EP0260312A1 - Method for blocking calcium channels - Google Patents

Abstract

Method for partially or totally blocking threshold calcium channels in cell membranes. The method involves exposing the cells to an effective amount of a low threshold calcium conductance blocking agent.

Description

METHOD FOR BLOCKING CALCIUM CHANNELS

Field of the Invention

This invention relates to methods and compositions for regulating calcium conductance across cell membranes. More particularly, this invention relates to methods and composi¬ tions for blocking "low-threshold" calcium chanηels and for treating the symptoms of tremors and other pathological condi¬ tions associated with, or controlled by, low-threshold calcium conductance.

Background of the Invention

Neurons of the central nervous system transmit informa¬ tion via electrical impulses. These impulses are generated by electrochemical potentials caused by the movement of charged particles across (i.e. through) the cell membrane. The size of the impulse transmitted is a function of the membrane conduc¬ tance. The term "conductance," as applied to transmission of ionic charge across a cell membrane, means the incremental cur¬ rent (or current-like) response exhibited through the cell mem- brane as a result of the application or a voltage (an incre¬ mental change in the electric field) across the membrane.

A calcium channel is a structure that spans the thickness of the lipid bilayer in cell membranes and allows calcium ions to move passively across this lipid bilayer ac- cording to the calcium electrochemical gradient between the interior of the cell (where calcium concentration is lower) and the extra-cellular fluid (where calcium concentration is high er) . These channels demonstrate a higher specificity for the movement of the calcium ion across this channel-like structure, although a calcium channel's selectivity with respect to other divalent cations is never perfect. Calcium channels can be activated by changes in the electric field of the cell mem¬ brane.

A calcium pump is a different structural moiety (proba¬ bly one or more macromolecules) of the cell membrane. This moiety spans the cell membrane and produces an active movement of calcium ions against their electrochemical gradient. Ion pumps are therefore different from ion channels in that they require energy to generate ionic movement since ionic flow through a pump occurs against the electrochemical gradient.

In a resting state, the interior of a nerve cell is negatively charged with respect to the extracellular medium or environment. This difference in potential, which is observed across the cell membrane, is reversed when an impulse passes along the nerve. For a brief period of time, the polarity of the nerve cell becomes positive. This sequence of events is known as an "action potential."

Direct stimulation of neurons in vitro produces action potentials having two main components: a fast spike, due to sodium conductance, and a slower, calcium-dependent spike (Llinas, R. and Sugi ori, M. J__ Physiol. 305:197-213 1980). Sodium-dependent potentials are the prominant feature of the somatic (or cell-body) response, while calcium-dependent action potentials are more apparent in the dendrites.

Llinas and Yaro (J. Phvsiol. 315: 549-567; J. Physiol. 315: 569-584, 1981) have previously disclosed that guinea-pig inferior olivary (I.O.) cells from the brainstem exhibit a calcium-dependent conductance which also has two components. The sodium- and calcium-dependent conductance is illustrated in Figure 1. Using techniques described below, the authors found that stimulation of these cells in vitro generates action po- tentials comprising a fast spike, 1 (due to sodium conductance) followed by an after-depolarization potential (ADP) , 2. The ADP was shown to be due to the activation of a high-threshold calcium conductance (HTCC) . The ADP was followed by an after- hyperpolarization potential (AHP) , 3. In turn, the AHP was followed by a rebound depolarization spike, 4. The rebound spike (RS) was shown to be due to the activation of low- threshold calcium conductance (LTCC) . The shaded areas in Fig. 1 represent the two types of voltage spikes due to the two types of calcium conductance. The ADP and AHP are tetrodo- toxin-insensitive (textrodoxin inhibits sodium conductance) and, therefore, they cannot be due to sodium conductance. The following can be considered an operative definition of the RS (or LTC spike) :

The RS is generated by the presence of LTCC as follows:

(a) Following tetrodoxin poisoning of the cell mem¬ brane, the RS occurs as the membrane is abruptly depolarized with square voltage pulses of increasing amplitude, if the membrane potential is more negative than -70mV. The threshold in the inferior olivary cells studied by the present inventio is -65mV.

(b) The RS occurs as a rebound action potential fol lowing hyperpolarization of the cell membrane from a restin potential. The firing level of the RS spike for the inferio olivary cells studied by the present inventors is the same a in (a) , i.e. -65mV.

(c) When a voltage clamp technique (described below) is used, the RS generates an ionic current that occurs at nega tive values of cell membrane potential, rises to a maximum, an is then inactivated — also within the negative potential rang (at about -45mV for the I.O. cells studied) .

HTCC is involved in dendridic action potentials, i certain components of heart action, and in synaptic transmis sion. LTCC appears to be a somatic response.

Tremor consists of a more or less regular rhythmic os cillation of a part of the body about a fixed point. The rat of this oscillation varies from individual to individual but, in a particular patient, the rate is fairly constant in al affected' parts of the ^"ody. Tremor may be caused by specifi pathological diseases (_.g. Parkinson's Disease) or be due to specific lesions in the central nervous system, or it may be of unknown origin.

From a functional point of view, tremor can be seen as a modification of the basic electrophysiological properties of cells comprising the central nervous system.

