Abstract
The primary toxicity associated with repeated oral administration of the PDE4 inhibitor IC542 to the rat is an inflammatory response leading to tissue damage primarily in the gastrointestinal tract and mesentery. Although necrotizing vasculitis is frequently seen with other PDE4 inhibitors, blood vessel injury was rare following IC542 administration and was always associated with inflammation in the surrounding tissue. The incidence and severity of the histologic changes in these studies correlated with elevated peripheral blood leukocytes, serum IL-6, haptoglobin, and fibrinogen, and with decreased serum albumin. By monitoring haptoglobin, fibrinogen and serum albumin changes in IC542-treated rats, it was possible to identify rats with early histologic changes that were reversible. Since PDE4 inhibition is generally associated with anti-inflammatory activity, extensive inflammation in multiple tissues was unexpected with IC542. Co-administration of dexamethasone completely blocked IC542-induced clinical and histologic changes in the rat, confirming the toxicity resulted from inflammatory response. In addition, IC542 augmented release of the proinflammatory cytokine IL-6 in LPS-activated whole blood from rats but not monkeys or humans. The effect of IC542 on IL-6 release from rat leukocytes in vitro is consistent with the proinflammatory response observed in vivo and demonstrates species differences to PDE4 inhibition.
Keywords
Introduction
The elevation of intracellular cAMP levels through the inhibition of phosphodiesterase type 4 (PDE4) family of isoenzymes effectively moderates some inflammatory responses by reducing the activity of monocytes and T cells (Giembycz et al., 1996; Manning et al., 1999; Hidi et al., 2000; Seldon et al., 2005). For example, selective PDE4 inhibitors inhibit TNF-α release in response to LPS, both in vitro and in vivo (Brideau et al., 1999; Manning et al., 1999; Gale et al., 2002; Alexandre-Moreira et al., 2005). PDE4 inhibitors have also been shown to either moderate or block inflammation in a number of preclinical disease models including asthma, endotoxic shock, and arthritis (Sekut et al., 1995; Bundschuh et al., 2001; Alexandre-Moreira et al., 2005; Moreita et al., 2005) and are reported to be efficacious in some human diseases including asthma and chronic obstructive pulmonary disease (Giembycz, 2001; Lipworth, 2005).
Although effective in moderating some inflammatory disease processes, multiple toxicities have been associated with repeated administration of PDE4 inhibitors. Early PDE4 inhibitors failed in clinical trials due to nausea and emesis (Dyke and Montana, 2002). PDE4 inhibitors have also been reported to cause severe toxicities in some animal species when administered at high doses (Larson et al., 1996; Losco et al., 2004). For example, rolipram, a prototypic PDE4 inhibitor, is reported to produce clinical signs of toxicity that include salivation, abdominal distension, weight loss and ataxia in rats with repeated dosing (Larson et al., 1996). Histologic evaluation of rolipram treated rats provided evidence of myocardial degeneration and necrosis, arteritis/periarteritis of the intramural and extramural coronary arteries, necrotizing vasculitis and inflammation of mesentery and interstitial areas of the liver, affecting medium-sized portal arteries and veins. The toxicities reported for rolipram and other early PDE4 inhibitors differ from more recent second-generation PDE4 inhibitors (Perry et al., 1998). These apparent differences may be the result of low potency (Sekut et al., 1995) and/or relatively poor selectivity of the early PDE4 inhibitors, leading to the inhibition of other PDE family members, such as PDE3.
IC542 is a highly selective and potent inhibitor of PDE4, inhibiting all 4 PDE4 isoforms (PDE4A, PDE4B, PDE4C and PDE4D) at low nanomolar concentrations. IC542 also has >1000-fold selectivity for PDE4 over the other PDE family members (PDE1-PDE3, PDE5, PDE7-PDE11). The oral administration of IC542 to rats induced many of the clinical observations described for rolipram at high doses (Larson et al., 1996). However, many of the important histopathologic changes seen in the tissues from IC542 treated rats are distinctly different from those seen with rolipram. Lesions seen with IC542 were characterized by severe inflammation of the mesentery, regions of the gastrointestinal tract and thymus. In contrast to what has been reported for rolipram and cilomilast, vascular injury was rarely seen and when present it was considered secondary to the inflammation in the surrounding tissue.
To fully characterize toxicities associated with IC542, investigational toxicity studies were conducted with the following objectives: (1) fully characterize toxicities associated with IC542 at high doses, (2) identify biomarkers that precede the development of histologic lesions, (3) determine if the use of an anti-inflammatory agent would prevent IC542-induced toxicity, (4) characterize the inflammatory response in rats for comparison with other species.
