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Neuroprotective effects of KCL-440, a new poly(ADP-ribose) polymerase inhibitor, in the rat middle cerebral artery occlusion model
Brain Research 1060 (2005) 73 – 80 www.elsevier.com/locate/brainres
Research Report
Neuroprotective effects of KCL-440, a new poly(ADP-ribose) polymerase inhibitor, in the rat middle cerebral artery occlusion model Yasuhiko Ikeda a, Kazuya Hokamura a, Tomoyuki Kawai b, Junichi Ishiyama b, Kumi Ishikawa b, Tsuyoshi Anraku b, Takashi Uno b, Kazuo Umemura a,* a
Department of Pharmacology, Hamamatsu University School of Medicine, Handayama, 1-20-1, Hamamatsu 432-8014, Japan b Discovery Res. Lab., Kyorin Pharmaceutical Co., Ltd., Tochigi, Japan Accepted 17 August 2005 Available online 3 October 2005
Abstract It is reported that ischemic brain injury is mediated by the activation of poly(ADP-ribose) polymerase (PARP). In this study, we examined the pharmacological profile of KCL-440, a new PARP inhibitor, and its neuroprotective effects in the rat acute cerebral infarction model induced by photothrombotic middle cerebral artery (MCA) occlusion. In an in vitro study, KCL-440 exhibited potency with regard to inhibition of PARP activity, with an IC50 value of 68 nM. An in vivo pharmacokinetic study showed that the brain concentration of KCL-440 was sufficient to inhibit PARP activity during the intravenous infusion of KCL-440 at the rate of 1 mg/kg/h. KCL-440 at various doses or saline was administered for 24 h immediately after the MCA occlusion. Administration of KCL-440 led to a dose-dependent reduction in the infarct size at 24 h after MCA occlusion. Infarct sizes were 44.8% T 3.0% (n = 8), 40.5% T 1.1% (n = 8), 38.2% T 1.4% (n = 8), 35.1% T 2.1% (n = 8), 34.2% T 2.3% (n = 7), 32.6% T 1.9% (n = 8), and 31.0% T 2.1% (n = 5) at doses of 0, 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h. When compared to the control group, a statistically significant difference was observed in the doses that were higher than 0.03 mg/kg/h. When the infusion of KCL-440 (1 mg/kg/h, n = 8) was started at 1 h after the MCA occlusion, a significant reduction in infarct size was observed; this was not observed when KCL-440 infusion was started 2 or 3 h after the MCA occlusion. Furthermore, increased poly(ADP-ribose) immunostaining was confirmed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment. These results suggest that KCL-440 is a possible neuroprotective agent with high blood – brain barrier permeability and high PARP inhibitory activity. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Poly(ADP-ribose) polymerase; Stroke; Neuroprotection
1. Introduction
Abbreviations: PARP, poly(ADP-ribose) polymerase; TCA, trichloroacetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; KCL-440, 4-(4-(N,N-dimethylaminomethyl)phenyl)-5-hydroxy isoquinolinone; PJ-34, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,Ndimethylactamide; 3-AB, 3-aminobenzamide; DPQ, 3,4-dihydro-5-[4-(1piperidinyl)butoxy]-1(2H)-isoquinolinone; DR2312, 2-methyl-3,5,7,8-tetrahydrothiopyrano(4,3-d)pyrimidine-4-one * Corresponding author. Fax: +81 53 435 2269. E-mail address: [email protected] (K. Umemura). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.046
Poly(ADP-ribose) polymerase (PARP) comprises a family of nuclear proteins that are activated by breaks in DNA strands, and it functions as a DNA repair enzyme. PARP cleaves NAD+ into nicotinamide and ADP-ribose to form long and branched (ADP-ribose) polymers that bind to numerous nuclear proteins, including histones and PARP itself [27]. However, overactivation of PARP, which is induced by excessive DNA damage, consumes NAD+; this leads to ATP depletion and consequent necrotic cell death [27]. It has been reported that the activation of PARP
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mediates ischemic brain injury [8]. In fact, PARP gene disruption rendered mice resistant to cerebral ischemia [7]. Moreover, several PARP inhibitors exerted neuroprotection in experimental models of cerebral ischemia [1,6,8,11,17 – 19,21]; these neuroprotective effects have been mainly attributed to PARP, which is considered to be the best characterized member of the PARP family. However, necrosis is not the only mechanism underlying PARP-mediated cellular suicide mechanisms. It was also reported that PARP played a crucial role in a caspase-independent apoptosis pathway that was mediated by an apoptosis-inducing factor [30]. KCL-440 is a newly developed PARP inhibitor. The objective of the present study was, first, to evaluate the PARP inhibitory activity and the blood – brain barrier permeability of this compound and, second, to study the neuroprotective effect of post-ischemic administration of KCL-440 in the rat acute cerebral infarction model induced by photothrombotic MCA occlusion. The neuroprotective effects that were observed were compared with those of the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one (known as edaravone). Using poly(ADP-ribose) immunohistochemistry, poly(ADP-ribosyl)ation was also studied in order to confirm the validity of the experimental model in assessing the neuroprotective effect of KCL-440. Some of these results have been published in the abstract form [12].
2. Materials and methods The Animal Care and Use Committee of Hamamatsu University School of Medicine approved all experimental protocol used in this study. 2.1. PARP assay The PARP enzyme assay was conducted in a total volume of 100 Al, consisting of 100 mM Tris –HCl (pH 8.0), 10 mM MgCl2, 10 Ag/ml activated DNA, 3 mM nicotinamide adenine dinucleotide, 10 kBq/ml adenosine-14C(U), 5 units/ml human recombinant PARP (Travigen), and various concentrations of the following test compounds: KCL-440
[4-(4-(N,N-dimethylaminomethyl)phenyl)-5-hydroxy isoquinolinone, chemical structure shown in Fig. 1]; PJ-34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylactamide]; and 3-AB (3-aminobenzamide). The reaction mixture was incubated at 25 -C; the reaction was terminated after 10 min by adding 1 ml of ice-cold 20% (w/v) trichloroacetic acid (TCA), and the mixture was stored overnight at 4 -C. The precipitate was collected on a glass fiber filter (Whatman GF/C) and washed three times with 5% TCA. Radioactivity was determined by scintillation counting. The experiments were carried out in triplicate. 2.2. H2O2 cytotoxicity assay in cortical cultures The cerebral cortex was isolated from 18-day-old rat embryos (Crj: CD(SD)IGS, Charles River Japan Inc.) and cut into small pieces. The dispersed cells were obtained by treatment with trypsin and subsequent mechanical agitation. The cell suspension was plated at density of 2 105 cells/cm2 on a 48-well plate that was coated with 0.1% polyethyleneimine in a DMEM/F12 medium containing a B27 supplement; the plate with the suspension was then placed in a CO2 incubator at 37 -C for more than 7 days. In the H2O2 cytotoxicity study, the culture medium was replaced with DMEM/F12 that contained B27 and various concentrations of the test compound. After preincubation for 1 h, the cells were washed twice with DMEM/F12 (B27-free) that contained the test compound and were then exposed to 0.2 mM H2O2 in the DMEM/F12 containing the test compound for 2 h in a CO2 incubator at 37 -C. Immediately after the incubation, the cells were washed twice with DMEM/F12 containing B27 and the test compound, and the cell viability was assessed using a standard colorimetric assay with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). A small aliquot (50 Al) of the 2.5 mg/ml MTT was added to the culture medium (250 Al) that contained B27 and the test compound in each well. After incubation for 4–5 h in the CO2 incubator at 37 -C, the reaction product, formazan, was solubilized with 0.04N HCl/isopropanol. The absorbance was measured at 540 nm. These experiments were carried out in duplicate. 2.3. Serum and brain concentrations
Fig. 1. Chemical structure of KCL-440.
