Journal of Gastroenterology and Hepatology (1992) 7, 586-590
A L I M E N T A R Y T R A C T AND P A N C R E A S
Adaptation of rat gastric mucosa to repeated doses of non-salicylate non-steroidal anti-inflammatory drugs M. V. S K E L J O , A. S . GIRAUD A N D N. D. Y E O M A N S The University of Melbourne Department of Medicine, Western Hospital, Footscray, Victoria, Australia
Abstract To determine whether gastric mucosal adaptation occurs to the damaging effects of repeated non-steroidal anti-inflammatory drug (NSAID) administration, we compared the extent of gastric damage in rats after single or repeated doses of four non-salicylate NSAID. With daily dosing, adaptation occurred only to repeated doses of the short-acting NSAID ibuprofen and diclofenac but not to indomethacin and naproxen, both of which have considerably longer half-lives. Adaptation to indomethacin was demonstrable if the dosage interval was lengthened and the drug was given on alternate days rather than daily. Histological examination of the gastric mucosa of diclofenac-treated rats showed a similar degree of superficial damage in the single and repeatedly dosed groups. However there was a highly significant reduction in the amount of deeper mucosal damage in the repeatedly dosed rats. Our findings show that under certain conditions of dosage, adaptation to non-salicylate NSAID is demonstrable. The pharmacokinetics of individual NSAID appear to be important in determining whether or not adaptation occurs. Histological examination showed that adaptation to one of the shorter acting NSAID, diclofenac, whs characterized by a significant reduction in deeper mucosal damage.
Key words: diclofenac, gastric mucosa, ibuprofen, inflammatory agents, physiological adaptation, rats.
INTRODUCTION Gastric mucosal adaptation to the damaging effects of repeated aspirin administration has been shown in both experimental animals’-3 and human^.^ In these studies a single dose of aspirin was found to produce more erosions or mucosal bleeding than did the last of a series of doses. Adaptation in the rat is demonstrable after 3 days of dosing and is lost if dosage is interrupted for a similar time.‘ Although there is some evidence for adaptation to indomethacin in man,5 the few studies with non-salicylate nonsteroidal anti-inflammatory drugs (NSAID) in animals have given conflicting results. Kuwayama et al. noted adaptation to a low dose (5 mg/kg) of indomethacin after daily dosing for 1 week.6 However other studies in the rat’ and pig8 have failed to demonstrate adaptation to injury during repeated dosing with indomethacin at doses 1%-2 times higher than those used by Kuwayama et aL6 Indeed, Bjarnason et al. found that indomethacin (8 mg/ kg) caused a 50% mortality rate in rats if given daily for 3 days.’ In view of this controversy, we have re-examined the question of whether adaptation to gastric injury occurs in the rat after repeated administration of indomethacin. We
indomethacin, naproxen, non-steroidal
anti-
also investigated whether adaptation occurs to three other commonly used NSAID with differing half-lives. T h e findings led us to examine the effect of altering the indomethacin dosage interval, and the histology of the adapted stomach.
METHODS Animals Male hooded Wistar rats weighing 340 k 42 g (mean ? s.d.) were maintained on a controlled feeding regimen to enable daily dosing of the animals in a fasted state. Controlled feeding was commenced 3 days before dosing. During this period, all rats were accustomed to handling and were dosed between 08:OO and 09:OOh with 0.5 m L of 1% methylcellulose. They were given free access to food between 09:OO and 18:OO h and were then starved until 08:OO h the next morning. All four NSAID (indomethacin 5 mg/kg bodyweight, naproxen 10 mg/kg, sodium diclofenac 10 mg/kg, and ibuprofen 10 mg/kg) were suspended in 0.5 m L of 1% methylcellulose and administered orally by gavage under brief halothane anaesthesia.
Correspondence: Professor N. D. Yeomans, Department of Medicine, Western Hospital, Footscray, Vic. 301 1, Australia. Accepted for publication 30 June 1992.
Gastric adaptation to NSAID
Daily NSAID dosing Groups of six rats were dosed for 8 days with either a daily dose of NSAID (repeated NSAID dose group), or seven daily doses of methylcellulose then a single NSAID dose on day 8 (single NSAID dose group). A vehicle-only group received eight doses of methylcellulose. The animals were killed 4 h after the single or final dose on day 8.
