NMDA-receptor

The anticonvulsant impact of antiepileptic medications is enhanced by LY 233053 (0.5 and 5 mg/kg), but it has no effect on the electroconvulsive threshold [1]. When used in conjunction with phenobarbital, fentanyl, or carbamazepine, LY 233053 (5 mg/kg) offers 50% of the maximum shock protection, however it may impair long-term memory [1]. Although LY233053 (intravenous bolus, 100 mg/kg; within 5, 30, or 60 minutes after reversible preset) was protective in preventing cardiovascular injury, New Zealand white rabbits will die if treatment is not started within 60 minutes of the injury [2].

---

Both LY235959 and LY 233053 ( < or = 0.5 and 5 mg/kg, respectively) did not influence the electroconvulsive threshold but potentiated the anticonvulsant action of all antiepileptics studied. The combined treatment of LY 233053 (5 mg/kg) with carbamazepine, diphenylhydantoin, or phenobarbital (providing a 50% protection against maximal electroshock) resulted in the impairment of long-term memory. No adverse effects were observed with combinations of LY235959 with these antiepileptics. The combined treatment of valproate with either LY235959 or LY 233053 was superior to valproate alone, as regards motor impairment, but not the impairment of long-term memory. Neither NMDA-receptor antagonist elevated the total plasma levels of antiepileptic drugs studied. Conclusions: It may be concluded that NMDA-receptor blockade leads to the enhanced anticonvulsive action of conventional antiepileptics against maximal electroshock-induced seizures. A pharmacokinetic interaction does not seem probable. [1]

---

Antagonists of excitatory amino acids appear to serve a neuroprotective role during ischemic conditions in a variety of in vivo and in vitro models. The usefulness of such agents in the clinical setting, however, may be limited by poor central nervous system (CNS) entry and intolerable side effects. The authors report high efficacy in reducing neurological damage and relatively limited side effects of LY 233053, a novel competitive glutamate antagonist, in two models of experimental CNS ischemia in the rabbit. [2]

---

Spinal Cord Ischemia [2]
In the spinal cord ischemia model, 14 control rabbits were subjected to spinal cord ischemia for a range of durations. The mean ET50 ± standard error of the mean of this control group was 25.2 ± 1.5 minutes. The group of 15 animals treated with 50 mg/kg LY 233053 had a mean ET50 of 31.1 ± 1.9 minutes, a statistically significant prolongation from that of control rabbits (t = 2.42). The group of 15 rabbits treated with 100 mg/kg LY 233053 had a mean ET50 of 39.4 ± 2.7 minutes, and the group of 10 animals treated with 200 mg/kg LY233053 had a mean ET50 of 40.9 ± 5.1 minutes. These ET50 values were also significantly prolonged compared to those of the control group (t = 4.62 and t = 2.95, respectively). These results are displayed in Fig. 1, which plots duration of spinal cord ischemia versus functional outcome for these groups.

---

Cerebral Ischemia [2]
Using the multiple cerebral emboli model, 15 rabbits were injected with a range of quantities of microspheres. The mean ES50 ± standard error of the mean of this control group was 0.42 ± 0.05 mg microspheres lodged in the brain. The group of 14 animals that received a single dose of 100 mg/kg LY 233053 had a mean ES50 of 0.32 ± 0.04 mg microspheres, a relative decrease compared to the control value that did not reach statistical significance (t = 1.51). The group of nine rabbits that received a total of 50 mg/kg LY 233053 in divided doses had a mean ES50 of 0.56 ± 0.14 mg microspheres, a relative increase compared to the control value that did not reach statistical significance (t = 0.94). The group of 14 animals receiving 100 mg/kg LY233053 in divided doses had a mean ES50 of 0.58 ± 0.04 mg microspheres. This represents a significant increase in the quantity of microspheres that could lodge in the brain without causing gross neurological deficit, compared to the control group (t = 2.46). These results are displayed in Fig. 2, which plots the quantity of microspheres recovered in the brain versus functional outcome for these groups.

