Abstract
CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, including the epidermal growth factor (EGF) receptor. Various inhibitors of the EGF receptor, including CI-1033, have resulted in cutaneous toxicity in humans as a common adverse event. In a chronic toxicity study in rats, CI-1033 produced cutaneous lesions with morphologic characteristics similar to that reported in man. Here the authors describe in detail the dermal changes observed, along with other noteworthy findings of that study. Male and female Wistar rats (15/sex/group) were administered CI-1033 for 27 weeks at 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m2, respectively) by gavage. Control animals (15/sex) received vehicle alone (aqueous 0.5% methylcellulose) in a dose volume of 5 mL/kg. Six animals/sex/dose were included for toxicokinetic evaluations. Skin lesions were the primary drug-related toxicity and occurred at ≥2.5 mg/kg in a dose-dependent fashion. The major gross lesions were papules that evolved into crusts and scales that were first observed in weeks 1 and 3, respectively. Alopecia developed in conjunction with the papular eruptions. Skin changes were most pronounced in females, possibly due to higher drug levels. In week 13, CI-1033 plasma AUC(0–24) values were 527 to 1980 ng·h/mL in males and 844 to 2920 ng·h/mL in females at 2.5 to 10 mg/kg. Microscopic changes could be described as 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration. Areas of hyperplasia on the tail were often associated with the development of new hair follicles. Pattern 2 was characterized by a suppurative to pyogranulomatous infundibular folliculitis. Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis; this correlated with alopecia. Elevations in bone marrow myeloid counts correlated with a peripheral leukocytosis, consistent with inflammatory changes in the dermis. In addition, hepatic cholestasis and epithelial atrophy in the gastrointestinal tract and vagina occurred at ≥2.5 mg/kg. In conclusion, CI-1033 produced cutaneous lesions involving the epidermis and hair follicle, and the morphologic characteristics were similar to that reported in clinical studies with various inhibitors of the EGF receptor. These changes are consistent with pharmacologic inhibition of the EGF receptor in these tissues and demonstrate that the rat can serve as an animal model for investigating the mechanisms for this toxicity.
Introduction
Neoplastic cells have been shown to contain various receptor proteins that recognize endogenous ligands that promote growth, phenotypic change, and metastasis. The erbB family of receptor tyrosine kinases constitutes a group of cell surface glycoprotein receptors that recognize various growth factors such as epidermal growth factor (EGF), TGF-α, heregulins, amphiregulin, and epiregulin. Receptor binding results in the initiation of intracellular signal transduction pathways that ultimately influence cell growth, survival, phenotype, and proliferation (Raymond et al., 2000; Adjei, 2000). Preclinical and clinical data have implicated these receptors, such as the EGF receptor (erbB-1 receptor; HER1), in the development, pathogenesis, and severity of various human solid tumors (Adjei, 2000; Gibbs, 2000; Hamid, 2004). Trastuzumab (Herceptin®), a monoclonal antibody specific for the erbB-2 (HER2/neu) receptor, was the first agent targeted against the erbB receptors to be approved for clinical use and is indicated for metastatic breast cancer (Goldenberg, 1999). Therapeutic agents such as trastuzumab, specifically targeted against tyrosine kinase receptors, may exhibit greater specificity toward tumor cells, thereby providing a greater therapeutic index upon use. In addition, these agents have demonstrated utility when combined with cytotoxic chemotherapy or radiation.
Several pharmacologic agents that are inhibitors of the EGF receptor have been approved for treating various solid tumors (e.g., non–small cell lung, colorectal carcinoma, pancreatic cancer). A significant number of patients administered these compounds (e.g., gefitinib or Iressa™, cetuximab or Erbitux™, erlotinib or Tarceva®) have developed dermatologic lesions such as acneiform eruption (Segaert and Van Cutsem, 2005; Johnson et al., 2005). Cutaneous toxicity is the most common adverse event associated with the use of EGF receptor inhibitors (Robert et al., 2005). Skin toxicity is believed to arise due to inhibition of the EGF receptor.
