Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations  were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding. In the peripheral nervous system (PNS), Schwann cells envelop axons and form insulating myelin sheaths. Defects in this fundamental structure result in chronic, severe neuropathological conditions, affecting nerve conduction velocity and neuron viability.A multilamellar membrane structure, myelin, enwraps selected axons in the nervous system, enabling the prompt transmission of nerve impulses along the axonal membrane. Myelin contains a compact lipid-rich compartment with a unique set of membrane-associated proteins, such as myelin protein zero (MPZ), peripheral myelin protein of 22 kDa (PMP22), myelin basic protein (MBP), and myelin protein P2, and more loosely packed regions that also encompass cytoplasm. Close interplay between myelin proteins is important for the formation of cytoplasmic channels within myelin2. Several types of CMT exist; the most common form, autosomal dominant CMT1, influences Schwann cells and myelin. In CMT1, motor and sensory nerve conduction velocities are remarkably reduced, leading to muscle weakness and atrophy in the feet and occasionally also in upper limbs. Demyelination and onion bulb formation are present in most CMT1 patients. Most commonly, CMT1a is caused by a duplication of or a point mutation in the pmp22 gene, and 70–80% of all CMT1 cases are associated with pmp22 mutations3. CMT1b results from mutations in the mpz gene and comprises 4% of all CMT1 cases. A number of other CMT1-associated genes have been described2. Recently, three point mutations in the pmp2 gene, coding for the myelin P2 protein, were linked to autosomal dominant CMT1. The I43N mutation was discovered in two families5 and T51P and I52T each in one family6. All patients with these mutations have reduced motor and sensory nerve conduction velocities, thinner myelin sheaths, and demyelinating axons with onion bulb formation. Symptoms also include progressive hand and foot muscle weakness and atrophy, as well as foot deformity and sensory loss in limbs6. Although the pathological features of different CMT1 types are comparable, the molecular pathomechanisms are different7.Charcot-Marie-Tooth disease (CMT) is one of the most common inherited human neuropathies , affecting both motor and sensory nerve conduction in the PNS8. It is located in compact myelin, stabilizing the multilayered lipid membrane assembly. P2 is a small 15-kDa protein with a β barrel structure covered by an α-helical lid10. It belongs to the conserved family of fatty acid-binding proteins (FABPs) and can transfer fatty acids from and to lipid membranes using a collision transfer mechanism, indicating a functional role in the lipid homeostasis of myelin11. P2 may also bind cholesterol; a CRAC cholesterol binding motif is found at its C terminus12.P2 is mainly expressed by Schwann cells in the PNSHere, we show that the crystal structures of CMT1-associated P2 mutant forms remain nearly unaltered, but the stability of the mutant proteins is remarkably reduced. Mutant proteins show decreased solubility, T51P triggering the most significant effect. T51P has a reduced membrane stacking activity, while I43N and I52T maintain their ability to bind and stack lipid membranes. On the other hand, protein dynamics, as well as fatty acid and membrane binding kinetics, of all P2 mutants differ from those of wild-type P2 (P2wt). The mutations may cause defects in protein folding and the regulation of functional conformational changes, and they provide clues into ligand binding dynamics in the FABP family.In order to explore the functional and structural effects of CMT1-linked mutations in P2, we expressed and purified all three mutant proteins; I43N, T51P, and I52T. We used a range of experimental techniques together with atomistic simulations to study the effects of the mutations on P2 structure, function, and dynamics.h) of monomeric T51P was also larger compared to the other variants  and maximum dimension (Dmax) of I43N and T51P slightly deviate from P2wt, while I52T gave similar results  data were collected to monitor possible variation in the solution structures of the patient mutants Fig. . Scatterts Table  differs 17, and it is noteworthy that all the CMT1-linked P2 mutations lie close in space to this gap . Spectra were recorded in water and in phosphate buffer Fig. . The intsn-glycero-3-phosphocholine (DMPC): 1,2-dimyristoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DMPG) or 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC): 1,2-dioleyl-sn-glycero-3-phosphatidyl-rac-glycerol (DOPG) vesicles, the spectra of P2 clearly changed ; T51P has the lowest Tm, +41 °C, and already at +38 °C, the conformation of T51P changed and unfolding started. I43N has a Tm at +48 °C and I52T at +52 °C. Therefore, both of these mutants also have decreased thermal stability compared to P2wt.We further studied the thermal stability of P2 mutants using conventional CD spectroscopy Fig. . Tm analRoot mean square fluctuation (RMSF) analysis from atomistic molecular dynamics (MD) simulations was used to examine P2 dynamics with and without bound palmitate inside the β barrel. Palmitate-bound P2wt is generally more dynamic than its empty counterpart. The most mobile parts of P2wt are located in the loops and, to some extent, in the helical lid Fig. ; these s10. At the hinge also lies a buried water molecule, which is conserved in FABPs and was suggested to be relevant for correct folding18. It is possible that the conserved Gly residues and the water molecule also play roles in functional conformational changes in FABPs, including P2. Rg for all eight systems  was estimated based on the MD simulations. The obtained values . For palmitate-bound P2 forms, the respective numbers of water molecules were 21.0, 22.1, 29.1, and 22.0. In both cases, T51P differs from the other variants, interestingly having less water molecules within the β barrel without than with palmitate inside. Here, the behaviour of the Ile mutants resemble each other but slightly differ from P2wt. These values are in accordance with studies on FABP, whereby faster exchange of water was observed for liganded FABP than for apo-FABP21. Also in the crystal structures of all mutant proteins studied here, a fatty acid, modelled as palmitate, is visible inside the β barrel. P2 captures abundant fatty acids during expression in E. coli10. With our earlier atomic-resolution crystallographic data, we modelled the fatty acid component as a mixture of palmitate and cis-vaccenate10.P2 belongs to the family of FABPs, and in all crystal structures of human P2, there is a fatty acid inside the β barrel22. The peak maxima clearly increase and shift in the presence of P2, indicating an interaction involving a non-polar environment between DAUDA and all P2 variants nts Fig. . This ph10, in which we earlier built in palmitate (C16:0) and cis-vaccenate (C18:1) with partial occupancies.Since the structure and dimensions of DAUDA diverge from natural fatty acids, we used liquid chromatography-mass spectrometry (LC-MS) to investigate the fatty acids bound to P2. Fatty acids with 16–20 carbons without or with a double bond were monitored. The total amount of bound fatty acids was similar between all the P2 variants, and C16 and C18 fatty acids together comprise nearly 100% of the total fatty acid content. We compared the amount of saturated and non-saturated C16 and C18 fatty acids, and noted the fatty acid content of all variants was nearly identical Fig. . P2 cont24.The binding of P2 onto lipid membrane surfaces was studied using surface plasmon resonance (SPR) with two different lipid compositions, differing only in the identity of the hydrocarbon chains of the lipids Fig. . SPR wasIn compact myelin, P2 is located in the cytoplasmic leaflet, interacting with two apposing membranes. An important feature of P2 is to bind lipid membranes and to stack them into multilayered systems. To test whether the mutant variants are able to attach lipid membranes together, we used a lipid vesicle aggregation assay and measured the increase in the turbidity of a lipid vesicle solution caused by P2. The results demonstrate that both Ile mutants can bind and stack lipid membranes as efficiently as P2wt Fig. . However6. P2 mutations are located near each other in the structure, in close proximity to the fatty acid binding pocket and the gap between the strands β4 and β5. Comparing the sequences of all 12 human FABPs, it can be seen that the mutated residues are conserved in most human FABPs, especially those with a collision-type ligand transfer mechanism10. Based on our work, all CMT1-associated P2 mutant forms maintain their overall fold in the crystal state, but their stability is significantly reduced, and fatty acid and lipid membrane binding altered, compared to P2wt.Recently, three P2 point mutations were shown to result in the most common heritable human peripheral neuropathy, CMT. All P2 patient mutations trigger an autosomal dominant form of demyelinating CMT1pmp22 gene, thought to lead to misfolding and aggregation of PMP22 in the ER of myelinating Schwann cells25. The three P2 mutations  studied here increase the tendency of P2 to form insoluble aggregates, when expressed in E. coli. Even though the prokaryotic expression system differs from the natural environment inside human Schwann cells, it can provide relevant information when comparing disease-associated forms of human proteins.Most forms of CMT1 are caused by duplication or mutation of the 10, may originate from the α-helical lid, which interacts with the head groups of lipids and may be partially buried inside the lipid membrane. The changes could also result from a membrane-induced opening of the β barrel, leading to possible ligand exchange with the membrane. The results indicate that all P2 variants interact with lipid membranes, but as seen with SPR, the mutants are different in their respective lipid binding dynamics.The secondary structure content of T51P in solution was lower than for the other variants. All forms of P2 showed conformational changes upon lipid vesicle binding. These changes, also seen before with P2wtImportantly, the thermal stability of all three CMT-linked P2 variants is dramatically reduced compared to P2wt. This finding supports the results from the solubility studies; reduced protein stability leads to partial or complete unfolding and the formation of protein aggregates. Overall, the T51P mutation has the most striking effect on the biophysical properties of P2. These results raise a question whether these mutants are able to fold properly in Schwann cells, or if the mutations lead to misfolding and protein accumulation.10, and differences are limited to 1–2 hydrogen bonds near the mutation site. Nevertheless, the fatty acid binding properties of all mutants differ from those of P2wt. Based on fluorescent probe assays, all three mutants bind fatty acids more effectively than P2wt, and T51P causes the highest change in DAUDA fluorescence. As DAUDA has a fairly large and rigid dansyl group at one end of the molecule, these results may suggest that T51P favours larger ligands than P2wt; the result can also reflect the increased open/close dynamics of the T51P variant. Different kinds of kinetics may come to play when the fatty acid ligand is replaced in this experiment, including affinities towards various ligands and different conformational dynamics.The crystal structures of the