Acra pharmacul. el iuxicul. 1979, 44, 103-109
From the Department of Clinical Pharmacology and Pharmacology, Linkoping University,
S-581 85 Linkoping, and AB Draco*. Research and Development Laboratories, Fack, S-221 01 Lund, Sweden
Physico-chemical Modification of Lidocaine Uptake in Rat Lung Tissue BY C. Post, R. C. C. Andersson. A. Ryrfeldt and E. Nilsson (Received may 22. 1978; Accepted August 14, 1978)
Abstr'at'r: The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of thequaternary analogue, methyllidocaine, was not changed in the pH interval 7.0-8.0. The uptake of the three other substances was about 3-4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed catiodbase ratio was bupivacaine>etidocaine>lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of14C-lidocainein lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced ''C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while thecationic form binds to accessible binding sites in the cell membranes. Key-wjurrls: Interaction
- local anaesthetics - lung uptake - pH - rat.
Previous studies on the distribution of lidocaine in tissues have shown that the lungs were able to accumulate lidocaine extensively (Akerman rf a/. 1966; Keenaghan & Boyes 1972; Post ei a/. 1976 & 1978; Bertler ff al. 1978). Further analysis showed that the accumulation was depressed by low temperature but not by anaerobic conditions. The extent of the uptake of lidocaine was also partly dependent on the level of Ca"-ions in the incubation medium. N o metabolism of lidocaine took place in the lung preparations tested, i.e. lung slices and isolated perfused lung of the rat (Post ef a/. 1978). Another parameter which, as far as muscle and nerve tissues are concerned, is of importance for the distribution and the effectiveness of local anaesthetics is the pH. An increased effectiveness of local
*
Subsidiary of AB Astra, Sweden.
anaesthetics at an alkaline rather than a neutral pH has been demonstrated (Skou 1954; Ryd 1961).The concentration and the effects of the local anaesthetics of e.g. lidocaine and procaine in amphibian nerve trunks are greater when they have been equilibrated in solutions at pH 8.2 than at pH 7.2 (Strobel & Bianchi 1970a & b). Quite apart from explaining these observations solely by the cationic form of the local anaesthetics, there is probably a contribution of the non-ionized form as well. To elucidate the relative contribution of the cationic versus the non-ionized form of different local anaesthetics concerning the distribution in lung tissue, we have investigated the uptake of lidocaine, etidocaine, bupivacaine and methyllidocaine at different pH values. Studies were also performed to compare the inhibition of lung uptake of lidocaine by bupivacaine and nortriptyline. The displacement of ''C-lidocaine from isolated
104
C. POST ET AL.
perfused rat lung by non-labeled lidocaine and nortriptyline was also studied. Materials and Methods Lung slices. Female Sprague-Dawley rats (150-200 g) were anaesthetized by intraperitoneal injection of 25 mg mebumale. The lungs were perfused by 5 ml of a Trisbuffer solution and were then removed, sliced and incubated as described in detail by Post er at. (1978). When determining the uptake of local anaesthetics at different pH's, the slices were preincubated in Tris buffer solutions of pH 7.0, 7.4 and 8.0 for 15 min. The labeled local anaesthetic compounds were then added and the incubation of the lung slices was performed for 90 min. No measureable changes of the p H were observed at the end of the incubation periods. In another series of experiments the uptake of 14C-lidocainein lung slices was studied in the presence of bupivacaine or nortriptyline after an incubation period of 90 min. Isolated perfused lung. Female Sprague-Dawley rats (200 g) were treated with heparin (300-400 IU) and anaesthetized with pentobarbital (mebumalum NFN) sodium (30-40 mg intraperitoneally). Lungs were removed from the animal and prepared for perfusion as described by Ryrfeldt & Nilsson (1978) and Post e/ a/. (1978). The lungs were perfused and ventilated for 15 min. in order to establish constant perfusion flow and ventilation, before they were perfused with a buffer containing dissolved drugs. The solution containing I4C-lidocaine was infused at the rate of 10 ml/min. and at a perfusion pressure of 15 cm H20. After 3 min. when a steady-state level of the extraction of ''C-lidocaine was reached, lidocaine (3.7 pnol) or nortriptyline (5.1 p n o l ) were injected into the lung-circulation, as a bolus of 0.1 ml during less than 3 sec. Samples were taken from the effluent and from the upper reservoir at selected times and saved for analysis. Assay of radioactiviry. Lung slices were digested in Soluenem -350 and radioactivity was measured as described by Post el al. (1978). Aliquots of the incubation medium and of the effluents were placed in scintillation vials and Instagel@-solution was added before counting the radioactivity in a Packard Tricarb 3375 liquid scintillation spectrometer. Calculations. Tissue uptake of local anaesthetics was expressed as relative distribution ( D % ) of radioactivity by the formula:
Dz'
D P W m g wet tissue DPM/pI medium x 100
(1)
The amount of lidocaine taken up during the infusion period was calculated according to the formula: 1
lidocaine in lung (at time=t)=JEXFXC,,Xdtl 0
(2)
(F=perfusion flow, C,.=influent concentration of lidocaine in the perfusion medium, tl =perfusion time), where lung extraction (E) is expressed as:
l c c,o ,~ ~ E % = cC
x 100
(Cout=concentration of lidocaine in the effluent from the lung)
(3)
T o calculate the amount of lidocaine displaced by nonlabeled nortriptyline and lidocaine, the following formula was used: (4) Displaced lidocaine=~Cd,,,l.XFXdt2 0
(Cd,,,I.=(effluent concentration in displacement peak)-(concentration before displacement peak), t2=duration of displacement Peak) The distribution of lidocaine between the neutral base (B) and the cationic form (HB'), was determined according to the Henderson-Hasselbalch equation: (5) Marerials. 14C-lidocaine (New England Nuclear (NEN),
Baltimore, USA); 'H-etidocaine (Astra AB, Sodertalje, Sweden); 'H-bupivacaine (Astra AB); i4C-methyllidocaine was synthesized according to Dr. R. Sandberg, Astra AB (personal communication) by adding "Cmethyl-iodine to dissolved lidocaine base.
