ABSTRACT
INTRODUCTION: M8891 is a selective and reversible inhibitor of methionine aminopeptidase 2 (MetAP2). We describe translational research to define the target pharmacokinetics (PK) of M8891 and associated pharmacodynamic (PD) levels, which were used to support efficacious dose selection in humans. METHODS: In vitro and in vivo PK characteristics were investigated in animal species, and data integrated using in vitro-in vivo correlation and allometric methods to predict the clearance, volume of distribution, and absorption parameters of M8891 in humans. In parallel, inhibition of MetAP2 activity by M8891 was studied in renal cancer xenografts in mice by measuring accumulation of Met-EF1α, a substrate of MetAP2. The corresponding PD effect was described by a turnover and effect compartment model. This model was used to simulate PD at the M8891 dose showing in vivo efficacy, i.e. significant tumor growth inhibition. Simulations of M8891 PK and associated PD in humans were conducted by integrating predicted human PK parameters into the preclinical PK/PD model. RESULTS: The target minimum PD level associated with efficacy was determined to be 125 µg Met-EF1α per mg protein. Integrating predicted human PK parameters into the preclinical PK/PD model defined a minimal M8891 concentration at steady-state (Ctrough) of 1500 ng/mL (3.9 µM) in humans as being required to produce the corresponding minimum target Met-EF1a level (125 µg per mg protein). CONCLUSION: The defined target PK and PD levels supported the design of the clinical Phase Ia dose escalation study of M8891 (NCT03138538) and selection of the recommended Phase II dose.
Subject(s)
Metalloendopeptidases , Models, Biological , Humans , Mice , Animals , Angiogenesis Inhibitors , Enzyme InhibitorsABSTRACT
BACKGROUND: To increase the chances of finding efficacious anticancer drugs, improve development times and reduce costs, it is of interest to rank test compounds based on their potential for human use as early as possible in the drug development process. In this paper, we present a method for ranking radiosensitizers using preclinical data. METHODS: We used data from three xenograft mice studies to calibrate a model that accounts for radiation treatment combined with radiosensitizers. A nonlinear mixed effects approach was utilized where between-subject variability and inter-study variability were considered. Using the calibrated model, we ranked three different Ataxia telangiectasia-mutated inhibitors in terms of anticancer activity. The ranking was based on the Tumor Static Exposure (TSE) concept and primarily illustrated through TSE-curves. RESULTS: The model described data well and the predicted number of eradicated tumors was in good agreement with experimental data. The efficacy of the radiosensitizers was evaluated for the median individual and the 95% population percentile. Simulations predicted that a total dose of 220 Gy (5 radiation sessions a week for 6 weeks) was required for 95% of tumors to be eradicated when radiation was given alone. When radiation was combined with doses that achieved at least 8 [Formula: see text] of each radiosensitizer in mouse blood, it was predicted that the radiation dose could be decreased to 50, 65, and 100 Gy, respectively, while maintaining 95% eradication. CONCLUSIONS: A simulation-based method for calculating TSE-curves was developed, which provides more accurate predictions of tumor eradication than earlier, analytically derived, TSE-curves. The tool we present can potentially be used for radiosensitizer selection before proceeding to subsequent phases of the drug discovery and development process.
Subject(s)
Antineoplastic Agents , Neoplasms , Radiation-Sensitizing Agents , Humans , Animals , Mice , Radiation-Sensitizing Agents/pharmacology , Radiation-Sensitizing Agents/therapeutic use , Neoplasms/drug therapy , Neoplasms/radiotherapy , Antineoplastic Agents/therapeutic use , Combined Modality TherapyABSTRACT
M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, ß5i, PSMB8) proteolytic subunit of the immunoproteasome, a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy modeling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). As M3258 only weakly inhibits LMP7 in mouse cells, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modeling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement with in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. SIGNIFICANCE STATEMENT: M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro-in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, while the extent and duration of PD response were more pronounced than estimated.
