Prediction of the Impact of CYP2C19 Polymorphism on Drug-Drug Interaction between Voriconazole and Tacrolimus Using Physiologically-Based Pharmacokinetic Modelling

Abstract Voriconazole increases tacrolimus blood concentration significantly when coadministrated. The recommendation of reducing tacrolimus to 1/3 in voriconazole package insert seems not to be satisfactory in clinical practice. In vitro studies demonstrated that the magnitude of inhibition depends on the concentration of voriconazole, while voriconazole exposure is determined by the genotype status of CYP2C19. CYP2C19 gene polymorphism challenges the management of drug-drug interactions(DDIs) between voriconazole and tacrolimus. This work aimed to predict the impact of CYP2C19 polymorphism on the DDIs by using physiologically based pharmacokinetics (PBPK) models. The precision of the developed voriconazole and tacrolimus models was reasonable by evaluating the pharmacokinetic parameters fold error, such as AUC0-24, Cmax and tmax. Voriconazole increased tacrolimus concentration immediately in all population. The simulated duration of DDIs disappearance after voriconazole withdrawal were 146h, 90h and 66h in poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers(EMs), respectively. The developed and optimized PBPK models in this study can be applied to assit the dose adjustment for tacrolimus with and without voriconazole.

Crystal structures and properties of two hydrated conglomerate forms of the heart-rate-lowering agent ivabradine hydrochloride.

Ivabradine hydrochloride (IVA-HCl) (systematic name: {[3,4-dimethoxybicyclo[4.2.0]octa-1(6),2,4-trien-7-yl]methyl}[3-(7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-3-yl)propyl]methylazanium), is a novel medication used for the symptomatic management of stable angina pectoris. In many recent patents, it has been claimed to exist in a very large number of polymorphic, hydrated and solvated phases, although no detailed analysis of the structural features of these forms has been published to date. Here, we have successfully crystallized the tetrahydrate form of IVA-HCl (form ß), C27H37N2O5+·Cl-·4H2O, and elucidated its structure for the first time. Simultaneously, a new crystal form of IVA-HCl, i.e. the hemihydrate (form II), C27H37N2O5+·Cl-·0.5H2O, was discovered. Its crystal structure was also accurately determined and compared to that of the tetrahydrate form. While the tetrahydrate form of IVA-HCl crystallized in the orthorhombic space group P212121, the new form (hemihydrate) was solved in the monoclinic space group P21. Detailed conformational and packing comparisons between the two forms have allowed us to understand the role of water in the crystal assembly of this hydrochloride salt. The stabilities of the two forms were compared theoretically by calculating the binding energy of the water in the crystal lattice using differential scanning calorimetry (DSC). The stability experiments show that the tetrahydrate is stable under high-humidity conditions, while the hemihydrate is stable under high-temperature conditions.

The Macronova in GRB 050709 and the GRB-macronova connection.

GRB 050709 was the first short Gamma-ray Burst (sGRB) with an identified optical counterpart. Here we report a reanalysis of the publicly available data of this event and the discovery of a Li-Paczynski macronova/kilonova that dominates the optical/infrared signal at t>2.5 days. Such a signal would arise from 0.05 r-process material launched by a compact binary merger. The implied mass ejection supports the suggestion that compact binary mergers are significant and possibly main sites of heavy r-process nucleosynthesis. Furthermore, we have reanalysed all afterglow data from nearby short and hybrid GRBs (shGRBs). A statistical study of shGRB/macronova connection reveals that macronova may have taken place in all these GRBs, although the fraction as low as 0.18 cannot be ruled out. The identification of two of the three macronova candidates in the I-band implies a more promising detection prospect for ground-based surveys.

A possible macronova in the late afterglow of the long-short burst GRB 060614.

Long-duration (>2 s) γ-ray bursts that are believed to originate from the death of massive stars are expected to be accompanied by supernovae. GRB 060614, that lasted 102 s, lacks a supernova-like emission down to very stringent limits and its physical origin is still debated. Here we report the discovery of near-infrared bump that is significantly above the regular decaying afterglow. This red bump is inconsistent with even the weakest known supernova. However, it can arise from a Li-Paczynski macronova--the radioactive decay of debris following a compact binary merger. If this interpretation is correct, GRB 060614 arose from a compact binary merger rather than from the death of a massive star and it was a site of a significant production of heavy r-process elements. The significant ejected mass favours a black hole-neutron star merger but a double neutron star merger cannot be ruled out.

A new series of macrolide derivatives with 4''-O-saccharide substituents.

A series of novel derivatives of macrolide with 4''-O-mono- or disaccharides were synthesized. The corresponding glycosyl trichloroacetimidates were used as the donors in the glycosylations. The in vitro antibacterial activities of 7a-f and 13-16 against a panel of susceptible and resistant pathogens were tested. The modification of 4''-O-mono- or disaccharides may lead to the understanding of interaction of the macrolide and the bacterial ribosome.

