Tenidap inhibits 5-lipoxygenase product formation in vitro, but this activity is not observed in three animal models.

OBJECTIVE AND DESIGN: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo. MATERIALS AND TREATMENT: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively. METHODS: 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA. RESULTS: Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models. CONCLUSIONS: Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.

Rheumatoid arthritis exhibits reduced acute phase and enhanced constitutive serum amyloid A protein in synovial fluid relative to serum. A comparison with C-reactive protein.

OBJECTIVE: There are 2 classes of serum amyloid A (SAA) protein, acute phase (A-SAA) and constitutive (C-SAA). Hepatic synthesis of A-SAA is dramatically upregulated by inflammatory cytokines, while C-SAA is constitutively produced in the absence of inflammation. A-SAA has been shown to attract monocytes, neutrophils, and T lymphocytes, but the function of C-SAA remains to be determined. SAA proteins have been found in both serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA), but have not been characterized with respect to isoform distribution. We determined the relative distribution of A-SAA and C-SAA in serum and SF of patients with RA and compared their abundance to the classic acute phase response protein, C-reactive protein (CRP). METHODS: A-SAA (isoforms SAA1, SAA2) and CRP were measured by commercially available ELISA kits. ELISA were developed for C-SAA (SAA4) and apolipoprotein AI (apo AI) in paired serum and SF from 56 patients with RA. RESULTS: Concentrations (mean +/- SD) of A-SAA (SAA1,2) in serum and SF are 124 +/- 247, 20 +/- 32 micrograms/ml; CRP 75 +/- 70, 33 +/- 37 micrograms/ml; C-SAA (SAA4) 106 +/-49, 91 +/- 39 micrograms/ml; and apo AI 1.19 +/- 0.32, 0.37 +/- 0.12 mg/ml, respectively. CRP correlated positively with A-SAA in serum or SF and negatively with apo AI in serum. There was no correlation with apo AI in SF. In contrast, there was no correlation between C-SAA and CRP, A-SAA, or apo AI in serum or in SF. Median concentrations of A-SAA in serum and SF (44, 10 micrograms/ml) and CRP (46, 20 micrograms/ml), respectively, markedly differed from the mean values, whereas median concentrations of C-SAA (104, 85 micrograms/ml) and apo AI (1.17, 0.37 mg/ml), respectively, did not. CONCLUSION: C-SAA concentrations vary in serum and SF independently of A-SAA and CRP levels. The lower concentration of A-SAA relative to C-SAA and CRP in SF suggests that A-SAA could be selectively catabolized in SF or alternatively not well transported into the synovial space.

Tenidap in patients with rheumatoid arthritis. A 4-week, placebo-controlled study.

The present double-blind, placebo-controlled study was conducted to compare the safety and efficacy of tenidap in patients with rheumatoid arthritis (RA). Patients with flare of active RA following NSAID withdrawal were randomized to receive either placebo (n = 67) or tenidap (n = 131; 40-200 mg/day). The mean changes from baseline in efficacy and biochemical variables were compared between treatment groups at endpoint (4 weeks). The improvements in four of the five primary efficacy variables were significantly greater in the tenidap group compared with the placebo group (p < 0.01). Tenidap was also associated with an 18% reduction in erythrocyte sedimentation rate (ESR) and a marked, 51%, reduction in serum C-reactive protein (CRP) level, both of which were significantly greater than the changes in the placebo group (p < 0.05). The percentage of patients who discontinued because of side effects was the same in both groups (3%). In conclusion, tenidap 40-200 mg/day was effective and well tolerated in the treatment of patients with RA for 4 weeks.

Effects of tenidap on superoxide-generating enzymes. Non-competitive inhibition of xanthine oxidase.

The anti-rheumatic drug tenidap has been shown previously to attenuate superoxide production by activated neutrophils. Given the importance of leukocyte as well as endothelial cell derived superoxide in mediating inflammatory responses, the effects of tenidap on mammalian enzymes capable of generating superoxide were determined. Tenidap had no effect on the generation of superoxide by NADPH oxidase reconstituted from fractionated neutrophil lysates. However, significant inhibition of superoxide production by mixtures of hypoxanthine and xanthine oxidase was observed in the presence of 3-30 micrograms/mL tenidap. The kientics of xanthine oxidase inhibition by tenidap were non-competitive; the Ki of tenidap for xanthine oxidase was 11 micrograms/mL (34 microM). No inhibition of xanthine oxidase was observed in the presence of other known inhibitors of cyclooxygenase. Inhibition of xanthine oxidase may be a heretofore unrecognized mechanism of the antirheumatic effects of tenidap.

