Biomarkers predict enhanced clinical outcomes with afatinib versus methotrexate in patients with second-line recurrent and/or metastatic head and neck cancer.

BACKGROUND: In the phase III LUX-Head & Neck 1 (LUX-H&N1) trial, second-line afatinib significantly improved progression-free survival (PFS) versus methotrexate in patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Here, we evaluated association of prespecified biomarkers with efficacy outcomes in LUX-H&N1. PATIENTS AND METHODS: Randomized patients with R/M HNSCC and progression following ≥2 cycles of platinum therapy received afatinib (40 mg/day) or methotrexate (40 mg/m2/week). Tumor/serum samples were collected at study entry for patients who volunteered for inclusion in biomarker analyses. Tumor biomarkers, including p16 (prespecified subgroup; all tumor subsites), EGFR, HER2, HER3, c-MET and PTEN, were assessed using tissue microarray cores and slides; serum protein was evaluated using the VeriStrat® test. Biomarkers were correlated with efficacy outcomes. RESULTS: Of 483 randomized patients, 326 (67%) were included in the biomarker analyses; baseline characteristics were consistent with the overall study population. Median PFS favored afatinib over methotrexate in patients with p16-negative [2.7 versus 1.6 months; HR 0.70 (95% CI 0.50-0.97)], EGFR-amplified [2.8 versus 1.5 months; HR 0.53 (0.33-0.85)], HER3-low [2.8 versus 1.8 months; HR 0.57 (0.37-0.88)], and PTEN-high [1.6 versus 1.4 months; HR 0.55 (0.29-1.05)] tumors. Afatinib also improved PFS in combined subsets of patients with p16-negative and EGFR-amplified tumors [2.7 versus 1.5 months; HR 0.47 (0.28-0.80)], and patients with p16-negative tumors who were EGFR therapy-naïve [4.0 versus 2.4 months; HR 0.55 (0.31-0.98)]. PFS was improved in afatinib-treated patients who were VeriStrat 'Good' versus 'Poor' [2.7 versus 1.5 months; HR 0.71 (0.49-0.94)], but no treatment interaction was observed. Afatinib improved tumor response versus methotrexate in all subsets analyzed except for those with p16-positive disease (n = 35). CONCLUSIONS: Subgroups of HNSCC patients who may achieve increased benefit from afatinib were identified based on prespecified tumor biomarkers (p16-negative, EGFR-amplified, HER3-low, PTEN-high). Future studies are warranted to validate these findings. CLINICAL TRIAL REGISTRATION: NCT01345682.

Afatinib versus methotrexate in older patients with second-line recurrent and/or metastatic head and neck squamous cell carcinoma: subgroup analysis of the LUX-Head & Neck 1 trial.

BACKGROUND: In the phase III LUX-Head & Neck 1 (LHN1) trial, afatinib significantly improved progression-free survival (PFS) versus methotrexate in recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients progressing on/after platinum-based therapy. This report evaluates afatinib efficacy and safety in prespecified subgroups of patients aged ≥65 and <65 years. PATIENTS AND METHODS: Patients were randomized (2:1) to 40 mg/day oral afatinib or 40 mg/m(2)/week intravenous methotrexate. PFS was the primary end point; overall survival (OS) was the key secondary end point. Other end points included: objective response rate (ORR), patient-reported outcomes, tumor shrinkage, and safety. Disease control rate (DCR) was also assessed. RESULTS: Of 483 randomized patients, 27% (83 afatinib; 45 methotrexate) were aged ≥65 years (older) and 73% (239 afatinib; 116 methotrexate) <65 years (younger) at study entry. Similar PFS benefit with afatinib versus methotrexate was observed in older {median 2.8 versus 2.3 months, hazard ratio (HR) = 0.68 [95% confidence interval (CI) 0.45-1.03], P = 0.061} and younger patients [2.6 versus 1.6 months, HR = 0.79 (0.62-1.01), P = 0.052]. In older and younger patients, the median OS with afatinib versus methotrexate was 7.3 versus 6.4 months [HR = 0.84 (0.54-1.31)] and 6.7 versus 6.2 months [HR = 0.98 (0.76-1.28)]. ORRs with afatinib versus methotrexate were 10.8% versus 6.7% and 10.0% versus 5.2%; DCRs were 53.0% versus 37.8% and 47.7% versus 38.8% in older and younger patients, respectively. In both subgroups, the most frequent treatment-related adverse events were rash/acne (73%-77%) and diarrhea (70%-80%) with afatinib, and stomatitis (43%) and fatigue (31%-34%) with methotrexate. Fewer treatment-related discontinuations were observed with afatinib (each subgroup 7% versus 16%). A trend toward improved time to deterioration of global health status, pain, and swallowing with afatinib was observed in both subgroups. CONCLUSIONS: Advancing age (≥65 years) did not adversely affect clinical outcomes or safety with afatinib versus methotrexate in second-line R/M HNSCC patients. CLINICAL TRIAL REGISTRATION: NCT01345682 (ClinicalTrials.gov).

