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Publication numberUS3898339 A
Publication typeGrant
Publication dateAug 5, 1975
Filing dateAug 14, 1973
Priority dateAug 14, 1973
Also published asCA1027863A1, DE2427732A1
Publication numberUS 3898339 A, US 3898339A, US-A-3898339, US3898339 A, US3898339A
InventorsHerbert J F Adams, Jr Murray R Blair, Robert N Boyes, Maxim I Lebeaux, Helen G Vassall
Original AssigneeAstra Pharma Prod
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Spinal anesthesia using small amounts of tetrodotoxin or desoxytetrodotoxin
US 3898339 A
Vertebrates can be anesthetized regionally by a subarachnoid administration of tetrodotoxin or desoxytetrodotoxin. The amount of tetrodotoxin which should be administered in a preferred embodiment ranges between 1 and 12 micrograms, whereas the amount of desoxytetrodotoxin ranges between 10 and 120 micrograms.
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United States Patent Adams et al.


Murray R. Blair, Jr., Sudhury; Robert N. Boyes, Auburn; Maxim I. Lebeaux, Shrewsbury; Helen G. Vassall, Worcester, all of Mass Astra Pharmaceutical Products, Inc., Worcester, Muss.

Filed: Aug. 14. I973 Appl. No.: 388.147


US. Cl 424/251; 424/!05 Int. Cl A61k 27/00 Field of Search .r 424/105, 251

References Cited OTHER PUBLICATIONS Chemical Abstracts, Vol. 77 (1972), p. l68634y.

Primary Exuml'ner-V. D. Turner Attorney Agent, or Firm-Brumbaugh, Graves, Donohue & Raymond [57] ABSTRACT 5 Claims, 1 Drawing Figure SPINAL ANESTHESIA USING SMALL AMOUNTS OF TETRODOTOXIN OR DESOXYTETRODOTOXIN The present invention relates to a method of obtaining spinal anesthesia (i.e., by injection of an anesthetic into the subarachnoid space which contains the cerebro-spinal fluid) and a pharmaceutical preparation which can be used with this method.

Toxins from marine sources of extraordinary potency have been known for many years. This invention particularly concerns novel uses for tetrodotoxin, and for its derivative, desoxytetrodotoxin.

Tetrodotoxin is obtained from the ovaries and eggs of several species of puffer fish of the suborder Gymnoa'onles. It is also found in certain species of California newts of the genus Taricha; and the toxin obtained from these species, known as tarichatoxin, is identical with tetrodotoxin. Tetrodotoxin has been purified, and its molecular structure is determined to be an amino perhydroquinazoline of the formula:

Tetrodotoxin and species in which it occurs are more fully described in P/mrnuuulugical Reviews, Vol. 18, No. 2, at pages 997-1049. Some of the effects of tetrodotoxin and its derivatives, e.g., desoxytetrodotoxin, are described in Chemical Abstracts. Vol. 37, p. l77O (I943) and Vol. 69, Column 9494w (1968).

Experiments with isolated nerves have shown that tetrodotoxin behaves in a fundamentally different manner from local anesthetics such as procaine and cocaine. In a voltage-clamped giant axon from the squid or lobster, the latter agents reduce both inward initial sodium current and outward potassium current. With tetrodotoxin, however, inward sodium current can be reduced or even obliterated, while the outward potassium current is totally unaffected. There are few if any other substances in which this unique action has been established.

Tetrodotoxin has not heretofore found any practical use as an anesthetic. While the compound can be used to induce nerve blocks in laboratory animals, the anes thetic dose is slightly below the lethal dose, which has precluded, as a practical matter, the use of the com pound as an anesthetic in its own right. M. H. Evans, Tuxicon, I968, Vol. 5, pp. 289294. tested its use topically on peripheral nerve branches and exposed dorsal and ventral spinal roots in anesthetized cats, and found blocks in the latter case but concluded that the impermcability of the connective tissue sheath of peripheral nerves seemed likely to preclude its use as a local anesthetic agent.

Combinations of tetrodotoxin with a local anesthetic compound have been found to possess unusual anesthetic properties. This is manifested most significantly in improved longevity of action of combinations of the toxin with local anesthetics. ln these combinations. tetrodotoxin is typically used in concentrations below those which produce reliable nerve blocks. The site of administration of the tetrodotoxin and anesthetic is outside the dura of the spinal canal, i.e., a peridural administration. It is theorized that the local anesthetic en ables the tetrodotoxin or desoxytetrodotoxin to penetrate the sheath surrounding the nerve roots and produce local anesthesia.

