Gastrointestinal Toxicity, Antiinflammatory Activity, and Superoxide

(DMF)2] to confirm the production of uric acid (and consequently superoxide). ..... complex, copper-aspirin, decreases its SOD activity with an increa...
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Chem. Res. Toxicol. 2003, 16, 28-37

Gastrointestinal Toxicity, Antiinflammatory Activity, and Superoxide Dismutase Activity of Copper and Zinc Complexes of the Antiinflammatory Drug Indomethacin Carolyn T. Dillon,† Trevor W. Hambley,† Brendan J. Kennedy,† Peter A. Lay,*,† Qingdi Zhou,† Neal M. Davies,‡ J. Ray Biffin,§ and Hubert L. Regtop§ Centre for Heavy Metals Research, School of Chemistry, University of Sydney, NSW, 2006, Australia, Faculty of Pharmacy, University of Sydney, NSW, 2006, Australia, and Biochemical Veterinary Research, Pty Ltd, Braemar, NSW, 2575, Australia Received August 16, 2002

Gastrointestinal (GI) toxicity is one of the major problems associated with antiinflammatory drugs. The complexation of the powerful antiinflammatory drug (IndoH) by metal ions, as a means of reducing GI toxicity, has been studied. The in vitro superoxide dismutase (SOD) activity, in vivo antiinflammatory activity, and gastrointestinal ulcerogenic properties of IndoH, [Cu2(Indo)4(DMF)2], and [Zn2(Indo)4(DMA)2] are reported. No SOD activity was observed for IndoH or [Zn2(Indo)4(DMA)2], but [Cu2(Indo)4(DMF)2] inhibited the reduction of nitroblue tetrazolium (NBT) at an IC50 value of 0.23 µM. All three compounds exhibited antiinflammatory activity in male Sprague-Dawley rats at an equivalent Indo dose of 10 mg/kg following oral administration of the drugs in 2% CMC solution. The severity of the toxicity (macroscopic ulcerations) in the stomach following oral dosing with [Zn2(Indo)4(DMF)2] was not significantly lower than that induced by IndoH (P ) 0.78). Gastric ulcerations induced by [Cu2(Indo)4(DMF)2] were significantly lower than those induced by IndoH or [Zn2(Indo)4(DMA)2] (P ) 0.0012 and P ) 0.0175, respectively) but significantly greater than the control (P ) 0.0013). The intestinal ulcerations induced by [Cu2(Indo)4(DMF)2] or [Zn2(Indo)4(DMA)2] were approximately 15 times lower than those of IndoH. A further indicator of gastrointestinal toxicity, caecal haemoglobin, increased in the following order: control < [Cu2(Indo)4(DMF)2] < [Zn2(Indo)4(DMA)2] < IndoH. [Cu2(Indo)4(DMF)2] exhibited the most promising results of the Indo complexes assayed, in that it exhibited SOD activity and the lowest gastrointestinal damage while also exhibiting antiinflammatory activity that was comparable to that for IndoH. Low-temperature EPR analyses also showed that the formulation used for [Cu2(Indo)4(DMF)2] administration was crucial to the integrity of the complex.

Introduction Indomethacin [IndoH,1 I, N-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3 acetic acid] is a commonly used and powerful nonsteroidal antiinflammatory drug that is believed to act by inhibiting cyclooxygenase and, consequently, prostaglandin synthesis (1). There are two established isoforms of cyclooxygenase (COX), COX-1 and COX-2, that play different roles in the inflammatory process (1-3). It is believed that COX-2 primarily produces the prostaglandins involved in inflammation and mitogenesis while COX-1 acts to maintain normal * To whom correspondence should be addressed. E-mail: lay_p@ chem.usyd.edu.au. Telephone: +61-2-9351 4269. Fax: +61-2-9351 3329. † Centre for Heavy Metals Research. ‡ Faculty of Pharmacy. § Biochemical Veterinary Research. 1 Abbreviations: CMC, carboxymethyl cellulose; COX, cyclooxygenase (oCOX, ovine-cyclooxygenase, hCOX, human-cyclooxygenase); DMA, N,N-dimethylacetamide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EPR, electron paramagnetic resonance; Hbpzbiap, 1,5-bis(1-pyrazolyl)-3-[bis(2-imidazolyl)methyl]azapentane); IC50, concentration at which 50% inhibition occurs; IndoH, N-[p-chlorobenzoyl)5-methoxy-2-methyl-1H-indole-3 acetic acid; MEM-PR, minimum essential medium without phenol red; NBT, nitroblue tetrazolium; NSAID, nonsteroidal antiinflammatory drugs; RPMI, Roswell Park Memorial Institute; SEM, standard error of the mean; SOD, superoxide dismutase.

physiological function by its control of the renal parenchyma, gastric mucosa, platelets, and most other mammalian tissues (1-3). The inhibition of COX-1 leads to gastrointestinal ulcerogenic toxicity, and as such, it is perceived that a drug that displays selective inhibition of COX-2 over COX-1 would be a safer and more effective

10.1021/tx020078o CCC: $25.00 © 2003 American Chemical Society Published on Web 12/10/2002