Synthesis of novel macrolide derivatives with imidazo[4,5-b]pyridinyl sulfur contained alkyl side chains and their antibacterial activity.

In an effort to find new antibiotics, a novel series of 14-membered macrolides with imidazo[4,5-b]pyridinyl sulfur contained alkyl side chains has been synthesized based on commercially available clarithromycin. Chemical transformation of hydroxy group at position C-3 afforded range of ketolides and acylides. Compared to telithromycin, compound 15a demonstrated improved in vitro activity against erythromycin-susceptible and -resistant strains.

Synthesis and antibacterial activity of 4''-O-heteroarylcarbamoyl derivatives of macrolide.

A series of novel 4''-position modified macrolide derivatives has been synthesized via a facile procedure. Their in vitro antibacterial activities against constitutively erythromycin-resistant strains were evaluated. Among the derivatives tested, compound 8a which has 11,12-carbamate and 4''-O-heteroarylcarbamoyl groups was found to have potent activity against most resistant bacteria.

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin.

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.

[Effects of methyl jasmonate and salicylic acid on phenylethanoid glycosides synthesis in suspension cultures of Cistanche deserticola].

The present study investigated the influence of the methyl jasmonate and salicylic acid elicitors on the formation of phenylethanoid glycosides (PeG) in the suspension cultures of Cistanche deserticola. The results showed that methyl jasmonate and salicylic acid enhanced greatly the accumulation of PeG and echinacoside (Echin), but their optimum elicitation dosage and addition time were different. The yields of PeG and Echin were significantly increased in the presence of 5 micromol/L methyl jasmonate on day 14 (up to 2.59-fold and 3.82-fold, respectively), whereas treated with 50 micromol/L salicylic acid on day 28, the maximum content of them were, respectively, 2.71 and 3.16-fold higher than the untreated cell cultures.

[Effects of physical and chemical factors on hairy root growth and flavonoids biosynthesis in the cultures of Saussurea medusa Maxim hairy root].

The effects of different physical and chemical factors on hairy root growth and flavonoids production were studied in suspension culture of Saussurea medusa hairy root in 1/2 MS medium. The results showed that the following culture conditions, nitrogen concentratiaon (involved NH4+ and NO3-), 30 mmol/L; the ratio of ammonium to nitrate, 5:25; the combination of 2% sucrose and 3% glucose; 0.5 mg/L GA3; 0.5 mg/L IBA; initial pH 5.8; light cycle, 18 h/d (3500lx); temperature, 24 degrees C; shaker revolutions per minute, 100 r/min, were favourable to hairy root growth and flavonoids production. Under the above culture conditions, up to 12.8 g/L (DW) of hairy root and 1922 mg/L of flavonoids were obtained after 21 days of culture. The content of total flavonoids in hairy root was 15%, which was about 25 times as that in the wild plantlet.

[Establishment of Saussurea involucrata hairy roots culture and plantlet regeneration].

Hairy root clones of Saussurea involucrata transformed with Agrobacterium rhizogenes strains R1601, R1000, and LBA9402 were established to investigate the flavonoid production. Opine synthesis and PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A. rhizogenes strain R1601 into the transformed root genome. The frequency of hairy root formation from root segments, which were pre-cultured 2 days in N6 solid medium without plant growth regulators, amounted to 100% following infection with R1601 strain of A. rhizogenes. The transformed roots were kept in hormone-free N6 liquid medium in the dark at 25 degrees C, 110r/min and routinely subcultured every 20 - 24 days. One hairy root clone, which grew vigorously with lateral branches, was periodically examined for the ability to produce flavonoid. The maximum of biomass and flavonoid yield achieved 66.7 g/L (fresh weight) and 102.3mg/g dry weight after incubation 20 days. The calli were induced from the hairy root culture in the presence of 0.5mg/L IBA and intact plantlets were regenerated from these calli. The regeneration plantlets from hairy roots, in which the flavonoid content were 53% in that of untransformed plants, weren't different in growth and morphology of the untransformed plantlets. Therefore plant regeneration from hairy roots may be also a means for producing transformed S. involucrata plants. Hairy root cultures of S. involucrata clearly showed higher flavonoid contents compared to the wild plant or the regeneration seedlings. As the wild S. involucrata grows only in special regions with peculiar climate, and cultivation of this species in a normal climate has been unsuccessful so far. The success in obtaining a method for high production of flavonoid might very well be one of the solutions for this problem in the future.

[Cloning and sequence analysis of MYB transcriptional regulator SmP gene of Saussurea medusa Maxim].

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.

[SSS method for screening cDNA library].

A quick and simple method subsection screening (SSS) method for screening cDNA library by PCR was established. With this method, cDNA phage plate was cut into several blocks and a couple of primers was designed according to target gene. And then the target genes were obtained by screening cDNA library. Comparing with other methods,this method has many advantages such as controlled range and clear target,and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. Thus,the CHI gene, F3'H gene, HSP gene and one HSP partial fragment, were obtained respectively in half month in our lab. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.