structures were modified by en- lak-Wojciechowska J., Michalak B., Pêkala E., Kieæ- Kononowicz K.: Impact of the aryl substituent kind larging annelated ring (from 5 to 7 membered) and and distance from pyrimido[2,1-f] purindiones on the by changing substituents (alkyl-aryl) (see figure). adenosine receptor selectivity and antagonistic pro- The synthesized compounds were evaluated in perties. Eur. J. Med. Chem., 2003, 00, 1–6. in vitro receptor binding studies for the affinity for 2. Hess S.: Recent advances in adenosine receptor an- A1 and A2A ARs in assays carried out in the rat tagonist research. Expert. Opin. Ther. Patents, 2001, 3 11, 1–29. brain membrane preparations using [ H] CCPA and 3. [3H] MSX-2, respectively. The selected compounds Kieæ-Kononowicz K., Drabczyñska A., Pêkala E., Mi- chalak B., Müller C.E., Schumacher B., Karolak-Woj- were additionally investigated in binding assays ciechowska J., Duddeck H., Rockitt S., Wartchow R.: with human recombinant A2B and A3 receptors. New developments in A1 and A2 adenosine receptor The most interesting derivatives exhibited submi- antagonists. Pure Appl. Chem., 2001, 73, 1411–1420.

O O (CH2)n (CH2)n X2 H C H C 3 N N 3 N N N X1 R O N N R O N N

CH3 CH3

n=2,3,4 X1 = CHR X2 =O

R = alkyl, aryl X1 =O X2 = CHR

ISSN 1230-6002 485 Abstracts of Symposium

Ca2+ EXCITOTOXICITY IN CEREBRAL ISCHEMIA DEPENDS ON ACTIN POLYMERIZATION: A POTENTIAL TARGET FOR PHARMACOTHERAPY

Klaus B. Fink1, Melanie Paehr1, Pierre Ch. Djoufack1, Carsten Weissbrich1, Claudia Capelleri1, Matthias Endres2 Department of Pharmacology, University of Bonn Medical School, D 53113 Bonn, Germany, Department of Neurology, Charité Hospital, Humboldt-University of Berlin, D 10098 Berlin, Germany

Cell injury after cerebral ischemia is mainly in- gion. K+ (15 mM)-induced release of endogenous duced by Ca2+ influx through NMDA receptors, glutamate in neocortex and hippocampus is in- voltage-dependent Ca2+ channels or impaired Ca2+ creased in gsn-deficient slices by 87.5% and 181%, extrusion through the Na+/Ca2+ exchanger. Volta- respectively, over wild-type. NMDA (300 mM)-in- ge-dependent Ca2+ channels and NMDA receptors duced [3H]noradrenaline release, which was used are linked to the actin cytoskeleton. The fungal as a reporter assay for NMDA receptor function, is toxin cytochalasin D disrupts and the endogenous increased in gsn-deficient neocortical slices by protein gelsolin (gsn) severs and caps filamentous 54% and decreased by cytochalasin D by 38%. As actin barbed ends which both result in uncoupling a consequence of increased Ca2+ influx and gluta- of the cytoskeletal link of these membrane ion mate release, the infarct area after 2 h focal cerebral channels. ischemia was 45% larger in gsn-deficient mice as Gsn-deficient neocortical and hippocampal syn- compared to wild-type mice which could be coun- aptosomes show 33.5% and 40.3% higher Ca2+ in- teracted by cytochalasin D treatment. flux than wild-type after K+ (30 mM) depolariza- It is concluded that slowed F-actin depolymeri- tion. K+-induced Ca2+ influx is mediated predomi- zation enhances neuronal vulnerability, and accel- nantly by P/Q-type and, in a minor part, by N-type erated F-actin depolymerization is a potential neu- voltage-dependent Ca2+ channels in either brain re- roprotective mechanism in cerebral ischemia.

ANTAGONISTIC PROPERTY OF BUPRANOLOL AT THE ATYPICAL b-ADRENOCEPTOR IS STEREOSELECTIVE BUT IS NOT RETAINED BY SEVEN ANALOGUES

Karsten Flau1, Barbara Malinowska2, Katarzyna Kieæ-Kononowicz3, Grzegorz Godlewski2, Hanna Koz³owska2, Markus Kathmann1, Eberhard Schlicker1 Department of Pharmacology and Toxicology, University of Bonn, D 53113 Bonn, Germany, Department of Experimental Physiology, Medical Academy, Mickiewicza 2a, PL 15-089 Bia³ystok, Poland, !Department of Chemical Technology of Drugs, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland

Bupranolol is an antagonist of the classical b-adre- nolol at the b4-receptor, seven bupranolol analo- noceptors (b1–b3) with additional antagonistic ac- gues were synthesized and their effects on the heart tivity at the atypical b-adrenoceptor (b4)inthe rate in pithed rats and the binding affinities for the 3 heart. For further investigation of structural proper- b1-receptor (labelled with H-CGP 12177) in the ties responsible for the antagonistic effect of bupra- rat brain cortex membranes were assayed.

486 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

BK-21, 22, 23, 25 and desmethylbupranolol in- prenalterol without affecting the chronotropic ef- creased heart rate by themselves, and the tachycar- fect of the b4-receptor agonist CGP 12177, which dia markedly decreased in the presence of low suggests that they are b1- but not b4-receptor an- doses of the b1-receptor antagonist CGP 20712. tagonists. Bupranolol stereoselectively antagonized The latter property and the high correlation of the the tachycardia caused by CGP 12177. potencies of the drugs with their affinities for b1 In summary, all analogues lost the antagonistic binding sites (r = 0.91) suggests the involvement effect of bupranolol at the b4-receptor in the heart. for b1-receptors. Bevantolol and BK-26, which had The stereoselective antagonistic effect of bupra- no effect on heart rate by themselves, antagonized nolol against CGP 12177 is a further piece of evi- the chronotropic effect of the b1-receptor agonist dence that the “b4-receptor” is really a receptor.

X R3 R2 Y OCH CH3 3 Increase in Inhibition of 3H- CGP 12177 binding R4 ONHR1 CCH3 heart rate (in presence of ICI 118,551; 70 nM)

OH CH3 OCH3

Compounds R R R! R" pED# pKE

(±)/(–)/(+)-Bupranolol X Cl H CH3 –/–/– 8.66±0.07 8.83±0.21 7.19±0.12 Desmethylbupranolol X Cl H H 8.4 8.98 ± 0.04

BK-21 X H H3C-O H 6.4 5.76 ± 0.13

BK-22 X CH3 H H 8.3 8.60 ± 0.08 BK-25 XHHH7.7 8.10±0.12

BK-26 X H Cl CH3 – 7.24 ± 0.06

BK-23 Y CH3 H H 7.3 8.37 ± 0.06

Bevantolol Y H H CH3 – 8.00 ± 0.08

IS MONOAMINE OXIDASE B ALONE OR TOGETHER WITH DIAMINE OXIDASE ENGAGED IN HISTAMINE METHYLATION PATHWAY?

Agnieszka W. Fogel1, Mercedes Unzeta2, Krystyna Sasiak1, Micha³ Maksymowicz3 Institute of Biogenic Amines, Polish Academy of Sciences, Tylna 3, PL 90-364£ódŸ, Poland, Department of Biochemistry, Faculty of Medicine, Universitat Autonoma de Barcelona, ES 08193 Bellaterra (Barcelona), Spain, !Institute of Medical Research Centre, Polish Academy of Sciences, Pawiñskiego 5, PL 02-106 Warszawa, Poland

Objective. The methylation pathway of hista- alone or with DAO [2] is involved in oxidative de- mine metabolism was investigated in portocavally amination in the periphery. shunted rats in which this pathway is known to be Materials and Methods. Portocavally shunted activated [1]. In the brain, which lacks diamine oxi- (PCA) and sham operated rats were used. The irre- dase, MAO B is the only enzyme degrading hista- versible inhibitor of MAO B, N-(2-propynyl)-2-(5- mine methylation product, t-methylhistamine. We benzylo-oxyindol)methylamine, PF 9601N, 1 mg/kg, attempted to answer the question whether MAO B 5 mg/kg ip (acute) or its 5 consecutive, ip injections

ISSN 1230-6002 487 Abstracts of Symposium at 0.5 mg/kg/dose every 3rd day (chronic), and tion pathway. Inhibition of oxidative deamination aminoguanidine (AMG), 10 mg/kg, were employed by AMG resulted in 2–2.5-fold increase in urinary to inhibit MAO B and DAO, respectively. Urine output of methylated histamine compounds and un- (24 h output) was collected in metabolic cages. Tis- metabolized histamine. After both inhibitors in com- sue and urinary histamine and t-methylhistamine bination (AMG +PF 9601N group), the excretion were assayed by radioenzymatic/fluorometric me- of t-methylhistamine and of histamine was similar thods and gas chromatography/mass spectrometry, to AMG group but t-methylimidazoleacetic acid respectively, urinary t-methylimidazoleacetic acid output was the same as in the control counterparts. t-MeImAA and creatinine were quantified by high Conclusion. It appears that, at least in the rat, performance liquid chromatography with ultravio- MAO B has an unquestioned position in histamine let detection, while tissue enzyme activities were metabolism both in the CNS as well as in the pe- measured by isotopic procedures [1]. riphery. Results. MAO B inhibition evoked by PF 9601N was over 80% in the brain and was accom- Acknowledgment. The study was supported by grant D13 COST. panied by the increased tissue t-methylhistamine. However, in the stomach, the maximum effect was REFERENCES 40% inhibition with no alteration in t-methylhista- mine concentration. PCA rats excreted 150% of 1. Fogel W.A., Michelsen K.A., Granerus G., Sasiak K., t-methylhistamine, 245% of t-methylimidazoleacetic Andrzejewski W., Panula P., Maslinski C.: Neuronal acid and 840% of unmetabolized histamine com- storage of histamine in the brain and tele-methylimi- dazoleacetic acid excretion in portocaval shunted rats. pared to sham operated rats. PF 9601N given alone, J. Neurochem., 2002, 80, 1–8. either acutely or chronically, had no significant ef- 2. Kusche J., Feussner K.D., Lorenz W.: Intestinal mo- fect on urinary output of these compounds due to noamine oxidase: does it have a role in histamine ca- presumed metabolic shift to the oxidative deamina- tabolism? Agent. Action., 1982, 12, 53–59.

PRESYNAPTIC INHIBITION BY ENDOTOXIN OF THE NEUROGENIC VASOPRESSOR RESPONSE IN RATS IN THE EARLY PHASE OF SEPTIC SHOCK

Grzegorz Godlewski1, Eberhard Schlicker2, Barbara Malinowska1 Department of Experimental Physiology, Medical Academy, Mickiewicza 2a, PL 15-089 Bia³ystok, Poland, Department of Pharmacology and Toxicology, University of Bonn, D 53113 Bonn, Germany

Various inflammatory mediators, e.g. hista- In pithed and vagotomized rats injected with mine, prostaglandins, nitric oxide contribute to the pancuronium (0.8 mmol/kg), electrical (1 Hz, 1 ms, cardiovascular collapse during sepsis. Recently, the 50 V, 10 s) stimulation of preganglionic sympa- endocannabinoid anandamide has also been shown thetic nerve fibers innervating the resistance ves- to cause vasodilation in response to bacterial endo- sels or injection of noradrenaline (1–3 nmol/kg) in- toxin through the activation of vascular cannabi- creased diastolic blood pressure (DBP) by about noid CB1 receptors [1]. The present study was un- 30 mmHg. Since injection of LPS (4 mg/kg iv) pro- dertaken to examine (i) whether lipopolysaccharide duced a profound hypotension throughout the ex- (LPS) affects the neurogenic vasopressor response periment, an infusion of vasopressin was used to via presynaptic mechanisms and (ii) to characterize obtain a stable value of basal DBP, comparable to the type of presynaptic receptor operating in the that in saline-treated animals. LPS inhibited the initial phase of septic shock. electrically induced vasopressor response. The in-

488 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS hibition occurred immediately after LPS admini- We conclude that LPS affects the neurogenic stration and amounted to about 40–50%. In con- vasopressor response via presynaptic receptors and trast, the rise in DBP evoked by noradrenaline was that cannabinoid CB1 and adrenergic a2 receptors not modified by endotoxin. The LPS-induced inhi- may be involved in the hypotension observed in the bition of the neurogenic vasopressor response was initial phase of septic shock. abolished by the cannabinoid CB1 receptor antago- nist SR 141716A (100 nmol/kg) and augmented REFERENCE by the a -adrenoceptor antagonist rauwolscine 2 1. (1 mmol/kg), whereas it was not affected by the Wagner J.A., Varga K., Kunos G.: Cardiovascular ac- tions of cannabinoids and their generation during cannabinoid CB2 receptor antagonist SR 144528 shock. J. Mol. Med., 1998, 76, 824–836. (100 nmol/kg) and the histamine H3 receptor an- tagonist clobenpropit (100 nmol/kg).

a1-ADRENOLYTIC PROPERTIES OF ANTIARRHYTHMIC ACTIVE ALKOXYPHENYLPIPERAZINE DERIVATIVES OF PHENYTOIN

Jadwiga Handzlik1, Dorota Maci¹g2, Heinz H. Pertz5, Sven Jähnichen5, Marek Bednarski3, Barbara Filipek3, Tomasz Dyl¹g4, Katarzyna Kieæ-Kononowicz1 Department of Chemical Technology of Drugs, Department of Pharmacobiology and !Laboratory of Pharmacological Screening, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, "Faculty of Chemistry, Jagiellonian University, Ingardena 3, PL 30-060 Kraków, Poland, #Institute of Pharmacy, Free University of Berlin, Germany

Several chemical modifications of an anticon- sive action in Wistar rats. Affinity for a1- and a2-adre- vulsant and antiarrhythmic drug phenytoin yielded noceptor was assessed in radioligand binding assay. new structures [1, 2], which changed acidic pro- Selectivity for a1-adrenoceptor (a1-AR) subtypes perty of phenytoin into more basic. As a result of the was tested in functional bioassays. Affinities for modifications, compound I (R =C2H5, R =H) a1A-ARs in rat tail artery and a1D-ARs in rat aorta was obtained which demonstrated antiarrhythmic were nearly identical (pA2 6.5–7.2), while affinities and antihypertensive activity and similar a1- and for a1B-ARs in guinea pig spleen were 4–16-fold a2-adrenoreceptor affinity. lower. Finally, an influence of the corresponding substituent on pharmacological properties was de- termined. R2 O OH N N N N R1 REFERENCES I O 1. Ciechanowicz-Rutkowska M., Stadnicka K., Kieæ- Kononowicz K., Byrtus H., Filipek B., Zygmunt M., 1 2 Treating the compound I (R =C2H5,R =H) Maci¹g D.: Structure-activity relationship of some as a lead, a series of alkoxyphenylpiperazine phe- new antiarrhythmic phenytoin derivatives., Arch. Pharm. Pharm. Med. Chem., 2000, 333, 357–364. nytoin derivatives was obtained I (R =CH3,C2H5, 2. Kieæ-Kononowicz K., Stadnicka K.,. Mitka A., Pêkala CH2COOCH3,CH2COOC2H5, CH(CH3)COOCH3, E., Filipek B.,. Sapa J,. Zygmunt M.: Synthesis, struc- CH(CH3)COOC2H5; R = OCH3,OC2H5). The ture and antiarrhythmic properties evaluation of new compounds were examined for their influence on basic derivatives of 5,5-diphenylhydantoin. Eur. J. normal ECG and antiarrhythmic and antihyperten- Med. Chem., in press.

