JPS63500525A - Methods for blocking calcium channels - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods for blocking calcium channelsTechnical field The present invention provides methods and methods for modulating calcium conductance across cell membranes. and compositions. More specifically, the present invention provides low-threshold old) Methods and compositions for blocking calcium channels and low threshold Values associated with calcium channel conductance or low threshold calcium Tremors and other diseases controlled by conductance The present invention relates to methods and compositions for treating conditions.

Background technology The nerves of the central nervous system transmit information through electrical impulses. These impa The electrical energy generated by the interaction of charged particles across (i.e., via) the cell membrane Caused by chemical potential. The magnitude of the transmitted impulse is determined by the membrane conductance It is a function of the tank. The term “conductance” refers to the measurement of ionic charges across cell membranes. used for charge transfer, by applying a voltage (incremental charge in an electric field) across the membrane refers to the incremental current (or current-like) response exhibited across the resulting cell membrane.

Calcium channels span the thickness of the lipid bilayer in cell membranes and Calcium ions are added to the intracellular (places where calcium concentration is low) and extracellular fluid (calcium the lipid bilayer following an electrochemical gradient of calcium between It has a structure that allows it to be moved passively. These channels are Although the selectivity of calcium channels for other divalent cations is imperfect, However, the movement of calcium ions across this channel-like structure is unclear. shows higher specificity. Calcium channels are electrically charged in the electric field of the cell membrane. It can be activated by

Calcium pumps are distinct structural parts of cell membranes (possibly one or more macromolecules) . This part spans the cell membrane and is sensitive to the electrochemical gradient of calcium ions. Causes active movement of calcium ions. Therefore, the ion pump Ion flow through the pump occurs against an electrochemical gradient; They differ from ion channels in that they require energy to generate .

In the resting state, the interior of a neuron is negatively banded with respect to the extracellular medium or environment. The electricity is on. The difference in potential observed across such a cell membrane is It is reversed when the impulse passes along the nerve. In the short term, nerve cells The polarity is positive. This series of events is known as a 1-axis potential. It is.

Direct stimulation of neuron in vitro is an action with two main components. generates potential. In other words, the sodium conductance-dependent fur spike and a more gradual calcium-dependent spike (Llina s, R, and Sugimori, M.

J, Physiol, 305: 197-2131980), sodium dependent Sexual potential is a hallmark of the trunk (or cell body) response. On the other hand, Karshi Um-dependent action potentials are more pronounced in dendrites.

Llfnas and Yarom (J, Physiol, 315:549-56 ? ; J, PhysioL, 315: 569-584.1981) previously Olive (1,0) cells in the brain stem of guinea pigs also contain calcium, which has two components. Discloses that it exhibits dependent conductance. sodium and calcium The dependent conductance is illustrated in FIG. The inventors have developed the technique described below. By using this technique, stimulation of these cells in vitro becomes the first spy. (sodium conductance dependence) and subsequent residual polarization potential. It was found that an action potential consisting of (ADP)2 is generated.

・This ADP depends on activation of high threshold calcium conductance (HTCC). It has been shown that there are ADP is connected to residual hyperpolarization potential (ARP)3. Ru. Subsequently, rebound polarization spike 4 continues in ARP. rebound spike (RS) is dependent on activation of low threshold calcium conductance (LTCC) It was shown that The shaded area in Figure 1 represents two types of calcium conductors. 2 shows two types of voltage spikes depending on the resistance. ADP and AHP are trodotoxin insensiteb (tetrodotoxin is a sodium conductor) therefore, they are dependent on the sodium conductance I can't do it. Below, we discuss the mechanism of RS (or LTC spike). .

That is, RS is generated as follows due to the existence of LTCC.

(a) Following tetrodotoxin poisoning of the cell membrane, RS If it is more negative than 0 mV, the membrane becomes suddenly polarized by positive pulses of increasing amplitude. It occurs as a result. The v4 value of the olive cells studied according to the present invention is -65 mV. Ru.

(b) RS is a rebound axis that follows hyperpolarization from the resting potential of the cell membrane. This occurs as the Ron potential increases. In the olive cells studied by the present inventors The firing level of the RS spike for It is 5mV.

(C) When the voltage clamp technique (described below) is used, the RS It occurs at negative values of the tensile, reaches a maximum, and then becomes inactivated, also at a negative point. Generating ionic currents within the tensile region (for the studied olive cells) at about -45+aV).

HTCC is a growth model with heart action in the axonal potential of dendrites. minutes, and even included in synalapse transmission.

LTCC appears to be a trunk response.

Tremors consist of more or less regular rhythmic vibrations around some fixed point in a part of the body. child The speed of these vibrations varies from person to person, but in a particular patient, the speed may vary depending on the body's It is fairly constant in all emotional areas. Tremors may be caused by a specific disease (e.g. Kinson's disease) or specific damage to the central nervous system or unknown organs. It occurs.

From a functional point of view, tremor is a fundamental electrophysiological phenomenon of the cells that make up the central nervous system. It can be regarded as a variation in properties.