It has been suggested (Llinas, R.R. in Movement Disorders: Tremor. pp. 165-182, Findley, L.J. and Capildeo, R. , eds., MacMillan, 1984; incorporated by reference) that the interplay between the high-threshold (or dendritic) calcium conductance and the low-threshold (or somatic) conductance could result in central oscillatory properties of nerve cells which have the same cyclic rhythmic frequence such as that found in physiological and abnormal tremor as well as in Parkinson's tremor. The present inventors have surprisingly found that cer¬ tain compounds (such as aliphatic alcohols) at extremely small amounts are capable of blocking (partially or completely) the low threshold calcium conductance (which generates the so-cal¬ led rebound calcium spike) . Significantly, these compounds, when used in small amounts, do not affect the high-threshold calcium conductance. Furthermore, the inventors have found that although lower alkyl alcohols have a blocking effect, the higher alcohols do so at extraordinarily low concentrations.

The prior art contains several references purporting to describe the effects of alcohols on calcium conductance, and in particular the effects of ethanol. In all instances known to the present inventors, however, the alcohols were used for a different purpose (to block HTCC, i.e., as "anaesthetics") and in amounts markedly exceeding those of the present invention. Requena et al (J__ Gen. Physiol. 85: 789-804. 1985) dis¬ close that exposure of squid axons to octanol, at a concentration of 10 -4M, correlated wi.th an apparent i.ncrease m. the observed intracellular calcium concentration in these axons. In other words, Requena et al state that octanol inter- feres with the ability of the calcium ions to leave the cell by crossing the cell membrane. This phenomenon is unrelated to blockage or non-blockage of calcium channels (high- or low threshold) .

As explained above, a calcium channel is a passive transport mechanism by which calcium ions move down their elec¬ trochemical gradient. In all cells, calcium concentration is low inside the- cell (e.g., 10~⁷M) and high in the extracellular medium (e.g., 10~³M) and so a calcium channel allows calcium to go into the cell.

By contrast, outward calcium transport takes place via "a calcium pump," an entirely different mechanism which trans- 0 ports calcium against a concentrational gradient (from the low concentration inside to the high concentration outside) . An ion pump is therefore an active membrane structure, usually an enzyme (e.g., sodium ATPase) which requires energy (ATP: adeno- sine triphosphate) to carry ions across the membrane. Requena

15 et al anaesthetize the cells and, therefore, "paralyze" the pump mechanism. In any event, Requena's observations concern a totally different phenomenon from that of the present invention and require different (markedly higher) octanol concentrations. If the present inventors measured the intracellular calcium

20 concentration after exposure of neuron cells to the alcohol in accordance with the present invention, they would observe a normal, or a lower-than-normal intracellular calcium concentra¬ tion, (i.e. an effect opposite to that said to have been ob¬ served by Requena et al.), which would be due to inability of

25 calcium to enter the cell through the blocked Ca channel. Furthermore, investigations conducted by the present inventors revealed no evidence of the presence of a low-threshold calciu channel in squid axons (unpublished observation) .

Similarly, Leslie et al. (J. Pharm. Exp. Ther. 225:571-

30 575. 1983) disclosed that ethanol, at 2.5 x lθ^"2 - 1.5 x 10^-1M_# inhibited voltage-dependent calcium uptake into synaptosomes isolated from rat brains. Aside from the high ethanol concentrations said to be used, there was no mention in thi publication of studies on low-conductance calcium channels.

35 Michaels et al. fBiochem. Phar . 31:963-969, 1983) des cribed the effects of ethanol (10 -1M) , propanol (10-2M) an butanol (10 M) on calcium-dependent fluxes in rat brain synap 1 tic membrane vesicles. All three alcohols inhibited calciu influxes in this experimental system.

Kitagawa et al. (Bioche . Biophys. Acta 798:210-215

-2

1984) disclose the use of butanol (5 x 10 M) or hexanol (5 x

5 10 ) as an inhibitors of calcium mobilization in bovine plate lets. The mechanism in this case is a calcium pump similar t that studied by Requena et al and the authors of the othe papers described above. Moreover, the LTCC has not been demon strated in platelets, and the mechanism by which calcium enter

1.0. platelets is not known.

It has been noted in the past that one or two drinks o an alcoholic beverage can abate the symptoms of familial tremo temporarily: Harrison's Principles of Internal Medicine, p.9 Isselbacher, K.J. et al eds. McGraw-Hill, New York, N.Y. 1980

15 One or two drinks of an alcohol-containing beverage would pro duce blood levels approximately on the order of 10~²M in etha nol, a concentration notably higher than that necessary in th present invention. Furthermore, the intoxicating and addictiv properties of ethanol are well-known.

20 In addition, the aforementioned empirical observation were never correlated with LTCC nor with the central nervou system. For these reasons, the above-cited phenomenon has onl superficial, if any, relevance to the present invention.