CI-1033 (canertinib; Figure 1) is an irreversible inhibitor of multiple members of the erbB family of receptors (panerbB receptor inhibitor) that was intended for oral administration in the treatment of solid tumors. When CI-1033 is present in the adenosine triphosphate pocket of the erbB receptor, the acrylamide side-chain at position C6 covalently modifies a cysteine residue, thereby irreversibly inactivating the intracellular kinase domain of the receptor (Allen et al., 2003). This compound is highly specific for the erbB receptors. For example, the IC50 for purified EGF receptor tyrosine kinase (erbB-1) was 1.7 nmol/L and in A431 human epidermoid cancer cells, suppression of EGF receptor tyro-sine kinase occurred at an IC50 of 7.4 nmol/L (Slichenmyer et al., 2001). In vitro inhibition of erbB-2, erbB-3, and erbB-4 receptors in MDA-MB-453 human breast cancer cells by CI-1033 was seen at IC50 values of 5–14 nmol/L (Slichenmyer et al., 2001). In animal models, significant tumor suppression was produced by oral CI-1033 administration against A431, H125 non–small cell lung, and SF-767 human glioblastoma tumors (Slichenmyer et al., 2001). CI-1033 was evaluated in phase I and II cancer clinical trials against various solid tumors but is no longer in development. Skin toxicity was a frequent event in the CI-1033 clinical trials (Allen et al., 2003; Nemunaitis et al., 2005).
The objective of this study was to evaluate the potential toxicity of CI-1033 when given orally to rats once daily for 6 months and was conducted in accordance with US FDA Good Laboratory Practice Regulations. Cutaneous toxicity was the primary toxicity observed, and the morphologic characteristics of the lesions were similar to that reported in clinical studies with other EGF receptor inhibitors. This article provides a description of the cutaneous changes observed in the rat and compares the results with published data regarding cutaneous toxicity in man produced by inhibitors of the EGF receptor. In addition, epithelial changes of various tissues and hepatic cholestasis were observed in the rat study and are therefore described.
Materials and Methods
Test Compound
CI-1033 was synthesized by Pfizer Global Research & Development as a dihydrochloride salt. Dosing suspensions were prepared approximately weekly in aqueous 0.5% methyl-cellulose at concentrations of 0.5, 1, and 2 mg/mL.
Animals
Male and female Wistar (Crl:[WI]BR) rats obtained from Charles River Laboratories (Wilmington, MA) were assigned to this study. Animals were 7 to 8 weeks old and weighed 218 to 277 g (males) and 165 to 205 g (females) at the start of dosing. Previous toxicity studies with CI-1033 have been conducted in Wistar rats, which are an acceptable animal model for detecting pharmacologic and toxicologic properties of experimental drugs. Clinically acceptable animals were assigned to dose groups using a computer-assisted randomization procedure. Animals were housed individually in stainless steel wire mesh cages. Standard procedures/conditions were applied for animal care, feeding, and maintenance of room, caging, and environment. Animals were fed powdered Lab Diet 5002 Certified Rodent Diet (PMI Nutrition International, Richmond, IN) ad libitum, and water was supplied ad libitum via an automatic watering system. Animals were fasted overnight prior to blood collection for clinical laboratory measurements at scheduled necropsy only. This study was conducted in accordance with current guidelines for animal welfare (National Research Council, 1996). The procedures used in this study were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.
Experimental Design
Animals (21 rats/sex/group) received CI-1033 for 27 weeks at doses of 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m2, respectively). Animals were dosed once daily (starting with day 1) by gavage with a dose volume of 5 mL/kg. Control animals (15/sex) received vehicle alone, which was aqueous 0.5% methylcellulose. All controls and the first 15 animals in each drug-treated group were designated for toxicologic assessment and scheduled for necropsy in week 28. The remaining 6 animals per drug-treated group were designated for toxicokinetic assessment, which occurred in week 13. Doses for this study were based on the results of a 13-week oral toxicity study of CI-1033 in Wistar rats at doses of 2.5, 5, and 10 mg/kg. In that study, no deaths occurred and dermal lesions were seen at ≥5 mg/kg. Decreased body weight and weight gain occurred in males only at 10 mg/kg. The low dose in the 6-month study was projected to be a no-adverse effect level, and the high dose was expected to produce mild to moderate toxicity without lethality.
