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Effects on intestinal microflora, gastrointestinal tolerability and antiinflammatory efficacy of diclofenac and nitrofenac in adjuvant arthritic rats
89
Pharmacological Research, Vol. 29, NO. I, 1994
EFFECTS ON INTESTINAL MICROFLORA, GASTROINTESTINAL TOLERABILITY AND ANTIINFLAMMATORY EFFICACY OF DICLOFENAC AND NITROFENAC IN ADJUVANT ARTHRITIC RATS LAURA CUZZOLIN, ANITA CONFORTI, MARTA DONINI, ALESSANDRA ADAMI, PIER0 DEL SOLDATO* and GIUSEPPINA BENONI
Institute of Pharmacology,
University of Verona, Italy and *Metgrove Ltd, UK SUMMARY
Since it is known that nitric oxide plays an important protective role in maintaining the tissue integrity and is cytotoxic for invasive micro-organisms, diclofenac and a new original diclofenac-derivate, nitrofenac (containing the nitric oxide group), was administered at doses of 0.3 and 3 mg kg-’ per OS to adjuvant arthritic rats. At the 14th, 21st and 28th days after arthritis induction, the antiinflammatory efficacy and the effects on intestinal microflora of the two drugs were evaluated; moreover, at the end of the study period, the gastrointestinal tract was examined macroscopically for any presence of lesions. Daily oral administration of diclofenac and nitrofenac a.t 3 mg kg-’ markedly and significantly inhibited arthritis development until the end of the study period. Some significant changes were observed in anaerobic and Gram-negative bacterial flora, particularly the total disappearance, in all treated rats, of Escherichia coli 1, also 7 days after the last drug administration. Finally, no ulcers or severe damage were observed macroscopically with either drug, even if some alterations in the mucosa and haemorrhagic effusions were more evident in rats treated with diclofenac at 3 mg kg-‘. In conclusion, in this chronic model a similar therapeutic efficacy of diclofenac and nitrofenac is shown in arthritic rats. The better gastrointestinal tolerability observed in nitrofenac-treated rats could be attributed to the release of nitric oxide. KEY WORDS:
arthritic
rats, intestinal
microflora,
gastrointestinal
tolerability,
diclofenac,
nitrofenac.
INTRODUCTION Many non-steroidal anti-inflammatory inflammation in the experimental Correspondence to: Dr Laura Cuzzolin, Roma, 37 134 Verona, Italy. 1043461
g/94/010089-10/$08.00/O
drugs animal,
(NSAIDs) cause gastrointestinal which may lead to ulceration,
Institute of Pharmacology,
University
01994
of Verona, Policlinico
The Italian Pharmacological
Borgo
Society
90
Pharmacological
Research.
Vol. 29, No. I, I994
perforation and death [ 1, 21. Adjuvant-induced arthritis reduces quantitatively the tolerance of the gastrointestinal tract to the lesion-inducing properties of several NSAIDs [3]. New and novel NSAIDs with greater therapeutic safety continue to be important pharmaceutical research goals today. Diclofenac has a potent anti-inflammatory action, mostly due to its ability to inhibit cycle-oxygenase activity [4]. Of course, this effect causes a concomitant deprivation of prostacyclin cellular content thus allowing some tissues (such as gastric and intestinal tissues) to become particularly sensitive to the noxious stimulus. It is known [S] that prostacyclin and nitric oxide play a very important protective role in maintaining the tissue integrity at cellular and extracellular levels. Nitrofenac is a new original diclofenac-derivate obtained through a molecular manipulation consisting of the insertion, in a side chain, of nitric oxide, probably released at the target site with the aim of counteracting the inflammatory process by inhibiting the effects of pathogenetic mediators. For this purpose it has been planned to study the release of nitric oxide at Prof. Wallace’s Institute (University of Calgary, Canada) and kinetic studies were performed in the Institute of Pharmacology in Verona. In Verona, during the first 24 h after a single oral dose of 5 mg kg-‘, the rats metabolism of nitrofenac produced the 23% of diclofenac and other metabolites, presumably belonging to the alcoholic metabolite compounds devoid of the NO moiety. Moreover, since several animal studies indicate that bacterial flora may play some role in the pathogenesis of NSAID-induced gastrointestinal lesions [2, 61 and that nitric oxide has been shown to be cytotoxic for invasive micro-organisms [7], the aim of this study was to evaluate the effect on intestinal microflora, the gastrointestinal tolerability and the efficacy of nitrofenac, compared to diclofenac in adjuvant arthritic rats.