disease-associated mutants are highly similar to P2wt, also regarding the fatty acid binding pocket26, but to our knowledge, it has not been observed for FABPs. It is obvious that several factors may affect such conformational changes, including the bound ligand, contact to a membrane surface, and the presence of mutations. The opening of the β barrel could also be linked to misfolding and/or increased β aggregation, as observed for the T51P mutant.RMSF analysis of MD simulations demonstrates altered dynamic properties for all disease mutants. Fatty acid-bound T51P is clearly more dynamic than P2wt and the other two mutants, and liganded T51P has more water molecules inside the β barrel than other palmitate-bound P2 variants. This is presumably the consequence of an increased flexibility of the α-helical lid and the β barrel structure of T51P, which at least to some extent arises from the lack of proper hydrogen bonds in the β sheet β1-4  protease cleavage site in the pTH27 vector9 was used as a template in mutagenesis to produce expression constructs with the c.T128A (p.I43N), c.C151A (p.T51P), and c.T155C (p.I52T) mutations. Mutagenesis was carried out using the QuikChange Site-Directed Mutagenesis protocol, and all mutations were confirmed by DNA sequencing. Proteins were expressed in E. coli BL21 RIPL (DE3) cells in ZYM-5052 autoinduction medium32 with 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol at +18 °C for 66 h. Cells were harvested and suspended in lysis buffer . Cells were lysed by sonication, and insoluble material was pelleted by centrifugation . The soluble fraction was then mixed with the HisPur Ni-NTA Resin (Thermo Fisher Scientific) at +4 °C for 1 h. The resin was washed twice with washing buffer  using centrifugation  and then transferred into a gravity-flow column and further washed with 50 ml of washing buffer. P2 was eluted with elution buffer . To cleave the His6 tag, 33 nmol of recombinant TEV protease33 were added. Imidazole was removed by dialysis through 6000–8000 MWCO dialysis tubing (SpectraPor) against 0.3 M NaCl, 1 mM DTT, 20 mM HEPES pH 7.5 at +4 °C for 16 h. Both TEV and the cleaved His6 tag were removed with reverse chromatography using HisPur Ni-NTA. Finally, P2 was purified with SEC using the dialysis buffer and a Superdex 75 pg 16/600 column  and concentrated with an Amicon Ultra 15, MWCO 10 kDa concentrator (Millipore). In T51P purification, all buffers, excluding the SEC buffer, also contained 10% glycerol to maintain the protein soluble during purification.Human P2 with an N-terminal His34. The structures were solved by molecular replacement using the human P2 structure (PDB entry 4BVM10) as a search model in Phaser35. The structures were refined and built using phenix.refine36 and Coot37. The refined coordinates and structure factors were deposited at the PDB with the entry codes 5N4M (I43N), 5N4P (I52T), and 5N4Q (T51P).P2 mutants were crystallised at +4 or +20 °C using the sitting drop vapour diffusion method, while the concentrations of the I43N, T51P, and I52T mutants were 8.5, 6.2, and 5.5 mg/ml, respectively. 0.3 M NaCl, 10% glycerol, 20 mM HEPES pH 7.5 was used as the protein buffer and 100 µl of 2.1 M DL-malic acid pH 7.25–7.5 as the reservoir solution, with 600-nl drops containing an equal volume of reservoir and protein. Diffraction data were collected at 100 K on beamline I03 and I04 at the Diamond Light Source, Didcot, UK, and processed using XDS38.Synchrotron SAXS data were collected on beamline P12 at the PETRAIII storage ring, DESY, Hamburg, Germany. 0.6–4.1 mg/ml P2wt, I43N, and I52T were studied in a buffer containing 0.3 M NaCl, 20 mM HEPES pH 7.5, and T51P in a buffer containing 0.3 M NaCl, 5% glycerol, 20 mM HEPES pH 7.5. Data were processed and further analysed using the ATSAS program package39. Soluble fractions were then calculated (%). DLS measurements were conducted as triplicates at +25 °C with protein concentrations of 0.6–0.9 mg/ml in a buffer containing 0.3 M NaCl, 20 mM HEPES pH 7.5, using the DynaPro Plate Reader II (Wyatt). After SEC, the proteins were stored on ice for 24 h before DLS measurements.Samples from soluble and insoluble fractions were taken and run on SDS-PAGE. Intensities of the P2 protein bands were determined using ImageJ2O. SRCD spectra were measured at +30 °C using 0.2 mm pathlength quartz cuvettes with protein concentrations of 17–21 µM (~0.3 mg/ml). Spectra for the P2 variants were also recorded in the presence of unilamellar vesicles containing a 1:1 molar ratio of DMPC and DMPG, or DOPC and DOPG. The molar protein:lipid ratio was 1:100. Thermal stability measurements were carried out using a Chirascan Plus CD spectropolarimeter (Applied Photophysics). Proteins were first diluted into 10 mM sodium phosphate buffer (pH 7.5) to final concentrations of 3.4 µM (50 µg/ml). CD spectra were then recorded at 195–260 nm with a ramping rate of 1 °C/min between +22–90 °C. The melting temperatures (Tm) were calculated using Global 3 (Applied Photophysics).SRCD was carried out on the DISCO  and UV-CD12  beamlines. Proteins were first dialysed into 10 mM sodium phosphate pH 7.5 or dH10 and converted to the CHARMM36 force field40. The topology for P2wt was directly obtained from the conversion. The point mutations  were constructed from P2wt. Water molecules were modeled using the TIP3P model41.The protein with bound palmitate was obtained from the PDB entry 4BVM3 and with 16000 water molecules, in accordance to our previous study42. Counter-ions  were included to neutralise the total charge of the system.Eight different systems were studied: P2wt and its point-mutated forms I43N, T51P, and I52T, all with and without a bound palmitate molecule inside the binding pocket. The proteins were simulated in solvated cubic simulation boxes with sizes of approximately 8 × 8 × 8 nm43 at a reference pressure of 1 bar with a coupling time constant of 2.0 ps and isothermal compressibility of 4.5 × 10−5 bar−1. All bonds were constrained with the LINCS algorithm44. Periodic boundary conditions were used. A cut-off radius of 1.0 nm was introduced for the neighbor list, the Lennard-Jones interactions, and the non-bonded interactions. The particle-mesh Ewald (PME) method45 with cubic interpolation was used for calculating long-range electrostatics, using a spacing of 0.16 nm for the Fourier grid.The MD simulations were carried out under NpT conditions. Temperatures were coupled using the velocity-rescale  method, with separate temperature coupling for protein and solvent. Reference temperatures of 300 K were used with coupling time constants of 2.0 ps. Pressure coupling was done isotropically with the isothermal Parrinello-Rahman barostat46. A time step of 2 fs was used in integrating the equations of motion. All systems were first energy-minimised with the steepest descent algorithm and then simulated for a total of 2.5 µs each. An equilibration period of 500 ns was removed from the beginning of each trajectory, using the final 2 µs for analysis. The trajectory coordinates were saved every 50 ps.All simulations were conducted using GROMACS 4.6.7The RMSF was calculated for each residue using the GROMACS tool g_rmsf. The RMSF of a residue shows its stability; the larger the value, the more mobile it is. Therefore, by calculating the RMSF, we can determine, which parts of the protein are most affected by the mutation and/or the presence of the ligand. Water analysis was done with the GROMACS tool trjorder by calculating the number of water molecules within 0.9 nm of the center of mass of the protein backbone (the radius of the P2 barrel is about 1.0 nm) at every time step over the course of the simulation.Fatty acid binding to P2 was studied with a fluorescent fatty acid analog, DAUDA. 10 µM DAUDA and 10 µM P2 were incubated for 1 h in 150 mM NaCl, 10 mM HEPES pH 7.5 at +23 °C. Fluorescence emission spectra were recorded with excitation at 345 nm and emission between 400–700 nm, using a Tecan Infinite M1000Pro plate reader.20-µl aliquots of protein solutions adjusted to the same concentration  were acidified with 1 µl formic acid and then precipitated with 20 µl of acetone. Samples came from 2–3 separate production batches for each variant, and each sample was analysed 1–2 times. After centrifugation, fatty acids were extracted into 20 µl of chloroform, the phases were separated by short centrifugation, and 5 µl of the chloroform phase was subjected to LC-MS using an Acquity UPLC system coupled to a SynaptG1 Q-TOF type mass spectrometer. The chromatography column was a BEH C18, 2.1 × 100, eluted with a gradient of acetonitrile . The mass spectrometer was operated in negative mode, collecting 0.2-s scans in centroid mode from m/z 50 to 1000. LC-MS was performed at the Proteomics and protein analysis core facility of Biocenter Oulu.SPR assays were performed using BiacoreT200  for P2wt as well as the I43N and I52T mutants. Due to its low stability and tendency to aggregate, T51P was not used in SPR. 1 mM DOPC:DOPG (1:1) and DMPC:DMPG (1:1) vesicles in a buffer containing 150 mM NaCl and 10 mM HEPES pH 7.5 were immobilised on an L1 sensor chip  according to the manufacturer’s instructions. The surface was then saturated with an injection of 1 µM bovine serum albumin (BSA). Lipid immobilisation and BSA injection were done for each SPR cycle. Duplicate injections of P2wt and the mutants at 0.1–10 µM, using 150 mM NaCl, 10 mM HEPES pH 7.5 as the running buffer, were carried out at +30 °C. Results were analysed with BiaEvaluation (GE Heathcare) using a kinetic two-state binding model as well as steady-state affinity. Due to the shape of the binding curves, fitting was also done using a sigmoidal 4-parameter modelFor a single-point assay, 0–10 µM P2 was mixed with 0.5 mM unilamellar vesicles containing a 1:1 molar ratio of DMPC:DMPG in 150 mM NaCl, 10 mM HEPES pH 7.5, and the samples were incubated for 35 min at +30 °C. The turbidity of the vesicle-protein solutions was measured with a Tecan Infinite M1000 Pro plate reader using absorbance at 450 nm at +30 °C. The assay was repeated 3–5 times with all concentrations and P2 forms.In order to study the time course of vesicle aggregation, 0.5 mM DMPC:DMPG vesicles were mixed with 5 µM P2 in buffer containing 150 mM NaCl and 10 mM HEPES (pH 7.5). A VersaMax microplate reader (Molecular Devices) was used to measure the turbidity at 1-min intervals, shaking before each measurement. The temperature was set at +30 °C and the wavelength at 460 nm. The difference of 10 nm in the wavelength in the above two experiments was not considered to affect the outcome, as the measurement follows turbidity of the sample.2O, samples were negatively stained with two drops of 2% uranyl acetate for 12 s in each drop and air-dried. TEM images were recorded using a Tecnai G2 Spirit 120 kV instrument equipped with a Quamesa CCD camera (Olympus Soft Imaging Solutions) at the EM core facility of Biocenter Oulu.34 µM (0.5 mg/ml) P2 was mixed with 740 µM (0.5 mg/ml) DMPC:DMPG (1:1) unilamellar vesicles, and the samples were incubated at +22 °C for 1 h. 4-µl samples were then pipetted onto glow-discharged carbon-coated copper grids, and after incubating for 1 min, excess solution was removed with filter paper. After 4 washes in droplets of dHThe crystal structure coordinates and structure factors are available at the PDB with the entry codes 5N4M (I43N), 5N4P (I52T), and 5N4Q (T51P). Other datasets analysed during the current study are available from the corresponding author upon reasonable request.