In order to determine the serum concentrations of KCL440, the drug was intravenously infused at a rate of either 1 or 10 mg/kg/h in Wistar rats. For deproteinization, the serum was added to 5 volumes of methanol; this solution was vortexed and then centrifuged at 20,000 g for 5 min. The supernatant was filtered and injected into an HPLC (Nanospace system, Shiseido, Japan), and UV detection was carried out at 340 nm. In another experiment, the brain was isolated immediately after 6 h of KCL-440 infusion at 10 mg/kg/h and homogenized in an ice-cold 100 mM phosphate buffer (pH 7.4). The quantification of KCL-440 in the brain homogenates was carried out under the same HPLC conditions as those for the analysis of the serum sample.
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2.4. Rat photothrombotic MCA occlusion model
2.6. Poly(ADP-ribose) immunohistochemistry
Male Sprague – Dawley rats (SLC, Japan), weighing 260 – 320 g, were anesthetized with halothane (4% induction/2% maintenance). The body temperature of the animals was maintained at 37.5 -C T 0.5 -C with a heating pad (K-module Model K-20, American Pharmaseal, USA). The left MCA was thrombotically occluded by a photochemical reaction [25]. Briefly, a catheter for the administration of either the drug or Rose Bengal was inserted into the femoral vein. The scalp and temporalis muscle were folded over, and subtemporal craniotomy was performed with a dental drill, under an operating microscope in order to open a 3-mm diameter oval bony window. The window was irradiated with green light (wavelength 540 nm) obtained by using a xenon lamp (L4887, Hamamatsu Photonics, Hamamatsu, Japan) that had heat absorption and a green filter. The irradiation was directed by a 3-mm diameter optic fiber mounted on a micromanipulator. The head of the optic fiber was placed on the window in the skull base at a distance of 2 mm above the artery. Photoillumination was performed for 10 min after an intravenous injection of Rose Bengal (20 mg/kg). After the MCA occlusion, the temporalis muscle and skin were closed in layers, and anesthesia was discontinued. The brain was rapidly excised 24 h after surgery under pentobarbital sodium anesthesia. After the separation of the cerebrum, the brain was coronally sectioned on a brain matrix (RBS-02, Neuroscience, Japan) into 2-mm thick slices starting from the frontal lobe. Six consecutive slices were then stained with 1% triphenyltetrazolium chloride (Katayama, Nagoya, Japan) and subsequently photographed. For each of these sections, infarct (white) and noninfarct (red) areas were determined using computerized image analysis on an Apple Macintosh G4 computer by using the public domain NIH image program (developed at the National Institute of Health and available on the Internet by anonymous FTP from zippy.nimh.nih.gov).
To elucidate the spatial localization of poly(ADP-ribose) following focal cerebral ischemia, the rats were anesthetized with sodium pentobarbital and transcardially perfused with 20 – 50 ml of saline, followed by perfusion with 10% paraformaldehyde (pH 7.4) at 2 h after cerebral ischemia. The brains of these rats were quickly excised and cryoprotected in 25% sucrose in phosphate-buffered saline at 4 -C overnight. Frozen coronal sections (12-Am thickness) were prepared. After quenching endogenous peroxidase with 0.5% H2O2 in methanol and blocking with 5% hose serum, the sections were incubated overnight at 4 -C with a monoclonal antibody against poly(ADP-ribose) (Alexis Biochemicals, Lausen, Switzerland). The sections were washed with phosphate-buffered saline and then incubated with a biotinylated antibody for 30 min and with an avidin – biotin complex for 30 min (Vectastain, ABC kit, Vector). Peroxidase staining was carried out using 3,3V-diaminobenzidine. To evaluate the inhibitory effects of KCL-440 on PARP activity in vivo, KCL-440 was administered at a dose of 1 g/kg/h for 2 h after cerebral ischemia; subsequently, the brain was excised, and poly(ADP-ribose) was stained immunohistochemically in the same manner as that of the control animals. 2.7. Drug administration KCL-440 at doses of 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h, dissolved in saline, was intravenously administered at a rate of 0.33 ml/kg/h for 24 h immediately after cerebral ischemia. In the case of the control group, saline was administered in the same manner. In the case of the positive control group, a free radical scavenger, 3-methyl-1phenyl-2-pyrazolin-5-one, at a dose of 1 mg/kg/h, was administered in the same manner. To elucidate the therapeutic time window, KCL-440 administration in a dose of 1 mg/kg/h was started at 1, 2, or 3 h after cerebral ischemia followed by a continuous infusion for 21 –23 h.
2.5. Evaluation of neurological symptoms 2.8. Statistical analysis In accordance with the method of Bederson et al. [5], neurological symptoms such as hemiplegia were evaluated in a posture test at 24 h after photothrombosis. This evaluation was carried out in a blind manner. In the postural reflex test, hemiparesis was observed in the animals. The degree of abnormal posture observed when the rats were suspended by their tails at a distance of 1 m above the floor was examined. These rats were scored according to the following criteria: 0, rats that extended both forelimbs straight, no observable deficit; 1, rats that attached the right forelimb to the breast and extended the left forelimb straight; 2, rats that demonstrated decreased resistance to a lateral push in addition to the behavior described in score 1 without circling; and 3, rats that twisted the upper half of their body in addition to the behavior described in score 2.
All data are expressed as mean T SE. Statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test for drug-treated groups versus the control group.
3. Results 3.1. In vitro activity of KCL-440 The in vitro inhibitory efficacies of KCL-440, PJ-34, and 3-AB were determined using the human recombinant PARP enzyme (Table 1). The IC50 value of KCL-440 was 68 nM, which was 4 and 1000 times more potent than that of PJ-34 and 3-AB, respectively. PARP is a nuclear enzyme and has
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Table 1 Inhibitory effects of KCL-440, PJ-34, and 3-AB on the PARP enzyme and their neuroprotective effects on H2O2-induced toxicity in rat cultured cortical neurons
PARP enzymatic activity H2O2-induced cytotoxicity
KCL-440 (nM)
PJ-34 (nM)
3-AB (AM)
68 73
250 170
72 410
an H2O2-induced cytotoxic role [31]. Therefore, the neuroprotective effects of the PARP inhibitor on H2O2-induced toxicity could reflect its potency with regard to the inhibition of cellular PARP. Thus, we examined the effects of these PARP inhibitors on H2O2-induced toxicity in cultured rat cortical neurons (Table 1). The PARP inhibitors that were tested exerted a protective effect against neuronal cell death in a concentration-dependent manner; this supports the role of PARP in the neuronal injury that was observed in the cortical cultures. The IC50 value of KCL440 was 73 nM, which is nearly the same value as the IC50 value on enzymatic activity, thus suggesting that KCL-440 easily penetrates and enters the nucleus of neuronal cells. 3.2. Serum and brain concentrations The serum concentration of KCL-440 approached steadystate levels within 1 h in rats that were intravenously infused at the rate of 1 mg/kg/h for 6 h (Table 2). One hour after the initiation of infusion, the serum concentration of KCL-440 was 0.11 Ag/ml, which is equivalent to 0.37 AM. The brain:serum concentration ratio after a 6-h infusion at 10 mg/kg/h was 0.8 (Table 2). These results indicated that the brain concentration of KCL-440 was sufficient to inhibit the PARP activity during the intravenous infusion at the rate of 1 mg/kg/h. 3.3. Neuroprotective effects of KCL-440 in the in vivo cerebral ischemia model Fig. 2 displays the image of the nontreated and treated animals, in which KCL-440 at a dose of 1 mg/kg/h was administered for 24 h after cerebral ischemia. KCL-440 treatment reduced cerebral damage, particularly in the cortex. Administration of KCL-440 led to a dose-dependent reduction in the cerebral infarct size at 24 h after the MCA occlusion (Fig. 3). Statistical significance was observed in the doses higher than 0.03 mg/kg/h. The neuroprotective effect of KCL-440 was found to be greater than that of 3Table 2 KCL-440 concentrations in serum and brain during intravenous infusion of KCL-440 Dose (mg/kg/h) Serum (Ag/ml) Brain Ratio (Ag/g tissue) (B/P) 1h 3h 6h 1 10 – , not tested.