Subject(s)
Multiple Myeloma , Humans , Rats , Mice , Animals , Dogs , Multiple Myeloma/drug therapy , Models, BiologicalABSTRACT
Pan-proteasome inhibitors (pPIs) significantly improve outcomes in patients with multiple myeloma; however, their indiscriminate inhibition of multiple proteasome and immunoproteasome subunits causes diverse toxicities, including thrombocytopenia. We investigated the mechanisms underlying the platelet depletion induced by the pPIs bortezomib, carfilzomib, and ixazomib. An established thrombocytopenia model was adapted for each compound (bortezomib, ixazomib, and carfilzomib) to compare the following two pharmacodynamic mechanisms: a reversible inhibition of new progenitor cell formation (the myelosuppression model) and a reversible effect on the function of megakaryocytes to bud new platelets (platelet formation model). Bortezomib, ixazomib, and carfilzomib plasma concentration profiles and platelet counts were extracted from the literature. Pharmacokinetic (PK) and thrombocytopenia models were developed to predict the PK of these drugs and to describe their effects on proliferating cells and platelet budding. The PK models reproduced the exposure of the three compounds at steady state well compared with those reported in the literature. Both the platelet formation and myelosuppression models seemed able to describe the platelet depletion caused by bortezomib, ixazomib, and carfilzomib. Estimated structural parameters in the myelosuppression model were in the range of the values reported in the literature, whereas the mean transit time estimated with the platelet formation model was 3-fold to 10-fold higher than the highest reported value. The model of drug-induced myelosuppression yielded estimates of structural parameters in the range of those previously reported. The platelet formation model captured the temporal variation reported in clinical studies.
Subject(s)
Antineoplastic Agents , Thrombocytopenia , Antineoplastic Agents/pharmacology , Blood Platelets , Bortezomib , Humans , Proteasome Inhibitors/adverse effects , Proton Pump Inhibitors , Thrombocytopenia/chemically inducedABSTRACT
A central question in drug discovery is how to select drug candidates from a large number of available compounds. This analysis presents a model-based approach for comparing and ranking combinations of radiation and radiosensitizers. The approach is quantitative and based on the previously-derived Tumor Static Exposure (TSE) concept. Combinations of radiation and radiosensitizers are evaluated based on their ability to induce tumor regression relative to toxicity and other potential costs. The approach is presented in the form of a case study where the objective is to find the most promising candidate out of three radiosensitizing agents. Data from a xenograft study is described using a nonlinear mixed-effects modeling approach and a previously-published tumor model for radiation and radiosensitizing agents. First, the most promising candidate is chosen under the assumption that all compounds are equally toxic. The impact of toxicity in compound selection is then illustrated by assuming that one compound is more toxic than the others, leading to a different choice of candidate.
Subject(s)
Neoplasms , Radiation-Sensitizing Agents , Humans , Neoplasms/drug therapy , Neoplasms/radiotherapy , Radiation-Sensitizing Agents/pharmacology , Radiation-Sensitizing Agents/therapeutic useABSTRACT
Tepotinib is a highly selective and potent MET inhibitor in development for the treatment of patients with solid tumors. Given the favorable tolerability and safety profiles up to the maximum tested dose in the first-in-human (FIH) trial, an efficacy-driven translational modeling approach was proposed to establish the recommended phase II dose (RP2D). To study the in vivo pharmacokinetics (PKs)/target inhibition/tumor growth inhibition relationship, a subcutaneous KP-4 pancreatic cell-line xenograft model in mice with sensitivity to MET pathway inhibition was selected as a surrogate tumor model. Further clinical PK and target inhibition data (derived from predose and postdose paired tumor biopsies) from a FIH study were integrated with the longitudinal PKs and target inhibition profiles from the mouse xenograft study to establish a translational PK/pharmacodynamic (PD) model. Preclinical data showed that tumor regression with tepotinib treatment in KP-4 xenograft tumors corresponded to 95% target inhibition. We therefore concluded that a PD criterion of sustained, near-to-complete (>95%) phospho-MET inhibition in tumors should be targeted for tepotinib to be effective. Simulations of dose-dependent target inhibition profiles in human tumors that exceeded the PD threshold in more than 90% of patients established an RP2D of tepotinib 500 mg once daily. This translational mathematical modeling approach supports an efficacy-driven rationale for tepotinib phase II dose selection of 500 mg once daily. Tepotinib at this dose has obtained regulatory approval for the treatment of patients with non-small cell lung cancer harboring MET exon 14 skipping.
