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A selective, non-peptide caspase-1 inhibitor, VRT-018858, markedly reduces brain damage induced by transient ischemia in the rat
Neuropharmacology 53 (2007) 638e642 www.elsevier.com/locate/neuropharm
A selective, non-peptide caspase-1 inhibitor, VRT-018858, markedly reduces brain damage induced by transient ischemia in the rat Jerard Ross a,b, David Brough a, Rosemary M. Gibson a,1, Sarah A. Loddick a,2, Nancy J. Rothwell a,* a
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester, M13 9PT, UK b Department of Clinical Neurosciences, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK Received 25 May 2007; received in revised form 27 June 2007; accepted 13 July 2007
Abstract Numerous preclinical studies have reported neuroprotective effects of new agents in animal studies. None of these agents has yet translated into a successful clinical trial and therefore to a new therapy. There are many possible reasons for this failure, including poor design of clinical trials, mismatch between preclinical and clinical protocols, and insufficient preclinical data. The enzyme caspase-1 has been implicated in neuronal death. Deletion of the caspase-1 gene, or administration of partially selective inhibitors, reduces neuronal injury induced by cerebral ischemia in rodents. We report here, for the first time, that VRT-018858, the non-peptide, active metabolite of the selective caspase-1 inhibitor pro-drug, pralnacasan, markedly reduced ischemic injury in rats. VRT-018858 was neuroprotective when delivered at 1 and 3 h (42% and 58% neuroprotection, respectively) but not 6 h after injury, and protection was sustained 7 days after the induction of ischemia (66% neuroprotection). These data confirm caspase-1 as an important target for intervention in acute CNS injury, and propose a new class of caspase-1 inhibitors as highly effective neuroprotective agents. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain; Caspase-1; Cerebral ischemia; IL-1
1. Introduction Caspases are a ubiquitously expressed family of cysteine proteases that are central to both apoptotic cell death and pro-inflammatory cytokine maturation. Caspase-1 was originally named interleukin-1b (IL-1b) converting enzyme (ICE), because it was identified as the protease responsible for maturation of inactive pro-IL-1b into an active IL-1b molecule (Thornberry et al., 1992). Caspase-1 is also essential for the cellular release of IL-1b and IL-1a, with the effects on IL-1a * Corresponding author. Tel.: þ44 161 275 5357; fax: þ44 161 275 5948. E-mail address: [email protected] (N.J. Rothwell). 1 Present address: Health and Safety Laboratory, Harpur Hill, Buxton SK17 9JN, UK. 2 Present address: AstraZeneca, Alderley Edge, Cheshire SK10 4TG, UK. 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.07.015
probably occurring through an indirect mechanism (Brough et al., 2003). Other caspase-1 substrates include the cytokines IL-18 (Gu et al., 1997), IL-1F7 (a new member of the IL-1 family, with unknown function; Kumar et al. (2002)), IL-33 (Dinarello, 2005) and a plasma membrane Ca2þ-ATPase (PMCA2; Schwab et al. (2002)). The caspase-1 substrate IL-1b is implicated in neurodegeneration, principally by acting through the type I IL-1 receptor (IL-1RI), and is reported to mediate diverse forms of experimental brain injury (Allan et al., 2005). The naturally occurring IL-1RI antagonist, IL-1 receptor antagonist (IL-1RA), is potently neuroprotective (Allan et al., 2005), and has been tested in phase II clinical trials in stroke (Emsley et al., 2005). However, IL-1RA is a large protein and selective, brain-penetrable caspase-1 inhibitors may offer therapeutic advantages.
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
Intracerebroventricular (icv) administration of the peptide N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) or zVAD-DCB, a pan-caspase inhibitor, significantly reduces ischemic brain damage in rats (Loddick et al., 1996) and improves functional outcome (Hara et al., 1997). A more selective caspase-1 inhibitor, based on the sequence Tyr-Val-Ala-Asp (YVAD), reported to inhibit the activity of caspases 1, 4, 5, 8 and 10 (Garcia-Calvo et al., 1998), similarly reduces brain injury after experimental, permanent focal ischemia in the rat (Rabuffetti et al., 2000). Together with the data from transgenic mice (Schielke et al., 1998), these inhibitor studies suggest that caspase-1 could be a major therapeutic target for reducing ischemic brain damage. The aim of this study was to test the effects of an active caspase-1 inhibitor, VRT-018858, the metabolite of the orally available pro-drug, pralnacasan (Rudolphi et al., 2003), on ischemic brain damage in the rat. We show that this caspase-1 inhibitor was protective, even when administered 3 h after induction of ischemia (58% neuroprotection), and that protection was sustained for up to 7 days (66% neuroprotection when administered at the onset of ischemia). 2. Materials and methods 2.1. Animals Experiments were conducted using male, SpragueeDawley rats (Charles River UK), in accordance with the UK Animals (Scientific Procedures) Act (1986). Surgical procedures were performed on animals under halothane anaesthesia (Fluothane, Zeneca, UK, 1e2% in a mixture of oxygen and nitrous oxide [1:2 ratio]). Body temperature was maintained at 37.0 0.5 C throughout anaesthesia by means of a homeothermic blanket.
