Characterization and translational development of IOA-289, a novel autotaxin inhibitor for the treatment of solid tumors.

Background: Autotaxin-lysophosphatidic acid (ATX-LPA) signaling has a predominant role in immunological and fibrotic processes, including cancer. Several ATX inhibitors and LPA receptor antagonists have been clinically evaluated, but none in patients with solid tumors. Many cancers are burdened with a high degree of fibrosis and an immune desert phenotype (so-called 'cold' tumors). In these cold tumors, the fibrotic stroma provides an intrinsic cancer-supporting mechanism. Furthermore, the stroma prevents penetration and limits the effectiveness of existing therapies. IOA-289 is a novel ATX inhibitor with a unique chemical structure, excellent potency and an attractive safety profile. Materials and methods: In vitro and in vivo pharmacology studies have been carried out to elucidate the pharmaceutical properties and mechanism of action of IOA-289. A phase I clinical study in healthy volunteers was carried out to determine the pharmacokinetics and pharmacodynamics of IOA-289 following a single oral dose. Results: In vitro and in vivo studies showed that IOA-289 is a potent inhibitor of ATX and, as a monotherapy, is able to slow progression of lung fibrosis and tumor growth in mouse models. In a clinical study, IOA-289 showed a dose-dependent increase in plasma exposure levels and a corresponding decrease in circulating LPA. Conclusions: Our data show that IOA-289 is a novel ATX inhibitor with a unique chemical structure, excellent potency and an attractive safety profile. Our data support the further development of IOA-289 as a novel therapeutic approach for the treatment of cancer, particularly those with a high fibrotic and immunologically cold phenotype.

Validation of an LC-MS/MS method for the quantification IOA-289 in human plasma and its application in a first-in-human clinical trial.

IOA-289 is a novel small molecule inhibitor of autotaxin developed as a first-in-class therapy of fibrotic pathologies including cancer. A method for quantitation of IOA-289 in human plasma was developed using a stable isotope labeled compound ([13C4]IOA-289) as internal standard. The analytes were extracted from human plasma by protein precipitation and the analysis was performed by liquid chromatography coupled with tandem mass spectrometric detection (LCMS/MS). The chromatographic separation was performed with a gradient elution from a BEH C18 column and under these conditions the retention time and the run time were 1 and 2 min, respectively. The assay was fully validated over the range 3-3000 ng/mL, proved to be accurate, precise and selective and was successfully applied to quantitate IOA-289 in plasma samples from subjects in a first-in-humanclinical trial.

A Phase 2 Study of Galunisertib (TGF-ß1 Receptor Type I Inhibitor) and Sorafenib in Patients With Advanced Hepatocellular Carcinoma.

INTRODUCTION: Inhibition of tumor growth factor-ß (TGF-ß) receptor type I potentiated the activity of sorafenib in preclinical models of hepatocellular carcinoma (HCC). Galunisertib is a small-molecule selective inhibitor of TGF-ß1 receptor type I, which demonstrated activity in a phase 2 trial as second-line HCC treatment. METHODS: The combination of galunisertib and sorafenib (400 mg BID) was tested in patients with advanced HCC and Child-Pugh A liver function without prior systemic therapy. Galunisertib dose was administered 80 or 150 mg b.i.d. orally for 14 days every 28 days in safety lead-in cohorts; in the expansion cohort, all patients received galunisertib 150 mg b.i.d. Objectives included time-to-tumor progression, changes in circulating alpha fetoprotein and TGF-ß1, safety, overall survival (OS), response rate, and pharmacokinetics (PK). RESULTS: Patients (n = 47) were enrolled from 5 non-Asian countries; 3 and 44 patients received the 80 mg and 150 mg b.i.d. doses of galunisertib, respectively. The pharmacokinetics and safety profiles were consistent with monotherapy of each drug. For the 150 mg b.i.d. galunisertib cohort, the median time-to-tumor progression was 4.1 months; the median OS was 18.8 months. A partial response was seen in 2 patients, stable disease in 21, and progressive disease in 13. TGF-ß1 responders (decrease of >20% from baseline) vs nonresponders had longer OS (22.8 vs 12.0 months, P = 0.038). DISCUSSION: The combination of galunisertib and sorafenib showed acceptable safety and a prolonged OS outcome.

