The matrix metalloproteinase inhibitor marimastat inhibits seizures in a model of kainic acid-induced status epilepticus

An intra-hippocampus injection of kainic acid serves as a model of status epilepticus and the subsequent development of temporal lobe epilepsy. Matrix metalloproteinase-9 (MMP-9) is an enzyme that controls remodeling of the extracellular milieu under physiological and pathological conditions. In response to brain insult, MMP-9 contributes to pathological synaptic plasticity that may play a role in the progression of an epileptic condition. Marimastat is a metalloproteinase inhibitor that was tested in clinical trials of cancer. The present study assessed whether marimastat can impair the development of epilepsy. The inhibitory efficacy of marimastat was initially tested in neuronal cultures in vitro. As a marker substrate, we used nectin-3. Next, we investigated the blood–brain barrier penetration of marimastat using mass spectrometry and evaluated the therapeutic potential of marimastat against seizure outcomes. We found that marimastat inhibited the cleavage of nectin-3 in hippocampal neuronal cell cultures. Marimastat penetrated the blood–brain barrier and exerted an inhibitory effect on metalloproteinase activity in the brain. Finally, marimastat decreased some seizure parameters, such as seizure score and number, but did not directly affect status epilepticus. The long-term effects of marimastat were evident up to 6 weeks after kainic acid administration, in which marimastat still inhibited seizure duration.

Scientific Reports | (2020) 10:21314 | https://doi.org/10.1038/s41598-020-78341-y www.nature.com/scientificreports/ been developed and clinically tested but have failed in clinical trials because of their side effects with chronic treatment [12][13][14][15][16] . The link between MMP-9 and epileptogenesis is supported by several lines of evidence. This enzyme is often excessively produced and released, within minutes to hours, in response to such epileptogenic insults as traumatic brain injury and status epilepticus that is induced by KA 17,18 . Furthermore, aberrant synaptic plasticity has been implicated in the pathogenesis of epilepsy 19 . The role of MMP-9 in synaptic plasticity has been well documented 17,20 . Several studies reported the functional involvement of MMP-9 in epileptogenesis. For example, Wilczynski et al. 21 provided genetic evidence that MMP-9 plays a pivotal role in epileptogenesis, in which MMP-9 knockout mice exhibited a delay in epilepsy development, whereas rats that overexpressed MMP-9 solely in neurons were more prone to develop seizures. Similarly, Pijet et al. 22 recently reported that the development of epilepsy following traumatic brain injury in mice was impaired in MMP-9 knockout mice, the number of seizures in MMP-9 KO mice after traumatic brain injury was significantly reduced and limited to 1-2 of seizures per month. While in MMP-9-overexpressing mice epilepsy development was augmented. The study also showed that increases in MMP-9 activity peaked 6-24 h after traumatic brain injury insult.
Marimastat is a broad-spectrum matrix inhibitor of several MMPs, including MMP-9 13 . Marimastat was the first MMP inhibitor that was tested in clinical trials 23 and shown to impair tumor progression in murine cancer models 13 . It was subsequently used in human patients with pancreatic, lung, breast, colorectal, and gastric adenocarcinoma cancer [24][25][26][27][28][29][30] . It was shown to have a favorable pharmacokinetic profile in humans with oral administration 31 . Importantly, it was well tolerated by patients with short-term treatment. However, longer or more chronic treatment was associated with side effects that eventually precluded its use in cancer treatment 27,[32][33][34] .
The present study investigated whether marimastat impairs seizures in an animal model. We evaluated its blood-brain barrier (BBB) permeability. We also tested whether it inhibits the cleavage of its protein substrate, nectin-3, which is considered to be MMP-9-dependent, to demonstrate the efficacy of marimastat in inhibiting the enzyme in the brain. Finally, using a mouse model of status epilepticus that was produced by an intrahippocampal injection of the glutamate analog KA, we tested whether marimastat can modulate seizure episodes and seizure intensity. Although no marimastat side effects have been recognized in animal models, to provide a translational value to our study and thus to avoid potential side effects that have been described with chronic treatment in humans, we administered marimastat only acutely.

