The pharmacology and toxicology of mepivacaine, a new local anesthetic

TOXICOLOGY

The F. P.

AND

APPLIED

2, 295-315

PHARMACOLOGY

(1960)

Pharmacology and Toxicology of Mepivacaine, a New Local Anesthetic LUDUENA,

JAMES

Sterling-Winthrop

0.

F.

HOPPE,

Research Received

COULSTON,

Institute, December

AND

Rensselaer,

New

H. P.

DROBECK

York

7, 1959

Recently, mepivacaine,l 1-methyl-2’,6’-pipecoloxylidide hydrochloride (see Fig. 1)) was introduced as a local anesthetic (DhunCr et al., 1957 ;

MEPIVACAINE

FIG.

1.

Structural

formula

for

mepivacaine.

Eckenstam et al., 1956). This new drug which has been studied clinically in Europe, and has now been introduced in this country, has been found effective in many types of local anesthetic procedures. In comparison with lidocaine, mepivacaine produces anesthesia of longer duration (Ekenstam et al., 1956). Frahm (1958) found that mepivacaine was slightly more active intradermally than lidocaine, but was less active topically. Mumford and Gray (1957) obtained adequate dental anesthesia in a high percentage of caseswith a solution of 2.05s mepivacaine HCl without a vasoconstrictor. Mepivacaine has been studied in this laboratory by various methods; the results are reported in this paper. 1 CarbocaineB Drug Inc.

HCl,

(brand

of mepivacaine)

295

is the registered

trademark

of Sterling

296

F. P.

LUDUENA

ET

AL.

METHODS

Local anesthetic activity was determined by intradermal injection in guinea pigs (Biilbring and Wajda, 1945; Luduena and Hoppe, 1956), cornea1 instillation (Luduena and Hoppe, 1955) and intraspinal injection in rabbits (Bieter ‘et al., 1936a,b; Luduena, 1957a). Concentrations graded at 0.3~log intervals were tested on eight or more animals. The mean anesthetic score or the mean duration of anesthesia was plotted against the log of the concentration, and the threshold anesthetic concentration, (TAC,) was estimated from the linear dose-effect curves. The TACB is the concentration expected to produce anesthesia of 5 minutes’ average duration or an average anesthetic score of 5. Mixed solutions of local anesthetics and vasoconstrictors were tested by the intracutaneous wheal method. Dilutions of the original solution graded at 0.15log intervals were tested on twelve or more guinea pigs (dilution test). The threshold anesthetic dilution, ( TADn) was calculated from the dilution-effect curve in the same manner as the TAC;, (Luduena, 1955). Duration of anesthesia was determined by intradermal injection in guinea pigs, using the technique and the scoring system devised by Biilbring and Wajda, with the exception that readings were taken every 10 minutes, from the time of injection, and continued until the mean anesthetic score was less than 50% of the maximal possible score (Luduena, 1957b). The mean score for each reading (maximal score = 6) was plotted against time. The mean duration represents the time in minutes at which the mean score reached 50% of maximal anesthesia (score = 3) as determined from the mean score-time curves by interpolation. The mean duration determined in this manner (mean,), is not identical to the mean duration of anesthesia of the individual wheals (mean,). In a previous study (Luduena, 1960) in which both means were calculated in sixteen experiments, the difference between the two values did not exceed 15%. Topical anesthetic activity was determined by instillation of solutions of concentrations graded at 0.3-log intervals into the conjunctival sac of rabbits (Luduena and Hoppe, 1955). Each solution was tested on ten rabbits. Spinal anesthesia was produced in rabbits by the method of Bieter et al. (1936a,b). Duration of urethral anesthesia and presence and absence of paraplegia were recorded.

STUDIES

ON

297

MEPIVACAINE

Acute Toxicity The acute toxicity determinations were made in male, albino, Webster mice weighing 18-22 g; New Zealand albino rabbits of mixed sex, weighing 2-3 kg; and guinea pigs, mixed sex, weighing 250-300 g. At least three dose-levels, graduated at logarithmic intervals, were used for each determination. The number of animals used for each dose level: mice, 10; rabbits, 5; and guinea pigs, 10. The LDso with its standard error was calculated at 24 hours after injection by the method of Miller and Tainter (1944). The acute intravenous toxicity of mepivacaine HCl, lidocaine HCl, and procaine HCI in distilled water was determined following injection at 10 ml/kg, at 1.0 ml per minute in the mouse. Each dose was adjusted to a volume of 10 ml/kg and the results expressed in terms of milligrams per kilogram body weight. The acute intravenous toxicity of solutions B and C (Table 1) was determined after injection of graded dilutions at 10 TABLE 1 MEPIVACAINE FORMULATIONS

Formula Mepivacaine HCI Levo-nordefrin basea Acetone sodium bisulfite Sodium chloride, reagent Water for injection USP, q.s. ad Initial pH

Solution 3.00 g 0.30 g 100.00 cc 4.75

A

Solution

B

2.00 p 1: 20,000 0.20 g 0.44 g 100.00

4.4

cc

Solution

C

2.00 g 0.5 g 100.00

cc

4.8

u Zevo-l-(3,4-Dihydroxyphenyl)-l-hydroxy-2-propylamine.

ml/kg in the mouse and was then expressed in terms of milliliters of undiluted solution per kilogram of body weight. The acute intravenous toxicity of solutions B and C was determined after injection of graded dilutions at 0.5-1.26 ml/kg per minute in the rabbit and I-1.58 ml/kg per minute in the guinea pig. The acute subcutaneous toxicity of mepivacaine HCl was determined following injection at 10 ml/kg in the mouse and 1 ml/kg in the rabbit and guinea pig. Each dose, in distilled water, was adjusted to a volume of 10 ml/kg for the mouse and 1 ml/kg for the rabbit and guinea pig and expressed in terms of milligrams per kilogram of body weight. The acute subcutaneous toxicity of solutions B and C, in terms of milliliters of undiluted solution per kilogram of body weight, was determined after

298

F.