2.2. Reagents General reagents were obtained from SigmaeAldrich (Poole, UK), unless stated otherwise. VRT-018858 was provided by Vertex Pharmaceuticals Inc. (Cambridge, USA).
2.3. Induction of cerebral ischemia Transient focal cerebral ischemia was induced in rats by unilateral middle cerebral artery occlusion (tMCAo) using the intraluminal thread method (Longa et al., 1989). Briefly, the right common carotid artery was exposed, and a poly-L-lysine-coated nylon monofilament thread was inserted into the external carotid artery and then into the internal carotid artery until resistance was felt (approximately 20 mm), at the point where it blocked the MCA. Laser Doppler flowmetry (Moor Instruments, UK) was used to monitor cerebral blood flow (CBF) during the surgical procedure and for 5 min after MCAo. Animals were allowed to recover from anaesthesia under a heating lamp. One hour after tMCAo, animals were briefly re-anesthetised and the thread withdrawn to permit reperfusion of the MCA. To permit icv administration of VRT-018858, guide cannulae were implanted stereotactically into the left lateral ventricle of the brain, 3e7 days before MCAo. Animals were randomised and injected icv with VRT-018858 as follows: (a) doseeresponse experiment: 2.5, 5, 10 or 20 mg VRT-018858 in 3 ml of 50 mM TriseHCl (pH 7.4) or vehicle (50 mM TriseHCl) immediately after ischemia, and again immediately after reperfusion (total dose 5, 10, 20 or 40 mg VRT-018858); (b) effect of VRT-018858 7 days after MCAo: 10 mg in 3 ml of 50 mM TriseHCl or vehicle (50 mM TriseHCl) immediately after ischemia and immediately after reperfusion; (c) therapeutic time-window effects: 20 mg VRT-018858 in 3 ml of 50 mM TriseHCl or vehicle (50 mM TriseHCl) 1, 3 or 6 h after MCAo. Investigators were blinded to treatments.
639
Animals were killed 24 h (a and c) or 7 days (b) after MCAo, and the brains were frozen on dry ice.
2.4. Data analysis Brain damage was assessed histologically, on a series of coronal sections (20e30 mm thick), at 390 or 500 mm intervals throughout the brain, stained with cresyl violet. Areas of dead tissue were clearly visualised by a complete lack of staining. Image analysis software (SigmaScan 5.0, SPSS Science, UK) was used to measure lesion volume. Lesion volumes were corrected for the effect of brain edema on lesion area. Brain edema was calculated as the difference between the ipsilateral and contralateral hemisphere volumes, divided by the contralateral hemisphere volume and expressed as a percentage of the contralateral hemisphere volume. All data are expressed as mean SD. Significant differences in lesion volumes between treatments were determined using analysis of variance (ANOVA) followed by Bonferroni post-hoc test for the doseeresponse study (a) and Student’s unpaired t-test for all other comparisons.
3. Results 3.1. Neuroprotective effect of VRT-018858 in transient MCAo Transient MCAo for 60 min induced reproducible cortical and sub-cortical (striatal and thalamic) lesions, analysed 24 h after reperfusion. The highest three doses of VRT-018858 (icv) caused a significant reduction in the total lesion volume compared to vehicle (206 58 mm3): 5 mg, 37% protection, NS; 10 mg, 46% protection, p < 0.001; 20 mg, 65% protection, p < 0.001 and 40 mg, 40% protection, p < 0.05 (Fig. 1A). The greatest effects of neuroprotection were observed in the cortex similar to our previously published work on IL-1RA (Mulcahy et al., 2003). Reductions in CBF at the time of tMCAo were comparable in all groups of animals and there was no significant difference between the body temperatures of vehicle- and VRT-018858-treated animals (data not shown). Within the first 60 min after tMCAo and drug treatment, VRT-018858 had no significant effect on blood gases recorded in a small number of animals under chronic anaesthesia (Table 1). 3.2. Sustained neuroprotection by VRT-018858 To investigate the effects of VRT-018858 on the development of brain damage after tMCAo, the drug was given in two 10 mg doses, the first immediately after tMCAo and the second immediately after reperfusion, and the lesion volumes were measured 7 days later. VRT-018858, compared to vehicle, caused a significant reduction in lesion volume (66%, p < 0.001, Fig. 1B). Reductions in CBF at the time of tMCAo were comparable in both groups (data not shown). There was no significant difference between the body temperatures of vehicle- or VRT-018858-treated animals at the time of reperfusion (data not shown). 3.3. Delayed administration of VRT-018858 is neuroprotective To characterise the therapeutic time window for VRT018858 administration, a single icv injection of VRT-018858
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
640
Lesion volume (mm3)
A
300
200
5 µg
Table 1 Arterial blood gas parameters of animals given VRT-018858 or vehicle at tMCAo ischaemia
10 µg
Time-point
Vehicle
20 µg
***
40 µg
*
*** 100
Partial pressure of O2 (kPa) Partial pressure of CO2 (kPa) pH
Control
VRT-018858
Pre-administration
60 min
Pre-administration
60 min
18 2
18 1
19 2
19 2
5 0.5
7 0.4
5 0.2
7 0.4
7.33
7.2
7.34
7.2
Animals underwent femoral artery cannulation followed by tMCAo and administration of VRT-018858 (20 mg) or vehicle icv. Data are mean SD. There were no significant differences between the groups. Statistical comparison was by ANOVA.