High-threshold (or dendritic) calcium conductance and low-threshold (trunk) conductance. Interactions between ductance contribute to physiological and abnormal tremors and Parkinson's disease It produces central oscillations in neurons with a periodic rhythm similar to that seen in the brain. Cheating was suggested (Llinas, R, R, Movement Dis orders: Tremor, pp, 165-182. Findley, L, J, and Capildo, R, eds, JacMillan, 1 984).

Therefore, very small amounts of certain compounds (e.g. aliphatic alcohols) may cause a low threshold. value of calcium conductance (resulting in the so-called rebound calcium spike) ) can be blocked (partially or completely) by the inventor. It was a very surprising discovery. Significantly, these compounds can be used in small amounts. If high threshold calcium conductance is not impaired. Furthermore, the present invention They found that lower alcohols have a blocking effect, but higher alcohols found that it has a blocking effect at extremely low concentrations.

The effect of alcohol on calcium conductance, especially the effect of ethanol. There are several citations to the prior art described. However, the inventors know Insofar, alcohol serves a different purpose (to block HTCC, (i.e., as an anesthetic) and the amount used is much higher than the dosage of the present invention. was.

Requena et al., J. Gen. Pbysiol, 85; 789-804 ．． In 1985, the squid axon was converted into an octatwo. The intracellular concentrations observed in these axons when exposed to a 10-'M concentration It is disclosed that the appearance of sium concentration is correlated with an increase in shape. Let me rephrase it For example, Requena et al. reported that octatools leave cells by crossing the cell membrane. It is claimed that the ability of calcium ions to be inhibited is inhibited. This phenomenon is caused by block or unblock (high or low threshold) the channel. do not have.

As mentioned above, calcium channels allow calcium ions to It is a passive transport mechanism down an electrical gradient. Calcium is present in every cell. M concentration is low within the cell (e.g. 10-'M) and high in the extracellular medium (e.g. , 10-”M), thus the calcium channel transports calcium into the cell. It is something that is complicated.

In contrast, outward calcium transport is carried out against the concentration gradient via calcium pumps. This is a completely different mechanism for transporting calcium (from low concentration on the inside to high concentration on the outside). ). Therefore, an ion pump is an active membrane structure that allows ions to flow across the membrane. Enzymes that require energy (ATP: adenosine triphosphate) to transport For example, sodium ATPase). Requena et al. anesthetized the cells , hence rendering the pump mechanism inoperable. In any event, Re The observation by Quena et al. is a completely different phenomenon from the observation by the present inventors, and is different ( Requires significantly higher octatool concentrations. In accordance with the present invention, the inventors have Measurement of intracellular calcium concentration after exposing Iron cells to alcohol , normal or subnormal intracellular calcium concentrations were observed (i.e., Requ. (opposite effect to that observed by ena et al.). Such observations are blocked by C This is due to the inability of calcium to enter cells through the a channel.

Furthermore, the study conducted by the present inventors shows that the low threshold in Squidaxon There is no evidence for the existence of calcium channels (unpublished observations) .

Similarly, Leslie et al. J, Pharm, Exp, Ther, 22 5: 571-575°1983, 2.5XIQ-”~L, 5X10- Voltage at which ethanol is applied to a synabutoseme isolated from a rat brain Discloses inhibiting calcium-dependent calcium. In this research report, high Although it has been described that high concentration ethanol is used, low conductance Nothing has been disclosed about the lucium channel.

Miehaels et al., Biochem, Pharm, 32:963-96 9. Effect of ethanol on rat brain synaptic membrane vesicles in 1983. (10”’M), the effect of propatool (10-”M) and the effect of butanol ( 10-'M). All three alcohols were added to this experimental system. Suppressed calcium influx.

Kitagawa et al., Biochem, Biophys, Acta 798: 210-215.1984 Inhibition of calcium movement in bovine platelets Butanol (5X10-"M) or hexanol (5X10-"M) as a factor ). The mechanism in this case is described by Requenar et al. and others. A calcium pump similar to that studied by the authors of the above-mentioned article. moreover , LTCC has not been shown in platelets, whereby calcium is The mechanism inside is also unknown.

Conventionally, genetic tremor symptoms can be temporarily caused by IN or two types of alcoholic beverages. It is known that it can be calmed down.

Barrison’++ Pr1nciple of Internal Me dicine, P, 961sselbacher, K, J, et al eds, McGrav-Hill, New York, N, Y, 1 See 98G. For one or two types of alcohol-containing beverages, the ethanol width produces blood levels of the order of 10-1, which is required in the present invention. significantly higher than the concentration. Furthermore, the toxicity and other properties of ethanol are sufficient. known to.

Furthermore, the aforementioned experimental observations do not correlate with LTCC, nor with the central nervous system. . These reasons include: - The above-mentioned phenomena are merely superficial phenomena. This does not mean that the substance of the present invention is foreseen in any way.

