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Test/Model Listings


  1. Standard Screening:

    TEST 1 - “Qualitative Mice (I.P) - Anticonvulsant Identification
    TEST 2 - “Qualitative Rats (P.O.) - Anticonvulsant Identification”
    TEST 3 - “Quantification Rats (P.O.) - Anticonvulsant Quantification”
    TEST 4 - “Quantification Mice (I.P.) - Anticonvulsant Quantification”
    TEST 5 - “Quantification Mice (P.O.) - Anticonvulsant Quantification”
    TEST 6 - “Differentiation Mice (I.P.) - Anticonvulsant Differentiation”
    TEST 7 - “6 Hz (Mice I.P.) - Mice Anticonvulsant Quantification”
    TEST 8 - “ID Rat (I.P.) - Anticonvulsant Identification”
    TEST 9 - “Tox Rat (I.P.) - Toxicity Screen”
    TEST 10 - “Quant Rat (I.P.) - Anticonvulsant Quantification”
    TEST 11 - “T/H (I.P.) - Toxicity and/or Hippocampal Kindling Screen (Rats)”
    TEST 12 - “ADT - Afterdischarge Threshold in Hippocampal Kindled Rats”
    TEST 13 - “Full Hippocampal (I.P.) - Hippocampal Kindled Rats”
    TEST 14 - “TTE (I.P.) - Threshold Tonic Extension (Mice)”
    TEST 15 - “I.V. Met - Metrazol Threshold”
    TEST 16 - “FRINGS - Audiogenic Quantification (Mice I.P.)”
    TEST 17 - “Mechanisms - Electrophysiology Studies”
    TEST 18 - “Flexible Format (Identification / Quantification)”
    TEST 19 - “P450 - Human Cytochrome P450 Inhibition Studies”
    TEST 21 - “Ames Mutagenicity Assay” – No longer utilized
    TEST 22 - “Formalin Test (Mice I.P.)”
    TEST 23 - “Sciatic Ligation Model in Rats”
    TEST 24 - “In Vitro Slice Electrophysiology Studies”
    TEST 25 - “Lamotrigine Resistant Amygdala Kindled Rat”
    TEST 26 - “Corneal Kindling (Focal Seizures)”
    TEST 28 - "In vitro Metobolic Gene Induction Studies"
    TEST 29 - "Learning & Memory – Morris Water Maze"
    TEST 31 - "Qualitative Mice (I.P) - Anticonvulsant Identification"

  2. Counter Measure Screening:

    TEST 71 - “Prevention of Pilocarpine-induced Status, Rats”
    TEST 72 - “Pilocarpine-induced Status - Acute Intervention, Rats”
    TEST 75 - “Status Electrophysiology”
    TEST 76 - “In Vitro Hippocampal Slice Culture Neuroprotection Assay”

  3. Other Special Studies:

    1) Evaluative Consideration for Unique Chemical Classes
    2) Pilot and Proof of Principle Studies
    3) Antiepileptogenic Study Designs (Chronic 24/7EEG Monitoring)
    4) New Model Development
    5) Model Validation

  4. Model Listings

    Standard Anti-Ictal Screening
    Models of Resistance Epilepsy
    Identification/Differentiation Purposes
    Screening for Related Indications


Test Descriptions

Test 1

Test 1 is an initial screen for anticonvulsant activity in the MES and scMET models combined with an initial assessment of toxicity in mice via i.p. injection. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 2

Test 2 is an initial screen for anticonvulsant activity in the and models combined with an initial assessment of toxicity in rats with oral drug administration. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 3

Test 3 is designed to quantify the anti-convulsant and toxic effects of a test compound in rats with oral dosing. The time to peak effect (TPE) is measured first at a fixed dose. The data for each time point is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. The ED50 for , and toxicity are measured at the TPE, and descriptive statistics are found in the 'ED50 Values' Section. The raw data used to determine these values are found under 'ED50 Biological Response'. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 4

Test 4 is designed to quantify the anti-convulsant and toxic effects of a test compound in mice after i.p. injection. The time to peak effect (TPE) is measured first at a fixed dose. The data for each time point is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. The ED50 for , , and toxicity are measured at the TPE, and descriptive statistics are found in the 'ED50 Values' Section. The raw data used to determine these values are found under 'ED50 Biological Response'. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 5

Test 5 is designed to quantify the anti-convulsant and toxic effects of a test compound in mice after oral dosing. The time to peak effect (TPE) is measured first at a fixed dose. The data for each time point is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. The ED50 for MES, scMET, and toxicity are measured at the TPE, and descriptive statistics are found in the 'ED50 Values' Section. The raw data used to determine these values are found under 'ED50 Biological Response'. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 6

Test 6 is designed to quantify the anti-convulsant effects of a test compound in the scBic and scPIC models. Mice treated orally with the test compound are tested at the previously determined TPE, and an ED50 may be determined accompanied by descriptive statistics found in the 'ED50 Values' section. The raw data used to determine these values are found under 'ED50 Biological Response'. The data for each dose is presented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 7

Test 7 is designed to quantify the effects of a test compound in the 6Hz model of epilepsy. Mice treated by i.p. injection with the test compound are tested at the previously determined TPE, and an ED50 may be determined accompanied by descriptive statistics found in the 'ED50 Values' section. The raw data used to determine these values are found under 'ED50 Biological Response'. The data for each dose is presented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 8

Test 8 is an initial screen for anticonvulsant activity in the MES and scMET models combined with an initial assessment of toxicity in rats via i.p. injection. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 9

Test 9 is an assessment of toxicity in rats via i.p. injection. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 10

Test 10 is designed to quantify the anti-convulsant and toxic effects of a test compound in rats after i.p. injection. The time to peak effect (TPE) is measured first at a fixed dose. The data for each time point is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. The ED50 for MES, scMET, and toxicity are measured at the TPE, and descriptive statistics are found in the 'ED50 Values' Section. The raw data used to determine these values are found under 'ED50 Biological Response'. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 11

Test 11 is a preliminary screen for efficacy of a test compound in the kindled rat model. Two animals are tested both before and after i.p. injection of the test compound. The data represent the seizure score (on a scale of 0 to 5, see below) and the afterdischarge duration. Both measurements are given as a range of observed values.

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Test 12

Test 12 is designed to test the effect of a test compound (administered via i.p. injection) on the afterdischarge threshold in the kindled rat model at a variety of doses and times. The seizure score and afterdischarge duration may be noted. Checked boxes indicate a value is significantly different from control.

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Test 13

Test 13 is designed to quantify the anti-convulsant effects of a test compound in the kindled rat model. Rats treated by i.p. injection with the test compound are tested at the previously determined TPE, and an ED50 may be determined accompanied by descriptive statistics found in the 'ED50 Values' section. The raw data for the ED50 determination is located under 'Dose Response,' where the data for each condition is presented as N/F, where N=number of animals protected (or toxic) and F=number of animals tested. The data for each dose and time point (including the average seizure score (0-5, see below) and afterdischarge duration ± SEM) is presented under 'Time Course'.