MATERIALS
AND METHODS
Adjuvant arthritis was induced in 50 male Sprague-Dawley rats weighing 185.50f 6.99 g by injection of 0.6 ml of heat-killed Mycohacterium hutyricum (Difco) suspended in 0.1 ml of mineral oil into the tail base. After 14, 21 and 28 days the arthritis development was evaluated by the same observer. Primary and secondary arthritic lesions were scored on an arbitrary scale as follows: left and right hind feet each O-7, left and right fore feet each 04.5, tail O-5, ears O-2, nose and eyes each O-l. Animals were weighed twice a week. Chemicals sodium was supplied by Ciba-Geigy; Diclofenac dichlorophenyl)amino]benzeneacetate-4 hydroxybutylnitric viscous oil, insoluble in water, molecular weight=412), kindly supplied by Metgrove Ltd (UK) (Fig. 1). Drug administration Animals were randomly
assigned
to five different
treatment
nitrofenac, 2-[(2,6ester (a light yellow was synthesized and
groups
(10 animals
91
Pharmacological Research, Vol. 29, No. I,1994
(a)
Fig. 1.
(b)
Structural formulae of diclofenac
(a) and nitrofenac
(b).
each group) as indicated below: the first group (controls) received methylcellulose per OS at 0.1 ml per 100 g body weight, the second and the third groups were treated with diclofenac at doses of 0.3 and 3 mg kg-’ body weight per OS, the other two groups received nitrofenac at 0.3 and 3 mg kg-’ per OS respectively. The drugs, suspended in methylcellulose 0.5%, were administered daily, from the 3rd to the 21st day after the arthritis induction, following a prophylactic protocol. At the 14th, 21 st and 28th days the anti-inflammatory activity of the drugs was expressed as a percentage of inhibition of arthritic score versus the control group. Microbiological study Stool specimens, taken before the arthritis induction and after 14, 21 and 28 days, were collected in sterile plastic containers and assayed immediately. Half a gram of stool was mixed in 4.5 ml of prereduced phosphate-buffered saline, serial ten-fold dilutions were made and 0.01 ml portions were inoculated on the following selective media: Wilkins-Chalgren anaerobe agar, total anaerobes; laked kanamycin-vancomycin agar, bacteroides; McConckey agar, enteric bacteria; mycosel agar, candida; mitis salivarius agar, streptococci; tomato juice agar, lactobacilli; reinforced clostridial medium, clostridia. The aerobic plates were incubated at 37°C for 24 h, and the anaerobic cultures at 37°C for 48 h in an anaerobic chamber. After incubation, different colony types appearing on the selective media were counted and isolated in pure culture. The species were then identified by colony morphology, Gram’s strain and standard biochemical techniques, with facultative Gram-negative bacilli, Gram-positive cocci and anaerobic bacteria identified respectively by API 20E, API STREP and ATB 32A (API System, La Balme les Grottes, France) [8]. All tests were performed in duplicate. Gastrointestinal tissue examination At the end of the study period, after 24 h of fasting, the animals were killed. The gastrointestinal tract was opened, the luminal content removed by washing with saline solution, the tissues carefully examined for any presence of lesions macroscopically and microscopically according to the usual technique [9].
92
Pharmarolo~ical
Statistical analysis The evaluation of the data comparison test.
was carried
Research,
out by applying
Vol. 29, No. 1, I994
Dunnett’s
multiple
RESULTS Adjuvant arthritis The arthritic scores and the inhibition of arthritic lesions in control rats and in rats receiving the different drug treatments are shown in Table I. Daily oral administration of diclofenac and nitrofenac at 3 mg kg-’ markedly and significantly inhibited arthritis development until the end of the study period (7 days after the last drug administration). With diclofenac the highest percentage of inhibition was observed on the 21st day with a 3 mg kg-’ dose (63%), while with nitrofenac this percentage was high on the 21st and 28th day with a 3 mg kg-’ dose (54-56%).