Studies of Cu- and Zn-Indomethacin

antiinflammatory agent (1). Recently, however, the promise of this class of drugs has been dampened by findings that they exacerbate preexisting ulcers and also cause renal damage, elevated blood pressure, and platelet aggregation (4-6). IndoH is described as a nonselective COX inhibitor, with IC50 ) 10 nM for COX-2 and 6 nM for COX-1 (79), or COX 1 selective inhibitor, with IC50 ) 0.05 µM for oCOX-1, IC50 ) 0.75 µM for hCOX-2 (10, 11), but not as a COX-2 selective inhibitor. Several groups have focused on chemically modifying IndoH in order to convert it from a nonselective COX inhibitor to a selective COX-2 inhibitor (8, 10, 11), which have resulted in drugs that demonstrate oral antiinflammatory activity in vivo, but nonulcerogenic behavior in animal models. An alternative approach to reducing the GI toxicity is to complex Indo to metal ions. Copper is also believed to possess antiinflammatory effects (12-14) and a number of Cu(II) complexes of NSAIDs have been synthesized and tested accordingly (14-20). CuAlgesic (BVR, Mittagong, Australia) is a registered veterinary drug, used as an antiinflammatory agent in dogs and horses in Australia, Asia, and South Africa. It contains [Cu2(Indo)4L2], II, as the active ingredient (21, 22), which has a dimeric structure similar to Cu(II) acetate monohydrate with four bridging carboxylates linking the Cu(II) centers (23). Therapeutic administration of CuAlgesic to dogs results in only mild gastrointestinal lesions while indoH is extremely GI toxic (24-26). Haemorrhages and erosions were also less severe than those produced by lowdose aspirin (24). Doses of [Cu2(Indo)4L2] five times the therapeutic levels are nontoxic in the GI tract and central nervous system of horses, while a similar excess dose of IndoH was toxic to the central nervous system (22). The rat has become an accepted model for the study of NSAID pharmacodynamic effects in humans, as the gastrointestinal damage, renal damage, and antiinflammatory activity parallel the clinical experience with these drugs in humans (27). Bertrand et al. (28) reported that Cu-Indo liposomes (15 mg/kg) exhibited similar antiinflammatory activity to that of IndoH (15 mg/kg) in the carrageenan-induced paw oedema assay in male Sprague-Dawley rats. The extent of ulcerogenic toxicity in the small intestine of the male Sprague-Dawley rats following treatment with Cu-Indo (20 mg/kg) or IndoH (20 mg/kg) at 8 and 24 h after administration was lower following treatment with Cu-Indo in both instances, but only the 24-h result was significantly lower (28). The intestinal damage was highly dependent on the form of administration of Cu-Indo. For instance, rats receiving 15 mg/kg Cu-Indo by oral dosing exhibited the following % ulcerations: DMF solution/RPMI 1640 medium, 20.19 ( 5.31%; liposomes 2.89 ( 0.92%; nanocapsules, 11.53 ( 4.18%. Despite these findings, the mechanism of action of [Cu2(Indo)4(DMF)2] remains unknown. Zinc increases the healing rate of ulcers (29-32) and Zn-Indo complexes, and [Zn2(Indo)4L2] (III) and cis-[Zn(Indo)2L2] have also been identified as potential antiinflammatory drugs (22, 30). Singla and Wadhwa showed that Zn-Indo is three times more potent as an NSAID than IndoH and 2.6 times more potent than the corresponding mixture of IndoH and zinc sulfate (30). AbouMohamed also confirmed that rats exhibited a greater antiinflammatory response when they were treated with a combination of IndoH and Zn administered either orally

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or subcutaneously than a corresponding treatment with either Indo or Zn alone (29). Increasing attention has been paid to the superoxide dismutase (SOD) activity of antiinflammatory drugs because superoxide is implicated in the promotion of arthritis by the degradation of hyaluronic acid, which is essential for maintaining internal joint homeostasis (3335). This is supported by the complete protection against this degradation by treatment with SOD (33, 36). Moreover, oxygen-derived free radicals reportedly play important roles in the pathogenesis of gastric mucosal injury (37) and SOD exists in the mucosal tissues of the gastrointestinal tract where it is believed to protect against such damage (38-42). Due to the diverse nature of the pathophysiological changes that occur with inflammation and arthritic diseases, it is difficult to predict the effectiveness of a particular drug solely based on investigations of one aspect of the drug. Here we present a series of in vivo and in vitro biological studies of IndoH, [Cu2(Indo)4(DMF)2] (DMF ) N,N-dimethylformamide) and [Zn2(Indo)4(DMA)2] (DMA ) N,N-dimethylacetamide) including investigations of the gastrointestinal toxicities and antiinflammatory activities in the rat and the SOD-“like” activities of IndoH, [Cu2(Indo)4(DMF)2], and [Zn2(Indo)4(DMA)2] to delineate possible mechanisms.

Experimental Procedures Compounds. IndoH was obtained from Sigma Aldrich, and [Cu2(Indo)4(DMF)2] was used as obtained from BVR Pty Ltd (23). [Zn2(Indo)4(DMA)2] was prepared as described previously (43). In Vitro SOD Activity. The SOD-like activities of IndoH, [Cu2(Indo)4(DMF)2], and [Zn2(Indo)4(DMA)2] were measured according to the method described by Younes and Weser (44). Spurious metal ions were removed from the glassware by soaking in decon-90 detergent (24 h), 1.5 M nitric acid (24 h), and distilled water (24 h) prior to the final rinses with milli-Q water. All plasticware and glassware were autoclaved at 121 °C for 20 min to deactivate any possible enzyme contaminates. The phosphate buffer (0.1 M, pH 7.8) used in this experiment was prepared by mixing the appropriate volumes of 0.1 M KH2PO4 and 0.1 M K2HPO4 (Ajax, AR grade) with the aid of a pH meter (Activon model 210). The SOD-mimetic analysis was performed by mixing the following solutions: 0.1 M phosphate buffer, pH 7.8, 1.1 mL; gelatin solution, 500 µL (1% in phosphate buffer, Sigma); catalase, 6 units/mL, 100 µL (Sigma); NBT, 4 mg/mL, 300 µL (Sigma); xanthine oxidase 0.2 units/mL, 250 µL (Sigma); and the test solution in DMSO (250 µL, DMSO ) dimethyl sulfoxide). Spectrophotometry was performed using a Hewlett-Packard 8452A diode array spectrophotometer in combination with a Hewlett-Packard 89090A temperature control unit. The above solutions were allowed to equilibrate at 25 °C and the initial spectrum was recorded. Xanthine solution (500 µL, 1 mM in phosphate buffer, Sigma) was added within seconds and the UV-vis spectra were recorded at 5 s intervals for 250 s. The reaction was monitored by the increase in absorption at 540 nm as the solution changed from yellow to purple. The activity was determined by linear regression of the plot of absorbance versus time and is quoted as ∆A540nm/min. Total inhibition was observed when there was no increase in absorption. As it is well documented that Cu2+ ions inhibit xanthine oxidase at mM concentrations (45), the activity of xanthine oxidase was monitored at 292 nm in the presence of [Cu2(Indo)4(DMF)2] to confirm the production of uric acid (and consequently superoxide). The assay was performed using the above protocol, although phosphate buffer was substituted for NBT according to the method of Ciuffi et al. (46).