Experimental Procedures
Animals were observed predose and approximately 1 hour postdose daily for clinical signs. Ophthalmic examinations were conducted pretest and the day prior to scheduled termination for animals designated for toxicologic assessment. Body weights were determined prior to dosing on day 1 and weekly thereafter. Food consumption was determined weekly and at scheduled termination. Hematological and serum chemistry parameters were evaluated in weeks 5, 9, 18, and 28 (scheduled termination) in animals designated for toxicologic assessment. Blood samples containing EDTA as an anticoagulant were analyzed for absolute and differential blood counts. Serum was analyzed for glucose, cholesterol, bilirubin, protein (total), albumin, globulin (calculated), albumin/globulin ratio (calculated), phosphorus, calcium, chloride, sodium, potassium, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), and urea nitrogen. Serum samples collected in week 28 were also assayed for bile acids (total). Bone marrow samples were collected from 5 animals/sex/group at scheduled necropsy in week 28 and evaluated by flow cytometry for total nucleated cell counts and differential. Overnight urine samples were collected from toxicologic assessment animals prior to necropsy in week 28 and evaluated by standard urinalysis procedures.
Complete necropsies were performed on all animals designated for toxicologic assessment. Animals were euthanized by carbon dioxide asphyxiation. The following organs were weighed at scheduled necropsy: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary, prostate, spleen, submandibular salivary glands, testes, thymus, and uterus. Organ-to-body weight and organ-to-brain weight ratios were calculated. Representative samples of the following tissues were collected at necropsy, fixed in 10% buffered formalin (except as noted), and processed for light microscopy: adrenals, bone marrow, brain, epididymides (Bouin’s fixative), esophagus, eyes (6% glutaraldehyde), Harderian gland, heart, kidneys, large intestine, liver, lungs, bone (distal femur with proximal tibia, and sternum), mammary gland, mesenteric lymph node, ovaries, pancreas, pituitary, prostate, sciatic nerve, seminal vesicles, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, submandibular salivary gland, testes (Bouin’s fixative), thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus, vagina, and gross lesions considered to be potentially drug-related. To better evaluate potential drug-related effects on the hair follicles, an 8-mm biopsy punch was used to take skin samples from the dorsum of all animals. The punch biopsies were fixed in 10% buffered formalin and transversely sectioned (sectioned parallel with the epidermis) so all of the hair follicles within the 8-mm sample could be evaluated.
All tissues from high-dose and control animals were sectioned, stained with hematoxylin and eosin (H&E), and evaluated microscopically. All potential drug-related gross lesions and potential target organs in the mid- and low-dose groups (skin, tongue, esophagus, stomach, large and small intestines, trachea, lung, liver, bone, bone marrow, mammary gland, skeletal muscle, female reproductive tract, and thyroid and parathyroid glands) were similarly sectioned, stained, and evaluated. Additionally, heart sections were stained by the Massons’ trichrome stain and kidney sections were stained with periodic acid Schiff stain. Kidney sections with green-gray pigment were stained with the acid-fast stain for lipofuscin, Prussian blue for iron, and Hall’s stain for bilirubin.
Toxicokinetic parameters were assessed in week 13 from animals designated for toxicokinetic assessment. Blood samples (approximately 1 mL) were collected at approximately 0.5, 1, 2, 4, 8, and 24 hours after dosing. Each drug-treated animal was used for 3 time points. The same animals were used for the following time points: 0.5, 2, and 8 hours, and 1, 4, and 24 hours. Blood samples were collected into a tube containing sodium heparin as the anticoagulant and immediately placed on ice. The samples were centrifuged under refrigerated conditions, the plasma separated and transferred to plastic tubes, 10 μL of 2% H3PO4 was added (to maintain analyte stability), and the sample was vortexed. Plasma samples were stored frozen at ≤–70°C prior to analysis. Following blood sampling, animals designated for toxicokinetic assessment were euthanized and discarded. No other procedures were conducted on these animals.