Table I Arthritic scores in arthritic rats and in rats receiving diclofenac or nitrofenac. The values are expressed as meanfs.E. Numbers in parentheses indicated percentage of inhibition of arthritic lesions Groups Arthritic Diclofenac (0.3 mg kg-‘) Diclofenac (3 mg kg-‘) Nitrofenac (0.3 mg kg-‘) Nitrofenac (3 mg kg-‘)
Day 14
Day 21
Day 28
10.83k2.16 6.70f2.07 (33) 4.60+1.81* (54) 7.1Ok1.96 (29) 4.77+0.81* (51)
13.55f3.01 8.75f2.57 (29) 4.60fl.37* (63) 7.55k1.87 (39) 6.33f1.10* (54)
12.44f3.10 8.00fl.98 (29) 5.20f0.66* (54) 6.40f 1.74 (44) 5.00+1.02* (56)
*p-Co.05
kicrobiological assay The changes in the intestinal aerobic microflora are shown in Fig. 2. Compared to base values, arthritic rats showed a significant decrease in lactobacilli until the 28th day; in animals treated with diclofenac this decrease in lactobacilli persisted, while the numbers of Gram-negative bacteria also decreased significantly (with a total disappearance of E. coli 1 also 7 days after the last drug administration with both doses; with nitrofenac, lactobacilli significantly decreased only with the high dose, while the marked reduction in enteric bacteria persisted with both doses. No changes were observed in Gram-positive cocci and candida. The anaerobic microflora was also affected by treatment, but the changes were moderate (Fig. 3). In arthritic rats a significant decrease was observed in total anaerobes and clostridia until the 28th day. Diclofenac administration maintained this significant decrease. Nitrofenac affected total anaerobes and clostridia only at the highest dose.
pharmacolo&%‘al Research,
Vol. 29, No. I, 1994
93
* IF
5 4 3 2
Proteus
morganiz -------ii
*-----*
@
t
Luctobacilli
4 1 t
;_.A
.
2-
lI 0
I 14
I 21
I 28
Time (days)
Fig. 2. Impact of diclofenac at 0.3 (0) and 3 mg kg-’ (0) and nitrofenac at 0.3 (0) and 3 mg kg-’ (U) on the aerobic intestinal microflora of arthritic rats (*) (mean geometric values).
Pharmacological Research, Vol. 29. No. I, 1994
94
Eubacteria
Bacteroide:
2lI 0
I 14
1 21
I 2
Time (days)
Fig. 3. Impact of diclofenac at 0.3 (0) and 3 mg kg-’ (0) and nitrofenac at 0.3 (Cl) and 3 mg kg-’ (m) on the anaerobic intestinal microflora of arthritic rats (*) (mean geometric values).
pharmacologicalResearch, Vol. 29, No. 1, 1994
9s
Table II Macroscopic evaluation of gastric lesions in arthritic rats and in rats treated with diclofenac and nitrofenac Arthritic
Diclofenac (0.3 mg kg- ‘)
Diclofenac (3 mg kg-‘)
Nitrofenac (0.3 mg kg-‘)
Nitrofenac (3 mg kg-‘)
1 normal 2 normal 3 normal 4 inflamed 5 normal 1 inflamed 2 inflamed 3 normal 4 normal 5 normal 1 normal 2 normal 3 normal 4 inflamed 5 inflamed 1 normal 2 normal 4 normal 5 inflamed
mucosa
mucosa mucoaa
6 inflamed 7 normal 8 normal 9 normal 10 inflamed
mucosa
6 inflamed 7 normal 8 inflamed 9 inflamed 10 inflamed
mucosa
mucosa
mucosa mucosa mucosa
mucosa mucosa
6 haemorrhagic effusions 7 haemorrhagic effusions 8 inflamed mucosa 9 haemorrhagic effusions 10 haemorrhagic effusions
mucosa
6 7 9 10
1 normal 2 normal 3 normal 4 normal 5 normal
inflamed inflamed normal normal
mucosa mucosa
6 normal 7 normal 8 inflamed mucosa 9 inflamed mucosa 10 haemorrhagic effusions
Gastrointestinal tolerability No ulcers were observed in arthritic and treated rats, even if in some rats treated with diclofenac and nitrofenac thin inflammatory reactions of the wall and haemorrhagic effusions were present after administration of the highest dose (Table II).