The conventional treatment for tremors is the administration of β-adrenergic blockers. (e.g. propatool hydrochloride and its derivatives). These drugs are It acts through a completely different mechanism than that of neuron cells, and is opposed to neuron cells. Has a negative effect on muscle cells. β-adrenergic blocking agents have many side effects (e.g. such as bronchodilation, mild lightheadedness, Bradycardia, hallucinations, and kidney disease. and liver abnormalities).

Furthermore, these drugs are not effective for all patients, and some patients ( (e.g. asthma) is harmful. Using the present invention, the concentration of alcohol administered can be extremely high. Because of its low energy consumption, side effects can be minimized to an extremely low level. Preferred alcohol concentration In most cases, only LTCC is affected, and in most patients, β- The present invention can be used in place of epinephrine for treatment. Furthermore, the present invention The use of alcohol according to the Wear.

Demonstration of invention The present invention has a plurality of objects as shown below, but the objects of the present invention are It is not limited.

Partially or completely blocks low-threshold calcium channels in mammalian cells. Provided are methods and compositions for

various types of tremors, including but not limited to enhanced physiological tremors; , essential tremor, severe essential tremor and labral tremor To provide methods and compositions for treating and the like.

Treating tremors caused by Parkinson's disease that can be used in addition to traditional therapy To provide methods and compositions for.

These and other objects of the invention will be apparent from the appended claims and accompanying drawings. This will be easily understood by those skilled in the art.

Brief description of the drawing Figure 1 shows the action potential obtained by electrical stimulation of olive cells. This is a diagram for illustrative purposes.

FIG. 2 is a transparent diagram showing two cross-sections of brain slice perfusion and the recording system according to the invention. This is a perspective view.

Figure 3 shows multiple oscilloscope tracings and alcohol low-threshold calculus. This is the effect on conductance and action potential.

Figure 4 shows the increase in current in the presence and absence of 101M butanol. This is an oscilloscope tracing obtained by stimulating olive cells.

Figure 5 shows the presence of 0.1% pentanol, the presence of 0.05% pentanol, and the presence of 0.05% pentanol. ．． External sulfur in the presence of 25% butanol, 0.5% propatool Oscilloscope series A-H showing the response of olivary cells to injection of sea urchin flow. This is trace tracing.

Figure 6 (A and B) shows various applied voltages using the voltage clamp technique. A series of oscilloscopes of ionic currents obtained from olive cells at pressure amplitudes. This is Roscope tracing. The graph on the right shows this vs. applied external voltage. Figure 3 is a plot of the ionic current difference (between alcohol and reference standards).

Figure 7 shows an octatool (10- Voltage response across the cell membrane in the absence (A) and presence (B) of Here is a series of oscilloscope tracings of the answer. The graph on the right (D) shows the injection current is a plot of the amplitude of the rebound calcium spike versus the value of .

Figure 8 shows electromyograms obtained from control or harmaline-treated rats. Oscilloscope tracings of the system (left) and autocorrelogram (right). .

BEST MODE FOR CARRYING OUT THE INVENTION The present invention blocks low threshold calcium conductance in mammalian cell membranes. 2. A method for reducing or decreasing said low threshold calcium conductance. LTCC blocking agents (e.g. (e.g., aliphatic alcohol). . Preferably, the agent adversely affects the high threshold calcium conductance of said cells. used at sufficiently low concentrations without causing adverse effects. Preferred agents are aliphatic alcohol, more preferably an alkyl alcohol of 03 to CIO.

Another feature of the invention is to suppress the expression of tremor in mammalian muscle cells. A method for inhibiting transmission of tremor signals from central nervous system cells to said muscle cells. LTCC blocking agents (e.g., fatty alcohols) in concentrations sufficient to cause harm. exposing the central nervous system of said mammal to. Preferably, the amount of said drug is , which interferes with high-threshold calcium conductance in both cell types. It is best to use an insufficient amount to

Additionally, another feature of the invention is to block LTCC or tremor. A composition useful for inhibiting LTCC blocking agents or shivering inhibitors. A composition comprising an effective amount and a physiologically acceptable carrier or diluent.

The inventors have demonstrated that certain drugs, such as aliphatic alcohols, are used in mammals, specifically specifically blocks so-called low-threshold calcium channels in the mammalian central nervous system. They found that it increased or decreased the This channel is located in the central nucleus, e.g. important for modulating the period of electrical discharge in the tube or taramas. known to be an ingredient. These drugs reduce the action potential of dendrites. cial, certain components of myocardial action potential and synalapse transmission. LT specifically without adverse effects (as measured) on HTCC contained It can also be used in amounts sufficient to block or reduce CC. therefore , the present invention provides a method for selectively blocking or reducing LTCC. , thereby allowing the isolation of HTCC. Therefore, one use of the present invention is in the isolation and study of HTCC unencumbered by LTCC, i.e. First, using tetrodotoxin to interfere with sodium conductance. It is similar to

According to the inventors, LTCC plays a fundamental role in clocking brain properties. perform the wari and provide the basic period for coordination of interlocking (a series of discontinuous movements) . Tremors have been described as an intensification of the fundamental period of vibration to a point that interferes with the coordination of movement. (Llinms, R, R, in Move@ent Diso rder: Tremor (Findley, L. J. & Capildo, R, Eds) PP, 165-182 Macmtllan 1984° (see). We further believe that similar drugs used in similarly low doses (e.g. For example, researchers have found that alcohols (e.g., fatty alcohols) suppress symptoms of tremor.

