Activation of P2Y receptor enhances high-molecular compound absorption from rat ileum.

While there are no reports concerning the effects of extracellular nucleotides on the intestinal absorption of drugs, it is well known that extracellular nucleotides are important regulators of intestinal epithelial ion transport. This report using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound is the first to investigate the effects of purine nucleotides on absorption of poorly absorbed drugs from intestine. ATP enhanced the absorption of FD-4 from rat ileum in a concentration-dependent manner. ADP also enhanced the absorption of FD-4. Other purine nucleotides (adenosine, AMP, UTP and UDP) did not show an absorption-enhancing effect. The absorption-enhancing effect by ATP was inhibited by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS), which are known P2 receptor antagonists. Additionally, 2-methylthio ATP (a P2Y receptor agonist) enhanced the absorption of FD-4, but alpha,beta-methylene ATP (a P2X receptor agonist) did not. These findings suggest that activation of the P2Y receptor may improve the absorption of water-soluble and high-molecular compounds from the ileum.

Optimization of antitumor efficacy and safety of in vivo cytokine gene therapy using RGD fiber-mutant adenovirus vector for preexisting murine melanoma.

We previously reported that RGD fiber-mutant adenovirus vector (AdRGD) was a very useful vector system for in vivo cytokine gene therapy for established murine B16BL6 melanoma. However, intratumoral administration of AdRGD expressing tumor necrosis factor alpha (AdRGD-TNFalpha) at high dose revealed not only the dramatic reinforcement of anti-tumor effect but also serious adverse effects, such as body weight reduction and sudden death, caused by high-level TNF-alpha leakage from the tumor into circulation. These results strongly suggested that the determination of 'limiting dose', which demonstrated therapeutic effectiveness without adverse effect, against each vector was important for the development of appropriate cytokine gene therapy. In the present study, we investigated the efficacy and the safety of AdRGD expressing interleukin-12 (AdRGD-IL12) in murine melanoma model, and determined its limiting dose. Moreover, we demonstrated that combination therapy using AdRGD-IL12 and AdRGD-TNFalpha at limiting doses or less could achieve more effective tumor regression without adverse effects. Therefore, we conclude that a combination of multiple AdRGD expressing cytokines having distinct anti-tumor mechanisms can contribute to the establishment of in vivo cytokine gene therapy for melanoma, which possesses both excellent efficacy and high safety.

Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors.

Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma.

Characterization of the influence of nitric oxide donors on intestinal absorption of macromolecules.

To characterize the influence of nitric oxide (NO) donors on the intestinal absorption of macromolecules, the relationship between the release rate of NO from NO donors and their absorption-enhancing effects and the effects of several scavengers and generators on the absorption-enhancing effects of NO donor were investigated. The t1/2 values of the NO release rate from 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-1-propanamine (NOC5), 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12) are 25, 5 and 100min, respectively. The absorption-enhancing effects of NO donors on the absorption of fluorescein isothiocyanate dextrans with an average molecular weight of 4400 (FD-4) are NOC5 > NOC7 > NOC12 in the colon. The lowest enhancing effect of NOC12 may be due to the slow rate of NO release. The enhancing effect of NOC7 rapidly disappeared compared with the effect of NOC5. The results raise the possibility that the difference between NOC5 and NOC7 on enhancing effect is related to the t1/2 of the NO release. The NOC7-induced enhancing effect was prevented by the co-administration of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide sodium salt (C-PTIO), an NO scavenger; tiron, an O2(-) scavenger; mannitol, an OH* scavenger, and deferoxamine, peroxynitrate scavenger. Pyrogallol, an O2(-) generator, potentiated the NOC7-induced enhancing effect. These results support a role for peroxynitrate, and possibly OH*, in the NO donor-induced intestinal enhancing effect.

[Variance of bioavailability of pharmaceutical preparations and analysis of factors affecting it].

Phenytoin (pulverized powder), mefenamic acid (capsule), and sulpiride (film-coated tablet) are currently available on the Japanese market. For absorption of these drugs from their pharmaceutical preparations, they must disintegrate and dissolve during passage through the gastrointestinal tract. The bioavailability of these drugs differ among different pharmaceutical preparations and even for the same preparation. This led to a review of the influence of the features of pharmaceutical preparations and the physicochemical properties of film coating materials as well as the physiologic factors affecting drug bioavailability. The influence of coadministered drugs and concomitant intake of beverages and food on the bioavailability of drugs from pharmaceutical preparations is also described.

Effect of fatty acid diesters on permeation of anti-inflammatory drugs through rat skin.

Four fatty acid diesters (diethyl succinate, diethyl adipate, diethyl sebacate, and diisopropyl adipate) were used to study their enhancement effect on the permeation of four non-steroidal anti-inflammatory drugs (NSAIDs: ketoprofen, indomethacin, diclofenac sodium, and ibuprofen) through rat abdominal skin. With the diester pretreatment, drug permeation increased and the lag times decreased. No relationship was observed between the solubilities of the drugs in the diesters and the diester enhancement effects. The enhancement effect decreased with an increase of the drug lipophilicity, but increased with an increase of the lipophilic index of the diester up to about 3.5, after which the enhancement effect decreased or remained constant. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was employed to investigate the biophysical changes in the stratum corneum lipids caused by the diesters. The FTIR results showed that treatment of the skin with diesters did not produce a blue shift in the asymmetric and symmetric C-H stretching peak positions. However, all of the above diesters showed a decrease in peak heights and areas for both asymmetric and symmetric C-H stretching absorbances in comparison with water treatment. These results suggested that the diesters were more effective for enhancing the penetration of hydrophilic drugs than lipophilic drugs, and the enhancing effect of lipophilic diesters was more effective than that of hydrophilic diesters. The enhancement effects of diesters may be due to their causing lipid extraction in the skin.

Tumor necrosis factor alpha-gene therapy for an established murine melanoma using RGD (Arg-Gly-Asp) fiber-mutant adenovirus vectors.

Although adenovirus vectors (Ad) provide high-level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie-adenovirus receptor, a primitive Ad-receptor, was very low in murine and human melanoma cells. We also found that fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting alpha(v)-integrins. In addition, intratumoral injection of RGD fiber-mutant Ad containing the tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) revealed dramatic anti-tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad-RGD-TNFalpha required one-tenth the dosage of Ad-TNFalpha to induce an equal therapeutic effect. These results suggest that alpha(v)-integrin-targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.