Styrene maleic acid copolymer-pirarubicin induces tumor-selective oxidative stress and decreases tumor hypoxia as possible treatment of colorectal cancer liver metastases.

BACKGROUND: Pirarubicin, a derivative of doxorubicin, induces tumor destruction via the production of reactive oxygen species (ROS) but is associated with cardiotoxicity. As a macromolecule (conjugated to styrene-maleic acid [SMA]), SMA-pirarubicin is selective to tumors resulting in improved survival with decreased systemic toxicity. Tumor destruction is, however incomplete, and resistant cells at the periphery of the tumor contribute to recurrence. Tumor hypoxia is a major factor in tumor resistance. Understanding the effect of oxidative stress induced by SMA-pirarubicin on the tumor microenvironment may be key to overcoming resistance. This study investigated the pattern of ROS production and tumor hypoxia after treatment with SMA-pirarubicin in a murine model of colorectal liver metastases. METHODS: Liver metastases were induced in male, CBA mice using a murine-derived colon cancer cell line. SMA-pirarubicin (maximum tolerated dose, 100 mg/kg) or pirarubicin, (maximum tolerated dose, 10 mg/kg) were administered intravenously 14 days after tumor induction. Systemic oxidative stress in serum, liver, and cardiac tissue was quantified using the thiobarbituric acid reactive substances assay. Flow cytometry and fluorescence microscopy were used to assess ROS production for 48 hours after treatment. Tumor hypoxia was quantified using immunohistochemistry for pimonidazole adducts. RESULTS: SMA-pirarubicin (100 mg/kg) induced ROS exclusively in tumors with minimal levels in serum and cardiac tissue. ROS levels were induced in a time-dependent and dose-dependent manner optimal between 4 and 24 hours after drug administration. Although tumor hypoxia was decreased overall, residual tumor cells adjacent to patent vessels were hypoxic. CONCLUSION: This study provides insight into the tumor microenvironment after chemotherapy. SMA-pirarubicin inhibits the growth of colorectal liver metastases by inducing ROS, which seems to be largely tumor selective. The temporal pattern of ROS production can be used to improve future dosing regimens. Furthermore, the observation that residual tumor cells are hypoxic clarifies the need for a multimodal approach with agents that can alter the hypoxic state to effect complete tumor destruction.

Physiological characterization of a mouse model of cachexia in colorectal liver metastases.

Loss of skeletal muscle mass and function (cachexia) is severe in patients with colorectal liver metastases because of the large increase in resting energy expenditure but remains understudied because of a lack of suitable preclinical models. Our aim was to characterize a novel preclinical model of cachexia in colorectal liver metastases. We tested the hypothesis that mice with colorectal liver metastases would exhibit cachexia, as evidenced by a reduction in liver-free body mass, muscle mass, and physiological impairment. Twelve-week-old male CBA mice received an intrasplenic injection of Ringer solution (sham) or murine colorectal cancer cells (MoCR) to induce colorectal liver metastases. At end-point (20-29 days), the livers of MoCR mice were infiltrated completely with metastases, and MoCR mice had reduced liver-free body mass, muscle mass, and epididymal fat mass compared with sham controls (P < 0.03). MoCR mice exhibited impaired rotarod performance and grip strength (P < 0.03). Histochemical analyses of tibialis anterior muscles from MoCR mice revealed muscle fiber atrophy and reduced oxidative enzyme activity (P < 0.001). Adipose tissue remodeling was evident in MoCR mice, with reduced adipocyte diameter and greater infiltration of nonadipocyte tissue (P < 0.05). These findings reveal the MoCR mouse model exhibits significant cachexia and is a suitable preclinical model of cachexia in colorectal liver metastases. This model should be used for identifying effective treatments for cachexia to improve quality of life and reduce mortality in patients with colorectal liver metastases.

Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor.

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of ß-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Blockade of the renin-angiotensin system improves the early stages of liver regeneration and liver function.