Results
The effect of p H on the accumulation of some local anaesthetics in lung slices. The uptake of local anaesthetics reach a steady state level after about 60 min. in rat lung slices (Post et al. 1978). In the present investigation the accumulation of ''C-lidocaine, 'H-etidocaine and 'H-bupivacaine was studied in rat lungslices. Fig. I summarizes the distribution of the drugs in lung BH' slices at different-ratios, determined according B to Henderson-Hasselbalch equation by varying the pH of the incubation solution. Since the pK,-values for these substances are 7.72, 7.74 and 8.05, respectively, the- [BH'l ratios for lidocaine and etidocaine
PI
are almost similar but different from that of bupivacaine. By increasing the pH of the local anaesthetic solution the percentage of the uncharged form (B) increases. The accumulation of the three anaesthetics increased markedly in an alkaline solution. The rank-order of uptake in lung BH' slices of the three substances was at the same B
LIDOCAINE AND LUNG
105
1800 100
\ -
--? 8C
1100
c
-"
c
0
E
0 ae
- 60
0
m
-
I
E
600
3
P e
.-P
(n
40
100
- 0.3
I
0
+
1
0.5 log. ( B H f B )
1.0
- 4.5
Fig. 1. The relative distribution ( D % ) of radioactivity between lung tissue and incubation medium after 90 min. incubation (X+S.E.M.; n=4) as a function of pH, i s . of the ratio between the amount of the cationic form (BH') and the uncharged base (B) of the local anaesthetics I4Clidocaine (A---A), 'H-etidocaine (*---a) and 'Hbupivacaine (B---B), all in a concentration of 2.78X IO'M.
- 4.0
+ -35 -30 log c o n c e n t r a t i o n imol/ll
Fig. 2. The effect of different concentrations of nonlabeled bupivacaine ( 0 - 0 ) (XfS.E.M.; n=4) and nortriptyline (-.) (X2S.E.M.; n=4) on the distribution of 2.78X IO-'M 14C-lidocaine relative to controls (100%) in rat lung slices, where no interacting substances has been added.
Efect of bupivacaine and nortriptyline on the accumulation of' ' 4 C-lidocaine in lung slices.
ratio bupivacaine>etidocaine>lidocaine. When comparing the accumulation of lidocaine with that of the cationic methyllidocaine at different pH's, it was found that the distribution of methyllidocaine was similar within the pH-range 7.0-8.0 and reached a steady-state level of approximately 130% (table 1). The distribution of lidocaine at pH 7.0, 7.4 and 8.0 varied, however, and was approximately 230, 360 and 800%, respectively (table I).
To obtain information about the interaction between lidocaine and other substances which bind to lung tissues, we have studied the accumulation of 14 C-lidocaine in the presence of the local anaesthetic bupivacaine and the tricyclic antidepressant nortriptyline. 14C-lidocainein a concentration of 2.78X 10-5Mwas incubated with increasing concentrations of bupivacaine and nortriptyline. As shown in fig. 2 the accumulation of I4C-
Table I.
The effect of various uH on the accumulation of lidocaine-'4C and meth~llidocaine-~~C into rat lung slices PH
7.0
7.4
8.0
0% (ixkS.E.M.) n=4
D % (nfS.E.M.) n=4
D % (n31s.E.M.) n=4
~~
Incubation time (min.) Lidocaine-I4C 2.78X10-5M Methyllidocaine-"C2.78X IO'M
____
~
3
30
90
135f2 227f4 232f6 50f2
92+ 1
130k1
3
30
90
3
_ _ _ ~ ____
30
90
1 9 6 f 8 3 8 4 f 2 8 3 6 0 f 1 4 2 6 7 f 1 6 688215 8 0 7 2 2 0 56f2
9722
129+5
5723
92f3
12022
106
C. POST ET AL.
lidocaine was markedly decreased in the presence
of both bupivacaine and nortriptyline. Nortriptyline was more effective in depressing the accumulation than bupivacaine. The concentrations of bupivacaine and nortriptyline, which inhibited the 14 C-lidocaine accumulation by 50%, were 6.5X 10-4M and 1.7X lo-", respectively. Displacement of I4C-lidocainefrom isolatedperfused rat lung by injections ofnorrriptyline and lidocaine.