Time course experiments with indomethacin Five groups of rats were fasted overnight in wire-bottom cages, to minimize coprophagy, and allowed free access to water. At 08:OO h the following morning they were dosed by gavage with indomethacin ( 5 mg/kg). All rats were then allowed to resume feeding and overnight (17:OO08:OO h) fasting until their individual times for sacrifice, to mimic exactly the controlled feeding programme in the daily dosing study. They were killed in groups of five, at 4, 8, 12, 24 or 48 h after dosing, and their stomachs removed for assessment of macroscopic damage.
587
measured and expressed as a percentage of the total mucosal length examined. Erosions which extended below the pits and into the gastric glands were classified as deep, whereas those that were confined to the surface and pits were designated superficial. T h e prevalence of both lesion types was expressed as a percentage of total mucosal sectioned length.
Statistical analysis
*
Data are expressed as the mean s.e.m. Differences between groups were analysed using analysis of variance (ANOVA) or the unpaired Student’s t-test. In both tests P < 0.05 was considered to be significant.
RESULTS Macroscopic damage Daily NSAID dosage
Alternate daily dosing with indomethacin In this experiment, three groups of rats received either eight doses of indomethacin ( 5 mg/kg; repeated dose group), seven doses of methylcellulose then one indomethacin dose (single dose group) or eight doses of methylcellulose (vehicle only group) every second day for 15 days. Their stomachs were removed, 4 h after dosing on day 15, for measurement of gastric damage.
Assessment of macroscopic damage Stomachs were removed, opened along the lesser curvature, rinsed with 0.15 mol/L NaCl and pinned flat. They were then photographed with high speed slide film. Macroscopic gastric mucosal injury was measured with a digital planimeter by tracing areas of damage on each coded photograph (observer blinded) and expressing the area damaged as a percentage of the total area of glandular stomach. Damage was defined as haemorrhagic areas of the mucosa which did not clear after rinsing.
The amount of macroscopic mucosal damage after single or repeated doses of indomethacin is shown in Fig. 1. After a single dose of 5 mg/kg, haemorrhagic lesions comprised about 1% of the mucosal area. In the group given repeated daily doses, about twice as much damage was found compared with the single dose group ( P < 0.01). In other words, rather than adaptation occurring, repeated dosing actually led to an increase in mucosal damage in these experiments with indomethacin. The results of the experiments with the other three non-aspirin NSAID are summarized in Table 1. With naproxen, mucosal damage was similar in the repeated daily dose and the single dose groups. However, after repeated doses of either diclofenac or ibuprofen, substantially less damage was found than in the corresponding
o)
m
5
’0
Histological assessment of damage
4.07
0
3.0-
? m ui 0
Two groups of rats received either seven doses of methylcellulose and one dose of sodium diclofenac (10 mg/kg) or eight daily doses of diclofenac, as described above. Their stomachs were removed 4 h after the first or final diclofenac dose on day 8, rinsed, pinned flat and immersed in phosphate buffered 4% formaldehyde solution overnight. T h e glandular mucosa was cut into 1 mm wide transverse strips which were subsequently cut into 3 mm lengths and embedded in paraffin for light microscopy. Five micrometre longitudinal sections were cut from the paraffin block and stained with haematoxylin and eosin. Sections were assessed blind, using an eyepiece micrometer, for a quantitative estimate of the proportion of damaged mucosa. The length of damaged mucosa was
&
1.0
c
c
o)
2
0.0
Figure 1 Macroscopic gastric mucosa in the rat after vehicle only (m); indomethacin 5 mg/kg as a single dose or the last of eight repeated daily doses Ks . Data are mean and s.e.m. for six rats per group. *P < 0.01, Student’s r-test. The severity of injury is significantly higher in the repeatedly dosed group, thus adaptation to indomethacin is not demonstrable if the drug is given daily.
a,,
M. V. Skeljo et al.
588 Table 1 Area of gastric mucosal damaget after single or repeated daily dosing with four non-salicylate NSAID.
0,
m
5 No. NSAID doses
NSAID administered (mg/kg) Long acting Indomethacin Naproxen 5 10
Short acting Diclofenac Ibuprofen 10 10
1
0.99k0.34
1.95k0.27
2.06k0.36
1.70k0.38
8
2.01 k0.27
1.82k0.28
0.13?0.09
O.OOtO.OO
*P 4 0.01
NS
*P < 0.01
*P < 0.01
* ANOVA. NS = not significant. Expressed as a percentage of total area of glandular stomach.
Single dose control groups. Macroscopic damage was reduced by 94% in the repeated dosage diclofenac group, and by 100% in the group given repeated doses of ibuprofen (Table 1).