Materials and Methods
Animals
In acute toxicity studies with IC542, male and female Sprague–Dawley rats developed comparable toxicities at the same dose levels, therefore only females were used in these investigative evaluations. Female Sprague–Dawley rats 7–8 weeks of age were obtained from Charles River Laboratories and maintained in the ICOS animal facility in accordance with the Guide for Care and Use of Laboratory Animals. Animals were gang housed in polycarbonate cages in a climate-controlled environment with a 12-hour light/dark cycle. With the exception of overnight fasting prior to scheduled necropsies, food and water were provided ad libitum during the study. All study protocols were approved by the ICOS Institutional Animal Care and use Committee.
IC542 Formulation and Administration
IC542, with an approximate purity of 96.7%, was used in these studies. For the various rat studies, a dosing solution was prepared weekly by dissolving IC542 in PEG400 at a concentration of 20 mg/mL. The formulated material was stored at ambient temperature, protected from light prior to use and the concentration was confirmed by HPLC analysis. Dosing was by oral gavage administered through 18-gauge disposable animal feeding needles (Popper and Sons, Inc., New Hyde Park, NY), using a volume of 5 mL/kg.
14-Day Study to Characterize the Toxicity of IC542 in the Rat
In an initial study to characterize the effects of IC542, 50 female rats received a daily oral IC542 dose of 100 mg/kg/day for up to 14 days. An additional 10 rats were given the vehicle control, PEG400, for up to 14 days. Rats were periodically sacrificed (n = 10/day for IC542-treated rats, n = 2/day for vehicle-treated rats) at predetermined timepoints to allow a full spectrum of histologic findings to be observed over the treatment period.
Necropsies were scheduled on Days 2, 5, 8, 12, and 15 for the collection of tissues and organs for histopathology. Over the course of the treatment period, a few rats developed clinical signs of severe toxicity, including abdominal distension or a moribund appearance and were euthanized prior to the scheduled necropsy. Blood samples were collected from all rats remaining on study at Days 1, 2, 3, 5, 8, 10, 12, and 15 to evaluate hematology and clinical chemistry parameters. At necropsy, additional serum was collected to assess levels of inflammatory cytokines.
Design of the Predictive Biomarker Study with IC542
Based on the results from the initial 14-day study, alterations in some hematologic and clinical chemistry parameters correlated with IC542-induced toxicities. The most notable changes were increased neutrophil counts, elevations in haptoglobin and fibrinogen levels and decreased albumin levels. To determine if changes in these parameters could serve as early and sensitive indicators or biomarkers of IC542-toxicities, a second toxicity study was performed. This study was designed to assess the potential of these biomarkers to identify animals with IC542-induced toxicities early and when still reversible.
Thirty-five female rats were dosed with IC542 at 100 mg/kg/day for up to 7 days, while an additional 10 rats received PEG400 as a vehicle control. Blood samples were collected from each animal on Days 0, 4, 5, 6, and 7 and assayed immediately for neutrophil counts, fibrinogen, haptoglobin and albumin levels. Based on historic laboratory data and the results from the 14-day study, fibrinogen levels ≥0.5 μg/mL, haptoglobin >40 mg/dL, and serum albumin <3.8 g/dL were considered clinically abnormal and indicative to IC542-induced toxicity. When one or more of the three parameters fell outside these predetermined cut-off levels, the animal was immediately assigned to 1 of 3 study subgroups, determined by the order that the rats were identified. For example, the first rat with abnormal fibrinogen, haptoglobin or albumin concentrations was assigned to Subgroup 1; the second rat was assigned to Subgroup 2, and so forth. This randomization procedure ensured that nearly equal numbers of rats were assigned to each subgroup for further study.
Rats assigned to Subgroup 1 continued to be dosed through Day 7 and were necrospsied on Day 8 to assess the extent of IC542-related histologic changes in target tissues when dosing continued. Rats assigned to Subgroup 2 were necrospsied immediately to determine the extent of histologic changes in target tissues at the time when one or more of the biomarkers was initially altered. Dosing of rats assigned to Subgroup 3 was immediately stopped, and animals were allowed a recovery period of at least 14 days prior to necropsy to evaluate the reversibility of IC542-induced changes. The remaining rats that had biomarker levels within the normal range over the 7-day treatment period were necropsied on Day 8 and tissues were evaluated for possible histologic changes.