Results
Marimastat inhibits MMP-9 activity in vitro. We first tested the inhibitory effects of marimastat in hippocampal neurons that were cultured in vitro on 7 days in vitro (DIV 7). To determine the minimum effective dose, marimastat was used at the following concentrations: 5 nM, 0.5 μM, 5 μM, 40 μM, and 100 μM. Cells were incubated with marimastat for 30 min in medium supplementation, and then the cultures were treated with glutamate to stimulate neuronal cell activity, leading to the release of MMP-9. Control cultures were treated with either glutamate or glutamate in the presence of MMP-9 inhibitor I (see "Materials and Methods"). MMP-9 activity was assessed by cleavage of the MMP-9 substrate nectin-3 37 . The partial degradation of nectin-3 occurs through proteolytic shedding of the extracellular N-terminal domain and subsequent cleavage of the intracellular domain. We evaluated the basal level of nectin-3 by immunoblotting and the presence of the cleaved fragment (~ 17 kDa) of this protein. As a reference protein, we used β-actin. We found that marimastat inhibited the MMP-9-dependent cleavage of nectin-3 at 0.5 μM, and nectin-3 cleavage was completely inhibited by 5 μM marimastat (Fig. 1).
Marimastat penetrates the blood-brain barrier. We initially tried to test BBB penetration with a low marimastat dose (3 mg/kg b.w.) but did not detect the compound in the brain at this dose. However, we found that marimastat was detectable in the brain tissue at 9 mg/kg b.w. Therefore, we decided to administer, in the following experiments, the drug at this concentration (intraperitoneally) three times during the first 24 h after the KA treatment. Blood plasma and hippocampus samples were collected 30, 60, 90, or 120 min after the injection. We performed quantitative analyses using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode. Marimastat was detected in both plasma and the brain (Fig. 2a). Initially high concentrations of marimastat in plasma at 30 min rapidly decreased over time, whereas its brain concentration remained constant at an average level of 80 ng/g. The brain-to-plasma (B/P) was calculated two ways. First, the B/P ratio was calculated for each post-injection time point separately as the ratio of the marimastat concentration in hippocampal extracts to blood plasma concentration (Fig. 2b). Notably, the B/P ratios for the 90 and 120 min time points exceeded 1 (1.53 and 2.85, respectively), demonstrating higher brain marimastat concentrations than plasma concentrations. Second, the ratio of areas under the kinetic curve (AUC brain /AUC plasma ) of the marimastat concentration in blood plasma and hippocampal tissue was also calculated (Fig. 2c). This ratio was 0.24, which also demonstrated the ability of marimastat to efficiently penetrate the BBB.

Marimastat blocks the MMP-9-dependent cleavage of nectin-3 in vivo.
To investigate the effects of marimastat in vivo, we used a pro-convulsive KA injection, a well-established mouse model of status epilepticus and subsequent epileptogenesis. In this model, KA is administered intraperitoneally, which leads to seizures that are indicative of pro-epileptogenic status epilepticus 38 . To evaluate the inhibitory effect of marimastat on MMP-9, the mice were injected intraperitoneally with marimastat (9 mg/kg) 1 h before KA administration. To assess whether marimastat inhibits MMP-9 in the brain, the hippocampus was isolated 6 h after the KA injection, and an immunoblotting assay was performed to detect the cleavage of nectin-3 protein. We found that Scientific Reports | (2020) 10:21314 | https://doi.org/10.1038/s41598-020-78341-y www.nature.com/scientificreports/ marimastat administration reduced the enzymatic cleavage of nectin-3 by 66%, reflected by the optical density of the 17 kDa nectin-3 fragment (p = 0.0022; Fig. 3).

Marimastat ameliorates seizures in epileptic mice.
The study design is presented in Fig. 4. In this part of the study, we evaluated the therapeutic effect of marimastat on KA-induced seizures. After the KA injection and electrode implantation ( Fig. 4a; for details, see "Material and Methods"), the mice were placed in Plexiglas cages and connected with commutators. Marimastat was injected three times: 30 min, 6 h, and 24 h after the KA injection (Fig. 4b). Video electroencephalography (EEG) was recorded at four stages: 0-2 h, first 24 h, first week (days 2-8), and for 2 weeks between weeks 4 and 6 after the KA injection (Fig. 4b). During the first 2 h, after the KA injection, the seizure number and latency to the first seizure were assessed. At the next stages (24 h, first week, and weeks 4-6), we studied such parameters as seizure duration (in seconds), seizure severity (according to the Racine scale), and seizure number (per animal/per day). Status epilepticus was observed in mice after KA injection during first 24 h after surgery. Mortality of the animals after intra-hippocampus KA administration was around 30% in both the control and marimastat-treated groups. Setting the onset and the end-point of a single seizure was based on EEG recordings and visual inspection. To assign each seizure to relevant point on Racine scale, EEG was compared to the video recording that was registered in parallel. During the first 2 h after KA administration, we observed no effect of marimastat on seizure number (hour 1, p > 0.05; hour 2, p > 0.05) or the latency to the first seizure (Fig. 5a). During the first day after the KA injection, marimastat significantly decreased seizure severity (p = 0.0278; Fig. 5b) and seizure number during the first 24 h (p = 0.0238; Fig. 5b), with no effect on seizure duration (p = 0.3506; Fig. 5b).
