Pharmacokinetics study of a supersaturatable self-microemulsifying drug delivery system for ellagic acid by UHPLC-Q-TOF-MS.

To evaluate the bioavailability of ellagic acid loaded super-saturatable self-microemulsifying drug delivery system (S-SMEDDS), its pharmacokinetic properties were studied in rats with an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. The plasma samples were treated by solid-phase extraction method, and gallic acid was used as the internal standard when determining the concentration of ellagic acid. Results showed that the established analytical method was sensitive and accurate, which is applicable to the pharmacokinetic study of ellagic acid. The drug was found to be absorbed rapidly in vivo, and the plasma concentration-time curve showed double peaks, indicating that ellagic acid were reabsorbed by entero-hepatic circulation after oral administration. Compared with ellagic acid suspension, the apparent clearance of ellagic acid-loaded S-SMEDDS and SMEDDS reduced significantly, and the AUC 0~t of them were 4.7 and 5.8-fold increase, respectively. Therefore, the bioavailability of ellagic acid-loaded S-SMEDDS was higher than that of the suspension and SMEDDS.

Insight into nucleophilic fragmentation mechanisms by glutamic acid side chain in singly protonated glutathione and related peptidyl ions.

Fragmentation mechanisms of the singly protonated glutathione (γ-ECG) and its synthetic analogue peptides (ECG and PPECG) have been investigated by liquid chromatography tandem-mass spectrometry and theoretical calculations. In the mass spectra, similar fragmentation patterns were observed for γ-ECG and ECG, but a completely different one was found in the case of PPECG. The E-C amide bond cleavage is the predominant pathway for the fragmentation of γ-ECG and ECG, whereas the additional N-terminal prolyl residues in PPECG significantly suppress the E-C amide bond cleavage. Theoretical calculations reveal that the fragmentation efficiencies of the E-C bonds in the protonated γ-ECG and ECG are much higher than that in the protonated PPECG, being attributed to their lower barriers of the potential energy; clearly the introduction of two prolyl residues can increase substantially the potential energy barrier. In the proposed mechanism, the protonated E-C amide bonds in the three peptides are first weakened followed by a nucleophilic addition by the glutamyl carboxyl oxygen atom in side chain, leading to the breaking of the E-C amide bonds. However, the processes of E-C bond fragmentation for three protonated analogs were not collaborative. Protonated amide bonds first fragment, then the nucleophilic addition by the side chain of glutamyl carboxyl oxygen atom takes places. On the other hand, the prolyl residues in PPECG can largely diminish the nucleophilic addition, resulting in a much lower efficiency of its E-C amide bond breaking. Distance analysis indicates that breaking the E-C amide bonds in the protonated γ-ECG, ECG, and PPECG ions could not occur without the assistance from the nucleophilic attack, highlighting an asynchronous collaborative process in the bond breakings.

Structure and magnetic properties of a copper(II) coordination polymer based on azide, pyridine and homophthalic acid.

The azide anion is a short bridging ligand that has been used extensively to construct magnetic coordination polymers, and fundamental magneto-structural correlations have been substantiated by theoretical calculations. The copper(II) coordination polymer poly[bis(µ-azido-κ(2)N(1):N(1))(µ4-homophthalato-κ(4)O:O':O'':O''')bis(pyridine-κN)dicopper(II)], [Cu2(C9H6O4)(N3)2(C5H5N)2]n, was synthesized from homophthalic acid (2-carboxyphenylacetic acid), pyridine and azide (N3(-)) by a hydrothermal reaction. Single-crystal structure analysis indicated that it features a one-dimensional chain structure which is comprised of (µ1,1-N3(-))(µ-syn-syn-COO(-))2- and (µ1,1-N3(-))2-bridged tetranuclear Cu(II) units. Magnetic measurements revealed that the compound exhibits dominant antiferromagnetic behaviour.

Humoral Immune Response against Nontargeted Tumor Antigens after Treatment with Sipuleucel-T and Its Association with Improved Clinical Outcome.

