In vitro metabolism considerations, including activity testing of metabolites, in the discovery and selection of the COX-2 inhibitor etoricoxib (MK-0663).

Characterization of the metabolites of the COX-2 inhibitor etoricoxib (MK-0663 and L-791,456) produced in vitro indicate formation of an N-oxide pyridine and hydroxymethyl pyridine that can further be glucuronidated or oxidized to an acid. Significant turnover is observed in human hepatocytes. Several CYPs are involved in the oxidative biotranformations and, from in vitro studies, etoricoxib is not a potent CYP3A4 inducer or inhibitor. Based on an in vitro whole blood assay, none of the metabolites of etoricoxib inhibits COX-1 or contributes significantly to the inhibition of COX-2.

In vitro metabolism of the COX-2 inhibitor DFU, including a novel glutathione adduct rearomatization.

The metabolic profile of DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone], a potent and selective COX-2 inhibitor, was characterized using in vitro microsomal and hepatocyte incubations. A single product, corresponding to p-hydroxylation, p-OH-DFU [(5,5-dimethyl-3-(3-fluoro-4-hydroxyphenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone)], was produced in rat microsomal incubations of DFU. In contrast, three metabolites were produced in incubations using suspensions of freshly isolated rat hepatocytes. Microsomal production of the p-O-glucuronide metabolite of DFU from synthetic p-OH-DFU was shown to have chromatographic and mass spectrometric properties identical to the earliest eluting hepatocyte metabolite (M1). The molecular weights of the other two hepatocyte metabolites were readily obtained using capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (HPLC/CF-LSIMS); however, the elemental composition of these metabolites was not. Unlike typical metabolic products, which produce readily identified increments in molecular weight, metabolites M2 and M3 produced molecular ions in positive- and negative-ion CF-LSIMS that were consistent with oxidation of DFU (+16 Da), followed by addition of glutathione (+306 Da) and subsequent loss of 20 and 18 Da, respectively. Capillary HPLC/high-resolution CF-LSIMS was used to generate accurate mass data for M2 and M3 that provided evidence that the losses of 20 and 18 Da, respectively, corresponded to a rearomatization through loss of HF or H(2)O. Isolation and NMR characterization provided the definitive structural proof for these metabolites. Overall, the metabolism of DFU in rat hepatocytes is proposed to proceed through an epoxide intermediate, which then either rearranges to the p-OH-DFU and is conjugated with glucuronic acid, or is trapped with glutathione, followed by rearomatization with loss of HF (M2) or H(2)O (M3).

Investigation of the in vitro metabolism profile of a phosphodiesterase-IV inhibitor, CDP-840: leading to structural optimization.

CDP-840 is a selective and potent phosphodiesterase type IV inhibitor, whose in vitro metabolism profile was first investigated using liver microsomes from different species. At least 10 phase I oxidative metabolites (M1-M10) were detected in the microsomal incubations and characterized by capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS). Significant differences in the microsomal metabolism of CDP-840 were found between rat and other species. The major route of metabolism in rat involved para-hydroxylation on the R4 phenyl. This pathway was not observed in human and several other species. The in vitro metabolism profile of CDP-840 was further examined using freshly isolated hepatocytes from rat, rabbit, and human. The hepatocyte incubations indicated more extensive metabolism relative to that in microsomes. In addition to the phase I oxidative metabolites observed in microsomal incubations, several phase II conjugates were identified and characterized by CF-LSIMS. Interspecies differences in phase II metabolism were also found in these hepatocyte incubations. The major metabolite in human hepatocytes was identified as the pyridinium glucuronide, which was not detected in rat hepatocytes. Simple structural modification on R4, such as p-Cl substitution, greatly reduced the species differences in microsomal metabolism. Furthermore, modifications on R3, such as the N-oxide, eliminated the N-glucuronide formation in human. These results not only helped in determining the suitability of animal species used in the preclinical safety studies but also provided valuable directions for the synthetic efforts in finding backup compounds that are more metabolically stable.

Synthesis, characterization, and activity of metabolites derived from the cyclooxygenase-2 inhibitor rofecoxib (MK-0966, Vioxx).

Metabolites of the COX-2 inhibitor rofecoxib (MK-0966, Vioxx) were prepared by synthetic or biosynthetic methods. Metabolites include products of oxidation, glucuronidation, reduction and hydrolytic ring opening. Based on an in vitro whole blood assay, none of the known human metabolites of rofecoxib inhibits COX-1 nor contributes significantly to the inhibition of COX-2.