In the second part of the experiment, we analyzed data from the next 7 days of EEG recording with regard to the appearance of spontaneous seizures using the same parameters. In contrast to the first 24 h, during the next week of observation, we found a difference only in seizure duration, in which marimastat shortened the duration of a single seizure (p = 0.0053; Fig. 5c). Seizure severity and number did not change (p = 0.6143 and p = 0.0900, respectively; Fig. 5c). To further analyze the therapeutic effect of marimastat on the appearance and parameters of seizures, we also evaluated chronic changes. We evaluated seizure appearance, duration, number, and severity (Fig. 6b). One month after epileptogenesis induction, marimastat inhibited the duration of a single seizure (p = 0.023; Fig. 6a,b). Moreover, marimastat slightly decreased seizure score and number, but these changes did not reach statistical significance (p = 0.1494 and p = 0.1108, respectively; Fig. 6).

Discussion
In the present study, marimastat potently inhibited MMP-9 activity that was evoked by glutamate in hippocampal cultures in vitro, in which it prevented proteolytic cleavage of the MMP-9 substrate nectin-3. Marimastat effectively penetrated the BBB and accumulated in the brain after peripheral administration, demonstrated by MS. Marimastat also inhibited proconvulsive KA-driven MMP-9 activity in the brain, reflected by a decrease in the proteolytic cleavage of nectin-3. Finally, marimastat impaired the development of seizures that were evoked by KA-driven status epilepticus. www.nature.com/scientificreports/ The major goal of the present study was to assess whether marimastat is useful for impairing seizure outcomes that are evoked by KA-induced status epilepticus. Epilepsy is a major, long-lasting, and debilitating complication following various brain insults. The latent period of epileptogenesis that might be supported by aberrant synaptic plasticity is believed to be responsible for the development of epilepsy. MMP-9 has been repeatedly shown to play a pivotal role in both physiological and aberrant synaptic plasticity and shown to contribute to epileptogenesis, in which it is rapidly and transiently activated by pro-epileptogenic insults and functionally involved in epilepsy development 17,36 . Marimastat has been previously tested in clinical trials for the prevention of tumor metastases, but it failed in the trials because chronic treatment resulted in side effects 36 . Therefore, we reasoned that the acute and time-limited administration of marimastat following pro-epileptogenic status epilepticus might be beneficial for preventing epilepsy development [see also 36 ].
In the present study, we first found that marimastat inhibited MMP-9 activity in neurons. Marimastat was previously shown to be a broad-spectrum MMP inhibitor and active against MMP-9, but this notion was not apparently tested in neurons. We also sought to validate inhibitor efficacy by testing substrate cleavage in the brain. A limited number of neuronal MMP-9 substrates have been identified to date. Among these, the partial proteolytic cleavage of β-dystroglycan was previously used as an indicator of MMP-9 activity 39 . Unfortunately, the appropriate antibody for β-dystroglycan is no longer available. Therefore, we chose another MMP-9 substrate, nectin-3 37 . Glutamate treatment, which is known to activate MMP-9, provoked the partial proteolysis of nectin-3, releasing the 17 kDa fragment that was visible by Western blot. The observed kinetics of production of the cleavage product was consistent with the dynamics of MMP-9 activation 37 , and marimastat prevented this cleavage. In previous studies and clinical trials, marimastat was used to block MMP activity in peripheral tissues, although it was also shown to affect gliomas in the brain 40 . However, no rigorous investigations of its permeability of the BBB to reach the brain have been conducted. Thus, we used HPLC-ESI-MS/MS to measure marimastat concentrations in blood plasma and brain tissue at different post-injection time points. HPLC-ESI-MS/ MS is currently the most frequently used analytical method in pharmacokinetic studies that seek to quantify the concentration of chemical substances in different tissues 41,42 , including BBB permeability studies 43 . We recently applied this analytical technique to examine the BBB penetration of a novel compound that inhibits gelatinase activity 44 . The AUC brain /AUC plasma ratio is a useful parameter for assessing the BBB permeability of a compound 45 . Compounds that are characterized by a AUC brain /AUC plasma ratio greater than 0.1 are considered to have sufficient access to the brain. The AUC brain /AUC plasma ratio for marimastat was 0.24, indicating its BBB permeability. The B/P ratio from the pharmacokinetic study was calculated separately for each post-injection time point. In a previous study, diazepam, which is considered to be a highly brain-penetrant compound, B/P ratios are  44 . Diazepam rapidly penetrates the BBB, but it is also quickly removed from both blood and the brain. In contrast, in the present study, plasma marimastat concentrations decreased over time but remained at a constant level in the brain. The following B/P ratios for marimastat were obtained: 0.07, 0.38, 1.54, and 2.85 at 30, 60, 90, and 120 min, respectively. Overall, we found that marimastat efficiently penetrated into the brain following the intraperitoneal injection and preferentially accumulated in brain tissue. In this context, it might be arguable that marimastat accumulates around the neurovasculature, with only limited penetration into brain parenchyma. However, such a phenomenon would not explain nectin-3 cleavage because this protein has been detected in parenchyma.