Melicope madagascariensis (Rutaceae) is an endemic plant species of Madagascar that was first classified as a member of the genus Euodia J. R. & G. Forst (Rutaceae) under the scientific name Euodia madagascariensis Baker. Based on morphological characteristics, Thomas Gordon Hartley taxonomically revised E. madagascariensis Baker to be M. madagascariensis (Baker) T.G. Hartley. Chemotaxonomical studies have long been used to help the identification and confirmation of taxonomical classification of plant species and botanicals. Aiming to find more evidences to support the taxonomical revision performed on E. madagascariensis, we carried out phytochemical investigation of two samples of the plant. Fractionation of the ethanol extracts prepared from two stem bark samples of M. madagascariensis (Baker) T.G. Hartley led to the isolation of seven known furoquinoline alkaloids 1–7 and two known methoxyflavones 8 and 9. The presence of furoquinoline alkaloids and methoxyflavones in the title species is in agreement with its taxonomic transfer from Euodia to Melicope. Antiprotozoal evaluation of the isolated compounds showed that 6-methoxy-7-hydroxydictamnine  showed weak antimalarial activity (IC50 = 35 µM) against the chloroquine-resistant strain Dd2 of Plasmodium falciparum. Skimmianine (4) displayed moderate cytotoxicity with IC50 value of 1.5 µM against HT-29 colon cancer cell line whereas 3,5-dihydroxy-3′,4′,7-trimethoxyflavone (9) was weakly active in the same assay (IC50 = 13.9 µM). Melicope J. R. & G. Forst (Rutaceae) comprises about 235 species distributed in the Madagascar and Mascarenes islands and Indo-Himalaya regions in the Hawaii and Marquesas islands to New Zealand. Melicope madagascariensis (Baker) T.G. Hartley is an endemic species to Madagascar, which has long been classified in the genus Euodia J. R. & G. Forst (Rutaceae) under the scientific name Euodia madagascariensis Baker before being assigned to the genus Melicope. This taxonomic revision introduced by Hartley in 2001 was based on morphological characteristics and concerned the other Madagascan plant species that were placed in the genus Euodia [The genus M. madagascariensis is a tree up to 10–20 m tall which is encountered in the rainforest of Madagascar and is traditionally employed as exhilarating agent, purgative, and in formulations for the treatment of liver, kidney and stomach disorders, bronchitis and mumps [Melicope are rich sources of furoquinoline alkaloids, methoxyflavones and acetophenones, some of which have elicited antiplatelet aggregation [M. madagascariensis reported the isolation of methoxyflavones [Euodia species already transferred into Melicope. As part of our ongoing projects aiming to characterize secondary metabolites of plants originated from Madagascar rainforest for biological and chemotaxonomical studies, we carried out phytochemical investigation of M. madagascariensis, which is one of the eleven Melicope species endemic to Madagascar. This paper deals with the isolation of nine known compounds including seven furoquinoline alkaloids 1–7 and two methoxyflavones 8 and 9 from two samples of the stem bark of this species. Herein, we discuss the chemotaxonomic significance as well as the antimalarial and cytotoxic activities of the isolated compounds.regation , cytotoxregation  and antiregation . A previflavones , a classM. madagascariensis was collected from Zahamena, Madagascar. Its ethanol extract (MG250) was first subjected to a modified Kupchan partitioning to give hexane-soluble, chloroform (CHCl3)-soluble and aqueous MeOH-soluble extracts. Fractionation of the CHCl3-soluble extract by successive open column chromatography over Sephadex LH-20 and silica gel yielded three furoquinoline alkaloids, pteleine (1) [2) [3) [A stem bark sample of eine (1) , evolitr (1) [2)  and 6-me [2) [3) .3-soluble extract obtained from the same method as above, to finally provide basic and neutral fractions. Subsequent separations of these two fractions by a combination of chromatography techniques  yielded four furoquinoline alkaloids identified as: skimmianine (4) [5) [6) [7) [8) [9) [1–9 , all the other compounds isolated in the present work have been already encountered in other Melicope species, such as M. semecarpifolia and M. pteleifolia [Further phytochemical investigation of the same plant was conducted on a larger collection of stem bark ST1375) samples collected from Moramanga, Madagascar. Fractionation of the ethanol extract of ST1375 was first carried out by performing selective isolation of alkaloids through conventional acid–base treatment of the CHClnine (4) , confusa (4) [5) , dictamn5 samples [6) [7) , and two [7) [8)  and 3,5- [8) [9) . This isEuodia, furoquinoline alkaloids are widespread in the Rutaceae family [Melicope [M. madagascariensis chemotaxonomically confirmed the classification of the present plant species in the genus Melicope of the Rutaceae family. Furthermore, polyoxygenated flavones (polymethoxy and methylenedioxy) have been considered to be characteristic chemical components of plant species of the genus Melicope inside the Rutaceae family [Euodia into Melicope. This study added one more species to the Melicope-producing methoxyflavones. Whether all of the Euodia species containing furoquinoline alkaloids and methoxyflavones should be moved to the genus Melicope or not is one of the major questions that can be unfolded from the present investigation. Apart from the morphological and phytochemical contributions, full genome sequencing of all species of the genus to be transferred would complete this taxonomical classification.With the exception of few species including those of the genus e family . In partMelicope , 15, 16.e family . The iso1–3 for their antimalarial activity against the multidrug-resistant strain Dd2 of Plasmodium falciparum showed that only heliparvifoline (3) exhibited growth inhibitory effect at relatively high concentration (IC50 = 35 µM). Positive control consisted of fosmidomycin (IC50 = 0.31 µM). Moreover, in vitro antimalarial activity against the chloroquine-susceptible HB3 (Honduras) and the chloroquine resistant W-2 (Indochina) strains of P. falciparum has been previously performed on some Rutaceous furoquinoline alkaloids such as skimmianine (4), haplopine (10), kokusaginine (11), acronycidine (12) and acronydine (13) [Plasmodium falciparum, the present paper added more data on the antimalarial activity of furoquinoline alkaloids of Rutaceous plants. In addition, compounds 4–9 isolated from the sample ST1375 were assayed against the human colorectal adenocarcinoma (HT-29) cell line. As results, compounds 5–8 were inactive in this assay while skimmianine (4) and 3,5-dihydroxy-3′,4′,7-trimethoxyflavone (9) exhibited moderate and weak activity with IC50 values of 1.5 µM and 13.9 µM, respectively.The sample MG250 was collected for the International Cooperative Biodiversity Group (ICBG)/Madagascar program that was mainly aimed at searching for antimalarial and antiproliferative agents from natural resources from Madagascar. Evaluation of compounds ine (13) . AlthougM. madagascariensis. The presence of furoquinoline alkaloids and methoxylated flavones in this plant provides support to its re-classification from the genus Euodia to Melicope. Heliparvifoline (3) displayed weak antimalarial activity (IC50 = 35 μM) against strain Dd2 of P. falciparum whereas skimmianine (4) and 3,5-dihydroxy-3′,4′,7-trimethoxyflavone (9) were identified as the most cytotoxic constituents against HT-29 colon cancer line.The current study extends the knowledge about the chemistry of 1H NMR spectra were recorded on Bruker Avance 400 and 500 spectrometers in CDCl3 or CD3OD with TMS as internal standard. Semi-preparative HPLC was performed on a Shimadzu instrument consisting of LC-20AB pump, SPD-20A Prominence detector and CBM–20Alite system controller. Sephadex LH-20 (Sigma), silica gel 60  and C18 reversed phase silica gel  were used for column chromatography.P. falciparum by using the SYBR Green I-based plate assay as previously reported [The antimalarial assay was carried out at Virginia Tech against the multidrug-resistant strain Dd2 of reported .Cytotoxicity assay against HT-29 human colon cancer cell line was performed at the College of Pharmacy, The Ohio State University, USA by using a sulforhodamine B reduction assay procedure as described in a previous paper .M. madagascariensis stem bark was collected in February 2000 from plants growing in the forest adjacent to the Zahamena National Park. A larger collection of the same plant material designated ST1375 was conducted in November 2009 in the Antsasaka forest of Moramanga. These two collection sites were about 250 km distant from each other in the Atsinanana region of Madagascar. Voucher specimens have been deposited at the herbarium of the Centre National d’Application de Recherches Pharmaceutiques (CNARP), Antananarivo, Madagascar under the codes RJO157 for MG250 and ST1375 B for ST1375.The first sample coded MG250 of n-hexane (3 × 200 mL portions). The aqueous MeOH layer was then diluted to 60 % aqueous MeOH by addition of water before partitioning with CHCl3 (3 × 200 mL portions). Finally, the aqueous MeOH phase was vacuum concentrated, suspended in H2O (75 mL) and extracted with n-BuOH saturated with water (3 × 75 mL portions). Elimination of all the solvents in vacuo provided dried n-hexane soluble fraction (143.5 mg), CHCl3-soluble fraction (538.6 mg), n-BuOH-soluble fraction (131 mg) and H2O-soluble fraction (630.8 mg). The CHCl3-soluble fraction was chromatographed over Sephadex LH-20 column eluted with CH2Cl2/MeOH, 1:1 and then silica gel column eluted with n-hexane/EtOAc, 7:3 to give compounds 1 (0.9 mg), 2 (0.8 mg) and 3 (1.2 mg).The dried and powdered sample MG250 (300 g) was macerated in EtOH for 48 h to yield 11.5 g of a crude EtOH extract. A portion (1.5 g) was suspended in 90 % aqueous MeOH (150 mL) and extracted with 3 fraction was subjected to a conventional acid–base extraction to afford a basic CHCl3-soluble fraction  and a neutral CHCl3-soluble fraction . Fraction A was first gel filtered on a Sephadex LH-20 column eluted with n-hexane/CH2Cl2/MeOH, 4:3:3 to give four pooled fractions (A1-A4). Fraction A2 was subjected to a combination of silica gel open column chromatography and HPLC on a semi-preparative C18 column  or silica gel preparative TLC to furnish 4 (1.4 mg) and 7 (0.9 mg). Fraction A4 (191.4 mg) was rechromatographed over silica gel open column eluted with n-hexane/EtOAc mixtures to give 5 (3.1 mg). A part of the fraction B (4 g) was flash chromatographed on silica gel column to furnish six subfractions (B1-B6). Subfraction B2 (171.5 mg) was separated by C18 open column chromatography and then by silica gel preparative TLC with CHCl3/MeOH, 50:1 as eluent to furnish 8 (3.4 mg), 9 (2.7 mg) and 6 (1.4 mg).The dried and powdered sample ST1375 (1.3 kg) was extracted as above to afford a crude EtOH extract (65.3 g). A portion (25 g) was liquid–liquid partitioned using same procedures described above. The resulting CHCl1H NMR spectra and comparison of data obtained with those published in the literature. The 1H NMR spectroscopic data of the isolated compounds can be obtained free of charge from the corresponding author.Structures of isolated compounds were established by the interpretation of their