0
VRT-018858
Lesion volume (mm3)
B
250
200
Vehicle VRT-018858
150
100
***
50
0
Fig. 1. Effects of different doses of VRT-018858 on total lesion volume (A), and a sustained protective effect (B) after transient ischemic brain injury. (A) Animals were injected icv with vehicle (3 ml, n ¼ 17) or VRT-018858 (2.5 mg, n ¼ 17; 5 mg, n ¼ 19; 10 mg, n ¼ 12 or 20 mg, n ¼ 10) immediately after tMCAo (60 min) and immediately after reperfusion (total dose injected ¼ 5 mg, 10 mg, 20 mg or 40 mg). Total lesion volume was measured 24 h after tMCAo. Data are presented as mean SD of a group of animals. *p < 0.05, ***p < 0.001 compared to vehicle-treated group (ANOVA with Bonferroni post-hoc). (B) Animals were subjected to tMCAo and injected icv with vehicle (n ¼ 8) or VRT-018858 (10 mg, n ¼ 7) immediately after tMCAo and immediately after reperfusion (total dose 20 mg). Total lesion volume was measured 7 days after tMCAo. ***p < 0.001 compared to vehicle-treated group (Student’s unpaired t-test).
(20 mg) or vehicle was administered 1, 3 or 6 h after tMCAo (i.e. 0, 2 or 5 h after reperfusion). Damage was assessed 24 h after tMCAo. VRT-018858 significantly reduced the lesion volume when administered 1 h (42%, p < 0.05) or 3 h (58%, p < 0.001) after tMCAo (Fig. 2A, B), compared to the vehicle-treated group, but had no significant effect when administration was delayed until 6 h after tMCAo (Fig. 2C). The greatest effects of neuroprotection were observed in the cortex (data not shown). Reductions in CBF at the time of tMCAo were comparable in all groups of animals (data not shown). There was no significant difference between the body temperatures of vehicle- or VRT-018858-treated animals (data not shown). 4. Discussion The objective of the experiments described here was to determine the role of caspase-1 in ischemic brain damage using
a low molecular weight inhibitor of caspase-1, and to assess the therapeutic potential of the compound. VRT-018858 is potently selective for group I or inflammatory caspases, with Ki values against caspase-1 and caspase-4 of 1.3 nM and 0.4 nM, respectively (Ku et al., 2001). VRT018858 exhibits >100-fold selectivity for caspase-1 and caspase-4 against other caspases (Ku et al., 2001). Although the substrates for human caspase-4 remain to be identified, it is suggested to be an orthologue of murine caspase-11, which is important for the activation of caspase-1 (Martinon and Tschopp, 2007). Therefore, we suggest that the neuroprotective effects of VRT-18858 reported here are due to inhibition of caspase-1-mediated IL-1b production and inflammatory responses. VRT-018858 caused marked inhibition of ischemic brain injury after transient focal cerebral ischemia when administered icv immediately or after a delay of 3 h, but failed to influence body temperature or carotid blood flow. Many potentially promising neuroprotective agents that have been evaluated in studies with experimental animals have failed in clinical trials (Kidwell et al., 2001). The reasons for these failures are diverse and complex, but include insufficient animal studies, undesirable side effects and sub-optimal design of clinical trials (Stroke Therapy Academic Industry Roundtable (STAIR, 1999)). Key issues for preclinical studies of neuroprotection in ischemic brain damage are the ‘‘therapeutic window’’ and the duration of the effect. We show here that VRT-018858 is effective when first administered icv 3 h after temporary MCAo (58% reduction in total lesion volume), but the neuroprotective effect is lost when administration is delayed until 6 h after ischemia (Fig. 2). Protection is maintained 7 days after VRT-018858 treatment (66% reduction in total lesion volume) (Fig. 1B), indicating that this compound prevents rather than delays the development of ischemic brain injury. Previous studies have reported that non-specific caspase inhibitors attenuate ischemic brain damage (Loddick et al., 1996; Rabuffetti et al., 2000) and injury is also reduced in mice in which the caspase-1 gene is deleted (Schielke et al., 1998). However, to date no studies have reported on a highly selective caspase-1 inhibitor. The primary actions of caspase-1 are to cleave the inactive pro-IL-1b to an active form (Thornberry et al., 1992) and to
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
Lesion volume (mm3)
A
300
200
Vehicle VRT-018858
*
100
Lesion volume (mm3)
300 Vehicle VRT-018858
200
***
Lesion volume (mm3)
This work was funded in part by the Medical Research Council (NR, RG, DB), The Royal Society (SL) and Salford Royal Hospitals NHS Trust (JR). The authors are grateful to Deborah Bentley for assistance with preparation of the manuscript. References
100
0
C