A randomized, phase II study of afatinib versus cetuximab in metastatic or recurrent squamous cell carcinoma of the head and neck.

BACKGROUND: Afatinib is an oral, irreversible ErbB family blocker that has shown activity in epidermal growth factor receptor (EGFR)-mutated lung cancer. We hypothesized that the agent would have greater antitumor activity compared with cetuximab in recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients, whose disease has progressed after platinum-containing therapy. PATIENTS AND METHODS: An open-label, randomized, phase II trial was conducted in 43 centers; 124 patients were randomized (1 : 1) to either afatinib (50 mg/day) or cetuximab (250 mg/m(2)/week) until disease progression or intolerable adverse events (AEs) (stage I), with optional crossover (stage II). The primary end point was tumor shrinkage before crossover assessed by investigator (IR) and independent central review (ICR). RESULTS: A total of 121 patients were treated (61 afatinib, 60 cetuximab) and 68 crossed over to stage II (32 and 36 respectively). In stage I, mean tumor shrinkage by IR/ICR was 10.4%/16.6% with afatinib and 5.4%/10.1% with cetuximab (P = 0.46/0.30). Objective response rate was 16.1%/8.1% with afatinib and 6.5%/9.7% with cetuximab (IR/ICR). Comparable disease control rates were observed with afatinib (50%) and cetuximab (56.5%) by IR; similar results were seen by ICR. Most common grade ≥3 drug-related AEs (DRAEs) were rash/acne (18% versus 8.3%), diarrhea (14.8% versus 0%), and stomatitis/mucositis (11.5% versus 0%) with afatinib and cetuximab, respectively. Patients with DRAEs leading to treatment discontinuation were 23% with afatinib and 5% with cetuximab. In stage II, disease control rate (IR/ICR) was 38.9%/33.3% with afatinib and 18.8%/18.8% with cetuximab. CONCLUSION: Afatinib showed antitumor activity comparable to cetuximab in R/M HNSCC in this exploratory phase II trial, although more patients on afatinib discontinued treatment due to AEs. Sequential EGFR/ErbB treatment with afatinib and cetuximab provided sustained clinical benefit in patients after crossover, suggesting a lack of cross-resistance.

Survival of patients with Stage III colon cancer is improved in hereditary non-polyposis colorectal cancer compared with sporadic cases. A Danish registry based study.

