Molecular Cloning and Characterization of a Human Mitochondrial Ceramidase

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism

Modeling of radiation therapy and radiosensitizing agents in tumor xenografts

III-36\ua0Tim\ua0Cardilin\ua0Modeling of radiation therapy and radiosensitizing agents in tumor xenografts\ua0Tim Cardilin (1,2), Joachim Almquist (1), Mats Jirstrand (1), Astrid Zimmermann (3), Floriane Lignet (4), Samer El Bawab (4), and Johan Gabrielsson (5)(1) Fraunhofer-Chalmers Centre, Gothenburg, Sweden, (2) Department of Mathematical Sciences, Chalmers University of Technology and Gothenburg University, Gothenburg, Sweden, (3) Merck, Translational Innovation Platform Oncology, Darmstadt, Germany, (4) Merck, Global Early Development - Quantitative Pharmacology, Darmstadt, Germany, (5) Division of Pharmacology and Toxicology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, SwedenObjectives:\ua0To conceptually and mathematically describe the treatment effects of radiation and radiosensitizing agents on tumor volume in xenografts with respect to short- and long-term effects.Methods:\ua0Data were generated in FaDu xenograft mouse models, where animals were treated with radiation given either as monotherapy (2 Gy per dose) or together with an early-discovery radiosensitizing agent (25 or 100 mg/kg per dose) that interferes with the repair of the DNA damage induced by irradiation. Animals received treatment following a clinically-relevant administration schedule with doses five days a week for six weeks. Tumor diameters were measured by caliper twice a week for up to 140 days. A pharmacodynamic tumor model was adapted from a previously-published model [1,2]. The improved model captures both short- and long-term treatment effects including tumor eradication and tumor regrowth. Short-term radiation effects are described by allowing lethally irradiated cells up to one more cell division before apoptosis. Long-term radiation effects are described by an irreversible decrease in tumor growth rate. The radiosensitizing agent was assumed to stimulate both processes. The model also includes a natural death rate of cancer cells. The model was calibrated to the xenograft data using a mixed-effects approach based on the FOCE method that was implemented in Mathematica [3]. Between-subject variability was accounted for in initial tumor volume, as well as in the short- and long-term radiation effects.Results:\ua0Data across all treatment groups were well-described by the model. All model parameters were estimated with acceptable precision and biologically reasonable values. Vehicle growth was approximately exponential during the observed time period with an estimated tumor doubling time of approximately 5 days. Tumor growth following radiation therapy resulted in significant tumor regression followed by either tumor eradication (2 animals) or slow regrowth (7 animals). The short- and long-term effects incorporated into the tumor model were able to account for both of these scenarios. A simple analysis shows that if the tumor growth rate is decreased below the natural death rate, the tumor will be eradicated. Otherwise, the tumor will regrow but at a slower rate compared to pre-treatment. The model predicts that each fraction of radiation (2 Gy) results in lethal damage in 15 % of viable cells, and that a total dose above 120 Gy will eradicate the tumor. Tumor growth following combination therapy with a lower dose (25 mg/kg) resulted in more cases of tumor eradication (6 animals) and fewer cases of regrowth (3 animals), whereas combination therapy with the higher dose (100 mg/kg) resulted in tumor eradication in all 9 animals. When radiation therapy was complemented by radiosensitizing treatment (100 mg/kg per dose), each fraction of 2 Gy was estimated to kill 25 % of viable cells, and the total radiation dose required for tumor eradication was decreased by a factor four to 30 Gy.Conclusions:\ua0A tumor model has been developed to describe the treatment effects of radiation therapy, as well as combination therapies involving radiation, in tumor xenografts. The model distinguishes between short- and long-term effects of radiation treatment and can describe different tumor dynamics, including tumor eradication and tumor regrowth at different rates. The novel tumor model can be used to predict treatment outcomes for a broad range of treatments including radiation therapy and combination therapies with different radiosensitizing agents.References:\ua0[1] Cardilin T, Almquist J, Jirstrand M, Zimmermann A, El Bawab S, Gabrielsson J. Model-based evaluation of radiation and radiosensitizing agents in oncology. CPT: Pharmacometrics & Syst. Pharmacol.\ua0(2017).[2] Cardilin T, Zimmermann A, Jirstrand M, Almquist J, El Bawab S, Gabrielsson J. Extending the Tumor Static Concentration Curve to average doses – a combination therapy example using radiation therapy. PAGE 25 (2016) Abstr 5975 [www.page-meeting.org/?abstract=5975].[3] Almquist J, Leander J, Jirstrand M. Using sensitivity equations for computing gradients of the FOCE and FOCEI approximations to the population likelihood. J Pharmacokinet Pharmacodyn (2015) 42: 191-209

