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Effect of experimental spinal cord injury on salicylate bioavailability after oral aspirin administration
J Pharmacol Toxicol 42 (1999) 93–97
Effect of experimental spinal cord injury on salicylate bioavailability after oral aspirin administration Griselda Fuentes-Laraa,c, Gabriel Guízar-Sahagúna,b, Patricia García-Lópezc,d,* b
a Proyecto Camina A.C., Mexico D.F., Mexico Unidad de Investigación Médica en Enfermedades Neurológicas, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico D.F., Mexico c Departamento de Farmacología y Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico D.F., Mexico dDivisión de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando 22, Tlalpan, 14000 Mexico D.F., Mexico Received 8 February 2000; accepted 8 February 2000
Abstract The purpose of the present work was to study whether spinal cord injury (SCI) alters salicylate bioavailability after oral aspirin administration. Female Sprague-Dawley rats were subjected to SCI at the T8 level by two procedures, contusion by the weight-drop method and severance by knife, and received a single oral aspirin dose (15 mg/kg) 24 h after injury. Blood samples were drawn and aspirin (ASA) and salicylic acid (SA) concentrations in whole blood were determined at selected times over a period of 240 min. Both SCI procedures produced similar alterations on salicylate bioavailability. ASA bioavailability was not significantly changed by SCI. On the other hand, SA peak concentrations were significantly reduced in 20% to 30%, compared with sham-lesioned controls. The area under the SA concentration against time curve was decreased in 10% to 25%, although this difference did not reach statistical significance. Results suggest that SCI at the T8 level decreases the rate, but not the extent, of aspirin absorption from the gastrointestinal tract. SCI-induced alterations in aspirin absorption appeared to be modest compared with those previously reported for other analgesic agents, such as paracetamol (acetaminophen). © 2000 Elsevier Science Inc. All rights reserved. Keywords: Aspirin; Salicylic acid; Salicylates; Bioavailability; Spinal cord injury
1. Introduction Spinal cord injury (SCI) is a catastrophic affliction that results not only in motor and sensory impairment, but also in major metabolic and systemic alterations (Guízar-Sahagún et al., 1998). Hence, SCI may change the kinetics of drug absorption, distribution, and elimination (Segal & Brunnemann, 1989). It has been reported that the pharmacokinetics of several drugs, such as paracetamol (acetaminophen) (Halstead et al., 1985), theophylline (Segal et al., 1985), dantrolene (Segal & Brunnemann, 1989), aminoglycosides (Gilman et al 1993; Segal et al., 1988), and lorazepam (Segal et al., 1991) are significantly altered in SCI patients in comparison with able-bodied subjects. Despite the evidence of significant pharmacokinetic changes in SCI, the criteria and strategies for optimizing drug therapy in this type of patient are seldom based on rational principles. Treatment strategies
* Corresponding author. Tel: 015 628 0426; Fax: 015 628 0432 E-mail address: [email protected]
are extrapolated, often uncritically, from clinical experience in able-bodied persons (Segal & Brunnemann, 1989). Clinical reports on drug kinetics in SCI are often anecdotal, because it is extremely difficult to perform systematic pharmacokinetic studies in SCI patients. This difficulty is due to the important interindividual variability in injury extent and location. Therefore, the use of experimental models appears to be a suitable strategy for understanding pharmacokinetic alterations due to SCI, as well as the pathophysiological mechanisms involved. We previously showed, by using an experimental model in the rat, that the oral bioavailability of paracetamol is significantly reduced by SCI (García-López et al., 1996, 1997). Aspirin is one of the most widely used drugs worldwide. Being an over-the-counter drug, aspirin can be consumed by virtually all patient populations, including patients with SCI. Therefore, we considered it of interest to study whether salicylate bioavailability after oral aspirin administration is altered by SCI. We studied the effects of SCI produced by two different experimental procedures, contusion and severance by knife (Das, 1989).