Window-of-opportunity trials to evaluate clinical activity of new molecular entities in oncology.

BACKGROUND: The introduction of molecular targeted agents (e.g., monoclonal antibodies or kinase inhibitors) and cancer vaccines has raised the question whether alternate clinical trial designs, including window trials, are better suited to evaluate such new molecular entities (NMEs) and improve their approval rates. In window trials, patients receive an NME for a window of time before starting standard treatment allowing the evaluation of an NME in tumors unperturbed by previous therapies. METHODS: A systematic literature search was conducted to identify window trials in adult and pediatric oncology. RESULTS: Twenty-nine window trials were identified and reviewed, 13 in pediatric and 16 in adult oncology. Most of the trials (20/29) tested cytotoxics known to have activity in other clinical situations. In contrast to trials with pretreated patients, the window trials established the antitumor activity of melphalan, topotecan, epirubicin and etoposide in untreated patients with rhabdomyosarcoma or small-cell lung cancer. In window trials with ineffective or modestly active NMEs, we found no indication of a significant negative effect on overall survival for participating patients. CONCLUSIONS: Provided close safety monitoring and careful patient selection, window trials are a safe option to investigate potential clinical activity of NMEs.

A window of opportunity phase II study of enzastaurin in chemonaive patients with asymptomatic metastatic colorectal cancer.

BACKGROUND: Preclinically, protein kinase C and AKT activation can be inhibited by enzastaurin and reduce tumor growth of colorectal cancer cells. In asymptomatic patients with metastatic colorectal cancer (mCRC), enzastaurin activity was evaluated by measuring the 6-month progression-free survival (PFS) rate in a window study design. PATIENTS AND METHODS: Chemonaive patients with asymptomatic mCRC who did not require immediate chemotherapy-induced tumor reduction received a 400-mg thrice daily loading dose of enzastaurin on day 1 of cycle 1, followed by 500 mg once daily for the remaining 28-day cycles. Progression was assessed on the basis of radiographic imaging, rise in carcinoembryonic antigen or lactate dehydrogenase (LDH) levels or by appearance of clinical symptoms. RESULTS: Twenty-eight patients received daily enzastaurin. The 6-month PFS rate was 28% [95% confidence interval (CI) 13%-45%] and median PFS was 1.9 months (95% CI 1.8-4.5 months). Twelve (43%) patients had stable disease with a median duration of 6.1 months. The survival rate at 20 months was 77% (95% CI 47%-92%). No grade 4 toxicity was reported and grade 3 toxic effects were observed in three patients with one patient showing probable drug-related elevation of liver transaminases. CONCLUSION: The window design in asymptomatic patients with mCRC can be safely applied to assess the activity and safety of novel cytostatic agents like enzastaurin.

Phase Ib safety and pharmacokinetic evaluation of daily and twice daily oral enzastaurin in combination with pemetrexed in advanced/metastatic cancer.

BACKGROUND: This phase Ib study evaluated the safety, pharmacokinetics, and activity of enzastaurin either 500 mg once daily (QD) or 250 mg twice daily (b.i.d.) in combination with pemetrexed. PATIENTS AND METHODS: Pemetrexed 500 mg/m(2) with folic acid and vitamin B(12) was given on day 1 every 21 days with enzastaurin 500 mg orally QD starting on day 5 of cycle 1 after a loading dose of 400 mg thrice daily on day 4. To evaluate whether a b.i.d. regimen results in higher enzastaurin exposures, the study was amended. After amendment, in cycle 1, patients received 500 mg enzastaurin QD on days 1-15 without initial loading dose and 250 mg b.i.d. on days 16-30; in subsequent cycles, patients received pemetrexed on day 1 every 21 days with enzastaurin b.i.d. RESULTS: Sixty-eight patients (42 preamendment and 26 postamendment) were assessed. Pemetrexed toxicity and pharmacokinetics did not appear to be altered by enzastaurin. Enzastaurin average steady-state plasma concentration (C(av,ss)) decreased by approximately 25% in the presence of pemetrexed. Enzastaurin C(av,ss) were approximately 40% higher in the b.i.d. versus QD regimen. Three patients (4.4%) with thyroid cancer of follicular/papillary type had partial response as defined by RECIST. CONCLUSIONS: Pemetrexed plus enzastaurin is well tolerated with preliminary evidence of anticancer activity, particularly in thyroid cancer.