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Test 14

Test 14 is designed to measure a compound’s efficacy in the TTE test. Mice treated by i.p. injection with the test compound are tested at the previously determined TPE, and an ED50 may be determined accompanied by descriptive statistics found in the 'ED50 Values' section. The raw data used to determine these values are found under 'ED50 Biological Response'. The data for each dose is presented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 15

Test 15 is designed to quantify a compound’s ability to shift the dose-response curve of Metrazol in the IV Metrazol model (see below). The raw data (shown under 'Response') gives relevant information for each animal tested, including the drug dose and the times to the first twitch and to clonus. These times are also converted to mg/kg of Metrazol. Under 'Analysis' the raw data is compiled to give the mean, standard error, and P value for each group.

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Test 16

Test 16 attempts to quantify the anti-convulsant effects of a test compound in the Frings model of audiogenic reflex seizures. Mice treated by i.p. injection with the test compound are tested at the previously determined TPE, and an ED50 may be determined accompanied by descriptive statistics found in the 'ED50 Values' section. The raw data used to determine these values are found under 'ED50 Biological Response'. The data for each dose is presented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 17

Test 17 tests the ability of a test compound to inhibit ion channel currents in vitro. For each condition tested, the table lists the drug concentration (μM), the number of cells, the holding potential (mV), and the normalized current. The normalized current is shown as a percentage of the current in untreated control cells. A checked box indicates that the normalized current is significantly different from the controls (p < 0.05).

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Test 18

Test 18 is used for experiments that do not fit our predefined test structure. Information in the header portion lists the species (mouse or rat) and route of administration. In each row, a model (e.g., MES, TOX, BIC) and dose (in mg/kg) is listed. For each time point investigated, the data is presented as N/F, where N=number of animals protected (or toxic for the TOX model) and F=number of animals tested.

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Test 19

Test 19 looks for drug-induced inhibition of some cytochrome P450 enzymes in vitro. For each enzyme isoform, activities are reported in nmol/mg protein/min in bold print. In parentheses below each activity is the percent inhibition relative to the activity determined at the same substrate concentration [S] in the absence of the test compound (i.e. at 0 μM on the first line). When available, an IC50 value (μM) is displayed in bold print in the bottom row for each substrate concentration. If there is sufficient data, the Ki and type of inhibition are displayed below the table.

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Test 21

Test 21 looks for potential mutagenicity using the Ames mutagenicity assay. Each experimental condition is run with duplicate samples (S1 and S2). The data presented on the results sheet are the number of revertant colonies, reported as percent of the positive control (benzo[a]pyrene). An additional positive control (acridine orange) is used to evaluate the efficacy of addition of the NADPH cofactor. The two negative controls appear at the top of the table (no addition and DMSO (vehicle)). If any drug concentration causes a doubling in the number of colonies in the vehicle control, the experiment will be repeated using an n of four to look for significance.

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Test 22

Test 22 tests a compound for inhibition of peripheral neuronal transmission using the formalin test. The results of the formalin test are given both as raw data ('Response') and in an aggregate, analyzed form ('Analysis'). In the Response section, each Trial involves sixteen animals, eight controls given an i.p. injection of vehicle and eight given the compound at a specified dose. Thus, for multiple doses of the compound, there will also be multiple control groups. The data recorded for each animal is the amount of time (s) spent licking the affected hind paw in a two minute period. These two minute periods occur at five minute intervals and continue for 45 minutes. Plotting the time spent licking versus time reveals a characteristic biphasic response. From this plot, we can then determine the area under the curve (AUC) for each animal during both the acute and inflammatory stages. The AUC for each phase is shown in the analysis section of the data sheet for both control and drug-treated animals. The AUC for each drug-treated animal is compared to the average result from the control group, yielding an average percent of control (reported with the SEM and p value). Significant reductions in this number indicate a reduction in licking and, presumably, a reduction of perceived pain.

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Test 23

Test 23 tests a compound for inhibition of peripheral neuronal transmission using the sciatic ligation model.

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Test 24

Test 24 is an In Vitro slice electrophysiology study. Investigational drug is applied at the concentration indicated in the report to evaluate its ability to eliminate spontaneous electrographic seizure-like activity. The results are reported as percentage values compared to control for burst rate and burst duration. Multiple slices were used in experiments. Based on statistics, the corresponding box is checked if the test data is significantly different from control.

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Test 25

Test 25 is a preliminary screen for efficacy of a test compound in the lamotrigine-resistant kindled rat model. A number of animals as indicated in the report are tested after i.p. injection of the test compound. The data represent the seizure score (on a scale of 0 to 5, see below) and the afterdischarge duration. Both measurements are given as an average of observed values. The corresponding values from a control group are also reported. If the test data is significantly different from control, the corresponding box is check.

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Test 26

Test 26 is a preliminary screen for efficacy of a test compound in the corneal kindled mouse model. Dosing and time point are reported. The test data is presented as N/F, where N=number of animals protected and F=number of animals tested. Individual seizure score (on a scale of 0 to 5) and the average seizure score are also reported.

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Test 28

Due to certain enzyme inducers could possibly cause serious drug-drug interaction, Test 28 (the in vitro enzyme induction model) is designed to evaluate if the test compound causes induction of several enzymes such as P-glycoprotein (MDR1), CYP1A2, 3A1, and 3A4. This is a 96-well based study using certain ""reporter"" cell lines with the promoter region connected with the firefly luciferase reporter gene for the above enzymes. The cultured cells will be exposed to a range of concentrations of the test compound for 24 hours and then be evaluated by the luminescenct assay. To avoid any impact caused by the cell number, the final results (fold of induction compared to the untreated control) will be presented after adjusted by cell viability.

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Test 29

Test 29, the Morris water maze model, is designed to assess spatial memory and learning in lithium-pilocarpine treated animals and to identify novel compounds that might ameliorate cognitive deficits and neuronal loss associated with pilocarpine-induced status epilepticus (SE). Pilocarpine-induced SE induces spontaneous seizures, long term deficits in learning, memory and neuronal loss, particularly in the hippocampal subfields CA1, CA3 and dentate hilus (Mello, et al., 1993; Cunha et al., 2009). Test 29 uses a round pool of water in which an escape platform is submerged beneath the surface. The task is to find the hidden platform using only extra maze visual cues (Morris, 1984). The learning of the animal can be assessed by the time it takes to find the platform (latency) over a number of trials. Further, this model is sensitive to the hippocampal damage induced by pilocarpine treatment (Liu et al., 1994). The cognitive assessment tasks and staining for hippocampal cell loss were performed in a blinded study to avoid any bias towards treatment conditions.

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Test 31

Test 31 is an initial screen for anticonvulsant activity in the MES and the 6Hz model of epilepsy combined with an initial assessment of toxicity in mice via i.p. injection. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted.