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2. Materials and methods 2.1. Animals Female Sprague-Dawley rats (240–260 g) were used. Twelve hours before drug administration, food was withheld, but animals had free access to water. The study was approved by the local Animal Care Committee. 2.2. Spinal cord injury Animals were subjected to SCI by two different procedures, contusion and severance by knife. Spinal cord contusion was performed by the weight-drop method of Allen (1911) modified for rats, as described previously (GarcíaLópez et al., 1995). Briefly, animals were anesthetized by the intramuscular injection of a mixture of ketamine (77.5 mg/kg) and xylazine hydrochloride (12.5 mg/kg). Under aseptic conditions, a laminectomy was performed at the T8 level. Rats were then placed on a stereotaxic device, and a stainless steel cylinder weighing 15 g was dropped from a height of 4 cm through a guided tube onto the exposed dura. When the presence of hematoma on the dorsal aspect of the spinal cord was corroborated, the aponeurotic plane and the skin were separately sutured with 5-0 nylon thread. For the severance-by-knife SCI, rats were anesthetized and a laminectomy was performed as heretofore described. The spinal cord was completely severed by a clean transversal cut performed with a scalpel at the T8 level (Das, 1989). Then, the aponeurotic plane and the skin were sutured. Postsurgical care was performed as described previously (GuízarSahagún et al., 1994). 2.3. Determination of salicylate bioavailability A single oral aspirin dose (15 mg/kg) suspended in 0.5% carboxymethyl cellulose was given by gavage. Blood samples (100–125 l) were drawn from the caudal artery at 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 360 min after aspirin administration. The total blood volume extracted did not exceed 2 ml. The concentration of aspirin (acetyl salicylic acid, ASA) and its active metabolite, salicylic acid (SA), were determined in whole blood by high-performance liquid chromatography with the use of 2-acetamidophenol as internal standard, as previously described (Cruz et al., 1999). Individual whole-blood concentration against time curves were constructed for both ASA and SA. The maximal concentration (Cmax), as well as the time to reach the maximal concentration (Tmax), were directly determined from these plots. The area under the concentration against time curve to the last concentration–time point (AUClast) was determined by the trapezoidal rule. Bioavailability was determined from the AUClast and Cmax values, according to current recommendations (Balant et al., 1991; Pabst & Jaeger, 1990). 2.4. Study design Three groups of seven rats each were studied. Animals in group 1 served as controls and were only subjected to lami-
nectomy. Animals in groups 2 and 3 were subjected to SCI by contusion and severance by knife, respectively. Salicylate bioavailability was studied 24 h after the surgical procedure. Comparisons between the control and the SCI groups were performed by analysis of variance followed by Dunnet’s test. 2.5. Drugs and reagents ASA, SA, and 2-acetamidophenol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile, chromatographic grade, was obtained from E. Merck (Darmstadt, Germany). All other reagents were of analytical grade. High-quality water employed to prepare solutions was obtained by using a Milli-Q Reagent Water System (Continental Water Systems, El Paso, TX, USA). 3. Results All animals exhibited normal locomotor activity before the initiation of the study. One day after SCI, by either contusion or severance by knife, rats showed complete flaccid paraplegia. On the other hand, sham-injured animals subjected only to laminectomy exhibited normal walk after they recovered from anesthesia. Whole-blood ASA levels are shown in Fig. 1. ASA was absorbed and eliminated very rapidly. In sham-lesioned animals, Cmax was observed 5 min after aspirin administration. ASA blood concentrations were below detection limits after 15 min. In rats with SCI by both contusion and severance by knife, Cmax was observed 10 min after aspirin administration. There was a trend toward a reduction in Cmax in SCI rats (Table 1). However, owing to the high variability observed, differences in Cmax did not reach statistical significance. AUClast values for ASA were similar in the three
Fig. 1. Aspirin (acetylsalicylic acid) blood concentrations observed after oral administration of a 15 mg/kg oral aspirin dose in rats subjected to spinal cord injury at the T8 level by contusion (white circles) and by severance by knife (white squares) and in sham-lesioned controls (black circles). Data are presented as mean ⫾ SEM of seven animals.
G. Fuentes-Lara et al. / J Pharmacol Toxicol 42 (1999) 93–97
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Table 1 Bioavailability parameters of ASA and SA observed after a single oral administration of a 15-mg/kg aspirin dose to rats subjected to SCI at the T8 level by two procedures, contusion by the weight-drop method and severance by knife, and to sham-lesioned controls. Acetylsalicylic acid
Sham lesion SCI by contusion SCI by severance by knife
Salicylic acid
Cmax (g/ml)
AUClast (g·h/ml)
Cmax (g/ml)
AUClast (g·h/ml)
3.4 ⫾ 0.6 2.4 ⫾ 1.0 2.1 ⫾ 0.3
24 ⫾ 5 21 ⫾ 7 24 ⫾ 4
21 ⫾ 1 14 ⫾ 2* 17 ⫾ 1*
5325 ⫾ 558 4102 ⫾ 426 4725 ⫾ 368
Data are expressed as mean ⫾ SEM of seven rats. *Significantly different from the sham-lesion group (p ⬍ 0.05), as determined by analysis of variance followed by Dunnet’s test.
groups of animals studied. The time course of ASA blood concentrations, as well as bioavailability parameters observed in the two SCI groups, contusion and severance by knife, were similar. Whole-blood SA levels are shown in Fig. 2. SA concentrations were considerably higher than for ASA at all sampling times. As a result, AUClast values for SA were about 200 times as great as those for ASA (Table 1). The rate of SA appearance in blood was slower in the two SCI groups (Tmax range 60–150 min) than in sham-lesioned controls (Tmax range 30–60 min). As a result, SA blood concentrations were lower in SCI animals than in controls during the first 120 min after aspirin administration but were similar thereafter. Cmax values were significantly reduced in both SCI groups compared with sham-lesioned controls (Table 1). AUClast values were slightly reduced in the two SCI groups compared with sham-lesioned controls (Table 1), but these differences did not reach statistical significance (p ⬎ 0.05). These results suggest that SCI decreased the rate, but not the extent, of drug absorption. The time course of SA blood concentrations, as well as bioavailability parameters,
Fig. 2. Salicylic acid blood concentrations observed after oral administration of a 15 mg/kg oral aspirin dose in rats subjected to spinal cord injury at the T8 level by contusion (white circles) and by severance by knife (white squares) and in sham-lesioned controls (black circles). Data are presented as mean ⫾ SEM of seven animals.