Modeling long-term tumor growth and kill after combinations of radiation and radiosensitizing agents

Purpose: Radiation therapy, whether given alone or in combination with chemical agents, is one of the cornerstones of oncology. We develop a quantitative model that describes tumor growth during and after treatment with radiation and radiosensitizing agents. The model also describes long-term treatment effects including tumor regrowth and eradication. Methods: We challenge the model with data from a xenograft study using a clinically relevant administration schedule and use a mixed-effects approach for model-fitting. We use the calibrated model to predict exposure combinations that result in tumor eradication using Tumor Static Exposure (TSE). Results: The model is able to adequately describe data from all treatment groups, with the parameter estimates taking biologically reasonable values. Using TSE, we predict the total radiation dose necessary for tumor eradication to be 110\ua0Gy, which is reduced to 80 or 30\ua0Gy with co-administration of 25 or 100\ua0mg\ua0kg\ua0−1\ua0of a radiosensitizer. TSE is also explored via a heat map of different growth and shrinkage rates. Finally, we discuss the translational potential of the model and TSE concept to humans. Conclusions: The new model is capable of describing different tumor dynamics including tumor eradication and tumor regrowth with different rates, and can be calibrated using data from standard xenograft experiments. TSE and related concepts can be used to predict tumor shrinkage and eradication, and have the potential to guide new experiments and support translations from animals to humans

selection of the recommended phase 2 dose for bintrafusp alfa a bifunctional fusion protein targeting tgf β and pd l1

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First-in-Man Phase I Trial of the Selective MET Inhibitor Tepotinib in Patients with Advanced Solid Tumors.

PurposeTepotinib is an oral, potent, highly selective MET inhibitor. This first-in-man phase I trial investigated the MTD of tepotinib to determine the recommended phase II dose (RP2D).Patients and methodsPatients received tepotinib orally according to one of three dose escalation regimens (R) on a 21-day cycle: R1, 30-400 mg once daily for 14 days; R2, 30-315 mg once daily 3 times/week; or R3, 300-1,400 mg once daily. After two cycles, treatment could continue in patients with stable disease until disease progression or unacceptable toxicity. The primary endpoint was incidence of dose-limiting toxicity (DLT) and treatment-emergent adverse events (TEAE). Secondary endpoints included safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor effects.ResultsOne hundred and forty-nine patients received tepotinib (R1: n = 42; R2: n = 45; R3: n = 62). Although six patients reported DLTs [one patient in R1 (115 mg), three patients in R2 (60, 100, 130 mg), two patients in R3 (1,000, 1,400 mg)], the MTD was not reached at the highest tested dose of 1,400 mg daily. The RP2D of tepotinib was established as 500 mg once daily, supported by translational modeling data as sufficient to achieve ≥95% MET inhibition in ≥90% of patients. Treatment-related TEAEs were mostly grade 1 or 2 fatigue, peripheral edema, decreased appetite, nausea, vomiting, and lipase increase. The best overall response in R3 was partial response in two patients, both with MET overexpression.ConclusionsTepotinib was well tolerated with clinical activity in MET-dysregulated tumors. The RP2D of tepotinib was established as 500 mg once daily. MET abnormalities can drive tumorigenesis. This first-in-man trial demonstrated that the potent, highly selective MET inhibitor tepotinib can reduce or stabilize tumor burden and is well tolerated at doses up to 1,400 mg once daily. An RP2D of 500 mg once daily, as determined from translational modeling and simulation integrating human population pharmacokinetic and pharmacodynamic data in tumor biopsies, is being used in ongoing clinical trials