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DOPAMINE AND ADDICTION: ROLE IN DRIVE, MOTIVATION AND (PHARMACO)THERAPY

Ursula Havemann-Reinecke Neurobiology of Psychosis and Addiction, Department of Psychiatry and Psychotherapy of the University of Goettingen, v. Sieboldstr. 5, D 37075 Goettingen, Germany

Experience of euphoria and “ecstasy” as well as serotonin, opioid peptides), kind of drug, age, de- drug reinforcing-, sensitization- and conditioning- velopmental and neurodegenerative factors, comor- mechanisms are described to be important for the bidity of other psychiatric diseases and environ- development and maintenance of psychic depend- mental elements that interact to produce profound ence on alcohol and other drugs. Especially chan- individual differences in both, initial and long-term ges in central dopaminergic neuronal activities in- responsiveness to addictive drugs. Experiences of duced by all addictive drugs seem to be critically “ecstasy” (intoxication) could be one important involved. Dopamine basically changes the drive factor, but are not necessary. Single representative and motivation of an individual towards addictive results from animal and clinical studies especially drugs by influencing basic mechanisms of incen- on (genetic) individual differences will be presen- tive and instinctive behavioral mechanisms leading ted to support this statement. Basing on the neuro- in part to craving, drug seeking and relapse behav- biological mechanisms of addiction, different clini- ior, which are all clinically difficult to treat. Pro- cal (pharmaco) treatment strategies (detoxification, nounced individual differences in vulnerability to substitution/maintenance, anti-craving) and the role addictive drugs and in the sensitivity of the dopa- of dopamine will be discussed. minergic neuronal activity (i.e. receptors) are de- scribed in animals (i.e. rats) [1] and humans. The REFERENCE occurrence of pathologic drive and motivation to 1. use an addictive drug (psychic dependence) with Germeyer S., Birke A., Schmitt U., Dahmen N., Hiemke C., Havemann-Reinecke U.: New dopamine drug seeking behavior is the outcome of a number D2 receptor polymorphisms in rats and association of variables including genic, neuronal (dopamine with apomorphine-induced stereotypies. Brain Res., and interactive transmitters, i.e. glutamate, GABA, 2002, 926, 1–9.

STAT3 SUPPRESSES AN ANTI-ANGIOGENETIC GENE PROGRAM AND IS IMPORTANT FOR MAINTENANCE OF CAPILLARIZATION IN THE ADULT MOUSE MYOCARDIUM

Denise Hilfiker-Kleiner1, Karol Kamiñski2, Martin Fuchs1, Arnd Schaefer1, Andres Hilfiker1, Gunnar Klein1, Zhao Ding3, Edith Podewski1, Helmut Drexler1 Department of Cardiology and Angiology, Medical School of Hannover, Germany, Department of Cardiology, Medical Academy, M. Sk³odowskiej-Curie 24a, PL 15-276 Bia³ystok, Poland, !Institute for Heart and Circulatory Physiology, University of Düsseldorf, Germany

Paracrine and autocrine mechanisms play an transcription (STAT3) in cardiomyocytes induces important role in myocardial vascularization. Over- expression of pro-angiogenic vascular endothelial expression of the signal transducer and activator of growth factor (VEGF) and may, therefore, be in-

490 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS volved in myocardial angiogenesis [1]. In the pres- blots, RT-PCR and Western blots, showed no dif- ent study, we investigated the function of STAT3 in ference in the expression of pro-angiogenic genes postnatal myocardial vascularization. such as VEGF, VEGF receptors (FLT-1, FLK), Using the Cre/LoxP system we generated mice Tie-1, c-met or CYR61. In contrast, increased ex- with a cardiac specific deletion of STAT3 (KO: pression of pro-fibrotic and anti-angiogenic genes, a-MHC-Cretg/+; stat3flox/flox). STAT3-deficient mice such as connective tissue growth factor (CTGF: developed reduced myocardial capillary density 194%, p < 0.05), thrombospondin-1 (TSP-1: 204%, with in the first three postnatal months (–30%, p < p < 0.05) and tissue inhibitor of metalloproteina- 0.05) as determined in H&E stained sections and se-1 (TIMP1: 402%, p < 0.05), p < 0.05) was ob- by immunohistochemical stainings using the endo- served. TSP-1 is known to induce together with thelial cell marker PECAM-1. Average size and FAS apoptosis in endothelial cells. Injection of density of coronary resistance vessels were similar agonistic FAS antibody for 24 h significantly in- in KO and wildtype (WT: stat3flox/flox) mice. Along creased number of apoptotic cells (assessed by this line, basal coronary flow (14 ± 3 vs. 17 ± 3 TUNEL Assay) in LVs of KO mice compared to ml/min) and peak coronary flow (46 ± 6 vs. 49 ± 5 WT animals (0.0033% vs. 0.0013%, p < 0.01). ml/min), known to be regulated primarily by coro- In summary, our observations establish a novel nary resistance vessels rather than capillaries were role for STAT3 in controlling paracrine circuits es- not altered in isolated Langendorff-perfused KO sential for postnatal maintenance of capillary vas- and WT hearts. There were no signs of hypoxia as culature in the heart. determined by the expression of hypoxia marker genes hypoxia inducible factor (HIF) and BNIP3 in REFERENCE KO or WT myocardium. In order to identify the 1. molecular mechanisms underlying the impaired Osugi T., Oshima Y., Fujio Y., Funamoto M., Ya- mashita A., Negoro S., Kunisada K., Izumi M., Naka- capillarization in KO mice, we performed a custom- oka Y., Hirota H., Okabe M., Yamauchi-Takihara K., made cDNA micro-array containing 648 genes, on Kawase I., Kishimoto T.: Cardiac-specific activation cDNA from left ventricles (5 of each genotype of signal transducer and activator of transcription 3 were pooled) of 12 weeks old KO and WT mice. promotes vascular formation in the heart. J. Biol. Micro-array data, in part confirmed by Northern Chem., 2002, 277, 6676–6681.

REGULATION OF THE PRO-ANGIOGENIC FACTOR CYR61 IN CARDIAC MUSCLE: IMPACT OF ISCHEMIA, BIOMECHANICAL STRESS, AND NEUROHORMONAL ACTIVATION

Denise Hilfiker-Kleiner1, Karol Kamiñski2, Agnieszka Kamiñska2, Andres Hilfiker1, Martin Fuchs1, Gunnar Klein1, Edith Podewski1, Kai C. Wollert1, Helmut Drexler1 Department of Cardiology and Angiology, Medical School of Hannover, Germany, Department of Cardiology, Medical Academy, M. Sk³odowskiej-Curie 24a, PL 15-276 Bia³ystok, Poland

Background. CYR61, a potent pro-angiogenic gated the regulation of CYR61 in cardiomyocytes factor, is induced in the vasculature by tissue in- in vitro and in the heart in vivo. jury, angiotensin II (AngII), and growth factor Methods and Results. AngII, signaling via the stimulation. As these conditions occur in myocar- AT 1-receptor, and adrenergic stimulation with phe- dial ischemia and pressure overload, we investi- nylephrine (PE) or norepinephrine (NE) induced

ISSN 1230-6002 491 Abstracts of Symposium

CYR61 expression in ventricular cardiomyocytes 6 h after the onset of ischemia. Similarly, a tran- (CM) isolated from 1- to 3-day-old rats, and pro- sient myocardial ischemia, followed by reperfusion moted release of CYR61 protein into the culture caused rapid, but very transient increase in abun- supernatant. AngII and PE-mediated induction of dance of CYR61 transcripts. Immunohistochemical CYR61 expression in CM was completely abol- staining doubtlessly proved that CM are the main ished by inhibition of MEK/ERK or protein kinase source of CYR61 after myocardial stress in vivo, C (PKC). Likewise, mechanical stretch induced and continue to express increased levels of CYR61 CYR61 expression in CM in vitro. Similarly, pres- as late as 6 months after myocardial infarction. sure overload in vivo up-regulated myocardial Conclusions. Pressure overload, ischemia, and CYR61 expression levels via AT 1-receptor and neurohormonal factors, such as AngII or adrenergic PKC-dependent mechanisms. Following myocar- stimuli, induce myocardial expression of CYR61, dial infarction in mice, CYR61 mRNA and protein a potent pro-angiogenic factor, supporting the no- expression was strongly induced, both in ischemic tion that CYR61 may play an important role in the and remote left ventricular myocardium, peaking adaptation of the heart to cardiovascular stress.

CANNABINOIDS ATTENUATE RECOGNITION MEMORY IN RATS

Piotr Kosiorek, Anna Hryniewicz, Izabela Bialuk, Robert £. Zbucki, Maria M. Winnicka Department of General and Experimental Pathology, Medical Academy, Mickiewicza 2c, PL 15-222 Bia³ystok, Poland

Cannabinoids are known to attenuate learning crimination between the familiar and a new object and memory in both humans and animals. In the presented in 1 h interval. Because cannabinoids at brain, they mainly bind to cannabinoid CB1 recep- the higher doses can produce motor inhibition and tors for which anandamide is a purported endoge- anxiogenic-like effect [1], the influence of both nous ligand. SR 141716, a specific antagonist of compounds on psychomotor activity and its anxio- CB1 receptors, dose-dependently reverses behav- genic property was evaluated in an open field and ioral, biochemical and pharmacological effects of in elevated plus maze test, respectively. CP 55,940 cannabimimetics. In animals, disruptive effect of and R-(+)-methanandamide, at both doses given cannabinoids on memory, reversed by SR 141716, once 15 min before presenting object A, signifi- was shown in behavioral tests based on condition- cantly attenuated recognition memory, measured ing. There is no data concerning the influence of by the difference in exploration of a new object B cannabinoids on recognition memory. However, and a duplicate of a familiar object A conducted 1 h one study suggested that CB1 receptor blockade by later. Moreover, CP 55,940 at the higher dose sig- SR 141716 might improve memory in the social nificantly attenuated ambulation, rearings and bar recognition test in rats [3]. This finding was con- approaches and at both doses also time of groom- firmed by improvement of recognition memory re- ing evaluated in an open field, performed immedi- ported in cannabinoid CB1 receptor knock-out ately after “object recognition” test, while R-(+)- mice [2]. Therefore, the purpose of the present methanandamide at both doses did not alter loco- study was to determine, whether a stable analogue motor and exploratory activity of rats. Moreover, of endogenous cannabinoid anandamide, R-(+)-me- there were no differences in performance between thanandamide (0.25 and 2.5 mg/kg, ip) and potent cannabinoid-injected and control rats in elevated CB1 receptor agonist, CP 55,940 (0.025 and 0.25 plus maze test, which may indicate that the effect mg/kg ip) affect recognition memory in rats evalu- observed in “object recognition“ test is memory ated in an “object recognition“ test, based on dis- specific. This is the first evidence that cannabi-

492 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS noids, a stable analogue of anandamide – R-(+)- pothalamic neurotransmitters. Pharmacol. Biochem. methanandamide, as well as, CB1 receptor agonist, Behav., 2001, 70, 123–131. 2. CP 55,940 impair recognition memory in rats. Reibaud M., Obinu M.C., Ledent C., Parmentier M., Böhme G.A., Imperato A.: Enhancement of memory Acknowledgment. The study was supported by AMB in cannabinoid CB1 receptor knock-out mice. Eur. J. grant No 3-24794. Pharmacol., 1999, 379, R1–R2. 3. Terranova J.P., Storme J.J., Lafon N., Pério A., Ri- REFERENCES naldi-Carmona M., Le Fur G., Soubrié P.: Improve- ment of memory in rodents by selective CB1 cannabi- 1. Arvéalo C., de Miguel R., Hernández-Tristán R.: Can- noid receptor antagonist, SR 141716. Psychopharma- nabinoid effects on anxiety-related behaviours and hy- cology, 1996, 126, 165–172.

DEPRESSIVE-LIKE BEHAVIOR IN RATS WITH NEONATAL 5-HYDROXYTRYPTAMINE DEPLETION

Wojciech Kostowski, Pawe³ Krz¹œcik Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Sobieskiego 1/9, PL 02-957 Warszawa, Poland

Little information exists about the subsequent trol groups while fluoxetine produced anti-immo- behavioral effects induced after developing neona- bility effect only in rats pretrated neonatally with tal rats had been depleted of serotonin (5-HT). It 5,7-DHT. We propose that the neonatal lesion of is known that neonatal 5,7-dihydroxytryptamine 5-HT neurons might be considered as an animal (5,7-DHT) treatment produces a marked 5-HT de- model of depression and a tool for looking into nervation in different brain areas [1, 2]. The present neuronal mechanism that may underlie the etiology study was performed to investigate the effect of of depression. treatment of newborn rats with 5,7-DHT on their subsequent behavior in forced swimming test. When 3-day-old rats were injected intracisternally with REFERENCES the neurotoxin, a marked depletion of brain 5-HT (but not noradrenaline and dopamine) was obser- 1. Jessa M., Krz¹œcik P., Kostowski W.: Neonatal treat- ved when animals were killed 4 months after the ment with 5,7-dihydroxytryptamine induces decrease treatment. The behavioral consequence of 5,7-DHT in alcohol drinking in adult rats. Pol. J. Pharmacol., 2001, 53, 109–116. administration to developing rats consisted in the 2. Sachs C., Jonsson G.: 5,7-Dihydroxytryptamine-indu- significant reduction of activity of adult animals in ced changes in the postnatal development of central the forced swimming test. Desipramine markedly 5-hydroxytryptamine neurons. Med. Biol., 1975, 53, increased activity of rats in both 5,7-DHT and con- 156–164.

