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Mechanisms of Creatine Kinase Release from Isolated Rat Skeletal Muscles Damaged by Propylene Glycol and Ethanol
Mechanisms of Creatine Kinase Release from Isolated Rat Skeletal Muscles Damaged by Propylene Glycol and Ethanol GAYLEA. BRAZEAU**' AND HO-LEUNG FUNG' Received March 21, 1989, from the 'Department of Pharmaceutics, School of Pharmacy, State University of New Yolk at Buffalo, Buffalo, NY 74260. Acce ted for publication August 22, 1989. *Present address: Department of Phannaceutics, University of Houston, Texas MedicarCenter, 1441 Moursund, Houston, TX 77030. - _Abstract Cl The organic cosolvents propylene glycol and ethanol are
found to cause skeletal muscle damage and creatine kinase release following intramuscular injection. The mechanisms of this organic cosolvent-induced enzyme release have not been elucidated. Cosolventinduced creatine kinase release was enhanced by the addition of calcium 10 the incubation medium, and inhibited, albeit modestly, by dibucaine, a nonspecific phospholipase A, inhibitor. The temporal pattern of creatine kinase release further suggested that cosolvent-induced enzyme release from skeletal muscles may be caused by an intracellular mechanism rather than by a direct solubilization of sarcolemma. This intracellular mechanism may involve the mobilization of calcium. - -
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Intramuscular administration of parenteral products is frequently associated with patient discomfort and evidence of skeletal muscle damage. l4 Many of these commercial products contain organic cosolvents to aid in the solubilization of lipophilic therapeutic agents. Propylene glycol, ethanol, and polyethylene glycol 400 are among the most commonly used organic cosolvents in intramuscular formulation^.^ We have previously shown that these organic cosolvents cause skeletal muscle damage (myotoxicity) as assessed by the release of creatine kinase, either in vitro or in vivo.' The mechanisms of organic cosolvent-induced skeletal muscle damage have yet to be elucidated. Previously, we have shown that organic cosolvent-inducedcreatine kinase release from a rat isolated skeletal muscle is not well correlated with physicochemical properties (viz., tonicity, dielectric constant, apparent pH, viscosity, or surface t e n ~ i o n )These .~ findings suggested that biochemical interactions between organic cosolvents and skeletal muscle fibers ought to be examined as possible mechanisms of cosolvent-induced myotoxicity. One possible mechanism is that organic cosolvents directly solubilize the constituents (viz., phospholipids, fatty acids, and proteins) of the sarcolemma, altering its integrity and thereby leading to the release of intracellular enzymes. Previous investigators have shown a rapid release of intracellular cytoplasmic enzymes when isolated muscles were incubated in the presence of detergents Iviz., deoxycholic acid (DCA)or Brij 58],%" or following a defined disruption of the membrane (i.e., muscle slice)." The major characteristic of this mode of cellular damage is a rapid release of intracellular enzymes followed by a decline to near normal levels once the damaging compound is removed from the incubation medium." A second possible mechanism responsible for enhanced enzyme release might involve an increase in intracellular calcium levels. Several investigators have suggested that skeletal muscle damage may result from increases in cytosolic calcium levels. ''-I3 Increased intracellular calcium levels may result from changes in the integrity of the sarcolemma, thereby leading to an increased flux of calcium from extracellular sources,11or from a redistribution of calcium from the OO22-3549/90/05OO-0393$0 1 .OO/O C 7990, American Pharmaceutical Association
intracellular sites of calcium sequestration (i.e., sarco lasmic reticulum and mitochondria) to the cytosolic space.lg14 An increase in intracellular cytosolic calcium levels within skeletal muscle has been reported to increase protein degradation16-" and to alter muscle histology and u l t r a ~ t r u c t u r e . ' ~ ~In~ ~addition, * ~ ~ ~ ~ increased intracellular calcium has been hypothesized to activate muscle phospholipases (e.g., phospholipase A2).,' Activation of these phospholipases leads to the release of membrane-bound fatty acids and the formation of lysophospholipids known to be deleterious to sarcolemma integrity. Furthermore, activation of phospholipase A2 could also release membrane-bound arachidonic acid which is subsequently converted to bioactive metabolites (e.g., prostaglandins, leukotrienea) by lipoxygenase and cyclooxygenase enzymes. These bioactive metabolites are also disruptive of skeletal muscle m e m b r a n e ~ , ~ ~ * ~ leading to the release of intracellular enzymes. This mechanism is characterized by increased enzyme release from isolated skeletal muscles incubated with agents [viz., the calcium ionophore calimycin (A23187)and the mitochondria1 poison 2,4-dinitrophenol (DNP)] known to increase intracellular calcium levels, and decreased enzyme release from A23187- or DNP-damaged muscles when inhibitors of phospholipase A,, lipoxygenase, or cycloox genase enzymes were included in the bathing media.24*2'*2 Y The onset of enzyme efflux is delayed in this mechanism compared with that caused by direct solubilization of the sarcolemma, and enzyme release is sustained aRer removal of the damaging agent from the incubation medium. In this study, we examined the possible applicability of these mechanisms (i.e., membrane solubilization versus intracellular calcium alteration) in cosolvent-induced enzyme release. The effect of added calcium in the incubation medium on organic cosolvent-induced creatine kinase release was investigated. Secondly, the temporal pattern of creatine kinase release following exposure to the organic cosolvents was compared with that following exposure to DCA, A23187, and DNP. Finally, the effect of a nonspecific phospholipase A, inhibitor, dibucaine (DBC), on propylene glycol-induced creatine kinase release was examined.