P. LUDUENA

ET

AL.

injection of graded dilutions at 10 ml/kg in the mouse, 2.6-10 ml/kg in the rabbit, and 2.6-10 ml/kg in the guinea pig. Half the acute intravenous LDSo of mepivacaine, lidocaine, and procaine was injected intravenously every half hour for eight consecutive doses in the mouse. Each compound was administered in aqueous solution as the hydrochloride salt. Subacute Toxicity The subacute toxicity was determined in male or female rhesus monkeys and in male albino Sprague-Dawley rats. Two formulations of mepivaCaine were injected into the test animals: one, solution B, contained mepivacaine at a 2.0%’ concentration and levo-nordefrin, 1:20,000; the other, solution A, contained mepivacaine at a 3.05% concentration and no levo-nordefrin. Complete formulations are presented in Table 1. Both formulations were administered intramuscularly or subcutaneously eighteen times in 21 days. Subacute toxicity in monkeys. Five monkeys were injected intramuscularly with solution A and another five monkeys were injected with solution B. The monkeys weighed between 2 and 3.5 kg each. The volume of each dose was 0.5 cc. The injected dose was approximately five to ten times the estimated human dose on a body weight basis. An additional group of five monkeys served as controls, and were injected intramuscularly with RNN,” a widely used commercial dental anesthetic. The injection sites were alternated between the hind legs so that each leg was injected every other day. Hematologic studies were done on the monkeys before the start of medication, and on the fifth and twenty-first days of the experiment. Urinalyses were done before medication and at the end of the experiment. Blood pressure determinations were done on the fifth and twenty-first days of the experiment, using a cuff sphygmomanometer. Two monkeys of each group were sacrificed at the end of the experiment. The injection sites and major organs were examined macroscopically, and portions of the tissues were fixed in Zenker-formalin. The tissues were embedded in paraffin, sectioned at 6u, and stained by the hematoxylin and eosin method. In addition, frozen sections of kidney and liver tissues were stained for fat by the Clark-Scharlach staining method. 2 RavocaineO HCl, 0.4% (brand of propoxycaine HCI), and Novocain@ 2.07% (brand of procaine HCl), with Neo-Cobefrin@ 1:20,000 (brand of levo-nordefrin). Registered trademarks of Sterling Drug Inc. Formula manufactured by Cooke-Waite Laboratories, Inc.

STUDIES

ON

MEPIVACAINE

299

Subacute toxicity in rats. Four groups, ten rats in each, were injected subcutaneously with one of the two mepivacaine formulations each: either at 0.1 cc/kg (approximately) or at 1.0 cc/kg. A fifth group, the controls, were injected with sterile distilled water at 1.0 cc/kg. Hematologic studies were done on the rats 2 days before the end of the experiment on the nineteenth day of medication. Systolic blood pressure of the rats was determined with a photoelectric tensometer at 2-3 hours after the injections on the nineteenth day of the experiment. At the end of the experiment the rats were sacrificed with ether. All major organs and the injection sites were examined macroscopically. Pieces of the tissues were prepared for histologic examination in the same manner as mentioned previously. Irritancy Intradermal irritancy was determined in rabbits by the trypan blue method (Hoppe et al., 1950). Five to ten rabbits per concentration and a minimum of three concentrations graded at 0.3-log intervals were used. The threshold irritant concentration, (TICa) was estimated from the dose-response curve obtained by plotting the mean irritation score against the log of the concentration (Luduena and Hoppe, 1952). The standard trypan blue test cannot be used to determine the irritancy of solutions containing vasoconstrictors because the local ischemia that follows the injection either prevents or greatly reduces the amount of trypan blue reaching the wheal. In the delayed trypan blue test (Hoppe, 1960), the dye is injected 3 hours instead of 30 minutes after the intradermal injections, as in the standard test. Solution B and a mixed solution of lidocaine were tested by the delayed trypan blue test. The mucous membrane irritancy of mepivacaine and lidocaine HCl was determined by the rabbit eye irritation method (Hoppe et al., 1950; Draize et al., 1944). Intramuscular irritation in the rabbit. Two groups of three adult albino rabbits were injected intramuscularly with I.O-ml doses of either 2.02 or 3.07c mepivacaine HCI. The formulations of both preparations are presented in Table 1. Each muscle injection site was used only once. The rabbits were sacrificed at l-, 2-, or ‘I-day intervals. The injection sites were examined macroscopically, fixed in Zenker-formalin, and embedded in paraffin. Histologic preparations were made in the usual way for microscopic examination.