Current treatment for tremors comprises administratio

25 of beta-adrenergic blockers (such as propranolol hydrochloride and its derivatives) . These drugs act via a mechanism totall different from that of the present invention, and affect muscl cells as opposed to neuron cells. Beta-adrenergic blocker cause a myriad of side-effects (e.g. , bronchodilation, light

30 headedness, bradycardia, hallucinations, and kidney and live abnormalties) . In addition, these drugs are not effective i all patients and are harmful to some (e.g. asthmatics) . Use o the present invention should lead to minimal side-effects du to the very low concentration of alcohols administered. At th

35 preferred alcohol concentrations, primarily only LTCC would b affected and a larger patient population could be treated usin the present invention instead of beta-adrenergic blockers. Furthermore, use of alcohols in accordance with the present invention could be made in conjunction with use of beta-block- ers.

Objects of the Invention

The present invention has several objects including, but not limited to, the following:

- to provide a method and composition for partial or complete blocking low-threshold calcium channels in mammalian cells.

- to provide a method and composition for treating different types of tremor including, but not limited to, en¬ hanced physiological tremor, essential tremor, severe essential tremor, and rubral tremor. - to provide a method and composition of treating Parkinson's tremor which can be used as an adjunct to conven¬ tional therapy.

These and other objects of the present invention will be apparent to those skilled in the art in light of the present description, accompanying claims, and appended drawings.

Brief Description of the Drawings

Fig. 1 is a diagram serving as an illustration of the action potential obtained by electrically stimulating I.O. cells.

Fig. 2 is a perspective view in two sections of a brain slice perfusion and recording system in accordance with the present invention.

Fig. 3 is a number of oscilloscope tracings and a graph showing the effect of alcohol on the low-threshold calcium con¬ ductance and action potential.

Fig. 4 is an oscilloscope tracing obtained by stimulat¬ ing I.O. cells with incremental increases in current both i the presence and absence of 10~⁴M butanol. Fig. 5 is a series of oscilloscope tracings, A throug H, showing the I.O. cell response to an external square curren injection, in the presence of 0.1% pentanol; in the presence o 0.05% pentanol; in the presence of 0.25% butanol; in the pre sence of 0.5% propanol.

Fig. 6 (A and B) is a series of oscilloscope tracing of the ionic current obtained from I.O. cells at differen voltage amplitudes applied using a voltage clamp technique. The graph on the right (C) is a plot of the difference in thi ionic current (between the alcohol and the control state) against the applied external voltage.

Fig. 7 is a series of oscilloscope tracings of th voltage response across the cell membrane in the absence (A)

—6 and presence (B) of octanol (at 10 M) pursuant to injection o a hyperpolarizing square current pulse (C) . The graph on th right (D) is a plot of the amplitude of the rebound calciu spike against the value of the injected current. Fig. 8 is an oscilloscope tracing of electromyogram (left) and autocorrelograms (right) obtained from control o harmaline-treated rats.

Summary of the Invention The present invention is directed to a method fo blocking or reducing the low-threshold calcium conductance i mammalian cell membranes comprising exposing said cells to a LTCC-blocking agent (such as an aliphatic alcohol) at a concen tration sufficient to block or reduce said low-threshold cal cium conductance selectively. Preferably, the agent will b used at a concentration sufficiently low so that it does no affect the high-threshold calcium conductance of said cells. Preferred agents are aliphatic alcohols and particularly pre ferred are C₃-C₁₀ alkyl alcohols. Another aspect of the present invention is directed t a method for inhibiting the manifestation of tremor in th muscle cells of a mammal comprising exposing central nervou system cells of said mammal to an LTCC-blocking agent (such a an aliphatic alcohol) at a concentration sufficient to imped transmission of a tremor signal from said central nervous sys tem cells to said muscle cells. Preferably, the amount of sai agent will be insufficient to interfere with the high-threshol calcium conductance in either type of cell.

Yet another aspect of the present invention relates to compositions useful in blocking LTCC or in inhibiting tremor comprising an effective amount of an LTCC-blocking or a tremor- inhibiting agent and a physiologically acceptable carrier or diluent.

Detailed Description of the Invention

The present inventors have discovered that certain agents, such as aliphatic alcohols, block or reduce specifi¬ cally the so-called low-threshold calcium channel in mammalian cells, and in mammalian central neurons in particular. This channel is known to be an important component in modulating the frequency of electrical discharges in central nuclei such as the inferior olive or thalamus. These agents may be used in amounts sufficient to block or reduce specifically the LTCC, without (measurably) affecting the HTCC which is involved in dendritic action potentials, in certain components of the myocardium action potential, and in synaptic transmission. Thus, the present invention provides a method for selectively blocking or reducing LTCC and thereby permits isolation of the HTCC. One use of the present invention is, therefore, in iso¬ lating and studying HTCC unencumbered by the LTCC, i.e. in a manner akin to that using tetrodotoxin to block sodium conduct- ance.