Plasma samples were analyzed for CI-1033 concentration using a validated LC/MS/MS method with a lower limit of quantitation of 2.50 ng/mL. Composite concentration-time profiles were constructed, and the toxicokinetic parameters of maximum concentration (Cmax), time to Cmax (tmax), and area under the concentration-time curve (AUC) were determined. Concentrations below the limit of quantitation were reported as zero and used in evaluation of mean concentrations and estimation of AUC. For the purpose of data analysis, plasma CI-1033 concentrations at 24 hours postdose were used as the concentration at time zero because steady-state conditions were assumed.
Statistical Analysis
Statistical analysis was conducted on data from animals designated for toxicologic assessment only and was conducted on body weight and weight change, food consumption, bone marrow, organ weight, and quantitative clinical laboratory data. Treatment comparisons were performed on rank-transformed data using a dose-trend test sequentially applied at the 2-tailed 1% and 5% significance levels within 1-factor analysis of variance. Dunnett’s test replaced the sequential trend test if the overall linear trend test was not significant at the 5% level and a quadratic trend was significant at the 1% level. All parameters were analyzed separately for each sex and time period.
Results
Clinical Responses
Drug-related deaths occurred in females at ≥5 mg/kg. One female at 5 mg/kg and 5 females at 10 mg/kg were euthanized early due to severe dermal lesions (between days 36 and 168). In addition, 1 female at 10 mg/kg was found dead on day 109. In general, clinical toxicity occurred to a greater extent in females. Primary drug-related clinical signs involved dermal changes of the tail and body and occurred at ≥2.5 mg/kg in a dose-dependent fashion (Table 1). Skin sores, composed of open or scabbed lesions on the tail and body, were first seen on day 6 (10 mg/kg). Dermatopathy, characterized by flaky or scaly appearance of skin, was observed on tail skin at all doses. Dermatopathy was first observed on day 21 (10 mg/kg) and occurred in a dose-dependent fashion. One 10 mg/kg male had generalized dermatopathy. Due to the severe nature of the tail skin lesions, tails of 5 females at 5 mg/kg, 3 females at 10 mg/kg, and 8 males at 10 mg/kg, were amputated beginning in week 13 to maintain the animals on study. Alopecia was associated with skin sores and was drug-related in females at ≥5 mg/kg and in 1 male at 10 mg/kg. Other clinical signs considered drug related occurred at ≥5 mg/kg and included chromodacryorrhea, rough pelage, urine staining, thinness, and/or soft feces. Drug-related ophthalmic changes were not observed. Decreased weight gain was seen periodically in males at 10 mg/kg, and increased food consumption occurred in females at ≥5 mg/kg (data not shown).
Clinical Laboratory Parameters
Summaries of select hematology and bone marrow and serum chemistry parameters are presented in Tables 2 and 3, respectively (week 28 data presented for brevity). White blood cell counts (WBC) were increased in both sexes at ≥2.5 mg/kg. Elevations in WBC were first observed in week 5. Increases in WBC varied from 14% to 193%, with the greatest elevations in females at 10 mg/kg in weeks 18 and 28. Males at 10 mg/kg had increases in neutrophils of 35% to 191% (at all time points) and increases in lymphocytes of 32% to 55% in weeks 18 and 28. Females at 10 mg/kg had increases in neutrophils of 320% to 440% (at all times) and lymphocytes of 102% in weeks 18 and 28. Animals at 10 mg/kg had slight decreases in red blood cell counts (RBC, 4%–10%), hemoglobin, and hematocrit. These mild changes were present following 1 month of exposure and persisted with similar magnitude following 6 months of exposure. Reticulocytes were also slightly increased in 10 mg/kg animals (up to 52%), indicating an appropriate bone marrow response to decreased RBC.