The extraction of i4C-lidocainereached a steadystate level after about 3 min. The total amount of lidocaine accumulated in the lung after 3 min. perfusion was about 2.84X lo-' mol. Nortriptyline and lidocaine injected into the lung circulation at that time, rapidly increased the 14C-lidocaine concentration in the effluent. Nortriptyline (5.1 pmol) released 2 2 f 2 % (X*S.E.M.; n=4) of the accumulated I4C-lidocaine into the outflowing perfusion solution within 45 sec. (fig. 3). Unlabeled lidocaine (3.7 pmo1)displaced 13%+5 (xfS.E.M.; n=4) of the accumulated 14C-lidocainewithin 30 sec. After the displacement period caused by lidocaine the lung extraction of i4C-lidocaineincreased (fig. 4) for some time until a new steady-state level was reached.
Discussion Three of the local anaesthetics tested are tertiary amines and they can exist both as free base and as cation in aqueous solution. The catiodbase BH' ratiois determined partly by the ambient pH B and partly by the pK, value ofthe substanceaccording to Henderson-Hasselbalch equation. By decreasing the pH more of the substance will exist in the cationic form. As is shown in fig. 1 the accumulation of I4C-lidocaine,'H-bupivacaine and 'H-etidocaine is 3-4 times lower at pH 7.0 than at pH 8.0. This is in accordance with the findings of Strobel & Bianchi (1970b) who showed that the uptake of lidocaine and procaine in amphibian nerves was higher at an alkaline pH than at a neutral pH probably because the local anaesthetic base form, which predominates at pH 8.0, has a high lipid solubility, in contrast to the cation form. BH' When comparing the lung uptake at a fixed B
ratio the rank-order of distribution was bupivacaine>etidocaine>lidocaine. The difference in distribution does not seem to be related in a simple manner to lipid solubility since the partition coefficients in a n-heptane/buffer system are etidocaine (142.0)>bupivacaine (27S)>lidocaine (2.9)(Boyes 1974). Although the partition coefficients may reflect relative penetration of compounds into biological membranes, the applicability to the biological system of the partition coefficients depends on the nature of the organic solvent used, when measuring the partition of compounds between buffer and a non-polar solvent. Since drug binding may play a subsidiary role to drug partitioning, it must be an advantage to use biological tissue instead of organic solvents when comparing the distribution properties of different drugs. The accumulation of 14C-methyllidocaine,which is a cationic substance, was not changed by changing the extracellular pH (table I). When comparing the lung uptake of lidocaine and methyllidocaine at pH 7.4, approximately 3 times more lidocaine was taken up. At this pH, the percentage of the uncharged form of lidocaine was about 52%. As judged from these results, the ion-binding of the cationic form of local anaesthetics to charged groups in the membranes, does not seem to be the main determinant of the accumulation in lung slices. Furthermore, the change in i4C-lidocaine distribution at different pH's was probably not due to changes of the binding properties of the extracellular binding sites for lidocaine. The accumulation of I4C-lidocaine in lung slices is inhibited by nortriptyline in a dose-dependent manner (fig. 2 ) . Tricyclic antidepressant drugs also effectively lowered the lung uptake of noradrenaline and 5-hydroxytryptamine (Iwasawa & Gillis 1974). Bickel & Steele (1974) have shown that imipramine and related substances are reversibly bound to membrane fractions of liver, lung, kidney, brain and skeletal muscle. It is therefore possible that nortriptyline and lidocaine share similar binding sites in the lung and that nortriptyline for this reason influences the accumulation process. Bupivacaine and lidocaine appear to compete for similar binding sites in the lung too, since bupivacaine inhibited the accumulation of I4C-lidocaine in tissue slices. These experiments were
107
LIDOCAINE AND LUNG
performed up to a bupivacaine concentration of 6.6X IO-”M. Higher concentrations of local ana-
esthetics are known to labilize the cell membranes. Morck ef al. (1976) have shown that bupivacaine (>1 mM) had a labilizing effect on rat liver lysosoma1 membranes, while lower concentrations had a stabilizing effect. The experiments on lung slices might indicate an interference between lidocaine and other substances on the process of accumulation in the lung. In isolated perfused lungs nortriptyline interfered with the ’4C-lidocaineuptake since a fraction of 14C-lidocaine was rapidly released into the effluent by a bolus injection of nortriptyline as is shown in fig. 3. It was also possible to release 14 C-lidocaine by unlabeled lidocaine. A typical experiment is shown in fig. 4. The type of displacement-curves for I4C-lidocaine by non-labeled lido-
caine and nortriptyline, indicate differences concerning displacing properties between the two substances. The effluent concentration of I4Clidocaine after a bolus injection of lidocaine decreased after the displacement-peak, which might be interpreted as lidocaine displaced by 14C-lidocaine. This phenomenon did not appear after a bolus injection of nortriptyline. It therefore seems probable that nortriptyline binds stronger to the lung tissue than lidocaine. The rapidity of the displacement might be indicative of a competition of the drugs on easily accessible binding sites in capillary endothelial cells of the lung. From the present data it is impossible to determine the specific sites of lidocaine and nortriptyline uptake in the lung. Interactions between lidocaine and nortriptyline on common phospholipid sites can be possible, since both local anaesthetics (Feinstein &
M.10
7-
6-
I
1
2
3 minutes
4
5
Fig. 3. Concentration of lidocaine in effluent from an isolated perfused rat lung as determined by assay ofradioactivity at different times duringa continuous infusion of ’‘C-lidocaine. Arrow indicates bolus injection (0.1 ml given during less than 3 sec.) 01’5.1 pmol nortriptyline. Area under the displacement peak represents 22 per cent displacement oftotally accumulated “C-hdocaine during the previous 3 min. Dotted line indicates lidocaine concentration in the influent.