According to the present inventors, the LTCC plays a basic role in the clocking properties of the brain, and pro¬ vides a basic frequency (continuous) for the coordination of movement (which is a series of discontinuous motions) . Tremor has been described as an exacerbation of the basic frequency of oscillation to the point of interference with the coordination of movement (Llinas, R.R. in Movement Disorders: Tremor (Findley, L.J. & Capildeo, R. Eds) pp. 165-182 Macmillan 1984, incorporated in this application by reference) . The present inventors have .further found that the same agents (e.g., aliphatic alcohols) used in the same low amounts also inhibit the symptoms of tremor. The importance of this finding is evident from the fac that enhanced physiological tremor, essential tremor, and th tremor produced in senile patients can be completely incapac itating. The method of the present invention is advantageou in that it does not affect HTCC of either the muscle or th neuron cells. The present invention provides a method fo treating the symptoms of tremor by acting on the central ner vous system rather than on the muscle that exhibits the tremo response, without affecting the neuron or muscle cell function that are associated with HTCC.

Nothing in the present disclosure or in the researc that culminated in the present invention can be construed t limit applicability of the LTCC-blocking effect of the prese invention to I.O. cells or even to neurons. Hence, the prese invention can be used with all cells that possess low-threshol calcium channels.

According to the present invention, aliphatic alcohol are effective in blocking LTCC and in inhibiting tremor extraordinarily low concentrations, which substantially reduc the risk of side-effects. Therefore, generally speaking, t lower the effective amount of particular alcohol, the mo desirable its use.

The degree of inhibition of the low-conductance calci channels appears to be related to the molecular weight of t alcohol (as well as the amount used) . As the molecular weig increases, the effective concentration of the alcohol necessa to reduce the low-conductance potential decreases. Hence, t calcium-conductance inhibition is greater as the alcohol m lecular weight is increased. This is demonstrated in detail Example 6, below, where it is shown that pentanol, at a 5-fo lower concentration, was more effective than propanol in i hibiting LTCC (compare Fig. 5, H with E) . Of course, the pr sent inventors do not wish to be bound by any theory, based the apparent relationship between LTCC blocking ability a alcohol molecular weight. The increasing Ca-channel-blocki ability of the higher alcohols may be due to many differe factors, such as the increasing hydrophobicity of alcohols wi increasing length of the hydrocarbon chain, and/or the lipid solubility of such alcohols.

The operability of the present invention has been dem¬ onstrated on living mammalian cells both in vitro and in vivo. The absence of any abnormal behavior in the test ani¬ mals employed to demonstrate the operability of the present invention provides a further strong indication that the risk of undesirable side-effects is very small.

In vitro, the cells are exposed to a culture or perfu- sion medium containing a LTCC-blocking effective amount of the alcohol. This amount is preferably sufficiently small so as not to interfere with HTCC. Of course, in view of the fact that different alcohols have different channel-blocking abili¬ ties, the amount will vary from alcohol to alcohol. Preferred highest molar concentrations (or preferred operative ranges for substantial and total blockage) for several of the alcohols used in the present invention are given in Table I below:

TABLE I

Alcohol From About To About

ethanol lθ^"3 lθ^"4 propanol 10^" 10 butanol lθ^"5 lθ^"6

—6 —7 pentanol 10 10 hexanol 10~ nd¹ heptanol 10 nd octanol 10~ nd nonanol 10 —6 nd decanol 10~ nd

With respect to the 10"⁶M figure given for hexanol through decanol above, it should be noted that the molarity could not be accurately determined. The perfusion solutions

^^■nd - not detectable 1. (or the parenterally administered compositions) employed in the experiments of the present inventors were made up by mixing an amount of alcohol with an amount of medium sufficient to make up a 10"⁶M solution, if all of the alcohol were soluble in the

5 medium. Hence, the concentrations employed were 10~⁶M at most. The minimum effective concentrations could not be specified because the solubility limits of these alcohols could not be accurately determined. Nevertheless, even if all of the C₆- alcohol was not soluble in the medium, it continued to be

10 effective, since control experiments employing medium that had not come in contact with alcohol did not result in either blockage of the LTCC rebound spike or in inhibition of tremor.

Moreover, the concentrations given in Table I above, are for complete blockage of LTCC. If partial blockage is

15 desired, (as is often the case in the treatment of tremor symp¬ toms) the amounts can be smaller, as determined by routine experimentation.

It is not to be assumed from the present discussion that the present invention is limited to use of aliphatic alco-

20 hols in solution. When alcohols are administered in vivo they can generally be administered orally, or parenterally in solid or liquid form with or without a carrier or diluent. Of course, particularly in case of oral administration, allowances should be made in the amount thus administered for any amount

25 of alcohol that is not absorbed in the alimentary tract, or that is metabolized before reaching the blood and the cerebro- spinal fluid.

Thus, the amount of the alcohol should be calculated to produce an effective LTCC-blocking (or LTCC-reducing) alcohol

30 concentration in the vicinity of the central nervous system cells, such as that given in Table I above. To this end, the concentration of the alcohol in the composition administered to the mammal will fall in the range at which it will produce the requisite alcohol concentration in the blood, (or in the cere-

35 brospinal fluid — CSF) as indicated in Table I, above for complete blockage. Preferably, the alcohol will be admini¬ stered in a composition, also comprising a physiologically acceptable carrier or diluent. Ringer's solution or isotonic saline are preferred diluents for parenteral administration.

Most preferred, is use of octyl alcohol in an amount sufficient to generate a blood or CSF (or perfusion medium) concentration of at most 10""⁶M.

The above amounts will both block LTCC and also inhibit tremors.