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ATYPICAL b-ADRENOCEPTORS RELAX THE HUMAN PULMONARY ARTERY

Hanna Koz³owska1, Miros³aw Koz³owski2, Urszula Szymska1, Eberhard Schlicker3, Barbara Malinowska1 Department of Experimental Physiology, Medical Academy, Mickiewicza 2a, PL 15-089 Bia³ystok, Poland, Department of Thoracic Surgery, Medical Academy, M. Curie-Sk³odowskiej 24a, PL 15-276 Bia³ystok, Poland, !Department of Pharmacology and Toxicology, University of Bonn, D 53113 Bonn, Germany

In addition to b2-adrenoceptors, the existence of of the agonists, respectively). These vasodilatory b3- and/or atypical b-adrenoceptors has been sug- effects were strongly inhibited (cyanopindolol) or gested in human umbilical artery [1] and in human abolished (CGP 12177) by bupranolol (10 mM), the internal mammary artery [2], respectively. In the non-selective b-adrenoceptor antagonist also known present study, we examined whether b-adrenoce- to block responses mediated via b3- or atypical b-adre- ptors different from b2- (and b1-) adrenoceptors noceptors. In endothelium-denuded rings, both cy- also relax the isolated human pulmonary artery, anopindolol and CGP 12177 did not cause any re- pre-contracted with phenylephrine (10 mM). The laxation. b2-adrenoceptor agonist, fenoterol (1 mM–1mM), In conclusion, we suggest that, in addition to produced a concentration-dependent relaxation of b2-adrenoceptors, also endothelial atypical b-adre- pulmonary rings. The vasodilatory effect obtained noceptors (or b3-adrenoceptors) relax the human for the highest concentration of the agonist was pulmonary artery. about 85%. The concentration response curve for fenoterol was shifted to the right by the b2-adren- oceptor antagonist ICI 118551 (1 mM) and by the REFERENCES b non-selective -adrenoceptor antagonist proprano- 1. Dennedy M.C., Houlihan D.D., McMillan H., Morri- m b lol (0.3 M). Two non-conventional partial -adre- son J.J.: Beta2- and beta3-adrenoreceptor agonists: hu- noceptor agonists, cyanopindolol (1 mM – 0.3 mM) man myometrial selectivity and effects on umbilical and CGP 12177 (1 mM – 1 mM) given in the pres- artery tone. Amer. J. Obstet. Gynecol., 2002, 187, ence of propranolol (0.3 mM), also produced a con- 641–647. 2. centration-dependent relaxation of phenylephrine- Shafiei M., Omrani G., Mahmoudian M.: Coexistence of at least three distinct beta-adrenoceptors in human preconstricted human pulmonary artery (maximally internal mammary artery. Acta Physiol. Hung., 2000, by about 75 and 40% for the highest concentration 87, 275–286.

INCIDENTAL LEWY BODIES AND NEURONAL PATHOLOGY IN ASYMPTOMATIC PATIENTS

Anna Krygowska-Wajs1, Dariusz Adamek2, Józef Ka³u¿a2 Department of Neurology and Department of Neuropathology, Collegium Medicum, Jagiellonian University, Botaniczna 3, PL 31-503 Kraków, Poland

Lewy bodies (LBs) and the loss of neurons in are found in small numbers in brains of clinically substantia nigra and locus coeruleus are the histo- normal elderly individuals [3], which appears to be pathologic hallmark of idiopathic Parkinson’s dis- a presymptomatic form of PD where a decrease in ease (PD), however, LBs are also detected in other the dopamine level has not reached 60% in the ba- neurodegenerative diseases, most notably in de- sal ganglia of the brain. The biological role of LBs mentia with LBs and Alzheimer’s disease [1]. LBs and other intracellular inclusions, the mechanisms

494 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS of the intracellular aggregation of insoluble protein senile type occurred more frequently than LBs. The deposits, and their implication for cellular dysfunc- increased frequency of neurodegenerative changes tion resulting in neurodegeneration are still unre- of senile type has been found in the brains of pa- solved [2]. tients with no symptoms of neurodegenerative dis- The purpose of this study was to determine the eases, which shows no apparent interaction be- frequency of LBs and neurodegenerative pathology tween LBs and the senile type pathology. This sug- in the brains of asymptomatic patients. We evalu- gests that degenerative process of senile type does ated brains of clinically normal elderly individuals, not seem to be related to nigral degeneration and who died of various causes but without symptoms LB formation. Further elucidation of neuronal pa- of parkinsonism or other neurodegenerative dis- thology in the early stage of PD will provide better eases ranging in age from 40 to 85 (average age insight into the pathogenesis of neurodegeneration 69). There were 11 males and 7 females and 4 pa- in PD and related disorders. tients affected by PD. All cases underwent a stan- dard morphological assessment including micros- Acknowledgment. Supported by statutory funds, grant copy of multiple cortical and subcortical sections to No. 501/KL/202/L, of the Collegium Medicum, Jagiello- detect neurodegenerative pathology. Immunocyto- nian University, Kraków, Poland. chemical examination was performed using mono- clonal antibodies against a-synuclein, tau protein, REFERENCES ubiquitin and b-amyloid. 1. Forno L.S.: Concentric hyalin intraneuronal inclu- The brains of 4 individuals with PD have shown sions of Lewy type in the brain of elderly persons (50 the presence of LBs and loss of neurons in substan- incidental cases). Relationship to parkinsonism. J. tia nigra and locus coeroleus. In the brains of Amer. Geriatr. Soc., 1969, 17, 557–575. 2. asymptomatic individuals, the tau pathology, a-sy- Jellinger K.A.: Recent developments in the pathology b of Parkinson’s disease. J. Neural Transm. Suppl., nuclein and -amyloid have been shown in the 2002, 62, 347–376. cerebral and cerebellar cortex, basal ganglia and 3. Van Duinen S.G., Lammers G.J., Maat-Schieman M.L., medulla oblongata. Most cases also had b-amyloid Ross R.A.C.: Numerous and widespread alpha-sy- positive senile plaques. LBs were found in 20% of nuclein-negative Lewy bodies in an asymptomatic pa- the studied individuals. Morphological changes of tient. Acta Neuropathol., 1999, 97, 533–539.

DESIGN, SYNTHESIS AND DEVELOPMENT OF 1-[3-(4-ARYLPIPERAZIN-1-YL)-PROPYL]-PYRROLIDIN-2-ONES AS POTENTIAL a1-ADRENOCEPTOR ANTAGONISTS

Katarzyna Kulig1, Barbara Malawska1, Barbara Filipek2, Dorota Maci¹g3, Jacek Sapa2, Ulrike Holzgrabe4, Cindy Spies1*, Annika Gippert1* Department of Pharmaceutical Chemistry, Laboratory of Pharmacological Screening, and !Radioligand Laboratory, Faculty of Pharmacy, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, "Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, Am Hubland, D 97074Würzburg, Germany

Adrenergic receptors (AR) are found through- tive a1-AR antagonist has intensified. Currently, out the body and play a dominant role in the control the presence of three a1-AR subtypes, a1A, a1B, of blood pressure, prostate function, and other pro- and a1D, has been established by means of molecu- cesses. In recent years, the search for a new selec- lar biology, radioligand binding and functional

ISSN 1230-6002 495 Abstracts of Symposium studies [1]. Search for new compounds, which substituted at the ortho-position of phenyl ring, and show high specificity to recognize target can be 1-[3-(4-arylpiperazin-1-yl)-propyl]-pyrrolidin-2-one carried out by several methods. In our investigation, derivatives, which displayed nanomolar affinity for the strategy based on modification of lead struc- a1- and a2-AR. ture, i.e. 1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)- propyl]-pyrrolidin-2-one was chosen [2]. REFERENCES The modifications included: 1. — introduction of various substituents into the Bock M., Patane M.: Toward the development of a1a-adrenergic receptor antagonists. Ann. Rep. Med. phenyl ring, Chem., 2000, 35, 221–230. — synthesis of 1-[3-(4-arylpiperazin-1-yl)-propyl]- 2. Malawska B., Kulig K., Filipek B., Sapa J., Maci¹g pyrrolidin-2-one derivatives, D., Zygmunt M., Antkiewicz-Michaluk L.: Synthesis, — synthesis of enantiomers of lead structure and antiarrhythmic and antihypertensive effects of novel its analogues. 1-substituted pyrrolidin-2-one and pyrrolidine deriva- The obtained compounds were tested in vitro tives with adrenolytic activity. Eur. J. Med. Chem., 2002, 37, 183–195. for a1- and a2-AR binding affinity, and in vivo for antiarrhythmic and hypotensive activity. It was found that some of them exhibited higher or com- * They were on leave from the Pharmaceutical Institute, Uni- parable with the lead structure pharmacological ac- versity of Bonn, Germany within the European Erasmus ex- tivity. Especially promising seem to be compounds change students’ program.

STUDIES ON ASSOCIATIVE AND NON-ASSOCIATIVE TYPE OF SENSITIZATION TO AMPHETAMINE AND MORPHINE

Klaus Kuschinsky, Martina Grönig, Awad Atalla Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Ketzerbach 63, D 35032 Marburg, Germany

After repeated administration of many addictive Neither L-NAME (10mg/kg ip) nor MK-801 drugs, such as psychostimulants and opioids, in (0.1 mg/kg ip) affected the development of sensiti- many cases no tolerance but a “reverse tolerance“, zation to morphine (3 mg/kg ip), neither of the as- a sensitization to the stimulant effects can be ob- sociative nor of the non-associative type. In con- served. This phenomenon can be in part (but not trast, L-NAME inhibited the expression of both the completely) explained by the presence of condi- associative and the non-associative type of sensiti- tional stimuli in association with drug administra- zation, whereas MK-801 did not influence either tion so that conditioned responses interact with type. MK-801 inhibited the expression of sensitiza- drug effects. In our previous study, it was shown tion to d-amphetamine (each dose during sensitiza- that an inhibition of nitric oxide synthase (NOS) tion and test dose of 1 mg/kg ip) only in rats sensi- could inhibit the expression (manifestation) of as- tized in the associative way. The latter findings and sociative but not of non-associative sensitization to the previous ones [1] suggest that both NMDA re- d-amphetamine [1]. Since NOS can be activated by ceptors and NOS might be involved in the expres- stimulation of glutamate-NMDA receptors, the ef- sion of the associative type of sensitization to d- fects of an inhibitor of NOS, L-NAME (NG-nitro- amphetamine. NOS but not NMDA receptors seem -L-arginine methyl-ester) or of a blocker of NMDA to be involved in the expression of both types of receptors, dizocilpine (MK-801) on sensitization sensitization to morphine. phenomena (measured as locomotor activity) were The results suggest that associative and non- studied. associative types of sensitization can be pharmaco-

496 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS logically influenced in different ways. Since sensi- REFERENCE tization phenomena, in particular the associative 1. Afanas’ev I., Ferger B., Kuschinsky K.: The associa- ones, seem to be relevant to addiction processes, tive type of sensitization to d-amphetamine is expressed these observations might be of therapeutic rele- as an NO-dependent dramatic increase in extracellular vance. dopamine in the nucleus accumbens. Naunyn-Schmied. Arch. Pharmacol., 2000, 362, 232–237.

SEARCH FOR THE MECHANISMS RESPONSIBLE FOR THE ANANDAMIDE-INDUCED INCREASE IN BLOOD PRESSURE IN URETHANE-ANESTHETIZED RATS

Grzegorz Kwolek1, Agnieszka Zakrzeska1, Grzegorz Godlewski1, Barbara Malinowska1, Eberhard Schlicker2, Manfred Göthert2 Department of Experimental Physiology, Medical Academy, Mickiewicza 2a, PL 15-089 Bia³ystok, Poland, Department of Pharmacology and Toxicology, University of Bonn, D 53113 Bonn, Germany

In urethane-anesthetized rats, intravenous injec- blocker and inhibitor of vanilloid responses ruthe- tion of the endogenous cannabinoid (CB) receptor nium red, by the calcium channel blocker nifedip- ligand anandamide (AND) and the vanilloid VR1 ine and by the non-competitive NMDA receptor receptor agonist capsaicin (CAP) induced tri- and antagonist MK-801. However, it was not modified biphasic changes in cardiovascular parameters, re- by the non-selective endothelin receptor antagonist spectively. Thus, the VR1 receptor-mediated tran- tezosentan, by the VR1 receptor antagonist cap- sient bradycardia and hypotension is followed by a sazepine and by adrenalectomy. Moreover, AND brief pressor response and then by a more pro- did not change the electrically evoked increase in longed CB1 receptor-dependent fall in blood pres- blood pressure in pithed rats, treated with the CB1 sure. CAP does not induce the third phase [1]. receptor antagonist SR 141716, both in the absence The aim of the present study was the search for and in the presence of capsazepine. the mechanism responsible for the brief pressor re- In conclusion, the AND-induced increase in sponse. We found that the AND (0.3–10 mg/kg)- blood pressure observed in urethane anesthetized and CAP (0.3 – 30 mg/kg)-induced increase in rats is propranolol-sensitive and dependent on the blood pressure was abolished in pentobarbitone- level of the sympathetic tone. However, its detailed anesthetized animals. The pressor responses to both mechanism still remains to be established. agonists were diminished in rats anaesthetized with urethane and then pithed and vagotomized in which an infusion of vasopressin adjusted blood pressure REFERENCE to the same level as in anesthetized animals. Pres- 1. sor responses were also reduced by the non-selec- Malinowska B., Kwolek G., Göthert M.: Anandamide and methanandamide induce both vanilloid VR1- and tive b1-/b2-adrenoceptor antagonist propranolol. cannabinoid CB1 receptor-mediated changes in heart Moreover, the increase in blood pressure evoked by rate and blood pressure in anaesthetized rats. Nau- AND was diminished by the non-selective channel nyn-Schmied. Arch. Pharmacol., 2001, 364, 562–569.