DISCUSSION Adjuvant arthritis is an accepted and well established standard test for predicting the clinical anti-inflammatory efficacy and ulcerogenicity of NSAIDs. From our data, it appears that diclofenac and nitrofenac are similar in the suppression of ongoing arthritis syndrome, reaching more than 50% inhibition with a 3 mg kg-’ dose. At the end of the study period (28th day) no ulcers or severe gastrointestinal damage were observed with the two studied drugs. It is important to underline that
96
Pharmacological
Research,
Vol. 29, No. I, 1994
the treatment was stopped on the 21st day and the gastrointestinal examination was made after 7 days from the end of the drug administration. Some significant changes in the bacterial flora pattern were observed. In other experiments non-arthritic rat were treated with diclofenac and nitrofenac at various doses for one month: no change in the microflora pattern compared to the controls was observed. E. coli, a proteolytic micro-organism, disappeared from the faecal content of the treated rats by the 14th day, while lactobacilli, saccharolytic flora, increased after nitrofenac administration. Anaerobes were maintained at control values with the lower dose of nitrofenac, while in arthritic rats and with high doses of both drugs anaerobe levels decreased. One consideration is that if the two examined drugs induced tissue damage during the treatment then this was no longer present 7 days after the end of drug administration. Some authors [IO] noted that in arthritic rats, at lower NSAID doses, the ulceration effect might be only that attributable to the last dose of the drug: omitting the drug dose on the last day before death might result in a lack of ulceration. Chronic drug administration for the demonstration of gastric lesions is not warranted since gastric lesions occur less frequently in animals given food or in animals that have ingested faeces [lo]. The lesions are not chronic in nature and there is virtually no evidence of damage 24 h after the last dose. The second consideration is that the E. coli growth is inhibited for more than 7 days after the end of treatment. In this experimental study, it was not possible to correlate the intestinal damage with the bacterial flora alterations, even if the importance of intestinal flora in the formation and maintenance of ulcers induced by indomethacin in rats has previously been shown [2, 1 I] by administering ulcerogenic doses. It is hypothesized that the described bacterial changes, in this study, are due to environmentally alterated conditions induced by the drugs such as acid secretion, mucosal blood flow and mucus secretion. Moreover, significant differences between the two drugs were not observed, except a minor influence of nitrofenac on anaerobes. It is probable that the partial release of nitric oxide by nitrofenac, before the intestinal absorption; induces a cytotoxic action against aerobes more than anaerobes. It is important to stress that nitric oxide is, in some cells, cytotoxic and in others cytostatic [ 121, suggesting that the sensitivity to nitric oxide varies from one cell to another. The nitric oxide-donor properties of nitrofenac are under study with kinetic experiments in the Institute of Pharmacology in Verona and in Prof. Wallace’s Institute, (University of Calgary, Canada), In conclusion, similar therapeutic efficacy of diclofenac and nitrofenac is shown in arthritic rats. No gastrointestinal ulcers are noted after administering 0.3 and 3 mg kg-’ doses for 21 days, but bacterial flora alterations due to various factors, probably related to nitric oxide for nitrofenac, were observed.
REFERENCES I. Fang W, Broughton A, Jacobson .I Dig Dis 1911; 22: 749-60.
ED. Indomethacin-induced
intestinal
inflammation.
