Comparative nonclinical assessments of the biosimilar PF-06410293 and originator adalimumab.

Adalimumab, a recombinant fully human monoclonal antibody targeting tumor necrosis factor (TNF), is approved in the United States and Europe to treat various inflammatory and autoimmune indications. Biosimilars are approved biologics highly similar, but not identical, to approved biotherapeutics. To support clinical development of PF-06410293, an adalimumab biosimilar, nonclinical studies evaluated the structural, functional, toxicologic, and toxicokinetic similarity to originator adalimumab sourced from the United States (adalimumab-US) and European Union (adalimumab-EU). Structural similarity was assessed by peptide mapping. Biologic activity was measured via inhibition of TNF-induced apoptosis and Fc-based functionality assessments. In vivo nonclinical similarity was evaluated in a toxicity study in cynomolgus monkeys administered subcutaneous PF-06410293 or adalimumab-EU (0 or 157 mg/kg/week). Peptide mapping demonstrated PF-06410293, adalimumab-US, and adalimumab-EU had identical amino acid sequences. Comparative functional and binding assessments were similar. Effects of PF-06410293 and adalimumab-EU were similar and limited to pharmacologically mediated decreased cellularity of lymphoid follicles and germinal centers in spleen. Toxicokinetics were similar; maximum plasma concentration and area-under-the-concentration-time curve ratio of PF-06410293:adalimumab-EU ranged from 1.0 to 1.2. These studies supported PF-06410293 entry into clinical development. Many regulatory agencies now only request nonclinical in vivo testing if there is residual uncertainty regarding biosimilarity after in vitro analytical studies.

Nonclinical assessments of the potential biosimilar PF-06439535 and bevacizumab.

Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non-small-cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF-06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab-EU) and United States (bevacizumab-US). Peptide mapping demonstrated the amino acid sequence of PF-06439535 was identical to bevacizumab-EU and bevacizumab-US. Biologic activity, measured via inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF-06439535 or bevacizumab-EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article-related effects were limited to physeal dysplasia of the distal femur. The potential for non-target-mediated toxicity of PF-06439535 was evaluated in rats administered intravenous PF-06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF-06439535 to bevacizumab, supporting entry into clinical development.

Key considerations in the preclinical development of biosimilars.

Biosimilar development requires several steps: selection of an appropriate reference biologic, understanding the key molecular attributes of that reference biologic and development of a manufacturing process to match these attributes of the reference biologic product. The European Medicines Agency (EMA) and the FDA guidance documents state that, in lieu of conducting extensive preclinical and clinical studies typically required for approval of novel biologics, biosimilars must undergo a rigorous similarity evaluation. The aim of this article is to increase understanding of the preclinical development and evaluation process for biosimilars, as required by the regulatory agencies, that precedes the clinical testing of biosimilars in humans.

Two-year carcinogenicity study in rats with a nonnucleoside reverse transcriptase inhibitor.

Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment.

Comparative nonclinical assessments of the proposed biosimilar PF-05280014 and trastuzumab (Herceptin(®)).

