CYP2D6 genotype predicts tamoxifen discontinuation and drug response: a secondary analysis of the KARISMA trial.

BACKGROUND: Guidelines regarding whether tamoxifen should be prescribed based on women's cytochrome P450 2D6 (CYP2D6) genotypes are conflicting and have caused confusion. This study aims to investigate if CYP2D6 metabolizer status is associated with tamoxifen-related endocrine symptoms, tamoxifen discontinuation, and mammographic density change. PATIENTS AND METHODS: We used data from 1440 healthy women who participated the KARISMA dose determination trial. Endocrine symptoms were measured using a modified Functional Assessment of Cancer Therapy - Endocrine Symptoms (FACT-ES) questionnaire. Change in mammographic density was measured and used as a proxy for tamoxifen response. Participants were genotyped and categorized as poor, intermediate, normal, or ultrarapid CYP2D6 metabolizers. RESULTS: The median endoxifen level per mg oral tamoxifen among poor, intermediate, normal and ultrarapid CYP2D6 metabolizers were 0.18 ng/ml, 0.38 ng/ml, 0.56 ng/ml and 0.67 ng/ml, respectively. Ultrarapid CYP2D6 metabolizers were more likely than other groups to report a clinically relevant change in cold sweats, hot flash, mood swings, being irritable, as well as the overall modified FACT-ES score, after taking tamoxifen. The 6-month tamoxifen discontinuation rates among poor, intermediate, normal, and ultrarapid CYP2D6 metabolizers were 25.7%, 23.6%, 28.6%, and 44.4%, respectively. Among those who continued and finished the 6-month tamoxifen intervention, the mean change in dense area among poor, intermediate, normal, and ultrarapid CYP2D6 metabolizers were -0.8 cm2, -4.5 cm2, -4.1 cm2, and -8.0 cm2 respectively. CONCLUSIONS: Poor CYP2D6 metabolizers are likely to experience an impaired response to tamoxifen, measured through mammographic density reduction. In contrast, ultrarapid CYP2D6 metabolizers are at risk for exaggerated response with pronounced adverse effects that may lead to treatment discontinuation.

FGF receptor genes and breast cancer susceptibility: results from the Breast Cancer Association Consortium.

BACKGROUND: Breast cancer is one of the most common malignancies in women. Genome-wide association studies have identified FGFR2 as a breast cancer susceptibility gene. Common variation in other fibroblast growth factor (FGF) receptors might also modify risk. We tested this hypothesis by studying genotyped single-nucleotide polymorphisms (SNPs) and imputed SNPs in FGFR1, FGFR3, FGFR4 and FGFRL1 in the Breast Cancer Association Consortium. METHODS: Data were combined from 49 studies, including 53 835 cases and 50 156 controls, of which 89 050 (46 450 cases and 42 600 controls) were of European ancestry, 12 893 (6269 cases and 6624 controls) of Asian and 2048 (1116 cases and 932 controls) of African ancestry. Associations with risk of breast cancer, overall and by disease sub-type, were assessed using unconditional logistic regression. RESULTS: Little evidence of association with breast cancer risk was observed for SNPs in the FGF receptor genes. The strongest evidence in European women was for rs743682 in FGFR3; the estimated per-allele odds ratio was 1.05 (95% confidence interval=1.02-1.09, P=0.0020), which is substantially lower than that observed for SNPs in FGFR2. CONCLUSION: Our results suggest that common variants in the other FGF receptors are not associated with risk of breast cancer to the degree observed for FGFR2.

Effects of three anti-TNF-alpha drugs: etanercept, infliximab and pirfenidone on release of TNF-alpha in medium and TNF-alpha associated with the cell in vitro.

Tumor necrosis factor-alpha (TNF-alpha) is a vital component of the inflammatory process and its aberrant over-expression has been linked to numerous disease states. New treatment strategies have sought to reduce circulating TNF-alpha, either with neutralizing anti-TNF-alpha binding proteins such as etanercept or via drugs that inhibit de novo TNF-alpha synthesis like pirfenidone. In the present study, we examined the effects of both classes of drugs on secreted and cell-associated TNF-alpha produced by THP-1 cells. All of the tested drugs significantly reduced secreted levels of bioactive TNF-alpha following stimulation with LPS as measured by bioassay. However, etanercept-treated cells had approximately six-fold higher levels of cell-associated TNF-alpha compared with that of the LPS-alone treatment group. Surprisingly, LPS+infliximab treated cells did not increase cell-associated TNF-alpha relative to the LPS-alone treatment. Pirfenidone significantly reduced both secreted and cell-associated TNF-alpha levels. These drug-related differences in cell-associated TNF-alpha may have broad implications in the future for the therapeutic uses of anti-TNF-alpha drugs in the management of TNF-alpha diseases.