observed in the two SCI groups, contusion and severance by knife, were similar. 4. Discussion The purpose of this work was to study salicylate bioavailability after oral aspirin administration in two models of SCI, contusion and severance by knife. The contusion model by the weight-drop method mimics the histopathological features of acceleration-related fracture/dislocation of the spine, producing contusion or compression of the spinal cord, as in most accidental injuries in humans. On the other hand, the severance-by-knife model resembles injuries produced by aggressions with puncturing or cutting devices (Das, 1989; Stover and Fine, 1987). SCI is not a static state. The primary injury due to the mechanical trauma is followed by a secondary injury that increases the original neural damage. This secondary injury has been attributed to the presence of multiple endogenous toxic substances released by the injured neurons within the lesion area (Holtz & Nystrom, 1990) as well as a disruption of the microcirculation (Hall & Wolf, 1986). In the contusion by the weightdrop method, the damage produced by the secondary injury is predominant. Conversely, in the severance-by-knife model, the damage produced by the primary injury is more important (Das, 1989). Despite these histopathological differences between the contusion and the severance-by-knife models, both procedures produced a similar locomotor impairment. This was also the case for the observed alterations in salicylate bioavailability after oral aspirin administration. It is therefore probable that the pathophysiological processes resulting in altered salicylate bioavailability during the acute phase of SCI depend only on the disruption of nervous transmission through the spinal cord. Whether this disruption is due mainly to the primary or to the secondary injury appears to be irrelevant. SCI at the T8 level, by either contusion or severance by knife, did not produce any significant alteration in ASA bioavailability, although there was a trend toward a reduction in Cmax and a prolongation in Tmax. ASA concentrations were very low. Moreover, ASA appearance and disappearance from the circulation were very rapid, and concentrations exhibited a high interindividual variability, owing to
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the fact that aspirin is readily absorbed from the stomach but is rapidly biotransformed into SA by esterases in the blood, gastrointestinal tract, and liver (Gibaldi, 1991; Higgs et al., 1987; Wentjes & Levy, 1988). Under these conditions, it is difficult to detect statistically significant differences in bioavailability between treatments. SA accounted for more than 99% of the total salicylate bioavailability after aspirin oral administration. Other aspirin metabolites, such as gentisic and salicyluric acids cannnot be detected under the conditions of the present study (Cruz et al., 1999). Gentisic acid concentrations can be detected in the rat blood only after the administration of considerably higher aspirin doses (Castañeda-Hernández et al., 1994). It then appears that, under the conditions used in the present study, SA bioavailability is the best indicator for aspirin absorption. Moreover, SA is considered to be the active metabolite of aspirin (Higgs et al., 1987). SCI by either contusion or severance by knife had a modest effect on SA bioavailability. AUClast, an indicator of the extent of drug absorption (Balant et al., 1991) was slightly, although not significantly, reduced. On the other hand, SCI significantly reduced Cmax, suggesting that SCI decreased the rate of aspirin absorption (Balant et al, 1991; Pabst & Jaeger, 1990). This is consistent with the observed trend toward a reduction in ASA Cmax and with the fact that Tmax was prolonged in SCI animals, for both ASA and SA. No attempt was made to perform any statistical comparison of Tmax values, because Tmax is not considered an adequate indicator for bioavailability (Pabst & Jaeger, 1990). The reduction in SA Cmax did not have a significant effect on AUClast. Because SA concentrations at times longer than 120 min were similar in SCI and sham-lesioned animals, no statistically significant differences were detected in AUClast. These data suggest that the extent of aspirin absorption was not significantly affected by SCI (Balant et al, 1991; Pabst & Jaeger, 1990). Our results hence suggest that SCI-induced alterations on aspirin bioavailability concern mainly the rate of absorption. It was not possible to make an adequate estimation of half-life with the experimental design used in the present study. The blood sampling schedule was designed to be able to detect differences in Cmax, a critical parameter to determine the rate of drug absorption (Pabst & Jaeger, 1990), for ASA and SA. Therefore, sampling was intense at short times. It was not possible to draw additional blood samples at longer times, because it will result in a bloodvolume reduction higher than 20%, which will likely result in significant modifications in drug volume of distribution in the rat (García-López et al., 1995). A modest effect of SCI on salicylate bioavailability was not expected on the basis of previously reported results with paracetamol. Low thoracic SCI at the T8 level by the weight-drop method significantly reduced paracetamol Cmax in about 50% and AUClast in about 30% (García-López et al., 1996, 1997) after oral administration of this drug. In the present study, we observed that SCI reduced SA Cmax in 20% to 30% and AUClast in 10% to 25%, the reduction in