Am
pharmacological
Research,
Vol. 29, No. 1, 1994
97
2. Kent TH, Cardelli RM, Stamler FW. Small intestinal ulcers and intestinal flora in rats given indomethacin. Am J Path01 1969; 45: 237-45. 3. Shriver DA, Dove PA, White CB, Sandor A, Rosenthale ME. A profile of the gastrointestinal toxicity of aspirin, indomethacin, oxaprozin, phenylbutazone and fentiazac in arthritic and Lewis normal rats. Toxic01 Appl Pharmacol 1977; 42: 75-83. 4. Wiesenberg-Boettcher I, Pfeilschifter J, Schweizer A, Sallmann A, Wenk P. Pharmacological properties of five diclofenac metabolites identified in human plasma. Agents Actions 199 1; 34: 135-7. 5. Radomski MW, Palmer RMJ, Read NG, Moncada S. Isolation and washing of human platelets with nitric oxide. Thromh RPS 1988; 50: 53746. 6. Satoh H, Guth PH, Grossman MJ. Role of food in gastrointestinal ulceration produced by indomethacin in the rat. Gastroenterology 1982; 83: 210-15. 7. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991; 43: 109-41. 8. Veilleux BG, Rowland I. Simulation of the rat intestinal ecosystem using a two-stage continuous culture system. J Gen Microbial 1981; 123: 103-15. 9. Hutcheson IR, Whittle BJR, Boughton-Smith NK. Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat. Br J Pharmacol 1990; 101: 815-20. 10. Otterness IG, Bliven ML. Laboratory models for testing nonsteroidal antiinflammatory drugs. In Lombardino JG ed. Non steroidal antiinflammatory drugs. New York: John Wiley, 1985: 223. 11. Benoni G, Del Soldato P, Cuzzolin L, Velo GP. Role of intestinal microflora in maintaining indomethacin-induced intestinal lesions in rats. In Rainsford KD, Velo GP eds. Side-effects of antiinflammatory drugs. Lancaster: MTP Press, 1987: 83-87. 12. Hibbs JB, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988; 157: 87-94.
Pharmacological Research, Vol. 29, NO. I, 1994
EFFECTS ON INTESTINAL MICROFLORA, GASTROINTESTINAL TOLERABILITY AND ANTIINFLAMMATORY EFFICACY OF DICLOFENAC AND NITROFENAC IN ADJUVANT ARTHRITIC RATS LAURA CUZZOLIN, ANITA CONFORTI, MARTA DONINI, ALESSANDRA ADAMI, PIER0 DEL SOLDATO* and GIUSEPPINA BENONI
Institute of Pharmacology,
University of Verona, Italy and *Metgrove Ltd, UK SUMMARY
Since it is known that nitric oxide plays an important protective role in maintaining the tissue integrity and is cytotoxic for invasive micro-organisms, diclofenac and a new original diclofenac-derivate, nitrofenac (containing the nitric oxide group), was administered at doses of 0.3 and 3 mg kg-’ per OS to adjuvant arthritic rats. At the 14th, 21st and 28th days after arthritis induction, the antiinflammatory efficacy and the effects on intestinal microflora of the two drugs were evaluated; moreover, at the end of the study period, the gastrointestinal tract was examined macroscopically for any presence of lesions. Daily oral administration of diclofenac and nitrofenac a.t 3 mg kg-’ markedly and significantly inhibited arthritis development until the end of the study period. Some significant changes were observed in anaerobic and Gram-negative bacterial flora, particularly the total disappearance, in all treated rats, of Escherichia coli 1, also 7 days after the last drug administration. Finally, no ulcers or severe damage were observed macroscopically with either drug, even if some alterations in the mucosa and haemorrhagic effusions were more evident in rats treated with diclofenac at 3 mg kg-‘. In conclusion, in this chronic model a similar therapeutic efficacy of diclofenac and nitrofenac is shown in arthritic rats. The better gastrointestinal tolerability observed in nitrofenac-treated rats could be attributed to the release of nitric oxide. KEY WORDS:
arthritic
rats, intestinal
microflora,
gastrointestinal
tolerability,
diclofenac,
nitrofenac.
INTRODUCTION Many non-steroidal anti-inflammatory inflammation in the experimental Correspondence to: Dr Laura Cuzzolin, Roma, 37 134 Verona, Italy. 1043461
g/94/010089-10/$08.00/O
drugs animal,
(NSAIDs) cause gastrointestinal which may lead to ulceration,
Institute of Pharmacology,
University
01994
of Verona, Policlinico
The Italian Pharmacological
Borgo
Society
90
Pharmacological
Research.