To test whether marimastat inhibits MMP-9 activity in the brain, we injected KA peripherally in mice. KA was previously shown to markedly activate MMP-9 18,21,45 . Furthermore, KA administration serves as a welldescribed model of status epilepticus-driven epileptogenesis. In the present study, KA administration produced nectin-3 cleavage, indicating an increase in MMP-9 activity, which was diminished by marimastat treatment. This result strongly suggested that marimastat effects were mediated by its ability to inhibit MMP-9. We cannot, however, exclude other marimastat targets. The direct testing of this notion by use of the drug in MMP-9 KO mice, subjected to KA treatment, is not workable, since only a very low number of MMP-9 KO mice undergo epileptogenesis 22 .
The less effective inhibition of nectin-3 cleavage in the brain than in neuronal cultures might be attributable to the complexity of the brain's response to KA, the pharmacokinetics of the penetration of marimastat into the brain, and the relatively low dose of marimastat that was tested, which was used to avoid potential side effects.
To reveal the whole spectrum of marimastat activity against the development of epilepsy, we determined behavioral seizure scores and various EEG features, such as seizure duration, seizure number, and seizure severity. Marimastat was effective against seizure severity and number during first 24 h after the KA injection, whereas marimastat inhibited seizure duration within the next 7 days and weeks.
In conclusion, the present study found that marimastat, a drug that was previously tested in human clinical trials for other disease conditions, may be useful for impairing the development of epileptogenesis and as an adjunct therapy for seizures. Despite the growing interest in addressing epileptogenesis to prevent the fullblown clinical manifestation of the disease, no anti-epileptogenic drug has yet emerged. Furthermore, we found a possible molecular mechanism of action of marimastat, in which it inhibited MMP-9 in the brain that was exposed to glutamate receptor agonist-induced neuronal excitation.   Inhibitor I is piperazine-based, a cell-permeable and highly, potent inhibitor of MMP-9 and MMP-13 (IC50 = 900 pM) but at much higher concentrations it also inhibits MMP-1, MMP-3 and MMP-7. As a control, non-stimulated neurons and cells that were stimulated only with glutamate were used. After stimulation, the cells were lysed (4 × sample buffer with 2-mercaptoethanol). The samples were incubated for 6 min (96 °C). Next, proteins were separated by electrophoresis, and Western blot analysis was performed. For analysis the same volume of each sample was used. For the experiments that evaluated the influence of marimastat on the MMP-9-dependent cleavage of nectin-3, the animals were injected with marimastat. Because of the fact that apparently, no antibody available that recognizes only the cleaved form of nectin-3 (17 kDA) in the brain tissue sections, nectin-3 cleavage was evaluated with use of western blot analysis, as described previously 37 . One hour later, KA was injected intraperitoneally (40 mM), and the mice were observed for the next 6 h for the seizures appearance. Next, the hippocampus was dissected and stored at − 80 °C. After tissue was thawed, the samples were homogenized in buffer that contained 10 mM CaCl 2 , 0.25% Triton X-100, protease inhibitor cocktail (cOmplete Mini EDTA-free; Roche) and centrifuged (6000×g, 30 min, 4 °C). The supernatant was recovered. The pellet (Triton X-100-insoluble) was re-suspended in a buffer that contained 50 mM Tris (pH 7.4) and 0.1 M CaCl 2 in water, incubated for 15 min (60 °C), and centrifuged ( Blood-brain barrier permeability. To evaluate the BBB permeability of marimastat, mice were injected intraperitoneally with marimastat, as described previously 44 . Thirty, 60, 90, or 120 min post-injection, the mice were anesthetized (sodium pentobarbital, 150 mg/kg), and venous blood was collected. The animals were perfused with ice-cold PBS, after which hippocampus tissue samples were collected (n = 5). Blood was centrifuged at 14,000×g for 30 min at RT in the presence of sodium citrate. The brain samples were weighed and homogenized in 500 ml of deionized water. Marimastat was extracted using acetonitrile/water (50/50, v/v, LC/MS grade, JT Baker). 