BACKGROUND: Partial hepatectomy is the preferred option for selected patients with colorectal cancer liver metastases (CRCLM). Sufficient liver regeneration (LR) is essential for a successful outcome in these patients. The blockade of the renin-angiotensin system (RAS) reduces the growth of several tumor types. The RAS also acts as a regulator of liver fibrosis and potentially LR. The angiotensin-converting enzyme (ACE) inhibitor, captopril, significantly inhibits the growth of CRCLM, but its effect on LR remains undefined. METHODS: After 70% of partial hepatectomy, mice were randomly assigned to control or captopril-treated groups. LR was measured by liver-to-body weight ratio on days 1, 2, 4, 6, and 8. Hepatocyte proliferation, apoptosis and cell size, hepatic stellate cell (HSC) count, and sinusoidal endothelial cell density were quantified. Matrix metalloproteinase 9 (MMP-9) protein levels, liver injury markers, and RAS messenger RNA levels were also determined. RESULTS: At day 2, captopril increased liver-to-body weight ratio (56.5 ± 1.7 captopril versus 49.3 ± 2.4 control, P = 0.027). This was associated with increased HSC count (65.4 ± 4.8 cells per 100,000 µm(2), 48.7 ± 2.3, P = 0.007) and MMP-9 levels (0.68 ± 0.12 AU, 0.12 ± 0.04, P = 0.014). The messenger RNA levels of angiotensin-converting enzyme (P = 0.045) and angiotensin 1 receptor (P = 0.039) were reduced by captopril at day 2. CONCLUSION: Captopril enhanced early LR. This effect was associated with increased HSC numbers and MMP-9 protein, whereas hepatocyte proliferation was lower than controls. Captopril may provide a beneficial treatment option for the management of patients with CRCLM.

P-21 activated kinase 1 knockdown inhibits ß-catenin signalling and blocks colorectal cancer growth.

The p21-activated kinase 1 (PAK1) plays important roles in cell growth, motility, and transformation. The aims of this study were to delineate the signalling mechanisms downstream of PAK1, and to investigate the importance of PAK1 for colorectal cancer (CRC) growth and metastasis in vivo. PAK1 knockdown in human CRC cell lines inhibited ß-catenin expression, ß-catenin/TCF4 transcriptional activity, and the expression of c-Myc. In mouse models PAK1 knockdown suppressed the growth and metastasis of human CRC cells by decreasing proliferation and increasing apoptosis. Our findings demonstrate for the first time the crucial role of PAK1 in CRC progression in vivo.

Lymphatic patterns of colorectal liver metastases.

BACKGROUND: Hematogenous spread is considered the predominant pathway for development of colorectal liver metastases (CRLM) and subsequent further tumor dissemination portal nodal involvement is also frequently observed in such cases, suggesting that lymphatics may have a role in the spread of CRLM. The role of lymphatics in the development of liver metastases is, however, controversial. The lymphatic patterns of CRLM were determined using a well established murine model. METHODS: CRLM were induced using a well established murine intrasplenic colorectal cancer model. Tumors were assessed at varying stages of development, and lymphatic patterns were determined in tumors and liver by immunohistochemistry staining for podoplanin and LYVE-1. Blood vessels were characterized using the vascular marker CD-34. Assessment was undertaken using digital microscopy and image analysis. RESULTS: Peri- and intratumoral lymphatic vessels were identified by podoplanin staining in all metastases and significantly increased with tumor growth. LYVE-1 staining was also noted but was variable. There was a concurrent significant increase in portal lymphatic staining within the normal liver with increasing growth of CRLM. CONCLUSION: There is increased expression of lymphatics within CRLM and normal liver with increasing tumor growth. Lymphatic development is likely to play a significant role in the intrahepatic and periportal dissemination of CRLM.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

In vitro and in vivo evaluation of tumor targeting styrene-maleic acid copolymer-pirarubicin micelles: Survival improvement and inhibition of liver metastases.

Pirarubicin is a derivative of doxorubicin with improved intracellular uptake and reduced cardiotoxicity. We have prepared a micellar formulation of pirarubicin using styrene-maleic acid copolymer (SMA) of mean molecular weight of 1.2 kDa, which exhibits a mean diameter of 248 nm in solution. Being a macromolecule, SMA-pirarubicin micelles exhibit excellent tumor targeting capacity due to the enhanced permeability and retention (EPR) effect. Here we report the antitumor activity of SMA-pirarubicin micelles on human colon and breast cancer cell lines in vitro, and a murine liver metastasis model in vivo. Metastatic tumor microvasculature, necrosis, apoptosis, proliferation, and survival were also investigated using immunohistochemistry for Ki-67, active caspase-3, and CD34, respectively. Drug cytotoxicity in vitro was assessed using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) assay. In vivo, SMA-pirarubicin was administered at 100, 150, or 200 mg/kg (pirarubicin equivalent). Tumor microvasculature was also assessed using scanning electron microscopy. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles were toxic against human colorectal and breast cancer cells in vitro. IC(50) was at or below 1 muM, free pirarubicin equivalent. In vivo, SMA-pirarubicin at 100 mg/kg reduced tumor volume by 80% and achieved a survival rate of 93% at 40 days after tumor inoculation. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles demonstrated potent antitumor activity in this liver metastases model, contributing to prolonged survival. Histological examination of tumor nodules showed significant reduction and proliferation of tumor cells (>90%). The present results suggest that investigation of the effect of multiple dosing at later time points to further improve survival is warranted.

