Risk of tumorigenicity in mesenchymal stromal cell-based therapies--bridging scientific observations and regulatory viewpoints.

In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.

Modelling the risk-benefit impact of H1N1 influenza vaccines.

BACKGROUND: Shortly after the H1N1 influenza virus reached pandemic status in June 2009, the benefit-risk project team at the European Medicines Agency recognized this presented a research opportunity for testing the usefulness of a decision analysis model in deliberations about approving vaccines soon based on limited data or waiting for more data. Undertaken purely as a research exercise, the model was not connected to the ongoing assessment by the European Medicines Agency, which approved the H1N1 vaccines on 25 September 2009. METHODS: A decision tree model constructed initially on 1 September 2009, and slightly revised subsequently as new data were obtained, represented an end-of-September or end-of-October approval of vaccines. The model showed combinations of uncertain events, the severity of the disease and the vaccines' efficacy and safety, leading to estimates of numbers of deaths and serious disabilities. The group based their probability assessments on available information and background knowledge about vaccines and similar pandemics in the past. RESULTS: Weighting the numbers by their joint probabilities for all paths through the decision tree gave a weighted average for a September decision of 216 500 deaths and serious disabilities, and for a decision delayed to October of 291 547, showing that an early decision was preferable. CONCLUSIONS: The process of constructing the model facilitated communications among the group's members and led to new insights for several participants, while its robustness built confidence in the decision. These findings suggest that models might be helpful to regulators, as they form their preferences during the process of deliberation and debate, and more generally, for public health issues when decision makers face considerable uncertainty.

Considerations for the chemistry, manufacturing and Controls (CMC) - quality package for COVID-19 vaccines- interim lessons learnt by the European medicines Agency (EMA).

The European Medicines Agency (EMA) has approved five pandemic COVID-19 vaccines (prior to April 2022) and many others are in the pipeline. The commentary describes how timely approval and rapid manufacturing capacity scale up could be achieved from our perspective. The commentary considers the need for: early, continuous engagement with the regulator for COVID-19 vaccines; understanding key Chemistry, Manufacturing and Controls (CMC) challenges in order to build a successful COVID-19 vaccine CMC dossier; investing in production and testing site readiness for COVID-19 vaccines; CMC Lifecycle and post-approval planning for COVID-19 vaccines as well as future directions including international regulatory cooperation. EMA's experience of the CMC scientific considerations, which facilitated both timely approvals (as Conditional Marketing Authorisations) and rapid increase in production capacity and supply, is of interest to healthcare professionals, academia, pharmaceutical industry and global regulators to communicate the flexibility and agility applied to COVID-19 vaccines by the EU regulatory system and how these activities can be optimised while complying with the strict quality standards in the EU.

Novel tetrahydroisoquinolin-ethyl-phenylamine based multidrug resistance inhibitors with broad-spectrum modulating properties.

PURPOSE: The ATP-binding cassette transporters P-glycoprotein (Pgp) and BCRP are implicated in multidrug resistance (MDR) of many tumors. Multi-targeted inhibitors such as cyclosporin A, have been shown to circumvent MDR in clinical trials. Here, we present the characterization of a novel class of effective and multi-targeted tetrahydroisoquinolin-ethyl-phenylamine based MDR inhibitors. METHODS: The novel MDR inhibitors, XR9577, WK-X-34, WK-X-50 and WK-X-84 were examined for cellular toxicity in several cell lines. Chemosensitivity and inhibition of BCRP-mediated mitoxantrone efflux were analyzed in BCRP-overexpressing MCF7/mx cells. Chemosensitivity towards daunorubicin and inhibition of Pgp-mediated efflux of (99m)Tc-Sestamibi were examined in Pgp-overexpressing A2780/Adr cells. Potential MRP-interactions were evaluated with 5-CFDA efflux assays in selectively transfected MRP-1, -2 and -3 cell lines. RESULTS: All WK-X-compounds showed significant BCRP inhibition in the MCF7/mx cells resulting in significant increases in mitoxantrone intracellular accumulation and 200-300 fold increases in mitroxantrone cytotoxicity. WK-X-34 and XR9577 were also potent inhibitors of Pgp, increasing (99m)Tc-Sestamibi accumulation with IC(50) values in the nM range. Daunorubicin cytotoxicity was also increased seven to eight-fold in cells co-treated with XR9577 or WK-X-34 (10 muM). These compounds did not appear to interact with the MRP transporters. As compared to cyclosporin A, these compounds showed reduced cellular toxicity and increased potency of BCRP and Pgp inhibition. CONCLUSION: The novel MDR inhibitors WK-X-34 and XR9577 demonstrate superior effectiveness in Pgp and BCRP inhibition, in vitro tolerance and specificity over cyclosporin A. The novel compounds might be the promising candidates for a broad-spectrum based approach to the circumvention of MDR in resistant tumors.