BACKGROUND AND OBJECTIVES: Trastuzumab (Herceptin(®)) is a humanized monoclonal antibody (mAb) that binds to the HER2 protein. PF-05280014 is being developed as a potential biosimilar to trastuzumab products marketed in the United States (trastuzumab-US) and European Union (trastuzumab-EU). Nonclinical studies were designed to evaluate the similarity of PF-05280014 to trastuzumab-US and trastuzumab-EU using in vitro structural and functional analyses, and in vivo pharmacokinetic and immunogenicity assessments. METHODS: Peptide mapping was utilized to determine structural similarity. Functional similarity was assessed via an in vitro tumor cell growth inhibition assay. CD-1 male mice were administered a single-dose (0, 1, 10, or 100 mg/kg) of PF-05280014, trastuzumab-US, or trastuzumab-EU. Mice were monitored for clinical signs and body weight changes over a 4-month period. At approximately 720, 1,080, 1,440, 2,160, and 2,880 h post-dose, terminal blood samples were collected and assayed for PF-05280014, trastuzumab-US, or trastuzumab-EU concentrations and anti-drug antibodies (ADA). Values for C max, area under the concentration time curve (AUC), clearance (CL), volume of distribution (V ss), half-life (t ½), and the presence of ADA were determined. RESULTS: In this report, peptide mapping of PF-05280014, trastuzumab-US, and trastuzumab-EU showed similar chromatographic profiles in a side-by-side analysis. The tumor cell growth inhibition of PF-05280014 was similar to trastuzumab-US and trastuzumab-EU. C max and AUC0-∞ values in mice were similar and dose-dependent across the mAbs at all doses, and CL and V ss values were similar and dose-independent. The CL values across doses ranged from 0.193 to 0.350 mL/h/kg (PF-05280014), from 0.200 to 0.346 mL/h/kg (trastuzumab-US), and from 0.193 to 0.335 mL/h/kg (trastuzumab-EU). V ss values across doses ranged from 84.9 to 120 mL/kg (PF-05280014), 86.7 to 130 mL/kg (trastuzumab-US), and 85.4 to 116 mL/kg (trastuzumab-EU). The incidence of ADA was low (~10%) and also similar across all dose levels and the three mAbs. The lower exposure generally observed in ADA-positive animals did not impact the overall PK interpretation. All animals survived to their scheduled terminal blood collection with no mAb-related differences in body weight gain or clinical signs. CONCLUSIONS: PF-05280014, trastuzumab-US, and trastuzumab-EU were well tolerated during the 4-month observation period following a single dose of up to 100 mg/kg. PF-05280014, trastuzumab-US, and trastuzumab-EU showed similar structural properties, tumor cell growth inhibition properties, and PK profiles. The incidence of ADA was low and similar across the three mAbs. The results of these studies support the development of PF-05280014 as a proposed biosimilar to Herceptin.

Comparative nonclinical assessments of the proposed biosimilar PF-05280586 and rituximab (MabThera®).

Comparative nonclinical studies were conducted with the proposed biosimilar PF-05280586 and rituximab-EU (MabThera®). In side-by-side analyses, peptide maps and complement-dependent cytotoxicity assay results were similar. Sexually-mature cynomolgus monkeys were administered PF-05280586 or rituximab-EU as a single dose of 0, 2, 10, or 20 mg/kg on day 1 and observed for 92 days (single-dose study) or as 5 weekly injections of 0 or 20 mg/kg and necropsied on day 30, the day after the 5th dose, or on day 121 (repeat-dose study). The pharmacokinetic and pharmacodynamic profiles for both molecules were similar. Marked depletion of peripheral blood B cells 4 days after dosing was followed by near or complete repletion (single-dose study) or partial repletion (repeat-dose study). In the single-dose study, anti-drug antibodies (ADA) were detected by day 29 in all animals administered PF-05280586 or rituximab-EU and persisted through day 85, the last day tested. In the repeat-dose study, ADA were detected on day 121 in 50% of animals administered PF-05280586 or rituximab-EU. Both molecules were well tolerated at all doses. In all endpoints evaluated, PF-05280586 exhibited similarity to rituximab-EU.

Effects of lersivirine on canine and rodent thyroid function.

Lersivirine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) being developed for the treatment of HIV-1 infection. Like other NNRTIs, lersivirine is a potent enzyme inducer in rodents capable of inducing a number of hepatic enzymes including those involved in its own metabolism. Preclinically lersivirine has been associated with hepatocellular hypertrophy and thyroid gland follicular cell hypertrophy in rats, mice, and dogs. In rodents, we show that development of thyroid hypertrophy is related to the classic mechanism, namely increased thyroxine (T4) clearance secondary to induction of uridine-diphosphoglucuronosyltransferase (UDPGT) in the liver and a resulting increase in thyroid-stimulating hormone. Similarly, lersivirine-exposed dogs exhibit a significant increase in hepatic UDPGT enzyme activity along with increased T4 clearance although clear effects on serum thyroid hormone levels were less apparent. These effects on thyroid hormonal clearance in the dog suggest that thyroid gland hypertrophy in this species is due to the same mechanism shown to occur in rodents although, as expected, dogs better adapt to these effects and therefore maintain relatively normal thyroid hormonal balance. It is also notable that the minimal thyroid follicular hypertrophy that occurs in dogs does not progress as is seen in rodents. As is the case with rodents, these adaptive changes in the dog are not considered indicative of a human health risk.

