How aligned are the perspectives of EU regulators and HTA bodies? A comparative analysis of regulatory-HTA parallel scientific advice.

BACKGROUND: In 2010, the European Medicines Agency (EMA) initiated a pilot project on parallel scientific advice with Health Technology Assessment bodies (HTABs) that allows manufacturers to receive simultaneous feedback from both the European Union (EU) regulators and HTABs on their development plans for medicines. AIMS: The present retrospective qualitative analysis aimed to explore how the parallel scientific advice system is working and levels of commonality between the EU regulators and HTABs, and among HTABs, when applicants obtain parallel scientific advice from both a regulatory and an HTA perspective. METHODS: We analysed the minutes of discussion meetings held at the EMA between 2010, when parallel advice was launched, and 1 May 2015, when the cutoff date for data extraction was set. The analysis was based on predefined criteria and conducted at two different levels of comparison: the answers of the HTABs vs. those of the regulators, and between the answers of the participating HTA agencies. RESULTS: The analysis was based on 31 procedures of parallel scientific advice. The level of full agreements was highest for questions on patient population (77%), while disagreements reached a peak for questions on the study comparator (30%). With regard to comparisons among HTABs, there was a high level of agreement for all domains. CONCLUSIONS: There is evident commonality, in terms of evidence requirements between the EU regulators and participating HTABs, as well as among HTABs, on most aspects of clinical development. Indeed, regardless of the question content, the analysis showed that a high level of overall agreement was reached through the process of parallel scientific advice.

A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development.

We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell-specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5(f/f) Ncr1-iCreTg animals. Stat5(f/f) Ncr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell-precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2-expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell-mediated tumor control against B16F10-melanoma cells. In contrast, T cell-mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function.

Leukemic challenge unmasks a requirement for PI3Kdelta in NK cell-mediated tumor surveillance.

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3Kdelta and therefore explored its impact on leukemia development. Using PI3Kdelta-deficient mice, we define a dual role of PI3Kdelta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3Kdelta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kdelta accelerated leukemia progression in vivo. However, the absence of PI3Kdelta also affected NK cell-mediated tumor surveillance. PI3Kdelta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kdelta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kdelta. Other tumor models confirmed that PI3Kdelta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kdelta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kdelta inhibitors as antileukemic agents in clinical trials.

Effects of duramycin on cardiac voltage-gated ion channels.

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 microM did not generate any effects on these currents. Concentrations >or=0.3 microM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (>or=3.3 microM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias.

Speeding the recovery from ultraslow inactivation of voltage-gated Na+ channels by metal ion binding to the selectivity filter: a foot-on-the-door?

Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K(+) inhibits C-type inactivation in Shaker K(+) channels by a "foot-in-the-door" mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I(US)), by ligand binding to the outer vestibule in voltage-gated Na(+) channels. Blocking the outer vestibule by a mutant mu-conotoxin GIIIA substantially accelerated recovery from I(US). A similar effect was observed if Cd(2+) was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 microM Cd(2+), the time constant of recovery from I(US) was decreased from 145.0 +/- 10.2 s to 32.5 +/- 3.3 s (P < 0.001). Recovery from I(US) was only accelerated if Cd(2+) was added to the bath solution during recovery (V = -120 mV) from I(US), but not when the channels were selectively exposed to Cd(2+) during the development of I(US) (-20 mV). These data could be explained by a kinetic model in which Cd(2+) binds with high affinity to a slow inactivated state (I(S)), which is transiently occupied during recovery from I(US). A total of 50 microM Cd(2+) produced an approximately 8 mV hyperpolarizing shift of the steady-state inactivation curve of I(S), supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I(US), suggesting that I(US) is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I(S)) occurs by a closure of the outer vestibule, whereas I(US) arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd(2+) to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I(US). Thus, Cd(2+) ions may act like a foot-on-the-door, kicking the I(S) gate to close.

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo.

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.

The catalytic PI3K isoforms p110gamma and p110delta contribute to B cell development and maintenance, transformation, and proliferation.