Time course of indomethacin damage It is possible that ongoing damage from the previous day's dose may have prevented adaptation being observed in the indomethacin study. The time course of the indo methacin damage was therefore examined. Macroscopic damage was found to increase three-fold between 4 and 12 h after a single 5 mg/kg dose, and then remained the same for the next 12 h (Fig. 2). Thus there was still substantial injury at 24 h. In the rats examined 48 h after dosing, healing lesions were still visible as white depressed areas in the mucosa, but quantitation was not attempted since the areas were no longer haemorrhagic.
Alternate daily indomethacin This study was performed in order to examine the possibility that continuing damage from the previous U
$+
3
2
3.0-
?
Figure 3 Severity of gastric damage in the rat after vehicle only (M);indornethacin (5 mg/kg) given as a single dose ; or repeatedlv Y on alternate days instead of daily. Each bar denotes the mean and s.e.m for eight rats. *P < 0.01, Student's t-test. Note that the pattern seen in Fig. 1 is reversed; with gastric damage significantly less in the repeated dose group, indicating that adaptation to indomethacin can be shown if the interval between doses is lengthened.
day's dose might have masked any adaptation to this agent. The results are shown in Fig. 3. When the dosage interval was extended to every second day, substantially less damage (> 90% reduction, P < 0.01) was found 4 h after the eighth alternate-daily dose than after a single indomethacin dose.
Histological studies The depth of histological damage in adapted rats treated with diclofenac compared with their single dose counterparts is shown in Fig. 4. T h e amount of superficial damage recorded in the vehicle-only group was higher than expected, but there was no significant difference between the two diclofenac-treated groups. However there was a striking difference in the amount of deeper
-
3.01
'"O]
25.0
Superficial damage
Deep damage (exlending pas1 1 s l parietal cell1
2.5
0.0
I 0
4
8
12
16
20
24
Time after indomethacin administration ( h )
Figure 2 Time course for the development of indomethacin induced gastric damage. Indomethacin ( 5 mg/kg) was administered at time zero. Mean ? s.e.m for five rats at each time point (*P< 0.001, ANOVA for comparison of four time points).
Figure 4 Depth of gastric mucosal damage in the rat after vehicle (m); single 3'; or repeated !%' daily dosing with diclofenac (10 mg/kg). Deep erosions extended beyond pits into the gastric glands (marked by the highest parietal cell). Mean k s.e.m for eight rats. *P < 0.001, ANOVA. There is a significant reduction in deeper mucosal damage in the repeatedly dosed rats.
Gastric adaptation
to
NSAID
damage, defined as those erosions which extended below the first parietal cell. In the single dose group, the mean length of sectioned mucosa which exhibited deep damage was 20.6 f 2.8%, compared with only 2.4 f 1.O% in the repeated dosage group (P < 0.001).
DISCUSSION This study shows, that under certain conditions of dosage, marked adaptation to repeated administration of nonsalicylate NSAID can occur. However there appear to be substantial differences between different NSAID, and the interval between doses appears to be an important factor. Adaptation to repeated daily dosing with indomethacin and naproxen did not occur. In contrast, daily dosing with diclofenac or ibuprofen led to marked adaptation. It is of interest that the latter are short-acting NSAID with plasma half-lives in humans of approximately 1-2 h and 2.0-2.5 h, respectively. Indomethacin and naproxen have much longer half-lives (6 and 12- 15 h). There is evidence to suggest that the half-life of indomethacin may be even longer in the rat.9,1”-12 One factor which may contribute to keeping systemic levels of indomethacin high for prolonged periods in rats is that it undergoes considerable enterohepatic circulation.”,’4 Perhaps because of its long half-life, the gastric damage induced by indomethacin in rats persisted for at least 24 h after a single dose. Similar observations have been made by Rainsford et al.I3 In contrast, the damage produced by diclofenac, as assessed by sequential changes in transmucosal potential difference, undergoes healing within a few hours (unpubl. data). It is plausible that the substantial residual damage from the previous day’s indomethacin dose may prevent adaptation from being apparent. Our finding that marked adaptation to indomethacin can be shown if the dosage interval is increased to 48 hourly is in keeping with this hypothesis. The present findings differ from those of Kuwayama et al., who found adaptation to gastric damage in rats dosed daily for 7 days with 5 mg/kg indomethacin. Some methodological differences between the two studies may explain this. It is not clear whether the rats in Kuwayama et d ’ s study were fed continuously. This could be an important consideration since feeding is known to alter both the degree’ and pattern of gastric