Concurrent Administration of Dexamethasone and IC542
Results from investigative toxicity studies with IC542 found that most of the important treatment-related effects of IC542 appeared to be closely linked to an inflammatory process. To further characterize the role of inflammation in IC542-induced toxicities, a study was performed to determine if dexamethasone could moderate toxicities associated with the compound. For this study female rats (n = 10/group) were dosed with IC542 at 100 mg/kg/day (p.o.), or concomitantly with 100 mg/kg/day IC542 and 0.5 mg/kg/day dexamethasone (administered i.p. at 1 mL/kg/day in saline). A third group of rats (n = 4), was dosed p.o with PEG400 and i.p. with saline, the vehicles for IC542 and dexamethasone, respectively. A fourth group of rats (n = 6), was treated with 0.5 mg/kg/day dexamethasone i.p. The treatment period was for 7 consecutive days. Clinical observations, body weights and serum levels of albumin and haptoglobin were monitored daily throughout the treatment period. Necropsies were conducted on Day 8 and target tissues were collected for histopathologic examination.
Histopathology
Tissues were collected and fixed in 10% neutral-buffered formalin, processed by routine histologic methods, trimmed, embedded in paraffin, sectioned to a thickness of 4–6 μm, and stained with hematoxylin and eosin (H&E) for histopathologic examination.
A histological examination was performed on target tissues identified in earlier dose-ranging studies with IC542 and those described in the literature as target organs for other PDE4 inhibitors. Tissues examined for evidence of treatment-related changes included: adrenal glands, gastrointestinal tract (cecum, colon, duodenum, ileum, and jejunum), heart, hind leg joints, and mesentery with associated vessels, mesenteric lymph nodes, spleen, and thymus with associated mediastinum.
Tissues were scored as follows based on the extent of tissue inflammation and tissue damage: unaffected (pathology score = 0), minimally affected (pathology score = 1), mildly affected (pathology score = 2), moderately affected (pathology score = 3) and severely affected (pathology score = 4).
Clinical Pathology
During the course of the rat studies, blood was periodically collected for hematology and clinical chemistry analyses. Blood samples for hematology determinations, approximately 0.125 mL volume, were collected in tubes containing EDTA (Greiner Bio-one MiniCollect, part no. 450404), mixed by inversion and analyzed on a Hemavet 850FS (CDC Technology). Analysis of hematology parameters included white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, mean corpuscle volume, mean corpuscular hemoglobin, mean corpuscle hemoglobin concentration, platelet count, mean platelet volume, red cell distribution width, percentages and absolute cell counts of neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
Samples for clinical chemistry determinations were placed in tubes containing a serum separator (Greiner Bio-one Mini-Collect, part no. 450401), allowed to clot at room temperature for approximately 30 minutes, centrifuged for 5 minutes at 10,000 rpm and the serum was collected. Serum samples were analyzed using a Dimension Clinical Chemistry Analyzer (Dade/Behring A-R.) for blood urea nitrogen, glucose, total protein, albumin, cholesterol, triglycerides, as-partate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total bilirubin, calcium, phosphorus, sodium, potassium, chloride, total CO2, and haptoglobin. Plasma fibrinogen levels were determined via denaturization (heat precipitation) method whereby total protein is first determined in plasma, then the samples are heated 3 minutes at 57°C and centrifuged at 5000 rpm for 5 minutes to precipitate the fibrinogen, and total protein again measured, the difference being the amount of fibrinogen in the sample.
Measurement of Plasma Cytokines and Other Biomarkers of Inflammation
Some plasma samples collected from the initial 14-day study to characterize the toxicities of IC542 were evaluated for multi-analyte profiles (Rules Based Medicine Inc, Austin, Texas). Using this system plasma samples were evaluated for 60 different plasma antigens that included cytokines, growth factors, acute phase proteins and other biomarkers of inflammation.
Whole Blood in Vitro Assays
A series of experiments was conducted to examine the effect of IC542 on TNF-α and IL-6 release by rat, monkey, and human leukocytes stimulated with LPS in vitro using whole blood assays. Peripheral blood was collected using heparin as an anticoagulant at a final concentration of 20 units/mL. Sprague–Dawley rats (n = 3/females) were anesthetized and exsanguinated by cardiac puncture. Peripheral blood was obtained from cynomolgus monkeys (n = 3/females) (SNBL, Everett, WA) and healthy human volunteers (n = 3/females). Blood samples were kept at room temperature until performing the assay. All whole blood assays were performed on the same day the blood was collected.
Blood samples for each determinant were added in triplicate to 96-well polypropylene plates (Costar No. 3790) in volumes of 200 μL/well. IC542 was diluted in assay media supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 50 mM 2-mercaptoethanol to equal 10× final test concentrations and added at a volume of 25 μL /well. DMSO, present in the IC542 stock solution, was kept constant at a final concentration of 0.13%. IC542 test concentrations were added to the appropriate wells of the assay plates and incubated for 1 hour at 37°C.