Class I PI3K-dependent signaling regulates cell proliferation, differentiation, and survival. Analysis of gene-deficient mice revealed specific roles for the hematopoietically expressed PI3K catalytic subunits, p110gamma and p110delta, in development and function of T and B lymphocytes. However, the functional redundancy between these two PI3K isoforms in the B cell lineage remains unclear. Here, we demonstrate that p110delta and p110gamma are expressed in B cells at early developmental stages. Normal B cell differentiation requires both isoforms, as p110gamma/p110delta double deficiency causes an increased percentage of CD43(hi)/B220(+)/CD19(-) cells as compared with single deficiency. Interestingly, initial transformation efficiency of B cell precursors was strongly reduced in double-deficient cells following transformation by p185 bcr-abl or v-abl oncogenes as compared with single-deficient cells. The requirement of p110gamma and p110delta in B cell development is underlined by reduced splenic B cell numbers of p110gamma/p110delta double-deficient mice and of lethally irradiated wild-type mice reconstituted with double-deficient BM. Moreover, the peripheral maintenance of p110gamma/p110delta double-deficient T and B cells was highly impaired following adoptive transfer of double-deficient splenocytes into wild-type mice. Functionally, LPS stimulation of splenocytes revealed proliferation defects resulting in decreased survival of p110gamma/p110delta double-deficient B cells, which correlated with impaired induction of D-type cyclins and Bcl-X(L). Surprisingly, this was not observed when purified B cells were analyzed, indicating a contribution of likely cell-extrinsic factor(s) to the impaired proliferation of double-deficient B cells. Thus, we provide novel evidence that p110gamma and p110delta have overlapping and cell-extrinsic roles in the development, peripheral maintenance, and function of B cells.

Identification of an indispensable role for tyrosine kinase 2 in CTL-mediated tumor surveillance.

We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.

Signal interception-based therapies--a double-edged sword in Bcr/abl-induced malignancies?

Imatinib was a major breakthrough in the treatment of Bcr/abl-positive leukemias. The effectiveness and value of this drug is limited by the emergence of resistance. Alternative drug targets may be identified by analyzing the downstream signaling network including the Jak/Stat-pathway, Ras-dependent signaling, PI3-kinases (PI3K), or the nuclear transcription factors onto which these pathways impinge. However, several factors limit the possible suitability of a drug target: (i) tissue-specific versus ubiquitous expression of the target; (ii) redundancy within the signaling network; and (iii) off-target effects on the immune system. Although the former two aspects are well appreciated as limiting factors, the latter has not been addressed so far. The advent of genetically engineered mice provides a sophisticated target validation in vivo as well as analysis of interactions between the immune system and tumor cells. Based on studies in such mouse models, we predict that many targeted compounds including PI3Kdelta-inhibitors, could act as double-edged swords because their beneficial action on tumor cells may be neutralized or even overwhelmed by their additional immunosuppressive effects.

C2C12 skeletal muscle cells adopt cardiac-like sodium current properties in a cardiac cell environment.

Intracardiac transplantation of undifferentiated skeletal muscle cells (myoblasts) has emerged as a promising therapy for myocardial infarct repair and is already undergoing clinical trials. The fact that cells originating from skeletal muscle have different electrophysiological properties than cardiomyocytes, however, may considerably limit the success of this therapy and, in addition, cause side effects. Indeed, a major problem observed after myoblast transplantation is the occurrence of ventricular arrhythmias. The most often transient nature of these arrhythmias may suggest that, once transplanted into cardiac tissue, skeletal muscle cells adopt more cardiac-like electrophysiological properties. To test whether a cardiac cell environment can indeed modify electrophysiological parameters of skeletal muscle cells, we treated mouse C(2)C(12) myocytes with medium preconditioned by primary cardiocytes and compared their functional sodium current properties with those of control cells. We found this treatment to significantly alter the activation and inactivation properties of sodium currents from "skeletal muscle" to more "cardiac"-like ones. Sodium currents of cardiac-conditioned cells showed a reduced sensitivity to block by tetrodotoxin. These findings and reverse transcription PCR experiments suggest that an upregulation of the expression of the cardiac sodium channel isoform Na(v)1.5 versus the skeletal muscle isoform Na(v)1.4 is responsible for the observed changes in sodium current function. We conclude that cardiomyocytes alter sodium channel isoform expression of skeletal muscle cells via a paracrine mechanism. Thereby, skeletal muscle cells with more cardiac-like sodium current properties are generated.