Synergism of interleukin 1 and interleukin 6 induces serum amyloid A production while depressing fibrinogen: a quantitative analysis.

OBJECTIVE: Production of the serum amyloid A (SAA) proteins in the liver of patients with arthritis can be increased from approximately 1 microgram/ml to > 1000 micrograms/ml, while fibrinogen (Fg) can be increased from 2 to 9 mg/ml. The increases appear to be regulated by mediators similar to those found in inflamed joints, e.g., interleukins 1 and 6 (IL-1 and IL-6, respectively). The sensitivity and dose response of SAA and Fg synthesis by hepatoma cells to IL-1 and IL-6 was investigated to understand the relationship between the inflammatory cytokines produced in inflamed joints and the acute phase protein response in the liver of arthritis patients. METHODS: SAA and Fg mRNA and protein production in human Hep3B cells stimulated by human monocyte conditioned medium (CM) containing known amounts of IL-1 and IL-6, or stimulated by corresponding concentrations of recombinant IL-1 and IL-6 was analyzed by ELISA and Northern blot hybridization techniques. RESULTS: Increases in SAA mRNA and protein were dose dependent in the presence of IL-1 and IL-6 at concentrations ranging from 0.1 and 1 ng/ml, respectively, to 10 and 100 ng/ml, respectively. In the presence of IL-1 receptor antagonist (IL-1ra), there was a 75% decrease in SAA production and > 100% increase in Fg production by cells stimulated with CM. CONCLUSION: Our results demonstrate that the thousand fold dynamic range associated with the acute phase SAA response requires IL-1 acting synergistically with cytokine(s) like IL-6. Optimum conditions for apoSAA production are suboptimal for Fg as indicated by the differential effects of IL-1ra.

Regulation of human normal and osteoarthritic chondrocyte interleukin-1 receptor by antirheumatic drugs.

OBJECTIVE: To determine the effect of antirheumatic drugs and corticosteroids on interleukin-1 receptor (IL-1R) levels in, and IL-1-stimulated metalloprotease synthesis and expression by, normal and osteoarthritic (OA) human articular chondrocytes. METHODS: IL-1R affinity and density of human chondrocytes were determined using radioligand binding experiments. Collagenase and stromelysin synthesis activities were analyzed by 14C-labeled type I collagen and Azocoll assays, respectively. Their messenger RNA (mRNA) levels were determined by Northern blot analysis. IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, and beta 2-microglobulin were measured using enzyme-linked immunosorbent assays. Protein synthesis was determined by 3H-leucine incorporation. RESULTS: Antirheumatic drugs reduced the IL-1R level in normal and OA chondrocytes in a dose-dependent manner. In normal chondrocytes, tenidap reduced the IL-1R level by 44% at 5 micrograms/ml to 88% at 100 micrograms/ml (50% inhibition constant [IC50] 10.1 micrograms/ml), indomethacin reduced IL-1R by 6% at 1.5 micrograms/ml to 43% at 60 micrograms/ml, and naproxen reduced IL-1R by 10% at 10 micrograms/ml to 41% at 300 micrograms/ml; the effects observed with indomethacin and naproxen occurred only when the drugs were used at levels above their therapeutic concentrations. In OA chondrocytes, the effect of indomethacin and naproxen on the IL-1R level was greatly reduced, whereas tenidap still had a marked effect (IC50 22.5 micrograms/ml). Dexamethasone and hydrocortisone had no consistent effect on the IL-1R level. At a therapeutic concentration (20 micrograms/ml), tenidap was found to reduce the IL-1R level in a time-dependent manner, with maximum inhibition (98%) by 48 hours. Tenidap was also found to markedly reduce collagenase and stromelysin synthesis and mRNA levels in IL-1-stimulated chondrocytes. CONCLUSION: The suppressive effects of tenidap on IL-1-stimulated metalloprotease synthesis and expression in OA and normal chondrocytes are likely related to a decrease in IL-1R levels. At therapeutic concentrations, tenidap has a greater effect on the IL-1R level than is seen with indomethacin or naproxen, and glucocorticoids have no effect on IL-1R.

Reduction of acute-phase proteins with tenidap sodium, a cytokine-modulating anti-rheumatic drug.