The cultures were stimulated with LPS and incubated for an additional 6–8 hours at 37°C. Due to species differences in sensitivity to LPS, the concentration of LPS (E. coli serotype 055:B5) was adjusted for maximal activation of TNF-α release for each of the three species: 10000, 10, and, 0.01 ng/mL LPS for rat, monkey, and human, respectively. Levels of TNF-α and IL-6 were measured by ELISA, using species-specific commercial kits and performed according to the manufacturer’s directions (TNF-α assays for human, monkey and rat BioSource International nos. KHC3012, KPC3012, and KRC3011. IL-6 for human and rat BioSource International nos., KHC0062 and KRC0062. IL-6 for monkey, BD Biosciences no. 551496). Absorbance at 450 nm (A450) was determined by a microplate reader (Molecular Devices Corporation, Spectra Max 250).
Using SOFTmax Pro software, version 3.0 (Molecular Devices Corporation), A450 values were corrected for the background absorbance of each assay plate and cytokine concentrations (pg/mL or units/mL) were calculated from a standard curve generated using a four parameter logistic equation. Cytokine concentrations were not extrapolated below the lowest standard control. Assay determinates below the cytokine concentration of the lowest standard control were assigned a concentration value of 0 pg or unit/mL.
Statistical Analysis
Mean hematology, clinical chemistry and cytokine values collected at necropsy from vehicle controls and the various IC542-treated subgroups were compared using SPSS, version 10.0 (Chicago, IL). Following a Levene’s test to determine if differences between group variances were significant (p ≤ 0.05), a 1-way analysis of variance (ANOVA) was conducted on the various hematology, clinical chemistry and cytokine parameters. Where significant differences were indicated by ANOVA (p < 0.05), a Dunnett’s t-test was used to compare between the treated and vehicle control groups.
Results
Toxicities Associated with Repeated Dosing of IC542 to Rats
Daily oral administration of IC542 at 100 mg/kg/day was found to produce severe toxicity in some rats over a 14-day treatment period, shown in Table 1. Clinical signs were first apparent in a few rats beginning at Day 3 and continued to be observed in some animals for the duration of the study. Important clinical signs seen in IC542-treated rats included decreased activity, distended abdomen, piloerection, evidence of diarrhea and swelling of a limb or neck. A loss of body weight was often seen in rats with clinical signs (data not shown).
The histologic evaluation found that inflammation, generally characterized by a marked neutrophilic infiltrate, was present in some tissues including the intestinal tract (duodenum, jejunum, ileum, cecum, and colon) and mesentery, shown in Table 2. In the gastrointestinal tract, IC542-related changes were generally most severe in the distal jejunum and ileum. Compared to vehicle-controls (Figure 1A), rats with gastrointestinal lesions graded as either minimal or mild had slightly increased numbers of neutrophils scattered within the mucosa, submucosa, and muscularis immediately surrounding the gut-associated lymphoid tissue (GALT) (Figure 1B). In rats with moderate or severe histologic changes in the gastrointestinal tract the neutrophilic infiltrate was multifocal to diffuse and located circumferentially in the mucosa, submu-cosa and muscular layers of the intestine (Figure 1C). In the rats with more severe lesions in the gastrointestinal tract, neutrophil localization adjacent to the mesentery was especially prominent (Figure 1D).
In the mesentery, lesions were characterized by variable numbers of macrophages and neutrophils, sometimes associated with degeneration and/or loss of adipocytes. Compared to controls (Figure 2A), mesenteric lesions graded minimal to mild were composed of few neutrophils around blood vessels with minimal changes to adipocytes (Figure 2B). Moderate lesions had significant numbers of infiltrating neutrophils and macrophages replacing some mesenteric adipocytes around blood vessels (Figure 2C). Severe lesions were characterized predominantly by large numbers of macrophages replacing most adipocytes (Figure 2D). Lesions in mesenteric arteries were very rarely observed and then only in animals with significant inflammation in the surrounding mesentery. During the course of the study six of the IC542-treated rats were euthanized prior to their scheduled necropsy date due to poor health and an additional 3 animals were found dead. For the early decedents, moderate to severe histopathological changes in the gastrointestinal tract and/or mesentery were considered the most likely cause of the morbidity and/or mortality.
Neutrophilic inflammation in the thymus and attached mediastinum, heart, and soft tissues surrounding the femoral-tibial joint were observed in some IC542-treated rats. Lymphoid depletion of the thymus and/or spleen was a common finding. Congestion associated with either hemorrhage or hyperplasia in the adrenal glands and atrophy of the salivary glands was also observed in some IC542-treated rats.