the effects of VRT-018858 are primarily anti-inflammatory. However, VRT-018858 may have significant advantages over IL-1RA, since caspase-1 cleaves a number of other proteins that may contribute to ischemic cell death, including several other cytokines, PMCA2 (Schwab et al., 2002), and calpistatin, the endogenous calpain inhibitor (Vaisid et al., 2005), and may therefore have several mechanisms of action in neuronal injury. Thus, the neuroprotective effects of VRT-018858 reported here suggest that caspase-1 is an attractive target for therapeutic intervention. Acknowledgements
0
B
641
300
Vehicle VRT-018858
200
100
0
Fig. 2. Effects of delayed administration of VRT-018858 on total lesion volume when administered 1 h (A), 3 h (B) and 6 h (C) after transient ischemic brain injury. Animals were subjected to tMCAo and injected icv with vehicleor VRT-018858 (20 mg) either 1 (vehicle n ¼ 9, VRT-018858 n ¼ 9, A), 3 (vehicle n ¼ 8, VRT-018858 n ¼ 9, B) or 6 h (vehicle n ¼ 8, VRT-018858 n ¼ 9, C) after tMCAo. Total lesion volume was measured 24 h after tMCAo. *p < 0.05, ***p < 0.001 compared to vehicle-treated group (Student’s unpaired t-test).
facilitate its cellular release (Brough et al., 2003), and its activity is also important for the cellular release of the related IL-1a (Li et al., 1995; Brough et al., 2003). IL-1 has been strongly implicated in ischemic brain injury and genetic ablation of both IL-1a and IL-b genes results in a marked inhibition (70%) of ischemic brain injury (Boutin et al., 2001). In addition, the selective endogenous IL-1 antagonist, IL-1RA, also markedly limits ischemic brain injury (Relton and Rothwell, 1992; Allan et al., 2005). Thus, we suggest that
Allan, S.M., Tyrrell, P.J., Rothwell, N.J., 2005. Interleukin-1 and neuronal injury. Nat. Rev. Immunol. 5, 629e640. Boutin, H., LeFeuvre, R.A., Horai, R., Asano, M., Iwakura, Y., Rothwell, N.J., 2001. Role of IL-1alpha and IL-1beta in ischemic brain damage. J. Neurosci. 21, 5528e5534. Brough, D., Le Feuvre, R.A., Wheeler, R.D., Solovyova, N., Hilfiker, S., Rothwell, N.J., Verkhratsky, A., 2003. Ca2þ stores and Ca2þ entry differentially contribute to the release of IL-1 beta and IL-1 alpha from murine macrophages. J. Immunol. 170, 3029e3036. Dinarello, C.A., 2005. An IL-1 family member requires caspase-1 processing and signals through the ST2 receptor. Immunity 23, 461e462. Emsley, H.C., Smith, C.J., Georgiou, R.F., Vail, A., Hopkins, S.J., Rothwell, N.J., Tyrrell, P.J., 2005. A randomised phase II study of interleukin-1 receptor antagonist in acute stroke patients. J. Neurol. Neurosurg. Psychiatry 76, 1366e1372. Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., Thornberry, N.A., 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273, 32608e32613. Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M.A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., Kurimoto, M., Tanimoto, T., Flavell, R.A., Sato, V., Harding, M.W., Livingston, D.J., Su, M.S., 1997. Activation of interferon-gamma inducing factor mediated by interleukin1beta converting enzyme. Science 275, 206e209. Hara, H., Friedlander, R.M., Gagliardini, V., Ayata, C., Fink, K., Huang, Z., Shimizu-Sasamata, M., Yuan, J., Moskowitz, M.A., 1997. Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. U.S.A. 94, 2007e2012. Kidwell, C.S., Liebeskind, D.S., Starkman, S., Saver, J.L., 2001. Trends in acute ischemic stroke trials through the 20th century. Stroke 32, 1349e1359. Ku, G., Ford, P., Raybuck, S.A., Harding, M.W., Randle, J.C, 2001. Selective interleukin-1beta converting enzyme (ICE/caspase-1) inhibition with pralnacasan (HMR 3480/VX-740) reduces inflammation and joint destruction in murine type II collagen-induced arthritis (CIA). Kumar, S., Hanning, C.R., Brigham-Burke, M.R., Rieman, D.J., Lehr, R., Khandekar, S., Kirkpatrick, R.B., Scott, G.F., Lee, J.C., Lynch, F.J., Gao, W., Gambotto, A., Lotze, M.T., 2002. Interleukin-1F7B (IL-1H4/ IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL18 receptor but does not induce IFN-gamma production. Cytokine 18, 61e71. Li, P., Allen, H., Banerjee, S., Franklin, S., Herzog, L., Johnston, C., McDowell, J., Paskind, M., Rodman, L., Salfeld, J., 1995. Mice deficient
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in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 80, 401e411. Loddick, S.A., MacKenzie, A., Rothwell, N.J., 1996. An ICE inhibitor, z-VAD-DCB attenuates ischaemic brain damage in the rat. Neuroreport 7, 1465e1468. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84e91. Martinon, F., Tschopp, J., 2007. Inflammatory caspases and inflammasomes: master switches of inflammation. Cell Death Differ. 14, 10e22. Mulcahy, N.J., Ross, J., Rothwell, N.J., Loddick, S.A., 2003. Delayed administration of interleukin-1 receptor antagonist protects against transient cerebral ischaemia in the rat. Br. J. Pharmacol. 140, 471e476. Rabuffetti, M., Sciorati, C., Tarozzo, G., Clementi, E., Manfredi, A.A., Beltramo, M., 2000. Inhibition of caspase-1-like activity by Ac-Tyr-ValAla-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J. Neurosci. 20, 4398e4404. Relton, J.K., Rothwell, N.J., 1992. Interleukin-1 receptor antagonist inhibits ischaemic and excitotoxic neuronal damage in the rat. Brain Res. Bull. 29, 243e246.