injury.” Another difference in design is that Kuwayama et al. used sodium bicarbonate to dissolve each indomethacin dose; this may have aided the development of adaptation by reducing the damage after each dose, since acid is known to augment NSAID-induced The mechanism or mechanisms that underlie adaptation are yet to be elucidated. The histological findings in this study show that rats given repeated doses of diclofenac had a marked reduction in the amount of deeper mucosal damage (into the gastric glands) even though there was only a modest reduction in the extent of superficial damage. Some candidate mechanisms for the adaptation might include increases in known defence factors such as prostaglandin production, phospholipid synthesis, bicarbonate or mucus secretion, or enhanced
589
mucosal blood flow. Alternatively, it may be that young, de-differentiated surface mucous cells, which migrate over areas of damage to restitute the surface,18319 are less susceptible to NSAID damage than their mature counterparts in the uninjured mucosa, or that restitution may occur more rapidly in the adapted rats. T h e mechanism of adaptation is unlikely to be a pharmacokinetic one since it has been shown in an earlier study that aspirin pharmacokinetics do not differ between adapted and non-adauted rats.’ The present results suggest that adaptation affords protection only against deeper mucosal damage while the degree of superficial damage remains the same as in the single dose group. This protection against the deeper, but not the superficial, injury has some parallels in the protection by exogenous prostaglandins, which reduce the deep mucosal injury by ethanol, while leaving the more superficial damage unchanged.20 Thus the data show that, with daily dosing, adaptation occurs only to short-acting non-salicylate NSAID. The pharmacokinetics of individual NSAID appear to be important in determining whether adaptation occurs in the rat. This may be in keeping with two recent studies which suggest that long half-life compounds tend to be more gastrotoxic in The interval between doses was crucial in demonstrating adaptation to indomethacin in our study. It may be clinically important to find out whether dosage frequency and NSAID half-life are also determinants of adaptation to NSAID in humans.
ACKNOWLEDGEMENTS This study was supported, in part, by a grant from the National Health and Medical Research Council of Australia. M.V.S. is the recipient of a Searle Australia Pty Ltd Gastroenterological Society of Australia Postgraduate Scholarship.
REFERENCES 1. HURI.EY J. W. & Cwh.nAi.i. L. A. T h e effect of salicylates upon the stomachs of dogs. Gastroenterology 1964; 46: 36-43. 2. ST J O H D. ~ €3. J., YEOMANSN. D., McDwMorr M. B. & r)E BOERW. G. K. M. Adaptation of the gastric mucosa to repeated administration of aspirin in the rat. Am. J. Dig. Dis. 1973; 18: 881-6. 3. EASTWOOD G. L. & QUIMHY G. F. Effect of chronic aspirin ingestion on epithelial proliferation in rat fundus, antrum, and duodenum. Gastroenterology 1982; 82: 852-6. 4. GRAHAM D. Y., SMITHL. J. & DOBHSS. M. Gastric adaptation occurs with aspirin administration in man. Dig. Dis. Sci. 1983; 28: 1-6. 5. SHORKOCK C. J., PRESCOTTK. J. & KELTS W. D. W. T h e effects of indomethacin on gastroduodenal morphology and mucosal p H gradient in the healthy human stomach. Gastroenterology 1990; 99: 334-9. H., EASTWOOD G.,M I ~ . A KS . E& Fc K i K A W A H. 6. KUWAYAMA Adaptation of gastric mucosa to repeated indomethacin administration in the rat. Gastroenterology 1986; 90: A1 506 (abstr.).
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7. BJAKNASON I., SMETHURST P., LEVIA. J. & PRICE A. B. Indomethacin induced chronic small intestinal ulceration in the rat. Gastroenterology 1989; 94: A37 (abstr.). 8. RAINSFORD K. D. & WILLISC. Relationship of gastric mucosal damage induced in pigs by antiinflammatory drugs and their effects on prostaglandin production. Dig. Dis. Sci. 1982; 27: 624-35. H. T., DIETZELK., NUERNBERC B. 9. BECKW. S., SCHNEIDER & BRUNEK. Gastrointestinal ulcerations induced by antiinflammatory drugs in rats. Arch. Toxicol. 1990; 64: 210-17. 10. FOWLERP. Phenylbutazone and indomethacin. Clin. Rheum. Dis. 1975; 1: 267-83. M., REMINCTON L. & KENSLER 11. YESAIRD. W.,CALLAHAN C. J. Role of the entero-hepatic cycle of indomethacin on its metabolism, distribution in tissues and its excretion by rats, dogs and monkeys. Biochem. Pharmacol. 1970; 19: 1579-90. 12. KENT T. H., CARDELLI R. M. & STAMIER F. W. Small intestinal ulcers and intestinal flora in rats given indomethacin. Am. J. Pathol. 1969; 54: 237-45. D. J. Comparative 13. RAINSFORD K. D., Fox S. A. & OSBOURNE effects of some non-steroidal anti-inflammatory drugs on the ultrastructural integrity and prostaglandin levels in the rat gastric mucosa: relationship to drug uptake. Scand. J. Gastroenterol. 1984; 19 (Suppl. 101): 55-68. H. T. 14. BRUNEK., DIETZEL K., NUERNBERC B. & SCHNEIDER Recent insight into the mechanism of gastrointestinal tract
15.