In contrast to what has been reported for other PDE4 inhibitors (Larson et al., 1996; Kerns et al., 2005), vascular injury was infrequently seen with IC542-treatment. In the few instances where the vasculature was involved, the changes were minimal and always associated with severe inflammation of the surrounding tissue, suggesting that they are secondary to the overall inflammatory process.
Identification of Potential Biomarkers Associated with IC542 Toxicity in Rats
Most of the IC542-treated rats with clinical signs had elevated neutrophil counts, increased haptoglobin and fibrinogen levels, and decreased albumin levels. The severity of histopathologic changes in the gastrointestinal tract correlated with changes in selected serum biochemistry, hematology and cytokine levels, presented in Table 3. Most hematology and clinical chemistry parameters in IC542-treated rats without histologic changes were comparable to vehicle-treated controls. However, a modest decrease in albumin and increases in haptoglobin and IL-6 levels, were seen in these rats. For rats with IC542-induced histological changes in the gastrointestinal tract, alterations in absolute and percent neutrophils, haptoglobin, fibrinogen, and albumin levels generally increased with the severity of the histological score. Increases in some cytokines, IL-6, MCP-1, and MCP-3, also correlated with the severity of histologic changes. While marked increases in IL-6 levels correlated with the severity of the histopathologic changes, this cytokine had high intersubject variability.
A similar analysis comparing the severity of histologic changes in the mesentery with hematology and clinical chemistry parameters is shown in Table 4. In IC542-treated rats without histologic lesions, many hematology and clinical chemistry parameters were comparable to vehicle-treated controls. However, modest changes in albumin, haptoglobin and IL-6 levels were again seen in the rats that lacked histopathologic changes. For IC542-treated rats with histologic lesions, absolute and percentage of neutrophils, and haptoglobin and fibrinogen levels increased with the severity of the histopathology score.
Predictive Biomarker Study
The results of the 14-day repeat-dose toxicity study suggested that elevations in haptoglobin and fibrinogen, and decreases in albumin levels may serve as early and sensitive biomarkers for identifying rats with IC542-induced histopathologic changes in the tissues. This hypothesis was tested in IC542-treated rats by daily monitoring haptoglobin, fibrinogen, and albumin levels. When levels for one or more of these potential biomarkers fell outside of the predetermined cutoff limits the rat was randomized into 3 subgroups. Rats randomized to Subgroup 1 continued to be dosed until Day 7, rats randomized to Subgroup 2 were immediately necropsied. For rats randomized to Subgroup 3, dosing was immediately stopped and the animals were allowed a 14-day recovery period before necropsy to assess reversibility.
Over the 7-day treatment period, plasma levels of the 3 biomarkers (haptoglobin, fibrinogen and albumin) remained within normal limits for 18 of the 35 rats administered IC542. The histopathologic examination of target tissues from these rats at Day 7 identified only minimal changes (Score 1) in the duodenum, jejunum, ileum, and thymus in some of these rats, shown in Table 5. Histologic changes were not observed in the mesentery, heart, other regions of the gastrointestinal tract, and blood vessels in this group of IC542-treated rats.
During the study 17 rats were found to have altered levels of one or more biomarker at some point during the 7-day treatment period. Of these rats, 16 had elevated haptoglobin levels exceeding the cutoff limit, 3 exceeded the fibrinogen cutoff limit, and 7 had albumin levels below the cutoff limit. Six of these rats were assigned to Subgroup 1 and continued to be dosed through Day 7, receiving between 2 to 4 doses of IC542 after one or more of the biomarkers was found to be outside the normal limits. Due to the development of severe clinical signs of toxicity, 2 rats from this group were euthanized prior to the scheduled Day 8 necropsy. All rats assigned to Subgroup 1 had histologic changes in the jejunum and ileum. Changes in the mesentery, cecum, colon, and thymus were also apparent in the majority of the rats assigned to Subgroup 1. The histologic changes in this subgroup were the most severe of the 3 groups. In Subgroup 1, minimal vascular inflammation was present in the mesentery and thymus of a single rat, while minimal vascular degeneration was seen in mesentery of 3/6 rats. In each case changes in the blood vessels were considered secondary to the extensive inflammation of the surrounding tissue.
For rats assigned to Subgroup 2, histologic changes were present in many of the same tissues, although the incidence and severity of the changes were reduced relative to Subgroup 1. Furthermore there was no evidence of vascular injury in Subgroup 2, again indicating that this infrequent histologic change is the result of extensive inflammation of the surrounding tissue. Histologic changes in Subgroup 3 after a 14-day recovery period were limited to parts of the gastrointestinal tract and thymus and generally considered minimal in severity. These findings indicate that IC542-related toxicities are highly reversible if dosing is stopped in response to alterations in the biomarkers.