108
C. POST E T A L .
1
2
3
5
4
minutes
Fig. 4.Concentration of lidocaine in effluent from an isolated perfused rat lung as determined by assay of radioactivity at different times during a continuous infusion of ''C-lidocaine. Arrow indicates bolus injection (0.1ml given during less than 3 sec.) of 3.7 pmol non-labeled lidocaine. Area under the displacement peak represents 13 per cent displacement of totally accumulated 14C-lidocaine during the previous 3 min. Dotted line indicates lidocaine concentration in the influent.
Paimre 1969) and tricyclic antidepressant drugs (Gilette 1973) have been shown to interact with phospholipids. In some celltypes of the lung the phospholipid content is very high. The lipophilic character and the existence of charged molecules at physiological pH (FriskHolmberg &van der Kleijn 1972) might be properties, which are ofimportance for the binding of organic compounds to the lung tissue. The isolated perfused lung is a model, which may be useful for the study of pharmacokinetically important competition of drugs for binding sites in the lung. Experiments are now in progress to further identify the pharmacokinetic behaviour of lidocaine and nortriptyline in vivo in animal and humans.
Acknowledgements Dr. Stig Allenmark is acknowledged for his synthesis of methyllidocaine. We are also indebted to the technical assistance of Miss Grete Storvold and Mrs. Inga-Lill Scherling and the secreterial aid of Mrs. Kerstin Nilsson. This investigation was supported by grants from the Medial Research Council (04X-4498).
References Akerman, B., A. Astrom, S. Ross & A. Telt: Studies o n the absorption, distribution and metabolism of labelled prilocaine and lidocaine in some animal species. Aria pharmacol. et ioxicol. 1966, 24, 389-403.
LIDOCAINE A N D LUNG Bertler, A,, D. H. Lewis, J. B. Lofstrom& C. Post: In vivo lung uptake of lidocaine in pigs. Acra Anaesth. Scand. 1978, 22, 530-536. Bickel, M. H. & J. W. Steele: Binding of basic and acidic drugs to rat tissue subcellular fractions. Chem. Biol. Interactions 1974, 8, I5 1- 162. Boyes, R. N.: Absorption, diffusion, fixation. metabolism and excretion of local anaesthetic agents in animal and man. In: Anesrhisiques locaux en anesthisie ei reanimaiion. Libraire Arnette, Paris, 1974, pp. 127-135. Feinstein, M. B. & M. Paimre: Pharmacological actionof local anaesthetics on excitation-contraction coupling in striated and smooth muscle. Fed. Proc. 1969, 28, 1643- 1648. Frisk-Holmberg, M. & E. van der Kleijn: The relationship between the lipophilic nature of tricyclic neuroleptics and antidepressants, and histamine release. Eur, J. Pharmacol. 1972, 18, 139-147. Gilette, J . R.: Overview of drug-protein binding. Ann. N . Y. Acrid Sci. 1973, 226, 6-17. Iwasawa. Y. & C. N. Gillis: Pharmacological analysis of norepinephrine and 5-hydroxytryptamine removal from the pulmonary circulation: differentiation of uptake sites for each amine. .1. Pharmacol. Exp. Therap. 1974. 188, 386-393. Keenaghan, J. B. & R. N. Boyes: The tissue distribution, metabolism and excretion of lidocaine in rats, guinea pigs, dogs and man. J. Pharmacol. Exp. Therap. 1972, 180, 454-463. Morck, E., P. Friberger & G. Aberg: Effects of four local
109
anaesthetics on the membrane stability of isolated rat liver lysosomes. Act0 pharmacol. er toxicol. 1976, 38, 24-30. Post, C., B. E. Alm, A. Bertler, D. H. Lewis & J . B. Lofstriim: Study of the pulmonary extraction of amines from the circulation. Microvasc. Res. 1976, 11, 423. Post, C., R. G . G. Anderson, A. Ryrfeldt & E. Nilsson: Transport and binding of lidocaine by lung slices and perfused lung of rats. Arta pharmacol. et toxicol. 1978, 43, 156-163. Ryd, J.: Local anaesthetics. An electrophysiological investigation of local anaesthesia of peripheral nerves with special reference to xylocaine. Actaphysiol. Scand. 1961, 51, SUPPI. 178, 1-171. Ryrfeldt, A. & E. Nilsson: Uptake and biotransformation of ibuterol and terbutaline in isolated perfused rat and guinea pig lungs. Biochem. pharmacol. 1978, 27, 301-305. Skou. J. C.: Local anesthetics. I. The blocking action of some local anaesthetics and of butyl alcohol on peripheral nerves. Acta pharmacol. et toxicol. 1954, 10, 28 1-29 1. Strobe1.G. E. & C. P. Bianchi: The effects of pH gradients on the action of procaine and lidocaine in intact and desheathed sciatic nerves. J. Pharmacol. Exp. Therap. 1970a. 172, 1-17. Strobel, G. E. & C. P. Bianchi: The effect of pH gradient on the uptake and distribution of ''C-procaine and lidocaine in intact and desheathed sciatic nerve trunks. J. Pharmacol. Exp. Therap. 1970b, 172, 18-32.