16. 17.
18.
19.
20.
21.
22.
ulceration. Scand. 3. Gastroenterol. 1987; 22 (suppl. 65): 135-40. S., INADAI., HIRATA T. & MAKIY. Indomethacin HIROSHI produces gastric antral ulcers in the refed rat. Gastroenrer010gy 1981; 81: 719-25. ROBERTA. Cytoprotection by prostaglandins. Gastroenterology 1979; 77: 761-7. WHITTLEB. J. R. Relationship between the prevention of rat gastric erosions and inhibition of acid secretion by prostaglandins. Eur. 3. Pharmacol. 1976; 40: 233-9. YEOMANS N. D., ST. JOHND. J. B. & DE BOERW. G. R. M. Regeneration of gastric mucosa after aspirin-induced injury in the rat. Am. 3. Dig. Dis. 1973; 18: 773-80. YEOMANS N. D. Electron microscopic study of the repair of aspirin induced gastric erosions. Dig. Dis. Sci. 1976; 21: 533-41. LACYE. R. & ITO S. Microscopic analysis of ethanol damage to rat gastric mucosa after treatment with a prostaglandin. Gastroenterology 1982; 83: 619-25. Ross1 A. C., Hsu J. P. & FAICH G. A. Ulcerogenicity of piroxicam: an analysis of spontaneously reported data. Br. Med. J. 1987; 249: 147. LAPORTE J. R., CARNEX., VIDAL. X., MORENO V. & JUANJ. Upper gastrointestinal bleeding in relation to previous use of analgesics and NSAIDs. Lancer 1991; 337: 85-9.
A L I M E N T A R Y T R A C T AND P A N C R E A S
Adaptation of rat gastric mucosa to repeated doses of non-salicylate non-steroidal anti-inflammatory drugs M. V. S K E L J O , A. S . GIRAUD A N D N. D. Y E O M A N S The University of Melbourne Department of Medicine, Western Hospital, Footscray, Victoria, Australia
Abstract To determine whether gastric mucosal adaptation occurs to the damaging effects of repeated non-steroidal anti-inflammatory drug (NSAID) administration, we compared the extent of gastric damage in rats after single or repeated doses of four non-salicylate NSAID. With daily dosing, adaptation occurred only to repeated doses of the short-acting NSAID ibuprofen and diclofenac but not to indomethacin and naproxen, both of which have considerably longer half-lives. Adaptation to indomethacin was demonstrable if the dosage interval was lengthened and the drug was given on alternate days rather than daily. Histological examination of the gastric mucosa of diclofenac-treated rats showed a similar degree of superficial damage in the single and repeatedly dosed groups. However there was a highly significant reduction in the amount of deeper mucosal damage in the repeatedly dosed rats. Our findings show that under certain conditions of dosage, adaptation to non-salicylate NSAID is demonstrable. The pharmacokinetics of individual NSAID appear to be important in determining whether or not adaptation occurs. Histological examination showed that adaptation to one of the shorter acting NSAID, diclofenac, whs characterized by a significant reduction in deeper mucosal damage.
Key words: diclofenac, gastric mucosa, ibuprofen, inflammatory agents, physiological adaptation, rats.