Four independent studies have investigated and compared the effects of tenidap sodium, naproxen and placebo on CRP in patients with active RA. One of these studies also investigated the effects of tenidap and naproxen on serum amyloid A (SAA) concentrations and ESR. The duration of the four studies ranged between 2 weeks and 24 weeks, and depending on the study, tenidap sodium was administered orally in doses of 40-120 mg/day and naproxen in doses of 1000 mg/day. In all four studies serum CRP concentrations in tenidap-treated patients had decreased significantly from baseline at the time of final assessment. The decrease in CRP concentration in tenidap-treated patients was observed as early as 1 week after initiation of therapy and was sustained for up to 6 months, the last assessment timepoint. CRP concentrations in naproxen-treated and placebo patients were essentially unchanged. The decreases from baseline observed in tenidap-treated patients were significantly greater than the changes observed in naproxen-treated or placebo patients. After 24 weeks of tenidap treatment the decrease in CRP was paralleled by significant decreases in SAA concentration and ESR. The finding that tenidap sodium rapidly, consistently and significantly lowered CRP serum concentrations differentiates tenidap sodium from the NSAID, naproxen. This could possibly have important therapeutic implications given that other long-term investigations have shown that reducing serum CRP and SAA concentrations correlates with a reduction in radiographically-assessed disease progression.

Human serum amyloid A protein. Complete amino acid sequence of a new variant.

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2'-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.

Inhibition of 5-lipoxygenase product formation and polymorphonuclear cell degranulation by tenidap sodium in patients with rheumatoid arthritis.

We studied the effect of tenidap sodium, a new antiinflammatory/antirheumatic drug (120 mg/day for 7 days), on eicosanoid production and neutrophil degranulation in patients with rheumatoid arthritis. Endogenous prostaglandin E2 levels and ex vivo production of leukotriene B4 (LTB4) were measured in synovial fluid samples obtained at baseline and 1 week later. We measured peripheral blood polymorphonuclear cell (PMN) degranulation following surface-bound IgG stimulation, a possible 5-lipoxygenase product-mediated event, by determining lactoferrin and elastase release into the culture fluid. We found decreased levels of endogenous prostaglandin E2 as measured by radioimmunoassay, and decreased ex vivo production of LTB4 by PMN as measured by high performance liquid chromatography, in synovial fluid samples from patients who took tenidap. Release of the granule proteins lactoferrin and elastase was decreased in PMN obtained from patients receiving tenidap, as well as in the PMN incubated in vitro with tenidap. Improvement in clinical measures paralleled the biochemical changes. The unique 5-lipoxygenase inhibitory property of tenidap, as measured by LTB4 production and degranulation, suggests that it may have clinical activity which differentiates it from nonsteroidal antiinflammatory drugs.

Tenidap, in contrast to several available nonsteroidal antiinflammatory drugs, potently inhibits the release of activated neutrophil collagenase.

Neutrophils contain a collagenase that is stored in a latent form within the specific granule. With cellular activation, the latent enzyme is activated in association with the production of a variety of oxidants, including hypochlorous acid. We evaluated 4 nonsteroidal antiinflammatory drugs (NSAIDs) currently on the market and the new antiinflammatory/antirheumatic drug tenidap for their effects on the release of activated collagenase. In contrast to the 4 NSAIDs, tenidap profoundly inhibited the release of activated collagenase. This inhibition was predominantly due to interference with activation of the latent enzyme, rather than interference with enzyme release. The inhibition of collagenase activation was associated with a profound reduction in myeloperoxidase activity and in hypochlorous acid production. These observations demonstrate that tenidap has properties that set it apart from conventional NSAIDs and suggest that it may be a particularly useful agent in the treatment of inflammatory rheumatic disorders.

Effects of gold sodium thiomalate and tenidap sodium (CP-66,248-2) on a model of macrophage differentiation using HL-60 cells.

We studied the effects of gold sodium thiomalate (GST) and a new antirheumatic drug, tenidap sodium ([Z]-5-chloro-2,3-dihydro-3-[hydroxy-2-thienylmethylene]-2-oxo-1H- indole-1-carboxamide, sodium salt), previously known as CP-66,248-2, in a model system of macrophage differentiation using a myelomonocytic cell line. HL-60 cells can be stimulated by vitamin D3 to differentiate along a monocytic pathway. Monocytic HL-60 cells express CD14 (Leu-M3), a macrophage surface marker, and develop the capacity to produce the second complement component (C2) in response to stimulation with cytokines such as gamma-interferon. The effects of GST and tenidap sodium were compared with the effects of dexamethasone and a variety of nonsteroidal antiinflammatory drugs in this model system. We found that GST inhibited the capacity of HL-60 cells to produce C2 but did not inhibit the expression of CD14. Tenidap sodium inhibited C2 production as well as CD14 expression, and it partially reversed the decrease in 3H-thymidine incorporation by HL-60 cells, which accompanies monocytic differentiation. At concentrations that inhibited C2 production by HL-60 cells, tenidap sodium did not inhibit C2 production by monocytes. Neither dexamethasone nor the other nonsteroidal antiinflammatory drugs tested possessed these activities. Thus, both GST and tenidap inhibit markers of monocytic differentiation in HL-60 cells, and this activity may relate to their antirheumatic activities.