PURPOSE: Antitumor activity of cancer immunotherapies may elicit immune responses to nontargeted (secondary) tumor antigens, or antigen spread. We evaluated humoral antigen spread after treatment with sipuleucel-T, an immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), designed to target prostatic acid phosphatase (PAP; primary antigen). EXPERIMENTAL DESIGN: Serum samples from patients with mCRPC enrolled in the placebo-controlled phase III IMPACT study (evaluable n = 142) were used to assess humoral antigen spread after treatment with sipuleucel-T. Immunoglobulin G (IgG) responses to self-antigens (including tumor antigens) were surveyed using protein microarrays and confirmed using Luminex xMAP. IgG responses were subsequently validated in ProACT (n = 33), an independent phase II study of sipuleucel-T. Association of IgG responses with overall survival (OS) was assessed using multivariate Cox models adjusted for baseline prostate-specific antigen (PSA) and lactate dehydrogenase levels. RESULTS: In patients from IMPACT and ProACT, levels of IgG against multiple secondary antigens, including PSA, KLK2/hK2, K-Ras, E-Ras, LGALS8/PCTA-1/galectin-8, and LGALS3/galectin-3, were elevated after treatment with sipuleucel-T (P < 0.01), but not control. IgG responses (≥ 2-fold elevation posttreatment) occurred in ≥ 25% of patients, appeared by 2 weeks after sipuleucel-T treatment, and persisted for up to 6 months. IgG responses to PSA and LGALS3 were associated with improved OS in sipuleucel-T-treated patients from IMPACT (P ≤ 0.05). CONCLUSIONS: Sipuleucel-T induced humoral antigen spread in patients with mCRPC. IgG responses were associated with improved OS in IMPACT. The methods and results reported may identify pharmacodynamic biomarkers of clinical outcome after sipuleucel-T treatment, and help in clinical assessments of other cancer immunotherapies. See related commentary by Hellstrom and Hellstrom, p. 3581.

Rapid preparation and characterization of methacrylate-based monoliths for chromatographic and electrophoretic separation.

Butyl-methacrylate-based porous monoliths were rapidly prepared in the fused-silica capillary with a 10-cm stripe of polyimide removed from its exterior. The photopolymerization could be carried out in 150 s using ethylene glycol dimethacrylate as a cross-linking agent; 1-propanol, 1,4-butanediol, and water as tri-porogenic solvents; and Irgacure 1800 as a photo-initiator. The effect of different morphologies on the efficiency and retention properties was investigated using pressure-assisted CEC (p-CEC), CEC, and low pressure-assisted liquid chromatography modes (LPLC). Baseline separation of the model analytes was respectively achieved including thiourea, toluene, naphthalene, and biphenyl with the lowest theoretical height up to 8.0 microm for thiourea in the mode of p-CEC. Furthermore, the influence of the tri-porogenic solvents on the morphology of methacrylate-based monoliths was systematically studied with mercury intrusion porosimetry and scanning electron microscopy.

Regulation of phase I and phase II steroid metabolism enzymes by PPAR alpha activators.

Peroxisome proliferators (PP) are a large class of structurally diverse chemicals that mediate their effects in the liver mainly through the peroxisome proliferator-activated receptor alpha (PPARalpha). Exposure to some PP results in alterations of steroid levels that may be mechanistically linked to adverse effects in reproductive organs. We hypothesized that changes in steroid levels after PP exposure are due to alterations in the levels of P450 enzymes that hydroxylate testosterone and estrogen. In testosterone hydroxylase assays, exposure to the PP, WY-14,643 (WY), gemfibrozil or di-n-butyl phthalate (DBP) led to compound-specific increases in 6beta and 16beta-testosterone and androstenedione hydroxylase activities and decreases in 16alpha, 2alpha-hydroxylase activities by all three PP. The decreases in 16alpha and 2alpha-testosterone hydroxylase activity can be attributed to a 2alpha and 16alpha- testosterone hydroxylase, CYP2C11, which we previously showed was dramatically down-regulated in these same tissues (Corton et al., 1998; Mol. Pharmacol. 54, 463-473). To explain the increases in 6beta- and 16beta-testosterone hydroxylase activities, we examined the expression of P450 family members known to carry out these functions. Alterations in the 6beta-testosterone hydroxylases CYP3A1, CYP3A2 and the 16beta-testosterone hydroxylase, CYP2B1 were observed after exposure to some PP. The male-specific estrogen sulfotransferase was down-regulated in rat liver after exposure to all PP. The mouse 6beta-testosterone hydroxylase, Cyp3a11 was down-regulated by WY in wild-type but not PPARalpha-null mice. In contrast, DEHP increased Cyp3a11 in both wild-type and PPARalpha-null mice. These studies demonstrate that PP alter the expression and activity of a number of enzymes which regulate levels of sex steroids. The changes in these enzymes may help explain why exposure to some PP leads to adverse effects in endocrine tissues that produce or are the targets of sex hormones.