The significance of this discovery is that increased physiological tremor, essential tremor, and This is clear from the fact that the tremor that occurs when thinking about age can be completely eliminated. . The method of the present invention has the advantage that HTCC of muscle or neuronal cells is not adversely affected. It's very effective. The present invention shows that neuron or muscle cells associated with HTCC are Acts on the central nervous system rather than on the muscles that exhibit a tremor response without being affected. The present invention provides a method for treating the symptoms of tremor by:

In the present disclosure or the research done in the present invention, the LTCC block of the present invention There is no reason to limit the applicability of the effect to olive cells or neurons.

Therefore, the present invention can be used in all cells with low threshold calcium channels. It is possible.

According to the present invention, the aliphatic alcohol is substantially secondary in blocking LTCC. Effective in suppressing tremors at very low concentrations without reducing the risk of action It is. Therefore, generally speaking, the lower the effective amount of a particular alcohol, the lower the The more desirable it is, the more desirable it is to use.

, the degree of inhibition of low-conductance calcium channels depends on the molecular weight of the alcohol (or It seems to be related to As molecular weight increases, lower conductance The effective strength of the alcohol required to reduce the potential is reduced. Therefore Therefore, calcium conductance suppression increases as the molecular weight of alcohol increases. I hear it. This suggests that pentanol, which has a 5-fold lower concentration in LTCC inhibition, It is shown in detail in Example 6 below that it is more effective than the tool. (Figure 5, compare H and E). Of course, we and the apparent correlation between alcohol molecular weight and alcohol molecular weight with some kind of theory. It's not a thing. The increased Ca channel blocking ability of higher alcohols For example, the increasing hydrophobicity of the alcohol with increasing hydrocarbon chain length. and/or due to the lipid solubility of alcohol.

The effects of the present invention are mainly achieved both in vitro and in vivo. Shown for complemented mammalian cells.

There is no abnormal behavior in the test animals used to demonstrate the effects of the present invention. Not doing so further suggests that the risk of unforeseen side effects is extremely low. It is something to do.

In vitro, cells receive an effective amount of alcohol to block LTCC. exposed to a culture or perfusion medium containing This amount is preferably HTCC The amount is low enough that it does not interfere with the Of course, various alcohols This quantity is important in that the alcohol has various channel blocking abilities. Varies depending on the rule. For some alcohols used in the present invention The preferred highest molecular concentration (or substantial and total block) is as follows: shown in the table.

Table 1 Alcohol upper limit lower limit Ethanol 10°S io-' Property tool 10-’ IO-’ Butanol 10-' 10-@ Pentanol 10-' 1G-' Hexanol 101 Undetectable Heptatool 10-” Undetectable Octatool 10-8 Undetectable Nonanol 10-6 Undetectable Decanol 10-@ undetectable Regarding 10-”M for hexanol to decanol above, the molar concentration is It could not be detected accurately. The perfusion solution used in our experiments (also (composition administered parenterally) if all the alcohol is soluble in the vehicle. To prepare a 10-”M solution, mix a sufficient amount of vehicle and alcohol to prepare a 10-”M solution. It was prepared by Therefore, the concentration used was the most dilute 10-”M. Since the solubility limits of these alcohols cannot be determined accurately, , the maximum dilution effective concentration cannot be determined. C@~CIO All alcohols are dissolved in the medium. Although not understood, a reference experiment using a medium that did not come into contact with alcohol is , effective because it does not block LTCC rebound spikes or suppress tremors. continue to be.

Furthermore, the concentrations shown in Table 1 above are for a complete block of LTCC. . If a partial block is desired (this may be the case in the treatment of tremors) ), the amount is small, as determined by routine experimentation.

The discussion herein does not imply that the invention is limited to aliphatic alcohols in solution. . When alcohols are administered in vivo, they can be administered either orally or parenterally. It can also be administered with or without a carrier or diluent. It may be in either solid or liquid form. Of course, especially oral When administered, it is not absorbed in the esophagus or reaches the blood and brain into the cerebrospinal fluid. The alcohol dose must be metabolized before it can be metabolized.

Thus, the amount of alcohol is an effective LTCC blocker in the vicinity of the central nervous system. (or LTCC reduction) alcohol concentration, e.g. as in Table 1 above. The resulting concentration should be calculated. For this, in the complete block above In Table 1, the concentration of alcohol in the composition to be administered to a mammal is Required concentration of alcohol in the blood (or brain and cerebrospinal fluid -- CSF) as indicated. This is the range in which this occurs. Preferably, the alcohol is also physiologically tolerable. It is administered in a composition containing a carrier or diluent.