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EPR Studies. Low-temperature EPR spectroscopic studies were performed to determine the nature of [Cu2(Indo)4(DMF)2] in the formulations administered in animal experiments. A series of monomeric Cu(II) standards were prepared from CuCl2 (BDH, AR grade) dissolved in a DMSO/water solution (1:10). [Cu2(Indo)4(DMF)2] (5 mg dissolved in 1 mL of DMF) was added to RPMI solution (2 mL, Sigma), and the solution was immediately frozen by slow immersion into liquid nitrogen. EPR spectroscopy was performed at 60 and 110 K using a He-cooled X-band Bruker EMX081 EPR spectrometer connected to an EMX 035M NMR gaussmeter. The same operating parameters were used for all the standards and the unknown solutions. These included microwave frequency, 9.45271 Hz; microwave power, 20.12 mW; receiver gain, 1.782 502 × 103; modulation amplitude, 5.95 G; conversion time, 20.48 ms; time constant 5.12 ms. Double integrations of the EPR signals obtained from the CuCl2 standards (4 spectra/concentration) were performed and a straight line in the region of 0-3 mg of CuCl2/5 mL was obtained with an R value of 0.998. The equation of the linear fit was used to determine the Cu(II) monomer content in the unknown solution. EPR spectra of various Cu(II) compounds in cell medium/ DMSO solution were collected at 110 K to determine if the type of monomeric Cu(II) species differed according to the species that was dissolved. A solution of [Cu2(Indo)4(DMF)2] (3 mM in DMSO, 1 mL) was added to MEM-PR solution (4 mL) and the solution was frozen immediately by immersion into liquid N2. Similarly, [Cu2(Ac)4(OH2)2] (3 mM in DMSO, 1 mL, Ac ) acetate) was added to MEM-PR solution (4 mL). Monomeric Cu(II), as CuCl2, was also added to MEM-PR solution by first dissolving in MEM-PR (4 mL) and then adding DMSO (1 mL) to prepare an equivalent solution. Attempts to identify the monomeric Cu(II) species by EPR spectroscopy were performed by reacting [Cu2(Indo)4(DMF)2] with the components of RPMI solution, namely D-glucose and amino acids. Reactions with D-glucose were performed by addition of [Cu2(Indo)4(DMF)2] (5 mg in 0.6 mL of DMSO) to D-glucose (2.5% in milli-Q water, 2.4 mL, final [D-glucose] ) 2% w/v). In a second experiment, EPR spectra were recorded in a mixture of DMSO and RPMI, whereby [Cu2(Indo)4(DMF)2] (5 mg in 1 mL of DMSO) was added to RPMI solution (2 mL). D-Glucose (2 mg) was then added, and the EPR spectrum was recollected to detect any increase in the monomeric Cu signal. A solution of [Cu2(Indo)4(DMF)2] (5 mg in 0.6 mL DMSO) was also reacted with the typical amino acid, glycine (25.5 mg in 2.4 mL milli-Q water), to determine if the signal was similar to that detected in the RPMI solution. In further experiments, [Cu2(Indo)4(DMF)2], [Cu2(Ac)4(OH2)2], or CuCl2 were added to a cell growth medium to establish any differences in the monomeric Cu(II) species formed at 110 K and thereby determine if Indo was still bound to Cu(II). Finally, [Cu2(Indo)4(DMF)2] (4.68 mg) was added to a 2% carboxymethylcellulose (CMC) solution (1 mL), and the solution was vortexed for approximately 10 min to obtain an evenly distributed suspension. The solution was pipetted into a quartz EPR tube and frozen by slow immersion in liquid nitrogen. EPR spectra were collected at 110 K for detection of both the monomer and dimer Cu(II) signals. Animals. Sprague-Dawley rats weighing 200-250 g were used throughout these studies (supplied by the laboratory animal services at the University of Sydney). Animals were housed in polypropylene cages and allowed free access to standard laboratory rat chow (Purina Rat Chow, Ralston Purina, St Louis MO) and tap water. Animals were housed in the animal care facility of the Faculty of Pharmacy at ambient temperature and humidity with a 12-h light-dark cycle. The experimental animal protocols were approved by the Animal Ethics Committee of the University of Sydney in July 1999, approval number L24/7-99/3/2972. In Vivo Antiinflammatory Activity. Rats (n ) 4 per group) were dosed orally with IndoH (10 mg/kg), [Cu2(Indo)4(DMF)2] (11.7 mg/kg), or [Zn2(Indo)4(DMA)2] (13.3 mg/kg) in 2% CMC (doses were calculated as equivalent concentrations of Indo). The

