144
`
`WALLACE ET AL.
`DRUG DEVELOPMENT RESEARCH 42:144–149 (1997)
`
`Research Overview
`Gastrointestinal-Sparing Anti-Inflammatory Drugs:
`The Development of Nitric Oxide-Releasing NSAIDs
`John L. Wallace,1* Susan N. Elliott,1 Piero Del Soldato,2 Webb McKnight,1
`Franco Sannicolo,3 and Giuseppe Cirino4
`1Department of Pharmacology, The University of Calgary, Calgary, Alberta, Canada
`2NicOx Ltd., Paris, France
`3Department of Organic Chemistry, University of Milan, Milan, Italy
`4Department of Experimental Pharmacology, University of Naples, Naples, Italy
`
`Strategy, Management and Health Policy
`
`Venture Capital
`Enabling
`Technology
`
`Preclinical
`Research
`
`Preclinical Development
`Toxicology, Formulation
`Drug Delivery,
`Pharmacokinetics
`
`Clinical Development
`Phases I-III
`Regulatory, Quality,
`Manufacturing
`
`Postmarketing
`Phase IV
`
`Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed medi-
`ABSTRACT
`cations, but their use continues to be limited by significant toxicity, particularly in the gastrointestinal tract
`and kidney. Better understanding of the pathogenesis of these adverse effects has led to the development of
`a series of derivatives of standard NSAIDs that are not only less toxic but more efficacious. The coupling of
`a nitric oxide-releasing moiety to a range of NSAIDs greatly reduces their ability to induce gastrointestinal
`damage, and greatly increases their tolerability in situations in which there is preexisting gastrointestinal
`inflammation. There is also evidence that these compounds are much better tolerated by the kidney. On the
`other hand, the analgesic and anti-thrombotic properties of NO-releasing NSAIDs significantly exceed those
`of the parent drugs. These compounds appear to represent a significant advance in the treatment of
`inflammation and pain and for prophylaxis of thrombotic conditions. Drug Dev. Res. 42:144–149, 1997.
`© 1997 Wiley-Liss, Inc.
`
`Key words:
`
`anti-inflammatory; ulcer; analgesic; anti-thrombotic
`
`INTRODUCTION
`The class of drugs known as ‘‘NSAIDs’’ (nonsteroi-
`dal anti-inflammatory drugs) are among the most com-
`monly used medications [Garner, 1992]. They are
`prescribed largely for their anti-inflammatory, antipyretic,
`and analgesic properties but are also very widely used in
`over-the-counter preparations for the same indications.
`Moreover, aspirin is increasingly used on a long-term
`basis for its well-documented anti-thrombotic effects. The
`major limitation to the use of NSAIDs is their ability to
`cause ulceration and bleeding in the gastrointestinal
`tract [Soll et al., 1991]. This effect of NSAIDs has been
`recognized for decades [Douthwaite and Lintott,
`1938]. More recently, it has become apparent that
`NSAIDs can also exert significant toxicity in the kid-
`ney [Segasothy et al., 1994].
`Over the past 40 years, numerous new NSAIDs
`
`© 1997 Wiley-Liss, Inc.
`
`have been marketed with the claim that they reduce gas-
`trointestinal toxicity. These approaches have included
`enteric coating of the drug to prevent absorption in the
`stomach, formulation as a pro-drug, to prevent contact
`between the active drug and the gastric mucosa, and de-
`livery by non-oral routes. The basis for these approaches
`was the observation that some NSAIDs, particularly those
`which are acidic, can directly damage gastric epithelial
`cells. Reducing the topical irritancy should therefore pre-
`vent the epithelial damage and, in turn, prevent ulcer-
`ation and bleeding. However, each of these approaches
`has failed in terms of reducing the truly significant ad-
`
`*Correspondence to: Dr. John L. Wallace, Department of Phar-
`macology and Therapeutics, The University of Calgary, 3330 Hospi-
`tal Drive, NW, Calgary, AB T2N 4N1, Canada. E-mail:wallacej@
`acs.ucalgary.ca
`
`Page 1 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`NO-RELEASING NSAIDS
`
`145
`
`verse effects of NSAIDs in the gastrointestinal tract [Gra-
`ham, 1990]. This is now recognized as being attributable
`to the fact that a central component of the mechanism
`underlying NSAID-induced ulceration and bleeding is
`their ability to inhibit prostaglandin synthesis in the gas-
`trointestinal mucosa. Since the desired effects of NSAIDs
`are also dependent upon suppression of prostaglandin
`synthesis [Vane, 1971], delivery of the drug to the sys-
`temic circulation at a dose that is effective invariably re-
`sulted, in some patients, in development of ulcers.
