Gary Cain

Effects of Anti-VEGF on Pharmacokinetics, Biodistribution, and Tumor Penetration of Trastuzumab in a Preclinical Breast Cancer Model

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. ©2012 AACR.

Combination Drug Scheduling Defines a “Window of Opportunity” for Chemopotentiation of Gemcitabine by an Orally Bioavailable, Selective ChK1 Inhibitor, GNE-900

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2–M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabines antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968–80. ©2013 AACR.

Toxicity Profile of Small-Molecule IAP Antagonist GDC-0152 Is Linked to TNF-α Pharmacology

Inhibitor-of-apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. Small-molecule IAP antagonists are currently being tested in clinical trials as novel cancer therapeutics. GDC-0152 is a small-molecule drug that triggers tumor cell apoptosis by selectively antagonizing IAPs. GDC-0152 induces NF-κB transcriptional activity leading to expression of several chemokines and cytokines, of which tumor necrosis factor alpha (TNF-α) is the most important for single-agent tumor activity. TNF-α is a pleiotropic cytokine that drives a variety of cellular responses, comprising inflammation, proliferation, and cell survival or death depending on the cellular context. As malignant and normal cells produce TNF-α upon IAP antagonism, increased TNF-α could drive both efficacy and toxicity. The toxicity profile of GDC-0152 in dogs and rats was characterized after iv dose administration once every 2 weeks for four doses. Findings in both species consisted of a dose-related, acute, systemic inflammatory response, and hepatic injury. Laboratory findings included elevated plasma cytokines, an inflammatory leukogram, and increased liver transaminases with histopathological findings of inflammatory infiltrates and apoptosis/necrosis in multiple tissues; a toxicology profile consistent with TNF-α-mediated toxicity. Dogs exhibited more severe findings than rats, and humans did not exhibit these findings, at comparable exposures across species. Furthermore, elevations in blood neutrophil count, serum monocyte chemoattractant protein-1, and other markers of inflammation corresponded to GDC-0152 exposure and toxicity and thus may have utility as safety biomarkers.

Pharmacokinetic drivers of toxicity for basic molecules: Strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.

Phosphorous Dysregulation Induced by MEK Small Molecule Inhibitors in the Rat Involves Blockade of FGF-23 Signaling in the Kidney

MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.

Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window

p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG.

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.

Balancing efficacy and safety of an anti-DLL4 antibody through pharmacokinetic modulation

Purpose: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. Experimental Design: The F(ab′)2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab′)2) was generated and assessed in efficacy and toxicity studies. Results: Anti-DLL4 F(ab′)2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab′)2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. Conclusions: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab′)2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans. Clin Cancer Res; 22(6); 1469–79. ©2015 AACR.

MEK and ERK Kinase Inhibitors Increase Circulating Ceruloplasmin and Cause Green Serum in Rats.

Inhibition of the mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is an attractive therapeutic approach for human cancer therapy. In the course of evaluating structurally distinct small molecule inhibitors that target mitogen-activated protein kinase kinase (MEK) and ERK kinases in this pathway, we observed an unusual, dose-related increase in the incidence of green serum in preclinical safety studies in rats. Having ruled out changes in bilirubin metabolism, we demonstrated a 2- to 3-fold increase in serum ceruloplasmin levels, likely accounting for the observed green color. This was not associated with an increase in α-2-macroglobulin, the major acute phase protein in rats, indicating that ceruloplasmin levels increased independently of an inflammatory response. Elevated serum ceruloplasmin was also not correlated with changes in total hepatic copper, adverse clinical signs, or pathology findings indicative of copper toxicity, therefore discounting copper overload as the etiology. Both ERK and MEK inhibitors led to increased ceruloplasmin secretion in rat primary hepatocyte cultures in vitro, and this increase was associated with activation of the Forkhead box, class O1 (FOXO1) transcription factor. In conclusion, increased serum ceruloplasmin induced by MEK and ERK inhibition is due to increased synthesis by hepatocytes from FOXO1 activation and results in the nonadverse development of green serum in rats.

Detrusor Myopathy in Young Beagle Dogs

Hematoxylin and eosin-stained sections of urinary bladder were examined microscopically from 449 male and female beagle dogs after 2- to 4-week toxicology studies. Degenerative lesions (detrusor myopathy) of the urinary bladder muscular tunic were present in 59 of 449 (13%) dogs. Myopathic lesions consisted of focal to multifocal areas of smooth muscle fiber atrophy with cytoplasmic basophilia and vacuolation, individual cell necrosis, enlarged smooth muscle nuclei and nucleoli, and occasional mitotic figures. Adjacent areas of arteritis and periarteritis were present in 10 of 59 (17%) beagles with detrusor myopathy suggesting a possible ischemic pathogenesis of the smooth muscle lesions. There was no significant difference in prevalence of myopathy in beagles administered vehicle or various test compounds. Prior urinary catheterization procedures appeared to augment the prevalence of myopathy; some lesions were adjacent to minor, iatrogenically traumatized urinary bladder mucosa. Muscle lesions were not observed in urinary bladders from mongrel dogs, monkeys, cats, rats, or microswine. When compared to crossbred dogs and other laboratory species, the beagle dog thus appears to be sensitive to development of detrusor myopathy.