AUClast not reaching statistical significance. It therefore appears that the effect of SCI on oral paracetamol bioavailability is more pronounced than that for aspirin, although both compounds are classified as nonsteroidal anti-inflammatory drugs. These differences are likely due to the different physiological mechanisms in drug absorption. Paracetamol is absorbed in the duodenum, its absorption from the stomach being negligible (Gibaldi, 1991). Hence, paracetamol absorption depends on gastric emptying (Bagnall et al., 1979; Heading et al., 1973). There is evidence that gastric emptying is impaired by SCI (Fealey et al., 1984; Segal et al., 1985), likely by a stimulation of nitric oxide release, resulting in an inhibition of gastrointestinal motility (GuízarSahagún et al., 1996). On the other hand, aspirin is an acidic drug and is therefore absorbed in the stomach (Gibaldi, 1991). Hence, aspirin absorption does not depend on gastric emptying; thus, the extent of the absorption of this drug is less affected by SCI. The reduced rate of aspirin absorption, suggested by the delayed appearance of SA in SCI rats, could be due to a vasoconstriction in the gastrointestinal wall, inasmuch as it is known that SCI results in the constriction of several vascular territories (Krassioukov & Weaver, 1995). Notwithstanding, other mechanisms cannot be discarded with the information available at present. In summary, salicylate bioavailability after oral aspirin administration is slightly altered by SCI at the T8 level. The rate, but not the extent, of aspirin absorption appeared to be significantly affected by SCI. The effect of SCI on aspirin absorption from the gastrointestinal tract is less pronounced than on that of paracetamol. Because over-the-counter analgesic agents, such as aspirin and paracetamol, are widely consumed by the general population, including SCI patients, SCI-induced alterations on the bioavailability of these drugs deserve further attention. Acknowledgments This work was supported by the Consejo Nacional de Ciencia y Tecnología (Grant No. 0807 PM9506). G. FuentesLara was supported by a fellowship from CONACYT. References Allen, A. R. (1911). Surgery of experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column: a preliminary report. J Am Med Assoc 57, 878–880. Bagnall, W. E., Kelleher, J., Walker, B. E., & Losowsky, M. S. (1979). The gastrointestinal absorption of paracetamol in the rat. J Pharm Pharmacol 31, 157–160. Balant, L. P., Benet, L. Z., Blume, H., Bozler, G., Breimer, D. D., Eichelbaum, M., Gundert-Remy, U., Hirtz, J. L., Mutschler, E., Midha, K. K., Rauws, A. G., Ritschel, W. A., Sansom, L. N., Skelly, J. P., & Vollmer, K. O. (1991). Is there a need for more precise definitions of bioavailability? Eur J Clin Pharmacol 40, 123–126. Castañeda-Hernández, G., Castillo-Méndez, M. S., López-Muñoz, F. J., Granados-Soto, V., & Flores-Murrieta, F. J. (1994). Potentiation by caffeine of the analgesic effect of aspirin in the pain-induced functional impairment model in the rat. Can J Physiol Pharmacol 72, 1127–1131.
G. Fuentes-Lara et al. / J Pharmacol Toxicol 42 (1999) 93–97 Cruz, L., Castañeda-Hernández, G., & Navarrete, A. (1999). Ingestion of chilli peper (Capsicum annuun) reduces salicylate bioavailability after oral aspirin administration in the rat. Can J Physiol Pharmacol 77, 441–446. Das, G. D. (1989). Perspectives in anatomy and pathology of paraplegia in experimental animals. Brain Res Bull 22, 7–32. Fealey, R. D., Szurszewski, J. H., Merrit, J. L., & DiMagno, E. P. (1984). Effect of traumatic spinal cord transection on human upper gastrointestinal motility and gastric emptying. Gastroenterology 87, 69–75. García-López, P., Pérez-Urizar, J., Ibarra, A., Guízar-Sahagún, G., FloresMurrieta, F. J., Grijalva, I., & Castañeda-Hernández, G. (1995) An experimental model for the study of pharmacokinetic alterations induced by spinal cord injury. Pharm Sci 1, 133–135. García-López, P., Pérez-Urizar, J., Ibarra, A., Grijalva, I., Madrazo, I., Flores-Murrieta, F. J., Castañeda-Hernández, G., & Guízar-Sahagún, G. (1996). Comparison between Sprague-Dawley and Wistar rats as an experimental model of pharmacokinetic alterations induced by spinal cord injury. Arch Med Res 27, 453–457. García-López, P., Pérez-Urizar, J., Madrazo, I., Guízar-Sahagún, G., & Castañeda-Hernández, G. (1997). Oral paracetamol bioavailability in rats subjected to experimental spinal cord injury. Biopharm Drug Dispos 18, 203–211. Gibaldi, M. (1991). Biopharmaceutics and Clinical Pharmacokinetcs, 4th ed. (pp. 40–60). Philadelphia and London: Lea & Febiger. Gilman, T. M., Brunnemann, S. R., & Segal, J. L. (1993). Comparison of population pharmacokinetic models for gentamicin in spinal cord injury and able-bodied patients. Antimicrob Agents Chemother 37, 93–99. Guízar-Sahagún, G., Grijalva, I., Madrazo, I., Franco-Bourland, R., Salgado-Ceballos, H., Ibarra, A., & Larriva-Sahd, J. (1994). Neuroprotection of completely lacerated spinal cord of adult rats by homotropic and heterotropic transplantation. Restor Neurol Neurosci 61, 61–70. Guízar-Sahagún, G., García-López, P., Espitia, A. L., Méndez, S., Castañeda-Henández, G., Madrazo, I., & Franco-Bourland, R. (1996). Histochemical evidence for the increased expression of nicotinamide adenine dinucleotide phosphate-dependent diaphorase in neurons of the myenteric plexus after acute spinal cord injury in adult rats. Neurosci Lett 206, 185–188. Guízar-Sahagún, G., Castañeda-Hernández, G., García-López, P., Franco-