Experimental Section Muscle Isolation and Incubation-Male
Sprague-Dawley rats
(200-350 g) were sacrificed and the extensor digitorum longus
muscles were isolated, removed, and placed into the carbogenated incubation medium (37 "C) as previously described.28At 30-min interv'als, the solutions (9.5 mL) were drained from the incubation vessels; this was followed by additionof fresh incubation medium and test compounds, when appropriate. A commercially available kit (SigmaChemical Company, St. Louis, MO) was used to assay creatine kinase. In paired muscle experiments, right and left muscles were randomized between controls and treatments. Role of Added Calcium on Organic Cosolvent-Induced Creatine Kinase Release-The effect of added calcium in the incubation medium on enzyme release was examined by using three incubation
Journal of Pharmaceutical Sciences I 393 Vol. 79, No. 5,May 7990
media. The control incubation medium was a balanced salt solution (BSS)containing 116 mM NaCl, 5.4 mM KCl, 5.6 mM dextrose, 26.2 mM NaHCO,, and 0.001 mM sodium phenol red in sterile water for irrigation. The pH was adjusted to 7.4 with 1 M HC1. The second incubation medium (BSS:CaCl,) was identical to the BSS, but with the addition of 2.5 mM CaC1,. The third incubation medium (BSS. NaCl) was identical to the BSS, but NaCl was added to match the tonicity in the BSS:CaCl, medium. Treated muscles were incubated in the presence of 6.3% (v/v) propylene glycol for 2 h. Contralateral muscles served as the control (no solvent). To further examine the effect of added calcium, a second series of experiments was conducted based on the protocol initially proposed by Jones and co-workers.8 The extensor digitorum longus muscles were individually mounted in the incubation baths via the tendons at resting length. Following a 30-min preincubation period, the muscle pairs were incubated for 30 min in the presence of propylene glycol or ethanol (7.4%,v/v, final concentration in the bath), using either the BSS or BSSCaCl, incubation medium. Preliminary studies showed that the extent of intracellular enzyme release induced by this concentration of the organic cosolvent in the incubation medium was similar to that produced by the direct injection of these cosolvents into the muscles.6 After the cosolvent solutions were drained from the incubation vessels, the muscles were rinsed 10 times with the incubation medium. Over the next 2 h, at 30-min intervals, the incubation medium was collected from the incubation vessel and replaced with fresh medium. The collected solutions were analyzed for creatine kinase activity within 1h. Possible assay interferences in the determination of creatine kinase activity by propylene glycol and ethanol at the above concentrations were ruled out in previous experiment^.^' The release of creatine kinase was expressed as the amount released per 30-min sampling interval. Similar experiments with 7.4% (v/v) polyethylene glycol 400 in the bath could not be carried out since creatine kinase activity was inhibited substantially by this solvent at this c o n c e n t r a t i ~ n . ~ ~ Other investigators have demonstrated that an incubation medium with reduced sodium content causes a greater influx of added calcium into muscle fibers via the Na+-Ca2+ exchange system within the s a r ~ o l e m r n a .31~ A ~ . third set of experiments was therefore conducted using an incubation medium with reduced sodium content. A pair of extensor digitorum longus muscles was incubated either in the BSS:CaCl, medium (116 mM NaC1) or in a reduced sodium (58 mM NaCl) BSS:CaCI, medium. The tonicity of the latter solution was maintained with the addition of sucrose. The rest of the protocol was identical to that described above. Temporal Pattern of Creatine Kinase Release-In order to determine whether the organic cosolvents cause the release of creatine kinase through a direct solubilization of the sarcolemma or through increases in cytosolic calcium, the temporal pattern of enzyme release was examined as described in the literature.’. 25 Following a 30-min preincubation period, one muscle was incubated in the presence of the damaging compound for 30 min, while the contralateral muscle served as a control. The muscles were rinsed 10 times with the incubation medium at the end of the 30-min period to remove the damaging compound. The remaining protocol was identical to that used in the calcium studies. The concentrations of DCA (500 pM), A23187 (20 pM), and DNP (1mM) were selected based on previous literature investigations.2P26 Three different concentrations of propylene glycol (5.3-lo%, v/v, final bath concentration) were investigated to determine if the temporal pattern of creatine kinase release was affected by concentration. The concentration of ethanol was identical to that used in the calcium studies. All experiments utilized BSS as the incubation medium. Possible interference in the determination of creatine kinase activity by the above agents was ruled out in previous experiments.” Inhibitor Studies-To evaluate the possibility that organic cosolvent-induced creatine kinase release may be modulated through the activation of phospholipase A,, we used a nonspecific phospholipase A, inhibitor, DBC, which has been shown to cause a dramatic decrease in DNP-induced lactate dehydrogenase release from isolated muscles.26 In the present study, creatine kinase release induced by 7.4% (dv) propylene glycol was examined in the presence of 100 pM DBC throughout the 3-h period. The contralateral muscle was incubated in an identical manner except for the omission of DBC (ethanol solvent control). The remaining experimental protocol was identical to that utilized in previous studies. A similar experiment was conducted using pairs of muscles exposed either to 300 pM DBC
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(treatment) or 300 pL of ethanol added to the bath (solvent for DBC). Due to significant interferences in the determination of creatine kinase activity, we were unable to study other inhibitors (viz., chlorpromazine, mepacrine, phenidone, nordihydroguaiaretic acid) that have been shown to reduce enzyme release following skeletal muscle damage.,’ Statistical Analysis-Data are presented as the mean and standard deviation. Statistical analysis was performed using one-way or two-way analysis of variance. Differences between the means were determined using Tukey’s test when appr~priate.~’
Results and Discussion Role of Added Calcium on Organic Cosolvent-Induced Creatine Kinase Release-Incubation of isolated muscles in the presence of 6.3% (v/v) propylene glycol for 2 h increased creatine kinase release -64-fold, 28-fold, and 27-fold over control muscles in the BSS, BSS:CaCl,, o r BSS:NaClmedium, respectively (Table I). Enzyme release from propylene glycoltreated muscles was significantly h i g h e r in the BSSCaCl, incubation medium (p < 0.05) compared w i t h the BSS and BSS:NaCl media. Since the BSS:CaCl, and the BSS:NaCl media are isotonic, these findings suggest a specific effect of calcium in affecting enzyme release, unrelated to a change in tonicity of the medium. In control muscles, there was n o significant difference in the cumulative creatine kinase release among the three media. A similar effect of added calcium w a s observed when the muscles were incubated for only 30 min in the presence of propylene glycol o r ethanol (Figure 1). Additional calcium in the incubation medium significantly enhanced (p < 0.05) the amount of creatine kinase release at 90 and 120 m i n for ethanol-exposed muscles and at 90,120,150, and 180 m i n for propylene glycol-exposed muscles. In an a t t e m p t to further increase intracellular calcium levels within the muscle fiber, the Na+-Ca2+ exchange system in the sarcolemma was inhibited or reversed b y using a low sodium incubation medium.30,31 It has recently been shown that the isotonic replacement of sodium b y Nmethylglucamine caused an increase in intracellular free calcium levels in an isolated rat skeletal muscle cell line.33In the present work, muscles exposed t o 7.4% (v/v) propylene glycol and incubated in the low sodium BSS:CaCl, medium showed a trend towards a slightly larger creatine kinase r e l e a s e compared w i t h the muscles i n c u b a t e d in the BSS:CaCl, medium, but n o statistical difference could be demonstrated (Figure 2). This finding might have a r i s e n from incomplete inhibition or reversal of the Na+-Ca2+ exchange system, since the sodium level in the incubation medium was only halved. It appears that propylene glycol a n d ethanol m a exert damaging m e c h a n i s m s similar t o bupivicaineY4 and DNPZ4,25 which also caused greater enzyme release in the presence of added calcium. To validate increases in muscle calcium levels, previous investigators have utilized atomic absorption spectrophotometry to measure agent-induced inTable I-Effect of Incubation Medla on Cumulatlve Creatlne Klnase Release from Propylene Glycol-Exposed or Control Muscles’
Incubation Media BSS BSS:NaCI BSS:CaCI,
Two-Hour Cumulative Creatine Kinase Release (Mean f SD) x lo2,U/L Propylene Glycol Control
0.048f 0.034 0.143 ? 0.075 0.381 2 0.293
3.05 2 1.35 3.86f 3.15 10.5f 1.49’
a Muscles were incubated in absence or presence of 6.3% (v/v) propylene glycol for 2 h, n = 4-6 in each experiment. Significantly different from BSS or BSS:NaCI (p < 0.05).
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Time (min) Figure l-Creatine kinase release per sampling interval (30 min) for muscles exposed to 7.4% (v/v) ethanol (Panel A) and 7.4% (v/v) propylene glycol (Panel 6 ) when incubated in the BSS (0)or the
BSS:CaCI, (0)media. The muscles were exposed to the organic cosolvents between 30 and 60 min. Each value is the mean and SD for 4-8 muscles; (*) indicates significant difference (p < 0.05).