Developmental toxicity of lersivirine in rabbits when administered throughout organogenesis and when limited to sensitive windows of axial skeletal development.

BACKGROUND: Lersivirine is a second-generation nonnucleoside reverse transcriptase inhibitor undergoing clinical development for the treatment of human immunodeficiency virus-1. An embryo-fetal development study was performed to evaluate the potential for maternal and developmental toxicity of lersivirine. METHODS: Pregnant New Zealand White rabbits were administered 0, 100, 250, and 500 mg/kg lersivirine by oral gavage once daily on gestation days (GDs) 7 to 19, followed by cesarean section on GD 29 and fetal evaluation. RESULTS: Maternal toxicity was noted at all dose levels (decreased food consumption and body weight gain), with fetal toxicity at 500 mg/kg (decreased fetal weights, increased postimplantation loss). Equivocal findings for axial skeletal malformations were observed in three fetuses at 500 mg/kg. To better understand if these malformations were related to treatment with lersivirine, a follow-up rabbit embryo-fetal development study was performed with 1000 mg/kg/day lersivirine (500 mg/kg BID, 12-hr interdose interval) for two different 3-day windows, GDs 8 to 10 or GDs 11 to 13, which represent the sensitive windows of axial skeletal development in rabbits. Control rabbits were administered vehicle following the same dosing regimen from GDs 8 to 13. Cesarean sections were performed on GD 29, and fetal skeletons were examined for the potential of lersivirine to cause skeletal malformations in rabbits. At maternal exposure levels higher than the initial study, lersivirine did not induce fetal skeletal malformations when administered in the sensitive windows of axial skeletal development. CONCLUSION: The results of these studies indicate that lersivirine did not exhibit any evidence of teratogenicity in rabbits.

Developmental toxicity study of lersivirine in mice.

Lersivirine is a second-generation nonnucleoside reverse transcriptase inhibitor undergoing clinical development for the treatment of HIV-1. An embryo-fetal developmental toxicity study was performed to evaluate the maternal and developmental toxicity of lersivirine in pregnant mice. Mated Crl:CD1(ICR) mice were administered 0, 150, 350, and 500 mg/kg lersivirine once daily by oral gavage on gestation days 6 to 17, followed by cesarean section on gestation day 18. The first 2 days of dosing for the high-dose group were done at 250 mg/kg to allow induction of hepatic metabolizing enzymes, after which the dose was increased to 500 mg/kg/day. This dosing paradigm allowed for maintenance of exposure in the high-dose group despite the considerable autoinduction that occurs in rodents following lersivirine treatment. Lersivirine did not cause an increase in external, visceral, or skeletal malformations. Intrauterine growth retardation, demonstrated by reduced fetal body weights and increased variations associated with delayed skeletal ossification, was noted at 350 and 500 mg/kg/day. The results of these studies indicate that lersivirine is not teratogenic in mice.

Trough insulin levels in bronchoalveolar lavage following inhaled human insulin (Exubera) in patients with diabetes mellitus.

BACKGROUND/AIM: There are few data regarding insulin levels in the lungs during diabetes therapy with inhaled insulin. We examined the disposition of inhaled human insulin (Exubera(®) [EXU] human insulin [recombinant DNA origin], Pfizer, New York, NY) in the lungs by measuring trough insulin levels in bronchoalveolar lavage (BAL) fluid after 12 weeks of EXU treatment. METHODS: After a 4-week run-in period of subcutaneous insulin therapy, 24 subjects with type 1 diabetes mellitus (T1DM) and 26 with type 2 diabetes mellitus (T2DM) continued their basal insulin regimen and received premeal subcutaneous (SC) insulin for 13 weeks, followed by 12 weeks of premeal EXU. BAL was performed approximately 12 h after the last insulin dose at (1) baseline, (2) following SC insulin, and (3) following EXU. RESULTS: Twenty patients with T1DM and 24 patients with T2DM completed all three bronchoscopies. BAL trough insulin levels were undetectable at baseline or following SC insulin. After EXU therapy, they increased to a median of 4.5 nM (1.6-9.0 nM) and 2.3 nM (0.5-9.4 nM) in T1DM and T2DM, respectively. BAL trough insulin levels did not correlate with treatment efficacy, adverse effects, plasma insulin levels, or changes in pulmonary function. A larger proportion of previous EXU doses was present in the BAL in patients with T1DM. We found no correlation between average daily insulin doses and BAL trough insulin levels. CONCLUSIONS: BAL trough insulin increased following EXU therapy, but this increase did not correlate with other clinical or laboratory parameters, suggesting no significant biological action. Further studies are warranted to better understand inhaled insulin deposition and clearance and possible effects of increased insulin levels on the lungs.