The present invention relies upon a subarachnoid spinal administration into the intact mammal of a composition which consists essentially of small amounts of tetrodotoxin or desoxytetrodotoxin. In this case, it is not therefore necessary to combine these compounds with (Zwitter'ion form) a local anesthetic, and it has been found that very low doses of the compounds give a longer duration of anesthesia than is obtainable with the use of certain prior art anesthetics of differing composition. For example. it is possible to administer a very low dose of 1-1 2 micrograms (pg) of tetrodotoxin or 10-120 micrograms of desoxytetrodotoxin and obtain a spinal block of between 3 and 24 hours in duration. Thus, subarachnoid anesthesia in mammals, including man, is contem plated. in the case of man, it is believed, based on animal tests, that a minimum dose of l #g of tetrodotoxin is effective to produce anesthesiav By contrast, subarachnoid administration of tetracaine (at larger doses of 3-20 mg) gives a block of from 1.5 to 3 hours and xylocainc (at mush larger doses of SO-ISO mg) gives blocks of from l to 2 hoursv P. C. Lund. Principles and Practice of Spinal Anesthesia, pp. 420 and 425, Charles C. Thomas (publisher), l97l. These known spinal anesthetics cause more irritation and have a greater local toxicity than the composition used in the present invention.

It has been found that the administration of the compounds used in the present invention should be deliv ered into the subarachnoid area of the spinal column for effective anesthesia to be induced. A particularly preferred dosage range is from 3 to 6 micrograms of tetrodotoxin and 30 to 60 micrograms of desoxytetrodotoxin.

procedure to be performed.

The following examples show the invention in practice:

EXAMPLE I water as the vehicle for the drug. lsobaric solutions are 5 usually prepared by using cerebrospinal fluid as the vehicle but sodium chloride added in a suitable concen- This example illustrates Pharmaceutical P tration to an aqueous solution of the compound can tiOnS and methods for P p g thcm which 3T6 yp also be used to obtain isobaricity. Hyperbaric solutions baric Solutions Containing 1 can be prepared by addition of varying amounts of glulo and Mg/ tClfOdOlOXifl hy r h ide; p cose (usually 5lO%) to a water solution of the com pounds. A hyperbaric solution, the most commonly The tetrodotoxin hydrochloride is dissolved in a Concentration [Lg/ml l 2 l 4 5 s 8 10 Component Amount Tetrodotoxin hydrochloride (mg) 10 I00 Hydrochloric acid.

Sodium hydroxide.

Waiter for injection USP XVIII If necessary to adjust pH If necessary to adjust pH Sufficient quantity to make l0 liters small amount of water and is diluted to a volume of 9.5 liters. The pH is adjusted and a sufficient volume of water is added to make a solution of l0 liters which is then sterilized.

EXAMPLE 2 This example illustrates pharmaceutical composiand thereby the extent of anesthesia in a manner that 30 lions and methods for preparing them which are isois well known in the art. The acidity of the solutions debaric solutions containing 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, scribed above is kept inside limits that give good stabil- 8.0, and 100 ug/ml tetrodotoxin hydrochloride; pH ity to the tetrodotoxin or desoxytetrodotoxin. 3,5 5

Concentration Lg/ml l 2 3 4 5 6 8 10 Component Amount Tetrodotoxin I0 20 3O 40 50 60 80 100 hydrochloride (mg) Sodium chloride 90 90 9o 90 90 90 90 USP XVII (gj Hydrochloric acid,

If necessary to adjust pH Sodium hydroxide,

If necessary to adjust pH Water for injection USP XVIII Sufi'icient quantity to make l0 liters.