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA).

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.

Characterization of macrophage dysfunction in rodent malaria.

Immunosuppression in malaria has been attributed, in part, to alterations in macrophage function. The present study was undertaken in an attempt to characterize the dysfunction and to determine if it is regional or if it occurs in different populations of macrophages. The resting O2 consumption of either hepatic, splenic, or peritoneal macrophages or polymorphonuclear neutrophils (PMNs) was unaltered by the malaria infection. However, the respiratory burst was significantly enhanced in the three macrophage populations but not in the PMNs. A significant increase in their phagocytic capacity and microbicidal activity was noted for hepatic and peritoneal but not splenic macrophages or PMNs. The malaria-infected mice had a marked decrease in serum antibody and splenic plaque response to bovine serum albumin (BSA) but not to keyhole limpet hemocyanin (KLH). Following an in vitro incubation with BSA, splenic macrophages from infected mice were not able to induce an antibody response when injected into normal mice. However, following incubation with KLH splenic macrophages could induce an adequate response in normal mice. This ability of macrophages from malaria-infected mice to transfer (or induce) a response in normal mice appeared to correlate with the amount of antigen digested, or perhaps retained, by the cells, i.e., BSA digestion was significantly less than KLH. These results indicate that the macrophage dysfunction in malaria is distinct depending on the tissue population that the macrophage is obtained from and that the impaired antibody response may be restricted to antigens requiring macrophage processing.

Biochemical and functional alterations in macrophages after thermal injury.

Biochemical and functional measurements of rat pulmonary alveolar macrophages were measured 4 h after 1 10-s, 26 to 28% total body surface area, full-thickness scald burn induced under ether anesthesia. Both phagocytic activity and capacity were significantly decreased to a comparable extent, whereas microbicidal activity was increased almost twofold in macrophages from the burned animals. Concurrent with the decreased phagocytic function was a marked impairment in chemotaxis and random migration of these cells when zymosan-activated serum was used as the chemoattractant. When biochemical parameters were examined, it was demonstrated that, on a per-cell but not total-protein basis, alveolar macrophages from burned animals had elevated levels of RNA, total protein beta-glucuronidase, acid phosphatase, and 5'-nucleotidase. These results raise the possibility that the increased pneumonitis in burned individuals may be due to more complex macrophage dysfunctions than impaired microbicidal activity, as was once thought. Alternatively, the biochemical and functional changes observed may be a reflection of a new population of macrophages appearing in the lungs after thermal injury.

Synthetic immunomodulators for prevention of fatal infections in a burned guinea pig model.

Individuals who have suffered severe trauma, such as burns, have a high incidence of infection associated with impaired host resistance. Nonspecific stimulators of host defense mechanisms, i.e., immunomodulators, may be of benefit in such situations. A small animal model (guinea pigs) was developed to study the efficacy of immunomodulators in burns. Anesthetized animals received a 20% total body surface area, full-thickness, scald burn. There was no mortality associated with this injury, but these animals were highly susceptible to challenge with Pseudomonas aeruginosa strain 1244 by direct injection into the burn wound within 24 hours of injury. This susceptibility persisted about 7 days. The standard model adopted was to injure animals, then challenge with 1 median lethal dose (LD50) of P. aeruginosa 96 hours after injury. Using this model, six synthetic immunomodulators were tested: CP-20,961, CP-46,665, muramyl dipeptide, thymopoietin pentapeptide (TP-5), levamisole, and lithium. Drug administration began 24 hours after injury and ended prior to challenge with P. aeruginosa at 96 hours. CP-20,961, muramyl dipeptide, levamisole, and lithium all had no beneficial effect on survival. A single dosage (0.3 mg/kg, I.V.) of CP-46,665, administered 24 hours postinjury, increased the survival rate from 50% to 85% and mean survival time (MST) from 8.2 days to 12.4 days. TP-5, given in four doses (0.1 mg/kg, I.V. each) every 24 hours, increased the survival rate from 40% to 80% and MST from 6.9 days to 11.6 days. These data show that immunomodulators could be of benefit in burns, but also that not all agents are effective in this particular situation.