300

F. P. LUDUENA

ET

AL.

RESULTS

Anesthesia Local anesthetic activity. Mepivacaine HCI was tested in parallel with procaine HCI using three concentrations of each drug and a total of 56 guinea pigs (Table 2). By plotting the mean score against the log of the concentration parallel dose-effect curves were obtained (Fig. 2). TABLE INTRADERMAL

Mepivacaine Mepivacaine Mepivacaine Procaine Procaine Procaine

HCI HCl HCl HCl HCI HCl

a Biilbring

and

Wajda’s

(GUINEA

PIGS) a

Number positive/ Number tested

Local anesthetic score (mean)

0.2 0.1 0.05

24/24 22/24

24.1 7.6 0.25

0.5 0.25 0.125

24/24 18/24

24.3 8.6

l/8

0.15

Concentration (5%)

Compound

2

ANESTHESIA

(1945)

2/a

Procaine HCI ratio

TAC, (%I 0.07

2.5

0.17

1.0

technique.

FIG. 2. Local anesthetic activity of mepivacaine hydrochloride hydrochloride by the intradermal wheal method in guinea pigs.

and

pro1 caine

STUDIES

ON

301

MEPIVACAINE

The threshold anesthetic concentration5 (TAG) of mepivacaine HCl and procaine HCl were 0.07% and 0.1770, respectively. By thk test, mepivacaine HCl is 2.5 times more active than procaine HCI. A solution of 2.0% mepivacaine HCl + levo-nordefrin 1: 20,000 (solution B) was tested in comparison with a dental anesthetic solution of lidocaine containing 1: 100,000 epinephrine. More than 200 guinea pigs were used. In the majority of the experiments the two solutions were tested in parallel. The results are presented in Table 3. The ANESTHESIA

INTRADERMAL

Solution mepivacaine 2% + levo-nordefrin

Dilution

Number positive/ Number tested

TABLE 3 (GUINEA

PIGS)

B: HCl, 1: 20,000 Local anesthetic score (mean)

l/20 l/28.2 l/40

S3/56 67/72 23/40

a Biilbring

and Wajda’s

28.3 14.2 3.8

,a DILUTION

Solution lidocaine 2 % + epinephrine

TAD, l/38

Dilution l/20 1128.2 l/40

TEST

L: HCl, 1: 100,000

Number positive/ Number tested

Local anesthetic score (mean)

56/56 49/64 22/24

24.8 14.4 4.8

TAD, l/39

technique.

threshold anesthetic dilution 5 (TADS) was approximately the same: 1:38 for solution B and 1:39 for the lidocaine solution. In other words, the local anesthetic activity of the two solutions was approximately the same, as determined by this test. At the TAD5 the concentration of mepivacaine HCl was O.OS$%. The intradermal TA&, for mepivacaine alone, was approximately 0.07%. The presence of the vasoconstrictor produced only a slight increase in activity. Solutions of mepivacaine and lidocaine containing epinephrine were tested in parallel for duration of anesthesia. The following values (in minutes) were obtained: Experiment 1 (10 guinea pigs). 2.0% lidocaine HCl: meanl, 31, and mean2 f S.E., 32 -t- 1.8; 2.0% mepivacaine HCl: meanl, 58, and mean2 f S.E., 66 k-7.3. Experiment 2 (8 guinea pigs). 2.070 lidocaine HCl + epinephrine 1: 100,000: meanI, 200, and mean, $ S.E., 215 t 17; 2.0% mepivacaine HCI + epinephrine 1:100,000: meanl, 256. (The mean2 value could not be calculated because two of the wheals raised with this solution were still anesthetized when the

302

F.

P. LUDUENA

ET

AL.

last reading was taken, 330 minutes after injection. Taking the minimal value for these two wheals, the mean:! 2 SE. would be 277 t 14. ) Cornea1 anesthesia. Mepivacaine HCI was tested at three concentration levels by instillation on the rabbit eye. Cocaine HCl was tested in the same manner on the other eye. The concentrations tested, the number of animals, the anesthetic scores, and the TACn values are shown in Table 4. The dose-effect curves from which the TAG values were obtained are shown in Fig. 3. TABLE CORNEAL

Compound Mepivacaine Mepivacaine Mepivacaine Cocaine Cocaine Cocaine

HCl HCI HCl HCl HCl HCI

4

ANESTHESIA

Concentration (%I

Number positive/ Number tested

0.5 0.25 0.125

9,:lO S/IO 3/10

0.5 0.25 0.125

lO/lO 7/10 3/10

0.1

Cone.