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Test 71

Test 71 is an initial screen for the pilocarpine induced status prevention (PISP) model. The investigational drug is administered at zero minute right after the first Stage III seizure. The data is resented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted. In addition the average weight change in 24 hours post the first Stage III seizure is reported for protected and non-protected rats respectively. If quantitative tests were performed, under the ED50 section the determinedED50 value and corresponding 95% confidence intervals are reported

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Test 72

Test 72 is a follow-up screen for the pilocarpine induced status prevention (PISP) model. The investigational drug is administered at 30 minutes after the first Stage III seizure. The data is resented as N/F, where N=number of animals protected and F=number of animals tested. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted. In addition the average weight change in 24 hours post the first Stage III seizure is reported for protected and non-protected rats respectively. If quantitative tests were performed, under the ED50 section the determinedED50 value and corresponding 95% confidence intervals are reported.

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Test 75

Test 75, the Status Electrophysiology model, is used to study the effects of test compounds on the electrographic properties of benzodiazepine-resistant status epilepticus (SE). Since nerve-agent exposure causes convulsive SE that becomes resistant to benzodiazepine therapy, the purpose of this test is to screen for compounds that block pilocarpine-induced electrographic seizures when administered at a time when benzodiazepines (e.g. diazepam) no longer block electrographic SE. Diazepam and other benzodiazepines are well-known to block convulsive SE if administered within 15-30 min, but they lose their effectiveness with increasing time after the onset of SE. This model is designed to identify those compounds that do or do not suppress electrographic SE when convulsive SE is blocked. Compounds that are found to block both convulsive and electrographic SE at a time when animals are known to be resistant to the benzodiazepines may offer some potential advantage over existing therapies for the treatment of SE.

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Test 76

Test 76 is an in vitro qualitative assessment of the ability of a test compound to prevent excitotoxic cell death. Organotypic hippocampal slice cultures are treated with N-methyl-D-aspartate (NMDA) or kainic acid (KA) to induce neuronal cell death. Propidium iodide (PI), a fluorescence compound, is used to determine the amount of cell death in the individual slices. Hippocampal slice cultures are treated with the excitotoxin alone, or where indicated, with the excitotoxin and either one or two investigational compounds at the concentrations indicated. If neuroprotection occurs as a consequence of the added compound, slice cultures will have a visibly reduced fluorescent intensity when compared to the slice cultures that have been treated with the excitotoxin alone.

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Models

Standard Anti-Ictal Screening

Qualitative Screens in MES, scMET and Toxcity (rat, mouse, i.p., p.o)

The test is an initial screen for anticonvulsant activity in the MES and scMET models combined with an initial assessment of toxicity. The data for each condition is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted. (See a sample report)

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Quantitative Screens in MES, scMET and Toxcity (rat, mouse, i.p., p.o)

The test is designed to quantify the anti-convulsant and toxic effects of a test compound. The time to peak effect (TPE) is measured first at a fixed dose. The data for each time point is presented as N/F, where N=number of animals protected and F=number of animals tested. For tests of toxicity (TOX), N=number of animals displaying toxic effects. The ED50 for MES, scMET and toxicity (TOX) are measured at the TPE, and descriptive statistics are found in the “ED50 Values” Section. The raw data used to determine these values are found under “ED50 Biological Response”. Codes in the C column refer to comments from the experiment and are defined in the comments section if necessary. Any deaths are noted. (See a sample report)

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Models of Resistance Epilepsy

6Hz psychomotor seizure test (mouse, i.p.) – Test 7

Some clinically useful AEDs are ineffective in the standard MES and scMET tests but still have anticonvulsant activities in vivo. In order to identify potential AEDs with this profile, some compounds may be tested in the minimal clonic seizure (6Hz or ‘psychomotor’) test. Like the maximal electroshock (MES) test, the minimal clonic seizure (6Hz) test is used to assess a compound’s efficacy against electrically-induced seizures but uses a lower frequency (6Hz) and longer duration of stimulation (3s). Test compounds are pre-administered to mice via i.p. injection. Individual mice are challenged with sufficient current delivered through corneal electrodes to elicit a psychomotor seizure in 97% of animals (32 mA for 3s) (Toman et al., 1952). Untreated mice will display seizures characterized by a minimal clonic phase followed by stereotyped, automatistic behaviors described originally as being similar to the aura of human patients with partial seizures. Animals not displaying this behavior are considered protected. Animals may also be evaluated using stimulation of 22 or 44 mA. References

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Focal Seizures in Hippocampal Kindled Rats – Test 11/13

Animals are kindled according to the procedure of Lothman et al. [11]. Briefly, one week after the surgery, the rats are stimulated with suprathreshold trains of 200 µAmps for 10 sec, 50 Hz, every 30 min for 6 hours (12 stimulations per day) on alternate days (4 to 5 stimulus days) until they are fully kindled. One week later the effect of a single dose of test substance (highest dose possible without MMI) given i.p. is assessed on the behavioral seizure score (BSS) and afterdischarge duration (ADD). A group of kindled rats (n = 6-8) are tested at 15, 45, 75, 105, 135, 165, and 195 min after drug administration. Results obtained at the various time points are compared with the last control stimulus delivered 15 minutes prior to drug administration. Thus, each animal serves as its own control. The BSS's are scored according to the following criteria [20]:

  • Stage 1 - mouth and facial clonus
  • Stage 2 - stage 1 plus head nodding
  • Stage 3 - stage 2 plus forelimb clonus
  • Stage 4 - stage 3 plus rearing
  • Stage 5 - stage 4 plus repeated rearing and falling

When a drug treatment is observed to significantly lower seizure score (3 or lower) and decrease afterdisharge, a dose-response study is initiated. The BSS and ADD for each dose is averaged at the TPE, the S.E.M. calculated, and the significance of the difference compared to control values determined. (Significant differences in BSS from control and treated groups are determined by the non-parametric Mann-Whitney U test). The ability of a candidate substance to reduce seizure severity is quantitated by varying the dose between 0 and 100 %, noting those animals having a BSS of 3 or less in the group, and calculating an ED50 by probit analysis.

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Afterdischarge Threshold in Hippocampal Kindled Rats – Test 12

After a one-week recovery period following the surgery, the animals are kindled to a Stage 5 behavioral seizure using a stimulus consisting of a 50Hz, 10 sec train of 1 msec biphasic 200 µA pulses delivered every 30 min for 6 hr (12 stimulations per day) on alternating days for a total of 60 stimulations (5 stimulus days) according to the procedures of Lothman [11] Drug testing is initiated after a one-week, stimulus-free period. On each day of a drug trial, the individual afterdischarge threshold of each rat is determined by increasing the current intensity in a stepwise fashion until the rat displays an electrographic afterdischarge with a duration of at least 4 sec. The initial stimulation is conducted at an intensity of 20 µA. The stimulus is then increased in 10 µA increments every 1 - 2 min until an afterdischarge is elicited. Fifteen minutes after the pre-drug threshold determination, a single dose of the test substance is administered to 2 animals. In this way the animals serves as its own control. The individual rat ADT is then redetermined at varying times (i.e., 1/4, 1, 2, and 4 hr) after drug administration. The seizure score and ADD is also recorded at the ADT. Seizures are scored according to the criteria as previously described by Racine. Individual seizure scores, ADDs, and ADTs are recorded; if 4 or more animals are used (in the case of an active compound) they are then averaged and the group mean and S.E.M. calculated.