Rôle des gangliosides dans les perturbations de la prolifération des péricytes rétiniens et des cellules mésangiales rénales (implication dans le développement de la rétinopathie et de la néphropathie diabétiques)

La perte des péricytes rétiniens est une des premières altérations de la rétinopathie diabétique. L'arrêt du cycle cellulaire, l'hypertrophie puis la disparition des cellules mésangiales rénales sont des caractéristiques typiques de la néphropathie diabétique. La voie de la glycation (formation des produits avancés de glycation ou AGE) et la voie de l'hexosamine sont deux hypothèses biochimiques proposées pour expliquer les altérations cellulaires des microcomplications du diabète. D'autre part, bien que les gangliosides aient souvent été décrits pour moduler la prolifération cellulaire, leur rôle potentiel dans les perturbations de prolifération caractéristiques de la rétinopathie et de la néphropathie diabétiques a été très peu étudié. L'objectif de ce travail de thèse a été de déterminer les effets des AGE et d'une activation de la voie de l'hexosamine, mimée par la glucosamine, sur la prolifération et le métabolisme des gangliosides des péricytes et de cellules mésangiales puis d'établir l'implication des gangliosides dans ces processus de régulation de la prolifération. Nos résultats ont montré que les AGE et la glucosamine inhibent la prolifération des deux types cellulaires étudiés. De plus, ils ont révélé que la glucosamine bloque le cycle cellulaire et induit l'hypertrophie des cellules mésangiales. D'autre part, les AGE comme la glucosamine sont capables de modifier le profil en gangliosides des cellules, en modulant les activités de leurs enzymes de biosynthèse. Enfin, nos observations suggèrent que les gangliosides sont impliqués dans l'inhibition de la prolifération et l'hypertrophie causée par les AGE et la glucosamine dans les péricytes et les cellules mésangiales. Elles présentent ainsi les gangliosides comme un nouveau mécanisme d'action des AGE et de la voie de l'hexosamine et permettent de proposer les gangliosides, en particulier ceux de la série-a, et la GM3 synthase, comme un mécanisme potentiel commun au développement de la rétinopathie et de la néphropathie diabétiques. Ce travail est un des premiers à suggérer l'implication des gangliosides dans les complications microvasculaires du diabète que sont la rétinopathie et la néphropathie diabétiques. Par l'intermédiaire des gangliosides, il ouvre des perspectives thérapeutiques nouvelles et communes pour le traitement des deux microcomplications.Loss of retinal pericyte is one of the earliest alteration of diabetic retinopathy. Cell cycle arrest, hypertrophy then death of renal mesangial cells are typical hallmarks of diabetic nephropathy. Glycation (advanced glycation end-products, AGE, formation) and the hexosamine pathway are two biochemical hypotheses proposed to explain cellular alteration occurring during diabetic vascular complication. On the other hand, although gangliosides have often been described as modulators of cellular proliferation, very few studies have explored their potential role in cell proliferation alteration of diabetic retinopathy and nephropathy. The aim of the present study was to determine the effects of AGE and hexosamine pathway activation, mimicked by glucosamine, on pericyte and mesangial cell proliferation and ganglioside metabolism and then to establish the implication of gangliosides in these regulation of proliferation process. Our result showed that AGE and glucosamine inhibit the proliferation of both cell types studied. Moreover, they revealed that glucosamine blocks the cell cycle and induces hypertrophy of mesangial cells. On the other hand, both AGE and glucosamine are able to affect cellular ganglioside profile by modulating their biosynthetic enzyme activities. Finally, our observations suggested that gangliosides are implicated in the inhibition of cell proliferation and hypertrophy caused by AGE and glucosamine in pericyte and mesangial cells. Thus, they present gangliosides as a novel mechanism of action of AGE and the hexosamine pathway and lead to propose gangliosides, especially a-series gangliosides and GM3 synthase, as a potential common mechanism of diabetic retinopathy and nephropathy development. This work is one of the first suggesting the implication of gangliosides in diabetic retinopathy and nephropathy. Through gangliosides, it offers new therapeutic prospects common to both microvascular complications.VILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