INTRODUCTION Gastric mucosal adaptation to the damaging effects of repeated aspirin administration has been shown in both experimental animals’-3 and human^.^ In these studies a single dose of aspirin was found to produce more erosions or mucosal bleeding than did the last of a series of doses. Adaptation in the rat is demonstrable after 3 days of dosing and is lost if dosage is interrupted for a similar time.‘ Although there is some evidence for adaptation to indomethacin in man,5 the few studies with non-salicylate nonsteroidal anti-inflammatory drugs (NSAID) in animals have given conflicting results. Kuwayama et al. noted adaptation to a low dose (5 mg/kg) of indomethacin after daily dosing for 1 week.6 However other studies in the rat’ and pig8 have failed to demonstrate adaptation to injury during repeated dosing with indomethacin at doses 1%-2 times higher than those used by Kuwayama et aL6 Indeed, Bjarnason et al. found that indomethacin (8 mg/ kg) caused a 50% mortality rate in rats if given daily for 3 days.’ In view of this controversy, we have re-examined the question of whether adaptation to gastric injury occurs in the rat after repeated administration of indomethacin. We
indomethacin, naproxen, non-steroidal
anti-
also investigated whether adaptation occurs to three other commonly used NSAID with differing half-lives. T h e findings led us to examine the effect of altering the indomethacin dosage interval, and the histology of the adapted stomach.
METHODS Animals Male hooded Wistar rats weighing 340 k 42 g (mean ? s.d.) were maintained on a controlled feeding regimen to enable daily dosing of the animals in a fasted state. Controlled feeding was commenced 3 days before dosing. During this period, all rats were accustomed to handling and were dosed between 08:OO and 09:OOh with 0.5 m L of 1% methylcellulose. They were given free access to food between 09:OO and 18:OO h and were then starved until 08:OO h the next morning. All four NSAID (indomethacin 5 mg/kg bodyweight, naproxen 10 mg/kg, sodium diclofenac 10 mg/kg, and ibuprofen 10 mg/kg) were suspended in 0.5 m L of 1% methylcellulose and administered orally by gavage under brief halothane anaesthesia.
Correspondence: Professor N. D. Yeomans, Department of Medicine, Western Hospital, Footscray, Vic. 301 1, Australia. Accepted for publication 30 June 1992.
Gastric adaptation to NSAID
Daily NSAID dosing Groups of six rats were dosed for 8 days with either a daily dose of NSAID (repeated NSAID dose group), or seven daily doses of methylcellulose then a single NSAID dose on day 8 (single NSAID dose group). A vehicle-only group received eight doses of methylcellulose. The animals were killed 4 h after the single or final dose on day 8.
Time course experiments with indomethacin Five groups of rats were fasted overnight in wire-bottom cages, to minimize coprophagy, and allowed free access to water. At 08:OO h the following morning they were dosed by gavage with indomethacin ( 5 mg/kg). All rats were then allowed to resume feeding and overnight (17:OO08:OO h) fasting until their individual times for sacrifice, to mimic exactly the controlled feeding programme in the daily dosing study. They were killed in groups of five, at 4, 8, 12, 24 or 48 h after dosing, and their stomachs removed for assessment of macroscopic damage.
587
measured and expressed as a percentage of the total mucosal length examined. Erosions which extended below the pits and into the gastric glands were classified as deep, whereas those that were confined to the surface and pits were designated superficial. T h e prevalence of both lesion types was expressed as a percentage of total mucosal sectioned length.
Statistical analysis
*
Data are expressed as the mean s.e.m. Differences between groups were analysed using analysis of variance (ANOVA) or the unpaired Student’s t-test. In both tests P < 0.05 was considered to be significant.
RESULTS Macroscopic damage Daily NSAID dosage
Alternate daily dosing with indomethacin In this experiment, three groups of rats received either eight doses of indomethacin ( 5 mg/kg; repeated dose group), seven doses of methylcellulose then one indomethacin dose (single dose group) or eight doses of methylcellulose (vehicle only group) every second day for 15 days. Their stomachs were removed, 4 h after dosing on day 15, for measurement of gastric damage.
Assessment of macroscopic damage Stomachs were removed, opened along the lesser curvature, rinsed with 0.15 mol/L NaCl and pinned flat. They were then photographed with high speed slide film. Macroscopic gastric mucosal injury was measured with a digital planimeter by tracing areas of damage on each coded photograph (observer blinded) and expressing the area damaged as a percentage of the total area of glandular stomach. Damage was defined as haemorrhagic areas of the mucosa which did not clear after rinsing.