If desired. addition of a vasoconstrictor, e.g. epinephrine, to the pharmaceutical preparation can also be made in manners that are well known in the art. The concentration of active ingredient is generally 1-1 2 mi crograms/ml for tetrodotoxin and 10-120 miero- 50 and thereafter a sufficient volume of water is added to grams/ml of desoxytetrodotoxin and volumes of apmake a solution of 10 liters which is then sterilized. proximately l-6 ml of solution are injected into the ce- EXAMPLE 3 rebrospmal fluid contained in the subarachnoid space This example illustrates pharmaceutical composi of the vertebrate to be anesthetized. The type of solugs ions 211nd methods for pmparing than. which are tion to be used, the concentration and volume to be adperbuflc sojumms Containing L0 20, 30 40 50 8'0 ministered are dependent on such factors as, for lll and Lg/m] tetmdmoxin hydrochloride. pH stance age and size of the patient and on the surgical Concentration ug/ml l 2 1 4 i 5 8 10 Component Amount Tetrodotoxin I (l 20 30 40 50 50 80 I00 hydrochloride (mgl Dextrose,

anhydrous 500 500 500 500 500 750 500 500 USP XVIII (gl Hydrochloric acid. 2N If necessary to adjust pH Sodium hydroxide,

Water for injection USP XVIII The tetrodotoxin hydrochloride is dissolved in a small amount of water and diluted to 9.0 liters. The pH is adjusted. The dextrose is added and dissolved. A suf ficient volume of water is added to make a solution of liters which is then sterilized.

Other forms of tetrodotoxin such as salts of other acids than hydrochloric acid or such as the base or zwitterion form can be used and solutions can be pre pared containing amounts equivalent to those of tetro dotoxin hydrochloride given in Examples l3.

In the case of desoxytetrodotoxin. compositions are made in the same manner as shown in Example l3 for tetrodotoxin. except that the concentrations of biotoxin are l0 to I00 #g/ml, because desoxytctrodotoxin is about one-tenth as active as tetrodotoxin.

minutes of the injection with recovery occurring before I7, I95 and 23 hours had elapsed from the time of the injection, respectively. in the animals. In two animals full recovery occurred within 28 hours, while in the third some ataxia was noted for an additional [2 to 24 hours with full recovery within 48 hours from the time of injection. The spread of the anesthesia towards the head (ccphalad spread) was on the average of 4 to 5 segments from the point of injection. Excessive cephalad spread is indicated by signs of Horners syndrome. head drop. abdominal breathing, cyanosis and front limb paresis. None of these signs were observed. Anesthesia in the lower region of the spinal column (caudal region) was complete. Table I sets forth the results obtained.

Table l Spinal Anesthesia in Sheep Drug: Tetrodotoxin Dose: 3 pg 0l.: 1 ml. Injection site: L6-Sl Duration Vulva or Spontane Anal Scrotum Digital our Muscle Full Sheep Wt. Onset Re Re Re- Activity Weight Re- No. Sex (k) (min) flex Pain flex Pain flex Pain Rear Leg Support covery 190 190 l90 l90 190 190 190 I F 29.3 4 90 min. hr.

hr. hr. hr. hr. hr. hr. hr.

420 420 420 420 420 420 420 2 M 28.9 4 60 min. 28 hr.

hr. hr. hr. hr. hr. hr. hr.

320 320 320 320 320 320 26 hr. 3 M 29.3 4 200 min. 195

Notes Time "I minutes except where indicated. Carrier: 7.5'1 dextrose in saline H=t.0|

EXAMPLE 4 EXAMPLE 5 Three sheep weighing between 28 and 29.5 kg and of approximately 4.5 to 5 months of age were treated with tetrodotoxin (3 micrograms). All three sheep were restrained with their lumbar and thoracic spine in horizontal position during the subarachnoid injection. which was made over a period of 5 to l0 seconds using a 22 gauge-3 inch spinal needle at the interspace between the sixth lumbar and first sacral segments. The sheep were then immediately tilted so that the slope of the lumbar and thoracic spine was positioned 10 to the horizontal (caudal inferior).