As stated above, the amount of the LTCC-blocking or tremor-inhibiting agent will vary according to the activity of the particular agent employed and according to the mode of administration. The frequency of administration may also vary according to the extent of the tremor symptoms and according to how often thoy occur.

Moreover, it may be desirable in certain cases to con- sider employing sustained delivery systems in order to maintain an LTCC-blocking or tremor-inhibiting concentration of the active agent in the patient's bloodstream.

Nevertheless, all of the above considerations concern optimization of the use of the present invention. The dosage and mode and form of administration can be fine-tuned by rou¬ tine and ordinary experimentation conducted by persons of ordi¬ nary skill in the field.

The present invention is further described below by reference to specific examples, which are intended to illus- trate the present invention without limiting its scope. In these examples, LTCC rebound spike was generated by each of the three methods involved in the operational definition given in the background section of the present application. Regardless of the method (voltage clamp, square current injection or depo- larization voltage) used to generate the RS, alcohols block this response, indicating that a single mechanism is at work and demonstrating the operability of the present invention.

Example l: Tissue Preparation Adult Hartley guinea pigs (400-600g) from Camm Research Institute, Wayne, New Jersey were decapitated, after ethe anesthesia, using a small animal guillotine. Immediately there after, two longitudinal sections were made along the latera edge of the squamous portion of the occipital bone. The resul ting bone slab was cut transversely, and pulled caudalward t expose the cerebellum and brain stem. Following transection o the cranial nerves and transverse section of the brain stem (a the inferior collicular level rostrally and at the level of C caudally) the brain stem was swiftly removed and placed i aerated Ringer's solution (containing 124 mM NaCl; 5 mM KC1 1.2 mM KH₂P0 ; 2.4 mM CaCl₂; 1.3 mM MgS0₄; 2.6 mM NaHC0₃ and 1 mM glucose) at about 5°C. It was then transected longitudi nally in a parasagittal plane and fixed to a Vibratome Model plate (available from Ted Pella, Inc., Tustin, CA.) in order t obtain thin longitudinal sections. From a single brain stem six 300-micron slices could be obtained. Following section ing, the slices were incubated in Ringer's solution for ap proximately one hour. The bathing medium was kept at roo temperature and a mixture of 95% 0₂ and 5% C0₂ was bubbled int the bath during this period. Brain stem slices were remove from the incubation bath after one hour but could be kept i good condition in the bath for periods of time up to 24 hours.

Example 2: Recording Chamber

Reference will be made to Figures 2A and 2B, which ar perspective views in section along line A-A of the perfusio and recording system used in the present experiments.

After incubation, a slice was transferred to recordin dish 1 where the tissue was continuously perfused with Ringer' solution at 37^βC. The perfusion system was gravity-fed, allow ing a routine flow of 0.5 ml/min. with a maximum of 2 ml/min The latter flow rate was used during solution exchange. Th central chamber 2 had a capacity of 2ml and the solution coul be completely exchanged in approximately ten minutes. Th saline flowed into the central chamber through three smal diameter channels 4 (one of which is shown in Fig. 2A) whic produced a close-to-laminar flow. Side-chambers 2a, whic communicated with chamber 2 via channels 4, were used as reser voirs for the perfusion saline. The outflow from the central chamber 2 was accomplished via a cotton wick system 3 which prevented turbulence by allowing continuous fluid movement (Fig. 2B) .

The brain slice was placed on a Sylgard plate 11 Corning Glass, Corning, N.Y.) at the bottom of the recording chamber 2 and secured with a bipolar stimulating electrode 5 pressing lightly on the brain tissue. The chamber was main¬ tained at 37^βC by a surrounding water bath 6, which kept it at the same temperature as the perfusing solution (37^βC) . The saline solution itself was temperature-regulated by passage through a heat exchanger 7 at 37^βC (Fig. 2A) .

The standard perfusion fluid was Ringer's solution; this medium was used during cutting and incubating, and during most of the recording time. Ringer's solution provided excel- lent pH-buffering properties at different temperatures. This was especially significant during the sectioning and incubation periods.

When desired, alcohols were added to the perfusion fluid by direct application into chamber 1 via the perfusion solution.

Example 3: Recording Techniques

Cells of the inferior olive were impaled with recording micropipettes 9 under direct vision using Hoffman modulation microscopy (Hoffman, R. J. Microsc.. 110: 205-222, 1977; incorporated by reference) which allowed good visualization of the unstained cells. In order to prevent vapor condensation on the objective lens of the microscope 14, a small suction spigot 8 was placed immediately to the side of its bottom surface (Fig. 1A) . Intracellular recordings were obtained with micro¬ pipettes, 9, filled with 3M potassium acetate or with 1M tetra- ethylammonium chloride (TEA) having an average D.C. resistanc of 60-80 milliohms. The micropipettes were connected to re¬ cording amplifier 12. Direct stimulation of the inferior oliv cells was implemented with a high-input impedance (10-- ohms) bridge amplifier. Capacity compensation allowed a frequenc response of 10-15 kHz, depending on the microelectrode proper ties.