Figure 24reatine kinase release per 30-min sampling interval for muscles exposed to 7.4% (Wv) propylene glycol between 30 and 60 min when incubated in the BSS:CaCI, medium (0)or in the low sodium BSS:CaCI, medium (0).Each value is the mean and SD for four pairs of muscles.
creases in total calcium levels in isolated skeletal muscles.27 However, this method cannot distinguish between bound and free intracellular calcium in this experimental system, and was therefore not attempted. Temporal Pattern of Organic Cosolvent-Induced Creatine Kinase Release-The temporal pattern of creatine kinase release following a 30-min exposure period to 20 pM A23187, 500 pM DCA, and 1 mM DNP and their ethanol solvent controls are shown in Figure 3. There are marked differences in the creatine kinase release pattern between the control and treated muscles (Figure 3). The patterns of creatine kinase release following a 30-min exposure to A23187, DCA, or DNP were similar to those reported by other investigators.". 9. 24* ', 27 Creatine kinase release increased rapidly after exposure to 20 pM A23187, then declined immediately after washout of A23187 (Figure 3A). Muscles subjected t o 500 p M DCA showed an immediate creatine kinase release during exposure, followed by a further increase in the subsequent 30-min period, and a decrease with time thereafter (Figure 3B). The results obtained from muscles exposed to 1 mM DNP were clearly different from those seen
Flgure 3-Creatine kinase release per 30-min sampling interval for muscles exposed to 20 pM A23187 and solvent control (Panel A), 500 pM DCA and solvent control (Panel B), or 1 mM DNP and solvent control (Panel C). The filled symbols are muscles exposed to the damaging compound and the open symbols are the solvent controls. The muscles were exposed to the damaging compound or solvent control between 30 and 60 min. Each value is the mean and SD for four pairs of muscles.
with DCA and A23187. There was little or no increase in creatine kinase release during the DNP exposure period, followed by a gradual increase in the amount of creatine kinase released over the next 2 h (Figure 3C). The temporal pattern of creatine kinase release following a 30-min exposure to 7.4% (v/v) propylene glycol is shown in Figures 1B and 4B. There is little or no release of creatine kinase during the 30-min exposure period. However, over the next 2 h, the amount of creatine kinase released per 30-min interval gradually increased (Figure 1B). This pattern of enzyme release was apparently independent of the concentration of propylene glycol (Figure 4B). Control muscles (Figure 4A) showed only small increases in creatine kinase release. There was no difference in the pattern of creatine kinase release in muscles exposed continuously to propylene glycol over a 2-h incubation or exposed to propylene glycol for only 30 min (data not shown), suggesting that the creatine kinase release pattern is not produced by rinsing propylene glycol out of the preparation. Similar to propylene glycol, creatine kinase release during the ethanol exposure period was small, but a dramatic rise was observed after the ethanol
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Time (min) Flgure U r e a t i n e kinase release per 30-min sampling interval for of normal muscles exposed to 0.5 mL (0),0.7 mL (0),and 1.0 mL (0) or saline added to the bath (Panel A) or 5.3%, v/v (0),7.470, vlv (O), 10.5%, v/v (0)propylene glycol (Panel 6 ) .The muscles were exposed to normal saline or propylene glycol between 30 and 60 min. The volume
of normal saline used is equal to the volume of organic solvent. Each value is the mean and SD for four pairs of muscles. Journal of Pharmaceutical Sciences / 395 Vol. 79, No. 5, May 1990
was removed (Figure 1A).The temporal pattern of propylene glycol- and ethanol-induced creatine kinase release was unaffected by the presence of added calcium in the incubation cu medium (Figure 1). 0 rl The pattern of propylene glycol- or ethanol-induced creatX ine kinase release was therefore similar to that of DNP, but distinctly different from those of DCA and A23187 (Figures 1 and 3). The dissimilar temporal patterns of enzyme release between A23187 and the organic cosolvents suggest that the latter probably do not increase cytosoliccalcium via increased permeability to calcium in the sarcoplasmic reticulum or mitochondria membrane (as is the case for A23187). Rather, they may act (like DNP) by inhibiting the activity of the intracellular and/or extracellular membrane-associated ion wu transport systems. The lag time between exposure to DNP or to the organic cosolvents and the release of creatine kinase could be explained by inhibition of the ion transport systems (both sodium and calcium) across intracellular and/or extraTime (min) cellular membranes, causing levels of intracellular sodium Figure Wreatine kinase release per 30-min sampling interval for and calcium to increase within the muscle fiber. Increased muscles exposed to 7.4% (v/v) propylene glycol. Control muscles (0) levels of sodium can lead to water influx and fiber swelling, were incubated in the presence of 100 pL of ethanol (solvent for DBC) thus resulting in the dilution of intracellular calcium levels. and treated muscles (0) were incubated in the presence of 100p M DBC Light and electron microscopy have demonstrated the presduring the entire experiment.The solid lines are experiments conducted ence of generalized edema in muscle fibers and swelling of the in the BSS:CaCI, medium and the dashed lines are experiments sarcoplasmicreticulum and mitochondria following exposure conducted in the BSS medium. Each value is the mean and SD for 6-8 to the local anesthetics. This possible dilution in cytosolic pairs of muscles; (*) denotes significant difference (p < 0.05). calcium levels could cause the activation of phospholipase A, (and the subsequent formation of lysophospholipids and reactive metabolites) to be delayed until sufficient calcium levels accumulate within the fiber. Moffatt and M i y a m ~ t o ~ ~ have suggested that the observed lag time in DNP-induced increases in the miniature endplate potential of the frog neuromuscular junction may be a function of the dilution of X I the resting intracellular calcium levels due to nerve terminal swelling. A similar phenomenon may be occurring in the \T organic cosolvent systems. M Effect of Phospholipase Inhibitor on Propylene Glycol\ m e, Induced Creatine Kinase Releas-Further evidence was sought to support the involvement of increased cytosolic , , , calcium and the activation of phospholipase A, as a mechanism of organic cosolvent-induced enzyme release. Studies M were conducted in which the nonspecific phospholipase