CD3zeta and CD28 down-modulation on CD8 T cells during viral infection.

Down-modulation of CD3zeta expression on CD8 T lymphocytes occurs, independently of other T-cell receptor (TCR)-CD3 components, in tumor-infiltrating lymphocytes, human immunodeficiency virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3zeta down-modulation was found, however, in CD8 T cells that proliferate in response to acute viral infections. In 3 otherwise healthy donors with acute gastroenteritis, infectious mononucleosis, and Epstein-Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3zeta to below the level of detection. The CD3zeta-T cells were also CD28- but expressed the activation markers HLA-DR and CD57. CD3zeta-CD28- T cells are effector CTL because they express perforin and produce IFN-gamma, but not IL-2, on activation and contain the viral-specific cytotoxic T lymphocyte (CTL). However, CD3zeta-CD28-T cells generally do not express CD25 after anti-CD3 and anti-CD28 stimulation and are not cytotoxic until they are cultured with IL-2 overnight. Cytotoxicity coincides with the re-expression of CD3zeta but not CD28. Down-modulation of CD3zeta and CD28 on effector CTL may control CTL triggering and proliferation to prevent immunopathogenesis.

Human immunodeficiency virus-specific circulating CD8 T lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation.

Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3zeta, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3zeta down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3zeta-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3zeta(-). CD8 T cells with down-modulated CD3zeta also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR(+) CD62L(-)). After T-cell activation, CD3zeta-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor alpha-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3zeta is not reexpressed even after IL-2 exposure.

Clonal expansion of antigen-specific CD8+ cytotoxic T lymphocytes is regulated by late exposure to serum to prevent apoptosis.

Although serum-free media have been used to expand lymphokine-activated killer cells, antigen-specific CD8 T cell cytotoxicity does not develop in vitro in the absence of serum. The immunodominant Vbeta17 response to an influenza A matrix protein epitope restricted by HLA A2.1 was used to study the serum requirement for CTL activation. Serum acts directly on T cells and not indirectly by activating APCs. In the absence of serum, the initial steps of T cell activation, including expression of CD69 and CD25, are unimpaired and some antigen-specific cytotoxicity may be generated in the first few days after stimulation. However, expression of late activation markers, such as HLA-DR and CD38, and clonal expansion of class I-restricted antigen-specific CTL does not occur if CTL are not exposed to serum within 4 days of antigen exposure. The antigen-specific CTL, but not unstimulated bystander T cells, undergo apoptosis if they are not exposed to serum within a few days of activation. Apoptosis of TCR-activated CTL does not appear to be Fas-mediated since it is not blocked by inhibiting the Fas pathway. Therefore, late exposure to an unidentified serum protein regulates the clonal expansion of TCR-activated CD8 CTL.

Expansion of CD57 and CD62L-CD45RA+ CD8 T lymphocytes correlates with reduced viral plasma RNA after primary HIV infection.

OBJECTIVE: CD8 T cells, expressing cell surface molecules distinct from those on resting and naive T cells, are increased in HIV infection. The association of increased CD38 and human leukocyte antigen DR (HLA-DR) CD8 T cells with poor prognosis has suggested that activated CD8 T cells may aggravate HIV infection. We examined whether other immunological parameters might influence the viral setpoint. DESIGN: Peripheral T cells from nine untreated patients, obtained after primary HIV infection when plasma HIV had stabilized, were examined for proteins expressed in activated versus resting, memory versus naive, and cytolytic versus non-cytolytic T cells. METHODS: The proportion of CD8 T cells that stain for CD38 and HLA-DR, CD28 and CD57 was compared with plasma viraemia and CD4 cell count. These parameters were also compared with the proportion of CD4 and CD8 T cells that express CD62L and CD45RA, present on naive cells and down-modulated in memory cells. Internal staining for the cytotoxic protein granzyme A was also examined. RESULTS: An increase in CD38 and CD38 HLA-DR CD8 T cells correlated with increased plasma viral RNA (P < 0.00002, P < 0.03, respectively). An increase in CD8 T cells expressing granzyme A was associated with lower CD4 cell counts (P < 0.04). However, the expansion of CD57 and CD62L CD45RA+ CD8 T cells was associated with a lower viral setpoint (P < 0.01, P < 0.02, respectively). CONCLUSION: Phenotypically defined activated CD8 T cells may have different functions in HIV infection. Activated CD8 T cells that are CD57 or CD62L(-)CD45RA+ may be beneficial, because their expansion in untreated patients correlates with a reduced viral setpoint after primary infection.

Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death.

Misselected CD8 cells that express T cell receptors (TCRs) that do not recognize class I major histocompatibility complex (MHC) protein can emerge from thymic selection. A postthymic quality control mechanism that purges these cells from the repertoire is defined here. The failure of mature CD8 cells to simultaneously engage their TCR and CD8 coreceptor triggers an activation process that begins with inhibition of CD8 gene expression through remethylation and concludes with up-regulation of surface Fas and Fas ligand and cellular apoptosis. Thus, inhibition of a death signal through continued TCR-CD8 coengagement of MHC molecules is a key checkpoint for the continued survival of correctly selected T cells. Molecular defects that prevent delivery of the death signal to mistakenly selected T cells underlie the expansion of double-negative T cells, which is the cellular signature of a subset of systemic autoimmune diseases.

Circulating CD8 T lymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex.

Although human immunodeficiency virus (HIV)-infected subjects without acquired immunodeficiency syndrome have a high frequency of HIV-specific CD8 T lymphocytes, freshly isolated lymphocytes frequently lack detectable HIV-specific cytotoxicity. However, this effector function becomes readily apparent after overnight culture. To investigate reasons for T-cell dysfunction, we analyzed T-cell expression of the cytolytic protease granzyme A and CD3 zeta, the signaling component of the T-cell receptor complex. An increased proportion of CD4 and CD8 T cells from HIV-infected donors contain granzyme A, consistent with the known increased frequency of activated T cells. In 28 HIV-infected donors with mild to advanced immunodeficiency, a substantial fraction of circulating T cells downmodulated CD3 zeta (fraction of T cells expressing CD3 zeta, 0.74 +/- 0.16 v 1.01 +/- 0.07 in healthy donors; P < .0000005). CD3 zeta expression is downregulated more severely in CD8 than CD4 T cells, decreases early in infection, and correlates with declining CD4 counts and disease stage. CD3 zeta expression increases over 6 to 16 hours of culture in an interleukin-2-dependent manner, coincident with restoration of viral-specific cytotoxicity. Impaired T-cell receptor signaling may help explain why HIV-specific cytotoxic T lymphocytes fail to control HIV replication.

The metabolic effects of pokeweed mitogen in mice.

Lectins are a family of proteins that stimulate cellular responses after binding to carbohydrate chains on plasma membranes. In the study described here, a mixture of lectins--pokeweed mitogen (PKW)--was shown to have insulinomimetic effects in mice. After receiving PKW (15 mg/kg intraperitoneally [IP]), serum glucose declined from 154 +/- 3 to 23 +/- 10 mg/dL by 24 hours later. Anorexia developed, and by 3 days, there was a significant decline in body weight. Carcass weights were 10% lower, and epididymal fat pad weights were 45% lower. When given for 16 days, PKW 3 mg/kg every other day caused a sustained 10% weight loss. Severe combined immune deficiency (SCID) mice were sensitive to PKW, showing that B and T lymphocytes were not required for the effects to develop. Cytokine antagonists attenuated the hypoglycemia and anorexia, but only by 50%. Further study showed that PKW has insulin-like effects in vitro. Glucose uptake was stimulated when murine C2C12 myotubes were exposed to an enriched fraction of PKW. These results demonstrated that PKW has both insulin-like activity and weight-reducing effects when administered to mice. The development of therapy for adult-onset diabetes or obesity based on lectins from pokeweed may be possible.

Verlukast (MK-0679) conjugation with glutathione by rat liver and kidney cytosols and excretion in the bile.