Rôle des sphingolipides endogènes dans les modifications de la prolifération des cellules mésangiales rénales en réponse aux produits avancés de glycation (AGE) (implication dans le développement de la néphropathie diabétique)

Les modifications de prolifération (hyperplasie, arrêt du cycle cellulaire, et hypertrophie), sont associées à de nombreuses pathologies touchant le glomérule rénal, et sont des caractéristiques typiques de la néphropathie diabétique. La voie de la glycation (formation des produits avancés de glycation ou AGE) constitue une hypothèse biochimique majeure évoquée dans ce type d'altérations cellulaires liées aux complications microvasculaires du diabète. D'autre part, bien que les sphingolipides, notamment les céramides, sphingosine, et sphingosine-1-phosphate aient souvent été décrits pour intervenir dans la régulation de la croissance cellulaire, leur rôle potentiel dans les perturbations de prolifération caractéristiques de la néphropathie diabétique n'a quasiment pas été étudié. L'objectif de ce travail de thèse a été de déterminer les effets des AGE sur la prolifération et le métabolisme des sphingolipdies des cellules mésangiales de rat en culture (RMC), puis de définir et préciser l'implication des sphingolipides dans ces processus de régulation de la prolifération. Une approche ex vivo dans des modèles d'animaux diabétiques a par ailleurs été développée afin d'appuyer ces hypothèses dans un contexte de néphropathie diabétique. Nos résultats ont montré que les AGE, par une interaction spécifique avec le récepteur RAGE exprimé par les RMC, modulent de façon bimodale la prolifération de ces cellules. Ainsi, les AGE induisent une activation de prolifération à faibles concentrations (<1 M), et inhibent au contraire la croissance des RMC à plus fortes concentrations (3-10 M). D'autre part, les AGE se sont révélés capables de moduler, de façon bimodale là encore, le métabolisme de conversion des céramides, en augmentant -à faibles concentrations- ou inhibant -à fortes concentrations- notamment l'activité et ou/l'expression de deux enzymes, la céramidase et la sphingosine-kinase. Du jeu de ces modulations enzymatiques résulte l'accumulation de dérivés sphingolipidiques du céramide. Les faibles concentrations d'AGE induisent la production de sphingosine-1-phosphate aux effets pro-mitogéniques, les fortes concentrations, au contraire, l'accumulation de glycosphingolipides et de sphingosine, capables d'inhiber la prolifération des RMC.Enfin, nos expériences dans des modèles animaux (rat STZ) suggèrent qu'une variation du métabolisme des sphingolipides participant à l'accumulation locale de sphingosine-1-phosphate est impliquée dans l'augmentation de la prolifération des cellules mésangiales glomérulaires apparaissant transitoirement in vivo dans les premiers stades de la néphropathie diabétique.Ce travail est un des premiers à suggérer l'implication des sphingolipides comme un nouveau médiateur de l'action des AGE dans les cellules mesangiales, et permet de proposer la production des différents sphingolipides résultant notamment de la modulation des céramidases et sphingosine kinase, comme un mécanisme potentiel participant au développement de la néphropathie diabétique.Advanced glycation end products (AGE) are generated by chronic hyperglycaemia and may cause cellular alteration leading to microvascular complications such as diabetic nephropathy (DN). Disregulation of mesangial cell proliferation is known to contribute to the development of DN. In this study, we investigated the effects of AGE on rat mesangial cells (RMC) proliferation. In addition, because sphingolipids (SPL), and in particular ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play important roles in the regulation of cell proliferation and cell death, we evaluated the involvement of SPL metabolism in the AGE response. On the other hand, ex vivo experiments using the streptozotocin (STZ)-diabetic rat model were undertaken in order to investigate the pathophysiological relevance of the cell studies results in the context of diabetic nephropathy. Our results showed that AGE induce bimodal effects on mesangial cells proliferation through a specific interaction with their receptor RAGE. Thus, after 72 h of treatment, low AGE concentrations (<1 M) induced a significant increase of RMC proliferation, whereas higher AGE concentrations (3-10 M) markedly reduced it. In parallel, AGE exerted biphasic effects on biosynthetic enzymes activities and/or expression, namely neutral ceramidase and sphingosine-kinase. Low AGE concentrations induced neutral ceramidase and sphingosine-kinase activation, whereas high AGE concentrations inhibited both activities. Surprisingly, neutral ceramidase activity inhibition by AGE did not result in changes of Cer levels. However, the AGE (10 M)-inhibitory effect on RMC proliferation was accompained by increased sphingosine levels and was specifically prevented by blocking glucosylceramide synthesis, suggesting that Sph and/or glycolipids are involved in mediating the effects of AGE at high concentrations. On the other hand, treatment of cells with low AGE concentrations led to an increase of S1P production, presumably associated to the observed activation of neutral ceramidase and sphingosine kinase. Taken together, these results show that AGE regulate mesangial cell growth by modulating Cer conversion into other bioactive SPL, namely, S1P, Sph, and glycolipids. On the other hand, our ex vivo observations on STZ-diabetic rats supported our cell studies results and suggested that glomerular S1P accumulation is likely implicated in the early and transient promotion of mesangial cells proliferation during the very early stages of diabetic nephropathy. In conclusion, our results present SPL metabolism as a novel mechanism of action of AGE. Further, this work is one of the first to propose SPL metabolism, especially regulation of S1P and Sph/glycosphingolipides levels as a potential mechanism involved in the development of diabetic nephropathy.VILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