Dillon et al. control cohort was dosed solely with 2% CMC solution. Inflammation was induced 1 h after dosing with the NSAID (or vehicle), by injecting with carrageenan (0.1 mL, 2% w/v in isotonic saline) into the plantar region of the hind paw. The thickness of the paw was measured at the ventral dorsal footpad using digital callipers prior to dosing and at 3 and 5 h after carrageenan injection. Similarly, volume displacement was measured prior to dosing and at 3 and 5 h after carrageenan injection by immersing the left hind paw (to the lateral malleus) into water. In Vivo Gastrointestinal Toxicity. Male Sprague Dawley rats (n ) 6) were orally dosed with IndoH (10 mg/kg), [Cu2(Indo)4(DMF)2] (11.7 mg/kg) or [Zn2(Indo)4(DMA)2] (13.3 mg/kg) suspended in 2% CMC solution (0.5 mL/rat). Rats were fasted for 24 h with access to water prior to assessment of gastric toxicity. Three hours after administration of the above-mentioned suspensions, the rats were euthanased and the stomach was excised and opened by incision along the greater curvature. The stomach was rinsed, submerged in 10% formaldehyde for 1 h and examined to determine the extent of macroscopic gastric toxicity, which is reported as the summation of the area of macroscopic ulcerations (mm2). For the assessment of damage to the small intestine, rats were allowed free access to food and water prior to and during the assay. At 24 h after dosing, the entire small intestine was excised and flushed with water to expel the intestinal contents. The intestine was examined from 10 cm distal to the ligament of Treitz to the ileocecal junction, and the toxicity is reported as the summation of the area of macroscopic ulcerations (mm2). Estimation of Caecal Hemoglobin Concentration. Caecal hemoglobin was assessed by adaptation of a method reported by Melarange et al. (47, 48) and the “Total Hemoglobin Sigma Diagnostics Kit” (Sigma). Caecums were removed from rats (n ) 4-6) 24 h after treatment with the vehicle (2% CMC solution) or 2% CMC solutions of IndoH (10 mg/kg), [Cu2(Indo)4(DMF)2] (11.7 mg/kg), or [Zn2(Indo)4(DMA)2] (13.3 mg/kg). The caecums were immediately frozen until required. Upon thawing, the whole caecums were vortexed with distilled water (5 mL) to produce a suspension of the caecal contents. The suspension was centrifuged for 10-20 min at 400g until a clear supernatant was obtained. The hemoglobin content was determined by addition of the supernatant (0.5 mL) to Drabkin’s Reagent (4.5 mL, containing NaHCO3, K3[Fe(CN)6] and KCN). After 30 min, the resultant solution was analyzed at 540 nm using a HewlettPackard Diode Array Spectrophotometer, and the hemoglobin content was determined from the cyanmethemoglobin standards (0-18 g/dL). Statistical Analyses. The results of the animal assays were assessed using the two-tailed t-test. In all cases, a value of P < 0.05 was considered significant (49-51).

Results SOD Activity. The results of the study of the reduction of NBT in the xanthine/xanthine oxidase system are summarized in Figure 1. A linear increase in the absorbance at 540 nm in the first 250 s was observed following the NBT reduction in the control solution containing DMSO, and this was associated with a color change from yellow to purple. When inhibiting solutions were tested, there was very little increase in the absorbance at 540 nm and the solution remained yellow. Total inhibition of the reduction of NBT was observed for 1 × 10-5 M [Cu2(Indo)4(DMF)2], and 50% inhibition was observed at approximately 2.3 × 10-7 M [Cu2(Indo)4(DMF)2]. Furthermore, monitoring of the spectra at 292 nm in the absence of NBT revealed the production of uric acid, which indicated that xanthine oxidase was not inhibited at 1 × 10-7 M [Cu2(Indo)4(DMF)2]. The IC50 was 7.4 × 10-7 M for [Cu2(Indo)4(DMF)2] inhibition of the enzyme,

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Figure 1. Results of the reduction of NBT in the xanthine/ xanthine oxidase system using the following test solutions: (a) IndoH; (b) [Zn2(Indo)4(DMA)2]; and (c) [Cu2(Indo)4(DMF)2].

Figure 3. X-Band EPR spectra collected at 110 K following the reactions of (a) [Cu2(Indo)4(DMF)2] (5 mg) in DMSO/RPMI solution (3 mL); (b) [Cu2(Indo)4(DMF)2] (5 mg) and D-glucose (2% w/v) in DMSO/water (3 mL); (c) [Cu2(Indo)4(DMF)2] (5 mg) and glycine (25.5 mg) in DMSO/water (3 mL) and (d) [Cu2(Indo)4(DMF)2] (11.7 mg) in 2% CMC solution (2 mL).

Figure 2. X-Band EPR spectra of (a) the standard CuCl2 (2 mg) in DMSO/water (5 mL) and (b) [Cu2(Indo)4(DMF)2] (5 mg) in DMF/RPMI (3 mL) collected at 60 K.

but there was still 30% activity of xanthine oxidase at 1 × 10-6 M [Cu2(Indo)4(DMF)2]. There was no evidence of SOD inhibition when IndoH was introduced into the system at concentrations as high as 10-4 M. Addition of IndoH solutions above this concentration resulted in IndoH precipitation and a slow color change from yellow to purple showing that a saturated solution of IndoH did not inhibit NBT reduction. No significant inhibition of the reduction of NBT was observed for [Zn2(Indo)4(DMA)2] for concentrations ranging from 10-7 M up to a supersaturated concentration of 10-3 M. EPR Studies. Figure 2 shows the EPR spectra obtained from the solutions of (a) CuCl2 in DMSO/water (2 mg CuCl2/5 mL) and (b) [Cu2(Indo)4(DMF)2] in DMF/ RPMI (5 mg/3 mL), collected at 60 K. Spectrum a (Figure 2) shows a typical monomeric Cu(II) signal with four hyperfine lines resulting from the 63Cu and 65Cu isotopes of I ) 3/2 where the g| ) 2.395, A| ) 138.0 × 10-4 cm-1, and g⊥ ) 2.079. Spectrum b also shows a signal typical of monomeric Cu(II) with g| ) 2.318, A| ) 193.2 × 10-4 cm-1, and g⊥ ) 2.054. There is also evidence of splitting of the g⊥ signal, which coincides with the fourth hyperfine signal at 3275.6 G. There was no signal at 50 or 4600 G to indicate the presence of dimeric Cu(II) species (23). Double integrations of the monomeric Cu(II) signals from CuCl2 standards (0-3 mg/5 mL) were performed using

