Piyanuch Wonganan

Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles.

Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells.

PEGylated adenoviruses: From mice to monkeys

Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression

Chemoresistance is a major issue for most gemcitabine-related chemotherapies. The overexpression of ribonucleotide reductase subunit M1 (RRM1) plays a key role in gemcitabine resistance. In this study, we synthesized a new highly acid-sensitive amphiphilic micelle material by conjugating hydrophilic polyethylene glycol with a hydrophobic stearic acid derivative (C18) using a hydrazone bond, which was named as PHC-2. A lipophilic prodrug of gemcitabine, 4-(N)-stearoyl gemcitabine (GemC18), was loaded into micelles prepared with PHC-2, a previously synthesized less acid-sensitive PHC-1, and their acid-insensitive counterpart, PAC. GemC18 loaded in acid-sensitive micelles can overcome gemcitabine resistance, and GemC18 in the highly acid-sensitive PHC-2 micelles was more cytotoxic than in the less acid-sensitive PHC-1 micelles. Mechanistic studies revealed that upon cellular uptake and lysosomal delivery, GemC18 in the acid-sensitive micelles was released and hydrolyzed more efficiently. Furthermore, GemC18 loaded in the highly acid-sensitive PHC-2 micelles inhibited the expression of RRM1 and increased the level of gemcitabine triphosphate (dFdCTP) in gemcitabine resistant tumor cells. The strategy of delivering lipophilized nucleoside analogs using highly acid-sensitive micelles may represent a new platform technology to increase the antitumor activity of nucleoside analogs and to overcome tumor cell resistance to them.

Species Differences in the Pharmacology and Toxicology of PEGylated Helper-Dependent Adenovirus

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.

Silencing of ribonucleotide reductase subunit M1 potentiates the antitumor activity of gemcitabine in resistant cancer cells

Gemcitabine is a deoxycytidine analog used for the treatment of a wide range of solid tumors. Its efficacy is however often reduced due to the development of resistance. Ribonucleotide reductase M1 subunit (RRM1) is a key determinant of gemcitabine resistance, and tumor cells that overexpress RRM1 are resistant to the cytotoxicity of gemcitabine. In the present study, we showed that RRM1-specific small interfering RNA (siRNA), when complexed with polyethylenimine, effectively downregulated the expression of RRM1 protein in mouse tumor cells that overexpress RRM1, both in vitro and in vivo. More importantly, systemic administration of the RRM1-specific siRNA significantly inhibited the growth of RRM1-overexpressing tumors in mice and sensitized the tumors to gemcitabine treatment. These findings suggest that silencing RRM1 expression using siRNA could potentially be an effective strategy to overcome gemcitabine resistance.

Drug-Virus Interaction: Effect of Administration of Recombinant Adenoviruses on the Pharmacokinetics of Docetaxel in a Rat Model

Modern cancer therapy combines recombinant viruses with traditional chemotherapeutic agents that are metabolized by hepatic cytochrome P450 3A4 (CYP3A4). A single dose of recombinant adenovirus (Ad) expressing β-galactosidase (AdlacZ) significantly alters CYP3A2, the correlate of CYP3A4, in rats for 14 days. Recombinant adenovirus expressing human p53 (Adp53) also suppresses CYP3A2. Plasma clearance of docetaxel (DTX) in animals given AdlacZ (3.38±0.22 l h−1 kg−1) was significantly lower than that of those given DTX alone (7.35±1.22 l h−1 kg−1, P⩽0.05). Area under the plasma concentration-time curve of DTX in rats given AdlacZ (2987.37±197.97 ng ml−1 h−1) was significantly greater than those given drug alone (1496.14±281.62 ng ml−1 h−1, P⩽0.05). Both viruses prolonged DTX half-life (t1/2). Ad infection may cause significant variability in the pharmacokinetics and pharmacodynamics of anti-cancer agents and should be considered when designing therapeutic regimens for patients with viral infection and those enrolled in clinical trials using recombinant viruses.

Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 × 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p ≤ 0.01). Helper-dependent adenovirus, with all viral genes removed, suppressed CYP3A2 (43%) and CYP2C11 (55%) within six hours. CYP3A2 remained significantly suppressed (47%, 14 days, p ≤ 0.01) while CYP2C11 returned to baseline at this time. CYP3A2 and 2C11 were reduced by 45 and 42% respectively 6 hours after treatment with PEGylated adenovirus, which has a low immunological profile (p ≤ 0.05). CYP3A2 remained suppressed (34%, p ≤ 0.05) for 14 days while CYP2C11 recovered. Inactivated virus suppressed CYP3A2 activity by 25–50% for 14 days (p ≤ 0.05). CYP2C11 was affected similar manner but recovered by day 14. Microarray and in vitro studies suggest that changes in cellular signaling pathways initiated early in virus infection contribute to changes in CYP.

