Raj K. Puri

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis.

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-β1 in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Rα2. Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-α. Second, it involves IL-13 signaling through IL-13Rα2 to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Rα2 expression reduced production of TGF-β1 in oxazolone-induced colitis and that prevention of IL-13Rα2 expression, Il13ra2 gene silencing or blockade of IL-13Rα2 signaling led to marked downregulation of TGF-β1 production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Rα2 signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-β1–mediated fibrosis.

Phase III randomized trial of CED of IL13-PE38QQR vs Gliadel wafers for recurrent glioblastoma †

Convection-enhanced delivery (CED) of cintredekin besudotox (CB) was compared with Gliadel wafers (GW) in adult patients with glioblastoma multiforme (GBM) at first recurrence. Patients were randomized 2:1 to receive CB or GW. CB (0.5 microg/mL; total flow rate 0.75 mL/h) was administered over 96 hours via 2-4 intraparenchymal catheters placed after tumor resection. GW (3.85%/7.7 mg carmustine per wafer; maximum 8 wafers) were placed immediately after tumor resection. The primary endpoint was overall survival from the time of randomization. Prestated interim analyses were built into the study design. Secondary and tertiary endpoints were safety and health-related quality-of-life assessments. From March 2004 to December 2005, 296 patients were enrolled at 52 centers. Demographic and baseline characteristics were balanced between the 2 treatment arms. Median survival was 36.4 weeks (9.1 months) for CB and 35.3 weeks (8.8 months) for GW (P = .476). For the efficacy evaluable population, the median survival was 45.3 weeks (11.3 months) for CB and 39.8 weeks (10 months) for GW (P = .310). The adverse-events profile was similar in both arms, except that pulmonary embolism was higher in the CB arm (8% vs 1%, P = .014). This is the first randomized phase III evaluation of an agent administered via CED and the first with an active comparator in GBM patients. There was no survival difference between CB administered via CED and GW. Drug distribution was not assessed and may be crucial for evaluating future CED-based therapeutics.

Direct Intracerebral Delivery of Cintredekin Besudotox (IL13-PE38QQR) in Recurrent Malignant Glioma: A Report by the Cintredekin Besudotox Intraparenchymal Study Group

PURPOSE Glioblastoma multiforme (GBM) is a devastating brain tumor with a median survival of 6 months after recurrence. Cintredekin besudotox (CB) is a recombinant protein consisting of interleukin-13 (IL-13) and a truncated form of Pseudomonas exotoxin (PE38QQR). Convection-enhanced delivery (CED) is a locoregional-administration method leading to high-tissue concentrations with large volume of distributions. We assessed the use of intracerebral CED to deliver CB in patients with recurrent malignant glioma (MG). PATIENTS AND METHODS Three phase I clinical studies evaluated intracerebral CED of CB along with tumor resection. The main objectives were to assess the tolerability of various concentrations and infusion durations; tissue distribution; and methods for optimizing delivery. All patients underwent tumor resection followed by a single intraparenchymal infusion (in addition to the intraparenchymal one following resection), with a portion of patients who had a preresection intratumoral infusion. RESULTS A total of 51 patients with MG were treated including 46 patients with GBM. The maximum tolerated intraparenchymal concentration was 0.5 microg/mL and tumor necrosis was observed at this concentration. Infusion durations of up to 6 days were well tolerated. Postoperative catheter placement appears to be important for optimal drug distribution. CB- and procedure-related adverse events were primarily limited to the CNS. Overall median survival for GBM patients is 42.7 weeks and 55.6 weeks for patients with optimally positioned catheters with patient follow-up extending beyond 5 years. CONCLUSION CB appears to have a favorable risk-benefit profile. CED is a complex delivery method requiring catheter placement via a second procedure to achieve accurate catheter positioning, better drug distribution, and better outcome.

The External RNA Controls Consortium: a progress report

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Medical applications of microarray technologies: a regulatory science perspective.

The potential medical applications of microarrays have generated much excitement, and some skepticism, within the biomedical community. Some researchers have suggested that within the decade microarrays will be routinely used in the selection, assessment, and quality control of the best drugs for pharmaceutical development, as well as for disease diagnosis and for monitoring desired and adverse outcomes of therapeutic interventions. Realizing this potential will be a challenge for the whole scientific community, as breakthroughs that show great promise at the bench often fail to meet the requirements of clinicians and regulatory scientists. The development of a cooperative framework among regulators, product sponsors, and technology experts will be essential for realizing the revolutionary promise that microarrays hold for drug development, regulatory science, medical practice and public health.