GmAOO

(RABBITS)

Mean duration of anesthesia (min)

TAC, (%b)

Cocaine HCl ratio (cocaine HCl = 1)

16.8 5.1 2.4

0.18

1.0

18.6 6.5 2.9

0.17

1.0

ml

FI c. 3. Topical anesthetic activity of mepivacaine hydr .ochloride by cornea1 application in the rabbit.

hydrochloride

and

cc

.ine

STUDIES

ON

MEPIVACAINE

303

Spinal anesthesia. Four concentrations of mepivacaine HCl (2, 3, 4, and gy,) were tested by the method of Bieter et al. (1936a,b; Luduena, 1957a). The SC/, solution was made up in distilled water. With the exception of one of the 45% solutions used, which was made up in saline and tested on six rabbits, the other solutions were made up in 4.5%) dextrose. The results obtained are shown in Table 5. Results obtained previously with procaine HCl and lidocaine HCI (Luduena, 1957a) are included in this table. Rabbits have very little spinal fluid (Bieter et al., 1936a,b) and, therefore, concentrated solutions of local anesthetics may produce local damage at concentration levels that are not harmful when injected in man by the same route. However, an estimate of relative intraspinal irritancy can be obtained by this method of administration. Previous studies have shown that the irritancy ranking of compounds by the trypan blue method is in general agreement with that obtained by intraspinal injection, although the TIC4 is lower than the smaller concentration that produces permanent paralysis. Increasing duration of anesthesia, measured by the time the urethral reflex was absent after injection, was produced by increasing the concentration of the solutions. Although the number of animals injected with lidocaine is small, the results appear to agree with those obtained by the trypan blue test, i.e., lidocaine is somewhat more irritating than mepivacaine HCl. Toxicity Acute toxicity. The acute toxicity of mepivacaine HCl alone and in a mixed solution with levo-nordefrin is shown in Table 6. Values for the LDS,, f S.E. of procaine, lidocaine, and a lidocaine solution containing epinephrine are included in this table. In mice, mepivacaine HCl is approximately twice as toxic as procaine HCI and 40% less toxic than lidocaine HCl. In the mouse, both mepivacaine HCl and lidocaine HCl are twice as toxic as procaine HCl. The toxicity of mepivacaine HCl, administered i.v. to mice, guinea pigs, and rabbits is not changed by the addition of one part of levonordefrin to 400 of the local anesthetic. Administered by subcutaneous injection in mice, the mepivacaine HCllevo-nordefrin mixture (solution B) is significantly more toxic than mepivacaine HCl alone. In rabbits and guinea pigs mepivacaine alone is more toxic, and this can be explained by a delay in the absorption

HClb

HCI

HCl

5 4

4

6 -

-

2

paralyzed

-

-

2

Delayed

-

Deaths Acute

5 IN RABBITS

which died after the third day, having been leg paralysis at the time they were sacrificed. lidocaine base was used in this experiment.

5

4

8

8

3.5 4.6

55.5 92.0

17419 22-85 67-115

36.4

6 59

2542

Range

3 4

31.4

Mean

Duration of urethral anesthesia (min)

10

Number of rabbits

TABLE ANESTHESIA

2

(%‘o,

a This group includes two rabbits survived up to 1 week and had hind 1, Calculated as the HCI. Actually

Lidocaine

Procaine

Mepivacaine

Compounds

Concentration

SPINAL

4

1

2

up to that

-

-

> 16 hours < 48 hours

--.

Hind

time

-

and

2

1

2

4

11

-

Number

rabbits

Permanent paralysis

leg paralysis (duration)

%

40

25

0 50

that

0 0 19 5011

$

z

z

2 2

r w

HCl

HCI

Mepivacaine Solution C Solution B

Mepivacaine Solution C Solution B

a The

Lidocaine

composition

of solutions

B and

C is shown

Subcutaneous Subcutaneous Subcutaneous

Guinea Guinea Guinea

in Table

Intravenous Intravenous

Intravenous

Subcutaneous Subcutaneous Subcutaneous

Rabbit Rabbit Rabbit pig pig pig

Subcutaneous Subcutaneous Subcutaneous

Mouse Mouse Mouse

Mouse Mouse

HCl

Mepivacaine Solution C Solution B

Intravenous Intravenous Intravenous

pig pig pig

Guinea Guinea Guinea

Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous

Mouse

HCl

Mepivacaine Solution C Solution B

1.

TABLE HCL,

Route of administration

OF MEPIVACAINE

Rabbit Rabbit Rabbit

HCl

HCI

Mepivacaine Solution C Solution B

Mouse Mouse Mouse

Species

TOXICITY

Solution L (lidocaine HCI 2% + epinephrine 1: 100,000) Procaine HCl

HCl

Mepivacaine Solution 0 Solution Ba

Compound or solution

ACUTE

6 LIDOCAINE

30 150

30

40 40 40

15 20 15

40 40 40

30 30 30

15 15 15

30 30 40

Number of animals

HCL,

1.0 z!z 0.1 -

-

20 i: 2 57 * 1

23 2 2

94 f 10 93 & 9 179 5 19

136&21

6.8 2 1.0 4.6 -t 0.45 8.9 -+ 0.95

110 -c 11 99 t 12

260 51 22 300-+30 184 2 16 4.9 r+ 0.6

15.0 -t 1.5 9.2 c+ 0.8

1.4

6.l 0

K G % % e z

2

20 k

24.5 2 2.4 24.5 -I 1.3

1.22 3- 0.12 1.20 f 0.06

s F; VI

21 k 1.6 20.5 xk 2.1

1.05 ” 0.08 1.02 2 0.1

2

22-+-2

-

32 & 2 31 ” 2 30 f 3.4

1.55 c+ 0.1 1.50 _’ 0.2

LD,, + S. E. calculated as Local anesthetic (w&c)

HCL

Undiluted solution (ml/kg)