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Spontaneous Bursting in Slices from KA-treated Rats (In Vitro Slice Electrophysiology Study) – Test 24

Because many epilepsy patients develop symptoms which are refractory to currently available AEDs, the ASP has added an in vitro model that may identify novel classes of anticonvulsants. This model examines the ability of test compound to block spontaneous electrographic seizure-like activity recorded in combined medial entorhinal cortex (mEC)-hippocampal (HC) brain slices obtained from rats that have been treated with kainic acid (KA). Extracellular field potential recordings made in Layer II of the mEC show spontaneous bursting activity upon introduction of 6mM KCl and 0.1 mM Mg2+ (Jones and Heinemann, 1988; Smith-Yockman et al., 2003) and both the frequency and duration of these bursts are variously affected by both traditional and non-traditional AEDs (Dreier and Heinemann, 1990; Dreier and Heinemann, 1991; Smith-Yockman et al., 2003; Walther et al., 1986; Zhang et al., 1995). KA treatment induces an initial insult resulting in status epilepticus, followed by a sustained latent period that subsequently gives way to the development of spontaneous seizures (Hellier et al., 1998). Thus, these experiments use brain slice preparations obtained from animals undergoing an epileptogenic process to evaluate the potential of novel anticonvulsants.

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Lamotrigine-Resistant Amygdala Kindled Rat Model – Test 25

The Lamotrigine-resistant kindled rat model was recently added to the ASP Program to expedite the discovery of candidate compounds that exert their anticonvulsant action via novel mechanisms or that might prove useful in the treatment of medically resistant seizures. Daily administration of Lamotrigine (LTG, 5 mg/kg) during the kindling acquisition phase does not prevent the development of kindling in the test animals but leads to a LTG-resistant state in the fully kindled rat (Postma et al., 2000). Subsequent studies have confirmed these original findings and extended them to include resistance to carbamazepine but not to valproic acid (Srivastava, 2003). These findings suggest that the presence of the Na+ channel blocker LTG during the epileptogenic process leads to a subsequent resistance to other Na+ channel blockers. Thus, this model may serve as a means to identify compounds which would treat therapy-resistant seizures. Results on the data sheet represent the responses of the LTG-resistant animals only and consist of control values (collected prior to dosing) and values obtained following a given dose of the compound. The average and SEM of the seizure scores and afterdischarge duration are noted, as are the number of animals protected from seizure (defined as a seizure score <3).

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Focal Seizures in Corneal Kindled Mice – Test 26

Mice are kindled electrically with 3 sec stimulation, 8 mA, 60 Hz, and corneal electrodes to a criterion of 10 consecutive Stage 5 seizures (facial clonus and head nodding progressing to forelimb clonus, and finally rearing and falling accompanied by a generalized clonic seizure as described by Racine [20]). Stage 5 is generally reached after twice daily stimulation for 8 days. With continued stimulation once a day, animals usually progress to a reproducible Stage 5 after 10-14 additional days. At least 72 hours after the mice have been kindled, the test substance is administered either i.p. or p.o. and, at the previously determined TPE, each animal is given the electrical stimulus indicated above. Following stimulation, the animals are observed for the presence or absence of the rearing and falling criteria of a Stage 5 seizure. Treated animals not displaying a Stage 3, 4, or 5 seizure are considered protected. The dose of the test substance is varied between the limits of 0 and 100% efficacy, and the ED50 and 95% confidence intervals calculated by probit analysis. Mean values and the S.E.M. are calculated for the length of clonus and seizure duration and p values are determined by the Student's t-test.

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Prevention of Pilocapine-induced Status (rat, i.p.) – Test 71/72

Compounds will be assessed for pharmacological evaluation of potential activity against nerve agents using the pilocarpine model of epilepsy as an introductory screen. This model shares many characteristics with nerve agent induced seizures since both initiation and early expression of nerve agent induced seizures are cholinergic followed by the recruitment of other neurotransmitter systems that serve to reinforce recurring seizure activity progressing to status epilepticus. The pilocarpine models are one of the most recognized animal models of status epilepticus (SE). The main features of the model consist of a large number of spontaneous recurrent seizures as well as development of mossy-fiber sprouting. The model has both acute induced SE and chronic spontaneous seizures. An important feature is the occurrence of spontaneous seizures post administration of the chemoconvulsant. In rodents there is induction of both ictal and interictal activity in hippocampal and cortical regions of the brain. Clinical manifestations include ataxia, akinesia and facial automatisms where symptoms quickly progress to full SE lasting up to twelve hours. This activity can be correlated closely with electrographic changes. Depending of the level of protection observed in the initial qualitative screen a series of evaluations using this chemoconvulsant may be employed to assess certain pharmacological characteristics of candidate compounds.

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Identification/Differentiation Purposes

Timed Intravenous Infusion of Metrazol (i.v. Met) Test – Test 15

The i.v. Met test provides a measure of a test substance's ability to raise or lower seizure threshold. Two doses of the test compound are usually employed in this test, the MES ED50 and the TD50 determined following i.p. quantification testing in mice. Randomly selected mice are injected i.p. 2 minutes apart with either the vehicle or the two test drug doses, maintaining the same order of dosing until 30 mice have been injected. At the previously determined TPE, 0.5% heparinized Metrazol solution is infused at a constant rate of 0.34 ml/min into a lateral tail vein of an unrestrained mouse. Infusion is by means of a Sage syringe pump (model 341A) and a 10 ml B-D plastic syringe connected to a length of No 20 P.E. tubing. A 27 gauge stainless steel needle with the hub removed is connected to the tubing and inserted into a vein, bevel side up, and secured to the tail by a narrow piece of adhesive tape. For the placement of the needle, the mouse is restrained in a cone shaped device with only the tail exposed. The rate selector is set on 4, and the switch is set at ml/min. At the start of the infusion, a hemostat clamped to the tubing to prevent backflow is removed, the infusion started, and two stopwatches started. The time in seconds from the start of the infusion to the appearance of the "first twitch" and the onset of sustained clonus is recorded. The times to each endpoint are converted to mg/kg of Metrazol for each mouse as follows:

mg/kg Met = Infusion time (T) x Rate of infusion (ml/min) x mg Met/ml x 1000 g
60 sec x Weight (W) of animal in g

= T x 0.34 x 5 x 1000 = 28.33 x T = mg/kg of Metrazol 60 x W W

The mean and S.E. for each of the 3 groups and the significance of the difference between the test groups and the control are calculated. An increase in mg/kg to first twitch or to clonus indicates the test substance increases seizure threshold; whereas a decrease indicates that the test substance decreases seizure threshold and is a proconvulsant.