A, O O inhibitor DBC was added to the incubation medium. Jackson 0 30 60 90 120 150 180 et al. have demonstrated that this inhibitor (and others such as mepacrine and c h l ~ r p r o r n a z i n e can ) ~ ~reduce * ~ ~ the amount Time (min) of lactate dehydrogenase and/or creatine kinase release from Figure GCreatine kinase release per 30-min sampling interval for DNP- or A23187-damaged skeletal muscles. The presence of muscles exposed to propylene glycol. Same experimental procedure as 100 pM DBC in the incubation medium caused a modest but in Figure 6, except the muscles were now exposed to either 300 pL of significant reduction in creatine kinase release (p < 0.05) a t ethanol solventcontrol (0)or 300 pM DBC (0). These experimentswere 120 and 150 min compared with the control muscles (Figure conducted in the BSS:CaCI, medium. Each value is the mean and SD 5). This modest effect could be a function of the DBC dose, the for six pairs of muscles; (*) denotes significant difference (p < 0.05). lack of DBC stability in the incubation medium, or the presence of other membrane effects from DBC.363s A similar release compared with the control a t later time points may reduction in propylene glycol-induced creatine kinase release have resulted from the inhibitory effect of DBC on phosphowas demonstrated a t 120, 150, and 180 min in muscles lipase A, activation. incubated in the presence of 300 pM DBC compared with Other literature data support our conclusion that the control muscles (p < 0.05, Figure 6). organic cosolvents may cause skeletal muscle damage via An interesting observation is the difference in the temporal increases in intracellular cytosolic calcium levels. For exampattern of propylene glycol-induced creatine kinase release in ple, ethanol has been suggested to cause increases in intrathe presence of 300 +M DBC (Figure 6). During the propylene cellular calcium levels,39inhibit calcium t r a n s p 0 r t , 4 ~and *~~ glycol exposure period and in the presence of DBC, there was enhance calcium permeability4, in skeletal sarcoplasmic a rapid and transient increase in creatine kinase release reticulum vesicles. Furthermore, it has been suggested that a compared with the control. Since other local anesthetics (e.g., defect in calcium homeostasis in myofibrils and organelles lidocaine and chlorpromazine) have been shown to disrupt causes ethanol-induced skeletal muscle degeneration in almembrane structure and cause lysis of membranes a t high coholic m y ~ p a t h yThe . ~ ~presence of ethanol may also directly concentrations,3Ms it is possible that the higher concentrainhibit the actions of other membrane-associated ion pumps. tion of DBC could have a direct deleterious effect on the Williams and co-workers have demonstrated that the inhibsarcolemma, leading to increased enzyme release. In contrast, itory effect of ethanol on the sarcolemma Na+,K+-ATPase is the subsequent reduction in the amount of creatine kinase 14334
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396 I Journal of Pharmaceutical Sciences Vol. 79, No. 5, May 1990
not due to a solubilization of the cardiac muscle membrane since the inhibitory effect is fully reversible once the membranes are rinsed free of the ethanol.44 The possible involvement of intracellular calcium i n causing tissue damage is not unique for skeletal muscle. Elevation in intracellular calcium has been shown to occur in he atotoxicity caused by h a l ~ t h a n e ; ~carbon tetrach1oride;'and a~etaminophen.4~
Conclusions In summary, our results suggest that propylene glycol and ethanol do not cause the release of creatine kinase via a direct solubilization of the sarcolemma. The increased release of creatine kinase following intramuscular administration of these solvents m a y b e due t o an intracellular mechanism, perhaps involving a disruption of intracellular calcium homeostasis within the skeletal muscle fiber. The nature of the interaction of organic cosolvents with the various membranes of the skeletal muscle fiber is at present undefined and warrants further investigation.
References and Notes 1. Greenblatt, D. J.; Duhme, D. W.; Koch-Weser, J. New E r g . J . Med. 1973,280,689. 2. Andersen, K. E.; Damsgaard, T. Acta Med. Scand. 1976, 199, 317-319. 3. Greenblatt, D. J.; Allen, M. D. JAMA 1978,240,542-544. 4. Korttila, K; Sothman, A,; Andemon, P. Actu Pharmol. Toxicol. 1976,39,104-117. 5. Yalkowsky, S.H.; Rubino, J. T. J.Pharm.Sci. 1985,74,416-421. 6. Brazeau, G. A.;Fung, H.-L. Pharm.Res. 1989,6,766-771. 7. Brazeau, G. A.; Fung, H.-L. J . Parent. Sci. Tech. 1989, 43, 144-149. 8. Jones, D. A,; Jackson, M. J.; Edwards, R. H. T. Clin. Sci. 1983, 65,193-201. 9. Jones, D. A.; Jackson, M. J.; McPhail, G.; Edwards, R. H. T. Clin. Sci. 1984,66,317-322. 10. Suarez-Kurtz, G.; Eastwood, A. B. A m . J . Physiol. 1981,241, C9W105. 11. Wrorremann. K.: Pena, S. D. J. Lancet 1976,ii. 672-674. 12. Pubhover, S.J.; Duncan, C. J.; Smith, J. L: J : Neuropath. Exp. Neurol. 1978.37,544557. 13. Duncan, C. J: Elperientia 1978,34,1531-1535. 14. Benoit, P. W.; Yagiela, J. A.; Fort, N. F. Toxicol. Appl. PharmaC O ~ .1980,52,187-198. 15. Kameyama, T.; Etlinger, J. D. Nature 1979,279.344-346. 16. Suaden. P.H.Biochem. J . 1980,190,593-603. 17. Rdemann, H. P.; Waxman, L.; Goldberg, A. L. J. Bwl. Chem. 1982,257,8716-8723. 18. Zeman, R. J.; Kame m a , T.; Matsumoto, K.; Berstein, P.; Etlinger, J. D. J . B w l Chem.1985,260,13619-13624.
19. Lewis, S.E. M.; Anderson, P.; Goldspink, D. F. Biochem. J . 1982, 204,257-264. 20. Hall-Craggs, E.C.B. Br. J.Exp. Path. 1980,61,139-149. 21. Yoshimura, T.; Tsqjihata, M: Satoh, A.; Mori, M.; Hazama, R.; Kinoshita, N.; Takashima, d.;Nagataki, S.Actu Neuropathul. (Bed) 1986,184-192. 22. Duncan, C. J. J . Cell Sci. 1987,87,581494. 23. Duncan, C. J. Med. Sci. Res. 1987,15,511-512. 24. Duncan, C. J.; Jackson, M. J . J. Cell Sci. 1987,87, 183-188. 25. Jackson, M. J.; Jones, D. A.; Edwards, R. H. T. Eur. J. Clin. Invest. 1984,14,369-374. 26. Duncan, C. J. Cell Tissue Res. 1988,253,457462. 27. Jackson, M. J.; Wa enmakers, Anton J. M.; Edwards, Richard H. T.Biochem. J . lf87,241,403407. 28. Brazeau, G.A.; Fung, H. -L. Pharm.Res. 1989,6,167-170. 29. Brazeau, G.A.; Fung, H. -L. Biochem. J . 1989,257,619-621. 30. Blaustein, M. P. Rev. Physwl. Biochem. Pharmacol.1974, 70, 3p82. Uhm. D. Y.: Dresdner. K. Science 1980.209.699-701. 31. Lee. C. 0.: 32. Zar' J. H: Biostatistkal Analysis; Prentice-Hall: Englewood Cliks, NJ, 1984;pp 162-235. 33. Klip, A.; Mack, E.; Ramlal, T.; Walker, D. Arch. Biochem. Bwphys. 1986,246,865-871. 34. Steer; J. H.; Mastaglia, F. L.; Papadimitriou, J. M.; Bruggen, I. Van J. Neurol. SCL.1986,205-217. 35. Moffatt. E. J.: Mivamoto. M. D. J. Pharmwol.EXD.Ther. 1988.