Curcuma xanthorrhiza is an immunomodulator that has similar biological effect as vitamin D. Combination of curcumin and vitamin D3 is expected to work synergistically. Curcumin contained in Curcuma xanthorrhiza supplementation on vitamin D3 administration to SLEDAI, IL-6, and TGF-β1 serum in SLE patients with hypovitamin D. To determine the effect of Curcuma xanthorrhiza 3 × 20 mg for 3 months (group II). SLEDAI, levels of vitamin D, IL-6, and TGF-β1 in serum were evaluated before and after the treatment. This was a double-blind RCT conducted in Saiful Anwar Hospital, Malang, in January 2016–March 2017. Subjects were SLE active (SLEDAI > 3) with levels of 25(OH)D3 ≤ 30 ng/ml and divided into two groups: those receiving cholecalciferol 3 × 400 IU and placebo 3 × 1 tablets (group I) and those receiving 3 × 400 IU cholecalciferol andβ1 serum among groups after the treatment. Decreased levels of serum IL-6 have a positive correlation with SLEDAI reduction. Conclusion. Curcuma xanthorrhiza supplementation on vitamin D3 had no effects on SLEDAI and serum levels of IL-6 and TGF-β1. This clinical trial is registered with NCT03155477. There were no significant differences in SLEDAI reduction, decreased serum levels of IL-6, and increased levels of TGF- SLE diseg cells .μg/day for 3 months in 39 SLE patients with hypovitamin D, and it showed an increase of serum vitamin D level  and significant improvement of Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)  and Fatigue Severity Scale (FSS)  after supplementation [β1 [3 therapy.Rifai et al. (2014) gave cholecalciferol 1200 IU/day or 30 entation . The distion [β1 . AlthougCurcuma longa and Curcuma xanthorrhiza—and has potential as an immunomodulator for the treatment of SLE [β, IL-6, IL-12, TNF-α, and IFN-γ [Curcumin is a polyphenol compound widely found in ginger plants—t of SLE . Curcumind IFN-γ . Curcumind IFN-γ  and it ind IFN-γ .Pratama et al. (2015) found there were a Th17 decrease and an increase of Treg cells after supplementation of 200 mg/kg/day  curcumin in SLE mice . Khajehd3 therapy, improve clinical symptoms, decrease disease progression, and reduce mortality rates in SLE patients with hypovitamin D by combining curcumin supplementation with vitamin D. Curcumin supplementation on vitamin D3 administration may affect the balance of TGF-β1/IL-6 cytokines at cellular level.The synergistic nature of curcumin with vitamin D in regulating immune cells provides an opportunity for researchers to improve the response of vitamin DThis was a double-blind randomized controlled trial conducted in Saiful Anwar Hospital, Malang, in January 2016–March 2017. This research was conducted after obtaining ethical approval from the Saiful Anwar Hospital, Malang.The sample size was calculated by the formula for unpaired numerical analytic research with one-way hypothesis. The minimum sample size required in this study is 34. The subjects who fulfilled inclusion criteria, were willing to participate in the research, and signed the informed consent were 40 patients.n = 20) and the group receiving 3 × 400 IU and curcumin (Curcuma xanthorrhiza) 3 × 20 mg for 3 months . The dosage of vitamin D given in this study was based on previous study by Rifai et al. (2014), who successfully increased level of vitamin D into normal levels [Research subjects were randomized using simple randomization. The subjects were divided into 2 groups: the group receiving 3 × 400 IU cholecalciferol and 3 × 1 tablet placebo  1997, active SLE (SLEDAI > 3), and 25(OH)D level < 30 ng/ml. Exclusion criteria were patients who were pregnant, taking supplements containing vitamin D and curcumin, had liver function disorders , and had impaired renal function (GFR < 25 ml/min) and severe infections such as tuberculosis, pneumonia, or HIV.β1. In examination of vitamin D levels Enzyme Immunoassay method was used , in examination of anti-dsDNA levels ELISA was used (Bioluminescenassay), in examination of C3, C4, IL-6, and serum TGF-β1 ELISA was used (Biolegend). Proteinuria was examined by using a urine spot sample with enzymatic-turbidimetric methods. SLE disease activity was assessed using SLEDAI score. Laboratory and SLEDAI examinations were performed at the beginning of the study and the end of the study, except for the serum calcium levels examined each month to determine the side effects of the drug.Fifteen cc of venous blood samples were taken for complete blood tests, liver function (AST/ALT), renal function (urea/creatinine), vitamin D (25(OH)D), calcium, anti-dsDNA, C3, C4, IL-6, and serum TGF- Curcuma xanthorrhiza, and placebo.Patients continue to receive the usual immunosuppressant drugs , as well as calcium, antihypertensive drugs, and other routine medications. A physician in the Rheumatology outpatient clinic who is not a member of the research team in different examination rooms administers regular drugs, cholecalciferol,n (%). The homogeneity test of variance uses Levene. The Shapiro-Wilk test is used to determine the normality of the data, assuming the normality of the data is fulfilled if p > 0.05. Different tests after the treatment in both study groups use a nonpaired/Mann–Whitney test. The influence between variables is tested using Spearman/Pearson correlation test. Data analysis uses Statistical Package for the Social Sciences Software version 22 . Differences and correlations are said to be significant when the value of p is <0.05.The results are presented in mean ± standard deviation, median (IQR 25–75%), and 3 and placebo group and 19 patients in vitamin D3 and Curcuma xanthorrhiza group (1 patient in this group dropped out because she did not take the medication routinely).Subjects who participated in this study were 39 patients and were divided into 2 groups: twenty patients in vitamin Dβ1 and protein-creatinine ratio , whereas, in group II, the average of vitamin D levels increased from 14.3 ± 6.5 ng/ml to 22.7 ± 5.4 ng/ml (p = 0.003). Although serum vitamin D levels differed significantly after the treatment, with higher levels of vitamin D in group I (p = 0.047), the delta of vitamin D levels (the difference between vitamin D levels after supplementation and before supplementation) did not differ significantly between the two groups . Five patients (26.3%) experienced a decrease in serum vitamin D levels in group II. Levels of vitamin D serum after supplementation in this study are still insufficient. p = 0.001), whereas, in group II, the mean SLEDAI score decreased from 14.8 ± 7.9 to 9.2 ± 7.4 (p = 0.003). SLEDAI score after treatment among groups did not differ significantly (p = 0.513). The delta of SLEDAI score reduction after treatment was not significantly different between the two groups . However, the activity of SLE disease in the study subjects after treatment was still moderate activity (SLEDAI score 6–10).β1, and ratio TGF-β1/IL-6 levels in both groups. The delta of decreased serum IL-6 levels and elevated TGF-β1 levels did not differ significantly after the treatment for the two groups (β1/IL-6 was significantly different among the two groups.o groups . Howeverp = 0.001), whereas, in group II, serum IL-6 levels decreased from 6.8 (4.7–15.4) pg/ml to 5.2 (3.2–6.4) (p = 0.013), respectively. Nevertheless, the mean difference (p = 0.061) and the amount of serum IL-6 levels decrease (p = 0.061) after the treatment was not found to be a significant difference. Several SLE patients in the vitamin D3 and Curcuma xanthorrhiza group experienced higher serum IL-6 levels (36.8%) more than in the vitamin D3 and placebo groups (15%), but it did not differ significantly.In group I, mean serum IL-6 levels decreased from 12.7 (6.6–18.1) pg/ml to 3.6 (2.6–4.8) pg/ml (β1 level increased from 289.1 (2488.8–341.9) pg/ml to 355.9 (328.8–427.1) pg/ml , whereas, in group II, mean serum TGF-β1 levels increased from 279.3 (249.4–313.1) pg/ml to 348.4 (292.1–4386.8) pg/ml (p = 0.011). The mean and elevated serum TGF-β1 levels after treatment in both groups did not have a significant difference . Several patients from the vitamin D3 and Curcuma xanthorrhiza group had a decrease of serum TGF-β1 levels after treatment (21.0%), whereas in the vitamin D3 and placebo group only one patient (5%) had a decrease of serum TGF-β1 levels after the treatment.In group I, the mean serum TGF-β1/IL-6 ratio increased from 35.2 ± 22.3 to 78.6 ± 48.7 (p = 0.003), whereas, in group II, the ratio of TGF-β1/IL-6 serum average increased from 30.5 ± 18.1 to 115.6 ± 58.0 (p = 0.000). In group I, there was a significantly greater difference of TGF-β1/IL-6 ratio of 85.1 ± 58.1, compared to group II (43.4 ± 56.2) (p = 0.029). It means that vitamin D can increase TGF-β1 level and decrease the level of IL-6 greater than the combination of vitamin D and Curcuma xanthorrhiza. The greater the ratio of TGF-β1/IL-6 is, the more improved the activity of the disease will be.In group I, the mean serum TGF-r = 0.569, p = 0.000). However, the strength of this correlation is moderate   . (Curcuma xanthorrhiza) 60 mg/day for 3 months was able to significantly increase serum 25(OH)D3 levels, but this increase has not yet resulted in normal levels of 25(OH)D3 serum (25(OH)D3 > 30 ng/ml). This may be due to the fact that patient's initial vitamin D levels were deficient (25(OH)D3 < 20 ng/ml) . Therefore, to achieve normal levels of 25(OH)D3, supplementation should be given over a longer period of 4–6 months or with larger doses, but not exceeding the tolerable upper intake of 50 μg/day equivalent to vitamin D3 2000 IU [3 first in SLE patients with vitamin D deficiency. Van Groningen et al. (2010) mentioned that, in patients with vitamin D deficiency, loading dose cholecalciferol is required to achieve 25(OH)D3 75 nmol/l with the calculation of the required dose (IU) = 40 × (75-serum 25(OH)D3) × body weight [Supplementation of cholecalciferol 1200 IU either with placebo or with curcumin 2000 IU . Or we cy weight . NHS D about 10 ng/ml 25(OH)D [The average increase level of 25(OH)D3 in both groups was approximately 8.4–11.4 ng/ml but slightly lower in the vitamin D 25(OH)D , so that3 and Curcuma xanthorrhiza, some patients (26%) experienced a decrease in serum 25(OH)D3 levels, whereas in the vitamin D3 and placebo groups none of them had a decrease of 25(OH)D serum levels. In the group of patients who had a decrease of serum 25(OH)D3 levels, all of them had LES with mucocutaneous manifestations and used methylprednisolone and chloroquine. Thus, a decrease in serum 25(OH)D3 levels may be due to the fact that the patient tends to avoid direct sun exposure either by limiting day-to-day activity, by applying sunscreen, or by using closed clothing to avoid flares due to photosensitivity.In the group given vitamin D2D3) into calcitriol 2D3) [2D3) into calcitriol 2D3) [The use of corticosteroids in the treatment of SLE also causes vitamin D deficiency because corticosteroids accelerate calcidiol catabolism (25(OH)(OH)2D3) . Chloroq(OH)2D3) . In addi(OH)2D3) , which a2 50,000 IU/week and calcium twice daily [SLEDAI scores in both groups decreased significantly after treatment. This is in accordance with Rifai et al. research in 2013 . Abou-Race daily . However3 and placebo groups (SLEDAI 5.9 ± 5.3) compared to the vitamin D3 and curcumin group (SLEDAI 5.6 ± 6.6), although it was not significant. The improvement of SLEDAI score in vitamin D3 and placebo group was relatively the same compared to previous research conducted by Rifai et al. (2013), which in their previous study obtained a decrease in SLEDAI score on vitamin D3 administration of 6.4 ± 3.1. Some patients did not improve the SLEDAI score in both groups.The improvement of SLEDAI scores was higher in the vitamin DFrom this study, we can conclude that the ability of vitamin D to decrease the score of SLEDAI ranged from 5 to 6. Immunosuppressant is still required, but at least the required dosage becomes smaller and may reduce side effects.The SLEDAI score after the treatment of both groups still showed moderate activity (SLEDAI score 6–10). This may be due to 25(OH)D serum levels which still do not sufficiently achieve (25(OH)D > 30 ng/ml) level. However, in Rifai et al.'s study (2013), although serum 25(OH)D levels have reached normal, SLEDAI scores have not yet achieved remission (SLEDAI score = 0) or mild activity (SLEDAI score 1–5). Disease activity in SLE is influenced by many factors, not only vitamin D status, but also environmental and biological factors . HoweverSerum IL-6 levels in both groups before the treatment were high  . The ser3 and curcumin (Curcuma xanthorrhiza active ingredient) capable of lowering serum IL-6 levels by inhibiting IL-6 production by blocking p38MKP5 [Serum IL-6 levels decreased significantly in both groups after treatment. The decrease of IL-6 is due to the work of vitamin D p38MKP5 , 28.Although vitamin D and curcumin reduced IL-6 levels, there was no significant difference in serum IL-6 levels of serum levels after the treatment in both groups. This may be caused by the fact that the curcumin dosage used was too small or the dose was adequate but the bioavailability was low, or probably due to the competition of curcumin against vitamin D in its bond to VDR so it does not produce a synergistic response . Curcuma xanthorrhiza is only about 1-2%, compared to curcumin obtained from Curcuma longa ranging from 3 to 8% [Bioavailability of curcumin administered per oral route is low, with rapid metabolism and elimination . Curcumi 3 to 8% .−4 M was able to compete against ligand 1,25D tied with VDR as much as 50%. It was concluded that curcumin did not potentiate the action of 1,25D-VDR [−5 M curcumin and 1,25D 10−8 M increased the expression of vitamin D responsive element (VDRE), RXRE, or GRE 2.16 times more than on cells that are given only 1.25D 10−8 M [Bartik et al. (2010) explained that curcumin concentration 10,25D-VDR . The comD 10−8 M .−6 to 10−5 M curcumin plasma concentrations, or the equivalent of 8 grams of curcumin per day) compared to VDR activation by 1,25D ligand so it can be concluded that curcumin is a weaker VDR ligand. Therefore, to improve the bioavailability of curcumin, it is necessary to prepare curcumin dosage forms which are added to certain substances such as piperine or converted into nanoparticles [Although vitamin D and curcumin are able to bind VDRs, the ability of curcumin to bind VDR to alternative pocket (VDR-AP) is greater, whereas vitamin D is more likely to bind VDR in the genomic pocket (VDR-GP) , 32. VDRarticles .γ (PPAR-γ) [γ is controversial. Narala et al. (2009) have conducted a study that curcumin is a ligand of PPAR-γ [γ are probably indirect or via other mechanisms.The anti-inflammatory effect of curcumin results from the activation of peroxisome proliferator-activated receptor-(PPAR-γ) , but thef PPAR-γ . Thus, c Curcuma xanthorrhiza may decrease IL-6 greater than vitamin D only and improves lupus disease activity.The magnitude of the decrease in interleukin-6 levels had an effect on the SLEDAI score after the treatment. The higher serum IL-6 levels of SLE patients will have more severe disease activity . Therefoβ significantly decreased [β1 levels had significant increases between the two groups, but there was no significant difference between serum TGF-β1 levels and magnitude elevated serum TGF-β1 levels after the treatment. This is because the work of curcumin inhibits TGF-β1, in contrast to vitamin D properties against TGF-β1. However, because the dose of curcumin used in this study is small, the effects of curcumin seem to be excluded by vitamin D.The more dominant cytokines in the SLE were IL-6 and IL-23, whereas the levels of TGF-ecreased . Serum Tβ1 in SLE patients. However, curcumin at certain doses and times can inhibit TGF-β1 activity in regulating MMP-9 and activation of SMAD-2, ERK1/2, and p38 in breast cancer cells [Until now, no studies have attempted to investigate the effect of curcumin on TGF-β is not clear yet [β1 levels did not correlate with SLEDAI score.Whether the mechanism of vitamin D will increase levels of TGF-lear yet . In thisIn this study, there were different SLE manifestation, disease activity, and varying degree of vitamin D levels. We did not divide subjects into 3 groups, so that we did not know the effect of curcumin and placebo supplementation towards the SLEDAI score and cytokine levels in SLE patients with hypovitamin D. Also, curcumin plasma concentration was not measured at the study.Curcuma xanthorrhiza supplementation on vitamin D3 in SLE patients with hypovitamin D did not have different effects on levels of SLEDAI, IL-6, and TGF-β1 in serum. Decreased levels of serum IL-6 have moderate correlation with a decrease in SLEDAI score.Preliminary research is needed to assess the amount of plasma concentration of curcumin in the preparation of curcumin to be used. It is also necessary to do research using curcumin with varying doses and formulas with high bioavailability. And research needs to be done with three different groups: curcumin and placebo groups, vitamin D and placebo groups, and a combination of vitamin D and curcumin.


However, the possible functional relationship between these membrane channels and their role in damaged renal cells remain unknown. In the present work, we found that MES-13 cells, from a cell line derived from mesangial cells, stimulated with angiotensin II (AngII) developed oxidative stress  and generated pro-inflammatory cytokines . The membrane permeability increased progressively several hours before the latter outcome, which was a response prevented by Losartan, indicating the involvement of AT1 receptors. Western blot analysis showed that the amount of phosphorylated MYPT (a substrate of RhoA/ROCK) and Cx43 increased progressively and in parallel in cells treated with AngII, a response followed by an increase in the amount in Panx1 and P2X7R. Greater membrane permeability was partially explained by opening of Cx43 hemichannels (Cx43 HCs) and Panx1 channels (Panx1 Chs), as well as P2X7Rs activation by extracellular ATP, which was presumably released via Cx HCs and Panx1 Chs. Additionally, inhibition of RhoA/ROCK blocked the progressive increase in membrane permeability, and the remaining response was explained by the other non-selective channels. The rise of activity in the RhoA/ROCK-dependent pathway, as well as in Cx HCs, P2X7R, and to a minor extent in Panx1 Chs led to higher amounts of TBARS and pro-inflammatory cytokines. We propose that AngII-induced mesangial cell damage could be effectively inhibited by concomitantly inhibiting the RhoA/ROCK-dependent pathway and one or more non-selective channel(s) activated through this pathway.Connexin43 (Cx43), pannexin1 (Panx1) and P2X In chronic kidney disease, hypertension is one of the most common complications that generate predispositions to other health problems, affecting several organs. Hypertensive nephropathy begins in the glomerulus by increasing intraglomerular pressure. These early events activate and damage mesangial cells, epithelial cells and podocytes in the glomerulus. In turn, these cells produce vasoactive and pro-inflammatory agents, which increase cell damage and promote fibrosis, reducing renal blood flow, permeability and, eventually, glomerular filtration .The renin-angiotensin-system is the prototype of a classic systemic endocrine network, whose actions in the kidney and the adrenal gland regulate blood pressure, intravascular volume and electrolyte balance . AngioteAngII interacts with AT1 and AT2 receptors, which are coupled to G-protein-dependent pathways. The AT1 receptor activates small G proteins, including Ras, Rac1, RhoA and Rho kinase (ROCK) , whereasThe RhoA/ROCK pathway has received considerable attention due to its involvement in a wide variety of pathological states . Recent Inflammation is a complex process, and it has recently been related to disorders in intercellular communication mediated by connexin gap junctions and hemichannels   as well Around two dozens of human and rodent Cx isoforms have been identified to date . Each Cx7Rs), which are permeable to cations, including Na+, K+ and Ca2+ [7R leads to Panx1 Ch opening [Extracellular ATP activates P2X7 receptors  and Panx1 have been found in the kidney [7Rs have also been detected in the kidney [7Rs interact, and whether they are regulated by AngII. To our knowledge only a few studies have described the participation of Cx HCs in renal damage [The kidneys contribute significantly to the homeostasis of the whole organism, and to fulfill these functions they require the coordinated action of its different cell types, including vascular and tubular cells. This process is poorly understood, but the involvement of intercellular communication is conceivable through Cx-based channels, Panx Chs and P2Xe kidney ,22,23, ae kidney ,24,25. Pl damage , and no + is a membrane-impermeant cationic dye, which becomes fluorescent when intercalated in nucleic acid strands normally found in the intracellular space [Since Etdar space , we used+ uptake (−7 M) for different times  progressively increased Etd+ fluorescence intensities or Etd+ uptake rates (+ uptake rate increased ~10-fold (from 2.9 ± 1.73 to 32.5 ± 1.30 AU/min). MES-13 cells treated for 72 h with AngII plus Losartan (20 μM)—an AT1 receptor blocker [+ uptake rate (3.0 ± 0.2 AU/min) similar to that of cells under control conditions   C. It waske rates C indicat−7 M). Moreover, and since open Panx1 Chs and P2X7Rs could increase membrane permeability and both are co-expressed in several cell types undergoing inflammatory responses [7R.AngII binding to AT1 receptor activates RhoA and Rho kinase (ROCK) , and Cx4esponses ,31, we a7R were not significantly different at 24 and 48 h, but were significantly increased at 72 h post-AngII treatment (Panx1 from 0.20 ± 0.03 AU at 0 h to 0.60 ± 0.06 at 72 h and P2X7R from 0.24 ± 0.04 AU at 0 h to 0.74 ± 0.10 AU at 72 h) , and increased even more at 48 h (0.65 ± 0.16 AU) and 72 h (1.2 ± 0.2 AU) . Similarat 72 h) B,C.