Most preferably, blood or C3F (or perfusion medium) at a maximum dilution of 1 G-@M Use sufficient octyl alcohol to produce a concentration.

The above amount blocks LTCC and also suppresses tremors.

As mentioned above, the amount of LTCC block or tremor suppressant will depend on the particular Varies depending on drug activity and administration method.

The frequency of administration also depends on the severity of the tremor symptoms and how many times the symptoms occur. The acupuncture changes.

Additionally, in some cases, LTCC block or tremor of the active agent in the patient's bloodstream. It is highly desirable to use a sustained delivery system to maintain inhibitory concentrations. stomach.

However, all of the above considerations concern optimizing the use of the present invention. . The amount, mode, and form of administration are routinely fine-tuned and This can be accomplished using no more than routine experimentation by one of ordinary skill in the art.

The present invention will be described in detail below based on specific examples, but the scope of the claims of the present invention shall not be limited. It's not. In the examples below, LTCC rebound spikes are defined herein as Each of the three methods included in Operational Definition described in the background section It occurred. Alcohol is used in the method used to generate RS (voltage). This response can be blocked regardless of diclamp, flow injection or polarizing voltage. Click. This shows that a single mechanism works and the effect of the present invention.

Example: Tissue preparation Camm Re5earch In5tit located in Wayne, New Chassis The head of an adult Hartley guinea pig (400~soog) obtained from ute. Small animals were decapitated using a guillotine after ether anesthesia. Immediately thereafter, the scales of the occipital bone Two long pieces were created along the lateral edges of the shaped section. Cut the resulting bone plate transversely and tear caudally to expose the cerebellum and brainstem. Transverse section of cranial nerve and According to the lateral fragment of the brainstem (beak-like at the level of the inferior colliculus and foot-like at the level of the CI) , the brainstem was quickly torn apart and diluted with Ringer's solution (124mM NaCl; 5mM KCI; 1.2mM KHtPO, ; 2.4mMCaClt; 1.3mM M g5O,;2.6mM? 1a) about 5 in IcOs and 10mM glucose) There was a separate device. It is then placed on the parasagittal surface to obtain elongated fragments. cut in the longitudinal direction on the asagittal plane) and Del G (Vibratome Model G) Plate (California) Tad Pe1la Inc. located in Stein.

(available from ). Six 300 micron slices from one brainstem I was able to get it. Following cutting, incubate slices in Ringer's solution for approximately 1 hour. I started. The heating medium was allowed to stand at room temperature, during which time a mixture of 95% COt and 5% COt was added to the buffer. It was bullish. After 1 hour, one brainstem slice was removed from the incubation bath. , the brainstem can be well preserved in the bath up to 24 hours later.

Example 2: Recording chamber See Figures 2A and 2B. These are the perfusion solutions used in this experiment. FIG. 2 is a perspective view of the recording system in a cross section taken along line A-A.

After incubation, the slices were transferred to recording dish 1 where they were assembled at a temperature of 87°C. Continuous perfusion of the tissue was performed. Irrigation system has a maximum of 2m and 17m, routine Gravity fed with a flow of 0.5 m1/mf,n. The latter flow – speed was used during fluid exchange. The central chamber 2 has a capacity of 2 ml and contains liquid is completely replaced in about 10 minutes. Three small diameter channels in the central chamber Physiological saline was injected through 4, creating a cloth laminate flow. (One of these is shown in Figure 2A).

Side chamber 2a communicates with chamber 2 via channel 4 to provide perfusion physiology. Used as a salt water reservoir. The outflow from central chamber 2 is Achieved through a cotton wick system 3, causing continuous fluid motion and turbulent flow (Figure 2B). Transfer the brain slices to the Sylgard drop at the bottom of recording chamber 2. Brain tissue was placed on a rate 11 Corning glass (Konink, New York). Hold it on the bipolar stimulation electrode 5 while lightly pressing it. surrounding water bath 6 to maintain the chamber at 37°C, which is the same temperature as the perfusion solution at 37°C. did. The temperature of the saline solution itself was controlled by passing it through a heat exchanger 7 at 37°C. (Figure 2A).

The standard perfusion solution is Ringer's solution, and this medium is used for cutting and incubation. and used during most of the recording time. Ringer's solution is excellent at various temperatures. It provided pH warming properties.

This was especially noticeable during cutting and incubation.

If desired, alcohol was added directly to Chamber 1 via the perfusion solution.

Example 3: Recording technology Observe and record olive cells directly using Hoffmann modulation. Insert Hof4a+an into recording micropipette 9.

See R, J. Microsc, 110: 205-222.1977;

), so that undamaged cells were visible on top. Steam to the objective lens of the microscope 14 A small saxilon spigot 8 is placed on the side of its bottom surface to prevent condensation. (Figure 1A). Intracellular recordings had an average resistance of 60-80 milliohms, C. Microfluidic solution filled with 3M potassium acetate or ethyl ammonium (ETA) Obtained using pipette 9. Micropipette to recording amplifier 12 Connected. Direct stimulation of olive cells uses high input impedance 101 It was given by a bridge amplifier of t ohms.