High incidence of skewed X chromosome inactivation in young patients with familial non-BRCA1/BRCA2 breast cancer.

BACKGROUND: A higher frequency of skewed X chromosome inactivation has been reported in a consecutive series of young patients with breast cancer compared with controls of a similar age. OBJECTIVE: To investigate the X inactivation pattern in patients with familial non-BRCA1/BRCA2 breast cancer (n = 272), BRCA1/BRCA2 germline mutations (n = 35), and sporadic breast cancer (n = 292). METHODS: X inactivation pattern was determined by polymerase chain reaction analysis of the highly polymorphic CAG repeat in the androgen receptor (AR) gene. The X inactivation pattern was classified as skewed when 90% or more of the cells preferentially expressed one X chromosome. RESULTS: Young patients with familial breast cancer had a significantly higher frequency of skewed X inactivation (11.2%) than young controls (2.7%) (p = 0.001). There was also a strong tendency for middle aged patients with sporadic breast cancer to be more skewed than middle aged controls (13.6% v 4.4%) (p = 0.02). No association between skewed X inactivation and breast cancer was found for the BRCA1/BRCA2 patients . CONCLUSIONS: Skewed X inactivation may be a risk factor for the development of breast cancer in both sporadic and familial breast cancer and may indicate an effect of X linked genes.

Pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production.

There are currently no effective, long-term drug therapies for the treatment of leiomyomas. Pirfenidone (Marnac, Inc.) is an antifibrotic agent that is being tested for use in patients with pulmonary fibrosis. Because leiomyomas are characterized also by increased cell proliferation and tissue fibrosis, we examined the effects of pirfenidone on cell proliferation and collagen expression in cultured myometrial and leiomyoma smooth muscle cells. Effects of pirfenidone on proliferation of myometrial and leiomyoma cells were measured using tritiated thymidine incorporation assays and changes in actual cell numbers. Possible cytotoxic effects were examined using lactate dehydrogenase assays and trypan blue exclusion. Effects on collagen type I and type III production were assessed by Northern blotting. Doses of pirfenidone tested were: 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. Serum-stimulated increases in DNA synthesis and cell proliferation by myometrial and leiomyoma cells were significantly inhibited in a dose-dependent manner by pirfenidone. Densitometric analysis of Northern blots showed significantly decreased expression of collagen type I and type III messenger RNAs in both leiomyoma and myometrial cells. Lactate dehydrogenase assays and trypan blue exclusion measurements showed no cytotoxic effect of pirfenidone at concentrations that inhibited cell proliferation and collagen production. Pirfenidone is an effective inhibitor of myometrial and leiomyoma cell proliferation in vitro and reduces the messenger RNA levels of collagen types I and III in a dose-dependent manner. This compound may prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas.

Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rats.

Fibrosis leads to chronic impairment of cardiac and renal function and thus reversal of existing fibrosis may improve function and survival. This project has determined whether pirfenidone, a new antifibrotic compound, and spironolactone, an aldosterone antagonist, reverse both deposition of the major extracellular matrix proteins, collagen and fibronectin, and functional changes in the streptozotocin(STZ)-diabetic rat. Streptozotocin (65 mg kg(-1) i.v.)-treated rats given pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone; approximately 200 mg kg(-1) day(-1) as 0.2 - 2g 1(-1) drinking water) or spironolactone (50 mg kg(-1) day(-1) s.c.) for 4 weeks starting 4 weeks after STZ showed no attenuation of the increased blood glucose concentrations and increased food and water intakes which characterize diabetes in this model. STZ-treatment increased perivascular and interstitial collagen deposition in the left ventricle and kidney, and surrounding the aorta. Cardiac, renal and plasma fibronectin concentrations increased in STZ-diabetic rats. Passive diastolic stiffness increased in isolated hearts from STZ-diabetic rats. Both pirfenidone and spironolactone treatment attenuated these increases without normalizing the decreased +dP/dt(max) of STZ-diabetic hearts. Left ventricular papillary muscles from STZ-treated rats showed decreased maximal positive inotropic responses to noradrenaline, EMD 57033 (calcium sensitizer) and calcium chloride; this was not reversed by pirfenidone or spironolactone treatment. STZ-treatment transiently decreased GFR and urine flow rates in isolated perfused kidneys; pirfenidone but not spironolactone prevented the return to control values. Thus, short-term pirfenidone and spironolactone treatment reversed cardiac and renal fibrosis and attenuated the increased diastolic stiffness without normalizing cardiac contractility or renal function in STZ-diabetic rats.