From the Department of Clinical Pharmacology and Pharmacology, Linkoping University,
S-581 85 Linkoping, and AB Draco*. Research and Development Laboratories, Fack, S-221 01 Lund, Sweden
Physico-chemical Modification of Lidocaine Uptake in Rat Lung Tissue BY C. Post, R. C. C. Andersson. A. Ryrfeldt and E. Nilsson (Received may 22. 1978; Accepted August 14, 1978)
Abstr'at'r: The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of thequaternary analogue, methyllidocaine, was not changed in the pH interval 7.0-8.0. The uptake of the three other substances was about 3-4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed catiodbase ratio was bupivacaine>etidocaine>lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of14C-lidocainein lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced ''C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while thecationic form binds to accessible binding sites in the cell membranes. Key-wjurrls: Interaction
- local anaesthetics - lung uptake - pH - rat.
Previous studies on the distribution of lidocaine in tissues have shown that the lungs were able to accumulate lidocaine extensively (Akerman rf a/. 1966; Keenaghan & Boyes 1972; Post ei a/. 1976 & 1978; Bertler ff al. 1978). Further analysis showed that the accumulation was depressed by low temperature but not by anaerobic conditions. The extent of the uptake of lidocaine was also partly dependent on the level of Ca"-ions in the incubation medium. N o metabolism of lidocaine took place in the lung preparations tested, i.e. lung slices and isolated perfused lung of the rat (Post ef a/. 1978). Another parameter which, as far as muscle and nerve tissues are concerned, is of importance for the distribution and the effectiveness of local anaesthetics is the pH. An increased effectiveness of local
*
Subsidiary of AB Astra, Sweden.
anaesthetics at an alkaline rather than a neutral pH has been demonstrated (Skou 1954; Ryd 1961).The concentration and the effects of the local anaesthetics of e.g. lidocaine and procaine in amphibian nerve trunks are greater when they have been equilibrated in solutions at pH 8.2 than at pH 7.2 (Strobel & Bianchi 1970a & b). Quite apart from explaining these observations solely by the cationic form of the local anaesthetics, there is probably a contribution of the non-ionized form as well. To elucidate the relative contribution of the cationic versus the non-ionized form of different local anaesthetics concerning the distribution in lung tissue, we have investigated the uptake of lidocaine, etidocaine, bupivacaine and methyllidocaine at different pH values. Studies were also performed to compare the inhibition of lung uptake of lidocaine by bupivacaine and nortriptyline. The displacement of ''C-lidocaine from isolated
104
C. POST ET AL.
perfused rat lung by non-labeled lidocaine and nortriptyline was also studied. Materials and Methods Lung slices. Female Sprague-Dawley rats (150-200 g) were anaesthetized by intraperitoneal injection of 25 mg mebumale. The lungs were perfused by 5 ml of a Trisbuffer solution and were then removed, sliced and incubated as described in detail by Post er at. (1978). When determining the uptake of local anaesthetics at different pH's, the slices were preincubated in Tris buffer solutions of pH 7.0, 7.4 and 8.0 for 15 min. The labeled local anaesthetic compounds were then added and the incubation of the lung slices was performed for 90 min. No measureable changes of the p H were observed at the end of the incubation periods. In another series of experiments the uptake of 14C-lidocainein lung slices was studied in the presence of bupivacaine or nortriptyline after an incubation period of 90 min. Isolated perfused lung. Female Sprague-Dawley rats (200 g) were treated with heparin (300-400 IU) and anaesthetized with pentobarbital (mebumalum NFN) sodium (30-40 mg intraperitoneally). Lungs were removed from the animal and prepared for perfusion as described by Ryrfeldt & Nilsson (1978) and Post e/ a/. (1978). The lungs were perfused and ventilated for 15 min. in order to establish constant perfusion flow and ventilation, before they were perfused with a buffer containing dissolved drugs. The solution containing I4C-lidocaine was infused at the rate of 10 ml/min. and at a perfusion pressure of 15 cm H20. After 3 min. when a steady-state level of the extraction of ''C-lidocaine was reached, lidocaine (3.7 pnol) or nortriptyline (5.1 p n o l ) were injected into the lung-circulation, as a bolus of 0.1 ml during less than 3 sec. Samples were taken from the effluent and from the upper reservoir at selected times and saved for analysis. Assay of radioactiviry. Lung slices were digested in Soluenem -350 and radioactivity was measured as described by Post el al. (1978). Aliquots of the incubation medium and of the effluents were placed in scintillation vials and Instagel@-solution was added before counting the radioactivity in a Packard Tricarb 3375 liquid scintillation spectrometer. Calculations. Tissue uptake of local anaesthetics was expressed as relative distribution ( D % ) of radioactivity by the formula:
Dz'
D P W m g wet tissue DPM/pI medium x 100
(1)
The amount of lidocaine taken up during the infusion period was calculated according to the formula: 1
lidocaine in lung (at time=t)=JEXFXC,,Xdtl 0
(2)
(F=perfusion flow, C,.=influent concentration of lidocaine in the perfusion medium, tl =perfusion time), where lung extraction (E) is expressed as:
l c c,o ,~ ~ E % = cC
x 100
(Cout=concentration of lidocaine in the effluent from the lung)
(3)
T o calculate the amount of lidocaine displaced by nonlabeled nortriptyline and lidocaine, the following formula was used: (4) Displaced lidocaine=~Cd,,,l.XFXdt2 0
(Cd,,,I.=(effluent concentration in displacement peak)-(concentration before displacement peak), t2=duration of displacement Peak) The distribution of lidocaine between the neutral base (B) and the cationic form (HB'), was determined according to the Henderson-Hasselbalch equation: (5) Marerials. 14C-lidocaine (New England Nuclear (NEN),
Baltimore, USA); 'H-etidocaine (Astra AB, Sodertalje, Sweden); 'H-bupivacaine (Astra AB); i4C-methyllidocaine was synthesized according to Dr. R. Sandberg, Astra AB (personal communication) by adding "Cmethyl-iodine to dissolved lidocaine base.