Carcinogenicity assessments of biotechnology-derived pharmaceuticals: a review of approved molecules and best practice recommendations.

An important safety consideration for developing new therapeutics is assessing the potential that the therapy will increase the risk of cancer. For biotherapeutics, traditional two-year rodent bioassays are often not scientifically applicable or feasible. This paper is a collaborative effort of industry toxicologists to review past and current practice regarding carcinogenicity assessments of biotherapeutics and to provide recommendations. Publicly available information on eighty marketed protein biotherapeutics was reviewed. In this review, no assessments related to carcinogenicity or tumor growth promotion were identified for fifty-one of the eighty molecules. For the twenty-nine biotherapeutics in which assessments related to carcinogenicity were identified, various experimental approaches were employed. This review also discusses several key principles to aid in the assessment of carcinogenic potential, including (1) careful consideration of mechanism of action to identify theoretical risks, (2) careful investigation of existing data for indications of proliferative or immunosuppressive potential, and (3) characterization of any proliferative or immunosuppressive signals detected. Traditional two-year carcinogenicity assays should not be considered as the default method for assessing the carcinogenicity potential of biotherapeutics. If experimentation is considered warranted, it should be hypothesis driven and may include a variety of experimental models. Ultimately, it is important that preclinical data provide useful guidance in product labeling.

Carcinogenic interactions between a single inhalation of 239PuO2 and chronic exposure to cigarette smoke in rats.

Rats were exposed once by inhalation to plutonium-239 dioxide ((239)PuO(2)), resulting in chronic alpha-particle irradiation of the lung, and exposed chronically to cigarette smoke to examine carcinogenic interactions between the two exposures. F344 rats were exposed to (239)PuO(2) to achieve an initial lung burden of 0.5 kBq and then exposed 6 h/day, 5 days/week to cigarette smoke at 100 or 250 mg particulate matter/m(3) for up to 30 months. Exposure to cigarette smoke increased the cumulative radiation dose to lung by slowing the clearance of (239)PuO(2). (239)PuO(2) alone did not affect survival, but the higher cigarette smoke exposure shortened survival in females. Combined exposure to (239)PuO(2) and cigarette smoke acted synergistically to shorten survival in both genders. The combined effects of cigarette smoke and (239)PuO(2) were approximately additive for lung hyperplasia and adenomas but were strongly synergistic for carcinomas. Differences between observed incidences and incidences predicted by survival-adjusted models accounting for increased radiation dose revealed a substantial component of synergy for carcinomas above that attributable to the radiation dose effect. The synergy for malignant lung tumors is consistent with findings from uranium miners and nuclear weapons production workers. These results bolster confidence in the epidemiological findings and have implications for risk assessment.

Tolterodine does not affect memory assessed by passive-avoidance response test in mice.