ISSN 1230-6002 497 Abstracts of Symposium

PRO- AND ANTI-APOPTOTIC EFFECTS OF NMDA RECEPTOR STIMULATION

W³adys³aw Lasoñ1, Ma³gorzata Kajta1,2, Cordian Beyer2 Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland, Department of Anatomy and Cell Biology, University of Ulm, Albert-Einstein-Allee 11, D 89061 Ulm, Germany

It has been commonly accepted that NMDA re- properties of NMDA, we investigated its effect on ceptors play a crucial role in neurotoxicity. Expo- protein kinase inhibitor (staurosporine)-induced sure of neuronal cells to high concentrations of caspase-3 activity and LDH release in primary NMDA results in calcium overload and necrotic mouse neocortical and hippocampal cell cultures at cell death, whereas at moderate concentrations it various stages of their development. triggers apoptotic processes in a variety of in vitro The data showed that NMDA (200 mM) stimu- models. On the other hand, stimulation of NMDA lated staurosporine (0.5 mM)-induced caspase-3 ac- receptors may also increase survival of neurons, es- tivity in cortical cells on 12 DIV and in hippocam- pecially at early stage of their development, and pal neurons on 2 DIV. In contrast, on 7 DIV NMDA receptor antagonists are known to induce NMDA inhibited staurosporine-induced caspase-3 apoptosis in both in vitro and in vivo conditions. activity in cortical and in hippocampal neurons. The anti-apoptotic and putative neuroprotective ef- This potentially anti-apoptotic effect of NMDA fects of NMDA are reflected in “preconditioning” was not followed by a decrease in staurosporine- phenomenon, where pretreatment of neuronal cells induced LDH-release up to 24 h post-treatment. with sublethal concentrations of NMDA via induc- Furthermore, the experiments revealed that besides tion of transcription and neurotrophic factors, e.g. stage of cell development and origin of cell culture NFkB, BDNF and heat shock proteins protects (hippocampus, neocortex), the pro- or anti-apopto- these cells against subsequent lethal concentrations tic effects of NMDA appear to depend on cellular of excitatory amino acids. In order to get a better composition of culture (mixed glia-neuronal or insight into mechanism of pro- and anti-apoptotic pure neuronal).

BLOOD CELLS AND COAGULATION

Wolfgang Lösche1, Stan Heptinstall2 Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Jena, Germany, Division of Cardiovascular Medicine, University Hospital Nottingham, UK

Blood coagulation is a process that prevents intravascular coagulation, and large amounts of TF blood loss after a blood vessel injury. However, in are found within the plaques. On the other hand, TF various pathological conditions intravascular co- circulating in the plasma, is believed to play an im- agulation occurs resulting in the formation of thrombi portant role in thrombus formation [2]. It has been that may occlude the blood stream. One of the main shown that both blood monocytes and platelets can causes of thrombus formation in cardiovascular release TF-bearing microvesicles (MV, Æ 0.2–0.6 disease is the rupture of an atherosclerotic plaque mm) that promote thrombin and fibrin formation. resulting in blood platelet adhesion and aggrega- TF-release from monocytes stimulated by endoto- tion as well as in thrombin and fibrin formation. xins or pro-inflammatory cytokines requires gene Tissue factor (TF) is the most important initiator of expression and protein synthesis. In contrast, plate-

498 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS let-TF is stored in and released within a few minu- This observation may explain why oral GPIIb/ tes from intracellular granules. Platelets and plate- IIIa-antagonists that were developed for secondary let-derived MV can transfer TF to leukocytes, in prevention in coronary artery disease had a poor particular to monocytes, and this transfer seems to anti-thrombotic efficiency as shown in recent clini- be important for its optimum procoagulant activity. cal trials. The interaction of platelets and platelet-derived MV with leukocytes is mediated by the platelet ad- hesion molecule CD62P and its receptor on the leu- REFERENCES kocyte surface, PSGL-1, and blocking the CD62P- 1. PSGL-1 interaction prevents the TF transfer from Muller I., Klocke A., Alex M., Kotzsch M., Luther T., Morgenstern E., Zieseniss S., Zahler S., Preissner K., platelets to leukocytes [1]. On the other hand, gly- Engelmann B.: Intravascular tissue factor initiates co- coprotein (GP) IIb/IIIa-antagonists such as eptifi- agulation via circulating microvesicles and platelets. ® ® batide (Integrilin ) or abciximab (Reopro ), that FASEB J., 2003, 17, 476–478. prevent the binding of fibrinogen to its receptor on 2. Rauch U., Nemerson Y.: Circulating tissue factor and the platelet surface and that are very potent inhibi- thrombosis. Curr. Opin. Hematol., 2000, 7, 273–277. tors of platelet aggregation enhance the TF transfer 3. Scholz T., Zhao L., Temmler U., Heptinstall S., Lösche from platelets to leukocytes due to an enhanced in- W:. The GPIIb/IIIa antagonist eptifibatide markedly potentiates platelet-leukocyte interaction and tissue teraction with CD62P-positive platelets and plate- factor expression following platelet activation in let-derived MV [3]. whole blood in vitro. Platelets, 2002, 13, 401–406.

SEARCH FOR NON-IMIDAZOLE HISTAMINE H3-RECEPTOR ANTAGONISTS

Dorota £a¿ewska1, Katarzyna Kieæ-Kononowicz1, Heinz H. Pertz2, Sigurd Elz3, Xavier Ligneau4, Jean-Charles Schwartz5, Holger Stark6, Walter Schunack2 Department of Chemical Technology of Drugs, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, Institut für Pharmazie, Freie Universität Berlin, Germany, !Institut für Pharmazie, Pharmazeutische Chemie I, Universität Regensburg, Germany, "Laboratoire Bioprojet, Paris, France, #Unité de Neurobiologie et Pharmacologie Moléculaire, Centre Paul Broca de l’INSERM, Paris, France, $Institut für Pharmazeutische Chemie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

Histamine is thought to exert its action in the histamine H3-receptor ligands was derived from CNS and periphery via the modulation of four histamine itself and contains an imidazole moiety. known so far distinct G-protein coupled receptors. However, as the imidazole is known to cause inter- The third subtype of histamine receptor acts as a action with cytochrome P450 (CYP 450), they may presynaptic autoreceptor regulating the synthesis be of the limited therapeutic value. Therefore, the and release of histamine from histaminergic neu- need for non-imidazole H3 ligands has been recog- rons. Additionally, H3-receptor plays an important nized. Recently, a lot of non-imidazole N-hetero- role as a heteroreceptor in the regulation of the re- cyclic compounds have been described and now, lease of other neurotransmitters, e.g. noradrenaline this class of compounds is quickly developing. and dopamine. As H3-receptor antagonists increase Piperidine ring was shown as a good replace- the histamine level in the brain, they might be use- ment for an imidazole one. [1] In our search for ful in the therapy of diseases of the central nervous non-imidazole histamine H3-receptor antagonists, system like dementia, epilepsy, morbus Alzheimer, we also chose this ring. Carbamate and ether de- or schizophrenia. So far, the majority of the potent rivatives of piperidine were prepared. [2, 3] Most

ISSN 1230-6002 499 Abstracts of Symposium of the compounds were evaluated in vitro for their REFERENCES antagonist activity in isolated organs of guinea pig 1. ileum. They proved to be moderate to good H3-re- Ganellin C.R., Leurquin F., Piripitsi A., Arrang J.-M., ceptor antagonists. Ether derivatives were also Garbarg M., Ligneau X., Schunack W., Schwartz J.-C.: Synthesis of potent non-imidazole histamine tested in vitro on cloned human H3-receptor and in H3-receptor antagonists. Arch. Pharm. Pharm. Med. vivo after per os administration to mice. Some of Chem., 1998, 331, 395–404. them were active at the nanomolar concentration 2. £a¿ewska D., Kieæ-Kononowicz K., Pertz H.H., Elz range and possessed high oral potency. Structure- S., Stark H., Schunack W.: Piperidine-containing his- activity relationships will be discussed for different tamine H3-receptor antagonists of the carbamate se- series of compounds. ries: the influence of the additional ether functionality. Pharmazie, 2002, 57, 791–795. Acknowledgment. This work was supported in part by 3. £a¿ewska D., Kieæ-Kononowicz K., Pertz H.H., Stark the State Committee for Scientific Research Warszawa, Po- H., Schunack W., Elz S.: Piperidine-containing hista- land (grant No. P05F 013 20) and the International Bureau mine H3-receptor antagonists of the carbamate series: of the BMBF, Bonn, Germany, and Committee for Scien- variation of the spacer length. Pharmazie, 2001, 56, tific Research, Warszawa, Poland (grant POL-030-98). 927–932.

ISOBOLOGRAPHIC ASSESSMENT OF INTERACTIONS BETWEEN TIAGABINE AND SOME NEWER ANTIEPILEPTIC DRUGS IN PENTETRAZOLE-INDUCED SEIZURES IN MICE

Jarogniew J. £uszczki1, Stanis³aw J. Czuczwar1,2 Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland, Isotope Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland

Although monotherapy in epilepsy has been es- dents is an experimental model of myoclonic sei- tablished as the treatment of choice “gold stan- zures and, to a certain extent, of absence attacks in dard”, there are still numbers of patients (about humans [3]. The exact types of interactions be- 30%) being inadequately medicated with current tween TGB and second generation AEDs (gaba- front-line antiepileptic drugs (AEDs) [2]. In such pentin, oxcarbazepine, loreclezole, felbamate and cases, only rational polytherapy may provide the vigabatrin) were determined with the isobolo- patients with adequate seizure control. In order to graphic analysis, which is an eligible method al- create the rationale for polytherapy in patients with lowing the assessment of synergy, antagonism or refractory seizures, the theoretically established additivity among AEDs tested. Furthermore, the AED-combinations might be primarily tested in adverse effects produced by these AEDs in combi- animal models of epilepsy for assessing the exact nations were evaluated in the chimney test. types of interactions in vivo [1]. Results indicate that TGB combined with ga- The aim of this study was to investigate the ex- bapentin or vigabatrin exerted the synergistic type act types of interactions between tiagabine (TGB) of interactions in the PTZ-induced seizure test in and some second generation AEDs in pentetrazole mice. Moreover, TGB co-administered with oxcar- (PTZ)-induced seizures in mice. Most recently, bazepine or loreclezole showed only additive inter- TGB, a novel AED, has been introduced into the actions, whereas the combinations of TGB with fel- therapy of partial complex seizures in humans. bamate were either additive, for the fixed-ratios of Therefore, it seemed important to evaluate the in- 1:3 and 1:1, or antagonistic for the combination of teractions of this drug in combinations with newer 3:1. Furthermore, TGB combined with the studied AEDs. It is widely accepted that the PTZ test in ro- AEDs, at the doses effective against PTZ-induced

500 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS convulsions, had no significant impact on motor REFERENCES performance evaluated in the chimney test in mice. 1. Deckers C.L., Czuczwar S.J., Hekster Y.A., Keyser Finally, one can speculate that TGB with gaba- A., Kubova H., Meinardi H., Patsalos P.N., Renier pentin or vigabatrin, affecting the same neurotrans- W.O., Van Rijn C.M.: Selection of antiepileptic drug mitter system (i.e. GABA-ergic) might be the ad- polytherapy based on mechanisms of action: the evi- vantageous AED combinations in patients with dence reviewed. Epilepsia, 2000, 41, 1364–1374. intractable seizures with or without secondary ge- 2. Kwan P., Brodie M.J.: Early identification of refrac- tory epilepsy. N. Engl. J. Med., 2000, 342, 314–319. neralization. If the experimental data could be 3. easily extrapolated into clinical practice, these Löscher W., Honack D., Fassbender C.P., Nolting B.: The role of technical, biological and pharmacological combinations would be an efficacious mode of factors in the laboratory evaluation of anticonvulsant treatment in patients with epilepsy, providing them drugs. III. Pentylenetetrazole seizure models. Epilepsy with the adequate seizure control. Res., 1991, 8, 171–189.

ADVANCES IN HUMAN CYTOGENETICS TOWARDS OUR UNDERSTANDING THE MOLECULAR BASIS OF HUMAN DISEASES

Alina T. Midro Department of Clinical Genetics, Medical Academy, Waszyngtona 13, PL 15-230 Bia³ystok, Poland

Rapid progress in human genetics, cytogenetics human diseases. We would like to present our own and clinical genetics influenced our understanding experiences coming from clinical observation of of molecular basis of many diseases. This opened a patients with rare entities underlain by changes at new view for medicine with pharmacology in- the molecular level of human genome, which have cluded. It means that we have received a good tool been obtained also on the basis of Polish-German with real perspectives for individualization of phar- collaboration. The patient with Russell-Silver s. and macological treatment for common diseases. Inter- reciprocal chromosome translocation t(1;17)(q31;q25) estingly, a lot of useful data on molecular structure de novo has been studied in collaboration with of human genome has been tapped from naturally group of Prof. Ingo Hansmann from Göttingen and occurring chromosome rearrangements observed in Halle [1]. Deletion of PTCH gene in the patient the patients with a distinct phenotype. Banding with Gorlin-Goltz s. caused by interstitial deletion techniques, which are used for diagnosis of karyo- 9q22.32-q33.2 and with Nail Patella s. due to fa- types, have been unchanged from the 1970s apart milial translocation t(9;17)(q34.11;p11.2) was dia- from the introduction of digital image processing. They are now combined with different fluorescence gnosed in collaboration with the Berlin group [3]. in situ hybridization methods and molecular analy- A new type of mutation of SRY gene was reco- sis giving direct link between microscope and gnized on the basis of collaboration with Prof. Gerd DNA sequence. Detailed phenotype analysis in pa- Scherer’s group from Freiburg, and undescribed so tients with gross chromosomal rearrangements di- far mutation in TRPS1 gene has been detected by agnosed by these techniques and/or in patients suf- Dr. Hermann-Josef Lüdecke from Essen [2].On the fering from one from among several thousand basis of twenty year collaboration with the group of monogenic syndromes has been a challenge till Prof. Sabine Stengel-Rutkowski from München, now to obtain a valuable information about clini- we created Polish Collection of Chromosomal cal-molecular relationship and, furthermore, to im- Translocations as a part of International Collection prove our understanding at the molecular basis of for further studies of genetic-phenotypic correla-

ISSN 1230-6002 501 Abstracts of Symposium tion based on naturally occurring chromosomal and phalangeal syndromes types I and III. Amer. J. Hum. genomic rearrangements. Genet., 2001, 68, 81–91. 3. Midro A.T., Panasiuk B., Tümer Z., Stankiewicz P., REFERENCES Silahtaroglu A., Lupski J.R., Zemanova Z., Stasie- wicz-Jarocka B., Hubert E., Tarasów E., Famulski W., 1. Dörr S., Ayala-Madriga M.L., Midro A.T., Giannaku- Zadro¿na-To³wiñska B., Wasilewska E., Kirchhoff dis J., Hansmann I.: Construction of a detailed physi- M., Kalscheuer V., Michalova K., Tommerup N.: In- cal and transcript map of the candidate region for terstitial deletion 9q22.32-q33.2 associated with addi- Russel-Silver syndrome on chromosome 17q23-q24. tional familial translocation t(9;17)(q34.11;p11.2) in Genomics, 2001, 71, 174–181. a patient with Gorlin-Goltz syndrome and features of 2. Lüdecke H.J., Schaper J., Meinecke P., et al.: Geno- nail-patella syndrome. Amer. J. Med. Genet., 2003, in typic and phenotypic spectrum in the tricho-rhino- press.