Results
The effect of p H on the accumulation of some local anaesthetics in lung slices. The uptake of local anaesthetics reach a steady state level after about 60 min. in rat lung slices (Post et al. 1978). In the present investigation the accumulation of ''C-lidocaine, 'H-etidocaine and 'H-bupivacaine was studied in rat lungslices. Fig. I summarizes the distribution of the drugs in lung BH' slices at different-ratios, determined according B to Henderson-Hasselbalch equation by varying the pH of the incubation solution. Since the pK,-values for these substances are 7.72, 7.74 and 8.05, respectively, the- [BH'l ratios for lidocaine and etidocaine
PI
are almost similar but different from that of bupivacaine. By increasing the pH of the local anaesthetic solution the percentage of the uncharged form (B) increases. The accumulation of the three anaesthetics increased markedly in an alkaline solution. The rank-order of uptake in lung BH' slices of the three substances was at the same B
LIDOCAINE AND LUNG
105
1800 100
\ -
--? 8C
1100
c
-"
c
0
E
0 ae
- 60
0
m
-
I
E
600
3
P e
.-P
(n
40
100
- 0.3
I
0
+
1
0.5 log. ( B H f B )
1.0
- 4.5
Fig. 1. The relative distribution ( D % ) of radioactivity between lung tissue and incubation medium after 90 min. incubation (X+S.E.M.; n=4) as a function of pH, i s . of the ratio between the amount of the cationic form (BH') and the uncharged base (B) of the local anaesthetics I4Clidocaine (A---A), 'H-etidocaine (*---a) and 'Hbupivacaine (B---B), all in a concentration of 2.78X IO'M.
- 4.0
+ -35 -30 log c o n c e n t r a t i o n imol/ll
Fig. 2. The effect of different concentrations of nonlabeled bupivacaine ( 0 - 0 ) (XfS.E.M.; n=4) and nortriptyline (-.) (X2S.E.M.; n=4) on the distribution of 2.78X IO-'M 14C-lidocaine relative to controls (100%) in rat lung slices, where no interacting substances has been added.
Efect of bupivacaine and nortriptyline on the accumulation of' ' 4 C-lidocaine in lung slices.
ratio bupivacaine>etidocaine>lidocaine. When comparing the accumulation of lidocaine with that of the cationic methyllidocaine at different pH's, it was found that the distribution of methyllidocaine was similar within the pH-range 7.0-8.0 and reached a steady-state level of approximately 130% (table 1). The distribution of lidocaine at pH 7.0, 7.4 and 8.0 varied, however, and was approximately 230, 360 and 800%, respectively (table I).
To obtain information about the interaction between lidocaine and other substances which bind to lung tissues, we have studied the accumulation of 14 C-lidocaine in the presence of the local anaesthetic bupivacaine and the tricyclic antidepressant nortriptyline. 14C-lidocainein a concentration of 2.78X 10-5Mwas incubated with increasing concentrations of bupivacaine and nortriptyline. As shown in fig. 2 the accumulation of I4C-
Table I.
The effect of various uH on the accumulation of lidocaine-'4C and meth~llidocaine-~~C into rat lung slices PH
7.0
7.4
8.0
0% (ixkS.E.M.) n=4
D % (nfS.E.M.) n=4
D % (n31s.E.M.) n=4
~~
Incubation time (min.) Lidocaine-I4C 2.78X10-5M Methyllidocaine-"C2.78X IO'M
____
~
3
30
90
135f2 227f4 232f6 50f2
92+ 1
130k1
3
30
90
3
_ _ _ ~ ____
30
90
1 9 6 f 8 3 8 4 f 2 8 3 6 0 f 1 4 2 6 7 f 1 6 688215 8 0 7 2 2 0 56f2
9722
129+5
5723
92f3
12022
106
C. POST ET AL.
lidocaine was markedly decreased in the presence
of both bupivacaine and nortriptyline. Nortriptyline was more effective in depressing the accumulation than bupivacaine. The concentrations of bupivacaine and nortriptyline, which inhibited the 14 C-lidocaine accumulation by 50%, were 6.5X 10-4M and 1.7X lo-", respectively. Displacement of I4C-lidocainefrom isolatedperfused rat lung by injections ofnorrriptyline and lidocaine.