Frequency response of 1G-15kHz with capacitive compensation depending on microelectrode performance was gotten.

Example 4: Low Threshold Calcium Diagram 3A-C Using Octatool Partrace) is a tetrodotoxin with a sodium-dependent spike of to-@M. (manufactured by Sigma Chemical Co., St. Louis, Mo.) after blocking a typical Olive (1,0°) nuclear cells exhibiting a calcium-dependent action potential. Intracellular recordings from cysts are shown. In this example and all examples, 160. cells carried out Isolated as in Example 1 and recorded as in Example 3. A, B , the top row in C (check standard performed in the absence of octanol) is Shows a pike-like LTCC rebound spike. The applied current is HTCC Since there is a low threshold for HTCC, there is no HTCC. Lower row in A, B, C The record of LTCC can be blocked by adding to-”M octatool to the bus. complete blockage of calcium-dependent rebound potential by Indicates the This also corresponds to the action potential in graph D on the right side of Figure 3. (closed-circuit) low-threshold action point in the reference standard. It shows the rate of increase in tensile versus membrane polarization. Low series of data points (square) is the low threshold action point after 1G-”M OctaTool is added to the perfusion medium. Indicates the rate of rise in tension. The difference between the graphs of the reference standard and Octatool is L. Figure 2 is a measurement of the effect of a 10-''M octatool block on TCC.

Example 5: Inhibition of LTCC by butanol Figure 4A shows tetrodotoxin Olive cells under conditions where sodium channels were blocked by the addition of The action potential observed in cells is shown. Injected square current A large increase in the axine potential response shown in Figure 4A It can be added while Once a sufficient threshold is reached, the action potential features A spike occurs. The first (leftmost) spike is the high-threshold calcium spike. the second (or rebound) spike is a low-threshold calcium spike. be.

Figure 4B shows the action potency that occurs with administration of 10-'M butanol. nsial and LTCC or rebound spike suppression. such effect occurs without any effect on the HTCC.

Example 6: Comparison of LTCC blocking ability of alcohol and related effects Figure 5 shows the absence of various types of alcohol (graphs A-D) and the presence of various concentrations. Polarization of the membrane following external square current injection in the presence of alcohol (E-H) Shown is a series of tracings A to H showing the response of olive cells to. vinegar At the rate of rise of the pike (the first derivative of the voltage shown in the middle record) The decrease indicates the blocking effect of alcohol on LTCC. This diagram shows high-end Alcohol is more effective than lower alcohols, in other words higher alcohols Shows effectiveness at lower concentrations.

The experiment giving the data shown in Figure 5 involved injecting a square current and The procedure was carried out in the same manner as described in Examples 1 to 3 as shown in this figure, except that It was.

In each of graphs A-H, the upper trace represents the axis observed across the membrane. tion potential. The middle trace is the action potential via the cell membrane. It shows the rate of change in the chart. The bottom trace shows the magnitude of the injected current.

In graphs A-D, cells exhibit characteristic low-threshold calcium spikes (mid-level code). In contrast, in graph F-H, The speed and magnitude of the change in action potential change with the addition of alcohol. Significantly adversely affected (decrease). In particular, in graph E, the action potential The signal lacks the characteristic spike appearance and resembles a subthread response.

Comparing graphs E-H, butanol is twice as effective as propatool; Butanol is five times more effective than butanol.

All of the above results were further demonstrated at an injection current of 0.5 nA in cells exposed to alcohol. This is a remarkable result. On the other hand, the reference standard was 0.25 nA in cells. It is. This means that pentanol is essentially 10 times more effective than butanol. However, this means that butanol is more than four times as effective as propatool. relative The effectiveness increases as the molecular weight of the alcohol increases. Octatool is Its solubility in aqueous media leads to highly effective concentrations for blocking LTCC. This is preferable because it responds to requests.

Example 7: Voltage for r,’rcc octatool guidance block clamp research The results in this example were recorded in olivary neurons from different parts of the nucleus. It was done. These conductances are similar to sodium conductance due to tetrodotoxy The potassium conductance is blocked by the addition of tetraethylene ammonia. Very low low threshold, value (− 7 hV). In these experiments, the membrane was measured after an ionic current was applied across the membrane. Keep voltage constant To do this, an electric current was injected into the cell. This current is converted into an ionic current across the membrane. Equally and in opposite directions, so that the frequency and spacing of the direct ionic current can be measured. The ionic current is a low-threshold calcium spike under normal conditions. cause a problem.

Figures 6A and 6B show a 5 mV depolarization from a holding potential of -80+eV. The magnitude of the low-threshold calcium ion current following the step is depolarized to -45 mV. and changes over time. These records were observed after putting the octatool in the bath. This shows the difference in ionic current caused by Therefore records A and B are 10-” The i Represents on-current. The internal current is first observed at -65 mV. For voltage process As the amplitude increases, the calcium current peaks at a membrane potential of -5 hV. It becomes 0 immediately at -42mV. -42mV is a calcium channel It is the potential to inactivate.