Pirfenidone prevents collagen accumulation in the remnant kidney in rats with partial nephrectomy.

Pirfenidone (PFD) is a new compound that prevents and even reverses the extracellular matrix accumulation in several organs as shown by experimental and clinical studies. In the present study, we examined the effect of PFD (500 mg/kg daily in the food) on the progression of chronic renal failure (CRF) in the 5/6 nephrectomy rat model. Proteinuria progressively increased in rats with renal ablation (C) at 12 weeks. Urinary protein excretion in PFD-treated rats (P) was numerically lower than C, but the difference did not reach statistical significance. In contrast, in the chronic phase, PFD improved renal function and reduced collagen accumulation detected by hydroxyproline content (OH-Pro) in the cortex of the remnant kidney. Although creatinine clearance decreased with time in C, the values in P were significantly better at 10 and 12 weeks. The OH-Pro in C at 12 weeks was significantly higher than that of no-ablation, sham-operated rats, whereas OH-Pro in CRF was lower in (P). Expression of mRNA for type IV and I collagen in the cortex also increased in C, but it was inhibited in (P). To study the role that TGF-beta plays in the regulatory process following CRF, we examined the expression of TGF-beta mRNA in this model. Levels of cortical TGF-beta mRNA in C were significantly elevated at 12 weeks. The increase was suppressed by PFD. These results demonstrate that PFD attenuates the development of CRF by preventing collagen accumulation in this model, and suggest that PFD can be clinically useful for treating CRF.

Pirfenidone diminishes cyclophosphamide-induced lung fibrosis in mice.

The deposition of excess or abnormal collagen characteristic of pulmonary fibrosis can disrupt gas exchange resulting in severe respiratory impairment. There currently are no effective pharmacologic agents available that inhibit the fibrotic process. Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is an investigational drug that, when administered at 0.5% (w/w) of the diet, decreases both histologic and biochemical evidence of lung fibrosis in hamsters treated intratracheally with bleomycin. The effectiveness of pirfenidone against lung fibrosis initiated by a systemically administered agent was investigated in mice treated intraperitoneally with 200 mg/kg cyclophosphamide (CP). Control and treated animals were fed a diet containing 0.277% (w/w) pirfenidone beginning 1 day after CP. Despite anorexia in the CP-treated mice the first day after treatment, they ingested a greater average pirfenidone dose over 20 days than saline-treated control mice (717 +/- 44 versus 564 +/- 30 mg/kg per day, respectively). Total lung hydroxyproline content, an index of fibrosis, was significantly lower 21 days after treatment with CP plus pirfenidone as compared to mice treated with CP alone. Although microscopic lung fibrosis scores were not significantly decreased by pirfenidone in CP-treated mice, the overall incidence of fibrosis was significantly decreased. Histologically, mice treated with CP showed fibrosis while mice treated with CP plus pirfenidone exhibited fewer abnormalities. The rate of hydroxyproline synthesis by lung tissue 9 days after treatment with CP was significantly elevated. This rate was not affected by pirfenidone treatment. Overall, these data support an antifibrotic effect of pirfenidone against CP-induced lung fibrosis in mice. The mechanism of its effect is not known, but appears to be unrelated to an inhibition of collagen synthesis.

Pirfenidone reduces in vitro rat renal fibroblast activation and mitogenesis.