`
`WHY NITRIC OXIDE?
`NSAIDs inhibit prostaglandin synthesis by inhib-
`iting the activity of the enzyme cyclo-oxygenase. Pros-
`taglandins play an important role in the gastrointestinal
`tract in that they mediate several components of mucosal
`defense (blood flow, mucus and bicarbonate secretion,
`mucosal immunocyte function) [Wallace and Tigley, 1995].
`Inhibition of gastrointestinal prostaglandin synthesis
`leads to decreased mucus and bicarbonate secretion,
`decreased blood flow, impaired repair of epithelial in-
`jury, and an increase in the number of leukocytes adher-
`ing to the vascular endothelium in the gastrointestinal
`microcirculation. It is this latter observation [Wallace,
`1993] that first suggested that nitric oxide (NO) might be
`capable of preventing NSAID-induced gastric injury. NO
`can inhibit leukocyte adherence to the vascular endot-
`helium [Kubes et al., 1991]. We had previously observed
`that prevention of NSAID-induced leukocyte adherence
`resulted in near-complete protection against gastric in-
`jury associated with these drugs in animals [Wallace et
`al., 1990, 1991, 1993]. Moreover, NO also exhibits many
`of the same actions in the stomach as prostaglandins;
`namely, stimulation of mucus secretion [Brown et al.,
`1992] and maintenance of mucosal blood flow [Whittle,
`1993]. Thus, it seemed logical that if NO could be deliv-
`ered in a controlled manner (i.e., slowly, so that systemic
`arterial blood pressure would not be affected), the detri-
`mental effects of NSAIDs in the gastrointestinal tract
`might be prevented (Fig. 1). The rate of delivery of NO,
`in terms of effectiveness in preventing NSAID-induced
`gastric damage, was underscored by our observations that
`some doses of standard NO donors (glyceryl trinitrate,
`sodium nitroprusside) were capable of reducing the ex-
`tent of gastric damage induced by an NSAID [Wallace et
`al., 1994c], but small increases in the dose led to an exac-
`erbation of injury (unpublished observations). Moreover,
`it was difficult to identify doses of these drugs which were
`effective in preventing gastric injury without altering
`systemic blood pressure. Nevertheless, the concept that
`NO was capable of reducing the severity of NSAID-in-
`duced gastric injury was proven [Wallace et al., 1994c].
`Protective effects of NO in other models of gastric injury
`had previously been demonstrated [MacNaughton et al.,
`
`Schematic diagram illustrating the rationale behind the develop-
`Fig. 1.
`ment of NO-releasing NSAIDs. As the compounds retain the ability to
`inhibit cyclo-oxygenase, they inhibit prostaglandin synthesis as effectively
`as the parent drugs. However, the release of NO counteracts the detri-
`mental effects of inhibition of prostaglandin synthesis in the gastrointesti-
`nal tract. The release of NO also appears to enhance the analgesic and
`anti-thrombotic efficacy of the drugs over that of the parent drugs.
`
`1989], but again, increasing the dose of the NO donor
`led to an exacerbation of injury and profound effects on
`systemic blood pressure [Lopez-Belmonte et al., 1993].
`
`NO-NSAIDS: REDUCED TOXICITY
`The hypothesis that NO-releasing derivatives of
`standard NSAIDs would have reduced toxicity has now
`been tested using derivatives of flurbiprofen, ketoprofen,
`naproxen, aspirin, and diclofenac [Wallace et al., 1994a,b,
`1995a, 1997; Davies et al., 1996]. When given as a single
`dose, all of the NO-NSAIDs have been shown to pro-
`duce significantly less gastric damage than standard
`NSAIDs [Wallace et al., 1994a,b, 1995a; Davies et al.,
`1996]. Figure 2 shows the effects of an NO-aspirin de-
`rivative (NCX-4016) in comparison to aspirin on the rat
`stomach. A similar reduction in toxicity is apparent if the
`compounds are given systemically rather than orally, in-
`dicating that the reduced injury observed with these com-
`pounds is not solely due to reduced ”topical irritant“
`properties. With repeated administration for up to three
`weeks, these compounds were also shown to produce sig-
`nificantly less gastrointestinal injury [Wallace et al., 1994a;
`Reuter et al., 1994; Davies et al., 1996].