The response to the tetrodotoxin was a rapid onset of anesthesia (within less than 4 minutes, usually within 2 to 3 minutes), with a long sensory block followed by complete recovery. Although the limitations of the normal working day interfered with exact measurements of the duration of the block, it was determined that the sensory blocks were still present after 190. 320 and 420 The procedure used in Example 4 was employed using a 6 microgram dose of tetrodotoxin. In the three sheep treated with this dose level. the onset of the block was rapid. The blocks lasted for longer than 6.5 hours but less than 24 hours. In one case the block lasted exactly 24 hours. Weight support required 48-72 hours for recovery and complete recovery usually required 72-86 hours. One animal had a persistent slight impairment of one joint that lasted 18 days but which eventually returned to normal. This was due to inflammation from causes other than drug-related causes. Cephalad segmental spread was slightly more exaggerated than the spread observed when animals were treated with 3 micrograms. Regression of two segments required 2-3 hours on the average. The Table given below summarizes the test results:

Table II Tetrodotoxin: 6 pg/ml (total dose 6 pg) VoL: l.(] ml. Duration Spontaneous heap Onset Anal Vulval-Scrotal Digital Rear-leg Weight Full Wt. Time Re- Pain Reflex Pain Reflex Pain Motor Activity Support Recovery flex No. 4 6 fi /z 6& 24 hr. 24 hr. 24 hr. 48 hr. 72 hr.

hr. hr. hr. hr. Fe- 3 min.


24 hr. 24 hr. 24 hr. 24 hr. 48 hr. 48 hr. 48 hr. 8.2k No. 5 l0 hr. l0 hr. l0 hr. l0 hr. l0 hr. I0 hr. l(J hr. 72 hr. 72 hr. Fe- 3-5 male min 24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 96 hr.

Table ll-Continued Tetrodotoxini 6 pg/ml (total dose 6 .Lg] Vol. |.U ml.

Duration Spontaneous heep Onset Anal Vulval'ScrotaI Digital Rear'leg ll/eight Full Wt. Time Pain Reflex Pain Reflex Pain Motor Activity Support Recovery No. Slight Fe- 3 min. 24 hr. 24 hr. 24 hr. 24 hr. 26 hr. 26 hr. 24 hr. 72 hr. knuckling male right fet- 6.8k

lock- I 8 days Not drug related Nu untoward systemic side cficcts were noted EXAMPLE 6 l5 scissa or X axis of FIG. I, the anesthesia wears off beginning antcriorly (cephalad) (because less drug has A l2 microgram dose was tested on one sheep weighreached these nerves) and progresses postcriorly (cauing 5L8 kg. Table III sets forth the results: dally) until total recovery occurs. Each point value in Table III Tetrodotoxin: I2 ug/ml (total dose I2 #gj Vol.1 1.0 ml. Duration Sheep Anal VuIvaI-Scrotal Digital Spontaneous Onset 4 Rear-leg Weight Full Weight Time Reflex Pain Reflex Pam Reflex Pain Motor Activity Support Recovery No. 7 10 hr. 10 hr. 10 hr. 10 hr. l0 hr. 48 hr. 96 hr.

3 min. 24 hr. 24 hr. 5 [.Bkg. hr. 20 hr. 20 hr. 20 hr.

FIG. 1 is a graph showing the spread and effect of the w FIG. I therefore represents the level of anesthesia at a subarachnoidally administered local anesthetic, versus I particular time and also the total number of spinal segtime after administration. ments which are blocked at that time. FIG. 1 compares Using the technique described in the foregoing Exthe level of anesthesia and the segmental regression amples 4-6, the local anesthetic drug to be tested was pattern obtained in sheep after administration of each injected into the subarachnoid space (which contains 39 of the three local anesthetics. Tetracainc, the most the cerebrospinal fluid), between the six lumbar and commonly used spinal anesthetic in humans, in these the first sacral vertebral interspace of the sheep. Each experiments anesthetized only a total of 6 or 7 segof three animals received I.() ml of 0.25% tetracaine or ments and regressed (the animal recovered) com- 0.5% tetracaine or 1.0 ml containing 6 (4g of tetrodopletely in between 3-5 hours. Tctrodotoxin, on the toxin. The most commonly used concentration of tetra- 40 other hand, at concentrations far below those of tetracaine in spinal anesthesia, which is the most commonly caine blocked a total of between 9-12 segments for at used anesthetic in humans, as indicated by Lund (prinleast 6 hours and did not regress completely for about cipals and Practice of Spinal Anesthesia, C. C. Thomas 24 hours.