The extraction of i4C-lidocainereached a steadystate level after about 3 min. The total amount of lidocaine accumulated in the lung after 3 min. perfusion was about 2.84X lo-' mol. Nortriptyline and lidocaine injected into the lung circulation at that time, rapidly increased the 14C-lidocaine concentration in the effluent. Nortriptyline (5.1 pmol) released 2 2 f 2 % (X*S.E.M.; n=4) of the accumulated I4C-lidocaine into the outflowing perfusion solution within 45 sec. (fig. 3). Unlabeled lidocaine (3.7 pmo1)displaced 13%+5 (xfS.E.M.; n=4) of the accumulated 14C-lidocainewithin 30 sec. After the displacement period caused by lidocaine the lung extraction of i4C-lidocaineincreased (fig. 4) for some time until a new steady-state level was reached.
Discussion Three of the local anaesthetics tested are tertiary amines and they can exist both as free base and as cation in aqueous solution. The catiodbase BH' ratiois determined partly by the ambient pH B and partly by the pK, value ofthe substanceaccording to Henderson-Hasselbalch equation. By decreasing the pH more of the substance will exist in the cationic form. As is shown in fig. 1 the accumulation of I4C-lidocaine,'H-bupivacaine and 'H-etidocaine is 3-4 times lower at pH 7.0 than at pH 8.0. This is in accordance with the findings of Strobel & Bianchi (1970b) who showed that the uptake of lidocaine and procaine in amphibian nerves was higher at an alkaline pH than at a neutral pH probably because the local anaesthetic base form, which predominates at pH 8.0, has a high lipid solubility, in contrast to the cation form. BH' When comparing the lung uptake at a fixed B
ratio the rank-order of distribution was bupivacaine>etidocaine>lidocaine. The difference in distribution does not seem to be related in a simple manner to lipid solubility since the partition coefficients in a n-heptane/buffer system are etidocaine (142.0)>bupivacaine (27S)>lidocaine (2.9)(Boyes 1974). Although the partition coefficients may reflect relative penetration of compounds into biological membranes, the applicability to the biological system of the partition coefficients depends on the nature of the organic solvent used, when measuring the partition of compounds between buffer and a non-polar solvent. Since drug binding may play a subsidiary role to drug partitioning, it must be an advantage to use biological tissue instead of organic solvents when comparing the distribution properties of different drugs. The accumulation of 14C-methyllidocaine,which is a cationic substance, was not changed by changing the extracellular pH (table I). When comparing the lung uptake of lidocaine and methyllidocaine at pH 7.4, approximately 3 times more lidocaine was taken up. At this pH, the percentage of the uncharged form of lidocaine was about 52%. As judged from these results, the ion-binding of the cationic form of local anaesthetics to charged groups in the membranes, does not seem to be the main determinant of the accumulation in lung slices. Furthermore, the change in i4C-lidocaine distribution at different pH's was probably not due to changes of the binding properties of the extracellular binding sites for lidocaine. The accumulation of I4C-lidocaine in lung slices is inhibited by nortriptyline in a dose-dependent manner (fig. 2 ) . Tricyclic antidepressant drugs also effectively lowered the lung uptake of noradrenaline and 5-hydroxytryptamine (Iwasawa & Gillis 1974). Bickel & Steele (1974) have shown that imipramine and related substances are reversibly bound to membrane fractions of liver, lung, kidney, brain and skeletal muscle. It is therefore possible that nortriptyline and lidocaine share similar binding sites in the lung and that nortriptyline for this reason influences the accumulation process. Bupivacaine and lidocaine appear to compete for similar binding sites in the lung too, since bupivacaine inhibited the accumulation of I4C-lidocaine in tissue slices. These experiments were
107
LIDOCAINE AND LUNG
performed up to a bupivacaine concentration of 6.6X IO-”M. Higher concentrations of local ana-
esthetics are known to labilize the cell membranes. Morck ef al. (1976) have shown that bupivacaine (>1 mM) had a labilizing effect on rat liver lysosoma1 membranes, while lower concentrations had a stabilizing effect. The experiments on lung slices might indicate an interference between lidocaine and other substances on the process of accumulation in the lung. In isolated perfused lungs nortriptyline interfered with the ’4C-lidocaineuptake since a fraction of 14C-lidocaine was rapidly released into the effluent by a bolus injection of nortriptyline as is shown in fig. 3. It was also possible to release 14 C-lidocaine by unlabeled lidocaine. A typical experiment is shown in fig. 4. The type of displacement-curves for I4C-lidocaine by non-labeled lido-
caine and nortriptyline, indicate differences concerning displacing properties between the two substances. The effluent concentration of I4Clidocaine after a bolus injection of lidocaine decreased after the displacement-peak, which might be interpreted as lidocaine displaced by 14C-lidocaine. This phenomenon did not appear after a bolus injection of nortriptyline. It therefore seems probable that nortriptyline binds stronger to the lung tissue than lidocaine. The rapidity of the displacement might be indicative of a competition of the drugs on easily accessible binding sites in capillary endothelial cells of the lung. From the present data it is impossible to determine the specific sites of lidocaine and nortriptyline uptake in the lung. Interactions between lidocaine and nortriptyline on common phospholipid sites can be possible, since both local anaesthetics (Feinstein &
M.10
7-
6-
I
1
2
3 minutes
4
5
Fig. 3. Concentration of lidocaine in effluent from an isolated perfused rat lung as determined by assay ofradioactivity at different times duringa continuous infusion of ’‘C-lidocaine. Arrow indicates bolus injection (0.1 ml given during less than 3 sec.) 01’5.1 pmol nortriptyline. Area under the displacement peak represents 22 per cent displacement oftotally accumulated “C-hdocaine during the previous 3 min. Dotted line indicates lidocaine concentration in the influent.