Selectivity filter residues contribute unequally to pore stabilization in voltage-gated sodium channels.

Mutations in the putative selectivity filter region of the voltage-gated Na+ channel, the so-called DEKA-motif, not only affect selectivity but also alter the channel's gating properties, suggesting functional coupling between permeation and gating. We have previously reported that charge-altering mutations at position 1237 in the P-loop of domain III (position K of the DEKA-motif in the adult rat skeletal muscle Na+ channel, rNa(v)1.4) dramatically enhanced entry to an inactivated state from which the channels recovered with a very slow time constant on the order of approximately 100 s (Todt, H., Dudley, S. C. J., Kyle, J. W., French, R. J., and Fozzard, H. A. (1999) Biophys. J. 76, 1335-1345). This state, termed "ultra-slow inactivation", may reflect a complex molecular rearrangement of the channel's pore region that involves both the extracellular and the cytoplasmic pore. Here, we tested whether charged DEKA-motif residues other than K1237 were also important determinants of a channel's gating properties. Therefore, we constructed the charge-neutralizing mutations D400A, E755A, and K1237A and studied the effects of these mutations on I(US). We found that, compared to wild-type rNa(v)1.4 channels, mutant D400A and K1237A but not E755A channels exhibited enhanced entry into ultra-slow inactivation. Selectivity for Na+ over K+, as judged from shifts in reversal potentials, was preserved in D400A, reduced in E755A, and completely lost in K1237A. These data suggest that an electrostatic interaction between the positively charged residue K1237 and the negatively charged residue D400 stabilizes the structure of the pore and thereby prevents I(US). Moreover, the interaction between K1237 and E755 appears to provide the basis for selective permeation of Na+ over K+.

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells.

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na+ channels in the C(2)C(12) murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca2+ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na+ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na+ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na+ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na+ channel isoform Na(v)1.5 compared with the skeletal muscle isoform Na(v)1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties.

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel.

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.

Comparison of transrectal sonography and double-contrast MR imaging when staging rectal cancer.

OBJECTIVE: The aim of this study was the prospective comparison of the diagnostic yield of transrectal sonography and double-contrast MR imaging for preoperative staging of rectal cancer. SUBJECTS AND METHODS. Thirty-nine rectal cancer patients (20 men, 19 women) underwent transrectal sonography performed with a 10-MHz endoanal probe and MR imaging (1.0 T or 1.5 T) using a whole-body coil. After rectal application of a superparamagnetic iron oxide MR contrast agent, T1- and T2-weighted images and gadolinium-enhanced double-contrast images were obtained. The results of examinations were compared with the histology of resected specimens. RESULTS: Histopathology showed four stage T1, 11 stage T2, 18 stage T3, and six stage T4 tumors using the TNM staging system. Nodal metastases were seen in 16 patients. Transrectal sonography could not be performed in 11 patients because of the high location of the tumor. In the remaining 28 patients, the accuracy of transrectal sonography for T stage was 64% overall (patients not receiving radiation, 69%; patients receiving radiation, 60%) and 70% for N stage. In 39 patients, double-contrast MR imaging correctly identified the T stage with an accuracy of 64% overall (patients not receiving radiation, 75%; patients receiving radiation, 53%) and the N stage with an accuracy of 62%. The assessment of rectal wall penetration (Dukes' classification A versus B) revealed a sensitivity, specificity, and accuracy of 93%, 71%, and 82%, respectively, for transrectal sonography and 100%, 60%, and 85% for MR imaging. CONCLUSION: If it is technically feasible, transrectal sonography is an accurate method for staging rectal cancer. In proximal or stenotic tumors, double-contrast MR imaging is the method of choice. Diagnostic accuracy of transrectal sonography and MR imaging is high for predicting bowel wall penetration.