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Subcutaneous Bicuculline (s.c. Bic) and Picrotoxin (s.c. Pic) Tests – Test 6

Bicuculline (Bic) and Picrotoxin (Pic) are convulsants which cause clonic seizures in a manner similar to Metrazol (i.e. by inhibiting GABA-ergic signaling). Bicuculline is a competitive antagonist of the GABAA receptor, while Picrotoxin’s antagonism is non-competitive. Anti-epileptic drugs (AEDs) are not always equally effective in protecting against seizures induced by these three compounds, and it is useful to differentiate novel AEDs pharmacologically. In the scBIC and scPIC tests, Adult male CF No 1 albino mice (18-25 g) are dosed with the test compound via i.p. injection in a volume of 0.01 ml/g of body weight. At the previously determined TPE, the animals are given a subcutaneous injection of either convulsant at the CD97 (a dose of 2.7 or 2.5 mg/kg, for Bic and Pic, respectively). The animals are placed in isolation cages to minimize stress (Swinyard et al., 1961) and observed for the next 30 minutes (for Bic) or 45 minutes (for Pic) for the presence or absence of a seizure. An episode of clonic spasms, approximately 3-5 seconds, of the fore and/or hindlimbs, jaws, or vibrissae is taken as the endpoint. Animals which do not meet this criterion are considered protected.

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Threshold Tonic Extension (TTE) Test - Test 14

The threshold tonic extension test is a nonselective, electroconvulsive seizure model that is usually used only with those compounds that are inactive in the MES and scMet tests. Twenty mice are pretreated i.p. with 100 mg/kg of the test substance (less if Test 1 indicates toxicity at that level). Groups of 4 animals each are tested at 1/4, 1/2, 1, 2 and 4 hr after treatment and are challenged with the CC97 for TTE (12.5 mA for 0.2 sec) delivered through corneal electrodes to elicit a threshold tonic extension seizure [18]. Mice not displaying a hindlimb tonic extension are considered protected. The ability of a test substance to block TTE seizures is quantitated by varying the doses between that which protects 100% of mice and that which protects 0% and quantitating an ED50 by probit analysis.

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Audiogenic Seizure Test (AGS) - Test 16

Male and female Frings audiogenic seizure-susceptible mice (18-30 g) are obtained in-house. Groups of 8 mice each are treated i.p. with varying doses of the test substance. At the time of peak effect as determined in the MES test, individual mice are placed in a round plexigrass jar (diameter, 15 cm; height, 18 cm) and exposed to a sound stimulus of 110 decibels (11 KHz) delivered for 20 sec. Mice are observed for 25 sec for the presence or absence of hindlimb tonic extension. Mice not displaying hindlimb tonic extension are considered protected. The ability of a test substance to block audiogenic seizures can be quantitated by varying the doses between that which protects 100% of mice and that which protects 0%.

The severity of a seizure may be quantitated by assigning a numerical score to the observed response, e.g. no response - 0; wild running for <10 sec - 1; wild running for >10 sec - 2; clonic seizure - 3; forelimb extension/hindlimb flexion - 4; tonic seizure - 5.

This test is model of reflex epilepsy which measures the ability of a test compound to block sound-induced seizures in genetically predisposed mice (Castellion et al., 1965). Male and female Frings mice (18-30 g) in groups of eight mice each are treated i.p. with varying doses of the test compound. At the previously determined time of peak effect, individual mice are placed in a round Plexiglas jar and exposed to a sound stimulus of 110 decibels (11 KHz) delivered for 20 sec. Mice are observed for 25 sec for the presence or absence of hindlimb tonic extension. Mice not displaying hindlimb tonic extension are considered protected. The ability of a test substance to block audiogenic seizures can be quantified by varying the doses between that which protects 100% of mice and that which protects 0%. References

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Mechanisms-Electrophysiology Studies - Test 17

To gain some insight into the potential mechanism of action of the candidate substance, its potential for interaction with voltage- and receptor-gated ion channels was examined using whole-cell patch-clamp electrophysiology techniques with primary neuronal cell cultures. For these studies, the test substance was dissolved in DMSO (50 mM stock) and diluted to make a final concentration of 100 µM. Final DMSO concentration was <1%.

  1. Transmitter-gated Channels

    Whole-cell voltage-clamp recordings obtained from murine cortical neurons in primary culture were used to characterize the possible interaction of test molecule with the NMDA and non-NMDA excitatory neurotransmitter receptors and the GABAA inhibitory receptor. Cortical cells were cultured from 15-gestational-day-old Swiss Webster mouse fetuses and were used 2-3 weeks after plating [1, 2, 3]. Recordings were carried out at room temperature (23° C) according to previously described techniques [4] in a control bathing solution containing 142 mM NaCl, 1.5 mM KCl, 1 mM CaCl2 , 10 mM HEPES, 10 mM glucose, and 20 mM sucrose (320 mOsm, pH 7.4). The bathing solution also contained 200-500 nM tetrodotoxin to block voltage-gated sodium channels. For NMDA receptor-mediated current experiments, the bathing solution contained either 0.1 or 1 µM glycine (a required co-agonist at the NMDA receptor). Whole cell voltage clamp recordings were obtained with an EPC-7 amplifier (List Instruments) using patch electrodes (2-3 MW) filled with an intracellular solution containing 153 mM CsCl, 10 mM EGTA, 10 mM HEPES, and 4 mM MgCl2 (290 mOsm, pH 7.4). Currents were filtered at 5 kHz, digitally sampled at 10 kHz, and acquired on computer using Clampex software (Axon Instruments). Currents were also acquired using a chart recorder.

    Cells were voltage clamped at -70 mV unless otherwise noted. A gravity-fed, three-barreled glass flow pipe was positioned 200-400 µm from the cell through which agonist, agonist plus test compound, and wash solution flowed continuously. A computer-controlled piezo-electric stepper system was employed to determine which solution flowed past the neurons. GABAA receptor currents were evoked using 5 µM GABA; whereas ionotropic glutamate currents were evoked using either 10 µM NMDA or 100 µM kainate. Agonists were applied for 1-5 sec and each application was separated by a 20-30 sec wash period. With this protocol, GABA and glutamate receptor currents were relatively stable for the duration of the recording period.

    The effects of the test molecule on GABA currents were evaluated in the following manner. Following at least three control applications of GABA (5 µM), the cell was perfused three times with test molecule (100 µM) plus GABA (5 µM) for 2-5 sec and then the perfusate was switched back to wash. A minimum of three additional GABA responses were obtained to monitor return of the GABA response to control levels. An identical protocol to that described for GABA was employed to assess the interaction of test molecule with the ionotropic glutamate receptors gated by NMDA (10 µM) and kainate (100 µM).

    The agonist-evoked current values were measured using Clampfit software (Axon Instruments). The effect of test molecule on agonist-evoked currents was determined by comparing the agonist currents in the presence of drug with control agonist responses in the absence of drug.