Subject(s)
Drug Evaluation, Preclinical , Models, Theoretical , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridazines/pharmacology , Pyrimidines/pharmacology , Administration, Oral , Animals , Clinical Trials, Phase II as Topic , Dose-Response Relationship, Drug , Humans , Mice , Piperidines/administration & dosage , Piperidines/pharmacokinetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Pyridazines/administration & dosage , Pyridazines/pharmacokinetics , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: This open-label, phase 1 trial (NCT02316197) aimed to determine the maximum-tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of peposertib (formerly M3814), a DNA-dependent protein kinase (DNA-PK) inhibitor in patients with advanced solid tumours. Secondary/exploratory objectives included safety/tolerability, pharmacokinetic/pharmacodynamic profiles and clinical activity. METHODS: Adult patients with advanced solid tumours received peposertib 100-200 mg once daily or 150-400 mg twice daily (BID) in 21-day cycles. RESULTS: Thirty-one patients were included (median age 66 years, 61% male). One dose-limiting toxicity, consisting of mainly gastrointestinal, non-serious adverse events (AEs) and long recovery duration, was reported at 300 mg BID. The most common peposertib-related AEs were nausea, vomiting, fatigue and pyrexia. The most common peposertib-related Grade 3 AEs were maculopapular rash and nausea. Peposertib was quickly absorbed systemically (median Tmax 1.1-2.5 h). The p-DNA-PK/t-DNA-PK ratio decreased consistently in peripheral blood mononuclear cells 3-6 h after doses ≥100 mg. The best overall response was stable disease (12 patients), lasting for ≥12 weeks in seven patients. CONCLUSIONS: Peposertib was well-tolerated and demonstrated modest efficacy in unselected tumours. The MTD was not reached; the RP2D was declared as 400 mg BID. Further studies, mainly with peposertib/chemo-radiation, are ongoing. CLINICAL TRIAL REGISTRATION: NCT02316197.
Subject(s)
DNA-Activated Protein Kinase/antagonists & inhibitors , Neoplasms/drug therapy , Pyridazines/administration & dosage , Pyridazines/adverse effects , Quinazolines/administration & dosage , Quinazolines/adverse effects , Adult , Aged , DNA-Activated Protein Kinase/metabolism , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Neoplasms/metabolism , Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Pyridazines/pharmacokinetics , Quinazolines/pharmacokineticsABSTRACT
Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-ßRII receptor (TGF-ß "trap") fused to a human IgG1-blocking PD-L1, showed a manageable safety profile and clinical activity in phase I studies in patients with heavily pretreated advanced solid tumors. The recommended phase 2 dose (RP2D) was selected based on integration of modeling, simulations, and all available data. A 1,200-mg every 2 weeks (q2w) dose was predicted to maintain serum trough concentration (Ctrough ) that inhibits all targets of bintrafusp alfa in circulation in > 95% of patients, and a 2,400-mg every 3 weeks (q3w) dose was predicted to have similar Ctrough . A trend toward an association between exposure and efficacy variables and a relatively stronger inverse association between clearance and efficacy variables were observed. Exposure was either weakly or not correlated with probability of adverse events. The selected intravenous RP2D of bintrafusp alfa is 1,200 mg q2w or 2,400 mg q3w.
Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , B7-H1 Antigen/antagonists & inhibitors , Drug Dosage Calculations , Immune Checkpoint Inhibitors/administration & dosage , Neoplasms/drug therapy , Transforming Growth Factor beta/antagonists & inhibitors , Administration, Intravenous , Animals , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Agents, Immunological/pharmacokinetics , B7-H1 Antigen/metabolism , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Computer Simulation , Disease Models, Animal , Drug Administration Schedule , Humans , Immune Checkpoint Inhibitors/adverse effects , Immune Checkpoint Inhibitors/pharmacokinetics , Mice , Models, Theoretical , Molecular Targeted Therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Research Design , Transforming Growth Factor beta/metabolismABSTRACT
PURPOSE: Tepotinib 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 METHODS: Patients 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. RESULTS: One 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. CONCLUSIONS: Tepotinib 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.
