Antinociception by fluoro-loxoprofen, a novel non-steroidal anti-inflammatory drug with less ulcerogenic effects, in rat models of inflammatory pain.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of inflammatory pain is limited by gastrointestinal complications. The rapid action of NSAIDs is associated with better pain relief. Previously, we demonstrated that fluoro-loxoprofen, a novel NSAID, has less ulcerogenic potential than other NSAIDs, attributable to its gastroprotective properties. The aim of this study was to investigate and compare the effects of fluoro-loxoprofen on inflammatory pain in rats with those of other NSAIDs. Oral administration of fluoro-loxoprofen, loxoprofen, and celecoxib resulted in equivalent analgesic action against yeast-induced inflammatory pain. The antinociceptive effect of fluoro-loxoprofen was maximized within 1 h after administration, which is less time than that observed for loxoprofen (2 h) and celecoxib (3 h). We confirmed that both fluoro-loxoprofen and loxoprofen suppressed the increases in prostaglandin E2 in inflamed paws. In addition to yeast-induced pain, fluoro-loxoprofen produced a similar effect against adjuvant-induced inflammatory pain, with faster peak analgesic effects than those observed for loxoprofen and celecoxib. Taken together, these results suggest that the analgesic effect of fluoro-loxoprofen is equivalent to that of loxoprofen and celecoxib. Moreover, the analgesic effect of fluoro-loxoprofen against inflammatory pain was more rapid than that of other NSAIDs, and this may be associated with its rapid absorption property.

Anethole restores delayed gastric emptying and impaired gastric accommodation in rodents.

Functional dyspepsia (FD), a functional gastrointestinal disorder, is characterized by persistent or recurrent postprandial upper abdominal discomfort and epigastric pain. The high prevalence of FD and associated healthcare costs suggests that treatment of this condition by methods other than prescribed medicines, such as natural products, could be beneficial. Delayed gastric emptying and impaired gastric accommodation play important roles in the development of FD. Anethole (1-methoxy-4-((E)-propenyl)-benzene), a major component of essential fennel oil, has been used as a flavoring, in alcoholic beverage production and in pharmaceutical formulations for many years. In this study, we examined the effects of anethole on delayed gastric emptying and impaired gastric accommodation in rodents. Oral administration of anethole improved clonidine-induced delayed gastric emptying but did not affect normal gastric emptying in mice. Fennel oil and Anchu-san (a Japanese herbal medicine containing anethole) also restored delayed gastric emptying. Furthermore, oral administration of anethole stimulated gastric accommodation in rats. These results suggest that anethole could be beneficial for the treatment of FD.

Aldioxa improves delayed gastric emptying and impaired gastric compliance, pathophysiologic mechanisms of functional dyspepsia.

Delayed gastric emptying and impaired gastric accommodation (decreased gastric compliance) play important roles in functional dyspepsia (FD). Here we screen for a clinically used drug with an ability to improve delayed gastric emptying in rats. Oral administration of aldioxa (dihydroxyaluminum allantoinate) partially improved clonidine- or restraint stress-induced delayed gastric emptying. Administration of allantoin, but not aluminium hydroxide, restored the gastric emptying. Both aldioxa and allantoin inhibited clonidine binding to the α-2 adrenergic receptor, suggesting that antagonistic activity of the allantoin moiety of aldioxa on this receptor is involved in the restoration of gastric emptying activity. Aldioxa or aluminium hydroxide but not allantoin restored gastric compliance with restraint stress, suggesting that aluminium hydroxide moiety is involved in this restoration. We propose that aldioxa is a candidate drug for FD, because its safety in humans has already been confirmed and its ameliorating effect on both of delayed gastric emptying and impaired gastric compliance are confirmed here.

Comparison of pharmacokinetics between loxoprofen and its derivative with lower ulcerogenic activity, fluoro-loxoprofen.