AND PROCAINE

EFFECT

Mepivacaine HCl Lidocaine HCl Procaine HCI

Compound

COMPARATIVE

REPEATED

16.0 10.0 30.0

Dose l/2 LD,,, (w/k)

OF

TABLE

7

o/10 o/10 o/10

1 O/IO o/10 l/10

2 o/10 o/10 z/10

3

4

o/10 o/10 3/10

at dose number 5

Mortality

o/10 o/10 3/10

Observed

o/10 l/10 3/10

6

INTRAVENOUS INJECTION OF HALF THE MOUSE INTRAVENOUS FOR EIGHT CONSECUTIVE DOSES IN THE MOUSE

7

-

o/10 2/10 3/10

LD,,

l/10 2/10 3/10

8

AT 3%MINUTE

l/10 2/10 3/10

Final mortality at 24 hours after injection

INTERVALS

F .

$

z

z 2

w

r

STUDIES

ON

307

MEPIVACAINE

of the mepivacaine from the subcutaneous depot due to vasoconstriction. An explanation for the toxicity pattern in mice is not immediately apparent. The addition of epinephrine 1: 100,000 to a 2.0% solution of lidocaine HCl does not increase the intravenous toxicity in mice. As shown in Table 7, repeated administration of one-half LDSo of mepivacaine, lidocaine, and procaine have very little cumulative effect, only one, two, and three out of each group of ten mice, respectively, dying within the period of 4 hours. It is obvious that in the case of mepivacaine and lidocaine the mice are able to dispose of one-half LDSo within 30 minutes by a mechanism (or mechanisms) other than pseudocholinesterase action. The rate of hydrolysis of the amide group by this enzyme is extremely slow in comparison with the ester linkage. Subacute toxicity in monkeys. All the monkeys injected intramuscularly with solution A or solution B eighteen times in 21 days were normal in appearance and behavior throughout the experiment. No significant changes were observed in the body weights of the monkeys. The average body weights are presented in Table 8. The values for the hemoglobin concentration, the hematocrit, total red and white blood cell counts, and the differential counts were normal throughout the TABLE THE

EFFECT AND

OF MEPIVACAINE RATS

8

ox

MEDICATED

BODY

WEIGHTS

18 TIMES

IN

21

OF MONKEYS DAYS

Average

body

weights

At Dose”

-~

Medication

(cc)

Number Species

group

At start

kg Solution Solution RNNb

B A (controls)

0.5 0.5 0.5

Monkeys Monkeys Monkeys

&l/kg 1 .O/kg o.l/kg 1 .O/kg l.O/kg

Rats Rats Rats Rats Rats

5 5 5

3.16

10 10 10 10 10

126

2 s4 2 so g

Solution Solution Solution Solution Dist. H,O

B B A A (controls)

a Administered h Ravocainem,

127 126 126 126

intramuscularly into monkeys and subcutaneously HCl 0.4% and Novocain@ 2.0% with Neo-Cobefrin@

21

days

Change (%I

kg 3.25 2.55 2.55 g 287 274 278 265 286 into

+ 2.8 + 0.2 + 2.0

+ 128 + 116 + 120 +

110

+ 127 rats. 1:20,000.

308

F. P.

LUDUENA

ET

AL.

experiment. The hematologic data of the premeditation counts and of the counts done at the end of the experiment are presented in Table 9. No pathologic changes attributabIe to medication with solution A or solution B (Table 1) were observed in the four monkeys sacrificed and autopsied at the end of the experiment. The frozen sections of liver and kidneys stained for fat were normal. No lesions were observed in the intramuscular injection sites of these monkeys except for small hemorrhagic spots in the most recent injection sites. The blood pressure readings of the medicated monkeys taken on the fifth and twenty-first days of the experiment were within the range of normal, not medicated monkeys. No significant differences were observed in the blood pressure readings of the medicated monkeys at 1 or 3 hours after the intramuscular injections as compared to the readings just prior to the injections. Subacute toxicity in rats. AlI the rats injected subcutaneously with solution A or solution B at a dose level of 0.1 cc/kg were normal in appearance and behavior throughout the experiment. The rats injected at the l.O-cc/kg dose level were normal except for three rats in the solution A and one rat in the solution B groups. These rats became dyspneic after an injection, but dyspnea was not observed after the injections on subsequent days. No obvious difference was observed between the average body weight gain of the rats medicated with the low dose of solution B and that of the control rats. The average body weight gain of the rats medicated with solution B at 1.O cc/kg or with solution A at 0.1 cc/kg or 1.0 cc/kg was shghtly reduced as compared to that of the control rats. These rats at 21 days gained on the average 110% to 120% body weight as compared to a 1275% body weight gain in the controls. These data are presented in Table 8. The average hemoglobin concentrations, the hematocrit, the total red and white blood cell counts, and the differential counts of the rats medicated with the mepivacaine preparations were normal at the end of the experiment. The hematologic data are presented in Table 9. The systolic blood pressure readings of the medicated rats were similar to those of the control rats. No microscopic or macroscopic lesions attributable to medication with mepivacaine were observed in the rats autopsied at the end of the experiment. However, small areas of inflammation in various stages of healing were observed in the subcutaneous tissue of the injection sites.