AIM: Patients with hereditary non-polyposis colorectal cancer (HNPCC) seem to have a better prognosis than those with sporadic colorectal cancer (CRC). The aim was to compare survival after Stage III CC in patients with HNPCC with those having sporadic CC. METHOD: A total of 230 patients with hereditary cancer from the Danish HNPCC Register and 3557 patients with sporadic CC from the Danish Colorectal Cancer Database, diagnosed during May 2001-December 2008, were included. HNPCC patients were classified according to mismatch repair mutation status and family pedigree. Sporadic cases had no known family history of cancer. Patient characteristics, geographical differences and survival data were analysed. RESULTS: The overall survival (OS) was better in HNPCC patients compared with sporadic CC after stratification for sex and age (P = 0.02; CI 1.04-1.7). The 5-year survival was 70% in HNPCC patients compared with 56% in sporadic CC (P < 0.001). No survival difference was found between HNPCC subgroups but a tendency to better OS was seen in patients with Lynch syndrome. No geographical differences in OS were found. The median follow-up was 3.9 (0-9.5) years for HNPCC vs 3.2 (0-9.6) years for sporadic CC. CONCLUSION: HNPCC patients with Stage III CC have a better OS compared with sporadic CC. No significant difference in OS was found within HNPCC subgroups.

A phase Ib, open-label study to assess the safety of continuous oral treatment with afatinib in combination with two chemotherapy regimens: cisplatin plus paclitaxel and cisplatin plus 5-fluorouracil, in patients with advanced solid tumors.

BACKGROUND: In this phase Ib, dose-escalation study, the oral irreversible ErbB family blocker afatinib (BIBW 2992) was combined with cisplatin (Cadila Healthcare Ltd, Ahmedabad, India) 50 or 75 mg/m(2)/paclitaxel (Bristol-Myers Squibb Pharmaceuticals Ltd, New York, USA) (Taxol)175 mg/m(2) (regimen A) or cisplatin 75-100 mg/m(2)/5-fluorouracil 750-1000 mg/m(2) (regimen B) in patients with advanced solid tumors. PATIENTS AND METHODS: The primary objective was to assess dose-limiting toxicities (DLTs) during cycle 1 for each regimen, from which the maximum tolerated dose (MTD) was determined. Patients received once daily oral afatinib 20, 30, 40 or 50 mg in 21-day cycles (3 + 3 design). RESULTS: The MTD for afatinib in regimens A (n = 26) and B (n = 21) was determined as 20 mg and 30 mg following DLTs in five and four patients in cycle 1, respectively. Most frequent adverse events (AEs, any grade) were diarrhea and nausea. Disease control was observed in 54% and 29% of patients in regimens A and B, respectively. Plasma sampling suggested no relevant pharmacokinetic interaction between afatinib and the chemotherapeutic agents. CONCLUSIONS: The MTD of afatinib was 20 mg with cisplatin-paclitaxel and 30 mg with cisplatin-5-fluorouracil. Pre-emptive management of side-effects is important to maintain adequate safety and tolerability. Both combinations showed antitumor activity across tumor types and lines of prior treatment.

A phase 1 trial of recombinant human IL-21 in combination with cetuximab in patients with metastatic colorectal cancer.

BACKGROUND: Pre-clinical data indicate enhanced anti-tumour activity when combining recombinant human interleukin-21 (rIL-21), a class 1 cytokine, with cetuximab, a monoclonal antibody, targeting the epidermal growth factor receptor. This phase 1 trial assessed the safety and tolerability of escalating doses of rIL-21 in combination with cetuximab in chemo-naïve patients with stage IV colorectal cancer. PATIENTS AND METHODS: Sequential cohorts of PS 0-1, asymptomatic patients, were treated weekly with cetuximab 250 mg m(-2) intravenously (i.v.) plus escalating i.v. doses of rIL-21 following an initial loading dose of cetuximab 400 mg m(-2). Initial treatment period was 8 weeks, with extension permitted in patients without disease progression. RESULTS: In all, 15 patients were included in this study. Adverse events related to rIL-21 or rIL-21 plus cetuximab included lethargy, nausea/vomiting, stomatitis, lymphopenia and pyrexia and were mainly ≤ grade 2. One dose limiting toxicity occurred (grade 3 diarrhoea). Maximum tolerated dose was not determined because of the premature study closure. Maximum administered dose was 100 µg kg(-1) rIL-21 weekly. In all, 60% of the patients had stable disease. Immune activation was confirmed by various T- and NK-cell activation biomarkers, including dose-dependent increases in serum sCD25. CONCLUSION: rIL-21 weekly combined with cetuximab is well tolerated at doses up to 100 µg kg(-1) and results in activation of immune response biomarkers.