  We recently reported that, compared to loxoprofen (LOX, an non-steroidal anti-inflammatory drug), the LOX derivative fluoro-loxoprofen (F-LOX) is less ulcerogenic but has similar anti-inflammatory activity. Our previous in vitro studies suggested that both LOX and F-LOX are pro-drugs, the active metabolites of which are their trans-alcohol forms. In this study, we compared the pharmacokinetics of F-LOX and LOX in rats. Overall, the pharmacokinetic characteristics of F-LOX, including the formation of metabolites in vivo and in vitro, were comparable to those of LOX. However, F-LOX disappeared from the plasma more rapidly than LOX, which could potentially explain its lower ulcerogenicity. However, we showed that F-LOX produced fewer gastric lesions than LOX, even when a higher plasma concentration of F-LOX was maintained. Similar to LOX, F-LOX was readily metabolized to its trans- and cis-alcohol forms, with a higher level of the trans-alcohol form being observed after oral or intravenous administration of the drug. The preferential formation of the trans-alcohol form was also observed after incubation of F-LOX with rat liver homogenates in vitro. These results suggest that, similar to LOX, F-LOX acts as a pro-drug and that there is a metabolic system that selectively produces its active metabolite.

Identification of a unique nsaid, fluoro-loxoprofen with gastroprotective activity.

We previously proposed that direct cytotoxicity of NSAIDs due to their membrane permeabilization activity, together with their ability to decrease gastric prostaglandin E(2), contributes to production of gastric lesions. Compared to loxoprofen (LOX), fluoro-loxoprofen (F-LOX) has much lower membrane permeabilization and gastric ulcerogenic activities but similar anti-inflammatory activity. In this study, we examined the mechanism for this low ulcerogenic activity in rats. Compared to LOX, the level of gastric mucosal cell death was lower following administration of F-LOX. However, the gastric level of prostaglandin E(2) was similar in response to treatment with the two NSAIDs. Oral pre-administration of F-LOX conferred protection against the formation of gastric lesions produced by subsequent administration of LOX and orally administered F-LOX resulted in a higher gastric pH value and mucus content. In the presence of a stimulant of gastric acid secretion, the difference in the ulcerogenic activity of F-LOX and LOX was less apparent. Furthermore, an increase in the mucus was observed in gastric cells cultured in the presence of F-LOX in a manner dependent of increase in the cellular level of cAMP. These results suggest that low ulcerogenic activity of F-LOX involves its both low direct cytotoxicity and protective effect against the development of gastric lesions. This protective effect seems to be mediated through an increase in a protective factor (mucus) and a decrease in an aggressive factor (acid).

Synthesis and biological evaluation of derivatives of 2-{2-fluoro-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic acid: nonsteroidal anti-inflammatory drugs with low gastric ulcerogenic activity.

We previously reported that 2-fluoroloxoprofen has lower gastric ulcerogenic activity than loxoprofen, a nonsteroidal anti-inflammatory drug (NSAID) without selectivity for COX-2. We synthesized derivatives of 2-fluoroloxoprofen and studied their properties. Compared to 2-fluoroloxoprofen, one derivative, 11a, exhibited higher anti-inflammatory activity and an equivalent ulcerogenic effect. These results suggest that 11a could be therapeutically beneficial for use as an NSAID.

Effects of ß-(1,3-1,6)-D-glucan on irritable bowel syndrome-related colonic hypersensitivity.

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain associated with altered bowel habits. Since the prevalence of IBS is very high and thus, involves elevated health-care costs, treatment of this condition by methods other than prescribed medicines could be beneficial. ß-(1,3)-D-glucan with ß-(1,6) branches (ß-glucan) has been used as a nutritional supplement for many years. In this study, we examined the effect of ß-glucan on fecal pellet output and visceral pain response in animal models of IBS. Oral administration of ß-glucan suppressed the restraint stress- or drug-induced fecal pellet output. ß-Glucan also suppressed the visceral pain response to colorectal distension. These results suggest that ß-glucan could be beneficial for the treatment and prevention of IBS.