Example 4: Blockage of Low-Threshold Calcium Conductance Wit Octanol Figure 3A-C (upper traces) shows intracellula recordings from inferior olivary (I.O.) nuclear cell demonstrating the typical calcium-dependent action potentia after the sodium-dependent spike was blocked with 10~⁶ tetrodotoxin (Sigma Chemical Co., St. Louis, MO). In this an all examples, I.O. cells were isolated as in Example l an recordings were made as in Example 3. The upper row in A, B, (controls conducted in the absence of octanol) shows the LTC rebound spike as the high spike. The HTCC is not presen because the applied current is subthreshold for the HTCC. Th records in the lower row in A, B, C show a complete blockage o the calcium-dependent rebound potentials due to the blockage o LTCC by the application of 10~⁶M octanol to the bath. This i also shown in the "action potential" v. "membrane potential graph D on the right of Figure 3 which demonstrates the rate o rise of the low-threshold action potential versus membrane pol arization in the control records (closed circles) . The lowe set of data points (squares) indicates the rate of rise of th low-threshold action potential after octanol at 10~⁶M is adde to the perfusion medium. The difference between the contro and the octanol graph is a measure of the blocking effect o 10~⁶M octanol on LTCC.

Example 5: Inhibition of LTCC by Butanol

Figure 4A depicts the action potential observed in I.O neurons under conditions where the sodium channel has bee blocked by the addition of tetrodotoxin. Incremental increase in injected square current are applied producing action poten tial responses shown in Fig. 4A. When a sufficient threshol value is reached, the characteristic spikes of the action po tential are produced. The first (left-most) spike is the high threshold calcium spike and the second (or rebound) spike i the low-threshold calcium spike. Figure 4B depicts the resultant action potential upon the administration of 10 M butanol and the inhibition of the LTCC or rebound spike. This effect occurs in the absence of any effect on the HTCC.

Example 6: Comparison of the LTCC-Blocking Ability and Relative Efficacy of Alcohols Figure 5 depicts a series of tracings, A through H, showing the I.O. cell response to an external square current injection following polarization of the membrane, both in the absence (graphs A-D) and in the presence (graphs E-H) of dif¬ ferent types and different concentrations of alcohol. Th reduction in the ratn of rise of the spike (first differential of the voltage, shown in the middle record) demonstrates th blocking effect of the alcohol on LTCC. This Figure illus trates that higher alkyl alcohols are more effective than lowe alcohols or, conversely, that higher alkyl alcohols are a effective but at lower concentrations.

The experiments giving rise to the data of Figure were conducted as described in Examples 1-3 except that square current was injected and different alcohols were used, as shown in this figure.

In each of the graphs A through H, the upper trace de picts the action potential observed across the membrane. Th middle trace shows the rate of change in the action potentia through the cell membrane. Finally, the bottom trace shows th magnitude of the injected current.

In graphs A-D, the cell is shown to have responded wit the characteristic low-threshold calcium spike (middle record) . By contrast, in graphs F through H, both the rate at which th action potential changes and its magnitude are significantl affected (decreased) by the addition of alcohol. Finally, i graph E, the action potential is lacking the characteristi spike appearance and resembles a subthreshold response. Comparing graphs E through H, it is observed that buta nol appears more than tavi.ce as effective as propanol, and tha pentanol appears more than five times as effective as butanol. The above results are all the more significant give that the injected current is 0.5nA in the alcohol-exposed cell but only 0.25nA in the control cells. This means that pentano is really over ten times as effective as butanol which is ove four times as effective as propanol. The relative effective ness increases with the molecular weight of the alcohol. Octa nol is preferred because its solubility in aqueous media cor responds closely to the most effective concentration for block ing the LTCC.

Example 7: Voltage Clamp Study of Octanol-Induced Blockage of LTCC The results presented in this Example were recorded i I.O. neurons from different parts of the nucleus. These con ductances were characterized by an inward current having a ver low threshold (-70mV) which could be observed after the sodiu conductance had been blocked by the addition of tetrodoxin an the potassium conductance had been blocked by addition of te traethyl-ammonium and cesium. In these experiments, the ioni current across the membrane was measured after a voltage clam pulse was applied across the membrane. In order to maintai the voltage constant, a current must be injected into the cell This current is equal and opposite to the ionic current acros the membrane and thus can be used to determine the size an duration of the ionic current directly. The ionic curren generates under normal conditions (not voltage-clamped) th low-threshold calcium spike.

The results in Figures 6A and 6B show the amplitude an time course of the low-threshold calcium ionic currents fol lowing depolarization steps of 5mV from a holding potential o -80mV to a value of -45mV. These records represent the differ ence in ionic current observed in the control state minus tha from the passive ionic current observed after octanol was in troduced in the bath. Thus, the records A and B illustrat those ionic currents that were blocked by octanol at 10^"- (wjr.'.-h completely blocked the ionic conductance) . An inwar currant is first observed at -65mV. As the voltage step is incrementally increased in amplitude, the calcium current reaches a peak at a membrane potential step of -50mV, and quickly reduces to null at -42mV, at which potential the calcium channels are inactivated. Figure 6C depicts a plot of ionic current against the applied external voltage. The value of this current is the difference between the ionic currents before and after octanol treatment. This difference is maximum at a membrane voltage between about -45 and about -60 millivolts. This graph charac- terizes the voltage dependence of the LTCC.