Influence of method of systemic administration of adenovirus on virus-mediated toxicity: Focus on mortality, virus distribution, and drug metabolism

INTRODUCTION Doses of 2 x 10(12) virus particles/kilogram (vp/kg) and higher of recombinant human adenovirus serotype 5 (HAdV-5) given via the tail vein induce significant toxicity and mortality in the rat. This was not observed when doses of 5.7 x 10(12) vp/kg were given through a surgically implanted jugular catheter. Here we assess how the manner by which HAdV-5 is introduced into the systemic circulation affects biodistribution, transgene expression, toxicity and mortality 0.25, 1, and 4 days after treatment in the rat. Animals were given 5.7 x 10(12) vp/kg of HAdV-5 expressing beta-galactosidase or saline through a jugular catheter or by direct tail vein injection. RESULTS All animals survived after jugular vein dosing. Tail vein injection of HAdV-5 increased the mortality rate to 42% (p< or =0.01). All deaths occurred within 4 h. Animals dosed through the jugular vein had significantly higher levels of transgene expression in the liver and spleen and significantly more viral genomes in these tissues and kidney and lung within the first 24 h of viral infection compared to those dosed by tail vein injection (p< or =0.01). There was no significant difference between the groups thereafter. Samples from animals that died contained even higher levels of viral genomes and serum transaminases were elevated on average by a factor of 4 at the time of death. There was no significant difference between the two dosing methods with respect to changes in hepatic cytochrome P450 expression and activity throughout the study. CONCLUSION These findings suggest that the method of systemic administration should be carefully considered when assessing toxicity data and other parameters at early time points after virus administration in the rat and possibly other animal models.

Evaluation of the HC-04 Cell Line as an In Vitro Model for Mechanistic Assessment of Changes in Hepatic Cytochrome P450 3A During Adenovirus Infection

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 μM) and phenobarbital (500 μM) increased activity by 230 and 124%, whereas ketoconazole (10 μM) and lipopolysaccharide (LPS) (10 μg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

142. Molecular and Macromolecular Alterations of Recombinant Adenoviral Vectors Do Not Eliminate Changes in Hepatic Drug Metabolism

The hepatic cytochrome P450 (CYP) enzyme system is responsible for the inactivation and clearance of numerous substrates and the conversion of drugs to their active metabolite(s). We have previously shown that a single dose of recombinant adenovirus (Ad) ranging from 5.7|[times]|106|[ndash]|5.7|[times]|1012 viral particles/kilogram (vp/kg) significantly alters hepatic CYP isoforms 3A2 and 2C11 for 14 days (Callahan et al, JPET, 2005, 312:492|[ndash]|501). We also found that the expression and function of these isoforms, selected for their predominance in drug metabolism (CYP3A2) and their responsiveness to inflammatory mediators (CYP2C11), is altered by certain transgene cassettes (Callahan et al, Mol. Ther., 2003, 7:S58). In an effort to determine the effect of viral gene expression and the innate immune response against virus capsid proteins on CYP, Sprague-Dawley rats were given 0.5mL (5.7|[times]|1012 vp/kg) of either: wild type Ad serotype 5 (WT), first generation Ad expressing E. coli beta-galactosidase (AdlacZ), PEGylated AdlacZ (PEGAd), a helper-dependent Ad expressing beta-galactosidase (HDAd), inactivated AdlacZ (UVAd), or phosphate buffered saline (Vehicle) via a jugular cannula. Animals were sacrificed at 0.25, 1, 4, and 14 days. In vitro catalytic activity, protein expression, mRNA, and ALT levels were measured at each timepoint. Six hours after administration of WT, CYP3A2 and 2C11 activity was significantly suppressed (37% and 39%, respectively) as compared to controls. This reduction continued through 14 days with levels falling to 67% (CYP3A2) and 79% (CYP2C11) of controls, P|[le]|0.01. mRNA corresponded to changes in CYP activity. ALT levels of animals treated with this virus were significantly elevated through the 4 day timepoint with respect to levels obtained from saline treated animals (P|[le]|0.05). Use of PEGAd, a vector with a low immunological profile (Croyle et al, Hum Gene Ther, 2002, 13:1887-1900), showed that capsid proteins alone are not responsible for CYP aberrations. CYP3A2 (45%) and 2C11 (42%) was initially suppressed 6 hours after administration (P|[le]|0.05). At 14 days CYP3A2 activity levels remained suppressed (34%, P|[le]|0.05) while CYP2C11 levels recovered. HDAd was administered to investigate the effects of viral gene expression on CYP. Within 6 hours after administration, CYP3A2 (43%) and CYP2C11 (55%) activity was suppressed (P|[le]|0.05). CYP3A2 levels remained suppressed through 14 days (47%, P|[le]|0.01) while CYP2C11 recovered. Preliminary data indicate these alterations occur at the transcriptional level. In contrast, AdlacZ suppressed CYP3A2 and 2C11 activity and mRNA and elevated ALT levels through the 14 day period. UVAd, an inactive control, suppressed CYP3A2 activity by 26% 1 day after administration (P|[le]|0.05) whereas CYP2C11 activity was unaffected throughout the study. These data show that modifications of adenoviral vectors at the molecular and macromolecular level do not eliminate aberrations in CYP following systemic administration and suggest that these changes are not simply the result of the innate immune response. Further studies investigating the role of altered signaling pathways and CYP gene regulatory elements during Ad infection are ongoing.