`A major clinical problem associated with the use of
`NSAIDs is the ability of these drugs to interfere with the
`healing of preexisting ulcers. For this reason, we assessed
`the effects of NO-NSAIDs in three models. First, we stud-
`ied the effects of an NO-NSAID in a model of colitis in
`the rat [Reuter et al., 1994]. In this model, standard
`
`Page 2 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`146
`
`WALLACE ET AL.
`
`Extent of gastric damage induced in the rat stomach 3 h after
`Fig. 2.
`oral administration of aspirin or equimolar doses of an NO-aspirin de-
`rivative (NCX-4016). Aspirin caused significant damage at doses of 30
`mg/kg or greater (u = P < 0.05, uu = P < 0.01 vs. the vehicle-treated
`group), while NCX-4016 did not cause damage at any dose tested.
`
`NSAIDs cause a marked exacerbation of colonic injury,
`leading to perforation and death. For example, in the case
`of diclofenac, treatment for one week at doses of 1, 5,
`and 10 mg/kg led to the death of 29%, 86%, and 100% of
`the rats, respectively. In contrast, treatment with equimo-
`lar doses of nitrofenac resulted in markedly less mortal-
`ity (0%, 0%, and 33%, respectively). The second model
`was one in which the effect of nitrofenac on healing of
`gastric ulcers in the rat were examined [Elliott et al.,
`1995]. While daily diclofenac administration for a week
`caused a significant reduction in body weight gain and a
`marked decrease in hematocrit, nitrofenac did not alter
`either of these parameters in comparison to the vehicle-
`treated control group. Moreover, nitrofenac actually ac-
`celerated ulcer healing. The third model we utilized was
`one in which intestinal injury is established by treatment
`with indomethacin, and the rats are then switched to a
`different NSAID, to the corresponding NO-NSAID, or
`to vehicle. This model mimics the clinical situation in
`which a patient develops gastrointestinal damage while
`taking an NSAID, and has to be switched to a second
`NSAID in the hope that it will be less injurious. The re-
`sults from one study performed with this model are shown
`in Figure 3. Intestinal injury was induced by two subcu-
`taneous injections of indomethacin (7.5 mg/kg) 24 h apart.
`The rats were left for 48 h, after which they were ran-
`domized to receive naproxen (20 mg/kg), an equimolar
`dose of NO-naproxen, or vehicle. The test drugs or ve-
`hicle were administered orally every 12 h for 4 days, af-
`ter which time the degree of intestinal ulceration was
`blindly scored. Indomethacin induced widespread intes-
`tinal ulceration. Considerable healing was observed in
`the group subsequently treated with vehicle. In contrast,
`
`Effects of naproxen and NO-naproxen on healing of intestinal
`Fig. 3.
`ulcers induced by indomethacin. Indomethacin was injected subcutane-
`ously (7.5 mg/kg) twice, separated by 24 h. 48 h after the second in-
`domethacin injection (day 3), the rats were randomized to receive
`naproxen, an equimolar dose of NO-naproxen, or vehicle. These drugs
`were given orally every 12 h for 4 days. 12 h after the final dose, the
`damage was scored. * = P < 0.05 compared to the vehicle-treated group.
`
`naproxen significantly delayed healing. On the other
`hand, NO-naproxen, which suppresses prostaglandin
`synthesis in the intestine to the same extent as naproxen,
`did not cause any retardation of ulcer healing.
`The fact that gastrointestinal toxicity was not ob-
`served following repeated administration of the NO-
`NSAIDs over periods of up to three weeks further
`supports the contention that these derivatives are not
`merely pro-drugs. Moreover, recent pharmacokinetic
`studies have demonstrated that the intact NO-NSAIDs
`can be detected in the plasma of rats for many hours af-
`ter administration [Davies et al., 1996; Reuter et al., 1997].
`Despite evidence that these agents generate NO in the
`rat in vivo, they do not significantly affect systemic blood
`pressure [Wallace et al., 1994a,b, 1995a].