  2. Voltage-gated Na+ Channels

    The effect of test molecule (100 µM) on voltage-gated Na+ channels was assessed using N1E-115 murine neuroblastoma cells and whole-cell voltage-clamp recording techniques. The N1E-115 neuroblastoma cell line was maintained at 35°C in Dulbecco's modified Eagles Medium supplemented with 5% fetal calf serum, 20 mM HEPES, 80 µg/ml gentamicin and 4 mM glutamine. Prior to electrophysiological studies, cells were plated and incubated for 3-5 days in a differentiation medium similar to above with reduced (2.5%) fetal calf serum and 2% DMSO. Recordings were carried out at room temperature in a bathing solution containing 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 5 mM HEPES. The buffer also contained 0.1 mM CdCl2 and 25 mM tetraethylammonium chloride to block voltage-gated Ca2+ and K+ channels, respectively. Whole cell recordings were obtained using patch electrodes (1-2 MW) filled with the intracellular solution described above. The currents were filtered at 10 KHz and acquired on computer using PClamp 6 (Axon Instruments). Series resistance and capacitive currents were compensated using the internal clamp circuitry. The series resistance was 3-5MW, and 40-50% of the series resistance was compensated. The test compound was applied as described above.

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Human Cytochrome P450 Inhibition Studies - Test 19

The cytochrome P450 enzymes found in the liver are the primary mode of metabolism for many drugs. It is therefore critically important to determine if a potential AED will inhibit one or more of these enzymes, which could lead to drug-drug interactions in vivo. Inhibition of various Cytochrome P450 isozymes can be measured in vitro using commercially available human liver microsomes. The ability of a potential AED to inhibit each cytochrome P450 isozyme-preferred activity has been examined using assays based on those reported in the literature but adapted as necessary to our laboratory equipment and needs. The cytochrome P450 isozymes detected by the ASP include CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. For inhibitory evaluation, human liver microsomes and the test compound under investigation were incubated under aerobic conditions with cytochrome P450 isozyme-selective substrates and excess NADPH at 37°C. Incubations were for time periods over which the reaction in the absence of AED, had been determined to be linear, and proportional to the protein concentration. The human liver microsomes used were selected from a bank of those commercially available to be especially rich in the amount of the cytochrome P450 isozyme under investigation. However, it is important to note that the microsomes contained all the other P450 isozymes in addition, and if the reaction/substrate is less than specific, the other isozymes may have contributed to a minor extent to the enzyme activity determined. Following incubation, the reaction was terminated, an internal standard added, the metabolic products extracted and separated by reverse phase HPLC. Each assay was performed in duplicate and the mean value calculated. The HPLC elutions were monitored by UV absorbance for all CYP reactions except those for CYP2A6, CYP2B6 and CYP2D6 where fluorescence monitoring was employed. Quantification was by comparison with authentic metabolites.

For each enzyme isoform, activities are reported in nmol/mg protein/min in bold print. In parentheses below each activity is the percent inhibition relative to the activity determined at the same substrate concentration [S] in the absence of the test compound (i.e. at 0 μM on the first line). When available, an IC50 value (μM) is displayed in bold print in the bottom row for each substrate concentration. If there is sufficient data, the Ki and type of inhibition are displayed below the table.

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Screening for Related Indications

Formalin Test (mouse, i.p) - Test 22

It tests a compound for inhibition of peripheral neuronal transmission using the formalin test. The results of the formalin test are given both as raw data (“Response”) and in an aggregate, analyzed form (“Analysis”). In the Response section, each Trial involves sixteen animals, eight controls given an i.p. injection of vehicle and eight given the compound at a specified dose. Thus, for multiple doses of the compound, there will also be multiple control groups. The data recorded for each animal is the amount of time (s) spent licking the affected hind paw in a two minute period. These two minute periods occur at five minute intervals and continue for 45 minutes. Plotting the time spent licking versus time reveals a characteristic biphasic response. From this plot, we can then determine the area under the curve (AUC) for each animal during both the acute and inflammatory stages. The AUC for each phase is shown in the analysis section of the data sheet for both control and drug-treated animals. The AUC for each drug-treated animal is compared to the average result from the control group, yielding an average percent of control (reported with the SEM and p value). Significant reductions in this number indicate a reduction in licking and, presumably, a reduction of perceived pain.

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Sciatic Ligation (rat, i.p) - Test 23

Partial Ligation of the Sciatic Nerve: Animals will be anesthetized with sodium pentobarbital and the depth of anesthesia monitored by their response to a tail pinch and observation of the depth of respiration. Sterile technique will be used throughout the surgery. The upper thigh will be shaved and wiped off with ethanol and betadine. A small incision will then be made in the skin. The underlying muscle of the upper thigh will be separated and the sciatic nerve exposed. The nerve is separated from the surrounding connective tissue and slightly elevated by a pair of fine, curved forceps. Approximately 1/3 to 1/2 of the nerve is tied off by passing a needle (7.0) and nylon suture through the nerve. The muscle and skin incision are closed off separately with 5.0 suture and the animals kept warm until they have recovered from the anesthesia. This procedure is routinely done on the right side (ipsilateral) while a sham surgery is performed on the left hind leg (contralateral). The latter involves a similar procedure with the exception that the sciatic nerve on this side is only exposed. The rats will be closely monitored daily for the development of infection or untoward effects of the surgery in which case the animals will be immediately euthanized. After an appropriate time for recovery (7 days) the animals will be tested for the development of mechanical allodynia (abnormal response to a non-noxious stimulus). The animals are each put in a bottomless plexiglass box placed on a wire mesh (1/4") platform. After 30 - 60 minutes in which to acclimate, a baseline mechanical sensitivity is determined. This procedure is done by applying a series of calibrated Von Frey fibres perpendicularly to the plantar surface of each hind paw and holding it there for about 6 secs with enough force to slightly bend the fibre. After a positive response (withdrawal of the foot) is noted a weaker fibre is applied. This is repeated until a 50% threshold for withdrawal can be determined. The allodynic threshold is then redetermined after intraperitoneal administration of an investigational AED. Testing will be conducted at the time-to-peak effect of the AED as determined in the acute seizure model.

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Hippocampal Slice Culture Neuroprotection Assay (in vitro) - Test 76

Introduction

Organotypic hippocampal brain slice cultures have been gaining importance as an early stage drug screening tool in the neuroprotection arena (for review, see Noraberg et al., 2005). It is possible, with the use of propidium iodide (PI), to quantitatively assess neuronal cell death in slice cultures following treatment with the excitotoxic agents, kainic acid (KA) or N-methyl-D-aspartate (NMDA). PI binds to the DNA of dead and dying cells and fluoresces. Using this assay, we can evaluate the ability of novel compounds to prevent such excitotoxic cell death when these compounds are added either in conjunction with, or following, treatment with agents that induce excitotoxic cell death.