Extending the Tumor Static Concentration curve to average doses - a combination therapy example using radiation therapy

Objectives: The recently developed concept of Tumor Static Concentration (TSC) is a valuable modeling tool for the quantitative analysis of combination therapies [2]. Here, we set out to extend TSC to situations where (average) doses are known but drug exposure data is not available.Methods: Data consisted of Patient-Derived xenografts from combination therapy studies using ionizing radiation and a probe compound. Modelling was based on a Tumor Growth Inhibition (TGI) model [3] modified for radiation treatment. Model parameters were estimated using a mixed-effects approach implemented in Mathematica 10 [1]. A TSC-like curve was derived from tumor stasis assumptions where one of the plasma concentrations was replaced with average radiation dose over time.Results: Drug exposure of the probe compound was successfully modeled using a one compartment exposure model. Initial attempts to model the combination efficacy data were not able to explain the effect from the combination arm. The TGI model was subsequently modified to account for potential interaction effects between the probe compound and radiation treatments. The radiation treatment-modified TGI model was then used to derive a TSC-like curve that determines all pairs of radiation doses and drug concentrations for which the tumor is kept in stasis. This curve exhibits significant curvature, reflecting the synergistic effects of administering the radiation therapy and drug together. The TSC-like curve can be used to improve the administration schedule of the treatment.Conclusions: A model-based method for evaluation of anticancer combination therapy was extended from the use of tumor static plasma concentrations to also include average drug doses. Although used for radiation therapy in this example, the method can also be applied for regular compounds when drug exposure data is not available