Vol. 29, No. I, I994
perforation and death [ 1, 21. Adjuvant-induced arthritis reduces quantitatively the tolerance of the gastrointestinal tract to the lesion-inducing properties of several NSAIDs [3]. New and novel NSAIDs with greater therapeutic safety continue to be important pharmaceutical research goals today. Diclofenac has a potent anti-inflammatory action, mostly due to its ability to inhibit cycle-oxygenase activity [4]. Of course, this effect causes a concomitant deprivation of prostacyclin cellular content thus allowing some tissues (such as gastric and intestinal tissues) to become particularly sensitive to the noxious stimulus. It is known [S] that prostacyclin and nitric oxide play a very important protective role in maintaining the tissue integrity at cellular and extracellular levels. Nitrofenac is a new original diclofenac-derivate obtained through a molecular manipulation consisting of the insertion, in a side chain, of nitric oxide, probably released at the target site with the aim of counteracting the inflammatory process by inhibiting the effects of pathogenetic mediators. For this purpose it has been planned to study the release of nitric oxide at Prof. Wallace’s Institute (University of Calgary, Canada) and kinetic studies were performed in the Institute of Pharmacology in Verona. In Verona, during the first 24 h after a single oral dose of 5 mg kg-‘, the rats metabolism of nitrofenac produced the 23% of diclofenac and other metabolites, presumably belonging to the alcoholic metabolite compounds devoid of the NO moiety. Moreover, since several animal studies indicate that bacterial flora may play some role in the pathogenesis of NSAID-induced gastrointestinal lesions [2, 61 and that nitric oxide has been shown to be cytotoxic for invasive micro-organisms [7], the aim of this study was to evaluate the effect on intestinal microflora, the gastrointestinal tolerability and the efficacy of nitrofenac, compared to diclofenac in adjuvant arthritic rats.
MATERIALS
AND METHODS
Adjuvant arthritis was induced in 50 male Sprague-Dawley rats weighing 185.50f 6.99 g by injection of 0.6 ml of heat-killed Mycohacterium hutyricum (Difco) suspended in 0.1 ml of mineral oil into the tail base. After 14, 21 and 28 days the arthritis development was evaluated by the same observer. Primary and secondary arthritic lesions were scored on an arbitrary scale as follows: left and right hind feet each O-7, left and right fore feet each 04.5, tail O-5, ears O-2, nose and eyes each O-l. Animals were weighed twice a week. Chemicals sodium was supplied by Ciba-Geigy; Diclofenac dichlorophenyl)amino]benzeneacetate-4 hydroxybutylnitric viscous oil, insoluble in water, molecular weight=412), kindly supplied by Metgrove Ltd (UK) (Fig. 1). Drug administration Animals were randomly
assigned
to five different
treatment
nitrofenac, 2-[(2,6ester (a light yellow was synthesized and
groups
(10 animals
91
Pharmacological Research, Vol. 29, No. I,1994
(a)
Fig. 1.
(b)
Structural formulae of diclofenac
(a) and nitrofenac
(b).
each group) as indicated below: the first group (controls) received methylcellulose per OS at 0.1 ml per 100 g body weight, the second and the third groups were treated with diclofenac at doses of 0.3 and 3 mg kg-’ body weight per OS, the other two groups received nitrofenac at 0.3 and 3 mg kg-’ per OS respectively. The drugs, suspended in methylcellulose 0.5%, were administered daily, from the 3rd to the 21st day after the arthritis induction, following a prophylactic protocol. At the 14th, 21 st and 28th days the anti-inflammatory activity of the drugs was expressed as a percentage of inhibition of arthritic score versus the control group. Microbiological study Stool specimens, taken before the arthritis induction and after 14, 21 and 28 days, were collected in sterile plastic containers and assayed immediately. Half a gram of stool was mixed in 4.5 ml of prereduced phosphate-buffered saline, serial ten-fold dilutions were made and 0.01 ml portions were inoculated on the following selective media: Wilkins-Chalgren anaerobe agar, total anaerobes; laked kanamycin-vancomycin agar, bacteroides; McConckey agar, enteric bacteria; mycosel agar, candida; mitis salivarius agar, streptococci; tomato juice agar, lactobacilli; reinforced clostridial medium, clostridia. The aerobic plates were incubated at 37°C for 24 h, and the anaerobic cultures at 37°C for 48 h in an anaerobic chamber. After incubation, different colony types appearing on the selective media were counted and isolated in pure culture. The species were then identified by colony morphology, Gram’s strain and standard biochemical techniques, with facultative Gram-negative bacilli, Gram-positive cocci and anaerobic bacteria identified respectively by API 20E, API STREP and ATB 32A (API System, La Balme les Grottes, France) [8]. All tests were performed in duplicate. Gastrointestinal tissue examination At the end of the study period, after 24 h of fasting, the animals were killed. The gastrointestinal tract was opened, the luminal content removed by washing with saline solution, the tissues carefully examined for any presence of lesions macroscopically and microscopically according to the usual technique [9].