Third-line therapy for metastatic colorectal cancer.

BACKGROUND: The past years' therapy for colorectal cancer has evolved rapidly with the introduction of novel cytotoxic agents such as irinotecan, capecitabine and oxaliplatin. Further advances have been achieved with the integration of targeted agents such as bevacizumab, cetuximab and recently, panitumumab. As a result, third-line treatment is now a necessary step in the optimal treatment of patients with metastatic colorectal cancer (MCRC). MATERIALS AND METHODS: We conducted a literature review of English language publications on third-line therapy for MCRC from January 2000 to April 2007. Data on median overall survival (mOS), median time to progression (mTTP) and response rate were recorded. RESULTS: We found 27 articles and 22 abstracts to fulfil the criteria. Patients who received regimens containing oxaliplatin and infusional 5-fluorouracil (5-FU) demonstrated mTTP up to 7 months and a mOS of 16 months. With irinotecan and 5-FU, mOS around 8 months were reported and with cetuximab combined with irinotecan, the highest mOS was 9.8 months. CONCLUSION: Third-line therapy in advanced colorectal cancer may improve mOS for patients with MCRC. Therefore, randomized studies should be conducted in the future.

Association of perceived physician communication style with patient satisfaction, distress, cancer-related self-efficacy, and perceived control over the disease.

The aim of the study was to investigate the association of physician communication behaviours as perceived by the patient with patient reported satisfaction, distress, cancer-related self-efficacy, and perceived control over the disease in cancer patients. Questionnaires measuring distress, self-efficacy, and perceived control were completed prior to and after the consultation by 454 patients attending an oncology outpatient clinic. After the consultation, the patients also rated the physicians' communicative behaviours by completing a patient-physician relationship inventory (PPRI), and the physicians were asked to estimate patient satisfaction. The overall results showed that higher PPRI scores of physician attentiveness and empathy were associated with greater patient satisfaction, increased self-efficacy, and reduced emotional distress following the consultation. In contrast, lower PPRI scores were associated with reduced ability of the physician to estimate patient satisfaction. The results confirm and expand previous findings, suggesting that communication is a core clinical skill in oncology.

Randomized trial of opioids versus tricyclic antidepressants for radiation-induced mucositis pain in head and neck cancer.

Patients who receive radiotherapy for head and neck cancer are likely to develop painful mucositis. The pain is characterized by a burning or stinging sensation similar to neuropathic pain sensations. The purpose of the present study was to compare the analgesic effect of a tricyclic antidepressant (TC), commonly used in the treatment of neuropathic pain, with the effect of opioids on radiation-induced mucositis pain. Forty-three patients receiving 66-68 Gy external radiation according to the DAHANCA guidelines (the Danish Head and Neck Cancer Study Group) were randomized to either morphine or TC when mucositis pain was insufficiently managed with weak analgesics. Patients with insufficient pain control in either treatment arm received supplementary medication from the opposite treatment arm. Pain was evaluated weekly using a VAS scale and the McGill Pain Questionnaire. The degree of mucositis and the degree of depression were measured at the same time intervals. Twenty-two patients entered the opioid arm and 21 the TC arm. Two patients in each arm were non-evaluable. VAS pain scores were significantly reduced in the opioid treatment arm one week after randomization (p = 0.01). Eight patients in the TC arm were managed with TC alone, but for 11 patients it was necessary to add morphine. The 20 evaluable patients in the morphine arm required no additional treatment. There were no significant differences in side effects between the two groups. Higher pain scores in the TC arm, but not in the opioid arm, were significantly correlated with higher BDI scores. Some head and neck cancer patients with radiation-induced nucositis pain may have sufficient pain control on TC alone. This might be useful in patients with relative counter-indications to opioid treatment.