PPARalpha activators down-regulate CYP2C7, a retinoic acid and testosterone hydroxylase.

Peroxisome proliferators (PP) are a large class of structurally diverse chemicals that mediate their effects in the liver mainly through the peroxisome proliferator-activated receptor alpha (PPARalpha). Exposure to PP results in down-regulation of CYP2C family members under control of growth hormone and sex steroids including CYP2C11 and CYP2C12. We hypothesized that PP exposure would also lead to similar changes in CYP2C7, a retinoic acid and testosterone hydroxylase. CYP2C7 gene expression was dramatically down-regulated in the livers of rats treated for 13 weeks by WY-14,643 (WY; 500 ppm) or gemfibrozil (GEM; 8000 ppm). In the same tissues, exposure to WY and GEM and to a lesser extent di-n-butyl phthalate (20,000 ppm) led to decreases in CYP2C7 protein levels in both male and female rats. An examination of the time and dose dependence of CYP2C7 protein changes after PP exposure revealed that CYP2C7 was more sensitive to compound exposure compared to other CYP2C family members. Protein expression was decreased after 1, 5 and 13 weeks of PP treatment. CYP2C7 protein expression was completely abolished at 5 ppm WY, the lowest dose tested. GEM and DBP exhibited dose-dependent decreases in CYP2C7 protein expression, becoming significant at 1000 ppm or 5000 ppm and above, respectively. These results show that PP exposure leads to changes in CYP2C7 mRNA and protein levels. Thus, in addition to known effects on steroid metabolism, exposure to PP may alter retinoic acid metabolism.

Molecular and immunological evaluation of the transcription factor SOX-4 as a lung tumor vaccine antigen.

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.

Opposing mechanisms of NADPH-cytochrome P450 oxidoreductase regulation by peroxisome proliferators.

Peroxisome proliferators (PPs) regulate a battery of rodent P450 genes, including CYP2B, CYP2C, and CYP4A family members. We hypothesized that other components of the P450-metabolizing system are altered by exposure to PPs, including NADPH-cytochrome P450 oxidoreductase (P450R), an often rate-limiting component in P450-dependent reactions. In this study, we determined whether exposure to structurally diverse PPs alters the expression of P450R mRNA and protein. Increases in P450R mRNA levels were observed in male and female F-344 rat livers and in male rat kidneys after chronic exposure of the animals to PPs. Paradoxically, under the same treatment conditions in male rats, liver P450R protein levels decreased after exposure to the PPs Wy-14,643 ([4-chloro-6-(2,3-xylidino)pyrimidynylthio]acetic acid) (WY) or gemfibrozil (GEM). The down-regulation of the P450R protein was sex- and tissue-specific in that exposure to PPs led to increases in P450R protein in female rat livers [di-n-butyl phthalate (DBP) only] and male rat kidneys (WY, GEM, DBP). In male wild-type SV129 mice, P450R mRNA levels increased in livers after exposure to WY and diethylhexyl phthalate (DEHP) and in male kidneys after exposure to DEHP. Induction of mRNA by PPs was not observed in the liver or kidneys of mice, which lack a functional peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of the effects of PPs in the rodent liver. In wild-type male mice, P450R protein was decreased in liver after WY and DEHP treatment and in kidneys after WY treatment. The down-regulation of the P450R protein was not observed in PPAR alpha-null mice. These studies demonstrate the complex regulation of P450R expression by PPs at two different levels, both of which are dependent upon PPAR alpha: up-regulation of transcript levels in liver and kidneys and down-regulation of protein levels in male rat and mouse liver by a novel posttranscriptional mechanism.

Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis.

To identify genes that are differentially over-expressed in Small Cell Lung Carcinoma (SCLC) we have used a combination of suppression subtractive hybridization and cDNA microarray to analyse the expression profiles of 2400 cDNAs clones. Genes that are over-expressed in SCLC were identified using 32 pairs of fluorescence-labeled cDNA samples representing various lung tumors and normal tissues. This comprehensive approach has resulted in the identification of 209 genes that are differentially over-expressed in SCLC. Quantitative real-time PCR was used to further validate the expression of 43 genes in SCLC tumors and various normal tissues. Discussed in this report are nine genes, which showed the most promising SCLC tumor to normal tissue differential expression profiles, including seven known and two novel genes. The large number of differentially expressed genes identified from this analysis and the characterization of these genes will provide valuable information in better understanding the biology of SCLC and help us in developing these gene products as potential targets for diagnostic as well as therapeutic usage.