Verlukast (MK-0679) is a potent leukotriene D4 antagonist that was under development for the treatment of bronchial asthma. A previously uncharacterized metabolite of verlukast was formed in incubations using rat liver cytosol fortified with glutathione (GSH). The metabolite was detected by HPLC and characterized by UV spectroscopy (photodiode array detection after HPLC) and capillary HPLC continuous flow-liquid secondary-ion mass spectrometry. After a large-scale incubation and isolation, it was further characterized by 500 MHz proton NMR. The metabolite is a 1,4-Michael addition product in which GSH has added to position 12 of the styryl quinoline double bond of verlukast. There is no apparent stereoselectivity because a mixture of the two possible isomers, in equal amounts, was observed by NMR. Although there was spontaneous chemical addition of GSH to verlukast (0.18 nmol/min), the reaction was shown to be enzyme-catalyzed in studies using three different preparations of rat liver cytosol at pH 7.4. Using Lineweaver-Burk analysis of experiments in which the effect of verlukast concentration on the rate of conjugation was studied, the apparent KM and Vmax were determined to be 107 +/- 22 microM (SD, N=3) and 0.66 +/- 0.21 nmol/min/mg protein, respectively. In similar studies with GSH as the variable substrate, the apparent KM and Vmax were 2.32 +/- 0.68 mM and 0.69 +/- 0.14 nmol/min/mg protein, respectively. Incubations with kidney cytosol produced the GSH, cysteinylglycine, and cysteine conjugates of verlukast. In bile collected from rats dosed intravenously with 50 mg/kg of verlukast, approximately 80% of the dose was recovered up to 4 hr postdose. The GSH conjugate accounted for 16.5% of the dose. The cysteinylglycine, cysteine, and N-acetylcysteine conjugates were observed and together accounted for 7.5%. Verlukast accounted for 14.5%, and the remainder of the metabolites (40.5%) were oxidation or acyl glucuronide metabolites.

Integrated application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry to discovery stage metabolism studies.

The application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) as part of an integrated approach for characterizing discovery stage in vitro metabolites, using a specific inhibitor for 5-lipoxygenase as a model compound, was investigated. CF-LSIMS demonstrated excellent sensitivity in detecting the metabolites in both the positive and the negative ion modes, with a good full-scan mass spectrum obtained when 5 pmol of metabolite was injected onto the capillary column. Strong pseudomolecular ions and key fragment ions were observed in the primary spectra of the parent drug and its three oxidative in vitro metabolites, allowing the site of metabolism to be pinpointed to particular substructures. This technique demonstrated versatility and offered a very rapid screening procedure for metabolite identification.

Enhanced susceptibility to human immunodeficiency virus infection in CD4+ T lymphocytes genetically deficient in CD43.

CD43 is a cell surface sialoglycoprotein expressed by most cells of hematopoietic origin, including all T lymphocytes. Elimination of CD43 expression by gene targeting in the CEM T cell line results in its increased homotypic adhesion and binding to HIV-1 gp120. Here we report that the CD43-negative CEM cells show increased susceptibility to HIV-1 infection and increased viral replication compared with the parental CD43+ CEM cell line. Increased HIV-1 replication also was observed in CEM cells with diminished CD43 expression secondary to functional inactivation of a single CD43 allele. The CD43- CEM cells were more susceptible to HIV-1-induced cytopathicity than their CD43+ counterparts. HIV-1 replication also was increased in the CD43- CEM cells after transfection with the infectious HIV molecular clone pNL4-3. These data suggest that factors that diminish CD43 expression on T lymphocytes may enhance their susceptibility to HIV-1 infection.

In vitro and in vivo biotransformations of the naphthalenic lignan lactone 5-lipoxygenase inhibitor, L-702,539.

It has been reported previously that the tetrahydropyranyl naphthtalenic lignan lactone L-702,539 is a potent nonredox, 5-lipoxygenase inhibitor that has the advantage that it can be dosed either as the lactone or as the corresponding nonactive hydroxy acid L-702,618 (opened lactone). Studies with hepatic microsomes from the rat, rhesus monkey, and human were undertaken in a phosphate buffer and suggested that the closure of the hydroxy acid L-702,618 to the lactone L-702,539 was an enzymatic process. The incubation of L-702,539 under oxidative conditions with these specific hepatic microsomes resulted in the formation of three significant metabolites (> 0.4 nmol/mg protein/hr) as determined by HPLC with UV detection. These metabolites were isolated from large microsomal incubations and were characterized by MS and NMR spectroscopy. Data showed that the lactone and tetrahydropyran portions of the molecule were both susceptible to hydroxylation, and the hydroxylated tetrahydropyran was further oxidized to the hydroxy acid. Analysis of plasma samples obtained from rat and rhesus monkeys following L-702,618 administration indicated that the in vivo metabolic pathway was similar to the one observed in vitro using hepatic microsomes. Studies conducted with microsomes from genetically engineered human cell lines expressing individual cytochrome P450s indicated that the isozyme responsible for the metabolism at the tetrahydropyran ring, was P4503A4. These findings were supported by studies conducted in human microsomes using an inhibitory P4503A4 antibody and troleandomycin, which is a potent P4503A inhibitor.