A new quantitative RT-PCR assay for thymidylate synthase mRNA in blood leukocytes applied to cancer patients and healthy controls.

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). TS mRNA and protein levels in colorectal tumours are among the most important determinants for tumour response to 5-FU. TS mRNA levels in blood leukocytes may give information on pharmacokinetic and pharmacodynamic actions of 5-FU on TS as it has previously been shown that inhibition of TS levels by 5-FU in bone marrow leukocytes resembles the degree of TS inhibition in colorectal tumours. The aim of this study was to develop a quantitative high-throughput RT-PCR assay for TS mRNA expression in blood leukocytes (CURT-PCR). Furthermore the TS mRNA levels in blood of patients with colorectal cancer and healthy controls was compared. TS mRNA levels in 17 patients with colorectal cancer did not differ from 20 matched controls whereas a group of 14 younger controls had significantly lower TS mRNA expression than patients and matched controls. In order to investigate the sensitivity of the assay towards cellular reactions such as proliferative stimuli, isolated blood leukocytes were stimulated with phytohemagglutinin both in mitogenic and non-mitogenic concentrations and an induction of TS mRNA expression was measured in both cases. TS activity and cellular proliferation also increased but only at mitogenic concentrations, suggesting that TS mRNA expression is an early leukocyte activation marker. This new CURT-PCR assay may allow improved studies of functional kinetics of drugs with impact upon TS. Further studies are required to establish the possible clinical benefit of TS mRNA measurements in blood leukocytes.

The effects of low-dose methotrexate on thymidylate synthetase activity in human peripheral blood mononuclear cells.

OBJECTIVE: Methotrexate (MTX) in low doses is widely used in the treatment of rheumatoid arthritis (RA) and it is not known whether its effects are due to immunosuppressive and/or anti-inflammatory actions. High concentrations of MTX inhibit the activity of thymidylate synthetase (TS) and dihydrofolate reductase essential for DNA synthesis. This study investigated the effects of low-dose MTX on TS activity and proliferation in human peripheral blood mononuclear cells (PBMC). METHODS: The MTX concentrations in our experiments were chosen according to the plasma concentrations measured in 8 RA patients treated with MTX. The effect of MTX on TS activity and DNA synthesis were measured in stimulated normal PBMC and in PBMC obtained from 6 RA patients treated with oral MTX before and 2 hours after intake of their weekly MTX dose. The effect of MTX on the TS mRNA concentration was also investigated in order to elucidate its effect on TS production. RESULTS: Low-dose MTX significantly inhibited TS activity and the proliferation of stimulated PBMC independent of the mode of activation. Interestingly, the concentration of TS mRNA in normal PBMC was upregulated by the presence of MTX. Finally, there was no difference between TS activity measured before and after MTX intake in 6 RA patients on long-term MTX treatment. CONCLUSION: We show that low concentrations of MTX inhibit TS activity in vitro. An in vivo effect cannot, however, be proven given our study design. The role of these in vitro findings is discussed, particularly in relation to the in vivo effects of MTX.

Changes in thymidylate synthase mRNA in blood leukocytes from patients with colorectal cancer after bolus administration of 5-fluorouracil.

5-fluorouracil (5-FU) is considered the standard antineoplastic drug of choice for metastatic colorectal cancer. It has been suggested that 5-FU administered as bolus infusion is cytotoxic mainly through an RNA damaging effect. We investigated the effect of i.v. bolus 5-FU 500-600 mg/m2 on the 5-FU target enzyme, thymidylate synthase (TS) mRNA, in blood leukocytes before and after courses 1 and 3 in 21 patients with colorectal cancer. TS mRNA expression was quantified using an RT-PCR assay with an internal RNA standard. Median TS mRNA expression decreased significantly 30 min after course no. 1 (p = 0.004), and both 15 min and 30 min after course 3 (p = 0.01). After course 1, the median TS mRNA expression decreased by 31% and after course 3 by 24%. Pharmacokinetic parameters were similar for individual patients during the two courses but did not correlate with the degree of TS mRNA inhibition. The present results indicate that TS mRNA in blood leukocytes may be an early indicator of an RNA damaging effect after i.v. bolus infusion of 5-FU.