PLANT COMPONENTS AND THEIR ROLE IN HUMAN CYTOGENETICS

Alina T. Midro1, Gesa Schwanitz2 Department of Clinical Genetics, Medical Academy, Waszyngtona 13, PL 15-230 Bia³ystok, Poland, Institute of Human Genetics, University of Bonn, Germany

Different plant components played a significant They lead to an equal distribution of the chromo- role in the development of chromosome diagnos- somes in the cell lumen and additionally improve tics in the last 50 years. In the beginning, phytohe- their spiralization. Furthermore, methods of cell magglutinin enabled the cultivation of T-lympho- synchronization through substances like vincristine cytes from peripheral blood, followed by concana- and vinblastine were successfully tested. Finally, valin which stimulates B-lymphocytes, both of different plant components played an important them transformating cells in Go-stage and starting role in chromosome staining and the selective their entrance into the cell cycle. Then, substances analyses of different structures of the cell. Exam- inhibiting the formation of the spindle apparatus, ples for different forms of application of plant com- such as colcemid and colchicine were introduced. ponents are given.

INCREASE IN MATRIX METALLOPROTEINASE-9 IN PERIPHERAL BLOOD OF MULTIPLE SCLEROSIS PATIENTS TREATED WITH HIGH DOSES OF METHYLPREDNISOLONE

Dagmara Mirowska1,2,3, Wojciech Wicha3, Andrzej Cz³onkowski2, Anna Cz³onkowska3,2, Florian Holsboer1, Frank Weber1 Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, D 80804Munich, Germany, Department of Experimental and Clinical Pharmacology, Medical University, Krakowskie Przedmieœcie 26/28, PL 00-927 Warszawa, Poland, !2nd Department of Neurology, Institute of Psychiatry and Neurology, Sobieskiego 1/9, PL 02-957 Warszawa, Poland

Increased blood-brain-barrier (BBB) perme- depends on several factors. Among them matrix ability during relapses of multiple sclerosis (MS) metalloproteinases (MMPs) are of great interest,

502 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS especially MMP-9 and MMP-2 and the natural tis- and comparable to that seen in the whole patient sue inhibitor of MMP-9 (TIMP-1). Intravenously population. There was, however, no significant im- (iv) administered methylprednisolone (MP) accele- provement between day 7 and month 3. Before rates the recovery from relapse in MS. The mecha- therapy, baseline TIMP-1 and MMP-2 levels were nism, however, is poorly understood. One of the significantly lower in MS patients than in healthy current concepts is that MP may stabilize the BBB controls. In contrast, MS patients showed higher by altering the balance of MMP-9 and TIMP-1. levels of MMP-9 than healthy controls. The differ- The aim of our study was to investigate, ence, however, was not statistically significant. whether different doses of iv given MP has any There was a statistically significant increase in short- and/or long-term effect on MMP-9, MMP-2 MMP-9 plasma levels on day 7 after starting ther- and TIMP-1 plasma levels. Thirty patients with apy followed by a decrease on day 14 and month 3 relapsing-remitting MS were treated with 1000 mg in group I. A similar trend was obvious in group II. (n = 17, group I) or 500 mg (n = 13, group II) of No significant effect of MP treatment, however, MP iv for five consecutive days. EDSS score was was noted on TIMP-1 and MMP-2 plasma levels. evaluated before starting therapy, after 7 days and In conclusion, our results demonstrate that at 3 months. MMP-9, MMP-2 and TIMP-1 levels a double dose of MP might not be superior with re- were determined before treatment, after 3, 7, 14 spect to clinical improvement and that MP in- days and after 3 months. During therapy with MP, creases MMP-9 levels in peripheral blood. There- we observed a statistically significant decrease in fore, the well-known effect of MP consisting in median EDSS score after 7 days and after 3 months restoring the BBB may possibly be related rather compared with the state before treatment. Improve- to some other mechanism than suppression of ment of EDSS was similar in group I treated with MMP-9, e.g. to suppression of adhesion molecules higher and group II treated with lower dose of MP or pro-inflammatory cytokines.

ANGIOTENSIN 1–9 INFLUENCES HEMODYNAMICS AND HEMOSTATIC PARAMETERS IN RATS

Andrzej Mogielnicki, Ewa Chabielska, Karol Kramkowski, W³odzimierz Buczko Department of Pharmacodynamics, Medical Academy, Mickiewicza 2c, PL 15-222 Bia³ystok, Poland

Ang-(1–9) is synthesized from angiotensin I by blood pressure, heart rate and blood flow in rats re- angiotensin-converting-enzyme-related carboxypep- ceiving 1 h infusion of Ang-(1–9) (200 pmol/kg/min) tidase (ACE2) [1] and can be a substrate for angio- was measured. The venous thrombosis was induced tensin II synthesis. On the other hand, Ang-(1–9) by vena cava ligation. After 2 h of continuous infu- may inhibit ACE, decreasing in this way formation sion of 0.9% NaCl vehiculum (VEH) or Ang-(1–9) of angiotensin II. It has been shown that Ang-(1–9) (10 and 100 pmol/kg/min), the blood samples were enhances nitric oxide release from endothelial cells collected, the thrombus was removed and its dry [2], which is a potent inhibitor of platelets aggrega- weight was measured after 24 h. Overall coagula- tion. tion, hemostasis and fibrinolysis potentials (OCP, Thus, the aim of the study was to investigate the OHP, OFP) were estimated in plasma. The in vitro influence of Ang-(1–9) on some hemostatic para- whole blood platelet aggregation in response to metrs in in vivo and in vitro studies as well as he- collagen (5 mM) was measured after 15 min of in- modynamic changes after its infusion in rat. cubation with Ang-(1–9) (10–7 M). Male Wistar rats with renal hypertension (two- In in vivo study, Ang-(1–9) showed hyperten- kidney, one-clip) were used in the experiment. The sive and prothrombotic activity. However, we also

ISSN 1230-6002 503 Abstracts of Symposium

Group n , Mean blood pressure (mmHg) , Blood flow (ml/min) , Heart rate

VEH 3 + 2.7 ± 0.7 0.4 ± 0.7 +32 ± 20 Ang-(1–9) – 200 4 +18.1 ± 2.1* 0.0 ± 0.7 –97 ± 44

Group n Thrombus OCP (%) OHP (%) OFP (%) Platelet weight (mg) aggregation (9)

VEH 14 1.0±0.3 97±3 28±4 71±4 5.3±0.2 Ang-(1–9) – 10 6 2.6 ± 0.8 101 ± 6 27 ± 4 74 ± 2 – Ang-(1–9) – 100 4 3.1 ± 2.7 105 ± 4 42 ± 4* 58 ± 4* – Ang-(1–9) 10–7 M11––––4.1±0.3** *p<0.05,**p<0.01vs.VEH provide evidence for antiaggregatory action of son K., Jeyaseelan R., Breitbart R.E., Acton S.: A no- Ang-(1–9) in in vitro study. vel angiotensin-converting-enzyme-related carboxy- peptidase (ACE2) converts angiotensin I to angio- tensin 1–9. Circ. Res., 2000, 87, E1–E9. REFERENCES 2. Jackman H.L., Massad M.G., Sekosan M., Tan F., Brovkovych V., Marcic B.M., Erdos E.G.: Angio- 1. Donoghue M., Hsieh F., Baronas E., Godbout K., Gos- tensin 1–9 and 1–7 release in human heart: role of selin M., Stagliano N., Donovan M., Woolf B., Robi- cathepsin A. Hypertension, 2002, 39, 976–981.

HIGH DENSITY LIPOPROTEINS: FROM THERAPEUTIC TARGET TO THERAPEUTIC AGENT

Jerzy-Roch Nofer, Gerd Assmann Institut für Klinische Chemie und Laboratoriumsmedizin und Institut für Arterioskleroseforschung, Westfälische Wilhelms-Universität, Münster, Germany

Despite the benefits of currently available plasma HDL cholesterol. Moreover, low level of therapeutic interventions, atherosclerotic vascular HDL strongly interacts with and aggravates the disease continues to be the major cause of mortality proatherogenic effects of other CHD risk factors. and morbidity in much of the Western world. This Raising HDL cholesterol either by adjustments of emphasizes the necessity to develop new strategies life style or by drug intervention was shown to be to augment currently used therapeutic strategies, beneficial in both primary and secondary CHD pre- such as lowering low density lipoproteins (LDL) vention. However, results of interventional trials cholesterol or limiting intestinal cholesterol ab- were confounded by the fact that in addition to sorption. High density lipoproteins (HDL) seem to HDL other risk factors such as LDL or trigly- be a promising target for development of new cerides were affected by applied therapeutics. therapies. Low levels of HDL cholesterol were re- Novel approaches directed at key points in HDL peatedly reported to be associated with an in- metabolic pathways, either through pharmacologi- creased risk of coronary heart disease (CHD). In cal manipulation or in more distant future through addition to genes, lifestyle factors, such as smok- gene therapy, may allow a more selective elevation ing, diet, and physical inactivity profoundly affect of HDL levels. Candidate targets for these thera-

504 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS pies include ATP-binding cassette transporter 1 peptides were shown not only to reduce cholesterol (ABCA1), a transmembrane protein that mediates accumulation in the arterial wall, but also to inhibit initial steps in the transport of cholesterol from the platelet activation and, thereby, to reduce formation arterial wall to the liver and, thereby, crucially con- of thrombi within arteriosclerotic plaques. Recent tributes to the regression of the arteriosclerotic studies have shown that HDL is a carrier of bioac- plaque, and cholesterol ester transfer protein (CETP), tive lysosphingolipis, such as sphingosine 1-phos- an essential component of the HDL cholesterol trans- phate (S-1-P), sphingosylphosphorylcholine (SPC), port machinery between arterial wall and the liver. and lysosulfatide (LSF). These compounds were The development of atherosclerosis is a com- demonstrated to exert an astonishing spectrum of plex process whose central elements include the potentially antiatherogenic properties, which include entrapment of LDL in the arterial wall, its modifi- inhibition of endothelial apoptosis, stimulation of cation, and the stimulation of proinflammatory nitric oxide production, inhibition of the expression gene expression leading to inflammatory cell re- of endothelial adhesive molecules, and inhibition cruitment, infiltration, and activation. HDL can in- of pro-oxidative processes. Although it may now teract with this process at multiple points, and these only be speculated which of these and other antia- interactions could be a rich source of new therapeu- therogenic properties of HDL will be exploited in tic approaches. Artificial HDL particles containing the therapy in the future, there can be no doubt that apolipoprotein A-I and phospholipids, some apo the time is ripe for the further developments of this A-I variants (apo A-I Milano), and apo A-I mimetic new frontier in antiatherogenic strategy.

INTERACTION BETWEEN STRIATAL NMDA AND ADENOSINE A2A RECEPTORS AS A POSSIBLE BASIS FOR ANTIPARKINSONIAN THERAPY

Wolfgang Nörenberg, Peter Illes Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Germany

The possible modulatory influences by adeno- In a Mg2+-free medium, CGS 21680 depressed the sine and its structural analogues on NMDA- or NMDA-receptor-mediated fraction of the EPSCs in AMPA-mediated neurotransmission in striatal me- a subpopulation of the investigated cells. NMDA dium spiny neurons were investigated by means of receptors occur in clusters anchored in the plasma the whole-cell patch clamp method in brain slices membrane. The binding of the NR1 subunit to actin of rats. NMDA- but not AMPA-induced inward via a-actinin-2 is essential for interaction with the currents were inhibited by the adenosine A2A re- cytoskeleton and an intact receptor function. ceptor agonist 2-[p-(2-carboxyethyl)-phenylethyl- Ca2+-dependent rundown of NMDA receptor chan- amino]-5’-ethylcarboxamido adenosine (CGS 21680) nels which is facilitated by the enhancement of ac- by postsynaptic interplay in a subset of medium tin depolimerization may be due to a competitive spiny neurons. This effect was reversed by the se- displacement of a-actinin-2 from the NR1 subunit 2+ lective A2A receptor antagonist 8-(3-chlorostyryl)- of the receptor by Ca /calmodulin. However, in caffeine. Confocal laser microscopy proved that addition to a direct binding site at the NR1 subunit 2+ CGS 21680-sensitive neurons possessed A2A re- of the NMDA receptor channel, Ca /calmodulin ceptors, while neurons insensitive to this agonist may also act via calmodulin kinase II (CAMKII) were devoid of them. A similar effect was observed and subsequent phosphorylation of the NR2B sub- when excitatory postsynaptic currents (EPSCs) unit. The intracellular mechanism of the crosstalk were evoked by intrastriatal electrical stimulation. between NMDA and adenosine A2A receptors in-

ISSN 1230-6002 505 Abstracts of Symposium volves the phospholipase C/inositol 1,4,5-trisphos- reducing the activity of the indirect output pathway phate/calmodulin and CAMKII pathway, whereas known to depress locomotor behavior they may al- the contribution of the adenylate cyclase/protein leviate the disturbance of motor coordination in kinase A and diacylglycerol/protein kinase C path- Parkinson’s disease. However, considering their ways could be excluded. Hence, agonists at striatal agonistic interaction with D2 dopamine receptors A2A receptors may selectively interfere with NMDA also located in this pathway, their action may be receptor-mediated excitatory amino acid neurotrans- more complicated in vivo. Therefore, the net effect mission from corticostriatal fibres onto enkephalin- of adenosine may depend on the balance between positive striatopallidal medium spiny neurons. By the two opposing modulatory functions.