The bone marrow myeloid to erythroid ratio (M:E) was increased 64% to 82% in females at ≥5 mg/kg. Total myeloid numbers were increased 40% in 10 mg/kg males and 22% to 64% in females at ≥5 mg/kg (determined by flow cytometry). Elevations in bone marrow myeloid counts are consistent with increased peripheral blood WBC and neutrophils. Megakaryocyte counts were increased 94% in males at 10 mg/kg (data not shown).
Serum chemistry changes consistent with cholestasis were observed. In week 28, total bilirubin was increased in males at 10 mg/kg and females at ≥2.5 mg/kg due to increases in direct (conjugated) bilirubin. Some animals at 5 and 10 mg/kg had total bilirubin levels increased as high as 15 to 23 times the concurrent mean control. In addition, serum bile acids were increased 264% in males at 10 mg/kg and 231% to 466% in females at ≥2.5 mg/kg in week 28. Moderate to large amounts of bilirubin in urine were detected in 1 male at 10 mg/kg and various females at ≥2.5 mg/kg, and correlated with high serum bilirubin levels (1.9–5.1 mg/dL) in these animals. Increases in serum ALT (up to 260%) and AST (up to 423%) were seen throughout the study in both sexes at ≥2.5 mg/kg. Increases in ALKP (up to 242%) occurred throughout the study at 10 mg/kg, and at 2.5 and 5 mg/kg in weeks 18 (females) and 28. Serum albumin was mildly decreased (up to 15%) in males at ≥5 mg/kg, and globulin was increased (up to 44%) in a dose-dependent fashion in females at ≥2.5 mg/kg. Globulin was also increased in males, primarily at 10 mg/kg (data not shown).
Toxicokinetics
CI-1033 plasma toxicokinetic parameters in week 13 are presented in Table 4. CI-1033 was rapidly absorbed with tmax of 0.5 to 1 hour. Cmax and AUC increased in a dose-dependent fashion in males and females, and the increase was generally linear. At all doses, values of Cmax and AUC were 30% to 56% and 23% to 60% higher, respectively, in females compared to males.
Anatomic Pathology
Drug-related dermal changes occurred in males and females at all doses, were dose-related in incidence and severity, and could be separated into 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration (Figure 3). Areas with epidermal hyperplasia on the tail were often associated with follicular neogenesis with early developing hair follicles observed (Figure 4). Pattern 2 was characterized by suppurative to pyogranulomatous infundibular folliculitis (Figure 5). Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis (Figure 6). This change correlated with alopecia observed grossly. The hair follicle deformity affected both the hair follicle and shaft. Follicles lost polarity and were growing laterally or downward. Some of these follicles had hair shafts that were brightly eosinophilic and devoid of a defined cortex and medulla. Deep dermal granulomatous inflammation correlated with the number of deformed hair follicles and appeared due to hair follicle deformity. The incidence of dermal changes (by pattern) affecting tail and haired skin are summarized in Tables 5 and 6, respectively. Epidermal changes (pattern 1) affecting haired skin were more common in females than males. Hair follicle deformity (pattern 3) was more pronounced in haired skin than tail skin in females.
Bone marrow hypercellularity was noted in 8 females at 10 mg/kg and correlated with the increase in myeloid cell counts as determined by flow cytometry. Elevations in white blood cells in peripheral blood were seen throughout the study in these animals, which are consistent with the changes in the bone marrow. These changes are consistent with the inflammatory lesions observed in the skin and represent an adaptive response by the bone marrow. Similar bone marrow and white blood cell changes were seen in rats administered a structurally related compound that also produced inflammatory lesions in skin (Brown et al., 2002).
Lymphoid hyperplasia of the mesenteric lymph nodes was noted in 3 high-dose females and varied from minimal to moderate. This change may have been secondary to the chronic inflammatory skin lesions. In humans, dermatopathic lymphadenopathy is a well-recognized sequel to chronic dermatitis (Shamoto et al., 1996). In this study, all rats with lymphoid hyperplasia had crusts, ulcers, and a deep folliculitis; however, because other rats had dermal lesions of similar severity but failed to demonstrate lymph node changes, a definitive correlation could not be made.