Rudolphi, K., Gerwin, N., Verzijl, N., van der, K.P., van den, B.W., 2003. Pralnacasan, an inhibitor of interleukin-1beta converting enzyme, reduces joint damage in two murine models of osteoarthritis. Osteoarthritis Cartilage 11, 738e746. Schielke, G.P., Yang, G.Y., Shivers, B.D., Betz, A.L., 1998. Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice. J. Cereb. Blood Flow Metab. 18, 180e185. Schwab, B.L., Guerini, D., Didszun, C., Bano, D., Ferrando-May, E., Fava, E., Tam, J., Xu, D., Xanthoudakis, S., Nicholson, D.W., Carafoli, E., Nicotera, P., 2002. Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis. Cell Death Differ. 9, 818e831. Stroke Therapy Academic Industry Roundtable (STAIR), 1999. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 30, 2752e2758. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunins, J., 1992. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768e774. Vaisid, T., Kosower, N.S., Barnoy, S., 2005. Caspase-1 activity is required for neuronal differentiation of PC12 cells: cross-talk between the caspase and calpain systems. Biochim. Biophys. Acta 1743, 223e230.
A selective, non-peptide caspase-1 inhibitor, VRT-018858, markedly reduces brain damage induced by transient ischemia in the rat Jerard Ross a,b, David Brough a, Rosemary M. Gibson a,1, Sarah A. Loddick a,2, Nancy J. Rothwell a,* a
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester, M13 9PT, UK b Department of Clinical Neurosciences, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK Received 25 May 2007; received in revised form 27 June 2007; accepted 13 July 2007
Abstract Numerous preclinical studies have reported neuroprotective effects of new agents in animal studies. None of these agents has yet translated into a successful clinical trial and therefore to a new therapy. There are many possible reasons for this failure, including poor design of clinical trials, mismatch between preclinical and clinical protocols, and insufficient preclinical data. The enzyme caspase-1 has been implicated in neuronal death. Deletion of the caspase-1 gene, or administration of partially selective inhibitors, reduces neuronal injury induced by cerebral ischemia in rodents. We report here, for the first time, that VRT-018858, the non-peptide, active metabolite of the selective caspase-1 inhibitor pro-drug, pralnacasan, markedly reduced ischemic injury in rats. VRT-018858 was neuroprotective when delivered at 1 and 3 h (42% and 58% neuroprotection, respectively) but not 6 h after injury, and protection was sustained 7 days after the induction of ischemia (66% neuroprotection). These data confirm caspase-1 as an important target for intervention in acute CNS injury, and propose a new class of caspase-1 inhibitors as highly effective neuroprotective agents. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain; Caspase-1; Cerebral ischemia; IL-1
1. Introduction Caspases are a ubiquitously expressed family of cysteine proteases that are central to both apoptotic cell death and pro-inflammatory cytokine maturation. Caspase-1 was originally named interleukin-1b (IL-1b) converting enzyme (ICE), because it was identified as the protease responsible for maturation of inactive pro-IL-1b into an active IL-1b molecule (Thornberry et al., 1992). Caspase-1 is also essential for the cellular release of IL-1b and IL-1a, with the effects on IL-1a * Corresponding author. Tel.: þ44 161 275 5357; fax: þ44 161 275 5948. E-mail address: [email protected] (N.J. Rothwell). 1 Present address: Health and Safety Laboratory, Harpur Hill, Buxton SK17 9JN, UK. 2 Present address: AstraZeneca, Alderley Edge, Cheshire SK10 4TG, UK. 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.07.015
probably occurring through an indirect mechanism (Brough et al., 2003). Other caspase-1 substrates include the cytokines IL-18 (Gu et al., 1997), IL-1F7 (a new member of the IL-1 family, with unknown function; Kumar et al. (2002)), IL-33 (Dinarello, 2005) and a plasma membrane Ca2þ-ATPase (PMCA2; Schwab et al. (2002)). The caspase-1 substrate IL-1b is implicated in neurodegeneration, principally by acting through the type I IL-1 receptor (IL-1RI), and is reported to mediate diverse forms of experimental brain injury (Allan et al., 2005). The naturally occurring IL-1RI antagonist, IL-1 receptor antagonist (IL-1RA), is potently neuroprotective (Allan et al., 2005), and has been tested in phase II clinical trials in stroke (Emsley et al., 2005). However, IL-1RA is a large protein and selective, brain-penetrable caspase-1 inhibitors may offer therapeutic advantages.