B A

Rats

1 .O/kg

l.O/kg

Monkeys Rats Rats Rats Rats

Monkeys Monkeys

Species

ON

0.5 &l/kg 1 .o/kg O.l/kg

0.5 0.5

of Experiment:

Dose” (cc)

cm MEPIVACAINE

10

5 10 10 10 10

5 5

group

Number in

AVERAGE

TABLE

44

46

45 46 4S

46

45

44

44 13.3

13.6

P 14.2

43

POE

9

E

P

E

h

E

13.4

6.6

15.5

16.2 18.1

20.3 16.4

17.6

20.9

17.5

6.7 7.4

17.8

17.9

14.4

16.0 15.4

5.9

6.1

5.7

6.8 6.6

5.7

5.6

5.8

16.4 15.4

14.9

14.8

14.1

AND

White blood cells ( 10s/mms)

OF MONKEYS

Red blood cells (lOa/mms)

COUNTS

Hemoglobin (g/100 cc)

BLOOD

46

(%b)

Hematocrit

PERIPHERAL

P

31

36

36

E

P

19

81

81

19

59 88

66

1

1

2

0

1 0

0 0

0

1

0

i 9 pig22

counts

18 TIMES

EPEPEPE 56

82 83

66

59

62

;

18 16

38 12

31

41

4 p

Differential

MEDICATED

i 2 gg

RATS

(1 Administered intramuscularly into monkeys and subcutaneously into rats. b P = premeditation blood counts. In rats, blood counts were done only at the end of the experiment. (’ See Footnote b of Table 8.

(controls) Solution B Solution B Solution A Solution A Dist. H.,O (contr&)

RNN”

Solution Solution

Days

EFFECT

Medication

THE

2

3

1

0

0

0 0 0

3

2

3

i : .-.s

(%)

0

0

0

0

0

0 0 0

0

0

0

& : -

IN 21 DAYS

310

F.

P.

LUDUENA

ET

AL.

Irritancy By the routine trypan blue test, mepivacaine HCl was found to be approximately 25-30s more irritating in rabbits than procaine HCl. [A TIC4 value of 4.6%’ for procaine HCl was obtained in a previous investigation (Luduena and Hoppe, 1952) .1 In a parallel test with TABLE INTRADERMAL

Mepivacaine Concentration (%o)

Mean irritation scorea

8.0

7.2

4.0

4.4 2.0

2.0

1.0 n5

0.2

-

IRRITANCY

10

(RABBITS),

HCI TICdb (%I

TRYPAN

Lidocaine Mean irritation scorea

-

BLUE

HCl TIC,0 (%I

TEST

Relative irritation (lidocaine HCI = 1.0)

-

3.3

-

-

6.0 4.8 1.6

1.9

= -

0.0

-

-

-

MC

0.58

-

a Each solution was tested on ten rabbits. b Threshold irritant concentration4. c M = mepivacaine HCl.

FIG.

dermally

4.

Irritancy of mepivacaine by the trypan blue test.

hydrochloride

and

lidocaine

hydrochloride

intra-

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lidocaine HCl (Table lo), the latter was found 70% more irritant than mepivacaine HCI (the dose-effect curves are shown in Fig. 4). A mixed solution of mepivacaine HCl + levo-nordefrin (solution B) tested by the delayed trypan blue test was found to be less irritating than a solution of 2% lidocaine HCl + epinephrine 1: 100,000, as measured by the irritation score and by the presence of petechiae (Table 11). [These two solutions were equally active when tested on a large number of guinea pigs by the intracutaneous wheal test (Table 3).] INTRADERMAL

IRRITANCY

TABLE 11 (RABBITS), Number of test areas

TRYPAN

BLUE

Average irritation score

TEST

Adjective rating

Solution

Procedure

Mepivacaine HCl 2% Solution A Solution B Lidocaine HCI 2.0% + epinephrine 1: 100,000 (solution L)

Routine Routine Delayed

5 5 5

2 .o 3.6 2.4

Mild Mild Mild

0 0 0.8

Delaved

20

4.3

Moderate

5.3

RABBIT

TABLE I2 EYE IRRITATION TEST. TOPICAL Concentratio@

Compound Mepivacaine Lidocaine

(%‘o, HCl

HCl

Petechiae

APPLICATION

Maximal irritation scoreb

Adjective rating

2.0 3.6 1.2 2.8

Mild Mild Mild Mild

10 20 10 20

a Each solution was tested on five eyes (five rabbits). the other compound was tested on the other eye. b Drake et al. (1944).

The

same

concentration

of

Mepivacaine HCl and lidocaine HCl in 10% and 20% solutions, applied to the rabbit eye (eye irritation test) produced only a slight degree of irritation (Table 12). Intramuscular irritation in the rabbit. No microscopic or macroscopic lesions were observed in the intramuscular injection sites of solution A or solution B in the rabbit at 7 days after the medication. One or two days after the injections, the intramuscular injection sites were char-

312

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ET

AL.

acterized by a slight inflammatory reaction, consisting of edema, occasional foci of coagulative necrosis, and slight leucocytic infiltration. DISCUSSION