METABOTROPIC GLUTAMATE RECEPTORS AS POTENTIAL TARGET FOR ANTIPARKINSONIAN THERAPY

Krystyna Ossowska Department of Neuro-Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

It is generally accepted that degeneration of do- [2] and MPEP – an antagonist of mGluR5 [3] or in- paminergic neurons of the nigrostriatal pathway trastriatal injections of AIDA – an antagonist of which leads to dramatic loss of striatal dopamine mGluR1 [4] inhibit parkinsonian-like symptoms in underlies symptoms of Parkinson’s disease. How- rats induced by haloperidol: muscle rigidity, cata- ever, in the course of this disease an overactivity of lepsy and hypolocomotion. The antiparkinsonian- glutamatergic neurotransmission develops which like effects of these compounds may result from may contribute to both the degenerative process normalization of the function of the striopallidal and appearance of parkinsonian symptoms. Gluta- pathway which was evidenced by expression of mic acid acts on two main groups of receptors: mRNA for preproenkephalin, a neuropeptide pres- (1) a group of ionotropic receptors (NMDA, AMPA/ ent in these neurons [4, Wardas et al., unpublished]. kainate) and (2) a group of metabotropic receptors Moreover, the blockade of mGluR5 by MPEP has (mGluR1-8). Ionotropic glutamate receptors are re- been found to reverse degeneration of dopaminer- sponsible for fast and relatively large changes in gic neurons of the nigrostriatal pathway produced membrane conductance, whereas stimulation of by methamphetamine, as measured by levels of mGluRs evokes a complex cascade of intracellular striatal dopamine and its metabolites in rats [1]. events which indirectly modulates neuronal excit- The present results suggest that inhibition of ability. Antiparkisonian agents are sought among glutamatergic transmission at the level of mGluRs antagonists of postsynaptic NMDA and group I produces both antiparkinsonian-like and neuropro- (mGluR1 and mGluR5) receptors, or agonists of tective effects in rodent models of Parkinson’s presynaptic group II (mGluR2/3) or group III disease. (mGluR4/7/8) receptors, which inhibit glutamate release. In contrast to NMDA receptor antagonists REFERENCES which produce many undesirable side-effects, 1. mGluRs ligands are devoid of such properties and, Go³embiowska K., Konieczny J., Wolfarth S., Ossow- ska K.: Neuroprotective action of MPEP, a selective therefore, seem to be more promising as putative mGluR5 antagonist, in the methamphetamine-induced antiparkinsonian drugs. dopaminergic neurotoxicity is associated with a de- Our recent studies indicate that systemic ad- crease in dopamine outflow and inhibition of hyper- ministration of LY 354740 – an agonist of group II thermia in rats. Neuropharmacology, 2003, in press.

506 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

2. Konieczny J., Ossowska K., Wolfarth S., Pilc A.: LY- subtype 5 (mGluR5) produces antiparkinsonian-like 354740, a group II metabotropic glutamate receptor effects in rats. Neuropharmacology, 2001, 41, 413–420. agonist with potential antiparkinsonian properties in 4. Ossowska K., Wardas J., Pietraszek M., Konieczny J., rats. Naunyn-Schmied. Arch. Pharmacol., 1998, 358, Wolfarth S.: The striopallidal pathway is involved in 500–502. antiparkinsonian-like effects of the blockade of group 3. Ossowska K., Konieczny J., Wolfarth S., Wieroñska J., I metabotropic glutamate receptors in rats. Neurosci. Pilc A.: Blockade of the metabotropic glutamate receptor Lett., 2003, 342, 21–24.

HISTAPRODIFEN AS A LEAD STRUCTURE FOR THE DESIGN OF NEW HISTAMINE H1-RECEPTOR AGONISTS

Heinz H. Pertz1, Kai Kramer1, Sonja Menghin1, Sigurd Elz2, Walter Schunack1 Institut für Pharmazie, Freie Universität Berlin, Germany, Institut für Pharmazie, Universität Regensburg, Germany

Histaprodifen (2-[2-(3,3-diphenylpropyl)-1H-imi- position of the pyridine ring. N=-[4-(2-pyridyl)bu- dazol-4-yl]ethanamine), an amalgam of a 2-substi- tyl]histaprodifen was equipotent with suprahista- tuted histamine fragment and a 3,3-diphenylpropyl prodifen (guinea pig ileum: pD2 8.2; guinea pig portion typical of H1-receptor antagonists, has re- aorta: pD2 7.9; rat aorta: pD2 6.7) and displayed cently been shown to act as a potent and selective low to moderate antagonist affinity for histamine histamine H1-receptor agonist in vitro [2]. Potency H2- and H3-receptors, respectively. Due to their high = = at H1-receptors was further enhanced by N -methyl- potency and selectivity, N -imidazolylalkyl and py- ation of histaprodifen [2]. Histaprodifen and methyl- ridylalkyl derivatives of histaprodifen may be valu- histaprodifen were also potent H1-receptor agonists able tools for the investigation of H1-receptor-me- in vivo [3]. Based on earlier findings that the intro- diated physiological and pathophysiological func- duction of bulkier groups at the N=-position might tions. increase agonist potency, we synthesized a series of N=-substituted histaprodifen derivatives of which suprahistaprodifen (N=-[2-(1H-imidazol-4-yl)-ethyl]- REFERENCES histaprodifen), an amalgam of histaprodifen and 1. Christophe B., Carlier B., Schunack W., Chatelain P., a histamine fragment, represents the most potent Peck M.J., Massingham R.: Histamine H1-agonist H1-receptor agonist described so far in the litera- properties of histaprodifen derivatives in guinea-pig ture [1]. Elongation of the alkyl spacer of suprahis- isolated trachea and ileum. Inflamm. Res., 2003, 52, Suppl. I, S51–S52. taprodifen and replacement of the basic imidazole 2. Elz S., Kramer K., Pertz H.H., Detert H., ter Laak ring by a lipophilic, weakly basic pyridine ring af- A.M., Kühne R., Schunack W.: Histaprodifens: syn- forded new compounds which were tested for ago- thesis, pharmacological in vitro evaluation, and mo- nist potency at contractile H1-receptors in guinea lecular modeling of a new class of highly active and pig ileum and aorta and at relaxant H1-receptors in selective histamine H1-receptor agonists. J. Med. rat aorta. While elongation of the alkyl spacer of Chem., 2000, 43, 1071–1084. 3. suprahistaprodifen decreased agonist potency, the Schlicker E., Koz³owska H., Kwolek G., Malinowska B., Kramer K., Pertz H.H., Elz S., Schunack W.: exchange of the terminal imidazole for a pyridine Novel histaprodifen analogues as potent H1-receptor ring showed that agonist potency was the highest agonists in the pithed and in the anaesthetized rat. when the spacer was butyl and attached to the ortho Naunyn-Schmied. Arch. Pharmacol., 2001, 364, 14–20.

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COMPARATIVE MOLECULAR SURFACE ANALYSIS (CoMSA): THEORETICAL ADVANTAGES AND PRACTICAL APPLICATION IN DRUG DESIGN

Jaros³aw Polañski, Rafa³ Gieleciak, Andrzej B¹k Department of Organic Chemistry, Institute of Chemistry, University of Silesia, Szkolna 9, PL 40-006 Katowice, Poland, e-mail: [email protected]

The considerable progress in drug development phore mapping of the HIV-1 integrase inhibitors can be observed in recent years. Completely new that enabled us both to rapidly screen the ex vivo strategies appeared in this field. Genomics, combi- activity of styrylquinoline inhibitors as well as to natorial chemistry, dynamic combinatorial chemis- precisely predict the activity of the newly synthe- try, high throughput screening are some of new sized compounds [3]. Using the same scheme, we trends in this area. There is also a need to improve designed new analogs which are now synthesized computational approaches to enable an efficient in our laboratory. We will also review an applica- processing of the molecular data both in classical tion of the similar method in the chemistry of alter- drug design and combinatorial procedures in order native sweeteners. to increase the chances of these schemes. The comparative molecular surface analysis (CoMSA) is a calculation scheme that includes REFERENCES self-organizing neural network applied for the com- 1. parison of the molecular surface features [1]. This Anzali S., Barnickel G., Krug M., Sadowski J., Wage- ner M., Gasteiger J., Polañski J.: The comparison of can be used for visualization, interpretation and mo- geometric and electronic properties of molecular sur- deling of (Q)SAR data. It was shown that a fuzzy faces by neural networks: application to the analysis molecular surface patterns generated in CoMSA can of corticosteroid globulin activity of steroids. J. Com- be a helpful tool in illustrating and describing differ- put.-Aid. Molec. Design, 1996, 10, 521–534. ent aspects of molecular similarity and diversity [2]. 2. Polañski J.: Molecular shape analysis. In: Chemoin- We will show that the method can also support phar- formatics from Data to Knowledge. Eds. Gasteiger J., Engel Th., Wiley-VCH, Weinheim, BRD, in press. macophore mapping strategies, differentiating typi- 3. Polañski J., Zouhiri F., Jeanson L., Desmaële D., cal drug-receptor, dye-cellulose or chemical mole- D’Angelo J., Mouscadet J.-F., Gieleciak R., Gasteiger cule-solvent environment interactions. J., Le Bret M.: Use of the Kohonen neural network for Additionally, we will discuss practical applica- rapid screening of ex vivo anti-HIV activity of sty- tion of this method to the alternative pharmaco- rylquinolines. J. Med. Chem., 2002, 45, 4647–4654.

SYSTEMATIC IDENTIFICATION OF GENETIC VARIANTS OF TARGETS FOR CNS-ACTIVE THERAPEUTICS

Peter Propping1, Yun Freudenberg-Hua1, Jan Freudenberg1, Markus M. Nöthen2 Institute of Human Genetics, University of Bonn, Germany, Department of Medical Genetics, University of Antwerp, Belgium

Pharmacogenetics becomes increasingly impor- variation between individuals. It is generally be- tant for the understanding of drug action and its lieved that the way drugs will be prescribed in the

508 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS future is going to be driven by individualized genetic could be mapped unambigously to the human ge- information. Progress in the field of pharmacogene- nome reference sequence. These 388 SNPs were tics was relatively slow in the last decades, because compared to polymorphisms annotated to the refer- traditional experiments in living individuals and ence sequence; 222 of the 388 SNPs are not in- their relatives are necessary. While the field started cluded in public SNP databases; 173 of the 388 with the study of genetic variation affecting drug SNPs are located in genomic regions annotated as metabolism, the advent of the human genome se- coding; 79 of the 173 SNPs are non-synonymous, quence enables to address genetic variation in genes thus leading to a variant gene product. Among that act as drug targets (pharmacodynamic level). them 4 variants lead to a premature stop codon. In a large-scale effort, 65 genes coding for pro- Since the study included 95 individuals rough esti- teins that may be targets for CNS-active therapeu- mates of the population frequencies of the identi- tics were resequenced in a stratified sample of 95 fied variants can be given. healthy Europeans. For this purpose, the complete It will be particularly interesting to examine the coding regions of the 65 genes including flanking functioning of the variant gene products and – if regions were amplified by PCR and sequenced on a they prove to be drug targets – to see whether the semi-automated ABI 377 machine. The sequenced variation affects drug action. All these studies can regions included e.g. the genes of various receptors first be performed in vitro. If the in vitro studies or their subunits, enzymes, ion channels, and puta- point to differerences in drug interaction, tests in tive disease genes. A total of 396 single nucleotide humans will become interesting. This approach polymorphisms (SNPs) were detected of which 388 will be an important new field of pharmacology.

CYCLOOXYGENASES IN MICE EXPERIMENTAL MODEL OF PARKINSON’S DISEASE INDUCED BY MPTP ADMINISTRATION

Adam Przyby³kowski1, Ilona Joniec1, Agnieszka Ciesielska2, Iwona Kurkowska-Jastrzêbska1, Andrzej Cz³onkowski1, Anna Cz³onkowska1,2 Department of Pharmacology, Medical University, Krakowskie Przedmieœcie 26/28, PL 00-927 Warszawa, Poland, 2nd Department of Neurology, Institute of Psychiatry and Neurology, Sobieskiego 1/9, PL 02-957 Warszawa, Poland

Cyclooxygenase-1 (COX-1) and COX-2 play on 7th day after MPTP lasting until 21st day, with an important role in pathology of various CNS dis- COX-1 expression not altered. PG production orders. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridi- raised significantly 24 h after MPTP. Changes in ne (MPTP) is a neurotoxin which causes neuropa- PG release did not correlate with changes in COX-2 thological, behavioral and biochemical changes expression. In immunohistochemical staining, COX-2 that closely mimics Parkinson’s disease. We inves- was present in neurons and glia on the 1st day after tigated the expression of COX-1 and COX-2, pros- intoxication but on 7th day COX-2 was observed taglandin (PG) production, dopamine (DA) deple- only in glial cells, probably astrocytes. tion in the striatum of C57Bl/6 mice after MPTP These results suggest that COX2 may be in- administration. We observed maximum overex- volved in MPTP-induced neuronal degeneration pression of COX-2 protein and corresponding gene and regeneration.

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ENDOGENOUS NEUROTOXINS OF THE TETRAHYDROISOQUINOLINE AND b-CARBOLINE FAMILIES IN PARKINSON’S DISEASE

Hans Rommelspacher, Ragnhild Bonnet, Gert Schulze Department of Clinical Neurobiology, UKBF, Free University of Berlin, Germany

The discovery in 1983 that the street drug con- BCs and TIQs interfere with several cellular taminant N-methyl-4-phenyl-1,2,3,6-tetrahydropy- mechanisms in dopaminergic neurons which may ridine (MPTP) caused profound parkinsonism in drug explain some of their neurotoxic actions. We have users ushered in new considerations about the etiol- recently found a strong inhibition of glycolytic en- ogy of Parkinson’s disease (PD). Of special interest zymes by several BCs. The interaction might sub- has been the remarkable structural overlap of N-me- sequently cause protein-protein associations con- b + thyl- -carbolinium ion with MPP , the neurotoxic tributing to the observed decrease in energy supply metabolite of MPTP. We found increased plasma to dopaminergic neurons in early stages of PD. levels of the b-carbolines (BC) norharman and har- man in patients with PD [1] and in crebro-spinal fluid Acknowledgment. Supported by BMBF: GFGZ 0110- (CSF) from de novo and L-DOPA treated patients [2]. 9202. Levels of N-methylated BCs were elevated in the CSF from patients with PD as well [3]. These and REFERENCES other findings at the cellular level suggest that BCs 1. Kuhn W., Müller T., Grosse H., Dierks T., Rom- are candidate neurotoxins to produce PD [4]. melspacher H.: Plasma levels of the b-carbolines har- Many studies have been published demonstrat- man and norharman in Parkinson’s disease. Acta Neu- ing that tetrahydroisoquinolines (TIQ) which are rol. Scand., 1995, 92, 451–454. synthesized in the brain from phenylethylamine or 2. Kuhn W., Müller T., Grosse H., Rommelspacher H.: dopamine contribute to the neurodegeneration of Elevated levels of harman and norharman in cerebro- dopaminergic neurons in the substantia nigra (SN). spinal fluid of parkinsonian patients. J. Neural The simple TIQ and 1-Me-TIQ was found in both Transm., 1996, 103, 1435–1440. 3. normal and PD brains. The presence of several Matsubara K., Kobayashi S., Kobayashi Y., Yamashita K., Koide H., Hatta M., Iwamoto H., Tanaka O., Ki- other TIQs was subsequently observed in CSF and + mura K.: Beta-carbolinium cations, endogenous MPP in the brain from patients with PD, i.e. N-Me-TIQ, analogs, in the lumbar cerebrospinal fluid of patients N-Me-norsalsolinol, N-Me-salsolinol, 1-phenyl-TIQ, with Parkinson’s disease. Neurology, 1995, 45, N-Me-1-phenyl-TIQ and 1-benzyl-TIQ. The TIQs 2240–2245. are subsequently oxidized by MAO to the isoqui- 4. Nagatsu T.: Amine-related neurotoxins in Parkinson’s nolinium cation which represents probably the ac- disease: past, present, and future. Neurotoxicol. Tera- tive neurotoxic compound [3]. tol., 2002, 24, 565–569.