Enzastaurin inhibits tumours sensitive and resistant to anti-EGFR drugs.

We investigated the antitumour effect and ability to overcome the resistance to anti-EGFR drugs of enzastaurin, an inhibitor of VEGFR-dependent PKCbeta signalling. Enzastaurin was evaluated alone and in combination with the EGFR inhibitor gefitinib, on growth and signalling protein expression in human cancer cells sensitive and resistant to anti-EGFR drugs, both in vitro and in nude mice. We demonstrated the marked inhibitory activity of enzastaurin against GEO colon and PC3 prostate cancer cells and their gefitinib-resistant counterparts GEO-GR and PC3-GR, accompanied by inhibition of pAkt and its effector pp70S6K, pGSK3beta and VEGF expression and secretion. Moreover, enzastaurin showed a cooperative effect with gefitinib in parental and in gefitinib-resistant cells. Remarkably, these results were confirmed in vivo, where enzastaurin showed antitumour activity and cooperativity with gefitinib in mice grafted with GEO and GEO-GR tumours, incrementing their median survival and inhibiting the aforesaid protein expression and secretion in tumour specimens. In conclusion, enzastaurin by interfering with signalling proteins implicated in EGFR drug resistance markedly cooperates with gefitinib in sensitive and gefitinib-resistant tumours, thus overcoming and reverting such resistance and providing a rational basis for its development in patients resistant to anti-EGFR drugs.

Development and validation of a phosphorylated SMAD ex vivo stimulation assay.

Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.

The role of protein kinase C-alpha (PKC-alpha) in malignancies of the gastrointestinal tract.

Drugs specifically designed to block cellular signalling proteins are currently evaluated as a new way to treat gastrointestinal tumours. One such "new targeted agent" is aprinocarsen, an antisense oligonucleotide that specifically blocks the mRNA of protein kinase C-alpha (PKC-alpha). Blocking PKC-alpha, an important cellular signalling molecule associated with tumour growth, is anticipated to result in tumour cell arrest and achieve clinical benefits. However, it is not known which patients may benefit most from a specific inhibition of PKC-alpha. Past experience with other novel targeted agents suggests that expression of the target molecule is an important factor for the success of such a specific therapy. Therefore, reviewing the specific role of PKC-alpha in various gastrointestinal tumours may contribute to focus the clinical development of selective or specific PKC-alpha inhibitors, such as aprinocarsen, on those patients with a distinctive PKC-alpha expression pattern.

Extensive and preferential Fas/Fas ligand-dependent death of gammadelta T cells following infection with Listeria monocytogenes.

In the spleens of mice infected intraperitoneally with the bacterium Listeria monocytogenes, both alphabeta and gammadelta T cells became rapidly activated, followed by a massive apoptotic death response predominantly within the gammadelta population. The death response involved two major splenic gammadelta T-cell subsets and was Fas/Fas ligand (Fas-L)-dependent. Among T cells isolated from the Listeria-infected spleen, Fas-L was almost exclusively expressed in gammadelta T cells. gammadelta T cells coexpressed Fas and Fas-L, suggesting activation-induced suicide as a mechanism of their death. In vivo treatment with an antibody specific for CD3epsilon induced activation, preferential Fas-L expression and apoptosis of gammadelta T cells, resembling the response pattern in listeriosis, whereas antibodies specific for T-cell receptor-beta (TCR-beta) or TCR-delta did not, suggesting that the complete response seen in listeriosis requires both gammadelta TCR engagement and additional stimuli. L. monocytogenes causes early nonspecific, Fas-independent lymphocyte death in heavily infected tissues. In contrast, the death response described here is selective, Fas-dependent and triggered at low local levels of bacteria, suggesting that it is controlled by interactions with other infection-activated host cells, and perhaps part of a regulatory circuit specifically curtailing gammadelta T cells.