`There is also evidence suggesting that NO-NSAIDs
`spare the renal system. We tested the effects of administra-
`tion of an NSAID (diclofenac) or the NO-releasing deriva-
`tive (nitrofenac) on renal blood flow in both healthy and
`cirrhotic rats. In both subgroups, diclofenac caused a sig-
`nificant reduction in renal blood flow, while nitrofenac had
`no effect. The effects of the two drugs on renal blood flow
`in healthy rats are shown in Figure 4. As the renal toxicity
`of NSAIDs is believed to be attributable to reduced blood
`flow secondary to suppression of prostaglandin synthesis
`[Segasothy et al., 1994], these results suggest that NO-
`NSAIDs may not produce the renal toxicity associated with
`the parent NSAIDs. This conclusion is supported by re-
`cent studies in which NO-NSAIDs were found to be sig-
`nificantly better tolerated in a rat model of kidney failure
`than native NSAIDs [Fujihara et al., 1995].
`
`Page 3 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`NO-RELEASING NSAIDS
`
`147
`
`generate NO when incubated with platelets, and to
`cause a significant increase in platelet cyclic GMP
`levels [Wallace et al., 1995a, 1997a]. Indeed, there is
`strong evidence that a significant component of the
`anti-platelet effects of NO-aspirin derivatives is due
`to NO generation [Wallace et al., 1995a]. These com-
`pounds also have the ability to suppress neutrophil
`adherence to the vascular endothelium [Wallace et al.,
`1997a], which might add to their anti-thrombotic ef-
`fects. This raises the possibility that an NO-releasing
`aspirin derivative may have utility in long-term use
`for the prevention of myocardial infarction and stroke.
`Aspirin is presently used for these indications [SALT
`Collaborative Group, 1991; Meade et al., 1992;
`Patrono, 1994], but despite the fact that low doses of
`this drug are used, there is still a significant increase
`in the incidence of gastrointestinal bleeding and other
`hemostatic complications [SALT Collaborative Group,
`1991; Meade et al., 1992; Cryer et al., 1995].
`
`NITRATE VS. NITRITE: A CRITICAL DIFFERENCE
`Several options existed with respect to the NO-
`releasing moiety to link to NSAIDs. For example, nitrites,
`nitrates, and S-thiol-glutathione are all well-characterized
`NO donors [Moncada et al., 1991]. The selection of a nitrate
`group was based on two key issues: stability and toxicity.
`Stability of a flurbiprofen nitrite ester and a
`flurbiprofen nitrate ester were compared using nuclear
`magnetic resonance. When dissolved in dimethylsulfox-
`ide and incubated at 20°C for 1 h, the nitrite-containing
`compound was found to undergo 25% hydrolysis, while
`no hydrolysis of the nitrate-containing compound oc-
`curred. Moreover, the native nitrate-containing com-
`pound was found to be stable at room temperature for
`more than a year.
`In terms of toxicity, the primary concern in choos-
`ing an appropriate NO-releasing group was mutagenic-
`ity. The Ames test, which measures the potential of a test
`substance to induce point mutations in Salmonella
`typhimurium, was used to compare the effects of sodium
`nitrite, sodium nitrate, S-nitroso-glutathione, and four
`nitrate-containing NO-NSAIDs (derivatives of flurbi-
`profen, aspirin, naproxen, and diclofenac). In this assay,
`mutagenic substances induce reversion of a histidine-
`deficient strain of S. typhimurium such that it is then able
`to grow and form colonies in a histidine-limited medium.
`Concentrations up to 5000 m g per plate of each drug were
`tested. As illustrated in Figure 5, sodium nitrite caused a
`significant increase in the number of revertants (over that
`seen with vehicle) at a concentration as low as 312.5 m g/
`plate, while S-nitroso-glutathione induced a positive re-
`sponse at ‡ 1250 m g/plate. On the other hand, sodium ni-
`trate and all four of the nitrate-containing NO-NSAIDs
`
`Effects of diclofenac (10 mg/kg) and an equimolar dose of its
`Fig. 4.
`NO-releasing derivative, nitrofenac, on renal blood flow in healthy rats.
`u = P < 0.05 compared to the corresponding basal period.