Atrophy of squamous epithelium of tongue, esophagus, and nonglandular stomach mucosa occurred at all doses in both sexes, was minimal to mild in severity, and was generally dose-related in incidence. Epithelial atrophy of the vagina, manifested as minimal to mild thinning of the squamous mucosa, was seen at ≥2.5 mg/kg.
Hepatocellular degeneration was observed mainly in the centrilobular region and characterized by single-cell necrosis, increased mitotic figures, increased sinusoidal cellularity, and less commonly as cytoplasmic vacuolation. Hepatocellular degeneration was dose-related in incidence and severity, and more prevalent in females (3, 4, and 6 rats at 2.5, 5, and 10 mg/kg, respectively) than males (1 and 2 rats at 5 and 10 mg/kg, respectively). Although histologic changes in liver were observed in individual animals, these changes did not consistently correlate with elevations in serum chemistries associated with liver function.
Abnormal pigmentation of the kidney, noted grossly as diffuse green-brown discoloration of the renal parenchyma, occurred in 1 female at 2.5 mg/kg, 2 females at 5 mg/kg, and 4 females and 1 male at 10 mg/kg. One female each at 5 and 10 mg/kg with kidney discoloration was also icteric. Microscopically, the pigment was most prominent in the cytoplasm of renal epithelial cells of the proximal cortical tubules. Special stains to define pigment composition were equivocal. Two females at 5 mg/kg, and 3 females and 1 male at 10 mg/kg with abnormal renal pigmentation, had increased liver and kidney weights. Elevations in liver serum chemistries occurred in these animals. Mean absolute kidney weights were increased 15% in females at 10 mg/kg (organ weight data not shown). Renal pigmentation secondary to bilirubinemia has been documented (Greaves, 2000, pp. 453, 580). In conclusion, these renal changes are considered secondary to hepatic changes and not due to direct drug-related effects on renal parenchyma.
Discussion
Oral administration of CI-1033 to rats for 6 months at ≥2.5 mg/kg resulted in dermal lesions as the primary drug-related toxicity. This was associated with steady-state CI-1033 plasma levels of Cmax ≥204 ng/mL and AUC(0–24) ≥527 ng·h/mL. Toxicity occurred to a greater extent in females, which may be related to the higher plasma drug levels in this sex (AUCs were 23% to 60% higher than males). Dermal changes were observed clinically as skin sores and dermatopathy, beginning in week 1, and correlated with microscopic changes involving the epidermis (pattern 1), upper hair follicle (pattern 2), and lower hair follicle (pattern 3). Elevations in white blood cells, due to increases in neutrophils and lymphocytes, were seen throughout the study and are consistent with inflammatory changes in the dermis.
CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, of which the epidermal growth factor (EGF) receptor is a member. Inhibition of receptor signaling results in alterations in intracellular signal transduction pathways that can affect cell growth, survival, proliferation, and/or differentiation (Raymond et al., 2000). Currently, several small molecule and monoclonal antibody inhibitors of the EGF receptor are approved for clinical use in oncology. Dermal toxicity is a commonly observed adverse event in patients administered these compounds, with reported incidence of greater than 50% (Robert et al., 2005). Folliculitis, sometimes referred to as an acneiform eruption or acne-like rash, is the most common dermal lesion observed with the EGF receptor inhibitors (Robert et al., 2005). Similar skin lesions to those observed in the current study have been described as side effects of gefitinib (Iressa™, ZD1839), an anticancer agent that inhibits the EGF receptor. A superficial, purulent, acneiform folliculitis (consistent with pattern 2) and hair growth abnormalities (consistent with pattern 3) were observed in skin sections from cancer patients administered gefitinib (Van Doorn et al., 2002). Epidermal atrophy, a clinical finding noted with gefitinib, was not present in CI-1033-treated rats (Albanell et al., 2002). Oral administration of the EGF receptor inhibitor erlotinib (Tarceva®, OSI-774) to cancer patients resulted in an acneiform-type lesion characterized by neutrophilic infiltration of dermal tissues, particularly the infundibular portion of the hair follicle (Hidalgo et al., 2001). These lesions occurred within 2 weeks of dosing. Intravenous