the Bruker WINEPR program (52). From the resultant standard curve, the monomeric Cu(II) concentration deduced from spectrum b was 5.28 ( 0.14 µmol/3 mL. The maximum concentration of monomeric Cu(II) in the solution added initially as dimeric [Cu2(Indo)4(DMF)2] was 5.88 µmol/3 mL, showing that approximately 90% of the Cu had dissociated to the monomeric form. The EPR spectra obtained for CuCl2 (0.6 mM), [Cu2(Ac)4(OH2)2] (0.6 mM), or [Cu2(Indo)4(DMF)2] (0.6 mM) in cell medium/DMSO solution resulted in identical monomeric Cu(II) signals (akin to that in Figure 2b) that differed only in intensity. Figure 3 shows the resultant EPR spectra collected at 110 K following the reaction of [Cu2(Indo)4(DMF)2] with the formulations and constituents of the formulations used for the animal assays. DMSO was used as the solvent for [Cu2(Indo)4(DMF)2] due to the ease and speed of dissolution. The EPR spectrum of [Cu2(Indo)4(DMF)2] (5 mg in 1 mL of DMSO) in RPMI solution (2 mL) is shown in Figure 3a. A similar spectrum was obtained to that following dissolution from DMF (Figure 2b) with the same distinguishing features and only minor shifts of the g and A values (g| ) 2.2532, A| ) 187.4 × 10-4 cm-1, and g⊥) 2.0556). The reaction of [Cu2(Indo)4(DMF)2] (5 mg in 0.6 mL of DMSO) with D-glucose (2.5% w/v in milli-Q water, 2.4 mL, final concentration ) 2% w/v) produced a weak monomeric Cu signal at g⊥ ) 2.0766 (accurate determination of g| was not achieved due to the low monomeric concentration) and a weak dimeric Cu signal at giso ) 1.4347 (Figure 3b). The results of EPR analysis of the reaction of [Cu2(Indo)4(DMF)2] (5 mg in 0.6 mL of DMSO) with glycine (25.5 mg in 2.4 mL of milli-Q water) are shown in Figure 3c. The spectrum exhibited similar features to those obtained from the addition of [Cu2(Indo)4(DMF)2] to RPMI solution with a strong signal due

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Figure 5. Gastrointestinal damage observed in rats following oral administration of a 2% CMC solution of IndoH (10 mg/kg), [Cu2(Indo)4(DMF)2] (11.7 mg/kg) or [Zn2(Indo 4(DMA)2] (13.3 mg/ kg) in (a) the stomach and (b) the small intestine. Each bar represents the mean ( SEM for six rats. Figure 4. Effect of oral administration of a 2% CMC solution (control), Indo (10 mg/kg), [Zn2(Indo)4(DMA)2] (13.3 mg/kg), or [Cu2(Indo)4(DMF)2] (11.7 mg/kg) on carrageenan-induced paw edema assessed as the increase in (a) paw diameter and (b) paw volume, compared to the baseline. Each bar represents the mean ( SEM for four rats.

to a monomer possessing g| ) 2.2658, A| ) 180.9 × 10-4 cm-1, and g⊥ ) 2.0556. The EPR spectrum of [Cu2(Indo)4(DMF)2] (11.7 mg) in 2% CMC solution (2 mL) at 110 K is shown in Figure 3d. There is a weak signal due to dimeric Cu(II) at giso ) 1.4380 and a dominant signal due to monomeric Cu(II) at g| ) 2.3631, A| ) 156.8 × 10-4 cm-1, and g⊥ ) 2.0689. Quantification of the signal due to the Cu(II) monomer showed that it represented only 3% of the total Cu concentration. In Vivo Antiinflammatory Activity. Figure 4 shows the results of the carrageenan-induced paw oedema assay as assessed by (a) paw diameter (using calliper measurements), and (b) paw volume (volume displacement method). At 3 h, the oedema peaked, and the most significant results were obtained. Consequently, only these results are shown. It is clear from Figure 4, panels a and b that the carrageenan-induced inflammation was reduced in the paws of all rats treated with Indocontaining compounds as compared to the control. Inhibition of inflammation as measured by paw diameter (Figure 4a) was greatest for [Zn2(Indo)4(DMA)2] (17.7 ( 3.4% increase in inflammation above baseline measurements) followed by IndoH (24.2 ( 2.0%) and [Cu2(Indo)4(DMF)2] (29.8 ( 2.2%). The significance of the reduction in paw diameter was confirmed by t-test comparisons of the control (48.3 ( 5.0%) versus the following treatments: IndoH, P < 0.0001; [Cu2(Indo)4(DMF)2], P ) 0.0001; [Zn2(Indo)4(DMA)2], P < 0.0001, but there were no significant differences among the three compounds. Similarly, inhibition of inflammation was reflected in the results of the paw volume whereby the percentage increase from baseline was [Zn2(Indo)4(DMA)2], 9.6 ( 5.2%; IndoH, 10.3 ( 7.1%; [Cu2(Indo)4(DMF)2], 14.9 ( 8.6% and control, 61.1 ( 10.2%. All treated rats exhibited significant reduction in the inflammation compared to the control rats: [Zn2(Indo)4(DMA)2], P ) 0.0036; IndoH, P ) 0.0063; [Cu2(Indo)4(DMF)2], P ) 0.0133 but there were no significant differences in the