99mTc-Sestamibi, a sensitive probe for in vivo imaging of P-glycoprotein inhibition by modulators and mdr1 antisense oligodeoxynucleotides.

PURPOSE: We tested the suitability of (99m)Tc-sestamibi to image the inhibition of P-glycoprotein (Pgp)-mediated multidrug resistance in tumor cells and xenografts after antisense treatment and/or inhibition with a novel Pgp modulator WK-X-34. PROCEDURE: Pgp inhibition was measured by daunorubicin transport assays and fluorescence microscopy in resistant A2780/Adr cells treated with WK-X-34 and antisense. A2780/Adr xenograft mice were dosed with mdr1 antisense oligodeoxynucleotides intratumorally for three days; next, mice were treated with WK-X-34, followed by (99m)Tc-sestamibi injection. Mice were imaged, sacrificed, and tissues collected. Images and isolated tissues were analyzed for (99)Tc distribution. Pgp expression was analyzed by immunofluorescence and reverse transcription-polymerase chain reaction. RESULTS: Both WK-X-34 and mdr1 antisense treatments significantly inhibited Pgp activity in vitro and in xenografts. Biodistribution results correlated with results from the (99m)Tc-sestamibi images. Mdr1 mRNA and Pgp were significantly down-regulated by antisense treatments. CONCLUSIONS: (99m)Tc-sestamibi is a sensitive probe to monitor Pgp inhibition by different mechanisms in vivo in tumor xenografts.

Cell-based therapies for cardiac repair: a meeting report on scientific observations and European regulatory viewpoints.

In the past decade, novel cell-based products have been studied in patients with acute and chronic cardiac disease to assess whether these therapies are efficacious in improving heart function and preventing the development of end-stage heart failure. Cardiac indications studied include acute myocardial infarction (AMI), refractory angina, and chronic heart failure (CHF). Increased clinical activity, experience, and multiple challenges faced by developers have been recognized at the regulatory level. In May 2014, the Committee for Advanced Therapies (CAT) discussed in an expert meeting various cell-based medicinal products developed for cardiac repair, with a focus on non-manipulated bone marrow cells, sorted bone marrow or apheresis, and expanded cells, applied to patients with AMI or CHF. The intention was to share information, both scientific and regulatory, and to examine the challenges and opportunities in this field. These aspects were considered from the quality, and non-clinical and clinical perspectives, including current imaging techniques, with a focus on AMI and CHF. The scope of this overview is to present the European regulatory viewpoint on cell-based therapies for cardiac repair in the context of scientific observations.

Functional comparison of single- and double-stranded mdr1 antisense oligodeoxynucleotides in human ovarian cancer cell lines.