0.11 –
0.1 –
0.12 2.36
– 1.99
– 0.8
Fig. 2. Effects of KCL-440 on the extent of cerebral damage, KCL-440 at a dose of 1 mg/kg/h reduced the damaged area, particularly in the cortex. (A) Control, (B) KCL-440 at a dose of 1 mg/kg/h. Scale bar = 10 mm.
methyl-1-phenyl-2-pyrazolin-5-one in the doses higher than 0.3 mg/kg/h. When KCL-440 (1 mg/kg/h, n = 8) was administered, starting from 1 h after the MCA occlusion, a significant reduction in infarct size was observed; however, this reduction was not observed when KCL-440 administration was started at 2 or 3 h after cerebral ischemia (Fig. 5). 3.4. Effect of KCL-440 on neurological deficits The animals in the control group exhibited impairment of the posture reflex. Administration of KCL-440 at doses of 0.03, 0.1, and 1.0 mg/kg/h, and 3-methyl-1-phenyl-2-pyrazolin-5-one significantly improved the neurological score (Fig. 4).
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Fig. 3. Effect of KCL-440 on cerebral infarct size. Each column represents mean T SE. Bold numbers inside the graph represent the number of animals. *P < 0.05 versus control, **P < 0.01 versus control.
3.5. Poly(ADP-ribose) immunohistochemistry Increased poly(ADP-ribose) immunostaining was observed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment (1 mg/kg/h) (Fig. 6).
4. Discussion In this study, KCL-440, a new PARP inhibitor, reduced brain damage in a dose-dependent manner in the rat cerebral
infarction model induced by photothrombotic MCA occlusion. KCL-440 also improved neurological symptoms. These neuroprotective effects can be attributed to its high blood –brain barrier permeability and neuroprotective activity, which was exhibited in vitro. It has been reported that several PARP inhibitors exert neuroprotective effects in stroke models [27]. Most of the compounds used in those studies were administered prior to the occurrence of cerebral ischemia [6,8,11,18,19,21]. 3-AB [6,8,19] and PJ-34 [1,18] are commonly used as PARP inhibitors. However, they have a relatively weak PARP inhibitory activity, as demonstrated in the present study. The
Fig. 4. Effect of KCL-440 on neurological deficits, *P < 0.05 versus control.
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Fig. 5. Therapeutic time window of the neuroprotective effect of KCL-440. KCL-440 was administered 1, 2, or 3 h after the MCA occlusion. Vehicle was administered 1 h after the MCA occlusion. Each column represents mean T SE. Bold numbers inside the graph represent the number of animals. *P < 0.05 versus control.
IC50 value of KCL-440 was 68 nM, which was 4 and 1000 times more potent than that of PJ-34 and 3-AB, respectively; further, using 50 AM of Km for NAD, the calculated value of K i was found to be 9.8 nM [10]. In addition, 3-AB does not pass the blood – brain barrier after peripheral administration [3,24], whereas a beneficial brain:serum concentration ratio of KCL-440 was observed after its continuous infusion of KCL-440. This pharmacokinetic property of KCL-440 makes it favorable for the purpose of intravenous administration. In the present study, KCL-440 was administered immediately after cerebral ischemia, and a marked reduction in extent of brain damage was observed. Furthermore, the
neuroprotective effect of KCL-440 was still significant when it was administered 1 h after ischemia. The therapeutic time window of this compound appears to be related to the activation of PARP. Tokime et al. reported that marked poly(ADP-ribose) immunoreactivity was detected 2 h after ischemia [24], which is consistent with the immunohistochemical results obtained in the present study. A similar relationship between the therapeutic time window of KCL440 and PARP activation has been reported in the transient focal ischemia model [4]; therefore, PARP inhibitors must be administered at least before the peak of PARP activation. Other papers have reported that post-ischemic administration of PARP inhibitors 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) and 2-methyl-3,5,7,8tetrahydrothiopyrano[4,3-d]pyrimidine-4-one (DR2312) induced neuroprotection [17,19]. Although the IC50 value of KCL-440 is the lowest, it is difficult to compare these compounds with KCL-440 in the different cerebral ischemic models in vivo and in vitro. The photoinduced thrombotic occlusion model involves the formation of a platelet- and fibrin-rich thrombus in the MCA at the irradiated site [20]. The photochemical approach to induce thrombotic MCA occlusion involves an intravenous Rose Bengal injection followed by irradiation of the MCA with green light. Rose Bengal is a photosensitive dye that, when photoactivated, produces reactive oxygen species, mainly singlet oxygen by a ‘‘photodynamic Type II’’ reaction or energy transfer [26]. Reactive oxygen species cause endothelial injury that is followed by platelet adhesion and aggregation to form fibrin nets; this results in the formation of a platelet- and fibrin-
Fig. 6. Immunohistochemistry of poly(ADP-ribose). At 2 h after the MCA occlusion, poly(ADP-ribose) formation is elevated (B) in the nuclei in the ischemic border zone but not (A) in the sham-operated animal. (C) Increase in poly(ADP-ribose) formation was reduced by KCL-440 treatment (1 mg/kg). Scale bar = 100 Am.
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rich thrombus at the irradiated site [20]. An important feature of this model is the continued cyclic flow reductions that are observed in the MCA [13,32]. Therefore, the present model can represent a new MCA occlusion model that is different from previously described models of permanent or ischemia/reperfusion MCA occlusion. In the clinical situation, approximately 60% of patients exhibit spontaneous recanalization of the MCA in the early phase of an ischemic stroke in the absence of thrombolytic therapy [2]. Similarly, rethrombosis after thrombolysis is also frequently observed in the cerebral artery [14,28]. This suggests that cyclic flow reductions may occur in the acute phase of human stroke. However, in permanent models [23], suture MCA occlusion models [15], and the embolic MCA occlusion model [29], spontaneous reperfusion has not been observed. In the model used in this study, a radical scavenger that was administered after the MCA occlusion was effective; however, it was not effective in permanent occlusion models, in which reperfusion of the MCA was not observed [22]. Therefore, the development of cerebral damage in this model is related to the generation of reactive oxygen species, which can damage DNA and activate PARP [9], within the brain tissue. Thus, in the model used in this study, the development of cerebral damage may be associated with PARP activation. In the present in vivo study, the neuroprotective effect of KCL-440 was compared with that of the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one that is known as edaravone. Since edaravone is already clinically used in Japan for the treatment of stroke patients, it is important to compare these two compounds. In the present study, the neuroprotective effect of KCL-440 was equivalent to that of edaravone, and the latter also exhibited neuroprotective effects in the same photothrombotic occlusion model as the one that was used in the present study [16]. This suggests the eligibility of this experimental model for the assessment of neuroprotectants. In conclusion, KCL-440 exhibited neuroprotective effects in vivo and in vitro; these effects appear to be related to its high blood – brain barrier permeability and PARP inhibitory action.
References [1] G.E. Abdelkarim, K. Gertz, C. Harms, J. Katchanov, U. Dirnagl, C. Szabo, M. Endres, Protective effects of PJ-34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke, Int. J. Mol. Med. 7 (2001) 255 – 260. [2] A.V. Alexandrov, C.F. Bladin, J.W. Norris, Intracranial blood flow velocities in acute ischemic stroke, Stroke 25 (1994) 1378 – 1383. [3] Y. Andersson, M. Bergstrom, B. Langstrom, Synthesis of 11C-labelled benzamide compounds as potential tracers for poly(ADP-ribose) synthetase, Appl. Radiat. Isot. 45 (1994) 707 – 714. [4] T. Anraku, T. Kawai, K. Ishikawa, J. Ishiyama, T. Uno, The broad therapeutic time window of KCL-440 bases on delayed activation of poly(ADP-ribose) polymerase after transient ischemic insults in rats, Stroke 36 (2005) 513 – 514.