administration of the anti-EGF receptor antibody, cetuximab (Erbitux™, C225), to cancer patients resulted in an acneiform follicular eruption within approximately 1 week into therapy (Busam et al., 2001). Skin biopsy results of erythematous follicular papules and pustules in a patient administered cetuximab revealed neutrophilic suppurative folliculitis (Kimyai-Asadi and Jih, 2002). Data from multiple studies with cetuximab and erlotinib have shown a positive relationship between skin rash and clinical response, including survival (Perez-Soler and Saltz, 2005). Clinical trial data with the various EGF receptor inhibitors indicate that skin rash typically occurs after 1 week of treatment and reaches a maximum intensity after 2 to 3 weeks, and in phase I dose-escalation studies, rash was dose-dependent, with higher drug exposure leading to higher incidence and/or severity (Perez-Soler and Saltz, 2005). These data are consistent with the 6-month rat study with CI-1033, in that cutaneous lesions were first observed in week 1, occurred in a dose-dependent fashion, and were more pronounced in female rats, which had higher plasma drug levels than the males. In addition, inflammatory changes in skin, including folliculitis, occur in both rats and man upon exposure to this class of drug.
CI-1033 has been evaluated in multiple phase I and II cancer clinical trials, with various treatment regimens, against a variety of solid tumors including non–small cell lung, head and neck, breast, colorectal, prostate, pancreas, and ovarian cancers (Rinehart et al., 2002; Campos et al., 2004; Nemunaitis et al., 2005). The most common adverse events were emesis, nausea, diarrhea, stomatitis, asthenia, and acneiform rash (Allen et al., 2003). In a CI-1033 phase I clinical trial in cancer patients, a dose of 250 mg/day produced skin rash and was associated with mean plasma Cmax of 183 ng/mL (Rowinsky et al., 2003). This exposure level was similar to that which produced skin toxicity (Cmax ≥ 204 ng/mL) in the 6-month rat study. This suggests similar sensitivity of both species to CI-1033-induced dermal toxicity. In a CI-1033 phase I trial involving daily dosing for 7 days, skin rash occurred at doses ≥95 mg and these doses were associated with decreases in tumor total EGF receptor protein expression and phosphorylated EGF receptors (Zinner et al., 2007). This suggests that skin toxicity occurs at CI-1033 exposure levels that elicit biologic activity at the EGF receptor. Although CI-1033 did produce some evidence of antitumor effects in the clinical trials, it was subsequently terminated from development due to insufficient efficacy.
The EGF receptor has been shown to play an important role in hair follicle physiology (Philpott and Kealey, 1994). Abnormalities in hair follicle morphology have been noted in transgenic mice with disruption of the EGF receptor allele (Murillas et al., 1995). Affected mice have short wavy hair that eventually results in permanent alopecia because the follicles are destroyed by an inflammatory reaction (Hansen et al., 1997). Patients administered various EGF receptor inhibitors have reported alterations in hair including alopecia of scalp hair and growth of facial hair and eyelashes (Robert et al., 2005). In the current study, CI-1033 produced significant changes in the hair follicles along with alopecia. Formation of new hair follicles (follicular neogenesis) was observed in areas of the tail with epidermal hyperplasia. While the mechanism for new follicle growth is not known, it may represent an adaptive response to the epidermal injury observed. Regeneration of hair follicles has been observed in scar tissue following dermal wounding in rabbits (Breedis, 1954). Hair follicle growth was observed in Wistar rats following dermal injury produced by topical hydrogen peroxide with ensuing epidermal hyperplasia (Umeda-Ikawa et al., 2002). In a recent study in mice, hair follicle formation occurred de novo following skin wounding and appeared to arise from epidermal cells (Ito et al., 2007). One of the major pathways for hair follicle development is through Wnt signaling, in which activation results in increased expression of a key effector protein, β-catenin (Van Mater et al., 2003). Increased β-catenin initiates adnexal genesis, growth, and development and is critical for de novo development of hair follicles in mice following cutaneous wounding (Lo Celso et al., 2004; Ito et al., 2007).