+ uptake in MES-13 cells. After 72 h, AngII-treated cells showed a significantly higher dye uptake rate compared to that of cells under control conditions   (+ uptake rates in cells treated with AngII for 48 h (11.4 ± 1.70 AU/min)  . However.34)  . This su AU/min)  were lik7Rs were also tested. We found a drastic reduction in Etd+ uptake rates in cells treated for 72 h with AngII, followed by 15 min treatment with La3+ (200 µM)—a known Cx HC and P2X7R blocker [7R blocker (3+ (3.50 ± 0.33 AU/min), CBX (3.80 ± 0.30 AU/min) or A740003 (3.90 ± 0.80 AU/min) were like cells under basal conditions (3.20 ± 0.30 AU/min). In contrast, a significant but not complete decrease in fluorescence intensity was observed (16.5 ± 1.30 AU/min) in MES-13 cells treated acutely with the mimetic peptide Gap27 (100 µM), which is a selective Cx43/Cx37 HC blocker [+ uptake rates of cells treated acutely with Fasudil or Y-27632 were about the same as those of cells treated with AngII for 72 h  compared to control cells (99 ± 1%)  A. In add99 ± 1%) B,C, henc−7 M), cells showed a reduced coupling incidence (44 ± 6%) compared to control cells , but Fasudil added the last 24 h to cells treated with AngII reduced the inhibitory effect of AngII on intercellular communication via GJs (incidence of coupling: 93.0 ± 3.4%)  on the functional state of GJs in AngII-treated MES-13 cells. After 72 h treatment with AngII (10 ± 3.4%) A. In add ± 3.4%) B,C as coAn increase in phosphorylation of MYPT was detected in MES-13 cells after 72 h of AngII stimulation (1.4 ± 0.1 AU) compared to control cells (control 0.45 ± 0.09 AU) or control cells treated with Fasudil (0.38 ± 0.10 AU) or Y-27632 (0.35 ± 0.06 AU) A. This s7R in MES-13 cells treated with AngII (10−7 M). An evident increase in the relative amount of Cx43 was found in MES-13 cells after 72 h treatment with AngII (1.0 ± 0.1 AU) compared to control cells (0.18 ± 0.04 AU), or control cells treated with Fasudil (0.17 ± 0.04 AU) or Y-27632 (0.16 ± 0.06 AU). But, Fasudil or Y-27632 added the last 24 h to MES-13 cells pretreated with AngII for 72 h drastically prevented a rise in Cx43 induced by AngII , reaching values close to those found in control cells  and Y-27632 (15 µM) on the amount of Cx43, Panx1 and P2Xol cells B. In conth AngII C,D. Ther7Rs participate in AngII-induced RhoA/ROCK activation, we evaluated the effects of selective blockers for each channel type—Gap27, PBC and A740003, respectively—applied during the last 24 h on the amount of phosphorylated MYPT in MES-13 cells treated with AngII (10−7 M) for 72 h. First, we found that these channel blockers applied for 24 h to cells under control conditions did not alter the amount of phosphorylated MYPT   . Moreove0.06 AU) . Therefo2+ influx as well as Ca2+ release from intracellular stores [7Rs as well as some Cx HCs are Ca2+ conductive channels [−7 M) for 72 h compared to control conditions (3.40 ± 0.40 µmol/L)  A. It was µmol/L) B. Moreov µmol/L) .Interestingly, treatment with Gap27 or A740003 24 h prior to the end of the 72 h post-treatment with AngII significantly reduced the amount of TBARS (Gap27: 3.04 ± 0.22 µmol/L and A740003: 3.70 ± 0.40 µmol/L). However, treatment with PBC caused only a partial reduction in the amount of TBARS (8.03 ± 0.82 µmol/L) A. Moreov7R) mainly through a RhoA/ROCK-dependent intracellular signaling pathway promoted by the activation of AT1 receptors. The generation of TBARS and pro-inflammatory cytokines was strongly dependent on the upregulation of functional Cx HCs as well as P2X7Rs, and to a lesser but still significant extent on Panx1 Chs  increases in amounts of Cx43, Panx1 and P2X7R; and (2) increases in the open probability of Cx43 HCs and Panx1 Chs. In favor of the role played by Cx HCs and Panx1 Chs in increasing AngII-induced membrane permeability is the fact that these two channels are permeable to Etd+ [The half-life of bioactive peptides such as AngII is likely to be about a few hours , and thentration ,41 as wentration , and thentration  and Panxntration ,49. Inte to Ca2+ ,43, it imembrane . In favo atrophy . Thus, o to Etd+ . Additio to Etd+ ,51. Inte to Etd+ ,24,25,52 to Etd+ . A simil to Etd+ . In agre to Etd+ ,55. Desp7Rs activated by extracellular ATP; and (3) PBC or low concentration of CBX—inhibitors of Panx1 Chs—and Gap27, inhibitor of Cx43 HCs, which are blockers of two membrane pathways through which ATP can be released to the extracellular milieu  apyrase that degrades ATP; (2) A740003, which is a selective inhibitor of P2Xr milieu .The AngII-induced generation of OS, IL-1β and TNF-α could explain decreases in intercellular communication mediated by GJs, since reduced redox potential and pro-inflammatory cytokines have also been shown to cause such effect in cortical astrocytes and in endothelial cells of the blood-brain barrier ,56,57,587Rs in different tissues undergoing inflammation has been demonstrated, and the inhibition or ablation of Cx HCs has been shown to confer significant tissue protection [The participation of Cx HCs, Panx1 Chs and P2Xotection ,61,62,63otection ), mainta+) bromide, lanthanum (La3+) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich ; Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA ; A740003—a specific P2X7Rblocker—was obtained from Tocris Biosciences . The monoclonal anti-α-tubulin antibody was obtained from Sigma-Aldrich ; the polyclonal anti-phosphorilated-MYPT1 (Thr696) antibody was obtained from Merck Millipore ; the monoclonal anti-MYPT1 antibody was obtained from BD Transduction Laboratories ; the monoclonal anti-unphosphorylated Cx43 antibody was obtained from Invitrogen ; a polyclonal anti-Panx1 antibody described previously [7R antibody was obtained from Abcam ; anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz Biotechnology Inc. .Angiotensin II (AngII) was obtained from Alomone ; Fasudil, Y-27632, ethidium (Etdeviously  was used−7 M) for different time periods  in a 2:1 mixture of DMEM and F-12 tissue culture media supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were kept at 37 °C in a 5% CO2/95% air atmosphere, at nearly 100% relative humidity. Fasudil (15 μM) or Y-27632 (15 μM) were added during the last 24 h to cell cultures treated with AngII for 72 h.The cell line MES-13, derived from mesangial cells , was cultured and treated with AngII  containing 5 μM Etd+, which is a membrane-impermeant cationic dye. In time-lapse experiments images were recorded (at regions of interest in different cells) every 30 s during 13 min using a Nikon Eclipse Ti inverted microscope  and NIS-Elements software. Basal fluorescence signal was recorded in cells only in the presence of Locke’s saline solution that contained divalent cations.Cx HC activity was evaluated by using the dye uptake method previously published . In brie+ (25 mM) was microinjected through a glass microelectrode to one cell. Dye transfer to neighboring cells was evaluated two minutes after injection. We routinely performed all dye coupling experiments in the presence of La3+ (150 µM) to block the Cx HCs and prevent Etd+ leakage through open hemichannels that would reduce the intercellular diffusion among coupled cells [MES-13 cells seeded on glass coverslips were bathed with Locke’s saline solution, and cultures were observed using an inverted microscope equipped with a xenon arc lamp and a Nikon B filter . Etded cells . The incg, Eppendorf Centrifuge 5415C, Hamburg, Germany), and supernatants were collected for Western blot analysis. Protein concentration was determined by using the Lowry’s method [w/v) and 1% BSA (w/v). Then, the PVDF membrane was incubated overnight at 4 °C with anti-Cx43 (1:1000), anti-p-MYPT1 (1:500), anti-MYPT1 (1:1000), anti-Panx1 (1:1000) or anti-P2X7R (1:1000) antibody, followed by incubation with rabbit or mouse secondary antibody conjugated to peroxidase (1:2000 both of them) for 1 h at room temperature. Then, the PVDF membrane was stripped and reblotted with the anti-α-tubulin antibody (1:5000) used as loading control, following the same procedure described above. After repeated rinses, immunoreactive proteins were detected by using ECL reagents  according to the manufacturer’s instructions. The bands detected were digitized and subjected to densitometry analysis using the software Image J .Cell cultures were placed on ice, washed twice with ice-cold PBS (pH 7.4), harvested by scraping in 80 μL of a solution containing a protease and phosphatase inhibitor cocktail . Lysates were centrifuged , thiobarbituric acid (0.8% TBA w/v), and acetic acid (20% v/v), and heated for 60 min at 90 °C. Precipitated material was removed by centrifugation, and the absorbance of the supernatant was evaluated at 532 nm. The amount of TBARS was calculated using a calibration curve obtained with malondialdehyde (MDA) as standard. MDA was obtained from Merck .The amount of TBARS was estimated using the method described by Ramanathan and collaborators  with sliIL-1β and TNF-α ELISA assays were performed to determine the amount of IL-1β and TNF-α in the extracellular medium under different conditions, following the manufacturer’s protocol . Results were normalized by protein amount in ng/mL.p < 0.05. Analyses were performed with the GraphPad Prism 5 software for Windows .Results were evaluated by ANOVA, and the Tuckey’s post-test was used to evaluate the difference between the two groups. Results below are expressed as the average of values from each independent experiment ± SE, and considered significantly different if


This Phase 3, open-label, rollover study (NCT01323244) investigated the efficacy and safety of simeprevir plus peginterferon α-2a (PegIFNα-2a) and ribavirin (RBV) in a well-characterized population of HCV genotype 1 (GT1)-infected treatment-experienced patients.n = 125), or had been exposed to HCV direct-acting antivirals (simeprevir or other) for up to 14 days in a selected Phase 1 study , were eligible. Phase 2/3 group patients were classified according to prior relapse, breakthrough, or non-response  to PegIFNα/RBV. Eight patients in the Phase 1 group received short-term (≤14 days) simeprevir.Patients who had failed PegIFNα/RBV treatment in the placebo arm of a previous Phase 2/3 simeprevir study  plus PegIFNα-2a/RBV for 12 weeks followed by PegIFNα-2a/RBV for 12 or 36 weeks  or 36 weeks fixed (Phase 2/3 group non-responders and Phase 1 group). The primary endpoint was sustained virologic response 12 weeks after planned end of treatment (SVR12).Phase 2/3 group: SVR12 rate was 69.6% (87/125) overall; 92.7% (51/55), 60.0% (6/10), 64.3% (18/28), and 36.7% (11/30) in patients with prior relapse, viral breakthrough, partial response, or null response, respectively. SVR12 rates were similar for patients with HCV GT1a (66.0% [33/50]) and GT1b infection (72.0% [54/75]) and among HCV GT1a-infected patients with/without a baseline Q80K polymorphism . The majority of RGT-eligible patients  met RGT criteria (89.2% [58/65]); of these, 89.7% (52/58) achieved SVR12. Overall, 16.0% (20/125) of patients experienced on-treatment failure and 14.4% (18/125) experienced post-treatment failure . Phase 1 group (simeprevir-naïve and -experienced patients combined): SVR12 rate was 37.5% (6/16). Safety and tolerability findings were comparable to those of the feeder studies.The majority of RGT-eligible patients met criteria for shortening treatment to 24 weeks in total. Simeprevir 150 mg QD with PegIFNα-2a/RBV led to a high SVR rate among prior relapsers with HCV GT1 infection. No new safety signals were noted.NCT01323244. .The online version of this article (doi:10.1186/s12879-017-2444-3) contains supplementary material, which is available to authorized users. Until 2011, the