FIG. 6C is a plot of ionic current versus applied external voltage. This electric The current value is the difference in ion current before and after Octatool treatment. This difference is approximately − Maximum occurs at membrane voltages between 45 and -60 millivolts. This graph is from LTCC. Voltage dependence is characteristic.

Example 8: Block of Reno subendocal>ftm1notion potential This embodiment will be described with reference to FIG. Figure 7 shows a square current pulse (C) Rebound calcium flux following membrane hyperpolarization in the absence of alcohol by injection of Pike is shown blocked by a 10-1 octatool (B).

In this experiment, olive cells were treated as described above.

The sodium spike was blocked by 10-@M tetrodotoxin.

Graph D on the right side of Figure 7 shows the rebound curve for the magnitude of the injected hyperpolarizing current. Showing the dependence of sium spikes.

In order to investigate the effect of LTCC blocking agent in vivo, Octa2 was administered to rats. Dilute the alcohol with saline and apply it around Neuron. The concentration of alcohol was adjusted to 10-”M (maximum). It was administered intraperitoneally at a concentration of mM. Trembling conditions induce tremors in mammals Pegamus harllara, which is known to be induced in rats by injection of harmaline induced by available from Sigma, St. Louis, Missouri).

The tremor thus evoked was physiologically equivalent or physiologically enhanced. It has periodicity.

Since destruction of olive cells eliminates tremors in harmaline-treated animals, olive Cells responsible for transmitting tremor signals to mammalian muscles It was found that (Llinas, et at, 5science 1 90:1230-1231.1975). Therefore, tremors are attracted by harmaline. Study of mammalian olive cells caused by tremor response in mammals provides an excellent in vivo model for

The tremor is Buchthal, F., et al., Acta Physio , 5cand, 39: 83-104.1957. more measurable.

278-300g Sprag - Dawley Rat Germantown, New York The electrical activity in the left platysma muscle of the Taconic Farm Unique while using two Teflon coated strand silver wires (exposed wire 2mm) was recorded. During the experiment, electromyogram (e m g) was measured continuously and the data was was analyzed at the following time points:

(1) Before drug administration: (2) After intraperitoneal administration of 15+og/kg harmaline: and (3) 10mM 1- Octatool (sonicated to 99% Octatool with saline) After intraperitoneal injection of 2c, the weight of the rat was 9.3% near the neuron cells. (all alcohol absorbed and total (assuming that it was not metabolized properly).

e m, g are Nicolet Explorer model 4060 digital oscilloscopes (N1colet 1nstruIlent located in Madison, Wyoming) Smooth identification was achieved using the following software: This device is compatible with AMG's &QQms Sexy It was also used to obtain the autocorrelogram of the The autocorrelogram is e Obtained by superimposing 800m5 section of mg . Emerging patterns are not easily recognized from the e m g signal itself. It can be used to determine the periodicity and amplitude characteristics of mg.

Reference reference e m g indicates an occasional period of low amplitude oscillation (Figure 8 upper pressure). The autocorrelogram shows a periodicity of 8 Hz in amg . However, the correlation function is low (Fig. 8, top). After harmaline injection, animals generally It indicates the period of trembling. The emg recorded during this period is the middle pressure in Figure 8. More shown below. The main period of the tremor is 7.5Hz, which is close to the reference standard value, and there is a difference in altitude. After the injection of 1-octanol, the trembling stopped and the base Linear muscle activity decreased below reference reference levels (tracing from lower pressure). major circumference The wave number is 10Hz, and the emg amplitude is obtained as an autocorrelogram (bottom right). This period of time required to amplify the signal in order to 10Hz period Although the periodicity (indicated by the arrow) is clear, the 60Hz periodicity due to the line voltage ( Amblifier noise) is the main correlation with large gain. lower cloth signal becomes a straight line if only the degree of the two upper autocorrelograms is amplified. It should be expressed as

If you simply want to reduce your tremors to normal or manageable levels, It is clear that the amount of alcohol can be reduced.

Figure 2 Figure 5 international search report

Claims (1)