The amount of macroscopic mucosal damage after single or repeated doses of indomethacin is shown in Fig. 1. After a single dose of 5 mg/kg, haemorrhagic lesions comprised about 1% of the mucosal area. In the group given repeated daily doses, about twice as much damage was found compared with the single dose group ( P < 0.01). In other words, rather than adaptation occurring, repeated dosing actually led to an increase in mucosal damage in these experiments with indomethacin. The results of the experiments with the other three non-aspirin NSAID are summarized in Table 1. With naproxen, mucosal damage was similar in the repeated daily dose and the single dose groups. However, after repeated doses of either diclofenac or ibuprofen, substantially less damage was found than in the corresponding
o)
m
5
’0
Histological assessment of damage
4.07
0
3.0-
? m ui 0
Two groups of rats received either seven doses of methylcellulose and one dose of sodium diclofenac (10 mg/kg) or eight daily doses of diclofenac, as described above. Their stomachs were removed 4 h after the first or final diclofenac dose on day 8, rinsed, pinned flat and immersed in phosphate buffered 4% formaldehyde solution overnight. T h e glandular mucosa was cut into 1 mm wide transverse strips which were subsequently cut into 3 mm lengths and embedded in paraffin for light microscopy. Five micrometre longitudinal sections were cut from the paraffin block and stained with haematoxylin and eosin. Sections were assessed blind, using an eyepiece micrometer, for a quantitative estimate of the proportion of damaged mucosa. The length of damaged mucosa was
&
1.0
c
c
o)
2
0.0
Figure 1 Macroscopic gastric mucosa in the rat after vehicle only (m); indomethacin 5 mg/kg as a single dose or the last of eight repeated daily doses Ks . Data are mean and s.e.m. for six rats per group. *P < 0.01, Student’s r-test. The severity of injury is significantly higher in the repeatedly dosed group, thus adaptation to indomethacin is not demonstrable if the drug is given daily.
a,,
M. V. Skeljo et al.
588 Table 1 Area of gastric mucosal damaget after single or repeated daily dosing with four non-salicylate NSAID.
0,
m
5 No. NSAID doses
NSAID administered (mg/kg) Long acting Indomethacin Naproxen 5 10
Short acting Diclofenac Ibuprofen 10 10
1
0.99k0.34
1.95k0.27
2.06k0.36
1.70k0.38
8
2.01 k0.27
1.82k0.28
0.13?0.09
O.OOtO.OO
*P 4 0.01
NS
*P < 0.01
*P < 0.01
* ANOVA. NS = not significant. Expressed as a percentage of total area of glandular stomach.
Single dose control groups. Macroscopic damage was reduced by 94% in the repeated dosage diclofenac group, and by 100% in the group given repeated doses of ibuprofen (Table 1).
Time course of indomethacin damage It is possible that ongoing damage from the previous day's dose may have prevented adaptation being observed in the indomethacin study. The time course of the indo methacin damage was therefore examined. Macroscopic damage was found to increase three-fold between 4 and 12 h after a single 5 mg/kg dose, and then remained the same for the next 12 h (Fig. 2). Thus there was still substantial injury at 24 h. In the rats examined 48 h after dosing, healing lesions were still visible as white depressed areas in the mucosa, but quantitation was not attempted since the areas were no longer haemorrhagic.
Alternate daily indomethacin This study was performed in order to examine the possibility that continuing damage from the previous U
$+
3
2
3.0-
?
Figure 3 Severity of gastric damage in the rat after vehicle only (M);indornethacin (5 mg/kg) given as a single dose ; or repeatedlv Y on alternate days instead of daily. Each bar denotes the mean and s.e.m for eight rats. *P < 0.01, Student's t-test. Note that the pattern seen in Fig. 1 is reversed; with gastric damage significantly less in the repeated dose group, indicating that adaptation to indomethacin can be shown if the interval between doses is lengthened.
day's dose might have masked any adaptation to this agent. The results are shown in Fig. 3. When the dosage interval was extended to every second day, substantially less damage (> 90% reduction, P < 0.01) was found 4 h after the eighth alternate-daily dose than after a single indomethacin dose.
Histological studies The depth of histological damage in adapted rats treated with diclofenac compared with their single dose counterparts is shown in Fig. 4. T h e amount of superficial damage recorded in the vehicle-only group was higher than expected, but there was no significant difference between the two diclofenac-treated groups. However there was a striking difference in the amount of deeper
-
3.01
'"O]
25.0
Superficial damage
Deep damage (exlending pas1 1 s l parietal cell1
2.5
0.0
I 0
4
8
12
16
20
24
Time after indomethacin administration ( h )
Figure 2 Time course for the development of indomethacin induced gastric damage. Indomethacin ( 5 mg/kg) was administered at time zero. Mean ? s.e.m for five rats at each time point (*P< 0.001, ANOVA for comparison of four time points).