inhibition of inflammation among the treatments with the Indo compounds. In Vivo Macroscopic Gastrointestinal Toxicity. Figure 5 shows the results of the gastrointestinal toxicity induced by IndoH, [Zn2(Indo)4(DMF)2], and [Cu2(Indo)4(DMA)2]. It is apparent in Figure 5a that the severity of the macroscopic ulcerations in the stomach is similar in IndoH- and [Zn2(Indo)4(DMF)2]-treated rats (P ) 0.7795) and that both treatments induced damage that significantly exceeded that of the control. Ulcerations induced by [Cu2(Indo)4(DMF)2] were significantly lower than those due to IndoH or [Zn2(Indo)4(DMA)2] (P ) 0.0012 and P ) 0.0175, respectively) but significantly greater than that of the control (P ) 0.0013). The intestinal ulcerations caused by [Cu2(Indo)4(DMF)2] or [Zn2(Indo)4(DMA)2] were markedly lower than those of IndoH with P < 0.0001 for both complexes (Figure 5b). There was no significant difference between the intestinal ulcerogenic activities of [Cu2(Indo)4(DMF)2] and [Zn2(Indo)4(DMA)2] (P ) 0.4178). Caecal Hemoglobin Content. Figure 6 shows the caecal hemoglobin concentration from rats that were administered a 2% CMC control solution and rats that have been treated with IndoH, [Cu2(Indo)4(DMF)2], or [Zn2(Indo)4(DMA)2] in CMC. Significant increases in the hemoglobin concentration were observed for all rats treated with IndoH or metal-Indo complexes compared to that of the control. The caecal hemoglobin induced by the treatments increased in the order: control (0.94 mg/ mL) < [Cu2(Indo)4(DMF)2] (1.38 mg/mL) < [Zn2(Indo)4(DMA)2] (2.40 mg/mL) < IndoH (5.40 mg/mL). The degree of caecal hemoglobin exhibited by [Cu2(Indo)4(DMF)2]treated rats was significantly lower than that induced by [Zn2(Indo)4(DMA)2] (P ) 0.008) or Indo (P < 0.0001). Furthermore, the damage induced by [Zn2(Indo)4(DMA)2] was significantly lower than that induced by IndoH (P ) 0.0018).

Discussion SOD Activity. SOD mimics have been hailed as potential therapeutics for treatment of a number of diseases including inflammation and diseases associated with inflammation, namely, rheumatoid arthritis and

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Table 1. SOD Activity of Some cu Complexes (54, 55)a,b,c copper complex

IC50 (µM)

copper complex

IC50 (µM)

cuprein [Cu(tyr)2] [Cu(lys)2] CuSO4 [Cu(φMeTIM)]e [Cu(TIM)]e [Cu2(bpzbiap)Cl3] [Cu(DIPS)2]d [Cu(DIPS)2]e [Cu(DIPS)2]

0.04 (55) 45 (44) 86 (44) 30 (58, 59) 16 (60) 210 (60) 0.255 (61) 2.85 (55) 8.9 (60) 2.9 (57)

[Cu(salicylate)2] [Cu(salicylate)2] [Cu(salicylate)2]d [Cu(Asa)2] [Cu(Asa)2]d [Cu(p-aminosalicylate)2] [Cu(p-aminosalicylate)2]d [Cu2(Ionazolac)4] [Cu2(Indo)4(DMF)2] [Cu2(Indo)4(DMF)2]f

4.6 (57) 16 (55) 1.3 (55) 23 (55) 2.15 (55) 28 (55) 3 (55) 1.5 (62) 2-25 (63) 0.23

a tyr ) tyrosine; lys ) lysine; φMeTIM ) 2,9-dimethyl-3,10-diphenyl-1,4,8,11-tetraacyclotetradeca-1,3,8,10-tetraene; TIM ) 2,3,9,10tetramethyl-1,4,11-tetraacyclotetradeca-1,3,8,10-tetraene; Hbpzpiap ) 1,5-bis(1-pyrazolyl)-3-[bis(2-imidazoyl)methyl]azapentane; DIPS ) diisopropylsalicylate; Asa ) acetylsalicylic acid; lonazolac ) 3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-acetic acid. b Assays were performed using xanthine/xanthine oxidase as the source of superoxide at pH 7.8, unless otherwise stated. c Second order rate constants for [Cu(tyr)2], k ) 2.7 × 109 M-1 s-1 (56), copper acetate-like structures, k ) 5 × 109 M-1 s-1 (59); [Cu(DIPS)2], k ) 2.9 × 109 M-1 s-1 (57); [Cu(salicylate)2], k ) 4.6 × 109 M-1 s-1 (57), and [Cu2(Indo)4(DMF)2] in water pH 7.0, k ) 6 × 109 M-1 s-1, in DMSO, k ) 1.1 × 109 M-1 s-1 (60). d Assays were performed using KO2 as a source of superoxide. e Assays were performed at pH 7.5. f This work.

Figure 6. The hemoglobin concentration obtained from rat caecums following oral administration of 2% CMC solution (control, n ) 3) or a 2% CMC solution of [Cu2(Indo)4(DMF)2] (11.7 mg/kg, n ) 8); [Zn2(Indo)4(DMA)2] (13.3 mg/kg, n ) 4) or IndoH (10 mg/kg, n ) 4). Each bar represents the mean ( standard deviation for three to eight rats.

osteoarthritis (53). The ability of SOD to remove toxic superoxide and consequently prevent the deleterious depolymerization of hyaluronic acid in the synovial fluid, suggested a role for SOD mimics for the treatment of arthritis and other inflammatory disorders (33-35). Of relevance here is the result that [Cu2(Indo)4(DMF)2] possesses SOD-like activity with an IC50 value of 0.23 µM, which compares to an IC50 concentration of 0.04 µM for native SOD [Table 1 (44, 54-63)]. Other studies performed by Deuschle and Weser (employing xanthine/ xanthine oxidase as the superoxide source) showed a 60% inhibition of NBT reduction at a [Cu2(Indo)4(DMF)2] concentration of 3 µM (62), which was reasonably consistent with the IC50 value obtained in this study. It is not surprising that Cu-Indo exhibits SOD activity as there is widespread evidence that Cu complexes possess SOD activity (44, 55, 58-67). For instance, the Cu(II) amino acid complexes [Cu(tyr)2] and [Cu(lys)2] induce 50% inhibition of the reduction of NBT by O2- at concentrations of 45 and 86 µM, respectively (44). On the basis of crystallographic studies of SOD, Tabbı` et al. synthesized an imidazole-bridged dinuclear complex of Cu(II) [Cu2(bpzbiap)Cl3] (Hbpzbiap ) 1,5-bis(1-pyrazolyl)3-[bis(2-imidazolyl)methyl]azapentane), which catalyzed the dismutation of superoxide with an IC50 of 0.255 µM (61). This was reportedly one of the most active in the literature. Importantly, [Cu2(Indo)4(DMF)2] compares