standard of care for chronic hepatitis C virus (HCV) was peginterferon-α (PegIFNα) with ribavirin (RBV). This combination led to sustained virologic response (SVR) rates of 38–66% in treatment-naïve patients infected with HCV genotype 1 (GT1) , 5.Direct-acting antiviral agents (DAAs) have significantly improved treatment options for chronic HCV infection; firstly, in combination with PegIFN and RBV and, more recently, as IFN-free DAA combination regimens. Simeprevir, an oral, once-daily (QD) HCV NS3/4A protease inhibitor with antiviral activity against HCV GT 1, 2, 4, 5, and 6 , is apprPrior to the availability of IFN-free therapies, in two Phase 2 studies, SVR rates of 81–86% and 67–80%, respectively, were achieved in treatment-naïve and treatment-experienced patients  with HCV GT1 infection treated with simeprevir plus PegIFNα/RBV (PILLAR ). In thrHere we report the results of a Phase 3 study that provided access to simeprevir in combination with PegIFNα-2a/RBV to HCV GT1-infected patients who had failed PegIFNα/RBV in the placebo group of one of the Phase 2/3 simeprevir studies mentioned above. In addition, a group of 16 patients who had been exposed to a DAA for up to 14 days in a Phase 1 study were included. This allowed evaluation of the efficacy and safety of simeprevir in a population that was well characterized in terms of prior response to PegIFN/RBV therapy. IFN-based treatment is still the standard of care in certain countries where IFN-free regimens are not yet available. The findings of this study may be of interest to healthcare professionals in such settings.OR had been exposed to a DAA for up to 14 days in a selected Phase 1 study (Phase 1 group) were enrolled. All patients were required to have completed the last study-related assessment in their previous study.Patients with HCV GT1 infection who had failed PegIFNα/RBV in the placebo arm of a Phase 2/3 simeprevir study (Phase 2/3 group: did not achieve undetectable HCV RNA levels at the end of treatment [EOT] or had relapsed [confirmed detectable HCV RNA] within 1 year after EOT) Patients in the Phase 2/3 group were recruited from two Phase 2 studies  based on their response to PegIFNα-2b/RBV in the feeder study.Patients in the Phase 1 group were recruited from the TMC649128HPC1002 Phase 1 study , in which patients had received an investigational nucleoside analogue NS5B inhibitor TMC649128 for 10 or 14 days, and from the TMC647055HPC1001 study (NCT01202825 [Cohort 8]), in which patients received the combination of simeprevir with an investigational non-nucleoside analogue NS5B inhibitor TMC647055 for 10 days .Key exclusion criteria included: hepatic decompensation, newly diagnosed liver disease of non-HCV etiology, hepatocellular carcinoma, co-infection with HCV non-GT1, HIV, or hepatitis B virus (hepatitis B surface antigen positive), any medical condition that contraindicated the use of PegIFNα or RBV, history of malignancy within 5 years of the screening visit, and previous treatment with any HCV DAA therapy, other than short-term (≤14 days) DAA treatment in the Phase 1 study. All patients provided written informed consent.This was a Phase 3, open-label, multicenter study . The study protocol and amendment(s) were reviewed by the Independent Ethics Committees and Institutional Review Boards at each study center. The study was conducted in accordance with the Declaration of Helsinki, and was consistent with Good Clinical Practice and applicable regulatory requirements.The study comprised a screening period (≤6 weeks), a 24- or 48-week treatment period, and a follow-up period of 24 weeks after the planned EOT. Patients in the Phase 2/3 group with previous relapse or breakthrough received simeprevir  for 12 weeks in combination with PegIFNα-2a  and RBV  followed by PegIFNα-2a/RBV for 12 or 36 weeks Fig. . The criPatients in the Phase 2/3 group who were classified as non-responders, and those in the Phase 1 group, received simeprevir 150 mg plus PegIFNα-2a/RBV for 12 weeks, followed by 36 weeks of PegIFNα-2a/RBV Fig. .Dose adjustments or treatment interruptions were permitted for PegIFNα-2a/RBV in response to AEs or tolerability issues; no dose adjustments of simeprevir were permitted. A single interruption (≤4 days) in simeprevir treatment was allowed in response to AEs.In accordance with the virologic stopping rules, all study medication was discontinued if HCV RNA plasma level was >1000 IU/ml at Week 4 or 12, or when HCV RNA was confirmed detectable at Week 24 or 36 . Patients enrolled under a later protocol amendment permanently discontinued all study medication when HCV RNA was ≥25 IU/ml at Week 4 or when HCV RNA was confirmed detectable at Week 12, 24, or 36.The primary efficacy endpoint was SVR12 .10 IU/ml increase in HCV RNA from the lowest level reached, or HCV RNA >100 IU/ml when previously <25 IU/ml); viral relapse (undetectable HCV RNA at EOT and HCV RNA ≥25 IU/ml during follow-up); normalized alanine aminotransferase (ALT) at EOT and SVR time points; and AEs and laboratory abnormalities.Secondary endpoints included SVR 24 weeks after the planned EOT (SVR24); undetectable HCV RNA (<25 IU/ml undetectable) and HCV RNA <25 IU/ml detectable at Weeks 4, 12, 24, 36, and 48; on-treatment failure (HCV RNA <25 IU/ml detectable or ≥25 IU/ml at EOT); viral breakthrough .IL28B; single nucleotide polymorphism rs12979860) status of the patient was unknown at the start of the study, IL28B genotyping was performed on blood samples obtained at screening.Standard population sequencing to assess for simeprevir resistance-associated substitutions in the HCV NS3/4A protease domain was performed at baseline for all patients and post-baseline for patients not achieving SVR. If the interleukin-28b  .Statistical analyses were performed using SAS® version 9.2. All analyses were performed on the intent-to-treat (ITT) population . No formal sample size determination was performed. It was calculated that the placebo groups in the previous Phase 2/3 studies could potentially provide 539 patients. Assuming that 50% of patients from the Phase 2/3 studies would fail treatment for virologic reasons, it was estimated that 270 patients would roll over into the current study. With this sample size, and an expected response rate of 50%, the width of the 95% confidence interval (CI) around the anticipated response was 11.9%. For a sample size of 135 patients, the width of the 95% CI was 16.9%. HCV GT1-infected patients who had received short-term (≤14 days) DAA therapy in the Phase 1 study were also considered for inclusion.Data from the Phase 2/3 group were analyzed overall and by subgroup according to treatment response in the feeder study. Safety and tolerability were evaluated for the overall population.IL28B genotype , HCV geno/subtype  and response to prior PegIFN/RBV treatment.The 95% CI was calculated for the proportion of patients achieving SVR12 based on the normal approximation. In addition, a logistic regression model was used to determine the proportion of patients achieving SVR12 and 95% CI in the Phase 2/3 group. This model was adjusted for baseline HCV RNA level, 10 HCV RNA levels from baseline at all time points.For all other response parameters in the Phase 2/3 group, 95% CIs were constructed around the observed values and the same logistic regression model as applied for the primary efficacy parameter was used. The time to achieve undetectable HCV RNA was estimated using Kaplan-Meier plots. Descriptive statistics were calculated for the change in logIL28B genotype and response in the feeder study.For the Phase 2/3 group, subgroup analyses were performed based on baseline HCV RNA levels, early viral response criteria, and baseline characteristics of race and age to investigate the possible effect on SVR12. In addition, a multivariate model applying cubic spline functions for continuous factors was fitted on the SVR12 data exploring the combined effect of several factors. Subgroup analyses were also performed based on the Due to the limited number of patients in the Phase 1 group, data were assessed descriptively.The study was conducted at 75 centers in 22 countries between December 5, 2011 and March 31, 2015. Baseline demographic and clinical characteristics are summarized in Table In total, 125 patients in the Phase 2/3 group received simeprevir treatment in the current study and represented the ITT population. The study was completed by 92.0% (115/125) of patients; the main reasons for study discontinuation were withdrawal of consent and lost to follow-up (both 4.0%). Thirteen patients discontinued simeprevir early . In total, 79.2% (99/125) completed treatment with PegIFNα-2a and/or RBV (24 or 48 weeks). Median simeprevir treatment duration was 12.0 weeks. Median PegIFNα-2a/RBV treatment duration was 24.1 weeks.Categorization according to prior treatment response was as follows: viral relapsers , viral breakthrough , or non-responder .– other than the Q80K polymorphism – were observed in 1 patient carrying D168E at baseline.The proportion of patients with baseline NS3 polymorphisms was 29.6% [37/125]. NS3 polymorphism Q80K was observed in 24.0% (12/50) of HCV GT1a-infected patients. None of the HCV GT1b-infected patients had a Q80K polymorphism. NS3 polymorphisms reducing simeprevir activity in vitro Sixteen patients in the Phase 1 group received simeprevir treatment in the current study and represented the Phase 1 ITT population. The study was completed by 75.0% (12/16) of patients; the main reasons for study discontinuation were withdrawal of consent (18.8%) and lost to follow-up (6.3%). One patient discontinued simeprevir early due to withdrawal of consent (6.3% [1/16]). In total, 56.3% (9/16) of patients in the Phase 1 group completed treatment with PegIFNα-2a and/or RBV (24 or 48 weeks). Median simeprevir treatment duration was 12.1 weeks. Median PegIFNα-2a/RBV treatment duration was 48.1 weeks.Fifty-percent of patients (8/16) were simeprevir treatment-naïve and 50.0% (8/16) were simeprevir treatment-experienced. Of the 8 patients who had not been previously exposed to simeprevir in a Phase 1 study, 6 were prior non-responders to PegIFN/RBV treatment, 1 was a prior relapser to PegIFN/RBV, and 1 was treatment-naïve.The proportion of patients with baseline NS3 polymorphisms was 50.0% (8/16). NS3 polymorphism Q80K was observed in 21.4% (3/14) of HCV GT1a-infected patients. None of the HCV GT1b-infected patients had a Q80K polymorphism. Among the 8 patients in the Phase 1 group with prior short-term exposure to simeprevir, 6 had an emerging R155K mutation that was still detected in 2 patients at baseline in the current study.The SVR12 rate was 69.6% . When patients were classified according to prior PegIFN/RBV response, the highest SVR12 rate was seen for prior relapsers (92.7% [51/55]). The SVR12 rate for prior viral breakthrough patients was 60.0% (6/10). For prior non-responders – including partial responders, null responders, and non-classifiable non-responders – SVR12 rates were 64.3% (18/28), 36.7% (11/30), and 50.0% (1/2), respectively. In the Phase 2/3 group, RGT criteria were met by the majority (89.2% [58/65]) of RGT-eligible patients. Of these, 89.7% (52/58) achieved SVR12.All patients achieving SVR12 also achieved SVR24; 