Effects of Dexamethasone on IC542-Induced Toxicities
Results indicate that the toxicity of IC542 is closely linked to a generalized inflammatory response with extensive neutrophil infiltration in the gastrointestinal tract, nearby mesentery, and thymus. To demonstrate that IC542-related toxicities are the result of an inflammatory response, rats were co-administered dexamethasone. Rats administered 100 mg/kg/day of IC542 for a 7-day treatment period developed the expected clinical observations, including weight loss, decreased activity, abdominal distension and diarrhea. Large alterations in neutrophil levels, haptoglobin, fibrinogen, and albumin levels were seen at Day 7, shown in Table 6. These rats also displayed the typical range of histopathologic changes in the gastrointestinal tract (Figure 3A), mesentery (Figure 4A), and other tissues, shown in Table 7. The co-administration of dexamethasone prevented the development of all clinical signs (data not shown), and inhibited all IC542-induced histopathologic changes, shown in Table 7 and Figures 3B and 4B. Thymic atrophy observed in the rats co-administrated IC542 and dexamethasone was attributed to the immunosuppressive effects of dexamethasone, since the incidence and severity was the same in rats treated with dexamethasone alone. Levels of haptoglobin, fibrinogen and albumin, as well as neutrophil counts in rat co-administered IC542 and dexamethasone were similar to vehicle controls, again demonstrating a good correlation between these biomarkers and histopathologic changes in the tissues.
Activity of IC542 on LPS-Activated Peripheral Blood Cells from Rats, Monkeys, and Humans
The inflammatory response in rats administered IC542 was unexpected, as inhibition of PDE4 has been shown to have potent anti-inflammatory activities in both in vitro and in vivo test systems. To further characterize the inflammatory response mechanisms in rats and evaluate the effect in other species, a series of in vitro studies was performed with LPS- activated whole blood. The in vitro assays evaluated IC542 concentrations over a concentration range that was comparable to plasma levels of IC542 measured in rats dosed with the compound. In these studies, IC542 effectively inhibited TNF-α release from LPS-activated rat, monkey and human whole blood in vitro in a concentration-dependent manner (Figure 5). Maximum TNF-α inhibition was observed at IC542 concentrations of 1 μM and 5 μM for rat and monkey whole blood, respectively. In humans, maximum inhibition was observed at IC542 concentrations of 1 to 10 μM.
The effects of IC542 on IL-6 release in rat, monkey, and human whole blood stimulated in vitro with LPS are illustrated in Figure 6. In contrast to TNF-α release, IC542 had little effect on LPS-induced IL-6 release in whole blood samples from healthy human donors. Increasing concentrations of IC542 also resulted in a modest decrease in IL-6 production in most nonhuman primate blood samples stimulated with LPS. However, in rats IC542 markedly enhanced LPS-induced IL-6 release in a concentration-dependent manner in the whole blood assays. Pretreatment with IC542 at the highest concentration examined (20 μM) resulted in increased IL-6 production with a mean % of control value 9-fold over untreated controls.
Discussion
This report describes important differences in the toxicities observed in rats treated with IC542 and other PDE4 inhibitors including rolipram and CI-1080. While clinical observations seen in rats administered high oral doses of IC542 were generally similar to what has been reported for rolipram, the histopathologic changes caused by the 2 molecules are distinct. The repeated administration of rolipram to rats is reported to produce histologic changes that include myocardial degeneration and necrosis, arteritis/periarteritis of intramural and extramural coronary arteries, necrotizing vasculitis and inflammation of the mesentery and interstitial areas of the liver (Larson et al., 1996). Focal necrosis in the stomach, and dilation/degeneration of the salivary glands were also observed in rolipram-treated rats. Vascular lesions in the mesentery similar to those induced by rolipram have also been reported in rats administered CI-1080 (Slim et al., 2002).
The most prevalent histologic change resulting from administration of IC542 to rats was a robust neutrophilic infiltration and associated tissue damage in the gastrointestinal tract and mesentery. A similar neutrophilic infiltration was observed in the heart and joint tissue of a few IC542-treated rats. This acute inflammatory response in the tissues has not been described as a feature of rolipram toxicity (Larson et al., 1996; Losco et al., 2004), but was reported for a different PDE4 inhibitor, CI-1080 (Slim et al., 2002). Other IC542-related findings included lymphoid depletion of the spleen and thymus, and atrophy of the salivary gland. IC542-induced histologic changes in the thymus, gastrointestinal tract, mesentery, and other affected tissues were always associated with a robust neutrophil infiltration. In contrast to what has been reported for other PDE4 inhibitors (Larson et al., 1996), vascular injury was infrequently seen with IC542-treatment. In the few instances where the vasculature was involved, the changes were generally considered minimal and were always associated with severe inflammation of the surrounding tissue, suggesting that these lesions are not a primary event, but rather secondary to the overall inflammatory process.