79
[5] J.B. Bederson, L.H. Pitts, M. Tsuji, M.C. Nishimura, R.L. Davis, H. Bartkowski, Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke 17 (1986) 472 – 476. [6] Y. Ding, Y. Zhou, Q. Lai, J. Li, V. Gordon, F.G. Diaz, Long-term neuroprotective effect of inhibiting poly(ADP-ribose) polymerase in rats with middle cerebral artery occlusion using a behavioral assessment, Brain Res. 915 (2001) 210 – 217. [7] M.J. Eliasson, K. Sampei, A.S. Mandir, P.D. Hurn, R.J. Traystman, J. Bao, A. Pieper, Z.Q. Wang, T.M. Dawson, S.H. Snyder, V.L. Dawson, Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia, Nat. Med. 3 (1997) 1089 – 1095. [8] M. Endres, Z.Q. Wang, S. Namura, C. Waeber, M.A. Moskowitz, Ischemic brain injury is mediated by the activation of poly(ADPribose) polymerase, J. Cereb. Blood Flow Metab. 17 (1997) 1143 – 1151. [9] H.C. Ha, S.H. Snyder, Poly(ADP-ribose) polymerase-1 in the nervous system, Neurobiol. Dis. 7 (2000) 225 – 239. [10] M. Ikejima, S. Noguchi, R. Yamashita, H. Suzuki, T. Sugimura, M. Miwa, Expression of human poly(ADP-ribose) polymerase with DNA-dependent enzymatic activity in Escherichia coli, Biochem. Biopys. Res. Commun. 163 (1989) 739 – 745. [11] A. Iwashita, N. Tojo, S. Matsuura, S. Yamazaki, K. Kamijo, J. Ishida, H. Yamamoto, K. Hattori, N. Matsuoka, S. Mutoh, A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia, J. Pharmacol. Exp. Ther. 310 (2004) 425 – 436. [12] T. Kawai, T. Anraku, J. Ishiyama, T. Uno, Acute application of KCL440, a novel Poly(ADP-ribose) polymerase inhibitor, leads to longterm neuroprotection in rat model of transient focal ischemia, Stroke 36 (2005) 514. [13] K. Kawano, Y. Ikeda, K. Kondo, K. Umemura, Increased cerebral infarction by cyclic flow reductions: studies in the guinea pig MCA thrombosis model, Am. J. Physiol. 275 (1998) R1578 – R1583. [14] R. von Kummer, R. Holle, L. Rosin, M. Forsting, W. Hacke, Dose arterial recanalization improve outcome in carotid territory stroke? Stroke 26 (1995) 581 – 587. [15] Y. Matsuo, T. Kihara, M. Ikeda, M. Ninomiya, H. Onodera, K. Kogure, Role of platelet-activating factor and thromboxane A2 in radical production during ischemia and reperfusion of the rat brain, Brain Res. 709 (1996) 296 – 302. [16] A. Mizuno, K. Umemura, M. Nakashima, Inhibitory effect of MCI-186, a free radical scavenger, on cerebral ischemia following rat middle cerebral artery occlusion, Gen. Pharmacol. 30 (1998) 575 – 578. [17] H. Nakajima, N. Kakui, K. Ohkuma, M. Ishikawa, T.A. Hasegawa, Newly Synthesized Poly(ADP-Ribose) Polymerase Inhibitor, DR2313 [2-Methyl-3,5,7,8-tetrahydrothiopyrano[4,3-d]-pyrimidine4-one]: pharmacological profiles, neuroprotective effects, and therapeutic time window in cerebral ischemia in rats, J. Pharmacol. Exp. Ther. 312 (2004) 472 – 481. [18] E.M. Park, S. Cho, K. Frys, G. Racchumi, P. Zhou, J. Anrather, C. Iadecola, Interaction between inducible nitric oxide synthase and poly(ADP-ribose) polymerase in focal ischemic brain injury, Stroke 35 (2004) 2896 – 2901. [19] K. Plaschke, J. Kopitz, M.A. Weigand, E. Martin, H.J. Bardenheuer, The neuroprotective effect of cerebral poly(ADP-ribose) polymerase inhibition in a rat model of global ischemia, Neurosci. Lett. 284 (2000) 109 – 112. [20] A.R. Saniabadi, K. Umemura, N. Matsumoto, S. Sakuma, M. Nakashima, Vessel wall injury and arterial thrombosis induced by a photochemical reaction, Thromb. Haemostasis 73 (1995) 868 – 872. [21] K. Takahashi, A.A. Pieper, S.E. Croul, J. Zhang, S.H. Snyder, J.H. Greenberg, Post-treatment with an inhibitor of poly(ADP-ribose) polymerase attenuates cerebral damage in focal ischemia, Brain Res. 829 (1999) 46 – 54.
80
Y. Ikeda et al. / Brain Research 1060 (2005) 73 – 80
[22] H. Takamatsu, K. Kondo, Y. Ikeda, K. Umemura, Neuroprotective effects depend on the model of focal ischemia following middle cerebral artery occlusion, Eur. J. Pharmacol. 362 (1998) 137 – 142. [23] A. Tamura, D.I. Graham, J. McCulloch, G.M. Teasdale, Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 1 (1981) 53 – 60. [24] T. Tokime, K. Nozaki, T. Sugino, H. Kikuchi, N. Hashimoto, K. Ueda, Enhanced poly(ADP-ribosyl)ation after focal ischemia in rat brain, J. Cereb. Blood Flow Metab. 18 (1998) 991 – 997. [25] K. Umemura, K. Wada, T. Uematsu, M. Nakashima, Evaluation of the combination of a tissue-type plasminogen activator, SUN9216, and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model, Stroke 24 (1993) 1077 – 1081. [26] G. Vandeplassche, M. Bernier, F. Thone, M. Borgers, Y. Kusama, D.J. Hearse, Singlet oxygen and myocardial injury: ultrastructural, cytochemical and electrocardiographic consequences of photoactivation of Rose Bengal, J. Mol. Cell. Cardiol. 22 (1990) 287 – 301. [27] L. Virag, C. Szabo, The therapeutic potential of poly(ADP-ribose) polymerase inhibitors, Pharmacol. Rev. 54 (2002) 375 – 429.
[28] R.C. Wallace, A.J. Furlan, D.J. Moliterno, G.H. Stevens, T.J. Masaryk, J. Perl, Basilar artery rethrombosis: successful treatment with platelet glycoprotein IIB/IIIA receptor inhibitor, Am. J. Neuroradiol. 18 (1997) 1257 – 1260. [29] Y. Yang, Q. Li, H. Miyashita, W. Howlett, M. Siddiqui, A. Shuaib, Usefulness of postischemic thrombolysis with or without neuroprotection in a focal embolic model of cerebral ischemia, J. Neurosurg. 92 (2000) 841 – 847. [30] S.W. Yu, H. Wang, M.F. Poitras, C. Coombs, W.J. Bowers, H.J. Federoff, G.G. Poirier, T.M. Dawson, V.L. Dawson, Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosisinducing factor, Science 297 (2002) 259 – 263. [31] J. Zhang, S. Lautar, S. Huang, C. Ramsey, A. Cheung, J.H. Li, GPI-6150 prevents H2O2 cytotoxicity by inhibiting poly(ADPribose) polymerase, Biochem. Biophys. Res. Commun. 278 (2000) 590 – 598. [32] B.Q. Zhao, Y. Suzuki, K. Kondo, K. Kawano, Y. Ikeda, K. Umemura, A novel MCA occlusion model of thrombotic ischemia with cyclic flow reductions: development of cerebral hemorrhage induced by heparin, Brain Res. Protoc. 9 (2002) 85 – 92.