Publishers, Springfield, Ill., (I971) p. 420) is 0.25% It is observed from these experiments, as illustrated 0.3%. Above this concentration the drug becomes ingraphically in FIG. I, that tctracaine at concentrations crcasingly irritating and is not generally used. In man of 0.25 and 0.5 percent would provide anesthesia and the injection is usually made between either the second pain relief in segments below 5-3 for only about 3 and third or third and fourth lumbar interspaccs. The hours. Tetrodotoxin. however, provided anesthesia and drug, after injection, mixes with the spinal fluid and is pain relief below 5-3 for at least I5 hours.

carried or spread anteriorly and postcriorly. Recovery of function in the animals tested was com- On the Y" axis (the ordinate) of FIG. I is a scheplete and no scquelac followed the long duration of anmatic representation of the segments of the spinal cord. B izl CuUSCd y lrodotoxin.

In the sheep, caudal to cephalad (posterior to anterior) Upon reading the foregoing description, persons in the segments of the spinal cord arc: coccygcal 3,2,]; the art will become aware of a number ofmodiflcations sacral 4,31,]; lumber 6,5,4,3 2,l; thora i that can be made to the invention described herein I3,l2,l l,l0,9,8,7,6, 5,4,3,2,I; cervical 7,65,43,11. without the exercise of inventive skill. These modifica- Thoracic 13 is the only one of the last two groups illustions are intended to be included within the scope of trated or represented in FIG. 1. the invention. and the foregoing is intended to be Depending on many factors such as on ent i merely illustrative otccrtain preferred embodiments of volume of solution, manipulation of the patient and in no the im'em'mn Th6 pp Claims dQlmL the Scope of herent characteristics of the drug, a variable degree of Pmmctlon Soughtduration and spread of anesthesia can be achieved. We claim:

This spread and its recovery or so-callcd regression is I. A process for inducing spinal anesthesia in a mampictorially represented as in FIG. 1. Above (or cepha mail comprising administration by injection into the lad to) each point there is no anesthesia and the spinal SU bllfaClmO'lCl PQC f the intact mammal from about nervgs um ff md b th d B l d l to] l to 6 milliliters of a pharmaceutical composition comeach point all spinal segments or nerves are blocked. prising a compatible vehicle and as the active ingredi- Over a period of time, which is represented on the abcnt a compound selected from the group consisting of tetroclotoxin in concentration of l to l2 micrograms per milliliter and desoxytetrodotoxin in concentration from 10 to I micrograms per milliliter of the vehicle.

2. The process as claimed in claim 1 wherein the amount of tetrodotoxin which is administered is from about 1 to l2 micrograms.

3. The process as claimed in claim I wherein the amount of desoxytctrodotoxin which is administered is

Non-Patent Citations
1 *Chemical Abstracts, Vol. 77 (1972), p. 168634y.
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US5846975 *Mar 11, 1995Dec 8, 1998Nanning Maple Leaf Pharmaceutical Co., Ltd.Use of amino hydrogenated quinazoline compounds and derivatives thereof for abstaining from drug dependence
US5942543 *Dec 16, 1996Aug 24, 1999Ernst; Amy A.Topical anesthetic comprising lidocaine, adrenaline, and tetracaine, and its method of use
US6030974 *Apr 2, 1998Feb 29, 2000The Regents Of The University Of CaliforniaTopical administration of a long-acting sodium channel blocker, e.g.,tetrodotoxin, saxitoxin; ocular surface anesthesia of long duration without impairing healing; contact lens delivery; excimer laser surgery for myopia
US7001734Nov 6, 1995Feb 21, 2006Merck & Co., Inc.Process for identifying para cation channel modulators
EP1930027A2 *Nov 19, 2001Jun 11, 2008Nanning Maple Leaf Pharmaceutical Co., Ltd.A composition of sodium channel blocking compound
WO1995024903A1 *Mar 11, 1995Sep 21, 1995Nanning Maple Leaf PharmaceutiThe use of amino hydrogenated quinazoline compounds and derivatives thereof for abstaining from drug dependence
WO1996014860A1 *Nov 6, 1995May 23, 1996Merck & Co IncProcess for identifying para cation channel modulators
WO2002041915A1 *Nov 19, 2001May 30, 2002Yuhong KangA composition of sodium channel blocking compound
U.S. Classification514/267, 424/559, 424/581
International ClassificationA61K31/495, A61K31/505, A61K9/00, A61K47/46
Cooperative ClassificationA61K9/0085, A61K31/495, A61K47/46
European ClassificationA61K47/46, A61K9/00M22, A61K31/495