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36. T k n g , T. Y.;Greenberg, M.; Kanehisa, M. I. Biochemistry 1977, 31153121. _ ~ ._ ~~ _ _ 37. Kwant, W. 0.; Seeman, P. Biochim. Bwphys. Acta 1969, 183, 530543. 38. Takeuchi, Y: Yamaoka, Y: Morimoto, Y.; Kaneko, I.; Fukumori, Y.; Fukuda, 'r. J . Phurm. hi.1989,78,3-7. 39. Katz, A. M. FASEB 1982,41,2456-2459. 40. Kondo, M.;Kasai, M. Biochim. Biophys. Acta 1973,311,3914399. 41. Almeida,, L. M.; Vaz, W. L. C.; Stumpel, J.; Madeira, V. M. C. Biochemutry 1986,25,4832-4839. 42. Ohnishi, S.T.; Flick, J. L.; Rubin, E. Arch. Biochem. Bwphys. 1984,233,588-594. 43. Baruah, J. K.; Washington, M.; Kinder, D. Exp. Pathol. 1988,33, 207-2 12. 44. Williams, J. W.; Tada, M.; Katz, A.M.; Rubin, Emanuel Biochem. Pharmacoll975,24,27-32. 45. Farrell, G. C.;Mahoney, J.; Bilous, M.; Frost, L. J. Pharmacol. Exp. Ther. 1988,247,751-756. 46. Long, R. M.; Moore, L. Biochem. Pharmacol. 1987,36, 12151221. 47. Saville, J. G.; Davidson, C. P.; DAdrea, G. H.; Born, C. K.; Hamrick, M. E. Bwchem. Pharmacol. 1988,37,2467-2471.
Acknowledgments S u p ~ r t e din part by a research ant from the Parenteral Drug Association. GAB was a Fellow o?the American Foundation for Pharmaceutical Education and the 1986-1987 Smithkline Beckman PharmaceuticdEIiopharmaceutics Fellow.
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found to cause skeletal muscle damage and creatine kinase release following intramuscular injection. The mechanisms of this organic cosolvent-induced enzyme release have not been elucidated. Cosolventinduced creatine kinase release was enhanced by the addition of calcium 10 the incubation medium, and inhibited, albeit modestly, by dibucaine, a nonspecific phospholipase A, inhibitor. The temporal pattern of creatine kinase release further suggested that cosolvent-induced enzyme release from skeletal muscles may be caused by an intracellular mechanism rather than by a direct solubilization of sarcolemma. This intracellular mechanism may involve the mobilization of calcium. - -
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Intramuscular administration of parenteral products is frequently associated with patient discomfort and evidence of skeletal muscle damage. l4 Many of these commercial products contain organic cosolvents to aid in the solubilization of lipophilic therapeutic agents. Propylene glycol, ethanol, and polyethylene glycol 400 are among the most commonly used organic cosolvents in intramuscular formulation^.^ We have previously shown that these organic cosolvents cause skeletal muscle damage (myotoxicity) as assessed by the release of creatine kinase, either in vitro or in vivo.' The mechanisms of organic cosolvent-induced skeletal muscle damage have yet to be elucidated. Previously, we have shown that organic cosolvent-inducedcreatine kinase release from a rat isolated skeletal muscle is not well correlated with physicochemical properties (viz., tonicity, dielectric constant, apparent pH, viscosity, or surface t e n ~ i o n )These .~ findings suggested that biochemical interactions between organic cosolvents and skeletal muscle fibers ought to be examined as possible mechanisms of cosolvent-induced myotoxicity. One possible mechanism is that organic cosolvents directly solubilize the constituents (viz., phospholipids, fatty acids, and proteins) of the sarcolemma, altering its integrity and thereby leading to the release of intracellular enzymes. Previous investigators have shown a rapid release of intracellular cytoplasmic enzymes when isolated muscles were incubated in the presence of detergents Iviz., deoxycholic acid (DCA)or Brij 58],%" or following a defined disruption of the membrane (i.e., muscle slice)." The major characteristic of this mode of cellular damage is a rapid release of intracellular enzymes followed by a decline to near normal levels once the damaging compound is removed from the incubation medium." A second possible mechanism responsible for enhanced enzyme release might involve an increase in intracellular calcium levels. Several investigators have suggested that skeletal muscle damage may result from increases in cytosolic calcium levels. ''-I3 Increased intracellular calcium levels may result from changes in the integrity of the sarcolemma, thereby leading to an increased flux of calcium from extracellular sources,11or from a redistribution of calcium from the OO22-3549/90/05OO-0393$0 1 .OO/O C 7990, American Pharmaceutical Association
intracellular sites of calcium sequestration (i.e., sarco lasmic reticulum and mitochondria) to the cytosolic space.lg14 An increase in intracellular cytosolic calcium levels within skeletal muscle has been reported to increase protein degradation16-" and to alter muscle histology and u l t r a ~ t r u c t u r e . ' ~ ~In~ ~addition, * ~ ~ ~ ~ increased intracellular calcium has been hypothesized to activate muscle phospholipases (e.g., phospholipase A2).,' Activation of these phospholipases leads to the release of membrane-bound fatty acids and the formation of lysophospholipids known to be deleterious to sarcolemma integrity. Furthermore, activation of phospholipase A2 could also release membrane-bound arachidonic acid which is subsequently converted to bioactive metabolites (e.g., prostaglandins, leukotrienea) by lipoxygenase and cyclooxygenase enzymes. These bioactive metabolites are also disruptive of skeletal muscle m e m b r a n e ~ , ~ ~ * ~ leading to the release of intracellular enzymes. This mechanism is characterized by increased enzyme release from isolated skeletal muscles incubated with agents [viz., the calcium ionophore calimycin (A23187)and the mitochondria1 poison 2,4-dinitrophenol (DNP)] known to increase intracellular calcium levels, and decreased enzyme release from A23187- or DNP-damaged muscles when inhibitors of phospholipase A,, lipoxygenase, or cycloox genase enzymes were included in the bathing media.24*2'*2 Y The onset of enzyme efflux is delayed in this mechanism compared with that caused by direct solubilization of the sarcolemma, and enzyme release is sustained aRer removal of the damaging agent from the incubation medium. In this study, we examined the possible applicability of these mechanisms (i.e., membrane solubilization versus intracellular calcium alteration) in cosolvent-induced enzyme release. The effect of added calcium in the incubation medium on organic cosolvent-induced creatine kinase release was investigated. Secondly, the temporal pattern of creatine kinase release following exposure to the organic cosolvents was compared with that following exposure to DCA, A23187, and DNP. Finally, the effect of a nonspecific phospholipase A, inhibitor, dibucaine (DBC), on propylene glycol-induced creatine kinase release was examined.