CYT997: a novel orally active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo.

CYT997 is a wholly synthetic compound that possesses highly potent cytotoxic activity in vitro through inhibition of microtubule polymerization. CYT997 blocks the cell cycle at the G(2)-M boundary, and Western blot analysis indicates an increase in phosphorylated Bcl-2, along with increased expression of cyclin B1. Caspase-3 activation is also observed in cells treated with CYT997 along with the generation of poly(ADP-ribose) polymerase. The compound possesses favorable pharmacokinetic properties, is orally bioavailable, and is efficacious per os in a range of in vivo cancer models, including some refractory to paclitaxel treatment. CYT997 exhibits vascular disrupting activity as measured in vitro by effects on the permeability of human umbilical vein endothelial cell monolayers, and in vivo by effects on tumor blood flow. CYT997 possesses a useful combination of pharmacologic and pharmacokinetic properties and has considerable potential as a novel anticancer agent.

Styrene maleic acid-pirarubicin disrupts tumor microcirculation and enhances the permeability of colorectal liver metastases.

BACKGROUND: Doxorubicin is a commonly used chemotherapy limited by cardiotoxicity. Pirarubicin, derived from doxorubicin, selectively targets tumors when encapsulated in styrene maleic acid (SMA), forming the macromolecular SMA pirarubicin. Selective targeting is achieved because of the enhanced permeability and retention (EPR) effect. SMA-pirarubicin inhibits the growth of colorectal liver metastases, but tumor destruction is incomplete. The role played by the tumor microcirculation is uncertain. This study investigates the pattern of microcirculatory changes following SMA-pirarubicin treatment. METHODS: Liver metastases were induced in CBA mice using a murine-derived colon cancer line. SMA-pirarubicin (100 mg/kg total dose) was administered intravenously in 3 separate doses. Twenty-four hours after chemotherapy, the tumor microvasculature was examined using CD34 immunohistochemistry and scanning electron microscopy. Tumor perfusion and permeability were assessed using confocal in vivo microscopy and the Evans blue method. RESULTS: SMA-pirarubicin reduced the microvascular index by 40%. Vascular occlusion and necrosis were extensive following treatment. Viable cells were arranged around tumor vessels. Tumor permeability was also increased. CONCLUSION: SMA-pirarubicin damages tumor cells and the tumor microvasculature and enhances tumor vessel permeability. However, tumor necrosis is incomplete, and the growth of residual cells is sustained by a microvascular network. Combined therapy with a vascular targeting agent may affect residual cells, allowing more extensive destruction of tumors.

Gastrin is not required for liver regeneration.

BACKGROUND: Although several growth factors are known to be essential for liver regeneration, the role of gastrin remains controversial. METHODS: Liver regeneration was examined in wild-type (WT) and gastrin-deficient (gastrin KO) mice at days 2 and 10 after partial (40%) hepatectomy by measurement of liver weight. Hepatocyte proliferation and circulating gastrin concentrations were measured at the same time points by immunohistochemistry and radioimmunoassay, respectively. RESULTS: There was no significant difference in the rate of liver regeneration between gastrin KO and WT mice. Hepatocyte proliferation in both groups was increased at day 2 but had returned to baseline values by day 10. At day 2, hepatocyte proliferation in the gastrin KO mice was significantly higher than in WT animals, whereas at day 10, proliferation was significantly greater in the WT mice. The circulating gastrin concentration in the WT mice was significantly lower at day 10 than in unoperated WT animals. CONCLUSION: This study suggests that gastrin is not essential for liver regeneration after partial hepatectomy.

Vaccination with in vitro grown whole tumor cells induces strong immune responses and retards tumor growth in a murine model of colorectal liver metastases.

In vitro adaptation of a murine colorectal cell line (MoCR) rendered it less aggressive and more immunogenic than the in vivo passaged parent tumor. Vaccination of syngeneic mice with the in vitro cultured tumor cells was shown to induce immune responses and protection against tumor challenge, thus overcoming the need for antigen selection and adjuvants. A syngeneic murine model of colorectal cancer (CRC) liver metastasis was used. In a prophylactic setting mice vaccinated with in vitro cultured tumor cells produced strong cellular immune responses and significant inhibition of tumor growth, compared to sham vaccinated controls. In a therapeutic setting however, vaccination exacerbated tumor growth, suggesting that the presence of tumor subverts the course of the immune response. The mechanisms of this subversion need to be investigated and counteracted for successful immunotherapy.