108
C. POST E T A L .
1
2
3
5
4
minutes
Fig. 4.Concentration of lidocaine in effluent from an isolated perfused rat lung as determined by assay of radioactivity at different times during a continuous infusion of ''C-lidocaine. Arrow indicates bolus injection (0.1ml given during less than 3 sec.) of 3.7 pmol non-labeled lidocaine. Area under the displacement peak represents 13 per cent displacement of totally accumulated 14C-lidocaine during the previous 3 min. Dotted line indicates lidocaine concentration in the influent.
Paimre 1969) and tricyclic antidepressant drugs (Gilette 1973) have been shown to interact with phospholipids. In some celltypes of the lung the phospholipid content is very high. The lipophilic character and the existence of charged molecules at physiological pH (FriskHolmberg &van der Kleijn 1972) might be properties, which are ofimportance for the binding of organic compounds to the lung tissue. The isolated perfused lung is a model, which may be useful for the study of pharmacokinetically important competition of drugs for binding sites in the lung. Experiments are now in progress to further identify the pharmacokinetic behaviour of lidocaine and nortriptyline in vivo in animal and humans.
Acknowledgements Dr. Stig Allenmark is acknowledged for his synthesis of methyllidocaine. We are also indebted to the technical assistance of Miss Grete Storvold and Mrs. Inga-Lill Scherling and the secreterial aid of Mrs. Kerstin Nilsson. This investigation was supported by grants from the Medial Research Council (04X-4498).
References Akerman, B., A. Astrom, S. Ross & A. Telt: Studies o n the absorption, distribution and metabolism of labelled prilocaine and lidocaine in some animal species. Aria pharmacol. et ioxicol. 1966, 24, 389-403.
LIDOCAINE A N D LUNG Bertler, A,, D. H. Lewis, J. B. Lofstrom& C. Post: In vivo lung uptake of lidocaine in pigs. Acra Anaesth. Scand. 1978, 22, 530-536. Bickel, M. H. & J. W. Steele: Binding of basic and acidic drugs to rat tissue subcellular fractions. Chem. Biol. Interactions 1974, 8, I5 1- 162. Boyes, R. N.: Absorption, diffusion, fixation. metabolism and excretion of local anaesthetic agents in animal and man. In: Anesrhisiques locaux en anesthisie ei reanimaiion. Libraire Arnette, Paris, 1974, pp. 127-135. Feinstein, M. B. & M. Paimre: Pharmacological actionof local anaesthetics on excitation-contraction coupling in striated and smooth muscle. Fed. Proc. 1969, 28, 1643- 1648. Frisk-Holmberg, M. & E. van der Kleijn: The relationship between the lipophilic nature of tricyclic neuroleptics and antidepressants, and histamine release. Eur, J. Pharmacol. 1972, 18, 139-147. Gilette, J . R.: Overview of drug-protein binding. Ann. N . Y. Acrid Sci. 1973, 226, 6-17. Iwasawa. Y. & C. N. Gillis: Pharmacological analysis of norepinephrine and 5-hydroxytryptamine removal from the pulmonary circulation: differentiation of uptake sites for each amine. .1. Pharmacol. Exp. Therap. 1974. 188, 386-393. Keenaghan, J. B. & R. N. Boyes: The tissue distribution, metabolism and excretion of lidocaine in rats, guinea pigs, dogs and man. J. Pharmacol. Exp. Therap. 1972, 180, 454-463. Morck, E., P. Friberger & G. Aberg: Effects of four local
109
anaesthetics on the membrane stability of isolated rat liver lysosomes. Act0 pharmacol. er toxicol. 1976, 38, 24-30. Post, C., B. E. Alm, A. Bertler, D. H. Lewis & J . B. Lofstriim: Study of the pulmonary extraction of amines from the circulation. Microvasc. Res. 1976, 11, 423. Post, C., R. G . G. Anderson, A. Ryrfeldt & E. Nilsson: Transport and binding of lidocaine by lung slices and perfused lung of rats. Arta pharmacol. et toxicol. 1978, 43, 156-163. Ryd, J.: Local anaesthetics. An electrophysiological investigation of local anaesthesia of peripheral nerves with special reference to xylocaine. Actaphysiol. Scand. 1961, 51, SUPPI. 178, 1-171. Ryrfeldt, A. & E. Nilsson: Uptake and biotransformation of ibuterol and terbutaline in isolated perfused rat and guinea pig lungs. Biochem. pharmacol. 1978, 27, 301-305. Skou. J. C.: Local anesthetics. I. The blocking action of some local anaesthetics and of butyl alcohol on peripheral nerves. Acta pharmacol. et toxicol. 1954, 10, 28 1-29 1. Strobe1.G. E. & C. P. Bianchi: The effects of pH gradients on the action of procaine and lidocaine in intact and desheathed sciatic nerves. J. Pharmacol. Exp. Therap. 1970a. 172, 1-17. Strobel, G. E. & C. P. Bianchi: The effect of pH gradient on the uptake and distribution of ''C-procaine and lidocaine in intact and desheathed sciatic nerve trunks. J. Pharmacol. Exp. Therap. 1970b, 172, 18-32.