Advance directives in skilled nursing facility residents transferred to emergency departments.

OBJECTIVE: Ten years have passed since Congress enacted the Patient Self-Determination Act to promote the use of advance directives (ADs). This study was performed to determine the frequency, type, demographic distribution, and utility of ADs that accompany residents of skilled nursing facilities (SNFs) transferred to emergency departments (EDs). METHODS: This was an observational, cross-sectional cohort of SNF residents, transferred to two urban, academic EDs. Chart review and physician interviews were conducted on consecutive patients arriving during 12-hour data collection shifts. RESULTS: Among 715 patients entered, 315 [44%, 95% confidence interval (95% CI) = 40% to 48%] had an AD. Advance directives were significantly more prevalent among white (50%) than African American (34%) or Hispanic (39%) patients (p < 0.001), and varied from 0% to 94% among SNFs. Of the 315 patients with ADs, do-not-resuscitate (DNR) orders were the most prevalent (65%, 95% CI = 58% to 69%). Although 75% (95% CI = 69% to 81%) of the DNR orders addressed cardiopulmonary resuscitation (CPR), only 12% (95% CI = 8% to 16%) addressed intubation. Among 39 patients who required intubation or CPR, 44% had ADs, 82% (95% CI = 57% to 96%) of which were deemed useful. CONCLUSIONS: Despite a decade of legislation promoting their use, ADs are lacking in most SNF residents transferred to EDs for evaluation and in most settings in which a clinical indication exists for intubation or CPR. Variation in their prevalence appears to be associated with both ethnicity and SNF origin. Although about three-fourths of DNR ADs addressed CPR, only about one in ten offered guidance regarding intubation. When available, ADs are used in most instances to guide emergency care.

Tumor necrosis factor-alpha negatively regulates airway hyperresponsiveness through gamma-delta T cells.

Tumor necrosis factor (TNF)-alpha is a potent cytokine with immunomodulatory, proinflammatory, and pathobiologic activities. Although TNF-alpha is thought to play a role in mediating airway inflammation and airway hyperresponsiveness (AHR), its function is not well defined. TNF-alpha-deficient mice and mice expressing TNF-alpha in their lungs because of a TNF-alpha transgene placed under the control of the surfactant protein (SP)-C promoter (SP-C/TNF-alpha-transgenic mice) were sensitized to ovalbumin (OVA) and subsequently challenged with OVA via the airways; airway function in response to inhaled methacholine was monitored. In the TNF-alpha-deficient mice, AHR was significantly increased over that in controls. In contrast, the transgenic mice failed to develop AHR. In addition, sensitized/ challenged TNF-alpha-deficient mice had significantly increased numbers of eosinophils and higher levels of interleukin (IL)-5 and IL-10 in their bronchoalveolar lavage fluid than were found for control mice. However, in SP-C/TNF-alpha-transgenic mice, both the numbers of eosinophils and levels of IL-5 and IL-10 were significantly lower than in sensitized/challenged transgene-negative mice. gammadelta T cells have been shown to be activated by TNF-alpha and to negatively regulate AHR. Depletion of gammadelta T cells in the TNF-alpha-transgenic mice in the present study increased AHR, whereas depletion of these cells had no significant effect in TNF-alpha-deficient mice. These data indicate that TNF-alpha can negatively modulate airway responsiveness, controlling airway function in allergen-induced AHR through the activation of gammadelta T cells.

gammadelta T cells as regulators of airway hyperresponsiveness.