The ability of hypoxia to modify the gene expression of thymidylate synthase in tumour cells in vivo.

PURPOSE: Hypoxic cells in tumours are resistant to 5-fluorouracil (5-FU). This in vivo study investigated the ability of hypoxia to regulate the gene expression of thymidylate synthase (TS), the target enzyme of 5-FU. MATERIALS AND METHODS: C3H mammary carcinomas, grown in the feet of female CDF1 mice, were used for all experiments. Mice were placed in a 10% oxygen environment for various time periods and the tumour oxygen status was determined with an Eppendorf oxygen electrode. The animals were then injected with BrdU (100 mg/kg, i.p.). Tumours were excised and immediately frozen (-80 degrees C) until isolation of total RNA. The mRNA was reversibly transcribed to complementary DNA and the resulting cDNA amplified in a multiplex PCR reaction, with beta-actin as the internal reference gene. RESULTS: One hour of low oxygen breathing made tumours significantly more hypoxic. This increase was maintained for a maximum incubation period of 48 h. In the same tumours, no change in TS gene expression was seen with up to 3 h of low oxygen breathing. At longer times it decreased, reaching significance at 12-24 h and remaining at this lower level for up to 48 h. BrdU labelling was significantly reduced after breathing low O2 for 24 h (p = 0.001). CONCLUSION: Hypoxia-induced down-regulation of TS gene expression was observed. This would be expected to make hypoxic tumour cells more sensitive to 5-FU. Other mechanisms must be responsible for the previously reported resistance to this drug.

The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors.

PURPOSE: To investigate the activity of combretastatin A-4 disodium phosphate in a transplanted C3H mouse mammary carcinoma and several murine spontaneous tumors. METHODS AND MATERIALS: The C3H mammary carcinoma was grown in the right rear foot of female CDF1 mice and treated when 200 mm3 in size. Spontaneous tumors (341-1437 mm3 in size) arose at different sites in female CDF1 mice that, 19-21 months earlier, had been irradiated. Oxygen partial pressure (pO2) distributions in the C3H tumors were measured with an Eppendorf oxygen electrode at various times after injecting combretastatin (100 mg/kg, i.p.) in restrained, nonanesthetized mice. Immediately after measurement, tumors were excised and necrotic fraction determined from histological sections. In the spontaneous tumors, pO2 was measured before and 3 h after giving combretastatin. The location of these spontaneous tumors required that measurements be made in anesthetised animals, achieved by injecting a mixture of hypnorm and diazepam. RESULTS: In untreated C3H tumors, the mean (+/- 1 SE) percentage of pO2 values < or = 2.5 mmHg was 32% (+/- 11). This was significantly (Student's t-test; p < 0.05) increased to 74% (+/- 4) within 1 h after injecting combretastatin, and remained at this level for at least 6 h, although some recovery was seen at 12 and 24 h. The necrotic fraction in control tumors was 1.9% (+/- 0.4) and this was significantly increased to 16.1% (+/- 3.7) 24 h after drug administration. In spontaneous tumors, the pO2 measurements indicated that 5 of 6 showed some response to combretastatin, although the degree of change was variable. CONCLUSIONS: Combretastatin increased tumor hypoxia and necrosis in the C3H mammary carcinoma, consistent with the induction of vascular damage. Drug-induced changes in pO2 were also found in spontaneous tumors, suggesting that the activity of this drug is not restricted to transplanted tumors alone.

Effect of induced hypercholesterolemia in rabbits on functional responses of isolated large proximal and small distal coronary arteries.