Methods

Hippocampal slice-culture preparation

Slice cultures are prepared as previously described 1, 2. Briefly, Sprague-Dawley rat pups (10-11 days) are anesthetized with pentobarbital, sacrificed, and their brains rapidly removed and placed on sterile filter paper. The brain is kept moist with sterile filtered dissection buffer. The brain is then bisected sagitally and the brainstem removed, revealing the cortex and underlying hippocampus. The tissue is then placed on a McElwain tissue chopper plate and then sliced into 400 um thick sections. Slices are placed into a small Petri dish and the hippocampi are then separated under microscopic control. Sections are then transferred into a six well culture plate containing a membrane insert (30 mm, Millipore). Each well contains four slices and 1 ml of media containing: 50% Opti-MEM, 25% horse serum, 25% Hank’s balanced salt solution and D-glucose (25 mM). The cultures are then placed in an incubator (5% CO2, 95% O2) and maintained at 36o. One day prior to the experiment (14 DIV), the culture medium is replaced with 1.0 ml of serum-free media containing Neurobasal medium, 25 mM D-glucose, 1 mM L-glutamine and 2% B27 supplement (Gibco).

Experimental Design

The initial assay determines if a test compound (100 μM) can prevent excitotoxic cell death induced by treatment with kainic acid (KA) or NMDA in an organotypic HC slice culture.

  1. Experiments are performed in a six-well plate, 4 organotypic hippocampal brain slices per well. After at least 14 DIV, 2 μM propidium iodide (PI) is added to all six wells to determine baseline cell death prior to toxicity experiments. Any slice demonstrating PI fluorescence is excluded from the study. Slices are imaged and photographed for analysis with an Axiovert 200 M Imaging System (Zeiss, Inc). Following this prescreening step, either KA (10 μM) or NMDA (10 μM) are added to all six wells and 100 μM of the test compound is added to three of the wells (n = 12 slices per condition). Slices are treated for 6 hours, at which point treatment is halted by replacing the media. PI uptake at 24 hours post-treatment is then evaluated by again acquiring a fluorescent image of individual slices in each condition 2, 3. At the end of this second imaging session, each slice is treated with glutamate (100 μM) to obtain a measure of total cell death for each slice. This allows us to reduce interslice variability by normalizing the percent of PI uptake (cell death) in each slice as a function of total possible PI uptake (cell death) for that same slice. The Axiovision software package (Zeiss, Inc) is used to calculate the mean optical density for each slice in a region of interest containing the CA1 and CA3 pyramidal cell body layers of the hippocampus. Thus, each slice is prescreened to insure that it is healthy and is then evaluated two times for cell death: once at the 24 hr time point after treatment with the excitotoxins and test compound and then again following glutamate treatment to completely kill the slice. Data is then represented as percent of total possible PI uptake (or cell death) for each slice in each condition. Figure 1 illustrates the type of data acquired and how the data is represented in bar graph form. In the experiment illustrated in Figure 1, NMDA (10 μM) was added to the top three wells of a six well plate (four slices per well) and the selective NMDA receptor antagonist, ((2R)-amino-5-phosphonovaleric acid (APV, 100 μM)) was added with NMDA (10 μM) in the bottom three wells. Significant neuroprotection, as indicated by a reduction in total PI uptake, was observed in slices treated with APV. Statistical differences between groups for the amount of PI uptake are determined by either a Student’s t-test or ANOVA and, where appropriate, with a Dunnett’s Multiple Comparison Post-Hoc test.
  2. If the test compound does not demonstrate significant neuroprotection when coapplied with either KA or NMDA, the compound is not subjected to further testing. If the test compound does exhibit significant neuroprotection in the initial screen, a number of subsequent experiments can then be performed.
  3. Concentration-Response Curve. This series of experiments attempts to determine the IC50 for the test compound in the PI neuroprotection assay. Using the protocol from (a), the test compound is coapplied with KA or NMDA (10 μM) for six hours at the following concentrations: 0, 10, 30, 100, and 300 μM. This is performed in two plates, so that a total of 8 brain slices will be exposed to each concentration of the test compound. Concentration- response curves will be evaluated to derive the IC50 using Probit analysis.
  4. Using the IC50 value obtained from the concentration-response studies (c), a time-dependent series of experiments may also be performed in the neuroprotection assay. The test compound can be applied to the wells at 0 hr, 0.25 hr, 0.5 hr, 1.0 hr, 2.0 hr and 3.0 hr after ΚΑ or NMDΑ (10 μΜ). This will determine how quickly after KA or NMDA administration the test compound must be delivered so as to maintain efficacy. Neuroprotection will be evaluated at 24 hr by assessing for damage with PI.

Interpretation

When organotypic slice cultures are treated for six hours with either KA (10 μM) or NMDA (10 μM), robust cell death occurs in the granule cell layer of the dentate gyrus and in the CA3 and CA1 pyramidal cell layers of the organotypic hippocampal slice preparation (see Figure 1) and this cell death can be quantified with PI. Treatment with agents known to prevent excitotoxic cell death (e.g., APV and MK-801) can significantly reduce the percentage of total PI uptake in a dose-dependent manner. Therefore, if novel compounds can prevent cell death, the amount of total PI uptake will be significantly reduced (see Figure 1). It is important to bear in mind that these experiments are performed in an in vitro cell culture system. Therefore, until confirmed in other experiments, it would be premature to conclude that compounds effective in this system would exhibit neuroprotection against excitotoxic agents in vivo.

APV attenuates NMDA-mediated

Special Capabilities and Studies under Development

  1. Evaluative Consideration for Unique Chemical Classes
  2. Pilot and Proof of Principle Studies
  3. Antiepileptogenic Study Designs (Chronic EEG Monitoring) – Drug cross-over designs
  4. Model Validation

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MES

The MES is a model for generalized tonic-clonic seizures and provides an indication of a compound’s ability to prevent seizure spread when all neuronal circuits in the brain are maximally active.These seizures are highly reproducible and are electrophysiologically consistent with human seizures.For all tests based on MES convulsions, 60Hz of alternating current (50 mA in mice and 150 mA in rats) is delivered for 2s by corneal electrodes which have been primed with an electrolyte solution containing an anesthetic agent (0.5% tetracaine HCL).In our initial screens, mice or rats are tested at various intervals following doses of 30, 100 and 300 mg/kg of test compound given by i.p. injection or through oral dosing (p.o.).Other doses can be used if indicated by previously known pharmacology or to determine an ED50. An animal is considered “protected” from MES-induced seizures upon abolition of the hindlimb tonic extensor component of the seizure (Swinyard et al., 1989; White et al., 1995a; White et al., 1995b). References

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scMET

Subcutaneous injection of the convulsant Metrazol (a GABAA receptor antagonist) produces clonic seizures in laboratory animals. The scMET test detects the ability of a test compound to raise the seizure threshold of an animal and thus protect it from exhibiting a clonic seizure in response to a normally convulsant dose of Metrazol. Animals are pretreated with various doses of the test compound given by i.p. injection or through oral dosing. At various times after dosing with the test compound, the dose of Metrazol which will induce convulsions in 97% of animals (CD97: 85 mg/kg mice or 56.4 mg/kg rats) is injected into a loose fold of skin in the midline of the neck. The animals are placed in isolation cages to minimize stress (Swinyard et al., 1961) and observed for the next 30 minutes for the presence or absence of a seizure. An episode of clonic spasms, approximately 3-5 seconds, of the fore and/or hindlimbs, jaws, or vibrissae is taken as the endpoint. Animals which do not meet this criterion are considered protected. References