PURPOSE: P-glycoprotein mediated multidrug resistance presents a major obstacle in the successful therapeutic treatment of solid tumors such as ovarian cancer. Among the more promising techniques used to overcome multidrug resistance in ovarian cancer, is the transcriptional suppression of P-glycoprotein by antisense oligodeoxynucleotides (ODNs). To design more potent antisense ODNs, we explored the concept that double-stranded antisense ODNs may offer advantages in stability and potency over single-stranded in analogy to double-stranded siRNA. METHOD: Single-stranded phosphorothioate antisense ODNs against the human mdr1 gene were compared to the duplex of the active antisense and sense sequence of the same length. Concentration dependant effects on P-glycoprotein (Pgp) expression and functionality were quantitatively compared in the Pgp overexpressing ovarian cancer cell line A2780/Adr and its parental cell line A2780. Antisense ODNs were (111)Indium- and fluorescein isothiocyanate-conjugated for stability, cellular uptake and nuclear localization studies. Duplex formation significantly enhanced transcriptional inhibition of Pgp surface expression and functionality. Cellular uptake and distribution to the nucleus was improved when utilized as double-stranded DNA. CONCLUSION: Novel findings from this study suggest that double-stranded antisense ODNs more effectively inhibit target protein expression and consequently enhance chemoresponsiveness through improvements in cellular uptake and distribution to the nucleus.

In vitro and in vivo evaluation of WK-X-34, a novel inhibitor of P-glycoprotein and BCRP, using radio imaging techniques.

Overexpression of the multidrug resistance proteins P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) results in treatment failure of many malignancies including ovarian cancer. Dual inhibition of Pgp and BCRP may restore the sensitivity of resistant cells to anticancer drugs. We report the synthesis and characterization of a novel anthranilic-acid based Pgp and BCRP modulator, WK-X-34. In vitro inhibition of Pgp activity was evaluated using 99mTc-Sestamibi and daunorubicin accumulation in Pgp overexpressing human ovarian cancer cells (A2780/Adr) and its sensitive counterpart (A2780/wt). Interaction with BCRP was examined with a mitoxantrone-efflux assay in BCRP-overexpressing MCF7/mx cells, with flow cytometry. Interactions with the multidrug resistance associated proteins (MRP) were evaluated in transfected MRP1, MRP2 and MRP3 cell lines, using a 5-CFDA efflux assay. In vivo 99mTc-Sestamibi imaging of human ovarian cancer xenografts was used to evaluate the in vivo efficacy of WK-X-34 in mice. Daunorubicin accumulation in A2780/Adr cells was inhibited by WK-X-34 at nanomolar concentrations (IC50: 82.1 +/- 6 nM). WK-X-34 inhibited mitoxantrone accumulation in BCRP-overexpressing cells at micromolar concentrations (IC50 = 26.5 +/- 4.6 microM), whereas WK-X-34 did not significantly alter 5-CFDA accumulation in MRP transfected cells. In vivo, uptake of 99mTc-Sestamibi was significantly increased in A2780/Adr xenograft tumors, brain and intestine (AUCs(0-4h) 136%, 147% and 138%; p < 0.05) in mice dosed with WK-X-34 (20 mg/kg i.p.). WK-X-34 selectively modulates Pgp and BCRP in vitro and in vivo in multidrug resistant ovarian cancer cells, and thus may have potential utility in the treatment of multidrug resistant tumors.

Induction of ABCC3 (MRP3) by pregnane X receptor activators.

The pregnane X receptor (PXR) mediates the induction of various genes by xenobiotics, including several ATP-binding cassette transporters. PXR is also activated by bile acids likely to prevent their accumulation to toxic levels; however, the role of PXR in the regulation of MRP3, an important bile acid efflux transporter, has not been elucidated. The impact of PXR activators on the hepatic expression of MRP3 was examined in vivo and in vitro. The human hepatoma cell lines HuH7 and HepG2 were treated with PXR activators including clotrimazole, rifampicin, 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one (RU486), metyrapone, nifedipine, lithocholic acid, and 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). Levels of MRP3 mRNA, as determined by reverse transcription-polymerase chain reaction, were induced 1.6- to 8-fold in a dose-dependent manner (p < 0.05). Corresponding decreases in the multidrug resistance-associated protein-dependent cellular retention of 5-carboxyfluorescein were also seen in the treated HuH7 cells. In vivo studies demonstrated increased PXR mRNA and induction of MRP3 mRNA in the livers of wild-type mice treated with the PXR activator RU486. On the other hand, MRP3 induction was not seen in the RU486-treated PXR-null mice. These results suggest that PXR activation may play a role in the regulation of MRP3 expression.