92
Pharmarolo~ical
Statistical analysis The evaluation of the data comparison test.
was carried
Research,
out by applying
Vol. 29, No. 1, I994
Dunnett’s
multiple
RESULTS Adjuvant arthritis The arthritic scores and the inhibition of arthritic lesions in control rats and in rats receiving the different drug treatments are shown in Table I. Daily oral administration of diclofenac and nitrofenac at 3 mg kg-’ markedly and significantly inhibited arthritis development until the end of the study period (7 days after the last drug administration). With diclofenac the highest percentage of inhibition was observed on the 21st day with a 3 mg kg-’ dose (63%), while with nitrofenac this percentage was high on the 21st and 28th day with a 3 mg kg-’ dose (54-56%).
Table I Arthritic scores in arthritic rats and in rats receiving diclofenac or nitrofenac. The values are expressed as meanfs.E. Numbers in parentheses indicated percentage of inhibition of arthritic lesions Groups Arthritic Diclofenac (0.3 mg kg-‘) Diclofenac (3 mg kg-‘) Nitrofenac (0.3 mg kg-‘) Nitrofenac (3 mg kg-‘)
Day 14
Day 21
Day 28
10.83k2.16 6.70f2.07 (33) 4.60+1.81* (54) 7.1Ok1.96 (29) 4.77+0.81* (51)
13.55f3.01 8.75f2.57 (29) 4.60fl.37* (63) 7.55k1.87 (39) 6.33f1.10* (54)
12.44f3.10 8.00fl.98 (29) 5.20f0.66* (54) 6.40f 1.74 (44) 5.00+1.02* (56)
*p-Co.05
kicrobiological assay The changes in the intestinal aerobic microflora are shown in Fig. 2. Compared to base values, arthritic rats showed a significant decrease in lactobacilli until the 28th day; in animals treated with diclofenac this decrease in lactobacilli persisted, while the numbers of Gram-negative bacteria also decreased significantly (with a total disappearance of E. coli 1 also 7 days after the last drug administration with both doses; with nitrofenac, lactobacilli significantly decreased only with the high dose, while the marked reduction in enteric bacteria persisted with both doses. No changes were observed in Gram-positive cocci and candida. The anaerobic microflora was also affected by treatment, but the changes were moderate (Fig. 3). In arthritic rats a significant decrease was observed in total anaerobes and clostridia until the 28th day. Diclofenac administration maintained this significant decrease. Nitrofenac affected total anaerobes and clostridia only at the highest dose.
pharmacolo&%‘al Research,
Vol. 29, No. I, 1994
93
* IF
5 4 3 2
Proteus
morganiz -------ii
*-----*
@
t
Luctobacilli
4 1 t
;_.A
.
2-
lI 0
I 14
I 21
I 28
Time (days)
Fig. 2. Impact of diclofenac at 0.3 (0) and 3 mg kg-’ (0) and nitrofenac at 0.3 (0) and 3 mg kg-’ (U) on the aerobic intestinal microflora of arthritic rats (*) (mean geometric values).
Pharmacological Research, Vol. 29. No. I, 1994
94
Eubacteria
Bacteroide:
2lI 0
I 14
1 21
I 2
Time (days)
Fig. 3. Impact of diclofenac at 0.3 (0) and 3 mg kg-’ (0) and nitrofenac at 0.3 (Cl) and 3 mg kg-’ (m) on the anaerobic intestinal microflora of arthritic rats (*) (mean geometric values).