100 µl and 50 µl of hippocampus and plasma samples were added to 500 µl and 250 µl of extraction buffer, respectively. The samples were incubated for 10 min, RT, 800 rpm. After centrifugation (17,500×g, RT, 10 min) pellets were discarded, and supernatants were transferred to LC autosampler vials. The HPLC-ESI-MS/ MS analysis was performed using an Agilent 1290 Infinity LC system coupled to an Agilent 6460 Triple Quadrupole Mass Spectrometer equipped with an ESI source operating in negative polarity. The mass spectrometer was operated in MRM mode following the optimization of working conditions for marimastat using a standard solution at 1 mg/l. The following m/z transitions were monitored: 330. 1% formic acid were used as eluents A and B, respectively. The mobile phase was delivered at 0.5 ml/min in isocratic mode with 50% of eluent B. The injection volume was 10 µl. The quantification of marimastat was achieved by the external matrix calibration curve method. Blank extracts were obtained from untreated animals following the same procedure. The calibration curve for brain aliquots was generated within the concentration range of (0.338-6.75) ng/ml (R 2 = 1). For plasma samples, the calibration curve was generated within two concentration ranges: (6.75-67.5) ng/ml (R 2 = 0.999) and (13.5-135) ng/ml (R 2 = 0.991). Considering the dilution factor and masses of tissues collected, the content of marimastat was calculated per gram or milliliter, respectively. The limit of quantification of marimastat (the lowest point of the calibration curve) was (31.64 ± 1.58) ng/g in brain samples and (20.24 ± 0.89) ng/ml in plasma samples.

Intracranial electrode implantation and electroencephalographic recording. The study design
is presented in Fig. 4b. For the experiments that evaluated the inhibitory effects of marimastat on nectin-3 and seizures, the mice were divided into two groups. Each group received an intra-hippocampus injection of KA (20 mM in 0.9% NaCl). Thirty minutes, 6 h, and 24 h after the KA injection, the mice were injected intraperitoneally with saline/marimastat (n = 6). The animals were anesthetized (domitor 0.5 g/kg; ketamine 8 g/kg, i.p.) and placed in a stereotaxic frame on a heating pad. The mice were injected with 70 nl of KA solution (flow rate: 50 nl/min) in the left CA1 field of the dorsal hippocampus at the following coordinates: anterior/posterior, − 1.8 mm; medial/lateral, + 1.7 mm, dorsal/ventral, − 2.3 mm from bregma. Immediately after the KA injection, four stainless-steel screw electrodes were placed in the skull (∅ 1.6 mm, Bilaney Consultants). Additionally, one bipolar hippocampal electrode (Bilaney Consultants) was placed in the injected hippocampus at the following coordinates: anterior/posterior, − 2.0 mm; medial/lateral, + 1.3 mm from bregma; dorsal/ventral, − 1.7 mm below dura. Cortical recording electrodes were placed bilaterally in the skull: two over the frontal cortex, two (reference electrode and ground electrode) over the cerebellum (Fig. 4a). After surgery, the mice were placed in Plexiglas cages (one mouse/cage) and connected to the recording system with commutators (SL6C, Plastics One, Roanoke, VA, USA). Video EEG was performed using the Twin EEG recording system that was connected to a Comet EEG PLUS with a 57-channel AS40-PLUS amplifier (Natus Medical) and filtered (high-pass filter cut-off 0.3 Hz, low-pass filter cut-off 100 Hz). Video EEG activity was monitored during: the first 2 h after KA administration, the first 24 h, the next 7 days, at weeks 4-6 for 2 weeks (Fig. 4b). The occurrence of seizures was evaluated by visual inspection of the EEG and video recordings. The following parameters were analyzed: seizure number, duration,

Scientific Reports
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