`
`NO-NSAIDS: EFFICACY
`Several of the NO-NSAIDs have been compared
`to the parent drugs in models of acute and chronic in-
`flammation [Wallace et al., 1994a,b; Cuzzolin et al., 1995;
`Davies et al., 1996]. In each case, these compounds were
`found to reduce inflammation at least as effectively as
`the parent NSAID. The NO-releasing diclofenac deriva-
`tive has also been shown to have virtually identical
`anti-pyretic activity to diclofenac [Wallace et al.,
`1995b]. The NO-naproxen derivative has significantly
`enhanced analgesic activity over the parent drug
`[Davies et al., 1996], while an NO-aspirin derivative
`was found to have markedly increased anti-thrombotic
`properties [Wallace et al., 1995a].
`The ability of NO-NSAIDs to suppress inflam-
`mation, pain, and fever is not surprising given the ob-
`servation that these derivatives retain the ability of
`the parent drugs to inhibit prostaglandin synthesis
`(with the exception of the NO-aspirin derivatives; see
`below) [Wallace et al., 1994a,b; Mitchell et al., 1994].
`The NO-NSAIDs themselves inhibit cyclo-oxygenase
`activity (types 1 and 2) without being metabolized,
`indicating that these derivatives are not just pro-drugs
`[Mitchell et al., 1994]. In the case of the NO-aspirin
`derivatives, however, the activity on cyclo-oxygenase
`activity is greatly reduced, perhaps contributing to the
`profoundly reduced gastric toxicity of these drugs
`[Wallace et al., 1995a]. Both NCX-4215 and NCX-4016
`have been shown to have weak inhibitory activity on
`platelet thromboxane synthesis [Wallace et al., 1995a,
`1997b]. With repeated dosing over several days, how-
`ever, the level of inhibition of thromboxane synthesis
`increases to levels comparable to equimolar doses of
`aspirin [Wallace et al., 1997b]. However, this weak
`effect on thromboxane synthesis does not account for
`the anti-thrombotic properties of the two NO-aspirin
`derivatives. These compounds have been shown to
`
`Page 4 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`148
`
`WALLACE ET AL.
`
`Effects of sodium nitrate, sodium nitrite, and S-nitroso-glutathione
`Fig. 5.
`on mutations of S. typhimurium. Results are shown as the number of
`
`revertants per plate following exposure to the test substances at a range
`of concentrations. * = P < 0.05 compared to the vehicle-treated group.
`
`failed to induce a positive response at any of the concen-
`trations tested.
`The differences in mutagenicity of nitrate- vs. ni-
`trite-containing compounds can also be considered in the
`context of the amounts of these substances likely to be
`ingested in comparison to World Health Organization
`recommendations [JECFA, 1996]. We calculated the
`amount of nitrate and nitrite that would be ingested per
`day based on maximum daily doses of the parent NSAID.
`These calculations were performed for nitrite- and ni-
`trate-containing NO-releasing derivatives of diclofenac,
`aspirin, flurbiprofen, naproxen, and ketoprofen. In the
`case of nitrite-containing NO-NSAIDs, ingestion of any
`of the compounds would result in nitrite ingestion far in
`excess of WHO recommendations. For example, with
`NO-diclofenac, NO-aspirin, and NO-naproxen, the ni-
`trite consumption would exceed WHO recommended
`maximal levels by threefold, tenfold, and 40-fold, respec-
`tively. On the other hand, in none of the five cases exam-
`ined would the amount of nitrate ingested with
`nitrate-containing NO-NSAIDs exceed WHO recom-
`mendations. For example, the amount of nitrate ingested
`with maximal doses of NO-diclofenac and NO-flurbi-
`profen would be 1/25th and 1/7th of the WHO recom-
`mended maximal levels, respectively.
`
`SUMMARY
`NO-releasing NSAIDs were developed on the ba-
`sis of observations that suggested that NO had the ca-
`pacity to interfere with several key steps in the
`pathogenesis of NSAID-induced gastrointestinal ulcer-
`ation. This novel class of compounds exhibits greatly re-
`duced gastrointestinal and renal toxicity. NO-NSAIDs
`exhibit reduced toxicity not only in the normal gas-
`trointestinal tract, but also in situations in which there
`
`was preexisting ulceration and inflammation. Moreover,
`these compounds have comparable anti-inflammatory and
`anti-pyretic activity to the parent drugs, but have en-
`hanced analgesic and anti-thrombotic properties. Nitrate-
`containing NO-NSAIDs have superior stability to
`nitrite-containing NO-NSAIDs. Moreover, the nitrate-
`containing NO-NSAIDs do not exhibit the mutagenicity
`seen with nitrite-containing NO-NSAIDs. Clinical trials
`are presently under way to examine the safety and effi-
`cacy of several NO-NSAIDs in humans.