Chimney test [1]
The effects of AEDs alone or in combination witheither LY235959 or LY 233053 on motor impairmentwere quantified with the chimney test of Boissier etal. In this test, animals had to climb backwardup the plastic tube (3-cm inner diameter, 25-cmlength). Motor impairment was indicated by the in-ability of the animals to climb backward up the tubewithin 60 s, and the results were shown as a percent-age of animals that failed to perform the test.

---

Estimation of the plasma levels of AEDs [1]
The animals were administered either saline plusone of the AEDs and LY235959 or LY 233053 plusone of these drugs. The mice were killed by decapita-tion at times scheduled for the convulsive test, andsamples of blood of -1 ml were collected into Ep-pendorf tubes. Samples of blood were centrifuged at 10,000 r/min for 3 min, and plasma samples of 70 pl weretransferred to Abbott System cartridges. The levelsof AEDs were estimated by immunofluorescence,by using an Abbott Tdx analyzer. Plasma levels were expressed asmeans t SD of at least seven determinations. Spinal Cord Ischemia Preparation [2]
New Zealand White rabbits, each weighing 2.5 to 3.0 kg, were anesthetized with halothane prior to invasive procedures. In the rabbit model of spinal cord ischemia, a 10-cm ventral midline incision was used to place a snare ligature around the infrarenal aorta.15 The snare device consisted of large-bore Tygon tubing which housed a loop of thin Tygon tubing to encircle the vessel. One end of the large tubing was exteriorized, allowing easy manipulation of the thin tubing within. Temporary spinal cord ischemia was accomplished by tightening the thin tubing for a predetermined duration, then releasing the snare to allow reperfusion. This ischemic duration was varied among the animals in each experimental group to provide a range of durations for study. Eighteen hours after ischemia, the animals were examined for the presence of paraplegia. A binary scoring system was utilized, with a blinded investigator rating the animals as either paraplegic or not paraplegic (the latter category included animals with minimal motor function).
Within each experimental group, the duration of ischemia producing a 50% probability of paraplegia in animals was termed “the ET50” (effective time = 50%). Three groups of animals were treated with LY 233053 intravenously 5 minutes after the onset of spinal cord ischemia, at doses of 50, 100, or 200 mg/kg. Efficacy of LY 233053 was assessed by shift of the ET50 in treated animals.

---

Cerebral Ischemia Preparation [2]
In the model with multiple cerebral emboli, the right common carotid arteries of rabbits were cannulated with small-gauge polyethylene tubing, and the animals were allowed to recover from anesthesia. A quantity of 50-µm microspheres, radiolabeled with 125I, were weighed and suspended in 100 µl of 0.05% polysorbate (Tween 80). The ratio of specific activity and weight of the microspheres was determined, and the microspheres were then transferred to a 0.5-ml gas-tight syringe for injection through the carotid catheter. The quantity of microspheres injected into each animal within an experimental group was varied to provide a range of microsphere doses for each group. Eighteen hours after injection of microspheres, a blinded investigator rated each animal with a binary scoring system as either functional (alert and able to right itself) or abnormal (dead or having a gross neurological deficit). The animals were then sacrificed, the brains were removed, and the weight of the microspheres that lodged in the brain was determined by measurement of brain specific activity.
Within each experimental group, the dose of microspheres producing a 50% probability of an abnormal rating in animals was termed “the ES50” (effective stroke = 50%). Three groups of animals were treated with LY 233053 by intravenous infusion. One group received a single 100-mg/kg dose 5 minutes after microsphere injection, the second group received 50 mg/kg in divided doses (40% at 5 minutes, 30% at 4 hours, and 30% at 10 hours following microsphere injection), and the third group received 100 mg/kg in divided doses (40% at 5 minutes, 20% at 3 hours, 20% at 6 hours, and 20% at 9 hours following microsphere injection). The therapeutic efficacy of LY 233053 was assessed by shift of the ES50.