Airway responsiveness (AR) is determined by complex mechanisms reflecting lung responses to airborne stimuli. Murine studies have identified a number of potential factors modulating AR and thus have contributed to the current understanding of these mechanisms. In allergic inflammation, immune cells, in particular alphabeta T cells, have emerged as important contributors to increased AR. We have found that in contrast to alphabeta T cells, gammadelta T cells can have a negative regulatory effect on AR. Here, we review the current studies on gammadelta T cells in allergic inflammation and discuss their role in modulating AR. We propose that gammadelta T cells exhibit different immune properties depending on the type of stimulus and inflammation. These differential immune properties appear to be associated with specific gammadelta T cell subsets, which control AR to airborne stimuli. In particular, our recent data indicate that the Vgamma4(+) T cell subset acts as an important negative regulator of AR and contributes to maintaining normal lung function in mice.

Activation of murine epidermal V gamma 5/V delta 1-TCR(+) T cell lines by Glu-Tyr polypeptides.

The physiologic role of gamma delta-T-cell-receptor-bearing cells and the T cell receptor ligands that they recognize is still poorly understood. Previous studies have suggested that one possible antigen for gamma delta-TCR(+) cells is the random copolymer poly-glutamic acid-tyrosine (poly-Glu-Tyr), because poly-Glu-Tyr-reactive gamma delta-TCR(+) hybridoma cells were produced from poly-Glu-Tyr-immunized mice. We have found, however, that clonal V gamma 5/V delta 1-TCR(+) epidermal T cell lines from nonimmune mice also respond to poly-Glu-Tyr by producing cytokines. Other amino acid homopolymers, copolymers, and tripolymers were not stimulatory for the V gamma 5/V delta 1-TCR(+) epidermal T cells, except for poly-glutamic acid-alanine-tyrosine (poly-Glu-Ala-Tyr). Of the poly-Glu-Tyr and poly-Glu-Ala-Tyr polymers, only those that contained Glu and Tyr in an equimolar ratio were stimulatory. The cytokine interleukin-2 was strictly required for the responses to poly-Glu-Ala-Tyr, whereas the responses to poly-Glu-Tyr were merely enhanced with interleukin-2. The response to poly-Glu-Tyr was also enhanced by crosslinking CD28 molecules with plate-bound anti-CD28 crosslinking antibody. This finding suggests that the poly-Glu-Tyr response has a partial dependence on CD28-mediated costimulation, a characteristic of TCR-dependent responses. Consistent with this observation, V gamma 5/V delta 1-TCR-loss variants of the epidermal T cell line could no longer respond to poly-Glu-Tyr. The unpredicted responses of epidermal gamma delta-TCR(+) T cells to poly-Glu-Tyr and poly-Glu-Ala-Tyr demonstrate that the functions of these cells potentially can be triggered by peptidic ligands, probably through a TCR-mediated process.

Inhibition of phosphodiesterase 4 attenuates airway hyperresponsiveness and airway inflammation in a model of secondary allergen challenge.

We compared for the first time the therapeutic potential of a specific phosphodiesterase 4 (PDE4) inhibitor, rolipram, with anti-VLA-4 and anti-IL-5 in a model of secondary allergen exposure of previously sensitized and challenged mice. To address these issues, mice were sensitized and challenged with ovalbumin (OVA) (primary challenge). Six weeks later, sensitized/challenged mice were reexposed to OVA (secondary challenge) and airway response (resistance [RL] and dynamic compliance [Cdyn]) to inhaled methacholine was monitored. After secondary OVA challenge, RL significantly increased as did the number of lung inflammatory cells and IL-4 and IL-5 production in bronchoalveolar lavage fluid (BALF). Administration of rolipram, in a dose-dependent manner, significantly prevented both changes in RL and Cdyn, as well as eosinophil, lymphocyte, and neutrophil accumulation in the BALF; IL-4 and IL-5 levels in BALF were also significantly reduced. In contrast, treatment with anti-VLA-4 and anti-IL-5 only prevented changes in RL and eosinophil numbers and IL-5 production in BALF. Further, goblet cell hyperplasia was suppressed only by treatment with rolipram. None of the treatments affected OVA-specific antibody levels. These studies confirm that IL-5 dependent eosinophilic inflammation plays an essential role in the development of certain aspects of airway function after rechallenge of sensitized mice and that lymphocytes and neutrophils are also important in the development of altered airway function. The use of agents that inhibit PDE4 may have an important role in the treatment of asthma in previously sensitized mice.