Antimuscarinics are first-line pharmacotherapy for the treatment of overactive bladder. However, because central nervous system cholinergic neurotransmission is involved in cognition, and the central nervous system-permeable antimuscarinics scopolamine and oxybutynin affect memory, cognitive impairment has been noted as a possible side effect of these drugs. We evaluated the effect of tolterodine, an antimuscarinic for overactive bladder, in a mouse passive-avoidance model of memory. Mice were chosen because like humans, mice but not rats, form the pharmacologically active 5-hydroxymethyl metabolite of tolterodine, DD01. In the passive-avoidance test, tolterodine at 1 or 3 mg/kg had no effect on memory; the latency to cross and percentage of animals crossing were comparable to controls. In contrast, scopolamine induced a memory deficit; the latency to cross was decreased, and the number of animals crossing was increased. Therefore, at a dose exceeding therapeutic exposure by six-fold, tolterodine had no effect on memory in the mouse passive-avoidance model, indicating that tolterodine does not disrupt cognitive function in this testing paradigm.

Immunological responses to exogenous insulin.

Regardless of purity and origin, therapeutic insulins continue to be immunogenic in humans. However, severe immunological complications occur rarely, and less severe events affect a small minority of patients. Insulin autoantibodies (IAAs) may be detectable in insulin-naive individuals who have a high likelihood of developing type 1 diabetes or in patients who have had viral disorders, have been treated with various drugs, or have autoimmune disorders or paraneoplastic syndromes. This suggests that under certain circumstances, immune tolerance to insulin can be overcome. Factors that can lead to more or less susceptibility to humoral responses to exogenous insulin include the recipient's immune response genes, age, the presence of sufficient circulating autologous insulin, and the site of insulin delivery. Little proof exists, however, that the development of insulin antibodies (IAs) to exogenous insulin therapy affects integrated glucose control, insulin dose requirements, and incidence of hypoglycemia, or contributes to beta-cell failure or to long-term complications of diabetes. Studies in which pregnant women with diabetes were monitored for glycemic control argue against a connection between IAs and fetal risk. Although studies have shown increased levels of immune complexes in patients with diabetic microangiopathic complications, these immune complexes often do not contain insulin or IAs, and insulin administration does not contribute to their formation. The majority of studies have shown no relationship between IAs and diabetic angiopathic complications, including nephropathy, retinopathy, and neuropathy. With the advent of novel insulin formulations and delivery systems, such as insulin pumps and inhaled insulin, examination of these issues is increasingly relevant.

Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways.

Although cigarette smoke has been epidemiologically associated with lung cancer in humans for many years, animal models of cigarette smoke-induced lung cancer have been lacking. This study demonstrated that life time whole body exposures of female B6C3F1 mice to mainstream cigarette smoke at 250 mg total particulate matter/m(3) for 6 h per day, 5 days a week induces marked increases in the incidence of focal alveolar hyperplasias, pulmonary adenomas, papillomas and adenocarcinomas. Cigarette smoke-exposed mice (n = 330) had a 10-fold increase in the incidence of hyperplastic lesions, and a 4.6-fold (adenomas and papillomas), 7.25-fold (adenocarcinomas) and 5-fold (metastatic pulmonary adenocarcinomas) increase in primary lung neoplasms compared with sham-exposed mice (n = 326). Activating point mutations in codon 12 of the K-ras gene were identified at a similar rate in tumors from sham-exposed mice (47%) and cigarette smoke-exposed mice (60%). The percentages of transversion and transition mutations were similar in both the groups. Hypermethylation of the death associated protein (DAP)-kinase and retinoic acid receptor (RAR)-beta gene promoters was detected in tumors from both sham- and cigarette smoke-exposed mice, with a tendency towards increased frequency of RAR-beta methylation in the tumors from the cigarette smoke-exposed mice. These results emphasize the importance of the activation of K-ras and silencing of DAP-kinase and RAR-beta in lung cancer development, and confirm the relevance of this mouse model for studying lung tumorigenesis.

Effects of strain and treatment with inhaled aII-trans-retinoic acid on cigarette smoke-induced pulmonary emphysema in mice.