favorably with these SOD mimics as the IC50 value for [Cu2(Indo)4(DMF)2] lies in the high activity region of the spectrum exhibited by Cu complexes (Table 1) (61). The lack of SOD activity of the redox-inactive Zn-Indo complexes is not surprising since, although both Cu and Zn are present in SOD, Zn is not essential for the catalytic activity of SOD but is believed to be important in the stabilization of the ternary and quaternary structures of the protein (68). The lack of SOD activities obtained for IndoH and the Zn-Indo complexes are consistent with results of Kensler and Trush (69), who found that diisopropylsalicylate (DIPS) and [Zn(DIPS)] also showed no SOD activity whereas [Cu(DIPS)] did. The question is whether the SOD activity of Cu-Indo is relevant to either the antiinflammatory or decreased GI toxicity exhibited by the drugs or whether some other factors are important. These issues are addressed in turn in the following sections. In Vivo Antiinflammatory Activity. The antiinflammatory activity (Figure 4) exhibited by [Zn2(Indo)4(DMA)2] (13.3 mg/kg) was consistent with studies reported by Singla et al. of [Zn(Indo)2] (13.68 mg/kg) whereby paw inflammation was only 15% compared to 80% for the controls and 33% for IndoH (12 mg/kg) (n ) 6) (30). In the work reported here, [Zn2(Indo)4(DMA)2] also showed greater antiinflammatory activity than IndoH, although the results were not significantly different, possibly due to the smaller sample size used. While the result for [Cu2(Indo)4(DMF)2] contradicts some reports that significantly greater antiinflammatory activity was observed for the Cu complexes over the parent ligands, the precision of this assay was not great enough to reveal any significant difference in the activities (12, 16). However, the results were consistent with those of other researchers who reported that the antiinflammatory activities of Cu complexes (e.g., ketoprofen, indomethacin, naproxen, niflumic acid, clopirac and aspirin) were similar to those exhibited by the parent drugs (15, 17). This is also consistent with recent work performed by Bertrand et al. where Cu-Indo liposomes exhibited similar antiinflammatory activity to that of IndoH (28). The similar antiinflammatory effects of Indo and its Cu and Zn drugs suggests that the metal complexes act as a means of introducing Indo into the blood stream with reduced GI toxicity but do not effect the mechanism of antiinflammatory action. In support of this are the EPR experiments that show that the Cu complex breaks down in biological medium and the observation that a similar

34

Chem. Res. Toxicol., Vol. 16, No. 1, 2003

complex, copper-aspirin, decreases its SOD activity with an increase in serum albumin concentration (70). Taken together, this suggests that the Cu-Indo complex is not transported intact to the site of inflammation. These results, however, show such complexes may form at the site of inflammation in vivo and, if this is the case, then the SOD activity may be an important factor in the efficacy of the drug (12, 71) irrespective of whether the Indo is delivered in the form of IndoH or the Cu(II) or Zn(II) complexes. Evidence to support this assertion is that both Indo and Cu(II) concentrate at the site of inflammation (72), and both are carried by serum albumin at different sites of the protein (72, 73). Moreover, synovial fluid contains lubricants that are ideal sites for the formation of Cu-Indo in vivo at the site of inflammation, which has relevance since the standard synthetic reaction of Cu(II) with Indo occurs in nonaqueous solvents (21-23). Taken together, this suggests a significant role for SOD activity of Cu-Indo in vivo in reducing inflammation irrespective of how the drug is delivered (i.e., IndoH, Cu-Indo or Zn-Indo). Gastrointestinal Toxicity. The lack of a significant improvement in the gastric ulcerogenic activity of [Zn2(Indo)4(DMA)2] compared to IndoH (Figure 5a) was also observed by Singla and Wadhwa (30) whereby the ulcerogenic effects, as measured by the lesion index, were 20.5 and 21.5 for IndoH (9.83 mg/kg, n ) 10) and ZnIndo (9.83 mg/kg, n )10), respectively. Although the ulcerogenic damage induced by [Cu2(Indo)4(DMF)2] was higher than that of the control (2% CMC solution), the complex was significantly less toxic in the stomach in comparison to IndoH or [Zn2(Indo)4(DMA)2] (Figure 5a). The greatest effects of the metal Indo complexes were evident in the small intestine whereby both complexes were far less damaging than IndoH (Figure 5b). These results were paralleled in the caecal hemoglobin assay (an indicator of the GI blood loss and ultimate GI toxicity) since [Cu2(Indo)4(DMF)2] induced markedly lower bleeding than either [Zn2(Indo)4(DMA)2] or IndoH (48). Consequently, [Cu2(Indo)4(DMF)2] was the least toxic drug in all the gastrointestinal assays studied. Low-temperature EPR analyses of [Cu2(Indo)4(DMF)2] (Figure 2) in the RPMI/DMF formulation used by Bertrand (28) showed immediate dissociation of [Cu2(Indo)4(DMF)2] to approximately 90% monomeric Cu(II) upon addition of the Cu complex to RPMI. This feature of CuNSAIDs was also observed following the addition of a DMF solution of [Cu2(niflumate)4L2] to water (74). The identical EPR signals obtained from the reactions of CuCl2, [Cu2(Ac)4(OH2)2], or [Cu2(Indo)4(DMF)2] in cell medium indicate that the monomeric Cu(II) species produced are the same, excluding the possibility that the Cu(II) species is a monomeric Cu-Indo complex. Furthermore, while some monomeric Cu(II) was formed from the isolated reaction of [Cu2(Indo)4(DMF)2] with glucose, the species formed from the reaction of [Cu2(Indo)4(DMF)2] with glycine showed closer similarities to the predominant species produced from [Cu2(Indo)4(DMF)2] and RPMI solution. This suggests that the amino acids present in RPMI act as ligands that bind to monomeric Cu(II) prior to administration of the formulation to the animals. It is, therefore, noteworthy that the advantages of using [Cu2(Indo)4(DMF)2] over IndoH (from DMF/RPMI solution) in the small intestine were not as pronounced in the study performed by Bertrand et al. (28). While this may be a result of the higher doses administered (20 vs

Dillon et al.