Stimulation of gastric ulcer healing by heat shock protein 70.

It is important in treatment of gastric ulcers to not only prevent further ulcer formation but also enhance ulcer healing. When cells are exposed to gastric irritants, expression of heat shock proteins (HSPs) is induced, making the cells resistant to the irritants. We recently reported direct evidence that HSPs, especially HSP70, are preventive against irritant-induced gastric ulcer formation. Gastric ulcer healing is a process involving cell proliferation and migration at the gastric ulcer margin and angiogenesis in granulation tissue. In this study, we have examined the role of HSP70 in gastric ulcer healing. Gastric ulcers were produced by focal and serosal application of acetic acid. Expression of HSP70 was induced in both the gastric ulcer margin and granulation tissue. Compared with wild-type mice, gastric ulcer healing was accelerated in transgenic mice expressing HSP70, and both cell proliferation at the gastric ulcer margin and angiogenesis in granulation tissue were enhanced. Oral administration of geranylgeranylacetone, an inducer of HSPs, to wild-type mice, either prior to or after ulcer formation, not only induced expression of HSP70 in the stomach but also accelerated gastric ulcer healing. On the other hand, oral administration of purified recombinant HSP70 prior to the ulcer formation, but not after formation, stimulated gastric ulcer healing. This study provides the first evidence that HSP70 accelerates gastric ulcer healing. The results also suggest that both the HSP70 produced prior to ulcer formation and released from damaged cells, and the HSP70 produced after ulcer formation are involved in this accelerated healing process.

Synthesis and biological evaluation of loxoprofen derivatives.

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory actions through an inhibitory effect on cyclooxygenase (COX). Two COX subtypes, COX-1 and COX-2, are responsible for the majority of COX activity at the gastrointestinal mucosa and in tissues with inflammation, respectively. We previously suggested that both gastric mucosal cell death due to the membrane permeabilization activity of NSAIDs and COX-inhibition at the gastric mucosa are involved in NSAID-induced gastric lesions. We have also reported that loxoprofen has the lowest membrane permeabilization activity among the NSAIDs we tested. In this study, we synthesized a series of loxoprofen derivatives and examined their membrane permeabilization activities and inhibitory effects on COX-1 and COX-2. Among these derivatives, 2-{4'-hydroxy-5-[(2-oxocyclopentyl)methyl]biphenyl-2-yl}propanoate 31 has a specificity for COX-2 over COX-1. Compared to loxoprofen, oral administration of 31 to rats produced fewer gastric lesions but showed an equivalent anti-inflammatory effect. These results suggest that 31 is likely to be a therapeutically beneficial and safer NSAID.

Properties and synthesis of 2-{2-fluoro (or bromo)-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic acid: nonsteroidal anti-inflammatory drugs with low membrane permeabilizing and gastric lesion-producing activities.

We previously proposed that membrane permeabilization activity of NSAIDs is involved in NSAID-induced gastric lesions. We here synthesized derivatives of loxoprofen that have lower membrane permeabilization activity than other NSAIDs. Compared to loxoprofen, the derivatives 10a and 10b have lower membrane permeabilization activity and their oral administration produced fewer gastric lesions but showed an equivalent anti-inflammatory effect. These results suggest that 10a and 10b are likely to be therapeutically beneficial as safer NSAIDs.

Suppression of expression of endoplasmic reticulum chaperones by Helicobacter pylori and its role in exacerbation of non-steroidal anti-inflammatory drug-induced gastric lesions.