Research Report
Neuroprotective effects of KCL-440, a new poly(ADP-ribose) polymerase inhibitor, in the rat middle cerebral artery occlusion model Yasuhiko Ikeda a, Kazuya Hokamura a, Tomoyuki Kawai b, Junichi Ishiyama b, Kumi Ishikawa b, Tsuyoshi Anraku b, Takashi Uno b, Kazuo Umemura a,* a
Department of Pharmacology, Hamamatsu University School of Medicine, Handayama, 1-20-1, Hamamatsu 432-8014, Japan b Discovery Res. Lab., Kyorin Pharmaceutical Co., Ltd., Tochigi, Japan Accepted 17 August 2005 Available online 3 October 2005
Abstract It is reported that ischemic brain injury is mediated by the activation of poly(ADP-ribose) polymerase (PARP). In this study, we examined the pharmacological profile of KCL-440, a new PARP inhibitor, and its neuroprotective effects in the rat acute cerebral infarction model induced by photothrombotic middle cerebral artery (MCA) occlusion. In an in vitro study, KCL-440 exhibited potency with regard to inhibition of PARP activity, with an IC50 value of 68 nM. An in vivo pharmacokinetic study showed that the brain concentration of KCL-440 was sufficient to inhibit PARP activity during the intravenous infusion of KCL-440 at the rate of 1 mg/kg/h. KCL-440 at various doses or saline was administered for 24 h immediately after the MCA occlusion. Administration of KCL-440 led to a dose-dependent reduction in the infarct size at 24 h after MCA occlusion. Infarct sizes were 44.8% T 3.0% (n = 8), 40.5% T 1.1% (n = 8), 38.2% T 1.4% (n = 8), 35.1% T 2.1% (n = 8), 34.2% T 2.3% (n = 7), 32.6% T 1.9% (n = 8), and 31.0% T 2.1% (n = 5) at doses of 0, 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h. When compared to the control group, a statistically significant difference was observed in the doses that were higher than 0.03 mg/kg/h. When the infusion of KCL-440 (1 mg/kg/h, n = 8) was started at 1 h after the MCA occlusion, a significant reduction in infarct size was observed; this was not observed when KCL-440 infusion was started 2 or 3 h after the MCA occlusion. Furthermore, increased poly(ADP-ribose) immunostaining was confirmed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment. These results suggest that KCL-440 is a possible neuroprotective agent with high blood – brain barrier permeability and high PARP inhibitory activity. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Poly(ADP-ribose) polymerase; Stroke; Neuroprotection
1. Introduction
Abbreviations: PARP, poly(ADP-ribose) polymerase; TCA, trichloroacetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; KCL-440, 4-(4-(N,N-dimethylaminomethyl)phenyl)-5-hydroxy isoquinolinone; PJ-34, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,Ndimethylactamide; 3-AB, 3-aminobenzamide; DPQ, 3,4-dihydro-5-[4-(1piperidinyl)butoxy]-1(2H)-isoquinolinone; DR2312, 2-methyl-3,5,7,8-tetrahydrothiopyrano(4,3-d)pyrimidine-4-one * Corresponding author. Fax: +81 53 435 2269. E-mail address: [email protected] (K. Umemura). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.046
Poly(ADP-ribose) polymerase (PARP) comprises a family of nuclear proteins that are activated by breaks in DNA strands, and it functions as a DNA repair enzyme. PARP cleaves NAD+ into nicotinamide and ADP-ribose to form long and branched (ADP-ribose) polymers that bind to numerous nuclear proteins, including histones and PARP itself [27]. However, overactivation of PARP, which is induced by excessive DNA damage, consumes NAD+; this leads to ATP depletion and consequent necrotic cell death [27]. It has been reported that the activation of PARP
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mediates ischemic brain injury [8]. In fact, PARP gene disruption rendered mice resistant to cerebral ischemia [7]. Moreover, several PARP inhibitors exerted neuroprotection in experimental models of cerebral ischemia [1,6,8,11,17 – 19,21]; these neuroprotective effects have been mainly attributed to PARP, which is considered to be the best characterized member of the PARP family. However, necrosis is not the only mechanism underlying PARP-mediated cellular suicide mechanisms. It was also reported that PARP played a crucial role in a caspase-independent apoptosis pathway that was mediated by an apoptosis-inducing factor [30]. KCL-440 is a newly developed PARP inhibitor. The objective of the present study was, first, to evaluate the PARP inhibitory activity and the blood – brain barrier permeability of this compound and, second, to study the neuroprotective effect of post-ischemic administration of KCL-440 in the rat acute cerebral infarction model induced by photothrombotic MCA occlusion. The neuroprotective effects that were observed were compared with those of the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one (known as edaravone). Using poly(ADP-ribose) immunohistochemistry, poly(ADP-ribosyl)ation was also studied in order to confirm the validity of the experimental model in assessing the neuroprotective effect of KCL-440. Some of these results have been published in the abstract form [12].
2. Materials and methods The Animal Care and Use Committee of Hamamatsu University School of Medicine approved all experimental protocol used in this study. 2.1. PARP assay The PARP enzyme assay was conducted in a total volume of 100 Al, consisting of 100 mM Tris –HCl (pH 8.0), 10 mM MgCl2, 10 Ag/ml activated DNA, 3 mM nicotinamide adenine dinucleotide, 10 kBq/ml adenosine-14C(U), 5 units/ml human recombinant PARP (Travigen), and various concentrations of the following test compounds: KCL-440
[4-(4-(N,N-dimethylaminomethyl)phenyl)-5-hydroxy isoquinolinone, chemical structure shown in Fig. 1]; PJ-34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylactamide]; and 3-AB (3-aminobenzamide). The reaction mixture was incubated at 25 -C; the reaction was terminated after 10 min by adding 1 ml of ice-cold 20% (w/v) trichloroacetic acid (TCA), and the mixture was stored overnight at 4 -C. The precipitate was collected on a glass fiber filter (Whatman GF/C) and washed three times with 5% TCA. Radioactivity was determined by scintillation counting. The experiments were carried out in triplicate. 2.2. H2O2 cytotoxicity assay in cortical cultures The cerebral cortex was isolated from 18-day-old rat embryos (Crj: CD(SD)IGS, Charles River Japan Inc.) and cut into small pieces. The dispersed cells were obtained by treatment with trypsin and subsequent mechanical agitation. The cell suspension was plated at density of 2 105 cells/cm2 on a 48-well plate that was coated with 0.1% polyethyleneimine in a DMEM/F12 medium containing a B27 supplement; the plate with the suspension was then placed in a CO2 incubator at 37 -C for more than 7 days. In the H2O2 cytotoxicity study, the culture medium was replaced with DMEM/F12 that contained B27 and various concentrations of the test compound. After preincubation for 1 h, the cells were washed twice with DMEM/F12 (B27-free) that contained the test compound and were then exposed to 0.2 mM H2O2 in the DMEM/F12 containing the test compound for 2 h in a CO2 incubator at 37 -C. Immediately after the incubation, the cells were washed twice with DMEM/F12 containing B27 and the test compound, and the cell viability was assessed using a standard colorimetric assay with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). A small aliquot (50 Al) of the 2.5 mg/ml MTT was added to the culture medium (250 Al) that contained B27 and the test compound in each well. After incubation for 4–5 h in the CO2 incubator at 37 -C, the reaction product, formazan, was solubilized with 0.04N HCl/isopropanol. The absorbance was measured at 540 nm. These experiments were carried out in duplicate. 2.3. Serum and brain concentrations
Fig. 1. Chemical structure of KCL-440.