Alterations in vascular architecture and permeability following OXi4503 treatment.

OXi4503 retards tumor growth in a dose-dependent manner and improves survival in a murine model of colorectal liver metastases. This agent causes extensive vascular shutdown by selectively altering the tubulin cytoskeleton within the endothelial cells of tumor vessels. The destruction of tumor vessels is incomplete, however, and tumor revascularization occurs after the treatment. This study evaluates the pattern of microcirculatory changes and alterations to the ultrastructural properties of the tumor vasculature that result from OXi4503 treatment. Male CBA mice were induced with liver metastases via an intrasplenic injection of a murine-derived colorectal cell line. After administering a single intraperitoneal dose of OXi4503, changes in tumor perfusion, microvascular architecture and permeability were assessed at various time points. One hour after a 100-mg/kg dose of OXi4503, a significant decrease in the percentage of tumor perfusion (63.96+/-1.98 in controls versus 43.77+/-2.71 in treated mice, P<0.001) was observed, which was still evident 5 days after the treatment. Substantial tumor microvascular damage and minimal normal liver injury were observed. Tumor vascular permeability was significantly elevated 45 min after the OXi4503 treatment (67.5+/-3.60 in controls versus 80.5+/-2.24 microg/g, P<0.05). The findings suggest that OXi4503 selectively targets tumor vessels and causes immediate microvascular destruction. Even at the maximum tolerated dose, however, residual patent tumor vessels were still present after treatment, implying incomplete tumor destruction. A combination of OXi4503 with other chemotherapeutic modalities might achieve complete tumor eradication and improve long-term survival.

Evaluation of the effect of SMA-pirarubicin micelles on colorectal cancer liver metastases and of hyperbaric oxygen in CBA mice.

Tetrahydropyranyladriamycin (THP or pirarubicin) destroys tumors via several mechanisms; one of which involves the production of ROS that requires molecular oxygen for its generation. SMA forms stable self-assembled associated micelles with pirarubicin (SMA-pirarubicin), and confers macromolecular characteristics to pirarubicin. This micellar macromolecular drug is selectively delivered to solid tumors via the EPR effect and its preferential tumor accumulation suppresses the systemic toxicity whilst its prolonged high concentration at the site of tumor enhances its efficacy much higher compared to free pirarubicin. Administration of SMA-pirarubicin micelle under HBO can further enhance the delivery of molecular oxygen that facilitates tumor selective generation of ROS, thus augmenting its antitumor potency. In this study, we evaluated the efficacy of SMA-pirarubicin micelles either as single drug or in combination with HBO in a mouse metastatic colorectal cancer model. At or below the maximum tolerated dose, SMA-pirarubicin remarkably reduced metastatic tumor nodules and it was far more effective than free pirarubicin. The data also suggests a potential benefit of combined therapy of HBO with micellar anthracyclins.

Effect of vascular targeting agent Oxi4503 on tumor cell kinetics in a mouse model of colorectal liver metastasis.

BACKGROUND: Oxi4503 has been shown to inhibit tumor growth and improve survival in an animal model of colorectal (CRC) liver metastases. This agent appears to selectively target the endothelial cytoskeleton with resultant vessel occlusion and tumor necrosis. MATERIALS AND METHODS: This study evaluated the pattern of tumor necrosis caused by Oxi4503, with particular emphasis on patterns of cell proliferation and apoptosis in a murine model of CRC liver metastases. RESULTS: A single dose of Oxi4503 caused immediate tumor vasculature collapse and subsequently tumor necrosis. There was widespread central necrosis with evidence of viable tumor cells at the periphery. Alterations in the number and spatial pattern of tumor cells undergoing apoptosis and the rate of cellular proliferation were also observed following treatment. Microvessel density was reduced following treatment, however patent vessels were still observed within the necrotic core. CONCLUSION: Although Oxi4503 caused significant tumor destruction, synergistic treatment with cytotoxic and/or anti-angiogenic agents should be considered in order to achieve complete tumor eradication and long-term survival.

Effect of ACE inhibitors and angiotensin II receptor antagonists in a mouse model of colorectal cancer liver metastases.