Figure 4 Depth of gastric mucosal damage in the rat after vehicle (m); single 3'; or repeated !%' daily dosing with diclofenac (10 mg/kg). Deep erosions extended beyond pits into the gastric glands (marked by the highest parietal cell). Mean k s.e.m for eight rats. *P < 0.001, ANOVA. There is a significant reduction in deeper mucosal damage in the repeatedly dosed rats.
Gastric adaptation
to
NSAID
damage, defined as those erosions which extended below the first parietal cell. In the single dose group, the mean length of sectioned mucosa which exhibited deep damage was 20.6 f 2.8%, compared with only 2.4 f 1.O% in the repeated dosage group (P < 0.001).
DISCUSSION This study shows, that under certain conditions of dosage, marked adaptation to repeated administration of nonsalicylate NSAID can occur. However there appear to be substantial differences between different NSAID, and the interval between doses appears to be an important factor. Adaptation to repeated daily dosing with indomethacin and naproxen did not occur. In contrast, daily dosing with diclofenac or ibuprofen led to marked adaptation. It is of interest that the latter are short-acting NSAID with plasma half-lives in humans of approximately 1-2 h and 2.0-2.5 h, respectively. Indomethacin and naproxen have much longer half-lives (6 and 12- 15 h). There is evidence to suggest that the half-life of indomethacin may be even longer in the rat.9,1”-12 One factor which may contribute to keeping systemic levels of indomethacin high for prolonged periods in rats is that it undergoes considerable enterohepatic circulation.”,’4 Perhaps because of its long half-life, the gastric damage induced by indomethacin in rats persisted for at least 24 h after a single dose. Similar observations have been made by Rainsford et al.I3 In contrast, the damage produced by diclofenac, as assessed by sequential changes in transmucosal potential difference, undergoes healing within a few hours (unpubl. data). It is plausible that the substantial residual damage from the previous day’s indomethacin dose may prevent adaptation from being apparent. Our finding that marked adaptation to indomethacin can be shown if the dosage interval is increased to 48 hourly is in keeping with this hypothesis. The present findings differ from those of Kuwayama et al., who found adaptation to gastric damage in rats dosed daily for 7 days with 5 mg/kg indomethacin. Some methodological differences between the two studies may explain this. It is not clear whether the rats in Kuwayama et d ’ s study were fed continuously. This could be an important consideration since feeding is known to alter both the degree’ and pattern of gastric injury.” Another difference in design is that Kuwayama et al. used sodium bicarbonate to dissolve each indomethacin dose; this may have aided the development of adaptation by reducing the damage after each dose, since acid is known to augment NSAID-induced The mechanism or mechanisms that underlie adaptation are yet to be elucidated. The histological findings in this study show that rats given repeated doses of diclofenac had a marked reduction in the amount of deeper mucosal damage (into the gastric glands) even though there was only a modest reduction in the extent of superficial damage. Some candidate mechanisms for the adaptation might include increases in known defence factors such as prostaglandin production, phospholipid synthesis, bicarbonate or mucus secretion, or enhanced
589
mucosal blood flow. Alternatively, it may be that young, de-differentiated surface mucous cells, which migrate over areas of damage to restitute the surface,18319 are less susceptible to NSAID damage than their mature counterparts in the uninjured mucosa, or that restitution may occur more rapidly in the adapted rats. T h e mechanism of adaptation is unlikely to be a pharmacokinetic one since it has been shown in an earlier study that aspirin pharmacokinetics do not differ between adapted and non-adauted rats.’ The present results suggest that adaptation affords protection only against deeper mucosal damage while the degree of superficial damage remains the same as in the single dose group. This protection against the deeper, but not the superficial, injury has some parallels in the protection by exogenous prostaglandins, which reduce the deep mucosal injury by ethanol, while leaving the more superficial damage unchanged.20 Thus the data show that, with daily dosing, adaptation occurs only to short-acting non-salicylate NSAID. The pharmacokinetics of individual NSAID appear to be important in determining whether adaptation occurs in the rat. This may be in keeping with two recent studies which suggest that long half-life compounds tend to be more gastrotoxic in The interval between doses was crucial in demonstrating adaptation to indomethacin in our study. It may be clinically important to find out whether dosage frequency and NSAID half-life are also determinants of adaptation to NSAID in humans.
ACKNOWLEDGEMENTS This study was supported, in part, by a grant from the National Health and Medical Research Council of Australia. M.V.S. is the recipient of a Searle Australia Pty Ltd Gastroenterological Society of Australia Postgraduate Scholarship.
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