Inflammatory mediator profile in urine and bladder wash fluid of patients with interstitial cystitis.

Interstitial cystitis is a syndrome of urinary urgency, frequency and suprapubic pain. We investigated the role of inflammatory mediators in 96 patients with histories and symptoms consistent with interstitial cystitis, and 13 controls from The New York Hospital-Cornell Medical Center, University of Washington and University of California at San Diego. Patients were classified into either group A (meets all criteria of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases for inclusion in research studies), group B (meets all of these criteria but without glomerulations) or an "other" group. A small number of group A patients had detectable interleukin-6 in the urine. Urinary concentrations of tumor necrosis factor, prostaglandins E2, D2 and F2 alpha, and thromboxane B2 were not different among either patient groups or controls. Urine specimens contained inhibitors of the bioactivity of interleukin-6 and tumor necrosis factors but no differences between patients or controls were found. No factors chemotactic for human neutrophils were detected in a small patient sample. Bladder wash fluid concentrations of prostaglandins E2, D2 and F2 alpha, and thromboxane were much lower than urinary levels. Bladder wash fluid interleukin-6 and tumor necrosis factor were not detectable. The results suggest that while a small subset of patients may have elevated levels of interleukin-6 the majority of patients do not appear to have elevated levels of inflammatory mediators in the urine or bladder wash fluid. Evaluation of patient bladder tissue may indicate changes not detectable in urine or bladder wash fluid. Alternatively, other etiologies must be considered in those patients.

NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.

Characterization of verlukast metabolites arising from an epoxide intermediate produced with hepatic microsomes from beta-naphthoflavone-treated rodents (P-4501A1).

Verlukast, (R)3-((((3-(2-(7-chloroquinolin-2-yl)-(E)-ethenyl)phenyl)-3- dimethylamino-3-oxopropylthio)methyl)thio)-propionic acid (also known as MK-0679 and L-668,019), is a potent leukotriene D4 antagonist. Verlukast was incubated with hepatic microsomes from beta-naphthoflavone (beta NF) or isosafrole-treated rodents to evaluate whether P-4501A1 or 1A2 mediated biotransformations could occur. With beta NF-induced mouse or rat microsomes, in which the induction of P-4501A1 had been proven by Western blot analysis, incubations produced new metabolites that were separated by reversed-phase HPLC and were initially characterized by UV (photodiode array). Metabolites were subsequently isolated and characterized by NMR and MS, and were assigned as the 5",6"-dihydrodiol and 6"-phenol (on the quinoline ring). The presumed 5",6"-epoxide intermediate was also detected and was characterized by UV (photodiode array) and MS. Microsomes from isosafrole-treated rodents produced the dihydrodiol to a much lesser extent and did not yield any other new metabolites. alpha-Naphthoflavone inhibited the dihydrodiol formation in incubations with microsomes from isosafrole- and beta NF-treated rats. In incubations with microsomes from beta NF-treated rats, to which the epoxide hydrolase inhibitor 3,3,3-trichloropropene 1,2-oxide had been added, the formation of dihydrodiol was inhibited, consistent with a microsomal epoxide hydrolase hydrolysis of the epoxide intermediate. When glutathione was added to incubations with microsomes from beta NF-treated rats, the dihydrodiol, phenol, and epoxide peaks were reduced in size and a new material, the glutathione adduct, was formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Toradol, an NSAID used for renal colic, decreases renal perfusion and ureteral pressure in a canine model of unilateral ureteral obstruction.

Toradol is a new parenteral, nonsteroidal anti-inflammatory drug which is efficacious in treating renal coli. In the present experiments, Toradol was administered to both control dogs and dogs with unilateral ureteral obstruction. In control dogs, Toradol had no effect on RBF or GFR, despite inhibition of renal prostaglandin synthesis (measured as urinary prostaglandin release). In contrast, RBF fell acutely by 35% (p < 0.001) within 15 minutes of Toradol administration in the setting of ureteral obstruction; contralateral RBF was unaffected. Ipsilateral ureteral pressure also fell. Changes in RBF and ureteral pressure, together with the known effects of NSAIDs on pain pathways, may contribute to the pain relief observed clinically with Toradol. However, the abrupt changes in renal hemodynamics brought on by Toradol to the obstructed kidney may compromise renal reserve, and Toradol should be used cautiously in treating renal colic.