Experimental Section Muscle Isolation and Incubation-Male
Sprague-Dawley rats
(200-350 g) were sacrificed and the extensor digitorum longus
muscles were isolated, removed, and placed into the carbogenated incubation medium (37 "C) as previously described.28At 30-min interv'als, the solutions (9.5 mL) were drained from the incubation vessels; this was followed by additionof fresh incubation medium and test compounds, when appropriate. A commercially available kit (SigmaChemical Company, St. Louis, MO) was used to assay creatine kinase. In paired muscle experiments, right and left muscles were randomized between controls and treatments. Role of Added Calcium on Organic Cosolvent-Induced Creatine Kinase Release-The effect of added calcium in the incubation medium on enzyme release was examined by using three incubation
Journal of Pharmaceutical Sciences I 393 Vol. 79, No. 5,May 7990
media. The control incubation medium was a balanced salt solution (BSS)containing 116 mM NaCl, 5.4 mM KCl, 5.6 mM dextrose, 26.2 mM NaHCO,, and 0.001 mM sodium phenol red in sterile water for irrigation. The pH was adjusted to 7.4 with 1 M HC1. The second incubation medium (BSS:CaCl,) was identical to the BSS, but with the addition of 2.5 mM CaC1,. The third incubation medium (BSS. NaCl) was identical to the BSS, but NaCl was added to match the tonicity in the BSS:CaCl, medium. Treated muscles were incubated in the presence of 6.3% (v/v) propylene glycol for 2 h. Contralateral muscles served as the control (no solvent). To further examine the effect of added calcium, a second series of experiments was conducted based on the protocol initially proposed by Jones and co-workers.8 The extensor digitorum longus muscles were individually mounted in the incubation baths via the tendons at resting length. Following a 30-min preincubation period, the muscle pairs were incubated for 30 min in the presence of propylene glycol or ethanol (7.4%,v/v, final concentration in the bath), using either the BSS or BSSCaCl, incubation medium. Preliminary studies showed that the extent of intracellular enzyme release induced by this concentration of the organic cosolvent in the incubation medium was similar to that produced by the direct injection of these cosolvents into the muscles.6 After the cosolvent solutions were drained from the incubation vessels, the muscles were rinsed 10 times with the incubation medium. Over the next 2 h, at 30-min intervals, the incubation medium was collected from the incubation vessel and replaced with fresh medium. The collected solutions were analyzed for creatine kinase activity within 1h. Possible assay interferences in the determination of creatine kinase activity by propylene glycol and ethanol at the above concentrations were ruled out in previous experiment^.^' The release of creatine kinase was expressed as the amount released per 30-min sampling interval. Similar experiments with 7.4% (v/v) polyethylene glycol 400 in the bath could not be carried out since creatine kinase activity was inhibited substantially by this solvent at this c o n c e n t r a t i ~ n . ~ ~ Other investigators have demonstrated that an incubation medium with reduced sodium content causes a greater influx of added calcium into muscle fibers via the Na+-Ca2+ exchange system within the s a r ~ o l e m r n a .31~ A ~ . third set of experiments was therefore conducted using an incubation medium with reduced sodium content. A pair of extensor digitorum longus muscles was incubated either in the BSS:CaCl, medium (116 mM NaC1) or in a reduced sodium (58 mM NaCl) BSS:CaCI, medium. The tonicity of the latter solution was maintained with the addition of sucrose. The rest of the protocol was identical to that described above. Temporal Pattern of Creatine Kinase Release-In order to determine whether the organic cosolvents cause the release of creatine kinase through a direct solubilization of the sarcolemma or through increases in cytosolic calcium, the temporal pattern of enzyme release was examined as described in the literature.’. 25 Following a 30-min preincubation period, one muscle was incubated in the presence of the damaging compound for 30 min, while the contralateral muscle served as a control. The muscles were rinsed 10 times with the incubation medium at the end of the 30-min period to remove the damaging compound. The remaining protocol was identical to that used in the calcium studies. The concentrations of DCA (500 pM), A23187 (20 pM), and DNP (1mM) were selected based on previous literature investigations.2P26 Three different concentrations of propylene glycol (5.3-lo%, v/v, final bath concentration) were investigated to determine if the temporal pattern of creatine kinase release was affected by concentration. The concentration of ethanol was identical to that used in the calcium studies. All experiments utilized BSS as the incubation medium. Possible interference in the determination of creatine kinase activity by the above agents was ruled out in previous experiments.” Inhibitor Studies-To evaluate the possibility that organic cosolvent-induced creatine kinase release may be modulated through the activation of phospholipase A,, we used a nonspecific phospholipase A, inhibitor, DBC, which has been shown to cause a dramatic decrease in DNP-induced lactate dehydrogenase release from isolated muscles.26 In the present study, creatine kinase release induced by 7.4% (dv) propylene glycol was examined in the presence of 100 pM DBC throughout the 3-h period. The contralateral muscle was incubated in an identical manner except for the omission of DBC (ethanol solvent control). The remaining experimental protocol was identical to that utilized in previous studies. A similar experiment was conducted using pairs of muscles exposed either to 300 pM DBC
*’
394 / Journal of Pharmaceutical Sciences Vol. 79, No. 5, May 1990
(treatment) or 300 pL of ethanol added to the bath (solvent for DBC). Due to significant interferences in the determination of creatine kinase activity, we were unable to study other inhibitors (viz., chlorpromazine, mepacrine, phenidone, nordihydroguaiaretic acid) that have been shown to reduce enzyme release following skeletal muscle damage.,’ Statistical Analysis-Data are presented as the mean and standard deviation. Statistical analysis was performed using one-way or two-way analysis of variance. Differences between the means were determined using Tukey’s test when appr~priate.~’
Results and Discussion Role of Added Calcium on Organic Cosolvent-Induced Creatine Kinase Release-Incubation of isolated muscles in the presence of 6.3% (v/v) propylene glycol for 2 h increased creatine kinase release -64-fold, 28-fold, and 27-fold over control muscles in the BSS, BSS:CaCl,, o r BSS:NaClmedium, respectively (Table I). Enzyme release from propylene glycoltreated muscles was significantly h i g h e r in the BSSCaCl, incubation medium (p < 0.05) compared w i t h the BSS and BSS:NaCl media. Since the BSS:CaCl, and the BSS:NaCl media are isotonic, these findings suggest a specific effect of calcium in affecting enzyme release, unrelated to a change in tonicity of the medium. In control muscles, there was n o significant difference in the cumulative creatine kinase release among the three media. A similar effect of added calcium w a s observed when the muscles were incubated for only 30 min in the presence of propylene glycol o r ethanol (Figure 1). Additional calcium in the incubation medium significantly enhanced (p < 0.05) the amount of creatine kinase release at 90 and 120 m i n for ethanol-exposed muscles and at 90,120,150, and 180 m i n for propylene glycol-exposed muscles. In an a t t e m p t to further increase intracellular calcium levels within the muscle fiber, the Na+-Ca2+ exchange system in the sarcolemma was inhibited or reversed b y using a low sodium incubation medium.30,31 It has recently been shown that the isotonic replacement of sodium b y Nmethylglucamine caused an increase in intracellular free calcium levels in an isolated rat skeletal muscle cell line.33In the present work, muscles exposed t o 7.4% (v/v) propylene glycol and incubated in the low sodium BSS:CaCl, medium showed a trend towards a slightly larger creatine kinase r e l e a s e compared w i t h the muscles i n c u b a t e d in the BSS:CaCl, medium, but n o statistical difference could be demonstrated (Figure 2). This finding might have a r i s e n from incomplete inhibition or reversal of the Na+-Ca2+ exchange system, since the sodium level in the incubation medium was only halved. It appears that propylene glycol a n d ethanol m a exert damaging m e c h a n i s m s similar t o bupivicaineY4 and DNPZ4,25 which also caused greater enzyme release in the presence of added calcium. To validate increases in muscle calcium levels, previous investigators have utilized atomic absorption spectrophotometry to measure agent-induced inTable I-Effect of Incubation Medla on Cumulatlve Creatlne Klnase Release from Propylene Glycol-Exposed or Control Muscles’
Incubation Media BSS BSS:NaCI BSS:CaCI,
Two-Hour Cumulative Creatine Kinase Release (Mean f SD) x lo2,U/L Propylene Glycol Control
0.048f 0.034 0.143 ? 0.075 0.381 2 0.293
3.05 2 1.35 3.86f 3.15 10.5f 1.49’
a Muscles were incubated in absence or presence of 6.3% (v/v) propylene glycol for 2 h, n = 4-6 in each experiment. Significantly different from BSS or BSS:NaCI (p < 0.05).