The EGF receptor is important for keratinocyte physiology because inhibition of receptor signaling can result in growth inhibition (Dvir et al., 1991; Powell et al., 1999). Human keratinocyte proliferation and terminal differentiation are mediated by the EGF receptor and include autocrine stimulation pathways (Peus et al., 1997). Apoptosis is produced in human keratinocytes incubated with specific inhibitors of the EGF receptor and is associated with down-regulation of the anti-apoptotic protein Bcl-XL (Stoll et al., 1998). This action is believed to occur in part due to decreased intracellular signaling via the MEK/MAPK pathway (Jost et al., 2001). Evaluation of skin biopsies from cancer patients administered gefitinib indicated increased apoptosis of epidermal cells with therapy, which is consistent with in vitro data demonstrating the importance of the EGF receptor for keratinocyte homeostasis (Albanell et al., 2002). Skin lesions consisting of ulcers and abscesses were seen in rats administered a structurally related analog of CI-1033 that is also an irreversible inhibitor of the erbB family of receptors (Brown et al., 2002). Based on these data, the dermal toxicity of CI-1033 in rats is consistent with inhibition of EGF receptor-mediated signal transduction pathways. Inhibition of receptor signaling appears to have a cytotoxic effect, which results in an ensuing inflammatory response in the skin. Therefore, the rat appears to provide an animal model for studying dermal toxicity produced by drugs that inhibit the EGF receptor.
Administration of CI-1033 to rats for 6 months resulted in epithelial atrophy of the gastrointestinal tract and vagina. Gastrointestinal epithelial atrophy (tongue, esophagus, stomach, intestines) was also seen in rats administered CI-1033 for up to 28 days and was reversible following cessation of dosing (Breider, 2000). The EGF receptor is present in the gastrointestinal tract (Tarnawski and Jones, 1998), and atrophy of epithelial tissues is consistent with the pharmacologic actions of CI-1033 (Raymond et al., 2000; Marti et al., 1989). Adverse events associated with the gastrointestinal tract were seen in the CI-1033 clinical trials (emesis, nausea, diarrhea, stomatitis) and appear consistent with the epithelial changes observed nonclinically. This provides further support for the rat serving as an appropriate animal model for assessing safety and exploring mechanisms of toxicity produced by EGF receptor inhibitors.
Toxicologically significant elevations in serum total bilirubin (conjugated) and bile acids were observed in the study, along with increases in ALT and AST, and alkaline phosphatase at ≥2.5 mg/kg. In addition, bilirubinuria, icterus, and abnormal renal pigmentation were observed in various animals. Elevations of biliary products in serum and urine, along with gross observations of icterus (jaundice) are indicative of cholestasis (Farrell, 1994; Cullen and Ruebner, 1991). Hepatocellular changes were observed microscopically (primarily in the centrilobular region) and characterized by single-cell necrosis, increased mitotic figures, increased sinusoidal cellularity, and less commonly as cytoplasmic vacuolation. These data indicate that administration of CI-1033 to rats resulted in cholestasis with minimal hepatic parenchymal injury. The relationship between hepatocellular injury and impaired biliary function is uncertain.
In conclusion, the primary drug-related toxicity following oral administration of CI-1033 to rats at ≥2.5 mg/kg was to the skin. The dermal changes involved the epidermis and hair follicle and are consistent with pharmacologic inhibition of the EGF receptor in these tissues. The morphologic characteristics of these lesions were similar to that reported in clinical studies with various inhibitors of the EGF receptor, demonstrating that the rat can serve as an animal model for studying this toxicity. Hepatic cholestasis and epithelial atrophy of the tongue, esophagus, nonglandular stomach mucosa, and vagina occurred at ≥2.5 mg/kg. A no-adverse-effect level was not identified.