BACKGROUND: Fibroblasts have been universally recognised in tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. Recently, pirfenidone (PF) has been shown to both ameliorate progressive fibrosis and reduce established scarring after ureteric obstruction (UUO) in the rat, suggesting that it is a novel anti-fibrotic agent. The objective of this study was therefore to determine if these effects include down-regulation of fibroblast function. METHODS: Cortical fibroblasts were obtained from outgrowth cultures of renal tissue isolated from kidneys 3 days after UUO and constituted 100% of cells studied. Functional studies examined the effects of 20 and 200 microg/ml PF on basal serum stimulated activity. Activation was examined by western blotting for alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Cell proliferation, collagenase activity and collagen production were determined from kinetic studies, zymography for MMP2 and [3H] proline incorporation in collagenous proteins respectively. RESULTS: Proliferation, as measured by [3H] thymidine incorporation, was reduced in dose dependent manner by 20 and 200 microg/ml PF (p<0.05; 200 vs 0 microg/ml). Likewise, 200 microg/ml PF reduced cell population growth over 5 days of culture (p<0.05 vs 0 microg/ml). PF (200 microg/ml) decreased alphaSMA and CTGF protein expression to 66+/-13 and 37+/-26% of basal levels respectively (both p<0.05 vs 0 microg/ml). Synthesis of collagen was unaffected by PF. Maximal dose of PF produced a modest reduction in MMP2 lytic activity (p=0.05). Effects of PF were independent of cell toxicity. CONCLUSIONS: Down-regulation of renal fibroblast activation and proliferation are specific actions of PF.

Effects of pirfenidone on the generation of reactive oxygen species in vitro.

Pirfenidone is a newly developed antifibrotic drug that has been reported to retard the progression of pulmonary fibrosis induced by bleomycin and cyclophosphamide in animal models of lung fibrosis. The present in vitro studies using noncellular and cellular systems evaluated the antioxidant and cytotoxic properties of this drug. The Fenton reaction [Fe(II) + H2O2 --> Fe(III) + *OH + OH-] and the xanthine/xanthine oxidase system were used as sources of hydroxyl (*OH) and superoxide anion (O2*-) radicals, respectively. Electron spin resonance spin trapping was used for free radical detection and measurement. The reaction rate of pirfenidone with *OH was found to be 1.63 x 10(10) M(-1) s(-1), which is comparable to several well-established antioxidants, such as ascorbate, glutathione, cysteine, azide, and lipoic acid. Compared to *OH radicals, the O2*- scavenging was less efficient 42.36 M(-1) s(-1) with pirfenidone. Pirfenidone was also effective in inhibiting zymosan-stimulated chemiluminescence. In a noncellular model of lipid peroxidation, pirfenidone inhibited crystalline silica-induced lipid peroxidation. The inhibition of crystalline silica-induced cytotoxic reactions and lipid peroxidation combined with the efficient antioxidant properties of pirfenidone indicate that this agent may express its antifibrotic effects partly through its ability to scavenge reactive oxygen species.

Management of radiation-induced moist skin desquamation using hydrocolloid dressing.

Moist skin desquamation has been of concern to radiation oncologists, nurses and patients since the inception of this mode of therapy. As radiation treatment machines became more sophisticated, severe reactions became less of a problem. However, with the increasing use of chemotherapy and radiation as combined modalities, moist skin reaction is occurring with greater frequency. A noncomparative study of 20 patients using a hydrocolloid occlusive dressing (Duoderm) was initiated. The purpose of the study was to determine whether moist occlusive healing would be beneficial. The dressing was evaluated on the basis of healing time, safety, wound temperature, bacterial growth, and comfort. Data were collected using photographs, bacterial cultures, temperature probes, and patient evaluations. Eighteen patients completed the study. All patients' skin reactions healed. There were no wound infections evident. Mean healing time was 12 days, with mean wound temperature relative to body core -0.8 degree C on day 1 and -1.2 degrees C on the healed site. Patient results on comfort were: 8 of 18 excellent, 7 of 18 good, 3 of 18 fair, and 0 of 18 poor. The results of this study indicate that a hydrocolloid occlusive dressing can be effective in the healing process of moist skin reaction that is due to radiation therapy.

Preventive effect of pirfenidone against experimental sclerosing peritonitis in rats.

We investigated the preventive effect of pirfenidone (PFD), a newly developed anti-fibrotic agent, on experimental sclerosing peritonitis (sp) which we had established in rats. Male Wistar rats (150-250 g) were divided into the following two groups; 7 rats were treated with daily intraperitoneal (i.p.) injection of 0.1% chlorhexidine gluconate (CH) and 15% ethanol solved in 2 ml of saline for 26 days as a control of SP model (CH group) and 6 rats were treated daily with peroral (p.o.) administration of 350 mg/kg of PFD in addition to the daily i.p. injection of aforementioned CH-ethanol-saline solution for 26 days (CH + PFD group). Macroscopic intraperitoneal changes and histological fibrotic changes were graded by scoring in a blind manner. Actual subserosal thickness was measured by observation under light microscope. Body weight gain was significantly greater in CH + PFD group than in CH group (P < 0.05). Macroscopic intraperitoneal adhesion changes in CH + PFD group were significantly fewer than in CH group (P < 0.05). Fibrotic changes were fewer and subserosal thickness in liver and intestine were smaller in CH + PFD group with statistical significance (p < 0.1). We concluded that PFD markedly inhibited or prevented fibrotic changes in the experimental sclerosing peritonitis induced by CH.