Due to earlier reports that PDE4 inhibitors produce vascular necrosis/arteritis, the blood vessels in various organs were thoroughly evaluated in IC542-treated rats. The livers of IC542-treated rats were considered normal with no evidence of the treatment-related vascular changes described with rolipram. An accumulation of inflammatory cells was noted in the hearts of a few IC542-treated rats, but there was no evidence of areritis/periarteritis of the coronary arteries. Rare instances of vascular injury, characterized as hemorrhagic or fibrinoid medial necrosis of small, medium and large arteries and arterioles, was observed in the mesentery and mediastinum of IC542-treated rats. These vascular changes were generally minimal and always associated with an extensive neutrophilic inflammation in the surrounding tissue. These data suggest that when vascular injury is seen in IC542-treated rats it is always secondary to the extensive inflammation in the surrounding tissue.
Selectivity for PDE4 relative to other PDE family members may account for the different histologic changes in rats administered various compounds classified as PDE4 inhibitors. At high dose levels, less selective PDE4 inhibitors will begin to inhibit the activity of other PDEs, and possibly produce toxicities unrelated to PDE4 inhibition. For example, the inhibition of PDE3 results in severe arterial medial necrosis and hemorrhage, due to prolonged vasodilatation and/or changes in hemodynamics resulting from changes in blood flow, heart rate and/or blood pressure (Dogterom et al., 1992; Joseph, 2000; Joseph et al., 1996; Kerns et al., 2005). A PDE4 inhibitor with low selectivity for PDE4 relative to PDE3 could produce vascular necrosis through PDE3 inhibition. IC542 inhibits PDE4 at low nanomolar concentrations, and is at least 1000-fold more selective for PDE4 than any other PDE family member including to PDE3. When compared to other PDE4 inhibitors described in the literature, including rolipram and cilomilast, IC542 is a more potent and selective inhibitor of PDE4 (Aoki et al., 2000; Hatzelmann et al., 2001). Therefore, IC542 would not necessarily produce the same histopathologic changes described for less potent and selective PDE4 inhibitors. It is also possible that the metabolism of a PDE4 inhibitor could yield a metabolite with reduced selectivity for PDE4 relative to other PDEs. Major metabolites of IC542 have been tested and found to be inactive against the PDE4 isoforms A, B, C, and D as well as the other major PDE family members (PDEs 1–3, 5, 7–11).
The severity of histopathologic changes seen in rats administered IC542 correlated with an increase in peripheral neutrophil counts and elevated levels of haptoglobin and fibrino-gen. Other changes closely associated with toxicity included detectable levels of specific proinflammatory cytokines in the serum (IL-6, MCP-1, and MCP-3) and a decrease in serum albumin levels. These changes in acute phase proteins, cytokine levels, and neutrophil levels are consistent with an inflammatory response, yet PDE4 inhibitors are considered to have anti-inflammatory activity. For example, PDE4 inhibitors have been reported to block neutrophil activation (Hatzelmann et al., 2001; Trifilieff et al., 2002), migration (Trifilieff et al., 2002), reduce the production of proinflammatory cytokines in vitro and in vivo (Reimund et al., 2001), and moderate inflammation in animal disease models (Griswold et al., 1998; Miotla et al., 1998). Although immune system activation by IC542 was unexpected, several published reports have described a proinflammatory effect of other PDE4 inhibitors in rats. The administration of the PDE4 inhibitor, rolipram, has been found to increase the number of circulating leukocytes in rats challenged with LPS (Turner et al., 1993), and increase TNF-α release by rat lung epithelium (Haddad et al., 2001), indicating that in some situations the inhibition of PDE4 can produce or exacerbate an inflammatory response in the rat.
Alterations in haptoglobin, fibrinogen, and albumin levels correlating with histopathologic changes suggest that these plasma proteins may serve as sensitive biomarkers that may be used to identify rats that have only minimal or no histological changes. Although high IL-6 levels correlate with IC542-induced toxicity, IL-6 levels among the affected animals were highly variable, probably the result of rapid clearance of the cytokine from circulation (Castell et al., 1998). However, because haptoglobin production increases in response to IL-6 and other inflammatory cytokines, the level of haptoglobin in the plasma is an indirect measure of IL-6 activity (Peppard et al., 1994; Banks et al., 1995; Wang et al., 2001). Additionally, both haptoglobin and the plasma protein fibrinogen have been reported to be a sensitive and useful marker of acute inflammation in the rat (Giffern et al., 2003). In the predictive biomarker study that monitored blood levels of haptoglobin, fibrinogen, and albumin levels, these 3 proteins proved to be early and sensitive biomarkers of IC542-induced toxicity. In IC542-treated rats with unaltered biomarkers, histologic changes of minimal severity occurred in a subset of rats. Rats that continued to be dosed with IC542, despite biomarker levels outside the normal range, developed much more severe histopathologic changes as compared to rats where IC542 dosing was stopped. Most importantly, when IC542 dosing was discontinued shortly after alterations were noted in one or more of the biomarkers, most of the histopathological changes were reversible over the 14-day recovery period.