[Claims] 1. Partially or totally blocks low-threshold calcium channels in cell membranes. a method for treating said cells with a low-threshold calcium conductance blocking agent. A method comprising exposing an effective amount of. 2. partially or completely blocks low-threshold calcium channels in mammalian cell membranes. A method for locking a low threshold calcium conductance within said cell. aliphatic alcohol in a concentration sufficient to partially or totally block A method consisting of exposing cells to cells. 3. The alcohol is a C2-C10 alkyl alcohol and mixtures thereof. 3. The method of claim 2, wherein the method is selected from the group consisting of: 4. the concentration is sufficient to have a significant effect on high threshold calcium conductance. 3. The method according to claim 2, wherein the method is not a concentration. 5. the concentration of the alcohol is about 10-3 M or less for ethanol; less than about 10-4 M for lopanol and about 10-5 M for butanol Claimed range of 10-8M or less for higher alcohols: The method described in Section 2. 6. The alcohol is octyl alcohol, and the alcohol concentration is 3. The method of claim 2, wherein the maximum density is about 10<-6>M in the vicinity of the cell. 7. 4. The method of claim 3, wherein said alcohol is octyl alcohol. 8. 3. The method of claim 2, wherein said cells are neuron cells. 9. 9. The method according to claim 8, wherein the forepaw neuron cells are olivary cells. 10. 3. The method of claim 2, wherein said method is performed in vivo. 11. a sufficient amount of said alcohol to produce said concentration in the vicinity of said cells; 11. The method according to claim 10, which comprises administering to said mammal. 12. an effective amount of said alcohol and a physiologically acceptable carrier or diluent. 12. The method of claim 11, comprising administering a composition comprising: 13. Claim 11, wherein the alcohol is administered parenterally. How to put it on. 14. Claim 11, wherein the alcohol is administered orally. the method of. 15. the composition is selected from the group consisting of Ringer's solution and isotonic saline; 13. The method according to claim 12, further comprising a diluent. 16. selectively partially or completely activates low-threshold calcium channels in mammals. A method for physically blocking C2-C10 alkyl alcohol and and mixtures thereof. the method comprising administering to said mammal an effective block amount of the drug. 17. To suppress the expression of tremors in mammals that require treatment of tremors. a method for reducing tremor, the method comprising: reducing the amount of calcium conductors with a low threshold of tremor-suppressing spinal efficacy; A method comprising administering a tans blocking agent to said mammal. 18. To suppress the expression of tremors in mammals that require treatment of tremors. This is a method for a tremor from the central nervous system of the mammal to the muscle cells of the forefoot mammal exhibiting tremor; an effective amount of alcohol to partially or totally block the transmission of electrical signals 2. A method comprising administering a drug to said mammal. 19. said amount produces a high threshold calcium in the central nervous system or muscle cells of said mammal. Claim 18: Not in sufficient amount to significantly disturb sium conductance Method described. 20. To suppress the expression of tremors in mammals that require treatment of tremors. This is a method for a low threshold calcium channel of said mammalian central nervous system having said low threshold calcium channel; Consists of selectively partially or totally blocking lucium channels Method. 21. The alcohol is ethanol or a higher alkyl alcohol. and mixtures thereof. 22. Claim No. 1, wherein the alcohol is a C3 to C10 alkyl alcohol. The method according to item 21. 23. 19. The method of claim 18, wherein said cells are olive cells. 24. Ethanol and higher alkyl alcohols as well as mixtures thereof. alcohol low threshold calcium channel blocker selected from the group consisting of compounds; and a physiologically acceptable diluent carrier. 19. The method of claim 18, which comprises administering to a patient. 25. 10-4M or less for propanol, 10-5M or less for butanol Below, regarding pentanol, alcohols below 10-8M and higher alcohols. and 10-8 M or less in the bloodstream or cerebrospinal fluid of the mammal. The procedure consists of administering said alcohol in an amount sufficient to produce a concentration of alcohol. The method according to item 21. 26. Claim 18, wherein the alcohol is administered orally. the method of. 27. Claim 18, wherein the alcohol is administered parenterally. How to put it on. 28. The method according to claim 22, wherein the alcohol is octyl alcohol. Law. 29. 26. The method of claim 25, wherein the alcohol is n-octanol. . 30. comprising an effective amount of said alcohol and a physiologically acceptable carrier or diluent. 19. The method of claim 18, comprising administering to said mammal a composition comprising: . 31. The composition is a liquid composition, and the diluent is Ringer's solution and an isotonic 31. The method of claim 30, wherein the method is selected from the group consisting of physiological saline. 32, which partially or completely suppresses low-threshold calcium conductance in mammalian cells. a composition for physically blocking an effective amount of a low threshold calcium conductor; A composition comprising a tank blocking agent and a carrier or diluent. 33. said drug is selected from the group consisting of aliphatic alcohols and mixtures thereof; 33. The composition according to claim 32. 34. 34. The set of claim 33, wherein the alcohol is an alkyl alcohol. A product. 35. the alcohol is an alkyl alcohol having 3 to 10 carbon atoms; A composition according to claim 34. 36. said amount has a significant effect on the high threshold calcium conductance of said cell. the composition selectively induces low-threshold calcium conductance. Claim 32 is useful for blocking partially or completely Composition of. 37. 37. The composition of claim 36, wherein said drug is alcohol. 38. the alcohol is octyl alcohol, and the amount is in the vicinity of the cell. in an amount sufficient to produce an octyl alcohol concentration of up to 10 M at A composition according to claim 37. 39. The alcohol is an alkyl alcohol having 3 to 10 carbon atoms. selected from the group consisting of about 1% of propanol in the vicinity of said cells 0-4M or less, about 10-5M or less for butanol, pentanol and A claim in an amount sufficient to produce less than about 10-5 M of higher alcohols. The composition according to item 37. 40. said action effective in reducing said tremor to a normal or manageable level; 19. The method of claim 18, wherein an amount of alcohol is administered to said mammal.