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
Intracerebroventricular (icv) administration of the peptide N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) or zVAD-DCB, a pan-caspase inhibitor, significantly reduces ischemic brain damage in rats (Loddick et al., 1996) and improves functional outcome (Hara et al., 1997). A more selective caspase-1 inhibitor, based on the sequence Tyr-Val-Ala-Asp (YVAD), reported to inhibit the activity of caspases 1, 4, 5, 8 and 10 (Garcia-Calvo et al., 1998), similarly reduces brain injury after experimental, permanent focal ischemia in the rat (Rabuffetti et al., 2000). Together with the data from transgenic mice (Schielke et al., 1998), these inhibitor studies suggest that caspase-1 could be a major therapeutic target for reducing ischemic brain damage. The aim of this study was to test the effects of an active caspase-1 inhibitor, VRT-018858, the metabolite of the orally available pro-drug, pralnacasan (Rudolphi et al., 2003), on ischemic brain damage in the rat. We show that this caspase-1 inhibitor was protective, even when administered 3 h after induction of ischemia (58% neuroprotection), and that protection was sustained for up to 7 days (66% neuroprotection when administered at the onset of ischemia). 2. Materials and methods 2.1. Animals Experiments were conducted using male, SpragueeDawley rats (Charles River UK), in accordance with the UK Animals (Scientific Procedures) Act (1986). Surgical procedures were performed on animals under halothane anaesthesia (Fluothane, Zeneca, UK, 1e2% in a mixture of oxygen and nitrous oxide [1:2 ratio]). Body temperature was maintained at 37.0 0.5 C throughout anaesthesia by means of a homeothermic blanket.
2.2. Reagents General reagents were obtained from SigmaeAldrich (Poole, UK), unless stated otherwise. VRT-018858 was provided by Vertex Pharmaceuticals Inc. (Cambridge, USA).
2.3. Induction of cerebral ischemia Transient focal cerebral ischemia was induced in rats by unilateral middle cerebral artery occlusion (tMCAo) using the intraluminal thread method (Longa et al., 1989). Briefly, the right common carotid artery was exposed, and a poly-L-lysine-coated nylon monofilament thread was inserted into the external carotid artery and then into the internal carotid artery until resistance was felt (approximately 20 mm), at the point where it blocked the MCA. Laser Doppler flowmetry (Moor Instruments, UK) was used to monitor cerebral blood flow (CBF) during the surgical procedure and for 5 min after MCAo. Animals were allowed to recover from anaesthesia under a heating lamp. One hour after tMCAo, animals were briefly re-anesthetised and the thread withdrawn to permit reperfusion of the MCA. To permit icv administration of VRT-018858, guide cannulae were implanted stereotactically into the left lateral ventricle of the brain, 3e7 days before MCAo. Animals were randomised and injected icv with VRT-018858 as follows: (a) doseeresponse experiment: 2.5, 5, 10 or 20 mg VRT-018858 in 3 ml of 50 mM TriseHCl (pH 7.4) or vehicle (50 mM TriseHCl) immediately after ischemia, and again immediately after reperfusion (total dose 5, 10, 20 or 40 mg VRT-018858); (b) effect of VRT-018858 7 days after MCAo: 10 mg in 3 ml of 50 mM TriseHCl or vehicle (50 mM TriseHCl) immediately after ischemia and immediately after reperfusion; (c) therapeutic time-window effects: 20 mg VRT-018858 in 3 ml of 50 mM TriseHCl or vehicle (50 mM TriseHCl) 1, 3 or 6 h after MCAo. Investigators were blinded to treatments.
639
Animals were killed 24 h (a and c) or 7 days (b) after MCAo, and the brains were frozen on dry ice.