The experiments described have shown that mepivacaine is a local anesthetic of greater potency and relatively less irritancy than procaine. The low irritancy of procaine is well known. When intradermal irritancy was measured by the trypan blue method, the moderate degree of skin injury which is expressed as the threshold irritant concentration, (TI&) was produced by procaine HCl at a concentration of 4.6% (estimated from the dose-effect curve, Luduena and Hoppe, 1952) and by mepivacaine HCI at a concentration of 3.3%. In other words, mepivacaine HCl is 25-30% more irritating. However, mepivacaine HCl is 2.5 times more active as a local anesthetic than procaine HCI (intradermal anesthesia, Biilbring and Wajda’s method), and therefore it could be used at lower concentrations than procaine to produce the same anesthetic effect. In regard to duration of action mepivacaine differs from lidocaine and procaine. Injected intradermally in guinea pigs the duration of anesthesia produced by mepivacaine was twice as long as that following the injection of lidocaine, both drugs being tested in parallel at the 2.Ocj: concentration level. When epinephrine was added to both solutions, a parallel test demonstrated that the anesthesia produced by the mepivacaine solution was 25r/c longer than that of the lidocaine-epinephrine solution. A longer duration of local anesthetic action is equivalent to a slower rate of diffusion of the anesthetic from the injected site. Vasoconstrictors added to local anesthetic solutions reduce the diffusion rate into the capillaries by decreasing the local circulatory rate. A slower diffusion of mepivacaine in comparison with procaine and lidocaine may be the explanation for the effective dental anesthesia obtained by various investigations with solutions of mepivacaine without a vasoconstrictor (Bezzemberger and Stellmach, 19.57; Harnisch, 1957; Berling, 1958). Relatively longer duration of local anesthetic action is commonly associated with a relatively greater irritancy. This is not the case when mepivacaine and the chemically related compound, lidocaine, are compared: the former is less irritating than lidocaine and significantly longer acting. The addition of a vasoconstrictor to mepivacaine solutions, as is the case with other local anesthetics, increases greatly its duration of action, but produces only a slight increase in activity. These results are similar

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313

to those obtained with solutions of propoxycaine containing vasoconstrictors (Luduena, 1960). Our experimental results showing that, by intravenous injection in mice, mepivacaine is almost twice as toxic as procaine and somewhat less toxic than lidocaine, confirm the results obtained by Eckenstam et al. (1956) and Ulfendahl (1957), but not those of Truant and Wiedling (1959), who found an LD5” value for lidocaine higher than those reported by other investigators. The experimental results described in this paper in regard to the local anesthetic activity, the low irritancy, and the duration of action of mepivacaine, are in general agreement with clinical results obtained on a large total number of cases by DhunCr et al. (1957), Griesser (1957), Dam and Guldmann (1957), Berling (1958), Frahm (1958), and other investigators (reviewed by Wessinger, 1960). SUMMARY The local anesthetic activity, toxicity, and irritancy of mepivacaine HCll (1-methyI2’,6’-pipecoloxylidide hydrochloride) and a solution (solution B) containing 2.0% mepivacaine HCl and 1:20,000 levo-nordefrin (levo-2,4-dihydroxyphenyl-3-hydroxy2-isopropylamine2) have been studied by various methods. By the intracutaneous wbeal test in guinea pigs mepivacaine HCl is 2.5 times more active than procaine HCl. The addition of levo-nordefrin produced a slight increase in the local anesthetic activity of the solution. Solution B was found as active as a solution of 2.0% lidocaine + epinephrine 1: 100,000 by the same route of administration (dilution test). At the 2.0% concentration level in solutions containing epinephrine l:lOO,OOO the duration of mepivacaine intradermal anesthesia was approximately 25% longer than that produced by lidocaine. Mepivacaine HCl was found to be as active as cocaine HCI by topical application to the rabbit eye. The topical TAC, was 0.18%. At 2, 3, 4, and 8% concentrations, mepivacaine HCI injected intraspinally in rabbits produced anesthesia lasting from 17 to 115 minutes (urethral anesthesia). The degree of intraspinal irritancy appears to be somewhat lower than that of lidocaine. By intravenous injection the following LD,, values for mepivacaine HCl in mice, rabbits, and guinea pigs were obtained: 32 -+ 2, 22 &2, and 20 -C 1.4 mg/kg, respectively. Mepivacaine is approximately twice as toxic as procaine HCl and 40% less toxic than lidocaine HCl in mice. The subcutaneous LD,, values were: 260 & 22, 110 -C- 11, and 94 51 10 mg/kg for mice, rabbits, and guinea pigs, respectively. Solution B and a 3% solution of mepivacaine HCl without levo-nordefrin (solution A) were well tolerated by monkeys and rats when injected intramuscularly or intravenously in single daily doses eighteen times in 21 days. No significant changes were observed in the body weights, blood counts, urinalyses, or blood pressures of the

314

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LUDUENA

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medicated animals at the end of the medication period. No changes attributable to medication with mepivacaine were observed at the autopsies of the medicated animals. By the trypan blue test the threshold irritant concentration4 of mepivacaine HCl was 3.3%. That of lidocaine HCl, which was tested in parallel, was 1.9%. LidoCaine HCl is therefore approximately 70% more irritant than mepivacaine HCl. Applied topically on the rabbit eye, mepivacaine HCI and lidocaine HCI were well tolerated in concentrations up to 20%. Solution A and solution B produced a slight, transient inflammatory reaction when injected intramuscularly into rabbits in l.O-cc doses. No lesions at the injection sites were observed at 7 days after the injection. In summary, mepivacaine HCl, which is 2.5 times more active than procaine HCI is only 25% more irritating and is well tolerated locally as well as systemically on repeated subcutaneous or intramuscular administration. ACKNOWLEDGMENTS The technical and Mr. Zigurd

assistance of Mr. Leon Duprey, Miss Mielens is gratefully acknowledged.