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Bourland, R., Grijalva, I., & Madrazo, I. (1998). Pathophysiological mechanisms involved in systemic and metabolic alterations secondary to spinal cord injury. Proc West Pharmacol Soc 41, 237–240. Halstead, L. S., Feldman, S., Claus-Walker, J., & Patel, V. C. (1985). Drug absorption in spinal cord injury. Arch Phys Med Rehab 66, 298–301. Hall, E. D., & Wolf, D. L. (1986). A pharmacological analysis of the pathophysiological mechanisms of posttraumatic spinal cord ischemia. J Neurosurg 64, 951–961. Heading, R. C., Nimmo, J., Prescott, L. F., & Tothill, P. (1973). The dependence of paracetamol absorption on the rate of gastric emptying. Br J Pharmacol 47, 415–421. Higgs, G. A., Salmon, J. A., Henderson, B., & Vane, J. R. (1987). Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate cyclooxygenase and antiinflammatory activity. Proc Natl Acad Sci USA 84, 1417–1420. Holtz, A., & Nystrom, B. (1990). Neuropathological changes and neurological function after spinal cord compression in the rat. J Neurotrauma 7, 155–167. Krassioukov, A. V., & Weaver, L. C. (1995). Episodic hypertension due to autonomic dysreflexia in acute and chronic spinal cord-injured rats. Am J Physiol 268, H2077–H2083. Pabst, G., & Jaeger, H. (1990). Review of methods and criteria for the evaluation of bioequivalence studies. Eur J Clin Pharmacol 38, 5–10. Segal, J. L., & Brunnemann, S. R. (1989). Clinical pharmacokinetics in patients with spinal cord injury. Clin Pharmacokinet 17, 109–129. Segal, J. L., Brunnemann, S. R., Gordon, S. K., & Eltorai, I. M. (1985). Decreased theophylline bioavailability and impaired gastric emptying in spinal cord injury. Curr Ther Res 38, 831–846. Segal, J. L., Brunnemann, S. R., Gordon, S. K., & Eltorai, I. M. (1988). Amikacin pharmacokinetics in patients with spinal cord injury. Pharmacotherapy 8, 79–81. Segal, J. L., Brunnemann, S. R., Eltorai, I. M., & Vulpe, M. (1991). Decreased systemic clearance of lorazepam in humans with spinal cord injury. J Clin Pharmacol 31, 651–656. Stover, S. L., & Fine, P. R. (1987). The epidemiology and economics of spinal cord injury. Paraplegia 25, 225–228. Wentjes, M. G., & Levy, G. (1988). Non-linear pharmacokinetics of aspirin in the rat. J Pharmacol Exp Ther 245, 809–815.
Effect of experimental spinal cord injury on salicylate bioavailability after oral aspirin administration Griselda Fuentes-Laraa,c, Gabriel Guízar-Sahagúna,b, Patricia García-Lópezc,d,* b
a Proyecto Camina A.C., Mexico D.F., Mexico Unidad de Investigación Médica en Enfermedades Neurológicas, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico D.F., Mexico c Departamento de Farmacología y Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico D.F., Mexico dDivisión de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando 22, Tlalpan, 14000 Mexico D.F., Mexico Received 8 February 2000; accepted 8 February 2000
Abstract The purpose of the present work was to study whether spinal cord injury (SCI) alters salicylate bioavailability after oral aspirin administration. Female Sprague-Dawley rats were subjected to SCI at the T8 level by two procedures, contusion by the weight-drop method and severance by knife, and received a single oral aspirin dose (15 mg/kg) 24 h after injury. Blood samples were drawn and aspirin (ASA) and salicylic acid (SA) concentrations in whole blood were determined at selected times over a period of 240 min. Both SCI procedures produced similar alterations on salicylate bioavailability. ASA bioavailability was not significantly changed by SCI. On the other hand, SA peak concentrations were significantly reduced in 20% to 30%, compared with sham-lesioned controls. The area under the SA concentration against time curve was decreased in 10% to 25%, although this difference did not reach statistical significance. Results suggest that SCI at the T8 level decreases the rate, but not the extent, of aspirin absorption from the gastrointestinal tract. SCI-induced alterations in aspirin absorption appeared to be modest compared with those previously reported for other analgesic agents, such as paracetamol (acetaminophen). © 2000 Elsevier Science Inc. All rights reserved. Keywords: Aspirin; Salicylic acid; Salicylates; Bioavailability; Spinal cord injury
1. Introduction Spinal cord injury (SCI) is a catastrophic affliction that results not only in motor and sensory impairment, but also in major metabolic and systemic alterations (Guízar-Sahagún et al., 1998). Hence, SCI may change the kinetics of drug absorption, distribution, and elimination (Segal & Brunnemann, 1989). It has been reported that the pharmacokinetics of several drugs, such as paracetamol (acetaminophen) (Halstead et al., 1985), theophylline (Segal et al., 1985), dantrolene (Segal & Brunnemann, 1989), aminoglycosides (Gilman et al 1993; Segal et al., 1988), and lorazepam (Segal et al., 1991) are significantly altered in SCI patients in comparison with able-bodied subjects. Despite the evidence of significant pharmacokinetic changes in SCI, the criteria and strategies for optimizing drug therapy in this type of patient are seldom based on rational principles. Treatment strategies
* Corresponding author. Tel: 015 628 0426; Fax: 015 628 0432 E-mail address: [email protected]
are extrapolated, often uncritically, from clinical experience in able-bodied persons (Segal & Brunnemann, 1989). Clinical reports on drug kinetics in SCI are often anecdotal, because it is extremely difficult to perform systematic pharmacokinetic studies in SCI patients. This difficulty is due to the important interindividual variability in injury extent and location. Therefore, the use of experimental models appears to be a suitable strategy for understanding pharmacokinetic alterations due to SCI, as well as the pathophysiological mechanisms involved. We previously showed, by using an experimental model in the rat, that the oral bioavailability of paracetamol is significantly reduced by SCI (García-López et al., 1996, 1997). Aspirin is one of the most widely used drugs worldwide. Being an over-the-counter drug, aspirin can be consumed by virtually all patient populations, including patients with SCI. Therefore, we considered it of interest to study whether salicylate bioavailability after oral aspirin administration is altered by SCI. We studied the effects of SCI produced by two different experimental procedures, contusion and severance by knife (Das, 1989).