10 mg/kg), the result is more likely to be due to effectively treating the animals with free IndoH as would occur when the DMF/RPMI solutions were used. In contrast, EPR analysis of the suspension of [Cu2(Indo)4(DMF)2] in 2% CMC solution (Figure 3) revealed the persistence of a dimer signal, showing that the complex was mainly delivered intact [the monomeric Cu(II) accounted for only 3% of the total Cu]. The incomplete dissolution of [Cu2(Indo)4(DMF)2] in the CMC solution and less extensive dissociation of the compound prior to administration, are the likely explanations for the lower GI toxicity observed in the current study. The higher degree of jejunoileal ulcerations observed by Bertrand following the use of various Cu-Indo formulations shows that the method of administration of [Cu2(Indo)4(DMF)2] is crucial to the ultimate efficacy and toxicity of the drug (28). The importance of the dimeric species also emphasizes the beneficial effects of the intact drug in comparison to uncomplexed IndoH, or administration of Cu(II) and IndoH. Thus, unlike chemical modifications of IndoH, the formation of the Cu complex does not reduce the GI toxicity by changing the selectivity of COX inhibition. Indeed, the only way that the large [Cu2(Indo)4L2] molecule can interact with the COX enzymes is for the Indo ligand to dissociate, since it is far too large to fit into the pockets of the enzymes. This has been shown by COX1 and COX2 selectivity that was the same for both the Cu complex and IndoH.2 The increased GI toxicity of both the Zn(II) and Cu(II) complexes in the stomach as opposed to the small intestine is no doubt due to some acid-catalyzed hydrolysis, which releases free IndoH in the stomach. Although such reactions are normally rapid, the lipophilic nature of the complexes means that they have low solubilities in water and a tendency to stay within the micelles of the formulations. These factors together mean that they are protected against acid hydrolysis for a sufficient length of time to be absorbed intact or to pass through to the small intestine. At the much higher pH value of the small intestine, the complexes are more stable, and hence, little or no GI toxicity is observed as the GIdamaging Indo is not released to any significant degree in this region of the GI tract before it is absorbed. The mechanism of reduced GI toxicity observed for [Cu2(Indo)4(DMF)2] compared to both free Indo and the Zn complex may also be due to the action of Cu(II) on a number of enzymic reactions. For example, it has been shown that Cu alters PG production, thromboxane (TXA2) production and SOD activity in the gastrointestinal tract (75, 76). It is well documented that SOD exists in the mucosa of the gastrointestinal tract (40) and it is believed that it is a key enzyme in the protective mechanism of gastric and duodenal mucosa against damaging species (38). Its importance in the human gastric mucosa has derived from observations of decreased SOD activity in the gastric ulcer (possibly due to disruption of the epithelial cells) and increased SOD activity associated with healing ulcers (77). It has also been shown by Franco and Velo (75), that the gastric mucosal SOD activity increases following oral administration of Cu complexes to rats. While there may be reduced SOD activity of Cu complexes in plasma due to their composition and binding 2 M. James, J. R. Biffin, and H. Regtop, (1996) IndoH and CuIndo showed similar selectivities for the inhibition of COX-1 and COX-2 on a ng/mL basis. The complexes were added to aqueous solution from DMSO.

Studies of Cu- and Zn-Indomethacin

of Cu(II) to other proteins (36), the gastrointestinal tract is the first contact point of the complex where it is less likely to have bound to proteins. It is likely, therefore, that the SOD activity exhibited by intact [Cu2(Indo)4(DMF)2] is responsible for the enhanced gastric mucosal SOD activity, thus protecting against ulceration caused by any released IndoH. This is also consistent with the greatly reduced protection in the stomach, where the more acidic environment will increase the release of IndoH and hence reduce the protective effect of the Cu(II) complex in comparison to the intestine. The key feature in both proposed mechanisms of reduced GI toxicity (lack of release of free Indo and SOD activity) relies on the lipophilic nature of the Cu(II)-Indo complex, which increases its stability in the micelle formulations used to deliver the drug to the stomach.

Concluding Remarks The pharmacological efficacy of any drug stems from the optimization of beneficial effects and the reduction of the toxic side effects. The latter remains a major goal in the development of antiinflammatory drugs. The antiinflammatory activity of Indo is not significantly inhibited by the complexation with a metal, and may even be slightly enhanced. Although the Cu complex possesses extremely high SOD activity, this did not result in obvious benefits to the observed antiinflammatory activity (although its formation at the site of inflammation in vivo and subsequent SOD activity is likely to be important for the mode of action of all Indo derivatives). The SOD activity may also be partly responsible for the markedly lower ulcerogenic activity observed by [Cu2(Indo)4(DMF)2]. In the present work, we have shown that the gastrointestinal toxicities associated with the administration of Zn-Indo or, more obviously, Cu-Indo are much lower than that of the noncomplexed IndoH and highlight the improved safety of these drugs. Furthermore, it is clear that the toxicity of the Cu complex is closely related to the form in which the drug is administered. EPR studies show that the most significant variation between formulations is the amount of monomeric Cu(II) and, consequently, the amount of free IndoH. There is no comparable technique available to measure the amount of monomeric Zn(II) present, so it is less clear as to the role(s) of the metal ion in this case.

Acknowledgment. The authors gratefully acknowledge an Australian Research Council (ARC) SPIRT grant and BVR for funding. We are also grateful for funding of the EPR instrumentation from an ARC RIEFP grant. The assistance of Drs. Ming Xie and Jane Weder in the EPR experiments is acknowledged gratefully.

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