We studied the effects of hypercholesterolemia on the vascular responses of proximal and distal parts of the rabbit coronary circulation in two consecutive studies. For 12 weeks, New Zealand White rabbits were fed a control diet or a diet with 1% cholesterol dissolved in either 3% coconut oil (study A) or ether (study B). Isolated proximal epicardial and distal intramyocardial coronary arteries from control and hypercholesterolemic rabbits were mounted for isometric tension recording in a double myograph. In study A for hypercholesterolemic rabbits (n = 12), the maximal relaxation and sensitivity to acetylcholine (ACh) were significantly decreased in proximal coronary segments contracted with 30 mmol/l potassium solution compared with segments from control rabbits (n = 13). The only change observed in distal coronary segments was a slight decrease in relaxation in response to low ACh concentrations (10(-8) and 3 x 10(-8) mol/l). However, in study B for proximal coronary and distal coronary segments from hypercholesterolemic rabbits (n = 13), the area under the ACh relaxation curve was increased compared with that of control rabbits (n = 12). Other parameters that were similarly affected in studies A and B include the following: 1) proximal coronary segments from hypercholesterolemic rabbits were more sensitive to sodium nitroprusside (SNP) than were those from control rabbits, but this was not true for distal coronary segments; 2) endothelial removal from arterial segments of control rabbits induced a significant increase in sensitivity and maximal relaxation to SNP of proximal coronary and distal coronary arteries; 3) in segments from hypercholesterolemic rabbits, the absence of endothelium did not alter the response of proximal coronary segments to SNP but did augment the relaxation of distal coronary segments to SNP; 4) the maximal response to 5-hydroxytryptamine in proximal coronary arteries from hypercholesterolemic rabbits was increased compared with those from control rabbits, whereas such changes were not observed in distal coronary arteries; and 5) histological examination showed the presence of atheromatous plaques in proximal coronary but not in distal coronary segments from treated animals. In conclusion, the present investigation demonstrates that induced hypercholesterolemia alters both the structure and function of proximal parts of the coronary circulation. In distal coronary arteries of hypercholesterolemic rabbits, the only change observed was an impaired endothelium-dependent cholinergic relaxation, but even this change appeared to be dependent on the manner in which cholesterol was added to the diet, although parallel studies are required to confirm this.

Functional properties in vitro of systemic small arteries from rabbits fed a cholesterol-rich diet for 12 weeks.

1. Recent evidence has suggested that the impairment of endothelium-dependent cholinergic relaxation in vitro, which is seen in atherosclerotic large arteries of animals and man, could be part of a general deleterious effect to the endothelium of hypercholesterolaemia. 2. This possibility has been investigated in vitro by measuring the response to acetylcholine, sodium nitroprusside, 5-hydroxytryptamine and noradrenaline in segments of aorta and of femoral, mesenteric and cerebral small arteries (internal diameter approximately 200 microns) from control rabbits (n = 12) and from rabbits fed a 1% (w/w) cholesterol and 3% (w/w) coconut oil diet (n = 12) for 12 weeks. 3. Thoracic aorta segments from the control rabbits exhibited a maximal relaxation in response to acetylcholine of 64 +/- 11% compared with 10 +/- 5% (P less than 0.01) for thoracic segments from cholesterol-fed animals. Cerebral, femoral and mesenteric small arteries exposed to acetylcholine (10(-9)-10(-4) mol/l) relaxed to the same degree as arteries from control rabbits. The responses to sodium nitroprusside and bradykinin of the small arteries from the cholesterol-fed rabbits remained unaffected. 5-Hydroxytryptamine evoked comparable contractions in the small arteries, while the sensitivity to noradrenaline of the femoral small arteries was significantly decreased and the response of cerebral small arteries to noradrenaline in cholesterol-fed rabbits slightly increased compared with control rabbits. 4. Aorta from the cholesterol-fed rabbits had extensive atheromatous lesions. Morphological measurements and histological examination showed unchanged thickness and light microscopic appearance of intima and media of small arteries from cholesterol-fed animals compared with control animals. 5. The present study indicates that hypercholesterolaemia in this rabbit model is followed by atherosclerotic lesions and changed function of large arteries, but that both function and structure of systemic small arteries are largely unaffected.