1056-8719/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 6 - 8 7 1 9 ( 0 0 ) 0 0 0 4 8 - 4
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2. Materials and methods 2.1. Animals Female Sprague-Dawley rats (240–260 g) were used. Twelve hours before drug administration, food was withheld, but animals had free access to water. The study was approved by the local Animal Care Committee. 2.2. Spinal cord injury Animals were subjected to SCI by two different procedures, contusion and severance by knife. Spinal cord contusion was performed by the weight-drop method of Allen (1911) modified for rats, as described previously (GarcíaLópez et al., 1995). Briefly, animals were anesthetized by the intramuscular injection of a mixture of ketamine (77.5 mg/kg) and xylazine hydrochloride (12.5 mg/kg). Under aseptic conditions, a laminectomy was performed at the T8 level. Rats were then placed on a stereotaxic device, and a stainless steel cylinder weighing 15 g was dropped from a height of 4 cm through a guided tube onto the exposed dura. When the presence of hematoma on the dorsal aspect of the spinal cord was corroborated, the aponeurotic plane and the skin were separately sutured with 5-0 nylon thread. For the severance-by-knife SCI, rats were anesthetized and a laminectomy was performed as heretofore described. The spinal cord was completely severed by a clean transversal cut performed with a scalpel at the T8 level (Das, 1989). Then, the aponeurotic plane and the skin were sutured. Postsurgical care was performed as described previously (GuízarSahagún et al., 1994). 2.3. Determination of salicylate bioavailability A single oral aspirin dose (15 mg/kg) suspended in 0.5% carboxymethyl cellulose was given by gavage. Blood samples (100–125 l) were drawn from the caudal artery at 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 360 min after aspirin administration. The total blood volume extracted did not exceed 2 ml. The concentration of aspirin (acetyl salicylic acid, ASA) and its active metabolite, salicylic acid (SA), were determined in whole blood by high-performance liquid chromatography with the use of 2-acetamidophenol as internal standard, as previously described (Cruz et al., 1999). Individual whole-blood concentration against time curves were constructed for both ASA and SA. The maximal concentration (Cmax), as well as the time to reach the maximal concentration (Tmax), were directly determined from these plots. The area under the concentration against time curve to the last concentration–time point (AUClast) was determined by the trapezoidal rule. Bioavailability was determined from the AUClast and Cmax values, according to current recommendations (Balant et al., 1991; Pabst & Jaeger, 1990). 2.4. Study design Three groups of seven rats each were studied. Animals in group 1 served as controls and were only subjected to lami-
nectomy. Animals in groups 2 and 3 were subjected to SCI by contusion and severance by knife, respectively. Salicylate bioavailability was studied 24 h after the surgical procedure. Comparisons between the control and the SCI groups were performed by analysis of variance followed by Dunnet’s test. 2.5. Drugs and reagents ASA, SA, and 2-acetamidophenol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile, chromatographic grade, was obtained from E. Merck (Darmstadt, Germany). All other reagents were of analytical grade. High-quality water employed to prepare solutions was obtained by using a Milli-Q Reagent Water System (Continental Water Systems, El Paso, TX, USA). 3. Results All animals exhibited normal locomotor activity before the initiation of the study. One day after SCI, by either contusion or severance by knife, rats showed complete flaccid paraplegia. On the other hand, sham-injured animals subjected only to laminectomy exhibited normal walk after they recovered from anesthesia. Whole-blood ASA levels are shown in Fig. 1. ASA was absorbed and eliminated very rapidly. In sham-lesioned animals, Cmax was observed 5 min after aspirin administration. ASA blood concentrations were below detection limits after 15 min. In rats with SCI by both contusion and severance by knife, Cmax was observed 10 min after aspirin administration. There was a trend toward a reduction in Cmax in SCI rats (Table 1). However, owing to the high variability observed, differences in Cmax did not reach statistical significance. AUClast values for ASA were similar in the three
Fig. 1. Aspirin (acetylsalicylic acid) blood concentrations observed after oral administration of a 15 mg/kg oral aspirin dose in rats subjected to spinal cord injury at the T8 level by contusion (white circles) and by severance by knife (white squares) and in sham-lesioned controls (black circles). Data are presented as mean ⫾ SEM of seven animals.
