In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds

The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.     

The absorption from the gut of quinine and chlorpheniramine given with various anionic agents

The absorption of quinine and chlorpheniramine with anionic agents was examined in the rat rectum, small intestine and stomach. The enhancement of absorption was related to partition behaviour, to the organic solvent, and surface activity of ion-pair complexes, but it seems that with results from these properties alone it is not possible to explain the apparent increase in the in situ absorption of amines. In the presence of anionic agents, the absorption of amines was enhanced at all sites examined and kinetically the uptake to the mucosal membrane was increased in the rectum and small intestine. The effect of anionic agents on the uptake of amines was greater in the rectum than in the small intestine and this may be related to differences in the nature of the gut wall.     

Effects of furanocoumarins in Kampo extract-based medicines on rat intestinal absorption of CYP3A and P-glycoprotein substrate drugs in vivo

While a great deal of information of drug-drug interactions is known, most concern Western drugs. Relatively little is known of the interactions between Western drugs and traditional drugs such as Kampo extract medicines (Japanese medicines modified from traditional Chinese medicines). This study investigated the effects of the marketed Kampo extract medicines, Senkyu-cha-cho-san and Sokei-kakketsu-to, on the intestinal absorption of CYP or P-glycoprotein (P-gp) in vivo. Midazolam, a CYP3A substrate drug, or talinolol, a P-gp substrate drug, was orally administered to rats with each of these Kampo extract medicines. Senkyu-cha-chosan or Sokei-kakketsu-to administered as a standard regimen did not obviously affect Cmax and area under the curve (AUC) of midazolam, although both Kampo extract medicines contained notopterol, a potent CYP3A4 inhibitor in vitro. The results implied a lack of potent drug-drug interactions between both Kampo extract medicines and CYP3A substrate drugs. Concomitant administration of each Kampo extract medicine unexpectedly showed the tendency to decrease Cmax and AUC of talinolol. Decreased intestinal absorption of talinolol might be caused, not by the inhibition of P-gp, but by the inhibition of organic anion transporting peptides by both Kampo extract medicines.     

Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.     

Estimation of bioavailability of salmon calcitonin from the hypocalcemic effect in rats (I): pharmacokinetic-pharmacodynamic modeling based on the endogenous Ca regulatory system

The hypocalcemic effect of salmon calcitonin (sCT) after intravenous administration was explained on the basis of an integrated pharmacokinetic-pharmacodynamic (PK-PD) model with the endogenous Ca regulatory system in the rat. The pharmacokinetics of sCT described by a conventional two-compartment model showed the extremely rapid elimination of sCT from plasma (MRT; 6.86 min). The hypocalcemic effect of sCT reached a peak from 0.5 to 1.5 hrs after administration, and the peak time tended to prolong with increasing doses. This delay in pharmacological effect of sCT against plasma concentration may be a result of a summation of multiple actions of the endogenous Ca regulatory system including feedback control. The plasma Ca regulation system in the rat was investigated by i.v. bolus administration of calcium gluconate and/or endogenous (rat) calcitonin (rCT). Since non-linearity in the relationship between Ca and rCT concentrations in plasma was observed, we assumed that rCT was secreted in accordance with the plasma Ca level via an exponential function. The pharmacokinetics of rCT was represented as a linear one-compartment model. To link the rCT level with plasma Ca level, an additional effect compartment was required to explain the delay in onset and decline of the pharmacological effect. This Ca regulation model explained the observed Ca and rCT profiles in plasma after administration of Ca and/or rCT. The plasma Ca levels after administration of sCT could be well described by the present integrated model. This suggested the potential for prediction of plasma sCT concentration only from the hypocalcemic effect after extravascular administration of sCT, using this PK-PD model.     

Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity.

To examine the systemic and mucosal immunity towards a liposomal antigen in an oral vaccine, we prepared ovalbumin (OVA)-encapsulating polyethyleneglycol (PEG)-modified liposomes and unmodified ones, and orally administered two different concentrations of them to mice. Unmodified liposomes tended to induce a stronger systemic immune response than the PEG-modified ones especially at the higher concentration of liposomes. Whereas at the lower liposome concentration the mucosal immune response was stronger for the PEG-modified liposomes than for the unmodified ones but nearly the same at the higher concentration. The relative amount of immunoglobulin G (IgG) against OVA in the plasma was 1.7-fold higher for a 12.5 micro mol phospholipid dose of PEG-liposomes encapsulating OVA than for a 5.0 micro mol one encapsulating the same amount of OVA. On the contrary, the relative amount of IgA in the intestinal wash was 2.6-fold higher for the 5.0 micro mol phospholipid dose than for the 12.5 micro mol one. These results indicate that OVA encapsulated in a small number of liposomes, especially the PEG-modified ones, is favorable for inducing a mucosal immune response and that the same amount of OVA in a large number of liposomes tends to improve the systemic immune response. A possible explanation for this tendency is the differential release rate of OVA from the liposomes at the intestinal mucosa. Our present study suggests that the dose of liposomes containing antigen is an important factor for controlling the response of systemic and mucosal immune systems.     

Enhanced percutaneous absorption of formoterol fumarate via pulsed iontophoresis. II. Effect of polarity, pulse frequency and duty

To clarify the effect of polarity, pulse frequency and duty on the iontophoretic transport of formoterol fumarate (FF), in vitro and in vivo studies were performed with guinea pigs. In the in vitro studies, the flux at steady state was enhanced by the factor 7.61 with anodal iontophoresis in comparison with control, whereas, with cathodal iontophoresis it was restrained by the factor 0.79. When pulse frequency was varied from 0 to 40 kHz, the flux at steady state was enhanced with an increase in pulse frequency. In the in vivo studies with varying the duty of pulse potential from 10 to 100%, the maximum plasma concentration of FF was obtained at a 30% duty. In contrast, the plasma concentration of FF achieved at 100% duty was not high. With stripped skin, no significant difference in the plasma concentration of FF was shown between iontophoresis and control. Consequently, it is suggested that iontophoresis contributes to FF penetration across stratum corneum.     

Usefulness of liposomes as an intranasal dosage formulation for topical drug application

The potential of liposomes as an intranasal dosage formulation for topical application was investigated in rats. When 5(6)-carboxyfluorescein (CF), a model absorbable drug, dissolved in phosphate-buffered saline (PBS) was administered intranasally, CF was rapidly absorbed into the systemic circulation and no adhesion of CF to the nasal mucosa was observed. The fraction of CF absorbed from the nasal mucosa reached about 48% 1 h after administration. On the other hand, only 3% of the dose was absorbed when CF was encapsulated in liposomes consisting of dipalmitoylphosphatidylcholine and cholesterol (DPPC-liposomes). In addition, the amount of CF adhering to the nasal mucosa after administration as DPPC-liposomes was 20- to 28-fold greater than that in PBS solution. In particular, positively charged liposomes markedly enhanced the adhesion of CF to the nasal mucosa. Differences in the lipid composition of liposomes did not affect the absorption of CF. However, the ability of liposomes to adhere to the nasal mucosa was consistent with the fluidity of the liposomal membrane. Furthermore, the action of liposomes on the anti-histaminic effect of diphenhydramine hydrochloride (DH) was studied in rats by measuring the amount of protein leaking into the nasal cavity under quasi-allergic conditions. The anti-histaminic effect of DH was strong but of short-duration when DH was administered as a PBS solution. However, liposomes prolonged the anti-histaminic effect of DH, suggesting that liposomes may adhere to the nasal mucosa and release DH slowly. In conclusion, liposomes suppress drug absorption into the systemic circulation and concurrently increase drug retention in the nasal cavity.