---

Physiological Effects [2]
Rectal temperatures were obtained at regular intervals for 2 hours in animals treated with a single injection of 100 mg/kg LY 233053 in the multiple cerebral emboli model. Blood pressure and pulse rate were monitored in animals not subjected to ischemia, following intravenous administration of either 50, 100, or 200 mg/kg LY233053.

The newly developed competitive NMDA antagonist, LY233053 (cis-(±)-4-((2H-tetrazol-5-yl)methyl)-piperidine-2-carboxylic acid), incorporates a tetrazole moiety in its chemical structure. The compound has shown high bioavailability and CNS penetration, with rapid onset and a short duration of effect [2].

Side Effects [2]
The spectrum of side effects associated with a particular glutamate antagonist will have increasing importance as clinical trials of these agents for CNS ischemia are considered. While extrapolation of animal data to humans in this area is exceedingly difficult, it should be noted that LY 233053, in doses that were effective for neuroprotection, had minimal effect on blood pressure and heart rate in these animals. Rectal temperature was also not affected, and altered thermoregulation could not have played a role in the drug's efficacy. The dependence of the drug's sedative effect on the degree of cerebral ischemia was notable. Animals with little or no cerebral ischemia (those animals in the spinal cord ischemia model or those harboring small quantities of microspheres in the model with multiple cerebral emboli) evidenced minimal sedation unless high doses of LY 233053 were administered. In contrast, animals with significant diffuse cerebral injury in the model with multiple cerebral emboli displayed marked sedation after injection of even small doses of the drug, compared to untreated animals in the same model with similar degrees of cerebral injury. This more potent sedative effect may be a peculiarity of the diffuse ischemia of the model and should be explored further in other models of stroke prior to clinical trials. Nevertheless, the period of sedation was much less with LY 233053 than with MK-801, which at comparable neuroprotective doses caused profound sedation for 24 hours or longer in the rabbit.

---

Drug Side Effects [2]
There was no significant effect of LY 233053 on temperature regulation in these animals (Table 1). Mean rectal temperatures slightly increased, but did not vary more than 0.8°F throughout the initial 2 hours after intravenous injection. Blood pressure was similarly unaffected even by the highest dose of LY233053 (Table 1).
An obvious side effect of the drug was sedation, but curiously, the degree of sedation caused by a particular dose was quite model-specific. In the spinal cord ischemia model, 50 mg/kg of LY 233053 had little apparent effect on the animal. Ataxia and mild sedation (that is, the animal was arousable with gentle tactile stimulation) resulted from doses of 100 mg/kg, and substantial ataxia and sedation were associated with doses of 200 mg/kg. However, in rabbits subjected to the diffuse cerebral injury of the model of multiple cerebral emboli, even relatively small doses such as 20 mg/kg LY233053 had pronounced sedative effects (defined as arousable only to vigorous tactile or painful stimulation). Sedation became apparent within 10 minutes after drug administration and persisted for 4 to 10 hours after the last dose, depending on the dose and degree of cerebral ischemia.

[1]. Competitive NMDA-receptor antagonists, LY 235959 and LY 233053, enhance the protective efficacy of various antiepileptic drugs against maximal electroshock-induced seizures in mice. Epilepsia. 1996 Jul;37(7):618-24.

[2]. Efficacy of LY233053, a competitive glutamate antagonist, in experimental central nervous system ischemia. J Neurosurg. 1992 Jan;76(1):106-10.

Our study demonstrated that competitive NMDAantagonists, LY 235959 and LY 233053, exerted ananticonvulsant efficacy, elevating the threshold forelectroconvulsions in mice. Both agents, adminis-tered in subthreshold doses, significantly enhanced the protective activity of valproate, carbamazepine,diphenylhydantoin, and phenobarbital against maxi-mal electroshock-induced seizures. A pharmacoki-netic interaction, in terms of total plasma levels atleast, does not seem probable, because plasma levelsof AEDs remained unchanged in the presence ofboth LY 235959 and LY 233053. [1]

---

This study represents the first investigation of LY 233053 as a therapeutic agent for CNS ischemia, and efficacy was demonstrated in two different experimental models of rabbits. The effect of the drug on preservation of neurological function was dose-dependent in the spinal cord ischemia model, a model of reversible ischemia of the CNS. In the model involving multiple cerebral emboli, a model of irreversible ischemia, efficacy was dependent not only on the total dose but also on the dosing schedule.