Models of emphysema produced by exposing animals to cigarette smoke (CS) have potential for use in testing treatments of this disease. To better characterize development of emphysema in an animal model, male and female mice of the B6C3F1 and A/J strains were exposed to CS at 250 mg total particulate material (TPM)/m3 for 15 weeks. Emphysema was evident in both strains of mice to differing degrees of severity. The CS-induced increase in the mean linear intercept (normalized to BW) of A/J mice was 51% greater than the control value, while CS-exposed B6C3F1 had an increase of 38% in this morphometric measurement of alveolar air space enlargement. In separate experiments, female B6C3F1 mice and male A/J mice were exposed to CS for 32 weeks and 15 weeks, respectively, and were then used to test the efficacy of all trans-retinoic acid (ATRA) treatments to ameliorate emphysema lesions. Following CS exposure, the B6C3F1 mice were treated once daily for 14 days in a 3-week period by nose-only inhalation exposure to aerosols of 180 or 1,800 mg-minutes ATRA/m3. The A/J mice were treated once daily, 4 days/week, for three weeks by either intraperitoneal injection of ATRA (0.5 or 2.5 mg/kg) or inhalation exposure to ATRA (3,600 or 18,000 mg-minutes/m3). Neither the injections nor inhalation exposures of ATRA in either strain of mouse caused reversal of the emphysema. In summary, CS-induced emphysema was more severe in A/J mice than in B6C3F1 mice. Treatment with ATRA did not reverse emphysema in either strain of CS-exposed mice.

Chronic inhalation exposure to mainstream cigarette smoke increases lung and nasal tumor incidence in rats.

An animal model of lung carcinogenicity induced by chronic inhalation of mainstream cigarette smoke would be useful for research on carcinogenic mechanisms, smoke composition-response relationships, co-carcinogenicity, and chemoprevention. A study was conducted to determine if chronic whole-body exposures of rats would significantly increase lung tumor incidence. Male and female F344 rats (n = 81 to 178/gender) were exposed whole-body 6 h/day, 5 days/week for up to 30 months to smoke from 1R3 research cigarettes diluted to 100 (LS) or 250 (HS) mg total particulate matter/m(3), or sham-exposed to clean air (C). Gross respiratory tract lesions and standard lung and nasal sections were evaluated by light microscopy. A slight reduction of survival suggested that the HS level was at the maximum tolerated dose as commonly defined. Cigarette smoke exposure significantly increased the incidences of non-neoplastic and neoplastic proliferative lung lesions in females, while nonsignificant increases were observed in males. The combined incidence of bronchioloalveolar adenomas and carcinomas in females were: HS = 14%; LS = 6%; and C = 0%. These incidences represented minima because only standard lung sections and gross lesions were evaluated. Mutations in codon 12 of the K-ras gene occurred in 4 of 23 (17%) tumors. Three mutations were G to A transitions and one was a G to T transversion. The incidence of neoplasia of the nasal cavity was significantly increased at the HS, but not the LS level in both males and females (HS = 6%, LS = 0.3%, C = 0.4% for combined genders). These results demonstrate that chronic whole-body exposure of rats to cigarette smoke can induce lung cancer.

Aberrant CpG island methylation of the p16(INK4a) and estrogen receptor genes in rat lung tumors induced by particulate carcinogens.

Recent studies by our laboratory indicate that the p16(INK4a) gene is frequently methylated in lung tumors induced by genotoxic carcinogens and that the frequency for methylation of the estrogen receptor alpha (ER) gene varies as a function of carcinogenic exposure. The purpose of the current investigation was to define the role of these two genes in lung tumors induced by the particulate carcinogens carbon black (CB), diesel exhaust (DE) or beryllium metal. Methylation of p16 was observed in 59 and 46% of DE and CB tumors, respectively. In contrast, the ER gene was inactivated in only 15% of DE or CB tumors. Methylation of the p16 and ER genes was very common (80 and 50%, respectively) in beryllium-induced lung tumors; both genes were methylated in 40% of the tumors. Bisulfite sequencing revealed dense methylation throughout exon 1 of the ER gene. The inhibitory effect of methylation on gene transcription was confirmed through RT-PCR expression studies in which p16 gene expression was 30-60-fold lower in methylated than unmethylated tumors. Residual expression in methylated tumors was consistent with contamination by stromal and inflammatory cells. Results indicate that tumors induced by these particulate carcinogens arise, in part, through inactivation of the p16 and ER genes. Furthermore, the inactivation of the p16 gene by these carcinogenic exposures supports a possible role for oxidative stress and inflammation in the etiology of human lung cancer.