In order to determine the serum concentrations of KCL440, the drug was intravenously infused at a rate of either 1 or 10 mg/kg/h in Wistar rats. For deproteinization, the serum was added to 5 volumes of methanol; this solution was vortexed and then centrifuged at 20,000 g for 5 min. The supernatant was filtered and injected into an HPLC (Nanospace system, Shiseido, Japan), and UV detection was carried out at 340 nm. In another experiment, the brain was isolated immediately after 6 h of KCL-440 infusion at 10 mg/kg/h and homogenized in an ice-cold 100 mM phosphate buffer (pH 7.4). The quantification of KCL-440 in the brain homogenates was carried out under the same HPLC conditions as those for the analysis of the serum sample.
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2.4. Rat photothrombotic MCA occlusion model
2.6. Poly(ADP-ribose) immunohistochemistry
Male Sprague – Dawley rats (SLC, Japan), weighing 260 – 320 g, were anesthetized with halothane (4% induction/2% maintenance). The body temperature of the animals was maintained at 37.5 -C T 0.5 -C with a heating pad (K-module Model K-20, American Pharmaseal, USA). The left MCA was thrombotically occluded by a photochemical reaction [25]. Briefly, a catheter for the administration of either the drug or Rose Bengal was inserted into the femoral vein. The scalp and temporalis muscle were folded over, and subtemporal craniotomy was performed with a dental drill, under an operating microscope in order to open a 3-mm diameter oval bony window. The window was irradiated with green light (wavelength 540 nm) obtained by using a xenon lamp (L4887, Hamamatsu Photonics, Hamamatsu, Japan) that had heat absorption and a green filter. The irradiation was directed by a 3-mm diameter optic fiber mounted on a micromanipulator. The head of the optic fiber was placed on the window in the skull base at a distance of 2 mm above the artery. Photoillumination was performed for 10 min after an intravenous injection of Rose Bengal (20 mg/kg). After the MCA occlusion, the temporalis muscle and skin were closed in layers, and anesthesia was discontinued. The brain was rapidly excised 24 h after surgery under pentobarbital sodium anesthesia. After the separation of the cerebrum, the brain was coronally sectioned on a brain matrix (RBS-02, Neuroscience, Japan) into 2-mm thick slices starting from the frontal lobe. Six consecutive slices were then stained with 1% triphenyltetrazolium chloride (Katayama, Nagoya, Japan) and subsequently photographed. For each of these sections, infarct (white) and noninfarct (red) areas were determined using computerized image analysis on an Apple Macintosh G4 computer by using the public domain NIH image program (developed at the National Institute of Health and available on the Internet by anonymous FTP from zippy.nimh.nih.gov).
To elucidate the spatial localization of poly(ADP-ribose) following focal cerebral ischemia, the rats were anesthetized with sodium pentobarbital and transcardially perfused with 20 – 50 ml of saline, followed by perfusion with 10% paraformaldehyde (pH 7.4) at 2 h after cerebral ischemia. The brains of these rats were quickly excised and cryoprotected in 25% sucrose in phosphate-buffered saline at 4 -C overnight. Frozen coronal sections (12-Am thickness) were prepared. After quenching endogenous peroxidase with 0.5% H2O2 in methanol and blocking with 5% hose serum, the sections were incubated overnight at 4 -C with a monoclonal antibody against poly(ADP-ribose) (Alexis Biochemicals, Lausen, Switzerland). The sections were washed with phosphate-buffered saline and then incubated with a biotinylated antibody for 30 min and with an avidin – biotin complex for 30 min (Vectastain, ABC kit, Vector). Peroxidase staining was carried out using 3,3V-diaminobenzidine. To evaluate the inhibitory effects of KCL-440 on PARP activity in vivo, KCL-440 was administered at a dose of 1 g/kg/h for 2 h after cerebral ischemia; subsequently, the brain was excised, and poly(ADP-ribose) was stained immunohistochemically in the same manner as that of the control animals. 2.7. Drug administration KCL-440 at doses of 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h, dissolved in saline, was intravenously administered at a rate of 0.33 ml/kg/h for 24 h immediately after cerebral ischemia. In the case of the control group, saline was administered in the same manner. In the case of the positive control group, a free radical scavenger, 3-methyl-1phenyl-2-pyrazolin-5-one, at a dose of 1 mg/kg/h, was administered in the same manner. To elucidate the therapeutic time window, KCL-440 administration in a dose of 1 mg/kg/h was started at 1, 2, or 3 h after cerebral ischemia followed by a continuous infusion for 21 –23 h.
2.5. Evaluation of neurological symptoms 2.8. Statistical analysis In accordance with the method of Bederson et al. [5], neurological symptoms such as hemiplegia were evaluated in a posture test at 24 h after photothrombosis. This evaluation was carried out in a blind manner. In the postural reflex test, hemiparesis was observed in the animals. The degree of abnormal posture observed when the rats were suspended by their tails at a distance of 1 m above the floor was examined. These rats were scored according to the following criteria: 0, rats that extended both forelimbs straight, no observable deficit; 1, rats that attached the right forelimb to the breast and extended the left forelimb straight; 2, rats that demonstrated decreased resistance to a lateral push in addition to the behavior described in score 1 without circling; and 3, rats that twisted the upper half of their body in addition to the behavior described in score 2.
All data are expressed as mean T SE. Statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test for drug-treated groups versus the control group.
3. Results 3.1. In vitro activity of KCL-440 The in vitro inhibitory efficacies of KCL-440, PJ-34, and 3-AB were determined using the human recombinant PARP enzyme (Table 1). The IC50 value of KCL-440 was 68 nM, which was 4 and 1000 times more potent than that of PJ-34 and 3-AB, respectively. PARP is a nuclear enzyme and has
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Table 1 Inhibitory effects of KCL-440, PJ-34, and 3-AB on the PARP enzyme and their neuroprotective effects on H2O2-induced toxicity in rat cultured cortical neurons
PARP enzymatic activity H2O2-induced cytotoxicity
KCL-440 (nM)
PJ-34 (nM)
3-AB (AM)
68 73
250 170
72 410
an H2O2-induced cytotoxic role [31]. Therefore, the neuroprotective effects of the PARP inhibitor on H2O2-induced toxicity could reflect its potency with regard to the inhibition of cellular PARP. Thus, we examined the effects of these PARP inhibitors on H2O2-induced toxicity in cultured rat cortical neurons (Table 1). The PARP inhibitors that were tested exerted a protective effect against neuronal cell death in a concentration-dependent manner; this supports the role of PARP in the neuronal injury that was observed in the cortical cultures. The IC50 value of KCL440 was 73 nM, which is nearly the same value as the IC50 value on enzymatic activity, thus suggesting that KCL-440 easily penetrates and enters the nucleus of neuronal cells. 3.2. Serum and brain concentrations The serum concentration of KCL-440 approached steadystate levels within 1 h in rats that were intravenously infused at the rate of 1 mg/kg/h for 6 h (Table 2). One hour after the initiation of infusion, the serum concentration of KCL-440 was 0.11 Ag/ml, which is equivalent to 0.37 AM. The brain:serum concentration ratio after a 6-h infusion at 10 mg/kg/h was 0.8 (Table 2). These results indicated that the brain concentration of KCL-440 was sufficient to inhibit the PARP activity during the intravenous infusion at the rate of 1 mg/kg/h. 3.3. Neuroprotective effects of KCL-440 in the in vivo cerebral ischemia model Fig. 2 displays the image of the nontreated and treated animals, in which KCL-440 at a dose of 1 mg/kg/h was administered for 24 h after cerebral ischemia. KCL-440 treatment reduced cerebral damage, particularly in the cortex. Administration of KCL-440 led to a dose-dependent reduction in the cerebral infarct size at 24 h after the MCA occlusion (Fig. 3). Statistical significance was observed in the doses higher than 0.03 mg/kg/h. The neuroprotective effect of KCL-440 was found to be greater than that of 3Table 2 KCL-440 concentrations in serum and brain during intravenous infusion of KCL-440 Dose (mg/kg/h) Serum (Ag/ml) Brain Ratio (Ag/g tissue) (B/P) 1h 3h 6h 1 10 – , not tested.