Relevance to Previous Studies [2]
Prior investigations into the neuroprotective effect of noncompetitive NMDA antagonists have demonstrated their efficacy using a wide variety of focal and global animal models of neuronal ischemia. In our laboratory, the noncompetitive antagonist MK-801 was shown to display potent therapeutic efficacy using both the spinal cord ischemia and the multiple cerebral emboli models. Experience with competitive antagonists in vivo, however, has until recently been limited to the gerbil or rat global ischemia model, in which most studies have demonstrated hippocampal protection; one study reported preservation of memory. Two recent anatomical studies reported a decrease in infarct size after permanent focal vascular lesion in rats treated with the competitive antagonists AP-77 or CGS-19755. The present study explored the potential of a competitive antagonist for preserving neurological function after both reversible and irreversible focal ischemia. The neuroprotective effects of LY233053 in the spinal cord ischemia and multiple cerebral emboli models were comparable to those of MK-801.

Dose Response [2]
The relationship of efficacy to dose differed in the two models. The mean ET50 increased from the control value of 25.2 to 31.1 minutes in the spinal cord ischemia model when 50 mg/kg LY 233053 was administered 5 minutes after ischemia. When the dose was doubled to 100 mg/kg, the ET50 further increased to 39.4 minutes. Doubling the dose again to 200 mg/kg did not result in any significant further increase in efficacy. Thus, in this reversible model of ischemia, an optimum dose range was found, using a bolus injection of the drug at a single time point following ischemia in each rabbit. The single dose had an adequate duration of effect to protect the spinal cord through the ischemic period until reperfusion occurred. However, in the model involving multiple cerebral emboli (a model producing diffuse irreversible ischemic injury), 100 mg/kg LY233053 given as a single injection did not have a protective effect. Presumably, the short half-life of this drug did not provide sufficient duration of effect when given as a bolus. When this same dose was given as four divided doses over a period of 9 hours, however, significant neuroprotection resulted. Despite these results, we do not believe that we have developed the optimum dosing regimen for LY233053 in this model. Continuous infusion over prolonged periods will likely provide even better neuroprotection than was demonstrated in this investigation.

This study demonstrates the therapeutic benefit of a competitive glutamate antagonist in two models of CNS ischemia using functional outcome as an index of efficacy. We have shown that LY 233053, a novel competitive antagonist with improved bioavailability and rapidity of onset of effect, has a level of efficacy comparable to that of a well-studied noncompetitive antagonist. Finally, in anticipation of emerging clinical trials of glutamate antagonists, we have provided preliminary information on the potential side effects of this drug, a spectrum that appears favorable in these animal models.[2]

C8H13N5O2

211.23

211.107

C, 45.49; H, 6.20; N, 33.16; O, 15.15

125546-04-5

3035974

Typically exists as solid at room temperature

1.398g/cm3

488.9ºC at 760mmHg

249.5ºC

2.25E-10mmHg at 25°C

1.581

-2.6

3

6

3

15

237

2

OC([C@@H]1C[C@H](CC2N=NN([H])N=2)CCN1[H])=O

FAAVTENFCLADRE-NTSWFWBYSA-N

InChI=1S/C8H13N5O2/c14-8(15)6-3-5(1-2-9-6)4-7-10-12-13-11-7/h5-6,9H,1-4H2,(H,14,15)(H,10,11,12,13)/t5-,6+/m0/s1

(2R,4S)-4-(2H-tetrazol-5-ylmethyl)piperidine-2-carboxylic acid

LY-233053 LY 233053; LY233,053; LY 233,053; LY-233,053; LY-235723; 4-((2H-Tetrazol-5-yl)methyl)piperidine-2-carboxylic acid; LY 235723; DTXSID60925169; ...; 125546-04-5; LY233053

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)