Example 8: Blockage of the Rebound Calcium Action Potential

This experiment will be described by reference to Fig ure 7, which shows the rebound calcium spike that follows mem brane hyperpolarization in the absence of alcohol (A) caused b injection of a square current pulse (C) is blocked by 10 —6 octanol (B) .

In this experiment, I.O. cells were treated as des cribeedd a<^•bove. The sodium spike was blocked by 10~ M tetrodo xin.

The graph D on the right of Figure 7 shows the depen dence of the rebound calcium spike on the amplitude of th injected hyperpolarizing current.

Example 9: Blockage of LTCC In Vivo By Higher

Alkyl Alcohols

In order to analyze the effects of LTCC-blocking agent in vivo, octanol was administered to rats. Alcohols wer diluted with physiological saline and sonicated to ensur mixing to create a concentration of alcohol around the neuron equivalent to 10 —6M (at most) . The alcohol was administere intraperitoneally at a concentration of 1 to lOmM. Tremo state was induced in the rats by injection of har aline, a alkaloid derived from Pegamus harmala which has been known t induce tremor in mammals. (Harmaline is available from Sigm

Chemical Co., St. Louis, MO.) The thus-induced tremor has periodicity similar to physiological or enhanced physiological tremor.

I.O. cells have been found to be responsible for trans mitting the tremor signal to mammalian muscles, since destruc tion of I.O. cells abolishes tremor in harmaline-treated ani als (Llinas, et al. , Science 190: 1230-1231, 1975). There fore, study of I.O. cells of mammals in which tremor has bee induced with harmaline provides an excellent in vivo model fo studying tremor response in mammals.

Tremor can be measured by electromyogram conducted ac cording to Buchthal, F. , et al., Acta Phvsio. Scand. 39: 83 104, 1957, incorporated by reference.

The electric activity in the left platysma muscle o 278 to 300 g Sprague-Dawley rats (from Taconic Farms, Germantown, N.Y.) was recorded differentially using two teflon coated stranded silver wires (2 mm of wire exposed) placed i the belly of the muscle. The electromyogram (emg) was recorde throughout the experiment and the data were analyzed at th following times: (1) before administration of any drugs; (2) after intraperitoneal injection of 15 mg/kg harmaline; and (3) after subsequent intraperitoneal injection of 2 σc of 10 mM 1 octanol (99% octanol sonicated in physiological saline) give to create a concentration of 9.3 micrograms/g of rat bod weight or 7 x 10 -5M m. the vi.ci.ni.ty of the neuronal cells (i all the alcohol was to be absorbed, and none metabolized) . Th emg was smoothed and differentiated (to further decrease th background noise) using a Nicolet Explorer Model 4060 digita oscilloscope (from Nicolet Instrument Corp. , Madison, WI) . This instrument was also used to obtain autocorrelograms o 800-ms sections of the emg's. Autocorrelograms were obtaine by superimposing 800-ms sections of the emg signals. Th emerging patterns (if any) can be used to determine frequenc and amplitude characteristics of the emg that may not b readily discernible from the naked emg signal.

Control emg's demonstrated occasional periods of lo amplitude oscillations (upper left, Figure 8) . Autocorrelatio revealed an 8 Hz periodicity in t e emg, but with a low cor relation function (upper right, Figure 8) . After harmaline injection, the animals demonstrated periods of generalized tremor. The emg recorded during such a period is illustrated in the middle left-hand trace of Figure 8; the tremor had a dominant frequency of 7.5 Hz, close to the control value, and showed a high degree of autocorrelation (middle right-hand trace). After injection of of 1-octanol, the tremor stopped and the baseline muscle activity was reduced below control levels (bottom left-hand trace) . The dominant frequency was at 10 Hz, and the emg amplitude was so low during this period that it was necessary to amplify the signal in order to obtain an autoσorrelogram (bottom, right) . Although a 10-Hz periodicity is evident (as shown by the arrows) , the 60-Hz frequency due to line voltage (amplifier noise) is the dominant correlation due to the large gain. If the bottom right signal had been ampli- fied only to the extent of the two upper autocorrelograms, it would have appeared as a straight line.

It is evident that, if one wanted to merely decreas the tremor to normal or manageable levels, the amount of alco¬ hol administered would have to be decreased.

Claims

WHAT IS CLAIMED IS:

1. A method for partially or totally blocking low threshold calcium channels in cell membranes comprising expos ing said cells to an effective amount of a low-threshold calcium-conductance blocking agent.

2. A method for partially or totally blocking low threshold calcium channel^~_in mammalian cell membranes com prising exposing said cells to an aliphatic alcohol at a con centration sufficient to partially or totally block low threshold calcium conductance in said cells.

3. The method of claim 2, wherein said alcohol i selected from the group consisting of C₂-Cχo alkyl alcohols an mixtures thereof.

4. The method of claim 2 wherein said concentration i insufficient to have a noticeable effect on the high-threshol calcium conductance of said cell.