To demonstrate that tissue injury associated with IC542 administration was the result of an inflammatory response, rats dosed with IC542 were co-administered dexamethasone. Concurrent dexamethasone treatment completely prevented both clinical observations and histologic lesions seen in rats administered IC542 alone. For rats co-administered IC542 and dexamethasone, biomarker levels remained near control values, further demonstrating that changes in these biomarkers correlate well with histopathologic changes. These results with IC542 do differ from a published study of CI-1080, a PDE4 inhibitor that induces marked vasculitis in the mesentery of treated rats (Slim et al., 2002).
Although the administration of dexamethasone moderated CI-1080 induced vascular injury, medial necrosis and degeneration of medial smooth muscle cells were still present in the absence of an inflammatory response. IC542 rarely produces vascular injury in rats and that injury is always associated with severe inflammation in the adjacent tissue. Furthermore, all histpathologic changes associated with IC542 are blocked by dexamethasone. In contrast, CI-1080 produces marked vasculitis in rats and this injury is only partially dependent on inflammation in the surrounding tissue. These results further highlight important differences between IC542 and many other compounds that are considered PDE4 inhibitors. It is tempting to speculate that the selectivity of CI-1080 for PDE4 relative to PDE3 may be low when compared to IC542 and vascular injury seen with CI-1080 results from PDE3 not PDE4 inhibition. Activity against PDE3 at high dose levels of CI-1080 could explain why the toxicities associated with this molecule are only partially responsive to dexamethasone.
To further investigate the inflammatory activity of IC542 and the upregulation of circulating cytokines, in vitro studies were undertaken to look at effects of IC542 on IL-6 production by activated PBLs in whole blood. As PDE4 inhibition in rats may not be reflective of other species, these studies were also done with monkey and human PBLs. As expected, IC542 inhibited TNF-α release in whole blood from all 3 species. However, IL-6 release from LPS-stimulated rat PBLs was markedly enhanced with increasing concentrations of IC542. In contrast, IL-6 release from stimulated monkey and human blood PBLs was either reduced or unaffected. This series of studies show that PDE4 inhibition in rat PBLs can augment some inflammatory responses, while reducing others. It is possible that low levels of endotoxin originating from the gastrointestinal tract that would normally be insufficient to activate an immune response in rats could elicit a robust response when PDE4 is inhibited. More importantly, the augmentation of IL-6 production was not seen in monkey or human PBLs. This suggests the rat may be much more sensitive than other species including humans to the effects of PDE4 inhibition.
Species differences in response to PDE4 inhibition are not completely unexpected. There are multiple studies showing that PDE4 distribution in some tissues differs between rodent and non-rodent species (Kajimoto et al., 1997; Imai et al., 1999; Perez-Torres et al., 2000). A recent study has found that levels of PDE4 enzyme activity are much higher in rats than humans in multiple tissues, making rats more susceptible to PDE4-inhibitor toxicities than other species (Bian et al., 2004). There is also evidence that the function of PDE4 in various tissues may vary between species. For example, in rats PDE3 and PDE4 are the dominant PDE isoforms involved in the regulation of basal L-type Ca+ channels in ventricular myocytes (Verde et al., 1999). However, in humans PDE3 is the dominant PDE that regulates activity of the Ca+ channel (Kajimoto et al., 1997). Collectively, these examples demonstrate that inhibition of PDE4 in rats would not necessarily be expected to produce similar effects in other species including humans.
In summary, the administration of high doses of IC542 results in toxicities that are attributed to an unexpected inflammatory response in the rat. The inflammation and associated tissue damage correlates with changes in acute phase proteins and can be monitored when histologic changes in the tissues are minimal and still fully reversible. Furthermore, the tissue damage is solely the result of a neutrophilic inflammation and can be completely suppressed by the co-administration of dexamethasone to moderate the inflammation. Differences in the toxicities observed with IC542 differ from what has been reported for other PDE4 inhibitors: IC542 does not cause arteritis or vasculitis in rats. It is likely that these differences may be related to the selectivity of various molecules for PDE4 versus other PDE family members. Finally, based on results from in vitro assays, the inflammatory response seen with IC542 may be unique to rats.