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Tox

To assess a compound’s undesirable side effects (toxicity), animals are monitored for overt signs of impaired neurological or muscular function. In mice, the rotorod (Dunham and Miya, 1957) procedure is used to disclose minimal muscular or neurological impairment. When a mouse is placed on a rod that rotates at a speed of 6 rpm, the animal can maintain its equilibrium for long periods of time. The animal is considered toxic if it falls off this rotating rod three times during a 1-min period. In rats, minimal motor impairment is indicated by ataxia, which is manifested by an abnormal, uncoordinated gait. Rats used for evaluating toxicity are examined before the test drug is administered, since individual animals may have peculiarities in gait, equilibrium, placing response, etc., which might be attributed erroneously to the test substance. In addition to MMI, animals may exhibit a circular or zigzag gait, abnormal body posture and spread of the legs, tremors, hyperactivity, lack of exploratory behavior, somnolence, stupor, catalepsy, loss of placing response and changes in muscle tone. References

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Pilocarpine Induced Status Prevention (PISP)

In the Test 71 screen, compounds are assessed for their ability to halt pilocarpine-induced convulsive status epilepticus (SE). The pilocarpine model is a well characterized model of status epilepticus (SE). This model shares many characteristics with nerve agent induced seizures since the seizures that result in both models are cholinergic mediated. Surviving rats display spontaneous recurrent seizures and mossy fibre sprouting. Clinical manifestations following an acute dose of pilocarpine include ataxia, akinesia and facial automatisms. These symptoms quickly progress to full SE which can last up to twelve hours. This activity can be correlated closely with electrographic seizure activity. Depending of the level of protection observed in the initial qualitative screen, a series of quantitative studies may be undertaken to ascertain the median effective (ED50) and median toxic (TD50) doses of the candidate compound.

Pilocarpine Induced Status Prevention (PISP) Model:

  1. Acute Toxicity:

    Acute motor impairment will be assessed following the intraperitoneal (i.p.) administration of doses starting at 100 and 300 mg/kg of the test substance. Individual Sprague Dawley rats are evaluated for acute toxicity over several time points following administration of test drugs (unless there is previously obtained i.p. toxicity data available). The results obtained from this initial study determine whether any dose adjustments may be required. The behavior of the animals are observed closely and recorded over a four hour period. Routinely, a minimum number of four (4) rats, two per dose will be employed in this acute screen. b. Status Intervention:

    To determine if the test substance can halt acute pilocarpine-induced status an initial qualitative efficacy screen is performed. A challenge dose of pilocarpine (50 mg/kg) is administered i.p. and animals observed until the first convulsive (e.g., Stage 3, 4, or 5) seizure (time zero). The seizure severity is determined using the well established Racine scale. At this point a minimally toxic dose of the candidate drug is administered to a group of 8 male albino Sprague Dawley rats (150-180 gm) via the i.p. route of administration. Efficacy is defined by the ability of an investigational drug to halt the further expression of pilocarpine induced convulsive seizures (e.g., Stage 3, 4, or 5). Compounds found to possess significant protection at time zero in T71 (time from the first stage 3, 4, or 5 seizure) are likely to proceed to further evaluation in the sustained status model (Test 72).

    In the Test 72, the investigational drug is administered 30 minutes after the first observed convulsive seizure. This is a more severe test of a candidate’s ability to halt the induced status. Compounds found to possess significant activity in Test 72 (30 minutes) may be advanced for quantification wherein the ED50 and TD50 and corresponding 95% confidence intervals are determined. A minimum of 4 doses with at least 8 rats per dose will be utilized in the quantification study. References

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References

  • Castellion AW, Swinyard EA and Goodman LS (1965) Effect of maturation on the development and reproducibility of audiogenic seizures in mice. Exp. Neurol. 13:206.
  • Dunham MS and Miya TA (1957) A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Amer. Pharm. Ass. Sci. Ed. 46:208-209.
  • Lothman EW and Williamson JM (1994) Closely spaced recurrent hippocampal seizures elicit two types of heightened epileptogenesis: a rapidly developing, transient kindling and a slowly developing, enduring kindling. Brain Res 649:71-84.
  • Morimoto K, Fahnestock M and Racine RJ (2004) Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog Neurobiol 73:1-60.
  • Orlof MJ, Williams HL and Pfeiffer CC (1949) Timed intravenous infusion of Metrazol and strychnine for testing anticonvulsant drugs. Proc. Soc. Exp. Biol. Med. 70:254.
  • Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32:281-94.
  • Swinyard EA, Clark LD, Miyahara JT and Wolf HH (1961) Studies on the mechanism of amphetamine toxicity in aggregated mice. J Physiol 132:97-102.
  • Swinyard EA, Woodhead JH, White HS and Franklin MR (1989) General principles: experimental selection, quantification, and evaluation of anticonvulsants, in Antiepileptic Drugs (R.H.Levy RHM, B. Melrum, J.K. Penry and F.E. Dreifuss ed) pp 85-102, Raven Press, New York.
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  • White HS, Johnson M, Wolf HH and Kupferberg HJ (1995a) The early identification of anticonvulsant activity: role of the maximal electroshock and subcutaneous pentylenetetrazol seizure models. Ital J Neurol Sci 16:73-7.
  • White HS, Woodhead JH and Franklin MR (1995b) General principles: experimental selection, quantification, and evaluation of antiepileptic drugs, in Antiepileptic Drugs (Levy RHM, R.H.; Meldrum, B.S. ed) pp 99-110, Raven Press, New York.
  • Dreier JP and Heinemann U (1990) Late low magnesium-induced epileptiform activity in rat entorhinal cortex slices becomes insensitive to the anticonvulsant valproic acid. Neurosci Lett 119:68-70.
  • Dreier JP and Heinemann U (1991) Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices. Exp Brain Res 87:581-96.
  • Hellier JL, Patrylo PR, Buckmaster PS and Dudek FE (1998) Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 31:73-84.
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  • Smith-Yockman MD, White HS and Wilcox K (2003) Proceedings of the AES Annual Meeting, Boston, Massachusetts. Epilepsia 44 Suppl 9:202.
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  • Zhang CL, Dreier JP and Heinemann U (1995) Paroxysmal epileptiform discharges in temporal lobe slices after prolonged exposure to low magnesium are resistant to clinically used anticonvulsants. Epilepsy Res 20:105-11.
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  • Postma T, Krupp E, Li XL, Post RM and Weiss SR (2000) LTG treatment during amygdala-kindled seizure development fails to inhibit seizures and diminishes subsequent anticonvulsant efficacy. Epilepsia 41:1514-21. Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32:281-94. Srivastava AKea (2003) Proceedings of the AES Annual Meeting, Boston, Massachusetts. Epilepsia 44 Suppl 9:42.
  • Racine, R.J., Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph. Clin. Neurophysiol., 1972. 32: p. 281-294.
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Last updated September 10, 2013