2.4. Data analysis Brain damage was assessed histologically, on a series of coronal sections (20e30 mm thick), at 390 or 500 mm intervals throughout the brain, stained with cresyl violet. Areas of dead tissue were clearly visualised by a complete lack of staining. Image analysis software (SigmaScan 5.0, SPSS Science, UK) was used to measure lesion volume. Lesion volumes were corrected for the effect of brain edema on lesion area. Brain edema was calculated as the difference between the ipsilateral and contralateral hemisphere volumes, divided by the contralateral hemisphere volume and expressed as a percentage of the contralateral hemisphere volume. All data are expressed as mean SD. Significant differences in lesion volumes between treatments were determined using analysis of variance (ANOVA) followed by Bonferroni post-hoc test for the doseeresponse study (a) and Student’s unpaired t-test for all other comparisons.
3. Results 3.1. Neuroprotective effect of VRT-018858 in transient MCAo Transient MCAo for 60 min induced reproducible cortical and sub-cortical (striatal and thalamic) lesions, analysed 24 h after reperfusion. The highest three doses of VRT-018858 (icv) caused a significant reduction in the total lesion volume compared to vehicle (206 58 mm3): 5 mg, 37% protection, NS; 10 mg, 46% protection, p < 0.001; 20 mg, 65% protection, p < 0.001 and 40 mg, 40% protection, p < 0.05 (Fig. 1A). The greatest effects of neuroprotection were observed in the cortex similar to our previously published work on IL-1RA (Mulcahy et al., 2003). Reductions in CBF at the time of tMCAo were comparable in all groups of animals and there was no significant difference between the body temperatures of vehicle- and VRT-018858-treated animals (data not shown). Within the first 60 min after tMCAo and drug treatment, VRT-018858 had no significant effect on blood gases recorded in a small number of animals under chronic anaesthesia (Table 1). 3.2. Sustained neuroprotection by VRT-018858 To investigate the effects of VRT-018858 on the development of brain damage after tMCAo, the drug was given in two 10 mg doses, the first immediately after tMCAo and the second immediately after reperfusion, and the lesion volumes were measured 7 days later. VRT-018858, compared to vehicle, caused a significant reduction in lesion volume (66%, p < 0.001, Fig. 1B). Reductions in CBF at the time of tMCAo were comparable in both groups (data not shown). There was no significant difference between the body temperatures of vehicle- or VRT-018858-treated animals at the time of reperfusion (data not shown). 3.3. Delayed administration of VRT-018858 is neuroprotective To characterise the therapeutic time window for VRT018858 administration, a single icv injection of VRT-018858
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
640
Lesion volume (mm3)
A
300
200
5 µg
Table 1 Arterial blood gas parameters of animals given VRT-018858 or vehicle at tMCAo ischaemia
10 µg
Time-point
Vehicle
20 µg
***
40 µg
*
*** 100
Partial pressure of O2 (kPa) Partial pressure of CO2 (kPa) pH
Control
VRT-018858
Pre-administration
60 min
Pre-administration
60 min
18 2
18 1
19 2
19 2
5 0.5
7 0.4
5 0.2
7 0.4
7.33
7.2
7.34
7.2
Animals underwent femoral artery cannulation followed by tMCAo and administration of VRT-018858 (20 mg) or vehicle icv. Data are mean SD. There were no significant differences between the groups. Statistical comparison was by ANOVA.
0
VRT-018858
Lesion volume (mm3)
B
250
200
Vehicle VRT-018858
150
100
***
50
0
Fig. 1. Effects of different doses of VRT-018858 on total lesion volume (A), and a sustained protective effect (B) after transient ischemic brain injury. (A) Animals were injected icv with vehicle (3 ml, n ¼ 17) or VRT-018858 (2.5 mg, n ¼ 17; 5 mg, n ¼ 19; 10 mg, n ¼ 12 or 20 mg, n ¼ 10) immediately after tMCAo (60 min) and immediately after reperfusion (total dose injected ¼ 5 mg, 10 mg, 20 mg or 40 mg). Total lesion volume was measured 24 h after tMCAo. Data are presented as mean SD of a group of animals. *p < 0.05, ***p < 0.001 compared to vehicle-treated group (ANOVA with Bonferroni post-hoc). (B) Animals were subjected to tMCAo and injected icv with vehicle (n ¼ 8) or VRT-018858 (10 mg, n ¼ 7) immediately after tMCAo and immediately after reperfusion (total dose 20 mg). Total lesion volume was measured 7 days after tMCAo. ***p < 0.001 compared to vehicle-treated group (Student’s unpaired t-test).