Both the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, and infection with Helicobacter pylori are major causes of gastric ulcers. Although some clinical studies suggest that infection with H. pylori increases the risk of developing NSAID-induced gastric lesions, the molecular mechanism governing this effect is unknown. We recently found that in cultured gastric cells, expression of endoplasmic reticulum (ER) chaperones (such as 150-kDa oxygen-regulated protein (ORP150) and glucose-regulated protein 78 (GRP78)) is induced by NSAIDs and confers protection against NSAID-induced apoptosis, which is important in the development of NSAID-induced gastric lesions. In this study we have found that co-culture of gastric cells with H. pylori suppresses the expression of ER chaperones. This suppression was regulated at the level of transcription and accompanied by a reduction in the level of activating transcription factor 6 (ATF6), one of the transcription factors for ER chaperone genes. In vivo, inoculation of mice with H. pylori suppressed the expression of ER chaperones at gastric mucosa both with and without administration of indomethacin. Inoculation with H. pylori also stimulated formation of indomethacin-induced gastric lesions and mucosal cell death. In addition, we found that heterozygous ORP150-deficient mice are sensitive to the development of indomethacin-induced gastric lesions and mucosal cell death. The results of this study suggest that H. pylori exacerbates NSAID-induced gastric lesions through suppression of expression of ER chaperones, which stimulates NSAID-induced mucosal cell death.

Low direct cytotoxicity of loxoprofen on gastric mucosal cells.

Pro-drugs of non-steroidal anti-inflammatory drugs (NSAIDs), such as loxoprofen are widely used for clinical purposes because they are not so harmful to the gastrointestinal mucosa. We recently showed that NSAIDs such as indomethacin and celecoxib have direct cytotoxicity (ability to induce necrosis and apoptosis in gastric mucosal cells) due to their membrane permeabilizing activities, which is involved in NSAID-induced gastric lesions. We show here that under conditions where indomethacin and celecoxib clearly induce necrosis and apoptosis, loxoprofen and its active metabolite loxoprofen-OH, do not have such effects in primary culture of guinea pig gastric mucosal cells. Loxoprofen and loxoprofen-OH induced apoptosis more effectively in cultured human gastric cancer cells than in the primary culture. Loxoprofen and loxoprofen-OH exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We thus consider that the low direct cytotoxicity of loxoprofen observed in vitro is involved in its relative safety on production of gastric lesions in clinical situation.

A role for HSP70 in protecting against indomethacin-induced gastric lesions.

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E(2) for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin.

Inhibition of both COX-1 and COX-2 and resulting decrease in the level of prostaglandins E2 is responsible for non-steroidal anti-inflammatory drug (NSAID)-dependent exacerbation of colitis.

A number of clinical studies have shown that non-steroidal anti-inflammatory drugs (NSAIDs) exacerbate inflammatory bowel disease; however the molecular mechanism whereby this occurs remains unclear. NSAIDs inhibit cyclooxygenase (COX), which has subtypes COX-1 and COX-2. In this study, we have examined the effect of various types of NSAIDs on the development of dextran sulfate sodium (DSS)-induced colitis, an animal model of inflammatory bowel disease. The DSS-induced colitis was worsened by administration of non-selective NSAIDs but not by COX-1 or COX-2 selective inhibitors. However, administration of a combination of both COX-1- and COX-2-selective inhibitors exacerbated the colitis. The intestinal level of PGE(2) dramatically decreased in response to administration of COX-1- and COX-2-selective inhibitors, and exogenously administered PGE(2) suppressed the exacerbation of colitis by NSAIDs. The expression of mucin proteins, which protect the intestinal mucosa, was suppressed by non-selective NSAIDs and this expression was restored by PGE(2), both in vivo and in vitro. Intestinal mucosal cell growth was inhibited by non-selective NSAIDs and this cell growth was restored by PGE(2), both in vivo and in vitro. This study provides evidence that inhibition of both COX-1 and COX-2 and the resulting dramatic decrease in the intestinal level of PGE(2) is responsible for NSAID-dependent exacerbation of DSS-induced colitis. Furthermore, expression of mucin proteins and intestinal mucosal cell growth seems to be involved in this exacerbation and its suppression by PGE(2).