`
`REFERENCES
`Brown JF, Hanson PJ, Whittle BJR (1992): Nitric oxide donors increase
`mucus gel thickness in rat stomach. Eur J Pharmacol 223:101–104.
`Cryer B, Luk G, Feldman M (1995): Effects of very low doses of aspi-
`rin (ASA) on gastric, duodenal and rectal prostaglandins (PGs) and
`mucosal injury (Abstract). Gastroenterology 108:A77.
`Cuzzolin L, Conforti A, Adami A, Lussignoli S, Memestrina F, Del
`Soldato P, Benoni G (1995): Anti-inflammatory potency and gas-
`trointestinal toxicity of a new compound, NO-naproxen. Pharmacol
`Res 31:61–65.
`Davies NM, Roseth AG, Appleyard CB, McKnight W, Del Soldato P,
`Calignano A, Cirino G, Wallace JL (1996): NO-naproxen vs.
`naproxen: Ulcerogenic, analgesic and anti-inflammatory effects.
`Aliment Pharmacol Ther 11: 69–79.
`Douthwaite AH, Lintott SAM (1938): Gastroscopic observation of the
`effect of aspirin and certain other substances on the stomach. Lan-
`cet 2:1222–1225.
`Elliott SN, McKnight W, Cirino G, Wallace JL (1995): A nitric oxide-
`releasing nonsteroidal anti-inflammatory drug accelerates gastric
`ulcer healing in the rat. Gastroenterology 109:524–530.
`Fujihara CK, Malheiros DMAC, Antunes E, Donato JL, Santos MM,
`Sena CR, De Nucci G, Zatz R (1995): Nitroflurbiprofen, a novel
`nonsteroidal anti-inflammatory drug, limits glomerular injury in the
`remnant model (Abstract). J Am Soc Nephrol 6:1012.
`Garner A (1992): Adaptation in the pharmaceutical industry, with par-
`ticular reference to gastrointestinal drugs and diseases. Scand J
`Gastroenterol 27(193):83–89.
`
`Page 5 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`NO-RELEASING NSAIDS
`
`149
`
`Graham DY (1990): The relationship between nonsteroidal anti-inflam-
`matory drug use and peptic ulcer disease. Gastroenterol Clin North
`Am 19:171–182.
`JECFA (1996): Toxicological evaluation of certain food additives and
`contaminants. 44th Meeting of the Joint FAO/WHO Expert Com-
`mittee on Food Additives, International Programme on Chemical
`Safety, World Health Organization.
`Kubes P, Suzuki M, Granger DN (1991): Nitric oxide: An endog-
`enous modulator of leukocyte adhesion. Proc Natl Acad Sci USA
`88:4651–4655.
`Lopez-Belmonte J, Whittle BJR, Moncada S (1993): The actions of
`nitric oxide donors in the prevention or induction of injury to the
`rat gastric mucosa. Br J Pharmacol 108:73–78.
`MacNaughton WK, Cirino G, Wallace JL (1989): Endothelium-derived
`relaxing factor (nitric oxide) has protective actions in the stomach.
`Life Sci 45:1869–1876.
`Meade TW, Roderick PJ, Brennan PJ, Wilkes HC, Kelleher CC
`(1992): Extracranial bleeding and other symptoms due to low
`dose aspirin and low intensity oral anticoagulation. Thromb
`Haemost 68:1–6.
`Mitchell JA, Cirino G, Akarasereenont P, Wallace JL, Flower RJ, Vane
`JR (1994): Flurbinitroxybutylester: A novel anti-inflammatory drug
`devoid of ulcerogenic activity, inhibits cyclo-oxygenase-1 and cyclo-
`oxgenase-2. Can J Physiol Pharmacol 72:270.
`Moncada S, Palmer RMJ, Higgs EA (1991): Nitric oxide: Physiology,
`pathophysiology, and pharmacology. Pharmacol Rev 43:109–142.
`Patrono C (1994): Aspirin as an antiplatelet drug. N Engl J Med
`330:1287–1294.