0.11 –
0.1 –
0.12 2.36
– 1.99
– 0.8
Fig. 2. Effects of KCL-440 on the extent of cerebral damage, KCL-440 at a dose of 1 mg/kg/h reduced the damaged area, particularly in the cortex. (A) Control, (B) KCL-440 at a dose of 1 mg/kg/h. Scale bar = 10 mm.
methyl-1-phenyl-2-pyrazolin-5-one in the doses higher than 0.3 mg/kg/h. When KCL-440 (1 mg/kg/h, n = 8) was administered, starting from 1 h after the MCA occlusion, a significant reduction in infarct size was observed; however, this reduction was not observed when KCL-440 administration was started at 2 or 3 h after cerebral ischemia (Fig. 5). 3.4. Effect of KCL-440 on neurological deficits The animals in the control group exhibited impairment of the posture reflex. Administration of KCL-440 at doses of 0.03, 0.1, and 1.0 mg/kg/h, and 3-methyl-1-phenyl-2-pyrazolin-5-one significantly improved the neurological score (Fig. 4).
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Fig. 3. Effect of KCL-440 on cerebral infarct size. Each column represents mean T SE. Bold numbers inside the graph represent the number of animals. *P < 0.05 versus control, **P < 0.01 versus control.
3.5. Poly(ADP-ribose) immunohistochemistry Increased poly(ADP-ribose) immunostaining was observed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment (1 mg/kg/h) (Fig. 6).
4. Discussion In this study, KCL-440, a new PARP inhibitor, reduced brain damage in a dose-dependent manner in the rat cerebral
infarction model induced by photothrombotic MCA occlusion. KCL-440 also improved neurological symptoms. These neuroprotective effects can be attributed to its high blood –brain barrier permeability and neuroprotective activity, which was exhibited in vitro. It has been reported that several PARP inhibitors exert neuroprotective effects in stroke models [27]. Most of the compounds used in those studies were administered prior to the occurrence of cerebral ischemia [6,8,11,18,19,21]. 3-AB [6,8,19] and PJ-34 [1,18] are commonly used as PARP inhibitors. However, they have a relatively weak PARP inhibitory activity, as demonstrated in the present study. The
Fig. 4. Effect of KCL-440 on neurological deficits, *P < 0.05 versus control.
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Fig. 5. Therapeutic time window of the neuroprotective effect of KCL-440. KCL-440 was administered 1, 2, or 3 h after the MCA occlusion. Vehicle was administered 1 h after the MCA occlusion. Each column represents mean T SE. Bold numbers inside the graph represent the number of animals. *P < 0.05 versus control.
IC50 value of KCL-440 was 68 nM, which was 4 and 1000 times more potent than that of PJ-34 and 3-AB, respectively; further, using 50 AM of Km for NAD, the calculated value of K i was found to be 9.8 nM [10]. In addition, 3-AB does not pass the blood – brain barrier after peripheral administration [3,24], whereas a beneficial brain:serum concentration ratio of KCL-440 was observed after its continuous infusion of KCL-440. This pharmacokinetic property of KCL-440 makes it favorable for the purpose of intravenous administration. In the present study, KCL-440 was administered immediately after cerebral ischemia, and a marked reduction in extent of brain damage was observed. Furthermore, the
neuroprotective effect of KCL-440 was still significant when it was administered 1 h after ischemia. The therapeutic time window of this compound appears to be related to the activation of PARP. Tokime et al. reported that marked poly(ADP-ribose) immunoreactivity was detected 2 h after ischemia [24], which is consistent with the immunohistochemical results obtained in the present study. A similar relationship between the therapeutic time window of KCL440 and PARP activation has been reported in the transient focal ischemia model [4]; therefore, PARP inhibitors must be administered at least before the peak of PARP activation. Other papers have reported that post-ischemic administration of PARP inhibitors 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) and 2-methyl-3,5,7,8tetrahydrothiopyrano[4,3-d]pyrimidine-4-one (DR2312) induced neuroprotection [17,19]. Although the IC50 value of KCL-440 is the lowest, it is difficult to compare these compounds with KCL-440 in the different cerebral ischemic models in vivo and in vitro. The photoinduced thrombotic occlusion model involves the formation of a platelet- and fibrin-rich thrombus in the MCA at the irradiated site [20]. The photochemical approach to induce thrombotic MCA occlusion involves an intravenous Rose Bengal injection followed by irradiation of the MCA with green light. Rose Bengal is a photosensitive dye that, when photoactivated, produces reactive oxygen species, mainly singlet oxygen by a ‘‘photodynamic Type II’’ reaction or energy transfer [26]. Reactive oxygen species cause endothelial injury that is followed by platelet adhesion and aggregation to form fibrin nets; this results in the formation of a platelet- and fibrin-
Fig. 6. Immunohistochemistry of poly(ADP-ribose). At 2 h after the MCA occlusion, poly(ADP-ribose) formation is elevated (B) in the nuclei in the ischemic border zone but not (A) in the sham-operated animal. (C) Increase in poly(ADP-ribose) formation was reduced by KCL-440 treatment (1 mg/kg). Scale bar = 100 Am.
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rich thrombus at the irradiated site [20]. An important feature of this model is the continued cyclic flow reductions that are observed in the MCA [13,32]. Therefore, the present model can represent a new MCA occlusion model that is different from previously described models of permanent or ischemia/reperfusion MCA occlusion. In the clinical situation, approximately 60% of patients exhibit spontaneous recanalization of the MCA in the early phase of an ischemic stroke in the absence of thrombolytic therapy [2]. Similarly, rethrombosis after thrombolysis is also frequently observed in the cerebral artery [14,28]. This suggests that cyclic flow reductions may occur in the acute phase of human stroke. However, in permanent models [23], suture MCA occlusion models [15], and the embolic MCA occlusion model [29], spontaneous reperfusion has not been observed. In the model used in this study, a radical scavenger that was administered after the MCA occlusion was effective; however, it was not effective in permanent occlusion models, in which reperfusion of the MCA was not observed [22]. Therefore, the development of cerebral damage in this model is related to the generation of reactive oxygen species, which can damage DNA and activate PARP [9], within the brain tissue. Thus, in the model used in this study, the development of cerebral damage may be associated with PARP activation. In the present in vivo study, the neuroprotective effect of KCL-440 was compared with that of the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one that is known as edaravone. Since edaravone is already clinically used in Japan for the treatment of stroke patients, it is important to compare these two compounds. In the present study, the neuroprotective effect of KCL-440 was equivalent to that of edaravone, and the latter also exhibited neuroprotective effects in the same photothrombotic occlusion model as the one that was used in the present study [16]. This suggests the eligibility of this experimental model for the assessment of neuroprotectants. In conclusion, KCL-440 exhibited neuroprotective effects in vivo and in vitro; these effects appear to be related to its high blood – brain barrier permeability and PARP inhibitory action.
References [1] G.E. Abdelkarim, K. Gertz, C. Harms, J. Katchanov, U. Dirnagl, C. Szabo, M. Endres, Protective effects of PJ-34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke, Int. J. Mol. Med. 7 (2001) 255 – 260. [2] A.V. Alexandrov, C.F. Bladin, J.W. Norris, Intracranial blood flow velocities in acute ischemic stroke, Stroke 25 (1994) 1378 – 1383. [3] Y. Andersson, M. Bergstrom, B. Langstrom, Synthesis of 11C-labelled benzamide compounds as potential tracers for poly(ADP-ribose) synthetase, Appl. Radiat. Isot. 45 (1994) 707 – 714. [4] T. Anraku, T. Kawai, K. Ishikawa, J. Ishiyama, T. Uno, The broad therapeutic time window of KCL-440 bases on delayed activation of poly(ADP-ribose) polymerase after transient ischemic insults in rats, Stroke 36 (2005) 513 – 514.