BACKGROUND AND AIMS: Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II type I receptor (AT1R) antagonists are commonly used as a treatment for hypertension. Recent experimental and population studies have suggested that these agents may exert an inhibitory effect on malignancy, possibly through anti-angiogenic pathways. The aim of this study was to investigate the effect of an ACE-I (captopril) and an AT1R antagonist (irbesartan) in colorectal cancer liver metastases. METHODS: The effect of captopril and irbesartan on tumor growth was investigated in a mouse model using quantitative stereological and histological analysis. Tumor microcirculation was assessed by microvascular resin casting. A survival study was also carried out. RESULTS: Both captopril and irbesartan markedly decreased tumor growth when compared to control (P = 0.003 and P = 0.004, respectively). However, there was no significant difference in survival or tumor necrosis for either of the drugs. Tumor microvasculature exhibited a reduction in central microvascular density, with constriction and tapering of vessels. CONCLUSION: Captopril and irbesartan caused a marked reduction in volume of colorectal cancer liver metastases and caused changes in tumor microvasculature. However, there was no difference in percentage tumor necrosis or improvements in survival. Further investigation is needed to identify the mode of action of these agents.

Effect of thalidomide on colorectal cancer liver metastases in CBA mice.

BACKGROUND AND AIMS: Thalidomide has undergone resurgence in the treatment of specific malignancies. One of the possible actions of thalidomide may be an antiangiogenic effect. This study investigates the effects of thalidomide on tumor growth and long-term survival in a murine model of colorectal liver metastases. METHODS: Liver metastases were produced in male CBA mice by intrasplenic injection of a dimethyl hydrazine induced MoCR colon cancer murine cell line. Thalidomide was administered daily at doses ranging from 50 to 300 mg/kg by intraperitoneal injection. Tumor growth was assessed using quantitative stereological analysis. The effect on long-term survival was determined at the maximum tolerated dose using Kaplan-Meier analysis. The microvascular effects of thalidomide were assessed by laser Doppler flowmetry (LDF) and microvascular resin casting. Immunohistochemistry was used to determine vascular endothelial growth factor (VGEF) and basic fibroblast growth factor (bFGF) expression. RESULTS: Thalidomide, (50-300 mg/kg) caused no significant reduction in tumor growth by day 21 following induction of liver metastases and caused systemic toxicity at a dose of 300 mg/kg. At a dose of 200 mg/kg given beyond 35 days, thalidomide significantly reduced tumor growth compared to control, (P = 0.029). No significant impact on survival was however observed (P = 0.93). LDF and microvascular resin casting showed no differences in blood flow or tumor microvascular architecture. VGEF and FGF were expressed in tumors, but remained unaltered by thalidomide administration compared to matched controls. CONCLUSIONS: Thalidomide caused a significant reduction in the volume of colorectal liver metastases during the late phase of tumor growth. There was however no improvement in survival. Tumor growth reduction in this model did not appear to be due to microvascular changes or altered expression of VGEF or basic FGF. Further investigation into potential mechanisms of action of thalidomide and its synergistic use with other therapies is required.

Aged gastric mucosa: Mechanisms of vulnerability.

The incidence, frequency of complications and mortality of gastric ulcer disease are increased four-fold in the elderly taking non-steroidal anti-inflammatory drugs (NSAID). There is controversy as to whether this reflects increased usage of NSAID or specific vulnerability associated with age. We have investigated two possible mechanisms for this increase in gastrointestinal effects in the elderly: (i) increased susceptibility to acute gastrotoxicity; and (ii) reduced adaptation to NSAID, in a model of young (2 month), mature (12 month) and aged (24 month) rats. Aspirin damaged 7.7% of the volume of gastric mucosa in the young rat. In mature and aged rats, this increased to 11.3% (P < 0.002 compared to control) and 21.9% (P < 0.005 compared to control), respectively. Thus, aspirin caused a three-fold increase in the severity of acute gastric mucosal injury in aged animals. However, indomethacin, ibuprofen and L745 337 did not produce any significant acute gastric mucosal damage in 2-, 12- or 24-month-old rats. Significant gastric adaptation to diclofenac treatment occurred in both aged and young rats as measured by gastric mucosal damage. The aged gastric mucosa adapted equally as well as the young gastric mucosa to diclofenac. The findings of this study provide only modest support to the hypothesis of increased vulnerability of the stomach in the aged. Aspirin was associated with greater damage in the aged. Adaptation to diclofenac-induced damage was not reduced in the aged and there was not an increased susceptibility to damage by the non-aspirin NSAID tested. The selective cyclo-oxygenase-2 inhibitor, L745 337, was the least toxic agent and may represent a group of NSAID which cause fewer gastrointestinal complications in the elderly.