FAMILY INVESTIGATIONS IN PROBANDS WITH REARRANGEMENTS IN THE ACROCENTRIC CHROMOSOMES Gesa Schwanitz1, Lieselotte Kalz1, Alina T. Midro2 Institute of Human Genetics, University of Bonn, D 53113 Bonn, Germany, Department of Clinical Genetics, Medical Academy, Waszyngtona 13, PL 15-230 Bia³ystok, Poland

The investigation comprises 100 probands and cific polymorphisms in the constitutive heterochro- their families. In all cases, a differentiated analysis matin and the rearrangement of euchromatin is ana- of the breakpoints is performed, combining cytoge- lyzed. Risk factors for carriers of different types of netic and molecular-cytogenetic methods. Family chromosomal rearrangement are given. investigations followed. The combination of spe-

510 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

DEVELOPMENT AND PHARMACOLOGICAL APPLICATIONS OF NOVEL DOPAMINE D3 RECEPTOR LIGANDS

Holger Stark1, Anneke Hackling1, Ulrich Mach1, Sandrine Ferry2, Robin Ghosh3, Sylvie Perachon2, Wolfgang Sippl3, Camille G. Wermuth4, Jean-Charles Schwartz2, Pierre Sokoloff2 Institut für Pharmazeutische Chemie, Biozentrum, Johann Wolfgang Goethe-Universität, Frankfurt/M, Germany, Unité de Neurobiologie et Pharmacologie Moléculaire de l´INSERM, Paris, France, !Heinrich Heine-Universität, Düsseldorf, Germany, "Prestwick Chemical, Illkirch, France

Classification of dopamine subreceptors has led pending on the basic moieties. The 1,2,3,4-tetra- into two families, i.e. the D1-like receptors (D1,D5) hydroisoquinoline derivative ST 198 is character- and the D2-like receptors (D2,D3,D4). Dopamine ized as competitive antagonist (pA2 = 7.5) showing D3 receptors have gained special interest since they about 60-fold D3 receptor-preference vs.D2 recep- are supposed to be involved in numerous central tors as well as low or negligible affinities for other regulatory processes and display a discrete local- dopamine subreceptors and numerous other amin- ization in the brain [2]. Their potential therapeutic ergic receptors. A novel in vivo D3 receptor screen- applications in psychiatric disorders, tardive dyski- ing model based on dizocilpine (MK 801)-induced nesia and drug abuse are suggested. In our medici- locomotion in mice showed oral potency of ST nal chemistry developments of potent and selective 198. In combination with compounds of other phar- D3 receptor ligands, we designed new leads sup- macological properties, St 198 is a useful tool for in ported by molecular modeling studies and in silico vitro and in vivo studies on dopamine D3 receptor- screening. dependent (patho)physiological functions [1, 3]. Interestingly, these compounds possess differ- ent efficacies from antagonists to full agonists de- REFERENCES 1. Bézard E., Ferry S., Mach U., Stark H., Leriche L., 100 Boraud T., Gross C., Sokoloff P.: Attenuation of levo- dopa-induced dyskinesia by normalizing dopamine D3 75 D2 receptor receptor function. Nat. Med., in press. 2. Hackling A.E., Stark H.: Dopamine D3 receptor li- 50 D3 receptor (Ki = 720 nM) gands with antagonist properties. Chembiochemistry, binding I]iodosulpride 25 (Ki =12nM) 2002, 3, 946–961.

125 3. [ 0 Weber B., Schlicker E., Sokoloff P, Stark H.: Identifi- cation of the dopamine autoreceptor in the guinea-pig -11 -10 -9 -8 -7 -6 -5 -4 retina as D2 receptor using novel subtype-selective an- log [ST 198] (M) tagonists. Brit. J. Pharmacol., 2001, 133, 1243–1248.

PRESYNAPTIC a2-AUTORECEPTORS ENTER THE FOURTH DECADE

Klaus Starke Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität Freiburg, Germany

a2-Autoreceptors were discovered in 1971 along cifically modulated exocytotic as opposed to carrier- with GABA and muscarinic autoreceptors, but only mediated release of noradrenaline. Their pharmacolo- the a2-autoreceptors were studied at depth in the gical properties made them the prototype a2-adreno- first decade after 1971. It was shown that they spe- ceptors. The second decade was characterized by

ISSN 1230-6002 511 Abstracts of Symposium a severe controversy about their physiological role At least three developments mark the beginning and even their existence [2]. The third decade of the fourth decade of a2-autoreceptor research. brought the differentiation of three a2-adrenoceptor First, it has been detected that the operation of pre- subtypes, a2A, a2B and a2C, and the cloning of their synaptic a2-autoinhibition is a prerequisite for the genes. The a2-autoreceptors turned out to be main- facilitation of noradrenaline release by angiotensin ly but not exclusively a2A. Work on mice in which II and bradykinin, and interruption of ongoing one or two of the three a2 genes had been disrupted autoinhibition was defined as a new mode of pre- (KO mice) confirmed this and identified the synaptic facilitation. Second, it has been recogni- a a non-a -autoreceptors as a . Observations in the zed that the third 2-subtype, 2B, also may func- 2A 2C a KO animals also confirmed that a -autoinhibition tion as an autoreceptor, at least when the 2A and 2 a a is a physiological modulatory mechanism, viz. like 2C genes have been disrupted. Third, an 2C-adre- noceptor polymorphism with hypofunctional a - administration of a2-adrenoceptor antagonists, KO 2C autoreceptors has been revealed as a risk factor for of the a2A gene shifted pulse number-noradrenaline release curves and pulse frequency-noradrenaline congestive heart failure. release curves from physiological, autoinhibited to REFERENCES nonphysiological, disinhibited levels [1]. Intestinal cholinergic and cerebral serotonergic neurons also 1. Starke, K.: Presynaptic autoreceptors in the third de- cade: focus on a2-autoreceptors. J. Neurochem., 2001, possess release-inhibiting a2-adrenoceptors. Like a a 78, 685–693. 2-autoreceptors, these “heteroreceptors” are 2A, 2. Starke K., Göthert M., Kilbinger H.: Modulation of in the case of the serotonergic neurons with an ad- neurotransmitter release by presynaptic autoreceptors. mixture of a2B. Physiol. Rev., 1989, 69, 864–989.

IN SEARCH FOR NEW ANTITUMOR COMPOUNDS

Tatiana Syrovets, Berthold Büchele, Klaus Belsner, Thomas Simmet Department of Pharmacology of Natural Products & Clinical Pharmacology, University of Ulm, Helmholtzstr. 20, D 89081 Ulm, Germany

Together with Prof. Dembiñska-Kieæ, we have bited cytotoxic activity; however, the latter com- discovered expression of the 5-lipooxygenase path- pound did not significantly inhibit 5-lipooxygenase way in brain tissue slices. In order to evaluate its activity indicating that the cytotoxicity occurred in- putative physiological or pathophysiological func- dependent of 5-lipooxygenase inhibition. Subse- tion, we were seeking for 5-lipooxygenase inhibi- quent studies revealed remarkable cytotoxic activi- tors able to penetrate the blood-brain barrier. Un- ties of various BAs in several cell lines derived fortunately, all synthetic inhibitors of this pathway from tumors resistant to chemotherapy such as ma- were polar in nature precluding any efficacy in the lignant gliomas (U87 MG, U373 MG), pancreatic CNS. Boswellic acids (BAs), natural compounds tumors (MIA PaCa-2, PANC-1) and prostate can- with a steroid-like lipophilic structure isolated from cers (PC-3, DU 145). BA-treated tumor cells showed the gum resin of frankincense, had also been de- early morphological changes that hinted to topo- scribed as specific non-redox 5-lipooxygenase in- isomerase as a potential target of the BA-mediated hibitors. Indeed, we could demonstrate that acetyl- cytotoxicity. Indeed, we could demonstrate that 11-keto-b-boswellic acid (AKbBA) inhibited the various pentacyclic triterpenes including several 5-lipooxygenase pathway in malignant human pri- acetyl-BAs (ABAs) act as potent dual catalytic in- mary glioma cells. Surprisingly, AKbBA as well as hibitors of the DNA-processing enzymes topo- acetyl-b-boswellic acid (AbBA) additionally exhi- isomerase I and IIa. These compounds do not sta-

512 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS bilize the cleavable complex, but prevent binding sensus oligonucleotides showed that ABAs did not of human topoisomerase I and IIa to substrate affect binding of recombinant c-Rel and p50 sug- DNA. Surface plasmon resonance (SPR) analysis gesting that inhibition of NF-kB must occur further revealed that the topoisomerases possess single upstream. Indeed, we could demonstrate that ABAs high affinity binding sites with KD in the nM range. inihibit IkB kinase, thereby preventing the degra- ABAs induce apoptosis as shown by release of cy- dation of the endogenous inhibitor of NF-kB pro- tochrome C from mitochondria and by expression teins. We have now isolated approximately 24 dif- of phosphatidylserine on the outer membrane, as ferent pentacyclic triterpenic acids with antitumor well as by appearance of a sub-G1 peak cell popu- efficacy, purified them to chemical homogeneity lation and finally by DNA laddering. Many tumor and applied molecular modeling techniques in or- cells evade chemotherapy by overexpressing NF-kB der to obtain structurally optimized antitumor com- that leads to induction of antiapoptotic genes. Inter- pounds. estingly, we recently discovered that distinct ABAs In conclusion, our data identify a novel pharma- inhibit the activation of transcription factor NF-kB. cological effect of BAs that might harbor consider- This can be shown in electrophoretic mobility shift able therapeutic potential both in antitumor therapy assays but also in kB4 enhancer element driven lu- as well as in the treatment of various inflammatory ciferase expression. SPR analysis with NF-kB con- disorders.

CB1 CANNABINOID RECEPTORS MEDIATE PRESYNAPTIC INHIBITION OF NEUROTRANSMISSION

Bela Szabo Institut für Pharmakologie, Albert-Ludwigs-Universität, Freiburg, Germany

Anatomical studies indicate that the CB1 can- of caudate-putamen medium spiny neurons and nabinoid receptor is widely distributed in the cen- substantia nigra pars reticulata neurons [3]; c) in tral nervous system [2]. In several brain regions, the globus pallidus: between axons of caudate- the CB1 receptor is localized in presynaptic axon putamen medium spiny neurons and globus palli- terminals. Our hypothesis was that activation of dus neurons; d) in the ventral tegmental area: be- these receptors modulates neurotransmission be- tween GABAergic afferent axons and dopaminer- tween the axon terminals and the postsynaptic neu- gic neurons; e) in the cerebellar cortex: between rons. basket cells and Purkinje cells. Activation of CB1 The studies were carried out on slices prepared receptors also inhibited glutamatergic neurotrans- from rat or mouse brains. The slices were electri- mission in the substantia nigra pars reticulata: be- cally stimulated and the resulting GABAergic in- tween axons originating in the subthalamic nucleus hibitory postsynaptic currents (IPSCs) or glutama- and substantia nigra pars reticulata neurons [1]. tergic excitatory postsynaptic currents (EPSCs) Several methods were used to determine whe- were recorded with patch-clamp techniques. ther the inhibition of neurotransmission was due to We observed that activation of CB1 receptors a pre- or postsynaptic action: analysis of post- inhibited GABAergic neurotransmission at the fol- synaptic currents elicited by exogenous muscimol lowing synapses (a–e): a) in the caudate-putamen: and glutamate, analysis of miniature IPSCs and between afferent axons and medium spiny neurons EPSCs and analysis of IPSCs and EPSCs evoked (the afferent axons most probably belong to fast by paired pulses. These experiments indicated that spiking parvalbumin-positive interneurons); b) in at all studied locations, the cannabinoids depressed the substantia nigra pars reticulata: between axons neurotransmission by inhibiting transmitter release

ISSN 1230-6002 513 Abstracts of Symposium from presynaptic axon terminals. Generally, we ob- glutamatergic neurotransmission in many regions served only minor (if any) cannabinoid effects on of the brain. The neurotransmission is inhibited somadendritic regions of neurons. with a presynaptic action. Endogenous cannabi- In the substantia nigra pars reticulata [3] and in noids released from postsynaptic neurons can also the cerebellar cortex, GABAergic neurotransmis- use this inhibitory mechanism. sion was inhibited by depolarization of the post- synaptic neuron. This phenomenon is called REFERENCES “depolarization-induced suppression of inhibition 1. (DSI)” and is attributed to a presynaptic action of Szabo B., Wallmichrath I., Mathonia P., Pfreundtner C.: Cannabinoids inhibit excitatory neurotransmission a retrogradely moving signaling molecule released in the substantia nigra pars reticulata. Neuroscience, from the postsynaptic neuron. DSI was prevented 2000, 97, 89–97. in our experiments by a CB1 receptor antagonist. 2. Tsou K., Brown S., Sanudo-Pena M.C., Mackie K., This is compatible with the view that DSI was me- Walker J.M.: Immunohistochemical distribution of diated by the endogenous cannabinoids ananda- cannabinoid CB1 receptors in the rat central nervous mide or 2-arachidonylglycerol. system. Neuroscience, 1998, 83, 393–411. 3. Wallmichrath I., Szabo B.: Cannabinoids inhibit stria- Summarizing, activation of CB1 cannabinoid tonigral GABAergic neurotransmission in the mouse. receptors leads to depression of GABAergic and Neuroscience, 2002, 113, 671–682.