pharmacologicalResearch, Vol. 29, No. 1, 1994
9s
Table II Macroscopic evaluation of gastric lesions in arthritic rats and in rats treated with diclofenac and nitrofenac Arthritic
Diclofenac (0.3 mg kg- ‘)
Diclofenac (3 mg kg-‘)
Nitrofenac (0.3 mg kg-‘)
Nitrofenac (3 mg kg-‘)
1 normal 2 normal 3 normal 4 inflamed 5 normal 1 inflamed 2 inflamed 3 normal 4 normal 5 normal 1 normal 2 normal 3 normal 4 inflamed 5 inflamed 1 normal 2 normal 4 normal 5 inflamed
mucosa
mucosa mucoaa
6 inflamed 7 normal 8 normal 9 normal 10 inflamed
mucosa
6 inflamed 7 normal 8 inflamed 9 inflamed 10 inflamed
mucosa
mucosa
mucosa mucosa mucosa
mucosa mucosa
6 haemorrhagic effusions 7 haemorrhagic effusions 8 inflamed mucosa 9 haemorrhagic effusions 10 haemorrhagic effusions
mucosa
6 7 9 10
1 normal 2 normal 3 normal 4 normal 5 normal
inflamed inflamed normal normal
mucosa mucosa
6 normal 7 normal 8 inflamed mucosa 9 inflamed mucosa 10 haemorrhagic effusions
Gastrointestinal tolerability No ulcers were observed in arthritic and treated rats, even if in some rats treated with diclofenac and nitrofenac thin inflammatory reactions of the wall and haemorrhagic effusions were present after administration of the highest dose (Table II).
DISCUSSION Adjuvant arthritis is an accepted and well established standard test for predicting the clinical anti-inflammatory efficacy and ulcerogenicity of NSAIDs. From our data, it appears that diclofenac and nitrofenac are similar in the suppression of ongoing arthritis syndrome, reaching more than 50% inhibition with a 3 mg kg-’ dose. At the end of the study period (28th day) no ulcers or severe gastrointestinal damage were observed with the two studied drugs. It is important to underline that
96
Pharmacological
Research,
Vol. 29, No. I, 1994
the treatment was stopped on the 21st day and the gastrointestinal examination was made after 7 days from the end of the drug administration. Some significant changes in the bacterial flora pattern were observed. In other experiments non-arthritic rat were treated with diclofenac and nitrofenac at various doses for one month: no change in the microflora pattern compared to the controls was observed. E. coli, a proteolytic micro-organism, disappeared from the faecal content of the treated rats by the 14th day, while lactobacilli, saccharolytic flora, increased after nitrofenac administration. Anaerobes were maintained at control values with the lower dose of nitrofenac, while in arthritic rats and with high doses of both drugs anaerobe levels decreased. One consideration is that if the two examined drugs induced tissue damage during the treatment then this was no longer present 7 days after the end of drug administration. Some authors [IO] noted that in arthritic rats, at lower NSAID doses, the ulceration effect might be only that attributable to the last dose of the drug: omitting the drug dose on the last day before death might result in a lack of ulceration. Chronic drug administration for the demonstration of gastric lesions is not warranted since gastric lesions occur less frequently in animals given food or in animals that have ingested faeces [lo]. The lesions are not chronic in nature and there is virtually no evidence of damage 24 h after the last dose. The second consideration is that the E. coli growth is inhibited for more than 7 days after the end of treatment. In this experimental study, it was not possible to correlate the intestinal damage with the bacterial flora alterations, even if the importance of intestinal flora in the formation and maintenance of ulcers induced by indomethacin in rats has previously been shown [2, 1 I] by administering ulcerogenic doses. It is hypothesized that the described bacterial changes, in this study, are due to environmentally alterated conditions induced by the drugs such as acid secretion, mucosal blood flow and mucus secretion. Moreover, significant differences between the two drugs were not observed, except a minor influence of nitrofenac on anaerobes. It is probable that the partial release of nitric oxide by nitrofenac, before the intestinal absorption; induces a cytotoxic action against aerobes more than anaerobes. It is important to stress that nitric oxide is, in some cells, cytotoxic and in others cytostatic [ 121, suggesting that the sensitivity to nitric oxide varies from one cell to another. The nitric oxide-donor properties of nitrofenac are under study with kinetic experiments in the Institute of Pharmacology in Verona and in Prof. Wallace’s Institute, (University of Calgary, Canada), In conclusion, similar therapeutic efficacy of diclofenac and nitrofenac is shown in arthritic rats. No gastrointestinal ulcers are noted after administering 0.3 and 3 mg kg-’ doses for 21 days, but bacterial flora alterations due to various factors, probably related to nitric oxide for nitrofenac, were observed.
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