(20 mg) or vehicle was administered 1, 3 or 6 h after tMCAo (i.e. 0, 2 or 5 h after reperfusion). Damage was assessed 24 h after tMCAo. VRT-018858 significantly reduced the lesion volume when administered 1 h (42%, p < 0.05) or 3 h (58%, p < 0.001) after tMCAo (Fig. 2A, B), compared to the vehicle-treated group, but had no significant effect when administration was delayed until 6 h after tMCAo (Fig. 2C). The greatest effects of neuroprotection were observed in the cortex (data not shown). Reductions in CBF at the time of tMCAo were comparable in all groups of animals (data not shown). There was no significant difference between the body temperatures of vehicle- or VRT-018858-treated animals (data not shown). 4. Discussion The objective of the experiments described here was to determine the role of caspase-1 in ischemic brain damage using
a low molecular weight inhibitor of caspase-1, and to assess the therapeutic potential of the compound. VRT-018858 is potently selective for group I or inflammatory caspases, with Ki values against caspase-1 and caspase-4 of 1.3 nM and 0.4 nM, respectively (Ku et al., 2001). VRT018858 exhibits >100-fold selectivity for caspase-1 and caspase-4 against other caspases (Ku et al., 2001). Although the substrates for human caspase-4 remain to be identified, it is suggested to be an orthologue of murine caspase-11, which is important for the activation of caspase-1 (Martinon and Tschopp, 2007). Therefore, we suggest that the neuroprotective effects of VRT-18858 reported here are due to inhibition of caspase-1-mediated IL-1b production and inflammatory responses. VRT-018858 caused marked inhibition of ischemic brain injury after transient focal cerebral ischemia when administered icv immediately or after a delay of 3 h, but failed to influence body temperature or carotid blood flow. Many potentially promising neuroprotective agents that have been evaluated in studies with experimental animals have failed in clinical trials (Kidwell et al., 2001). The reasons for these failures are diverse and complex, but include insufficient animal studies, undesirable side effects and sub-optimal design of clinical trials (Stroke Therapy Academic Industry Roundtable (STAIR, 1999)). Key issues for preclinical studies of neuroprotection in ischemic brain damage are the ‘‘therapeutic window’’ and the duration of the effect. We show here that VRT-018858 is effective when first administered icv 3 h after temporary MCAo (58% reduction in total lesion volume), but the neuroprotective effect is lost when administration is delayed until 6 h after ischemia (Fig. 2). Protection is maintained 7 days after VRT-018858 treatment (66% reduction in total lesion volume) (Fig. 1B), indicating that this compound prevents rather than delays the development of ischemic brain injury. Previous studies have reported that non-specific caspase inhibitors attenuate ischemic brain damage (Loddick et al., 1996; Rabuffetti et al., 2000) and injury is also reduced in mice in which the caspase-1 gene is deleted (Schielke et al., 1998). However, to date no studies have reported on a highly selective caspase-1 inhibitor. The primary actions of caspase-1 are to cleave the inactive pro-IL-1b to an active form (Thornberry et al., 1992) and to
J. Ross et al. / Neuropharmacology 53 (2007) 638e642
Lesion volume (mm3)
A
300
200
Vehicle VRT-018858
*
100
Lesion volume (mm3)
300 Vehicle VRT-018858
200
***
Lesion volume (mm3)
This work was funded in part by the Medical Research Council (NR, RG, DB), The Royal Society (SL) and Salford Royal Hospitals NHS Trust (JR). The authors are grateful to Deborah Bentley for assistance with preparation of the manuscript. References
100
0
C
the effects of VRT-018858 are primarily anti-inflammatory. However, VRT-018858 may have significant advantages over IL-1RA, since caspase-1 cleaves a number of other proteins that may contribute to ischemic cell death, including several other cytokines, PMCA2 (Schwab et al., 2002), and calpistatin, the endogenous calpain inhibitor (Vaisid et al., 2005), and may therefore have several mechanisms of action in neuronal injury. Thus, the neuroprotective effects of VRT-018858 reported here suggest that caspase-1 is an attractive target for therapeutic intervention. Acknowledgements
0
B
641
300
Vehicle VRT-018858
200
100
0
Fig. 2. Effects of delayed administration of VRT-018858 on total lesion volume when administered 1 h (A), 3 h (B) and 6 h (C) after transient ischemic brain injury. Animals were subjected to tMCAo and injected icv with vehicleor VRT-018858 (20 mg) either 1 (vehicle n ¼ 9, VRT-018858 n ¼ 9, A), 3 (vehicle n ¼ 8, VRT-018858 n ¼ 9, B) or 6 h (vehicle n ¼ 8, VRT-018858 n ¼ 9, C) after tMCAo. Total lesion volume was measured 24 h after tMCAo. *p < 0.05, ***p < 0.001 compared to vehicle-treated group (Student’s unpaired t-test).
facilitate its cellular release (Brough et al., 2003), and its activity is also important for the cellular release of the related IL-1a (Li et al., 1995; Brough et al., 2003). IL-1 has been strongly implicated in ischemic brain injury and genetic ablation of both IL-1a and IL-b genes results in a marked inhibition (70%) of ischemic brain injury (Boutin et al., 2001). In addition, the selective endogenous IL-1 antagonist, IL-1RA, also markedly limits ischemic brain injury (Relton and Rothwell, 1992; Allan et al., 2005). Thus, we suggest that
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