STRUCTURE-ACTIVITY RELATIONSHIP STUDIES ON 5-ARYLIDENE HYDANTOIN DERIVATIVES

Ewa Szymañska1, Katarzyna Kieæ-Kononowicz1, Bettina Popa3, Marek ¯ylewski2 Department of Chemical Technology of Drugs and Department of Organic Chemistry, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, !Pharmaceutical Institute Poppelsdorf, University of Bonn, Germany

The large number of epidemic infections due to 2-amino/hydrazino/hydrazido hydantoin derivati- Mycobacterium tuberculosis and Mycobacterium ves was synthesized and examined in vitro against avium complex as well as the increasing resistance M. tuberculosis [2]. For the most active compounds of bacteria to currently available therapy stimulate (ortho, meta and para chloro/fluoro benzylidene intensive search for new active antituberculotics. hydantoin derivatives with MIC £ 3.13 mg/ml) re- The list of pharmacophores includes isonicotinic sults on the cytotoxity, macrophage activity, single acid hydrazides, pyrazinoic acids, b-lactams, fluoro- drug resistant MIC and MBC assays as well as in quinolones, compounds containing a thiocarbonyl vivo tests were reported previously [1]. group (thioamides, thioureas), compounds contain- As a continuation of our studies on structure- ing an alkylthio group bound to an electron-defi- activity relationships, the analysis of topological cient atom of carbon, oxazolidinones, nitroimida- and physicochemical properties of the investigated zoles and others. compounds was performed. The results of molecu- The interesting results of the antimycobacterial lar modeling and conformational analysis of seve- in vitro screening, carried out among 5-arylidene- ral structures are described. The lipophilicity of the hydantoin derivatives, containing the thiocarbonyl obtained derivatives was determined by reverse- or alkylthio group as possible elements of the phar- phase thin-layer chromatography and compared macophore, prompted us to continue our investiga- with their partition coefficients, calculated by means tions in this field. The large series of 5-arylideno- of computer program (Alchemy 2000 SciLogP

514 Pol. J. Pharmacol., 2003, 55, 479–518 THIRTY YEARS OF COOPERATION BETWEEN GERMAN AND POLISH PHARMACOLOGISTS

Application, HyperChem v.4.5, CaChe v.5.01, Pal- REFERENCES las v.1.2). 1. Antimycobacterial Activity of 5-Arylidene Aromatic To improve the aqueous solubility of the ob- Derivatives of Imidazolidyn-2,4-dione, XXII Corso tained compounds, the attempt of preparation of in- Avanzato in Chimica Farmaceutica, Urbino 2002. clusion solid complexes with b-cyclodextrin was 2. Tuberculosis Antimicrobial Acquisition & Coordinat- performed, using kneading method. The effects of ing Facility, sponsored by the NIAID of the U.S. Na- complexation were investigated by NMR methods. tional Institutes of Health; [online] http:/www.taacf.org.

CEREBRAL DOPAMINE SYSTEMS AND THEIR MALFUNCTIONS: PARKINSONISM, SCHIZOPHRENIA AND ADDICTION

Jerzy Vetulani Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Dopaminergic system regulates several behav- Those neurons modulate the striatal output system ioral activities. It remains under excessive short- by stimulation, via D1 receptors, the direct (stimu- term feedback and long-term homeostatic control latory) pathway and inhibition, via D2 receptors, effected by autoreceptors and is regulated by sev- the indirect (inhibitory) pathway. Their degenera- eral other neurotransmitters. The important part in tion decreases the stimulatory effect of the thala- dopaminergic neuron control is played by an extra- mus on cortical motoneurons, which leads to the cellular pool of dopamine released from the den- symptoms of Parkinson’s disease. drites of neighboring dopaminergic neurons. The The aberrant activities of mesolimbic and me- human dopaminergic system differs from that in socortical dopamine systems are the cause of the lower animals, particularly in extension of cortical symptoms of schizophrenia. Because of aberrations dopaminergic projections. in the development of the cortex, the mesolimbic The dopaminergic system consists of four well- dopaminergic system becomes hypersensitive, and defined neuronal groups and their projections, this leads to induction of the productive symptoms forming nigrostriatal, mesolimbic, mesocortical of schizophrenia. In contrast, the mesocortical sys- and tuberoinfundibular tracts. The system consists tem becomes hypoactive, and this causes hypofron- of relatively small number of cells (approximately tality and negative symptoms of schizophrenia. 450 000 in the human midbrain) but plays a pivotal The mesolimbic dopamine system is also in- role in regulation of various activities of the central volved in reward behavior, and it is suggested that nervous system. The nigrostriatal system is primar- its hypoactivity leads to a compensatory seeking ily responsible for fine tuning of motor signals by for all types of reward, including substance abuse. basal ganglia, the mesolimbic system participates Addiction is now regarded as a disease of the re- in controlling emotional activity and reward, the ward system, in which dopaminergic projections mesocortical system plays an important role in cog- from ventral tegmental area to the nucleus accum- nition, and tuberoinfundibular system is involved bens and amygdala play a crucial role. in hormonal regulation. The dopaminergic neurons The knowledge about the involvement of dopa- are prone to neurodegeneration, as they release do- mine in pathologies such as Parkinson’s disease, pamine, whose metabolism by MAO leads to for- schizophrenia and substance abuse permits a search mation of reactive oxygen species. The neurode- for pharmacotherapy of those conditions, although generation affects primarily the dopaminergic neu- in most cases the presently available treatments are rons in the pars compacta of substantia nigra. only symptomatic.

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ANTIDEPRESSANT DRUGS: PROF. J. MAJ’S CONTRIBUTION TO THE UNDERSTANDING OF THEIR MECHANISM OF ACTION

Krzysztof Wêdzony Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Depression is a devastating brain disorder which, repeated, but not acute, administration of various according the estimates of epidemiological and antidepressants enhanced the locomotor hyperac- prognostic studies, will be a major cause of disabil- tivity induced by indirect dopaminergic agonists ity in the population of young people between (e.g. amphetamine) and direct agonists activating 15–35 years in the coming 15 years. Hence, depres- dopaminergic receptors of the D2 and D3 family. sion and its treatment pose a serious challenge to The increased responsiveness to dopaminergic neuropsychopharmacology. Research in this field receptor agonists suggests enhanced dopaminergic has been carried out in three major directions: the neurotransmission, which was further confirmed by development of novel drugs, the understanding of autoradiographic and saturation binding studies their mechanism of action and, the search for phar- with dopamine receptors, as well as by investiga- macological agents which may facilitate the effec- tions into the expression of mRNA encoding their tiveness of the available antidepressants. Regarding synthesis. The nature of the mechanism of the the mechanism of action, our attention was focused above hypersensitivity of the dopaminergic system, on two major mechanisms: the up-regulation of ad- as well as the “anatomical selectivity” of antide- renergic neurotransmission associated with a1-adre- pressants towards the mesolimbic and mesocortical nergic receptors, and the activation of dopaminer- dopaminergic systems are still an open question, gic neurotransmission, associated in particular with since antidepressants seem to be devoid of any af- the mesolimbic and the mesocortical dopaminergic finity for dopaminergic transporter or dopaminer- systems. Up-regulation of noradrenergic neuro- gic receptors. It cannot be ruled out that the neuro- transmission was observed after repeated treatment transmitters primarily influenced by antidepressant with various antidepressant drugs blocking the up- drugs, e.g. noradrenaline or serotonin, may “tune take of noradrenaline and serotonin. The above- up” dopaminergic neurotransmission. As to the mentioned activation was observed at a functional other neurotransmitters that may amplify the func- level as enhancement of the exploratory activity in- tion of antidepressant drugs, attention should be duced by intraventricular administration of pheny- paid to glutamatergic neurotransmission, since an- lephrine or methoxamine to rats. The functional tidepressants given repeatedly enhance rats’ re- data were further supported by the results of bind- sponsiveness to such non-competitive antagonists ing studies which showed increased affinity of of NMDA receptors as MK-801 and amantadine. a1-adrenergic receptors for agonists, but no consis- The effects discussed above are interesting from tent changes in their number occurred. Importantly, the viewpoint of not only theory, but also therapeu- the above effects were observed only after re- tic strategies, since clinical observations show that peated, but not acute, administration of antidepres- amantadine may facilitate the effectiveness of anti- sants, which is in line with their clinical profile of depressants, especially in drug-resistant patients. action. In parallel with the alterations in noradren- In conclusion, the three major issues concern- ergic neurotransmission, repeated administration of ing the action of antidepressant drugs and their ef- various antidepressant drugs also activated dopa- fectiveness have been presented: the up-regulation minergic neurotransmission in the mesolimbic and of a1-adrenergic and dopaminergic neurotransmis- mesocortical dopaminergic systems regulating re- sion with concomitant decreases in the glutamater- ward, cognitive functions and locomotion. Again, gic tone.

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SEARCH FOR IMIDAZOLE HISTAMINE H3-RECEPTOR ANTAGONISTS

Ma³gorzata Wiêcek1, Katarzyna Kieæ-Kononowicz1, Heinz H. Pertz2, Sigurd Elz3, Xavier Ligneau4, Jean-Charles Schwartz5, Holger Stark6, Walter Schunack2 Department of Chemical Technology of Drugs, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland, Institut für Pharmazie, Freie Universität Berlin, Germany,! Institut für Pharmazie, Universität Regensburg, Germany, "Laboratoire Bioprojet, Paris, France, #Centre Paul Broca de l’INSERM, Paris, France, $ Institut für Pharmazeutische Chemie, Johann Wolfgang Goethe-Universität, Frankfurt, Germany

Histamine H3 receptors are located presynapti- This joint research project resulted in design cally on various kinds of central nervous system and synthesis of few series of carbamates contain- (CNS) neurons, regulating synthesis and release of ing N-cycloalkyl- or phenylalkyl-, branched alkyl- histamine (via autoreceptors), and other neuro- unsaturated- and (un)substituted phenoxyalkyl- transmitters such as noradrenaline, acetylcholine, substituents. The obtained compounds were tested dopamine and serotonin (via heteroreceptors). for their effects at histamine H3 receptor in vitro Moreover, H3 receptors can also play a modulating (on isolated guinea pig ileum or on rat synapto- role in peripheral neurotransmission. Now it is somes and cloned human H3 receptor expressed on known that this receptor belongs to the superfamily CHO cells) and in vivo in the CNS after oral ad- of G-protein coupled receptors, and since 1999 ministration to mice. Moreover, in vitro activities at genes encoding the human, rat, and guinea pig re- histamine H1 and H2 receptors were evaluated. ceptors have been cloned [1]. Most of the novel compounds tested showed mode- The aim of our work was the search for hista- rate to excellent histamine H3 antagonist activity mine H3 receptor antagonists in the group of and good selectivity. 3-(1H-imidazol-4yl)propanol carbamates (see figu- re). The structure of these compounds is in accor- Acknowledgment. This work was supported in part by dance with general construction pattern for hista- the State Committee for Scientific Research Warszawa, Po- mine H receptor antagonists described first by land (grant No 4 P05F 011 18) and the International Bureau 3 of the BMBF, Bonn, Germany and Committe of Scientific Stark et. al. in 1996 [2]. Research, Warszawa, Poland (grant POL-030-98).

REFERENCES O 1. Lovenberg T.W., Roland B.L., Wilson S.J., Jiang X., N Pyati J., Huvar A., Jackson M.R., Erlander M.G.: Clon- O NH R ing and functional expression of the human histamine H3 receptor. Mol. Pharmacol., 1999, 55, 1101–1107. N 2. Stark H., Schlicker E., Schunack W.: Developments of H histamine H3-receptor antagonists. Drugs Fut., 1996, 21, 507–520.

ISSN 1230-6002 517 Abstracts of Symposium

EFFECT OF ADENOSINE RECEPTOR ANTAGONISTS ON EFFICACY OF THE DRUGS AND SUBSTANCES PROTECTING AGAINST 3-NITROPROPIONIC ACID-EVOKED SEIZURES AND NEURODEGENERATION

Beata Zuchora1, Marian Wielosz1, Ewa M. Urbañska1,2 Department of Pharmacology and Toxicology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland, Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland

Increasing evidence suggests that an impairment cross blood-brain barrier, did not alter protective of oxidative phosphorylation may underlie neu- effects of 2-CADO and R-PIA. Moreover, amino- ronal death in neurodegenerative diseases [3]. The phylline and DPCPX, but not 8-(p-sulfophenyl)- mitochondrial toxin, 3-nitropropionic acid (3-NPA) theophylline, were found to attenuate anticonvul- is an irreversible inhibitor of succinate dehydroge- sive activity of diazepam, phenobarbital, valproic nase, an enzyme which is present in both Krebs acid and gabapentin in seizures caused by systemic cycle and the mitochondrial complex II. Chronic application of 3-NPA. The presented results indi- administration of relatively low doses or local ap- cate that intrastriatal injections of adenosine recep- plication of 3-NPA were demonstrated to evoke se- tor agonists, 2-CADO and R-PIA, decreased bio- lective neuronal loss within striatum and neurologi- chemical results of local 3-NPA administration, re- cal symptoms resembling Huntington’s disease [1]. sulting in attenuation of decline in GAD’s activity. Adenosine, an endogenous neuromodulator, ex- The obtained effect was reversed by co-administra- erts potent neuroprotective effects in vivo and in vi- tion of an adenosine receptor antagonist, 8-(p-sulfo- tro [3]. phenyl)theophylline. The aim of the present study was to evaluate the The obtained results indicate that impairment of effect of adenosine receptor antagonists on efficacy adenosinergic neurotransmission may contribute to of the drugs and substances protecting against the development of seizures and neuronal loss fol- 3-NPA-evoked seizures and neurodegeneration. lowing 3-NPA administration. Moreover, protec- The studies were carried out on male Albino Swiss tive effects of diazepam, phenobarbital, valproic mice and male Wistar rats. Convulsions were evo- acid and gabapentin seem to be related to the cen- ked by systemic administration of 3-NPA in mice. tral adenosine A1 receptor stimulation. Striatal damage was induced by local stereotaxic injection of 3-NPA in rats and its extent was evalu- ated by the glutamate decarboxylase activity REFERENCES (GAD) measurement. 1. Beal M.F.: Neurochemistry and toxin models in The results presented here indicate that nonse- Huntington’s disease. Curr. Opin. Neurol., 1994, 7, lective adenosine receptor antagonist, aminophyl- 542–547. line, as well as selective adenosine receptor antago- 2. De Mendonca A., Sebastiao A.M, Ribeiro J.A.: Ade- nist, DPCPX, reversed the protective action of nosine: does it have a neuroprotective action after all? Brain Rev., 2000, 33, 258–274. adenosine receptor agonists, 2-CADO and R-PIA, 3. in seizures evoked by systemic 3-NPA administra- Schapira A.H.: Mitochondrial involvement in Parkin- son’s disease, Huntington’s disease, hereditary spastic tion. In contrast, A1/A2 adenosine receptor antago- paraplegia and Friedreich’s ataxia. Biochim. Biophys. nist 8-(p-sulfophenyl)theophylline, which does not Acta, 1999, 1410, 159–170.

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