Subject(s)
Neoplasms/drug therapy , Piperidines/pharmacokinetics , Piperidines/therapeutic use , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridazines/pharmacokinetics , Pyridazines/therapeutic use , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Adult , Aged , Aged, 80 and over , Dose-Response Relationship, Drug , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Nausea/chemically induced , Neoplasms/pathology , Patient Safety , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Tissue Distribution , Treatment Outcome , Vomiting/chemically induced , Young AdultABSTRACT
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 Gy, which is reduced to 80 or 30 Gy with co-administration of 25 or 100 mg kg-1 of 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.
Subject(s)
Models, Biological , Neoplasms/radiotherapy , Radiation-Sensitizing Agents/administration & dosage , Animals , Dose-Response Relationship, Drug , Female , Humans , Mice , Mice, Nude , Radiotherapy Dosage , Species Specificity , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Evofosfamide is a cytotoxic small-molecule prodrug preferentially activated under hypoxic conditions. The cytotoxicity of evofosfamide impacted the generation of in vitro drug-drug interaction (DDI) data, especially in vitro induction results. Therefore, a novel physiologically based pharmacokinetic (PBPK) approach was used, which involved available in vitro and clinical data of evofosfamide and combined it with induction data from the prototypical cytochrome P450 (CYP)3A inducer rifampicin. The area under the concentration-time curve (AUC) ratios of midazolam were above 0.80, indicating that induction of CYP3A by evofosfamide administered weekly is unlikely to occur in humans. Moreover, static and PBPK modeling showed no clinically relevant inhibition via CYP2B6, CYP2D6, and CYP3A4. In conclusion, PBPK models were used to supplement in vitro information of a cytotoxic compound. This approach may set a precedent for future studies of cytotoxic drugs, potentially reducing the need for clinical DDI studies and providing more confidence in the clinical use of approved cytotoxic compounds for which DDI information is sparse.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Drug Interactions , Nitroimidazoles/metabolism , Phosphoramide Mustards/metabolism , Prodrugs/metabolism , HumansABSTRACT
Radiotherapy is one of the major therapy forms in oncology, and combination therapies involving radiation and chemical compounds can yield highly effective tumor eradication. In this paper, we develop a tumor growth inhibition model for combination therapy with radiation and radiosensitizing agents. Moreover, we extend previous analyses of drug combinations by introducing the tumor static exposure (TSE) curve. The TSE curve for radiation and radiosensitizer visualizes exposure combinations sufficient for tumor regression. The model and TSE analysis are then tested on xenograft data. The calibrated model indicates that the highest dose of combination therapy increases the time until tumor regrowth 10-fold. The TSE curve shows that with an average radiosensitizer concentration of 1.0 µg/mL the radiation dose can be decreased from 2.2 Gy to 0.7 Gy. Finally, we successfully predict the effect of a clinically relevant treatment schedule, which contributes to validating both the model and the TSE concept.
Subject(s)
Models, Biological , Neoplasms/radiotherapy , Radiation-Sensitizing Agents/therapeutic use , Animals , Combined Modality Therapy , Humans , Neoplasms/drug therapy , Predictive Value of Tests , Radiation-Sensitizing Agents/administration & dosage , Radiotherapy/methods , Xenograft Model Antitumor AssaysABSTRACT
Combination therapies are widely accepted as a cornerstone for treatment of different cancer types. A tumor growth inhibition (TGI) model is developed for combinations of cetuximab and cisplatin obtained from xenograft mice. Unlike traditional TGI models, both natural cell growth and cell death are considered explicitly. The growth rate was estimated to 0.006 h-1 and the natural cell death to 0.0039 h-1 resulting in a tumor doubling time of 14 days. The tumor static concentrations (TSC) are predicted for each individual compound. When the compounds are given as single-agents, the required concentrations were computed to be 506 µg · mL-1 and 56 ng · mL-1 for cetuximab and cisplatin, respectively. A TSC curve is constructed for different combinations of the two drugs, which separates concentration combinations into regions of tumor shrinkage and tumor growth. The more concave the TSC curve is, the lower is the total exposure to test compounds necessary to achieve tumor regression. The TSC curve for cetuximab and cisplatin showed weak concavity. TSC values and TSC curves were estimated that predict tumor regression for 95% of the population by taking between-subject variability into account. The TSC concept is further discussed for different concentration-effect relationships and for combinations of three or more compounds.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Models, Biological , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Carcinoma, Non-Small-Cell Lung/pathology , Cetuximab/administration & dosage , Cisplatin/administration & dosage , Dose-Response Relationship, Drug , Female , Humans , Lung Neoplasms/pathology , Mice , Mice, Nude , Xenograft Model Antitumor Assays/methodsABSTRACT
Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Antineoplastic Agents/administration & dosage , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Mediator Complex/antagonists & inhibitors , Protein Kinase Inhibitors/administration & dosage , Animals , Anti-Inflammatory Agents/adverse effects , Anti-Inflammatory Agents/toxicity , Antineoplastic Agents/adverse effects , Antineoplastic Agents/toxicity , Disease Models, Animal , Heterografts , Humans , Hyperplasia/drug therapy , Mice , Neoplasms/drug therapy , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/toxicity , Treatment OutcomeABSTRACT
SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.