’
w
A
B
A
1
I
0
60
120
180 0
60
120
180 0
60
120
180
Time (min)
Time (min) Figure l-Creatine kinase release per sampling interval (30 min) for muscles exposed to 7.4% (v/v) ethanol (Panel A) and 7.4% (v/v) propylene glycol (Panel 6 ) when incubated in the BSS (0)or the
BSS:CaCI, (0)media. The muscles were exposed to the organic cosolvents between 30 and 60 min. Each value is the mean and SD for 4-8 muscles; (*) indicates significant difference (p < 0.05).
Figure 24reatine kinase release per 30-min sampling interval for muscles exposed to 7.4% (Wv) propylene glycol between 30 and 60 min when incubated in the BSS:CaCI, medium (0)or in the low sodium BSS:CaCI, medium (0).Each value is the mean and SD for four pairs of muscles.
creases in total calcium levels in isolated skeletal muscles.27 However, this method cannot distinguish between bound and free intracellular calcium in this experimental system, and was therefore not attempted. Temporal Pattern of Organic Cosolvent-Induced Creatine Kinase Release-The temporal pattern of creatine kinase release following a 30-min exposure period to 20 pM A23187, 500 pM DCA, and 1 mM DNP and their ethanol solvent controls are shown in Figure 3. There are marked differences in the creatine kinase release pattern between the control and treated muscles (Figure 3). The patterns of creatine kinase release following a 30-min exposure to A23187, DCA, or DNP were similar to those reported by other investigators.". 9. 24* ', 27 Creatine kinase release increased rapidly after exposure to 20 pM A23187, then declined immediately after washout of A23187 (Figure 3A). Muscles subjected t o 500 p M DCA showed an immediate creatine kinase release during exposure, followed by a further increase in the subsequent 30-min period, and a decrease with time thereafter (Figure 3B). The results obtained from muscles exposed to 1 mM DNP were clearly different from those seen
Flgure 3-Creatine kinase release per 30-min sampling interval for muscles exposed to 20 pM A23187 and solvent control (Panel A), 500 pM DCA and solvent control (Panel B), or 1 mM DNP and solvent control (Panel C). The filled symbols are muscles exposed to the damaging compound and the open symbols are the solvent controls. The muscles were exposed to the damaging compound or solvent control between 30 and 60 min. Each value is the mean and SD for four pairs of muscles.
with DCA and A23187. There was little or no increase in creatine kinase release during the DNP exposure period, followed by a gradual increase in the amount of creatine kinase released over the next 2 h (Figure 3C). The temporal pattern of creatine kinase release following a 30-min exposure to 7.4% (v/v) propylene glycol is shown in Figures 1B and 4B. There is little or no release of creatine kinase during the 30-min exposure period. However, over the next 2 h, the amount of creatine kinase released per 30-min interval gradually increased (Figure 1B). This pattern of enzyme release was apparently independent of the concentration of propylene glycol (Figure 4B). Control muscles (Figure 4A) showed only small increases in creatine kinase release. There was no difference in the pattern of creatine kinase release in muscles exposed continuously to propylene glycol over a 2-h incubation or exposed to propylene glycol for only 30 min (data not shown), suggesting that the creatine kinase release pattern is not produced by rinsing propylene glycol out of the preparation. Similar to propylene glycol, creatine kinase release during the ethanol exposure period was small, but a dramatic rise was observed after the ethanol
A
R ,"
6.0 -
(I) v1
$
3.0
-
p: ?c 0.0 0
so
120
180 0
60
120
180
Time (min) Flgure U r e a t i n e kinase release per 30-min sampling interval for of normal muscles exposed to 0.5 mL (0),0.7 mL (0),and 1.0 mL (0) or saline added to the bath (Panel A) or 5.3%, v/v (0),7.470, vlv (O), 10.5%, v/v (0)propylene glycol (Panel 6 ) .The muscles were exposed to normal saline or propylene glycol between 30 and 60 min. The volume
of normal saline used is equal to the volume of organic solvent. Each value is the mean and SD for four pairs of muscles. Journal of Pharmaceutical Sciences / 395 Vol. 79, No. 5, May 1990
was removed (Figure 1A).The temporal pattern of propylene glycol- and ethanol-induced creatine kinase release was unaffected by the presence of added calcium in the incubation cu medium (Figure 1). 0 rl The pattern of propylene glycol- or ethanol-induced creatX ine kinase release was therefore similar to that of DNP, but distinctly different from those of DCA and A23187 (Figures 1 and 3). The dissimilar temporal patterns of enzyme release between A23187 and the organic cosolvents suggest that the latter probably do not increase cytosoliccalcium via increased permeability to calcium in the sarcoplasmic reticulum or mitochondria membrane (as is the case for A23187). Rather, they may act (like DNP) by inhibiting the activity of the intracellular and/or extracellular membrane-associated ion wu transport systems. The lag time between exposure to DNP or to the organic cosolvents and the release of creatine kinase could be explained by inhibition of the ion transport systems (both sodium and calcium) across intracellular and/or extraTime (min) cellular membranes, causing levels of intracellular sodium Figure Wreatine kinase release per 30-min sampling interval for and calcium to increase within the muscle fiber. Increased muscles exposed to 7.4% (v/v) propylene glycol. Control muscles (0) levels of sodium can lead to water influx and fiber swelling, were incubated in the presence of 100 pL of ethanol (solvent for DBC) thus resulting in the dilution of intracellular calcium levels. and treated muscles (0) were incubated in the presence of 100p M DBC Light and electron microscopy have demonstrated the presduring the entire experiment.The solid lines are experiments conducted ence of generalized edema in muscle fibers and swelling of the in the BSS:CaCI, medium and the dashed lines are experiments sarcoplasmicreticulum and mitochondria following exposure conducted in the BSS medium. Each value is the mean and SD for 6-8 to the local anesthetics. This possible dilution in cytosolic pairs of muscles; (*) denotes significant difference (p < 0.05). calcium levels could cause the activation of phospholipase A, (and the subsequent formation of lysophospholipids and reactive metabolites) to be delayed until sufficient calcium levels accumulate within the fiber. Moffatt and M i y a m ~ t o ~ ~ have suggested that the observed lag time in DNP-induced increases in the miniature endplate potential of the frog neuromuscular junction may be a function of the dilution of X I the resting intracellular calcium levels due to nerve terminal swelling. A similar phenomenon may be occurring in the \T organic cosolvent systems. M Effect of Phospholipase Inhibitor on Propylene Glycol\ m e, Induced Creatine Kinase Releas-Further evidence was sought to