TGFBR1(*)6A and Int7G24A variants of transforming growth factor-beta receptor 1 in Swedish familial and sporadic breast cancer.

Two common variants in transforming growth factor-beta receptor 1 (TGFBR1), TGFBR1(*)6A and Int7G24A, A allele, have been shown to act as low-penetrance tumour susceptibility alleles in several common cancers, including breast cancer. We evaluated the TGFBR1 9A/6A and Int7G24A variant frequencies in two breast cancer cohorts; a population-based cohort of breast cancer with defined family history (n=459) and in breast cancer patients from a familial cancer clinic (n=340) and in 856 controls from the Stockholm region. The familial patients from both cohorts were further divided into high- and low-risk familial breast cancer based on pedigree analysis. There was no overall association with either variant and breast cancer risk. The TGFBR1(*)6A allelic frequency was, however, higher in low-risk familial breast cancer (0.138), compared to controls (0.106; P=0.04). No significant difference was found in the high-risk familial (0.102) or sporadic cases (0.109; P=0.83 and 0.83, respectively). TGFBR1(*)6A carrier status was further associated with a high-grade sporadic breast cancer (odds ratio: 2.27; 95% confidence interval: 1.01-5.11; P=0.049). These results indicate that the TGFBR1(*)6A variant may be associated with an increased risk of low-risk familial breast cancer and might be a marker for poorly differentiated breast cancer. The Int7G24A variant was not associated with breast cancer risk or clinical presentation of the disease including prognosis in our material.

Pirfenidone for chronic progressive multiple sclerosis.

Current treatment of secondary progressive multiple sclerosis is unsatisfactory in stabilizing or reversing the disabilities associated with the disease. Pirfenidone is a new non-peptide drug which has been shown in vitro and in vivo to decrease synthesis of Tumor Necrosis Factor-alpha (TNF-alpha) and block receptors for TNF-alpha. Since TNF-alpha seems to be a key cytokine in demyelination, a pilot study of oral pirfenidone was undertaken in an open-label baseline vs treatment protocol over a 2-year period in 20 patients. Fourteen (14120) patients (70%) remained in the study for 2 years. Three (3/20) patients dropped out early because of gastrointestinal adverse reactions, and another three patients dropped out for personal reasons after 1 year (not because of adverse reactions). The remaining patients did not manifest any other drug-related adverse reactions and complications. Improvement or stabilization occurred in most patients at about 3 months, and it was sustained at 6, 12 and 24 months as evaluated by both primary and secondary outcome measures. Magnetic resonance imaging foiled to reveal any new lesions. Thus, pirfenidone appears to offer protection against the usual slow progression of the disease. Most patients experienced a distinct decrease in their neurological disability. These findings indicate that an extensive multi-center double blind and placebo controlled trial is warranted.

Pharmacokinetics of orally administered pirfenidone in male and female beagles.

Pirfenidone, a promising antifibrotic agent, was administered orally to dogs at 0, 40, 140, and 400 mg/kg/day. Serum was collected for pirfenidone assay at 0, 26 and 39 weeks of treatment. From the pirfenidone concentrations, pharmacokinetic parameters were determined for each dog at each treatment interval. The only significant differences because of gender were for concentration maxima. Unsurprisingly, there were many significant differences because of dose in concentration maximum and area under curve (AUC), and significant, positive linear correlations of both parameters with dose. There were few significant differences in time of maximal concentration and no correlation with dose. The mean +/- SE clearances were 1.99 +/- 0.13, 1.64 +/- 0.13 and 1.78 +/- 0.14 L/h/kg for doses of 40, 140, and 400 mg/kg, respectively, with no significant differences attributable to dose. There was an unexplained pattern in maximal concentration and AUC with regard to duration of treatment, with the parameters being highest at week 0, lowest at week 26, and intermediate at week 39. Clearance had the reverse pattern; time of maximal concentration had no pattern.