5. The method of claim 2, wherein the concentration o said alcohol is up to about 10~³M for ethanol; up to about 10 ⁴M for propanol; up to about 10"⁵M for butanol; up to about 10 ⁶M for pentanol; and at most about 10"⁶M for higher alcohols.

6. The method of claim 2, wherein said alcohol i octyl alcohol, and the concentration of said alcohol is at mos about 10"⁶M in the vicinity of said cells.

7. The method of claim 3, wherein said alcohol i octyl alcohol.

8. The method of claim 2, wherein said cells ar neuron cells.

9. The method of claim 8, wherein said neuron cells are inferior olivary cells.

10. The method of claim 2, wherein said method is con¬ ducted in vivo.

11. The method of claim 10 comprising administering said alcohol to said mammal in an amount sufficient to generate said concentration in the vicinity of said cells.

12. The method of claim 11, comprising administering to the mammal a composition comprising an effective amount of said alcohol and a physiologically acceptable carrier or di¬ luent.

13. The method of claim 11, comprising administering said alcohol parenterally.

14. The method of claim 11, comprising administering said alcohol orally.

15. The method of claim 12, wherein said composition comprises a diluent selected from the group consisting of Ringer's solution, and isotonic saline solution.

16. A method for selectively partially or totally blocking low-threshold calcium channels in a mammal comprising administering to said mammal a low-threshold calcium-channel effective blocking amount of a compound selected from the group of C₂ - C₁₀ alkyl alcohols and mixtures thereof.

17. A method for inhibiting the manifestation of tremor in a mammal in need of such treatment, the method com¬ prising: administering to said mammal a tremor-inhibiting effective amount of a low-threshold calcium conductance block¬ ing agent.

18. A method for inhibiting the manifestation of tre mor in a mammal in need of such treatment comprising:

administering to said mammal an aliphatic alcoho in an amount effective to partially or totally impede th transmission of a tremor-generating electrical signal from th central nervous system of said mammal. to the muscle cells o said mammal that would otherwise exhibit said tremor.

19. The method of claim 18, wherein said amount is insufficient to interfere noticeably with the high-threshol calcium conductance in either the central nervous system cell or the muscle cells of said mammal.

20. A method for inhibiting the manifestation o tremor in a mammal in need of such treatment comprising select ively partially or totally blocking the low threshold calciu channel of the central nervous system cells of said mammal pos sessing said low-threshold calcium channel.

21. The method of claim 18, wherein said alcohol i selected from the group consisting of ethanol, higher alky alcohols, and mixtures thereof.

22. The method of claim 21, wherein said alcohols ar ^c3 " ^c10 alkyl alcohols.

23. The method of claim 18, wherein said cells ar inferior olivary cells.

24. The method of claim 18 comprising administering t said mammal a composition comprising a low-threshold calciu channel blocking effective amount of an alcohol selected fro the group consisting of ethanol, higher alkyl alcohols an mixtures thereof, and a physiologically_.acceptable carrier o diluent.

25. The method of claim 21 comprising administering an amount of said alcohol sufficient to generate a concentration of said alcohol in the bloodstream or cerebrospinal fluid of said mammal up to about 10"⁴M for propanol; up to about 10^"5M for butanol; up to about 10"⁶M for pentanol; and at most about 10"⁶M for higher alcohols.

26. The method of claim 18 comprising administering said alcohol orally.

27. The method of claim 18 comprising administering said alcohol parenterally.

28. The method of claim 22 wherein said alcohol is octyl alcohol.

29. The method of claim 25, wherein said alcohol is n- octanol.

30. The method of claim 18 comprising administering to said mammal a composition comprising an effective amount of said alcohol and a physiologically acceptable carrier or dilu¬ ent.

31. The method of claim 30, wherein said composition is a liquid composition and said diluent is selected from the group consisting of Ringer's solution and isotonic saline.

32. A composition for partially or totally blocking low-threshold calcium conductance in mammalian cells comprising an effective amount of a low-threshold calcium conductance blocking agent and a carrier or diluent.

33. The composition of claim 32, wherein said agent is selected from the group consisting of aliphatic alcohols and mixtures thereof.

34. The composition of claim 33, wherein said alcohols are alkyl alcohols.

35. The composition of claim 34, wherein said alcohols are alkyl alcohols having from three through ten carbon atoms.

36. The composition of claim 32, wherein said amount is insufficient to have a noticeable effect on the high- threshold calcium conductance of said cells, said composition being useful for selectively partially or totally blocking low- threshold calcium conductance.

37. The composition of claim 36, wherein said agent is an alcohol.

38. The composition of claim 37, wherein said alcohol is octyl alcohol and said amount is sufficient to generate a concentration of said octyl alcohol in the vicinity of said cells of at most about 10"⁶M.

39. The composition of claim 37, wherein said alcohol is selected from the group consisting of alkyl alcohols having from three to ten carbon atoms, and said amount is sufficient to generate a concentration in the vicinity of said cells of at most about 10"⁴M for propanol; about 10"⁵M for butanol; about 10~-M for pentanol and higher alcohols.

40. The method of claim 18 comprising administering to said mammal an amount of said alcohol effective to reduce said tremor to normal or manageable levels.