94.6% (87/92) of patients achieving SVR4 achieved SVR12. Considering both groups of prior non-responder patients together , the SVR12 rate was 81.1% (73/90) in patients with rapid virologic response .SVR12 rates according to baseline characteristics are summarized in Table In the multivariate analysis that considered all factors, baseline homeostatic model assessment of insulin resistance (HOMA-IR), response to prior PegIFN/RBV treatment, complete early virologic response (<25 IU/ml undetectable at Week 12), and HCV RNA <25 IU/ml at Week 4 were the characteristics identified as having a statistically significant effect on the probability of achieving SVR12 , METAVIR F0–F2 (vs. F3–F4), and for patients with HOMA-IR <2 (vs. HOMA-IR ≥2 to ≤4 or >4)  of patients in the Phase 2/3 group (of which 10/12 patients had been PegIFN/RBV null responders in the feeder study). None of the patients with viral breakthrough achieved SVR12. All but 1 of the patients with viral breakthrough met a virologic stopping rule; these comprised 7 patients at Week 4, and 4 patients at Weeks 12, 24, or 36. The proportion of patients with on-treatment failure was 16.0% (20/125) (Table Post-treatment failure was observed in 14.4% (18/125) of patients, with the majority of patients experiencing viral relapse (12.0% [15/125]). Missing data at the SVR12 assessment time point was the reason for post-treatment failure in 2.4% (3/125) patients. Fifteen of 104 patients (14.4%) with undetectable HCV RNA at EOT had a viral relapse. In all patients with virologic failure, available sequencing data did not suggest any cases of reinfection.Twenty-seven of 33 patients (81.8%) with treatment failure and available sequencing data had emerging substitutions at NS3 amino acid positions 80, 122, 155, and/or 168 at the time of failure (Table The majority of patients (71.2% [42/59]) had normalization of their ALT during treatment. Median time to normalization of ALT was 8.0 (95% CI 2.0–24.1) weeks.The SVR12 rate was 37.5% , and no difference in SVR12 rate was observed between simeprevir-naïve and experienced patients (both 3/8 [37.5%]) ). Missing data at the SVR12 assessment time point was the reason for post-treatment failure in 2/16 (12.5%) of patients. Five of 12 patients (41.7%) with undetectable HCV RNA at EOT had a viral relapse. In all patients with virologic failure, available sequencing data did not suggest any cases of reinfection.Seven of 8 patients 87.5%) with treatment failure and sequencing data available had emerging substitutions at NS3 amino acid positions 80, 122, 155, and/or 168 at the time of failure (Table % with trThe majority of patients (5/6 [83.3%]) had normalization of their ALT during treatment. Median time to normalization of ALT was 24.1 (95% CI 2.1–36.1) weeks.The majority (90.8%) of patients experienced at least one AE during the simeprevir plus PegIFNα-2a/RBV treatment phase. The most common AEs (>20% of patients) were fatigue 32.6%), neutropenia (22.7%), and influenza-like illness (22.0%). The majority of AEs were Grade 1 or 2 in severity; Grade 3 or 4 AEs were reported in 29.1% of patients and were considered at least possibly related to simeprevir in 6.4% of patients , pruritus, anemia, photosensitivity conditions, neutropenia, and dyspnea Table . The majNone of the AEs of special/clinical interest were serious and only a small number resulted in permanent discontinuation of study treatment: Grade 3 hyperbilirubinemia (increased bilirubin AE) led to permanent discontinuation of simeprevir in 1 patient (0.7%) (protocol-driven) and a Grade 3 photosensitivity reaction  led to permanent discontinuation of simeprevir, PegIFNα-2a, and RBV in 1 patient . The patient with Grade 3 hyperbilirubinemia, considered probably related to simeprevir, also experienced Grade 1 rash, Grade 3 pruritus, Grade 2 ALT, and Grade 1 aspartate aminotransferase (AST) AEs. The Grade 3 photosensitivity reaction which led to discontinuation of simeprevir, PegIFNα-2a, and RBV was considered to be very likely related to simeprevir and was subsequently reported as resolved following treatment discontinuation.The most frequently noted laboratory parameter abnormalities (>25% of patients) during the simeprevir plus PegIFNα-2a/RBV treatment phase for which Grade ≥3 was noted included decreases in neutrophil and precursor count , hyperbilirubinemia , and decreases in platelet count . Increases in direct and indirect bilirubin levels above normal limits (any grade) were reported in 46.4% and 30.7% of patients, respectively. Decreases in neutrophil and precursor count were the only graded treatment-emergent laboratory abnormalities for which Grade 3 or 4 abnormalities were observed in >10% of patients (19.3%). Grade 3 hyperbilirubinemia was observed in 9 (6.4%) patients and Grade 4 hyperbilirubinemia in 1 (0.7%) patient.Grade 3 ALT or AST AEs were experienced by 2.1% (3/141) of patients during the simeprevir plus PegIFNα-2a/RBV treatment phase . Grade 3 ALT and AST events were not considered to be related to the study drug. No Grade 4 ALT/AST AEs were reported during treatment with simeprevir.This open-label study was designed to provide access to simeprevir treatment in combination with PegIFNα-2a and RBV for a well characterized treatment-experienced patient population with HCV GT1 infection in terms of baseline characteristics and prior response to PegIFN/RBV, after they had completed participation in a prior study and at a time when current therapeutic IFN-free alternatives were not yet available.The Phase 2/3 group comprised a high proportion of prior relapsers (44.0%); null responders and partial responders (each approximately 25%) to PegIFNα/RBV. Fewer than 10% of patients had experienced viral breakthrough. The SVR12 rate (primary efficacy endpoint) was 69.6% (87/125); all patients who achieved SVR12 also went on to achieve SVR24. The majority (89.2%) of patients eligible for RGT met RGT criteria and received a shorter 24-week duration of PegIFNα-2a/RBV therapy. The SVR12 rate in this group of patients was 89.7%. There was a small difference in the number of patients (50/51 [98.0%]) who qualified for shorter treatment duration using the original RGT as compared to those who qualified for shorter treatment duration with the modified criteria in the same group of patients (45/51 [88.2%]) retrospectively. SVR12 was achieved in 45 of 50 (90%) patients who qualified for shorter treatment duration with the original RGT versus 41 of the 45 (91.1%) patients from the same group who qualified for shorter treatment with the modified RGT retrospectively.The virologic response for non-responders in the Phase 2/3 group (50%) was comparable to the overall SVR12 rate reported with simeprevir plus PegIFNα-2a/RBV (54%) and telaprevir plus PegIFNα-2a/RBV (55%) in a previous non-responder HCV GT1-infected population in the ATTAIN study .While the HCV geno/subtype influences response to HCV therapy and higher rates of virologic failure are seen in GT1a-infected patients compared with GT1b , the SVRPhase 3 studies evaluating the combination of simeprevir with PegIFN/RBV indicated lower SVR rates in HCV GT1a-infected patients with a Q80K baseline polymorphism compared to those without Q80K polymorphisms –14. In tThe proportion of patients with on-treatment failure in the Phase 2/3 group was 16.0%. The incidence of viral breakthrough was <10% and 10/12 patients with viral breakthrough were prior null responders to PegIFN/RBV treatment. Consistent with the simeprevir resistance profile observed in HCV GT1-infected patients who failed simeprevir plus PegIFNα/RBV in Phase 2/3 studies , the majThis observation raises the possibility that patients who fail simeprevir plus PegIFNα/RBV could be retreated with a simeprevir-containing regimen. However, the rapid development of IFN-free combination DAA regimens has provided more effective retreatment options, which are recommended by current treatment guidelines , 20.IL28B rs12979860 CC genotype [Consistent with the established association between improved response (SVR) to PegIFN/RBV and presence of the genotype , 22, a hgenotype .The tolerability profile of simeprevir in the current study was comparable to that reported in the previous Phase 2/3 studies investigating simeprevir in combination with PegIFNα-2a/RBV in patients with HCV GT1 infection , 12–14. In the Phase 1 group, half of the patients (8/16) had not been previously exposed to simeprevir in a Phase 1 study and, of these, the majority  were prior non-responders to PegIFN/RBV treatment. The SVR12 rate was 37.5% (6/16); all patients who achieved SVR12 also went on to achieve SVR24.Equal proportions of patients with and without prior exposure to simeprevir achieved SVR12 (3/8 patients each). Among the 8 subjects who were not previously exposed to simeprevir, 2 of the 6 prior non-responders to PegIFN/RBV treatment achieved SVR12, one prior relapser achieved SVR12, and one treatment-naïve patient did not achieve SVR12. Patients with prior simeprevir exposure were treatment-naïve or prior relapsers to PegIFN/RBV. Given the small sample size in these subgroups, it is difficult to draw conclusions for a comparison of the SVR rates to historic controls.IL28B CC genotype. Among the group of 8 patients in the Phase 1 group who had previously been exposed to simeprevir, 2 subjects discontinued treatment early due to AEs. One of these 8 simeprevir-experienced patients had the favorable IL28B CC genotype and this patient achieved SVR12. Of note, the R155K substitution was found emerging in 6/8 patients during previous short-term exposure to simeprevir in the Phase 1 study and was still detected in 2 patients using population sequencing at baseline in the current study but may have still been present as minority substitution in other patients at baseline. The presence of this substitution could have also impacted the treatment response in this group of simeprevir-experienced patients.Multiple factors may have contributed to the lower virologic response rate in the Phase 1 group. Missing data at the SVR12 assessment time point was the reason for post-treatment failure in 12.5% of subjects (2/16) in the Phase 1 group. In addition, among the group of 8 patients in the Phase 1 group who had not previously been exposed to simeprevir, 1 patient (prior non-responder) discontinued treatment early at Week 36 due to AEs and 2 patients discontinued treatment early due to consent withdrawal (one prior non-responder on Day 5 and one simeprevir-naïve patient at Week 16). None of these 8 simeprevir-naïve patients had the favorable baseline The proportion of patients with on-treatment failure (18.8%) was similar to that of the Phase 2/3 group. The incidence of viral breakthrough was <10% and 10/12 patients with viral breakthrough were prior null responders to PegIFN/RBV treatment.n = 16). In addition, when the Phase 2/3 group was analyzed according to response to prior treatment, patient numbers were relatively low in some of the groups . Similar limitations in terms of patient numbers also applied to other subgroups, for example, grouping according to IL28B genotype.A limitation of the study was the small sample size in the Phase 1 group . (DOCX 22 kb)Additional file 2:List of ethics committees. (DOCX 67 kb)