Cross-platform comparability of microarray technology: Intra-platform consistency and appropriate data analysis procedures are essential

BackgroundThe acceptance of microarray technology in regulatory decision-making is being challenged by the existence of various platforms and data analysis methods. A recent report (E. Marshall, Science, 306, 630–631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676–5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology.ResultsWe reanalyzed Tans dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tans study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. and Marshall.ConclusionOur results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures. Thus, we are progressively coordinating the MAQC project, a community-wide effort for microarray quality control.

Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4.

Previously, Puri et al. (Puri, R. K., M. Ogata, P. Leland, G. M. Feldman, D. Fitzgerald, and I. Pastan. 1991. Cancer Res. 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin. In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4. By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM. Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb. IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line. The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines. Tumor cell growth, as measured by [3H]thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner. A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4. Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro. In fact, IL-4 caused modest stimulation of their growth. Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.

Poor drug distribution as a possible explanation for the results of the PRECISE trial

OBJECT Convection-enhanced delivery (CED) is a novel intracerebral drug delivery technique with considerable promise for delivering therapeutic agents throughout the CNS. Despite this promise, Phase III clinical trials employing CED have failed to meet clinical end points. Although this may be due to inactive agents or a failure to rigorously validate drug targets, the authors have previously demonstrated that catheter positioning plays a major role in drug distribution using this technique. The purpose of the present work was to retrospectively analyze the expected drug distribution based on catheter positioning data available from the CED arm of the PRECISE trial. METHODS Data on catheter positioning from all patients randomized to the CED arm of the PRECISE trial were available for analyses. BrainLAB iPlan Flow software was used to estimate the expected drug distribution. RESULTS Only 49.8% of catheters met all positioning criteria. Still, catheter positioning score (hazard ratio 0.93, p = 0.043) and the number of optimally positioned catheters (hazard ratio 0.72, p = 0.038) had a significant effect on progression-free survival. Estimated coverage of relevant target volumes was low, however, with only 20.1% of the 2-cm penumbra surrounding the resection cavity covered on average. Although tumor location and resection cavity volume had no effect on coverage volume, estimations of drug delivery to relevant target volumes did correlate well with catheter score (p < 0.003), and optimally positioned catheters had larger coverage volumes (p < 0.002). Only overall survival (p = 0.006) was higher for investigators considered experienced after adjusting for patient age and Karnofsky Performance Scale score. CONCLUSIONS The potential efficacy of drugs delivered by CED may be severely constrained by ineffective delivery in many patients. Routine use of software algorithms and alternative catheter designs and infusion parameters may improve the efficacy of drugs delivered by CED.

Safety, Tolerability, and Tumor Response of IL4-Pseudomonas Exotoxin (NBI-3001) in Patients with Recurrent Malignant Glioma

SummaryPurpose: This was an open-label, dose-escalation trial of intratumoral administration of IL-4Pseudomonas exotoxin (NBI-3001) in patients with recurrent malignant glioma. Patients and methods: A total of 31 patients with histologically verified supratentorial grades 3 and 4 astrocytoma were studied. Of these, 25 patients were diagnosed with glioblastoma multiforme (GBM) while six were diagnosed with anaplastic astrocytoma. Patients were over 18 years of age and had Karnofsky performance scores ≥60. Patients were assigned to one of four dose groups in a dose-escalation fashion: 6 µg/ml × 40 ml, 9 µg/ml × 40 ml, 15 µg/ml × 40 ml, or 9 µg/ml × 100 ml of NBI-3001 administered via convection-enhanced delivery intratumorally using stereotactically placed catheters. Patients were followed with serial MRI scans and clinical assessments every four weeks for the first 16 weeks and then every eight weeks until week 26. Results: No drug-related systemic toxicity, as evident by lack of hematological or serum chemical changes, was apparent in any patients; treatment-related adverse effects were limited to the central nervous system. No deaths were attributable to treatment. Drug-related grade 3 or 4 toxicity was seen in 39% of patients in all dose groups and 22% of patients at the maximum tolerated dose of 6 µg/ml × 40 ml. The overall median survival was 8.2 months with a median survival of 5.8 months for the GBM patients. Six-month survival was 52% and 48%, respectively. Gadolinium-enhanced magnetic resonance imaging of the brain showed areas of decreased signal intensity within the tumor consistent with tumor necrosis following treatment in many patients. Conclusions: NBI-3001 appears to have an acceptable safety and toxicity profile when administered intratumorally in patients with recurrent malignant glioma.

MSC-Based Product Characterization for Clinical Trials: An FDA Perspective

Proposals submitted to the FDA for MSC-based products are undergoing a rapid expansion that is characterized by increased variability in donor and tissue sources, manufacturing processes, proposed functional mechanisms, and characterization methods. Here we discuss the diversity in MSC-based clinical trial product proposals and highlight potential challenges for clinical translation.