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Table 1 Bioavailability parameters of ASA and SA observed after a single oral administration of a 15-mg/kg aspirin dose to rats subjected to SCI at the T8 level by two procedures, contusion by the weight-drop method and severance by knife, and to sham-lesioned controls. Acetylsalicylic acid
Sham lesion SCI by contusion SCI by severance by knife
Salicylic acid
Cmax (g/ml)
AUClast (g·h/ml)
Cmax (g/ml)
AUClast (g·h/ml)
3.4 ⫾ 0.6 2.4 ⫾ 1.0 2.1 ⫾ 0.3
24 ⫾ 5 21 ⫾ 7 24 ⫾ 4
21 ⫾ 1 14 ⫾ 2* 17 ⫾ 1*
5325 ⫾ 558 4102 ⫾ 426 4725 ⫾ 368
Data are expressed as mean ⫾ SEM of seven rats. *Significantly different from the sham-lesion group (p ⬍ 0.05), as determined by analysis of variance followed by Dunnet’s test.
groups of animals studied. The time course of ASA blood concentrations, as well as bioavailability parameters observed in the two SCI groups, contusion and severance by knife, were similar. Whole-blood SA levels are shown in Fig. 2. SA concentrations were considerably higher than for ASA at all sampling times. As a result, AUClast values for SA were about 200 times as great as those for ASA (Table 1). The rate of SA appearance in blood was slower in the two SCI groups (Tmax range 60–150 min) than in sham-lesioned controls (Tmax range 30–60 min). As a result, SA blood concentrations were lower in SCI animals than in controls during the first 120 min after aspirin administration but were similar thereafter. Cmax values were significantly reduced in both SCI groups compared with sham-lesioned controls (Table 1). AUClast values were slightly reduced in the two SCI groups compared with sham-lesioned controls (Table 1), but these differences did not reach statistical significance (p ⬎ 0.05). These results suggest that SCI decreased the rate, but not the extent, of drug absorption. The time course of SA blood concentrations, as well as bioavailability parameters,
Fig. 2. Salicylic acid blood concentrations observed after oral administration of a 15 mg/kg oral aspirin dose in rats subjected to spinal cord injury at the T8 level by contusion (white circles) and by severance by knife (white squares) and in sham-lesioned controls (black circles). Data are presented as mean ⫾ SEM of seven animals.
observed in the two SCI groups, contusion and severance by knife, were similar. 4. Discussion The purpose of this work was to study salicylate bioavailability after oral aspirin administration in two models of SCI, contusion and severance by knife. The contusion model by the weight-drop method mimics the histopathological features of acceleration-related fracture/dislocation of the spine, producing contusion or compression of the spinal cord, as in most accidental injuries in humans. On the other hand, the severance-by-knife model resembles injuries produced by aggressions with puncturing or cutting devices (Das, 1989; Stover and Fine, 1987). SCI is not a static state. The primary injury due to the mechanical trauma is followed by a secondary injury that increases the original neural damage. This secondary injury has been attributed to the presence of multiple endogenous toxic substances released by the injured neurons within the lesion area (Holtz & Nystrom, 1990) as well as a disruption of the microcirculation (Hall & Wolf, 1986). In the contusion by the weightdrop method, the damage produced by the secondary injury is predominant. Conversely, in the severance-by-knife model, the damage produced by the primary injury is more important (Das, 1989). Despite these histopathological differences between the contusion and the severance-by-knife models, both procedures produced a similar locomotor impairment. This was also the case for the observed alterations in salicylate bioavailability after oral aspirin administration. It is therefore probable that the pathophysiological processes resulting in altered salicylate bioavailability during the acute phase of SCI depend only on the disruption of nervous transmission through the spinal cord. Whether this disruption is due mainly to the primary or to the secondary injury appears to be irrelevant. SCI at the T8 level, by either contusion or severance by knife, did not produce any significant alteration in ASA bioavailability, although there was a trend toward a reduction in Cmax and a prolongation in Tmax. ASA concentrations were very low. Moreover, ASA appearance and disappearance from the circulation were very rapid, and concentrations exhibited a high interindividual variability, owing to
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the fact that aspirin is readily absorbed from the stomach but is rapidly biotransformed into SA by esterases in the blood, gastrointestinal tract, and liver (Gibaldi, 1991; Higgs et al., 1987; Wentjes & Levy, 1988). Under these conditions, it is difficult to detect statistically significant differences in bioavailability between treatments. SA accounted for more than 99% of the total salicylate bioavailability after aspirin oral administration. Other aspirin metabolites, such as gentisic and salicyluric acids cannnot be detected under the conditions of the present study (Cruz et al., 1999). Gentisic acid concentrations can be detected in the rat blood only after the administration of considerably higher aspirin doses (Castañeda-Hernández et al., 1994). It then appears that, under the conditions used in the present study, SA bioavailability is the best indicator for aspirin absorption. Moreover, SA