support the involvement of increased cytosolic , , , calcium and the activation of phospholipase A, as a mechanism of organic cosolvent-induced enzyme release. Studies M were conducted in which the nonspecific phospholipase A, O O inhibitor DBC was added to the incubation medium. Jackson 0 30 60 90 120 150 180 et al. have demonstrated that this inhibitor (and others such as mepacrine and c h l ~ r p r o r n a z i n e can ) ~ ~reduce * ~ ~ the amount Time (min) of lactate dehydrogenase and/or creatine kinase release from Figure GCreatine kinase release per 30-min sampling interval for DNP- or A23187-damaged skeletal muscles. The presence of muscles exposed to propylene glycol. Same experimental procedure as 100 pM DBC in the incubation medium caused a modest but in Figure 6, except the muscles were now exposed to either 300 pL of significant reduction in creatine kinase release (p < 0.05) a t ethanol solventcontrol (0)or 300 pM DBC (0). These experimentswere 120 and 150 min compared with the control muscles (Figure conducted in the BSS:CaCI, medium. Each value is the mean and SD 5). This modest effect could be a function of the DBC dose, the for six pairs of muscles; (*) denotes significant difference (p < 0.05). lack of DBC stability in the incubation medium, or the presence of other membrane effects from DBC.363s A similar release compared with the control a t later time points may reduction in propylene glycol-induced creatine kinase release have resulted from the inhibitory effect of DBC on phosphowas demonstrated a t 120, 150, and 180 min in muscles lipase A, activation. incubated in the presence of 300 pM DBC compared with Other literature data support our conclusion that the control muscles (p < 0.05, Figure 6). organic cosolvents may cause skeletal muscle damage via An interesting observation is the difference in the temporal increases in intracellular cytosolic calcium levels. For exampattern of propylene glycol-induced creatine kinase release in ple, ethanol has been suggested to cause increases in intrathe presence of 300 +M DBC (Figure 6). During the propylene cellular calcium levels,39inhibit calcium t r a n s p 0 r t , 4 ~and *~~ glycol exposure period and in the presence of DBC, there was enhance calcium permeability4, in skeletal sarcoplasmic a rapid and transient increase in creatine kinase release reticulum vesicles. Furthermore, it has been suggested that a compared with the control. Since other local anesthetics (e.g., defect in calcium homeostasis in myofibrils and organelles lidocaine and chlorpromazine) have been shown to disrupt causes ethanol-induced skeletal muscle degeneration in almembrane structure and cause lysis of membranes a t high coholic m y ~ p a t h yThe . ~ ~presence of ethanol may also directly concentrations,3Ms it is possible that the higher concentrainhibit the actions of other membrane-associated ion pumps. tion of DBC could have a direct deleterious effect on the Williams and co-workers have demonstrated that the inhibsarcolemma, leading to increased enzyme release. In contrast, itory effect of ethanol on the sarcolemma Na+,K+-ATPase is the subsequent reduction in the amount of creatine kinase 14334
51 z[y,
396 I Journal of Pharmaceutical Sciences Vol. 79, No. 5, May 1990
not due to a solubilization of the cardiac muscle membrane since the inhibitory effect is fully reversible once the membranes are rinsed free of the ethanol.44 The possible involvement of intracellular calcium i n causing tissue damage is not unique for skeletal muscle. Elevation in intracellular calcium has been shown to occur in he atotoxicity caused by h a l ~ t h a n e ; ~carbon tetrach1oride;'and a~etaminophen.4~
Conclusions In summary, our results suggest that propylene glycol and ethanol do not cause the release of creatine kinase via a direct solubilization of the sarcolemma. The increased release of creatine kinase following intramuscular administration of these solvents m a y b e due t o an intracellular mechanism, perhaps involving a disruption of intracellular calcium homeostasis within the skeletal muscle fiber. The nature of the interaction of organic cosolvents with the various membranes of the skeletal muscle fiber is at present undefined and warrants further investigation.
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19. Lewis, S.E. M.; Anderson, P.; Goldspink, D. F. Biochem. J . 1982, 204,257-264. 20. Hall-Craggs, E.C.B. Br. J.Exp. Path. 1980,61,139-149. 21. Yoshimura, T.; Tsqjihata, M: Satoh, A.; Mori, M.; Hazama, R.; Kinoshita, N.; Takashima, d.;Nagataki, S.Actu Neuropathul. (Bed) 1986,184-192. 22. Duncan, C. J. J . Cell Sci. 1987,87,581494. 23. Duncan, C. J. Med. Sci. Res. 1987,15,511-512. 24. Duncan, C. J.; Jackson, M. J . J. Cell Sci. 1987,87, 183-188. 25. Jackson, M. J.; Jones, D. A.; Edwards, R. H. T. Eur. J. Clin. Invest. 1984,14,369-374. 26. Duncan, C. J. Cell Tissue Res. 1988,253,457462. 27. Jackson, M. J.; Wa enmakers, Anton J. M.; Edwards, Richard H. T.Biochem. J . lf87,241,403407. 28. Brazeau, G.A.; Fung, H. -L. Pharm.Res. 1989,6,167-170. 29. Brazeau, G.A.; Fung, H. -L. Biochem. J . 1989,257,619-621. 30. Blaustein, M. P. Rev. Physwl. Biochem. Pharmacol.1974, 70, 3p82. Uhm. D. Y.: Dresdner. K. Science 1980.209.699-701. 31. Lee. C. 0.: 32. Zar' J. H: Biostatistkal Analysis; Prentice-Hall: Englewood Cliks, NJ, 1984;pp 162-235. 33. Klip, A.; Mack, E.; Ramlal, T.; Walker, D. Arch. Biochem. Bwphys. 1986,246,865-871. 34. Steer; J. H.; Mastaglia, F. L.; Papadimitriou, J. M.; Bruggen, I. Van J. Neurol. SCL.1986,205-217. 35. Moffatt. E. J.: Mivamoto. M. D. J. Pharmwol.EXD.Ther. 1988.
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36. T k n g , T. Y.;Greenberg, M.; Kanehisa, M. I. Biochemistry 1977, 31153121. _ ~ ._ ~~ _ _ 37. Kwant, W. 0.; Seeman, P. Biochim. Bwphys. Acta 1969, 183, 530543. 38. Takeuchi, Y: Yamaoka, Y: Morimoto, Y.; Kaneko, I.; Fukumori, Y.; Fukuda, 'r. J . Phurm. hi.1989,78,3-7. 39. Katz, A. M. FASEB 1982,41,2456-2459. 40. Kondo, M.;Kasai, M. Biochim. Biophys. Acta 1973,311,3914399. 41. Almeida,, L. M.; Vaz, W. L. C.; Stumpel, J.; Madeira, V. M. C. Biochemutry 1986,25,4832-4839. 42. Ohnishi, S.T.; Flick, J. L.; Rubin, E. Arch. Biochem. Bwphys. 1984,233,588-594. 43. Baruah, J. K.; Washington, M.; Kinder, D. Exp. Pathol. 1988,33, 207-2 12. 44. Williams, J. W.; Tada, M.; Katz, A.M.; Rubin, Emanuel Biochem. Pharmacoll975,24,27-32. 45. Farrell, G. C.;Mahoney, J.; Bilous, M.; Frost, L. J. Pharmacol. Exp. Ther. 1988,247,751-756. 46. Long, R. M.; Moore, L. Biochem. Pharmacol. 1987,36, 12151221. 47. Saville, J. G.; Davidson, C. P.; DAdrea, G. H.; Born, C. K.; Hamrick, M. E. Bwchem. Pharmacol. 1988,37,2467-2471.
Acknowledgments S u p ~ r t e din part by a research ant from the Parenteral Drug Association. GAB was a Fellow o?the American Foundation for Pharmaceutical Education and the 1986-1987 Smithkline Beckman PharmaceuticdEIiopharmaceutics Fellow.
Journal of Pharmaceutical Sciences I 397 Vol. 79, No. 5, May 1990