79
[5] J.B. Bederson, L.H. Pitts, M. Tsuji, M.C. Nishimura, R.L. Davis, H. Bartkowski, Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke 17 (1986) 472 – 476. [6] Y. Ding, Y. Zhou, Q. Lai, J. Li, V. Gordon, F.G. Diaz, Long-term neuroprotective effect of inhibiting poly(ADP-ribose) polymerase in rats with middle cerebral artery occlusion using a behavioral assessment, Brain Res. 915 (2001) 210 – 217. [7] M.J. Eliasson, K. Sampei, A.S. Mandir, P.D. Hurn, R.J. Traystman, J. Bao, A. Pieper, Z.Q. Wang, T.M. Dawson, S.H. Snyder, V.L. Dawson, Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia, Nat. Med. 3 (1997) 1089 – 1095. [8] M. Endres, Z.Q. Wang, S. Namura, C. Waeber, M.A. Moskowitz, Ischemic brain injury is mediated by the activation of poly(ADPribose) polymerase, J. Cereb. Blood Flow Metab. 17 (1997) 1143 – 1151. [9] H.C. Ha, S.H. Snyder, Poly(ADP-ribose) polymerase-1 in the nervous system, Neurobiol. Dis. 7 (2000) 225 – 239. [10] M. Ikejima, S. Noguchi, R. Yamashita, H. Suzuki, T. Sugimura, M. Miwa, Expression of human poly(ADP-ribose) polymerase with DNA-dependent enzymatic activity in Escherichia coli, Biochem. Biopys. Res. Commun. 163 (1989) 739 – 745. [11] A. Iwashita, N. Tojo, S. Matsuura, S. Yamazaki, K. Kamijo, J. Ishida, H. Yamamoto, K. Hattori, N. Matsuoka, S. Mutoh, A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia, J. Pharmacol. Exp. Ther. 310 (2004) 425 – 436. [12] T. Kawai, T. Anraku, J. Ishiyama, T. Uno, Acute application of KCL440, a novel Poly(ADP-ribose) polymerase inhibitor, leads to longterm neuroprotection in rat model of transient focal ischemia, Stroke 36 (2005) 514. [13] K. Kawano, Y. Ikeda, K. Kondo, K. Umemura, Increased cerebral infarction by cyclic flow reductions: studies in the guinea pig MCA thrombosis model, Am. J. Physiol. 275 (1998) R1578 – R1583. [14] R. von Kummer, R. Holle, L. Rosin, M. Forsting, W. Hacke, Dose arterial recanalization improve outcome in carotid territory stroke? Stroke 26 (1995) 581 – 587. [15] Y. Matsuo, T. Kihara, M. Ikeda, M. Ninomiya, H. Onodera, K. Kogure, Role of platelet-activating factor and thromboxane A2 in radical production during ischemia and reperfusion of the rat brain, Brain Res. 709 (1996) 296 – 302. [16] A. Mizuno, K. Umemura, M. Nakashima, Inhibitory effect of MCI-186, a free radical scavenger, on cerebral ischemia following rat middle cerebral artery occlusion, Gen. Pharmacol. 30 (1998) 575 – 578. [17] H. Nakajima, N. Kakui, K. Ohkuma, M. Ishikawa, T.A. Hasegawa, Newly Synthesized Poly(ADP-Ribose) Polymerase Inhibitor, DR2313 [2-Methyl-3,5,7,8-tetrahydrothiopyrano[4,3-d]-pyrimidine4-one]: pharmacological profiles, neuroprotective effects, and therapeutic time window in cerebral ischemia in rats, J. Pharmacol. Exp. Ther. 312 (2004) 472 – 481. [18] E.M. Park, S. Cho, K. Frys, G. Racchumi, P. Zhou, J. Anrather, C. Iadecola, Interaction between inducible nitric oxide synthase and poly(ADP-ribose) polymerase in focal ischemic brain injury, Stroke 35 (2004) 2896 – 2901. [19] K. Plaschke, J. Kopitz, M.A. Weigand, E. Martin, H.J. Bardenheuer, The neuroprotective effect of cerebral poly(ADP-ribose) polymerase inhibition in a rat model of global ischemia, Neurosci. Lett. 284 (2000) 109 – 112. [20] A.R. Saniabadi, K. Umemura, N. Matsumoto, S. Sakuma, M. Nakashima, Vessel wall injury and arterial thrombosis induced by a photochemical reaction, Thromb. Haemostasis 73 (1995) 868 – 872. [21] K. Takahashi, A.A. Pieper, S.E. Croul, J. Zhang, S.H. Snyder, J.H. Greenberg, Post-treatment with an inhibitor of poly(ADP-ribose) polymerase attenuates cerebral damage in focal ischemia, Brain Res. 829 (1999) 46 – 54.
80
Y. Ikeda et al. / Brain Research 1060 (2005) 73 – 80
[22] H. Takamatsu, K. Kondo, Y. Ikeda, K. Umemura, Neuroprotective effects depend on the model of focal ischemia following middle cerebral artery occlusion, Eur. J. Pharmacol. 362 (1998) 137 – 142. [23] A. Tamura, D.I. Graham, J. McCulloch, G.M. Teasdale, Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 1 (1981) 53 – 60. [24] T. Tokime, K. Nozaki, T. Sugino, H. Kikuchi, N. Hashimoto, K. Ueda, Enhanced poly(ADP-ribosyl)ation after focal ischemia in rat brain, J. Cereb. Blood Flow Metab. 18 (1998) 991 – 997. [25] K. Umemura, K. Wada, T. Uematsu, M. Nakashima, Evaluation of the combination of a tissue-type plasminogen activator, SUN9216, and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model, Stroke 24 (1993) 1077 – 1081. [26] G. Vandeplassche, M. Bernier, F. Thone, M. Borgers, Y. Kusama, D.J. Hearse, Singlet oxygen and myocardial injury: ultrastructural, cytochemical and electrocardiographic consequences of photoactivation of Rose Bengal, J. Mol. Cell. Cardiol. 22 (1990) 287 – 301. [27] L. Virag, C. Szabo, The therapeutic potential of poly(ADP-ribose) polymerase inhibitors, Pharmacol. Rev. 54 (2002) 375 – 429.
[28] R.C. Wallace, A.J. Furlan, D.J. Moliterno, G.H. Stevens, T.J. Masaryk, J. Perl, Basilar artery rethrombosis: successful treatment with platelet glycoprotein IIB/IIIA receptor inhibitor, Am. J. Neuroradiol. 18 (1997) 1257 – 1260. [29] Y. Yang, Q. Li, H. Miyashita, W. Howlett, M. Siddiqui, A. Shuaib, Usefulness of postischemic thrombolysis with or without neuroprotection in a focal embolic model of cerebral ischemia, J. Neurosurg. 92 (2000) 841 – 847. [30] S.W. Yu, H. Wang, M.F. Poitras, C. Coombs, W.J. Bowers, H.J. Federoff, G.G. Poirier, T.M. Dawson, V.L. Dawson, Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosisinducing factor, Science 297 (2002) 259 – 263. [31] J. Zhang, S. Lautar, S. Huang, C. Ramsey, A. Cheung, J.H. Li, GPI-6150 prevents H2O2 cytotoxicity by inhibiting poly(ADPribose) polymerase, Biochem. Biophys. Res. Commun. 278 (2000) 590 – 598. [32] B.Q. Zhao, Y. Suzuki, K. Kondo, K. Kawano, Y. Ikeda, K. Umemura, A novel MCA occlusion model of thrombotic ischemia with cyclic flow reductions: development of cerebral hemorrhage induced by heparin, Brain Res. Protoc. 9 (2002) 85 – 92.