is considered to be the active metabolite of aspirin (Higgs et al., 1987). SCI by either contusion or severance by knife had a modest effect on SA bioavailability. AUClast, an indicator of the extent of drug absorption (Balant et al., 1991) was slightly, although not significantly, reduced. On the other hand, SCI significantly reduced Cmax, suggesting that SCI decreased the rate of aspirin absorption (Balant et al, 1991; Pabst & Jaeger, 1990). This is consistent with the observed trend toward a reduction in ASA Cmax and with the fact that Tmax was prolonged in SCI animals, for both ASA and SA. No attempt was made to perform any statistical comparison of Tmax values, because Tmax is not considered an adequate indicator for bioavailability (Pabst & Jaeger, 1990). The reduction in SA Cmax did not have a significant effect on AUClast. Because SA concentrations at times longer than 120 min were similar in SCI and sham-lesioned animals, no statistically significant differences were detected in AUClast. These data suggest that the extent of aspirin absorption was not significantly affected by SCI (Balant et al, 1991; Pabst & Jaeger, 1990). Our results hence suggest that SCI-induced alterations on aspirin bioavailability concern mainly the rate of absorption. It was not possible to make an adequate estimation of half-life with the experimental design used in the present study. The blood sampling schedule was designed to be able to detect differences in Cmax, a critical parameter to determine the rate of drug absorption (Pabst & Jaeger, 1990), for ASA and SA. Therefore, sampling was intense at short times. It was not possible to draw additional blood samples at longer times, because it will result in a bloodvolume reduction higher than 20%, which will likely result in significant modifications in drug volume of distribution in the rat (García-López et al., 1995). A modest effect of SCI on salicylate bioavailability was not expected on the basis of previously reported results with paracetamol. Low thoracic SCI at the T8 level by the weight-drop method significantly reduced paracetamol Cmax in about 50% and AUClast in about 30% (García-López et al., 1996, 1997) after oral administration of this drug. In the present study, we observed that SCI reduced SA Cmax in 20% to 30% and AUClast in 10% to 25%, the reduction in
AUClast not reaching statistical significance. It therefore appears that the effect of SCI on oral paracetamol bioavailability is more pronounced than that for aspirin, although both compounds are classified as nonsteroidal anti-inflammatory drugs. These differences are likely due to the different physiological mechanisms in drug absorption. Paracetamol is absorbed in the duodenum, its absorption from the stomach being negligible (Gibaldi, 1991). Hence, paracetamol absorption depends on gastric emptying (Bagnall et al., 1979; Heading et al., 1973). There is evidence that gastric emptying is impaired by SCI (Fealey et al., 1984; Segal et al., 1985), likely by a stimulation of nitric oxide release, resulting in an inhibition of gastrointestinal motility (GuízarSahagún et al., 1996). On the other hand, aspirin is an acidic drug and is therefore absorbed in the stomach (Gibaldi, 1991). Hence, aspirin absorption does not depend on gastric emptying; thus, the extent of the absorption of this drug is less affected by SCI. The reduced rate of aspirin absorption, suggested by the delayed appearance of SA in SCI rats, could be due to a vasoconstriction in the gastrointestinal wall, inasmuch as it is known that SCI results in the constriction of several vascular territories (Krassioukov & Weaver, 1995). Notwithstanding, other mechanisms cannot be discarded with the information available at present. In summary, salicylate bioavailability after oral aspirin administration is slightly altered by SCI at the T8 level. The rate, but not the extent, of aspirin absorption appeared to be significantly affected by SCI. The effect of SCI on aspirin absorption from the gastrointestinal tract is less pronounced than on that of paracetamol. Because over-the-counter analgesic agents, such as aspirin and paracetamol, are widely consumed by the general population, including SCI patients, SCI-induced alterations on the bioavailability of these drugs deserve further attention. Acknowledgments This work was supported by the Consejo Nacional de Ciencia y Tecnología (Grant No. 0807 PM9506). G. FuentesLara was supported by a fellowship from CONACYT. References Allen, A. R. (1911). Surgery of experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column: a preliminary report. J Am Med Assoc 57, 878–880. Bagnall, W. E., Kelleher, J., Walker, B. E., & Losowsky, M. S. (1979). The gastrointestinal absorption of paracetamol in the rat. J Pharm Pharmacol 31, 157–160. Balant, L. P., Benet, L. Z., Blume, H., Bozler, G., Breimer, D. D., Eichelbaum, M., Gundert-Remy, U., Hirtz, J. L., Mutschler, E., Midha, K. K., Rauws, A. G., Ritschel, W. A., Sansom, L. N., Skelly, J. P., & Vollmer, K. O. (1991). Is there a need for more precise definitions of bioavailability? Eur J Clin Pharmacol 40, 123–126. Castañeda-Hernández, G., Castillo-Méndez, M. S., López-Muñoz, F. J., Granados-Soto, V., & Flores-Murrieta, F. J. (1994). Potentiation by caffeine of the analgesic effect of aspirin in the pain-induced functional impairment model in the rat. Can J Physiol Pharmacol 72, 1127–1131.
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