`Reuter BK, Cirino G, Wallace JL (1994): Markedly reduced intestinal
`toxicity of a diclofenac derivative. Life Sci 55:PL1–PL8.
`Reuter BK, Davies NM, Wallace JL (1997): Nonsteroidal anti-inflam-
`matory drug-enteropathy in rats: Role of permeability, bacteria and
`enterohepatic circulation. Gastroenterology 112:109–117.
`The SALT Collaborative Group (1991): Swedish Aspirin Low-dose Trial
`(SALT) of 75 mg aspirin as secondary prophylaxis after cerebrovas-
`cular ischaemic events. Lancet 338:1345–1349.
`Segasothy M, Samad SA, Zulfigar A, Bennett WM (1994): Chronic
`renal disease and papillary necrosis associated with the long-term
`use of nonsteroidal anti-inflammatory drugs as the sole or predomi-
`nant analgesic. Am J Kidney Dis 24:17–24.
`Soll AH, Weinstein WM, Kurata J, McCarthy D (1991): Nonsteroidal
`anti-inflammatory drugs and peptic ulcer disease. Ann Intern Med
`114:307–319.
`
`Vane JR (1971): Inhibition of prostaglandin synthesis as a mechanism
`of action for aspirin-like drugs. Nat New Biol 231:232–235.
`Wallace JL (1993): Gastric ulceration: Critical events at the neutro-
`phil-endothelium interface. Can J Physiol Pharmacol 71:98–102.
`Wallace JL, Tigley AW (1995): New insights into prostaglandins and
`mucosal defence. Aliment Pharmacol Ther 9:227–235.
`Wallace JL, Keenan CM, Granger DN (1990): Gastric ulceration in-
`duced by nonsteroidal anti-inflammatory drugs is a neutrophil-de-
`pendent process. Am J Physiol 259:G462–G467.
`Wallace JL, Arfors K-E, McKnight GW (1991): A monoclonal anti-
`body against the CD18 leukocyte adhesion molecule prevents in-
`domethacin-induced gastric damage in the rabbit. Gastroenterology
`100:878–883.
`Wallace JL, McKnight W, Miyasaka M, Tamatani T, Paulson J, Ander-
`son DC, Granger DN, Kubes P (1993): Role of endothelial adhesion
`molecules in NSAID-induced gastric mucosal injury. Am J Physiol
`265:G993–G998.
`Wallace JL, Reuter B, Cicala C, McKnight W, Grisham MB, Cirino
`G (1994a): Novel nonsteriodal anti-inflammatory drug derivatives
`with markedly reduced ulcerogenic properties in the rat. Gastro-
`enterology 107:173–179.
`Wallace JL, Reuter B, Cicala C, McKnight W, Grisham MB, Cirino G
`(1994b): Adiclofenac derivative without ulcerogenic properties. Eur
`J Pharmacol 257:249–255.
`Wallace JL, Reuter BK, Cirino G (1994c): Nitric oxide releasing
`NSAIDs: A novel approach for reducing gastropathy. J Gastroenterol
`Hepatol 9:S40–S44.
`Wallace JL, McKnight W, Del Soldato P, Cirino G (1995a): Anti-throm-
`botic properties of a nitric oxide-releasing, gastric sparing aspirin
`derivative. J Clin Invest 96:2711–2718.
`Wallace JL, Pittman QJ, Cirino G (1995b): Nitric oxide-releasing
`NSAIDs: A novel class of GI-sparing anti-inflammatory drugs.
`Agents Actions 46:S121–S129.
`Wallace JL, McKnight W, Wilson TL, Del Soldato P, Cirino G (1997a):
`Reduction of shock-induced gastric damage by a nitric oxide-releasing
`aspirin derivative: Role of neutrophils. Am J Physiol 273:G1246–1251.
`Wallace JL, McKnight W, Wilson TL, Cirino G, Del Soldato P, Davies
`NM (1997b): Nitric oxide releasing aspirin derivative (NCX 4016):
`A gastric-sparing anti-thrombotic drug (Abstract). Gastroenterol-
`ogy 112:A325.
`Whittle BJR (1993): Neuronal and endothelium-derived mediators in
`the modulation of the gastric microcirculation: Integrity in the bal-
`ance. Br J Pharmacol 110:3–17.
`
`Page 6 of 6
`
`Patent Owner Ex. 2063
`Mylan v. Pozen
`IPR2017-01995
`
`