ABSTRACT
The Hexosamine Pathway (HP) is one hypothesis proposed to explain glucose toxicity and the alterations observed during the course of diabetic microvascular complication development. Glucosamine is a precursor of UDP-N-Acetylglucosamine (UDP-GlcNAc), the main product of the HP that has often been used to mimic its activation. The transfer of a UDP-GlcNAc residue onto proteins (O-GlcNAc modification) represents the final step of the HP and is considered as a major mechanism by which this pathway exerts its signalling effects. While it is well accepted that the HP promotes extracellular matrix accumulation in the context of diabetic nephropathy, its involvement in the perturbations of cell cycle progression and hypertrophy of renal cells has been poorly investigated. Nevertheless, in a growing number of studies, the HP and O-GlcNAc modification are emerging as important regulators of cell cycle progression. This review will focus on the role of glucosamine and O-GlcNAc modification in cell cycle regulation in the context of diabetic nephropathy. Special emphasis will be given into the role of the HP as a potential mediator of the effects of high glucose on the perturbations of renal cell growth.
Subject(s)
Cell Cycle/physiology , Glucosamine/physiology , Hyperglycemia/metabolism , Mesangial Cells/physiology , Animals , Humans , Hypertrophy/metabolism , Hypertrophy/pathology , Mesangial Cells/cytology , Mesangial Cells/pathologyABSTRACT
Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.
Subject(s)
Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amidohydrolases/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Catalysis/drug effects , Ceramidases , Cloning, Molecular , Consensus Sequence , Conserved Sequence , Gene Expression , Humans , Hydrogen-Ion Concentration , Metals/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Neutral Ceramidase , Rats , Saccharomyces cerevisiae , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.
Subject(s)
Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Lysophospholipids/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Amidohydrolases/metabolism , Animals , Cell Proliferation , Ceramidases , Diabetic Nephropathies/chemically induced , Male , Neutral Ceramidase , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Rats , Rats, Wistar , Streptozocin/pharmacology , Time FactorsABSTRACT
Alterations in proliferation and hypertrophy of renal mesangial cells are typical features of diabetic nephropathy. The HP (hexosamine pathway) has been proposed as a biochemical hypothesis to explain microvascular alterations due to diabetic nephropathy; however, involvement of HP in the regulation of mesangial cell growth or hypertrophy has been poorly studied. Although gangliosides are known to regulate cell proliferation, their potential role in mesangial cell-growth perturbations has hardly been explored. In the present study, we investigated the effects of the HP activation, mimicked by GlcN (glucosamine) treatment, on mesangial cell growth and hypertrophy and the potential implication of gangliosides in these processes. Our results indicate that GlcN induced hypertrophy of mesangial cells, as measured by an increase in the protein/cell ratio, and it caused cell-cycle arrest by an increase in the expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Furthermore, GlcN treatment resulted in a massive increase in the levels of gangliosides G(M2) and G(M1). Treatment of cells with exogenous G(M2) and G(M1) reproduced the effects of 0.5 mM GlcN on p21(Waf1/Cip1) expression, cell-cycle arrest and hypertrophy, suggesting that gangliosides G(M2) and G(M1) are probably involved in mediating GlcN effects. These results document a new role of the HP in the regulation of mesangial cell growth and hypertrophy. They also suggest a potential new mechanism of action of the HP through modulation of ganglioside levels.