Lois

Jewell,

Mrs.

Ellen

Miller,

REFERENCES BERLING, C. (1958). Carbocaine in local anesthesia in the oral cavity. Odontologisk Revy. 9, 254-267. BEZZEMBERGER, O., and STELLMACH, R. (1957). Klinische Untersuchungen iiber des Deb. zahniirztl. Z. 12, 1596-1597. neues Lokalanasthetikum “Carbocain.” BIETER, R. N., CUNNINGHAM, R. W., LENZ, O., and MCNEARNEY, J. J. (1936a). Threshold anesthetic and lethal concentrations of certain spinal anesthetics in the rabbit. J. Pharmacol. Exptl. Therap. 57, 221-244. BIETER, R. N., MCNEARNEY, L. J., CUNNINGHAM, R. W., and LENZ, 0. (1936b). On the duration of spinal anesthesia in the rabbit. J. Pharmacol. Exptl. Therap. 57, 264-273. BULBRING, E., and WAJDA, I. (1945). Biological comparison of local anesthetics. J. Pharmacol. Exptl. Therap. 85, 78-84. DAM, W., and GULDMANN, N. (1957). Carbocaine-a new local anesthetic. Acta Anaesthesiol. &and. 1, 101-104. DHUN~R, K. G., OLJELUND, O., and AAGESEN, G. (1957). Carbocain-dZ-N-methylpipecolic acid-2,6-dimethylanilide-a new local anesthetic agent. Acta Chir. Sand. 112, 350-35s. DRAIZE, J. H., WOODARD, G., and CALVERY, H. 0. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exptl. Therap. 82, 377-390. ECKENSTAM, B., EGNER, B., ULFENDAHL, L. R., DHUN~R, K. G., and OLJELUND, 0. (1956). Trials with Carbocaine; a new local anaesthetic drug. &it. J. Anaesthesia 28, 503-506. FRAHM, M. (1958). Beitrage zur pharmakologischen Auswertung neuer Lokalanalgetica. Anaesthetist 7, 44-46.

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GRIESSER, G. (1957). ijber ein neues Lokalanaesthetikum. Deut. med. Wochschr. 82, 2071-2073. HARNISCH, H. (1957). Die ijrtliche Betiubung ohne gefbskontrahierenden Zusats. Zahniirztl. Welt u. Zahniirztl. Reform 58, 271-273. HOPPE, J. 0. (1960). Delayed trypan blue test. To be published. HOPPE, J. O., ALEXANDER, E. B., and MILLER, L. C. (1950). The use of the trypan blue and rabbit eye tests for irritation. J. Am. Pharm. Assoc. Sci. Ed. 39, 147-151. LUDUENA, F. P. (1955). Ravocaine and Sympocaine, new local anesthetics. Anesthesiology 16, 751-769. LUDUENA, F. P. (1957a). Experimental spinal anesthesia. Arch. intern. pharmacodynomie 109, 143-156. LUDUENA, F. P. (1957b). Local effect of various drugs on the duration of infiltration anesthesia produced by dental anesthetic solutions. J. Dental Research 36, 613-622. LUDUENA, F. P. (1960). Effect of some vasoconstrictors on intradermal anesthesia. J. Dentat Research in press. LUDUENA, F. P., and HOPPE, J. 0. (1952). Local anesthetic activity, toxicity and irritancy of 2.alkoxy analogs of procaine and tetracaine. J. Pharmacol. Exptl. Therap. 104, 40-53. LUDUENA, F. P., and HOPPE, J. 0. (1955). Cornea1 anesthetic activity and toxicity of some alkoxy analogs of thiocaine and related compounds. J. Am. Pharm. Assoc. Sci. Ed. 44, 393496. LUDUENA, F. P., and HOPPE, J. 0. (1956). 2-Alkoxy benzoate and thiol-benzoate derivatives as local anesthetics. J. Pharmacol Exptl. Therap. 117, 89-96. MILLER, L. C., and TAINTER, M. L. (1944). Estimation of the ED,, and its error by means of logarithmic-probit graph paper. Proc. Sot. Exptl. Biol. Med. 57, 261-264. MUMFORD, J. M., and GRAY, T. C. (1957). Dental trial of Carbocaine; a new local anaesthetic. Brif. 1. Anaesfhesiu 29, 210-216. TRUANT, A. P., and WIEDLING, S. (1959). A contribution to the pharmacological and toxicological evaluation of a new local anaesthetic, dl-N-methylpipecolyl-2,6xylidide. Acta Chir. &and. 116, 351-361. ULFENDAHL, H. R. (1957). Some pharmacological and toxicological properties of a new local anesthetic, Carbocaine. Acta Anesthesiol. &and. 1, 81-86. WESSINGER, G. D. (1960). Mepivacaine-a potent new local anesthetic. To be published.