Effect of biofield therapy in the human brain.

OBJECTIVES: The effects of Okada Purifying Therapy (OPT), a form of subtle energy (biofield) therapy that originated in Japan, were investigated. Electroencephalograms and the Profile of Mood States scores were measured using a crossover design during OPT and placebo sessions. PARTICIPANTS: Nineteen (19) healthy Japanese adults (mean age±standard deviation: 40.8±11.2 years; 10 females) with no previous experience of biofield therapy participated in this study. METHODS: Each session lasted 15 minutes. A single-blind, randomized design with a protocol consisting of regular cycles with eyes open followed by eyes closed was used. The power spectral value was calculated in θ (4.0-7.9 Hz), α (8.0-12.9 Hz), and ß (13.0-29.9 Hz) frequency ranges. RESULTS: The power spectral value of the α band at F(p1), F(p2), F(7), F(z), F(8), C(3), C(z), C(4), and P(z) increased significantly in the OPT session compared with the placebo session. Mood state was improved after both sessions, and no significant difference was found between the two sessions. CONCLUSIONS: OPT was more effective in increasing α waves in the frontal and central cortex than a placebo treatment.

Bootstrap method-based estimation of the minimum sample number for obtaining pharmacokinetic parameters in preclinical experiments.

Empirically, 3-6 samples at each sampling time point have been used for most preclinical one-point sampling experiments without any theoretical justification. The purpose of the present study is to propose a practical approach to determine the minimum sample number (N(min)) based on Monte Carlo simulation and a bootstrap resampling. A computer program MOMENT(BS), in which a bootstrap resampling algorithm is used to estimate mean and standard deviations of pharmacokinetic parameters, such as area under the curve and mean residence time, was applied to estimate N(min). A new simulation program, MONTE1, was developed to generate simulated data for bootstrap resampling using the model parameters including inter- and/or intra-individual variations. Then, an index, S(2)CV calculated as the sum of the squared coefficient of variation is proposed to determine the N(min). The proposed approach was applied to the actual data in preclinical experiments, and the usefulness of the approach was suggested. An issue that one-point sampling data cannot separately assess inter- and intra-individual variability is discussed.

Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors.

Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetics of short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) driven by U6, H1 or tRNA promoter (pU6-shLuc, pH1-shLuc, and ptRNA-shLuc) was studied in melanoma cells expressing firefly luciferase. A bootstrap method-based moment analysis was performed to statistically and quantitatively evaluate the profile of gene silencing. The analysis showed that pU6-shLuc induced a significantly greater and longer gene silencing than that produced by other promoter-driven shRNA expression vectors. In addition, it was found that pU6-shLuc was at least 100-fold more potent in gene silencing than siRNA targeting the same gene on a numerical basis. These statistical considerations demonstrated that U6 promoter-driven shRNA expressing pDNA is the most effective in inducing gene silencing effect as far as the intensity and duration of RNAi effect is concerned.

Improved anti-cancer effect of interferon gene transfer by sustained expression using CpG-reduced plasmid DNA.

Plasmid DNA (pDNA) expressing mouse interferon (IFN)-beta or IFN-gamma (pCMV-Mu beta and pCMV-Mu gamma, respectively) has been shown to be effective in inhibiting the growth of colon carcinoma CT-26 cells in the liver (Kobayashi et al., Molecular Therapy 2002;6:737-44). The therapeutic effect of such IFN gene transfer could be significantly increased by the sustained expression of IFNs. In the present study, CpG-reduced pDNA encoding IFN-beta or IFN-gamma (pGZB-Mu beta and pGZB-Mu gamma, respectively) was constructed. pCMV-Mu beta and pCMV-Mu gamma were used as conventional CpG-replete pDNAs. Each pDNA was injected into the tail vein of mice by the hydrodynamics-based procedure. An injection of pGZB-Mu beta resulted in very high IFN-beta activities in the serum for at least 24 hr after injection, whereas the IFN-beta activity after pCMV-Mu beta injection declined quickly. About a 14-fold greater amount of IFN-beta was produced from pGZB-Mu beta than from pCMV-Mu beta. pGZB-Mu beta markedly inhibited the pulmonary metastasis of CT-26 cells. Similar, but more marked results were obtained with pGZB-Mu gamma: it increased the area under the concentration-time curve by more than a 60-fold and the mean residence time of IFN-gamma 4-fold compared with pCMV-Mu gamma. The survival time of the pGZB-Mu gamma-treated mice was significantly (p<0.05) longer than that of the saline- or pCMV-Mu gamma-treated mice. These results indicate that long-term expression of IFN can be achieved by CpG-reduced pDNA and sustained IFN gene expression results in enhanced therapeutic effects of IFN gene transfer against tumor metastasis.

Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo.

Mammalian cells have an activity of mutagenic repair for DNA double-strand breaks (DSBs), microhomology-mediated end joining (MMEJ), in which DNA ends are joined via microhomologous sequences flanking the breakpoint. MMEJ has been indicated to be undertaken without Ku proteins, which are essential factors for non-homologous end joining (NHEJ). On the other hand, recent studies with cell-free (in vitro) systems indicated the involvement of Ku proteins in MMEJ, suggesting that MMEJ could be also undertaken by a Ku-dependent pathway. To clarify whether Ku proteins are essential in MMEJ in vivo, linearized plasmid DNAs with microhomologous sequences of 10bp at both ends were introduced as repair substrates into Ku80-proficient and Ku80-deficient CHO cells, and were subjected to MMEJ and NHEJ. Activities of MMEJ and NHEJ, respectively, of the cells were evaluated by mathematical modeling for the increase in fluorescence of GFP proteins produced from repaired products. The Ku80 deficiency caused approximately 75% reduction of the MMEJ activity in CHO cells, while it caused is > or =90% reduction of the NHEJ activity. Therefore, it was indicated that there is a Ku-dependent pathway for MMEJ; however, MMEJ is less dependent on Ku80 protein than NHEJ. The fraction of MMEJ products increased in proportion to the increase in the amounts of substrates. The results suggest that the increase in DSBs makes the cell more predominant for MMEJ. MMEJ might function as a salvage pathway for DSBs that cannot be repaired by NHEJ.

Moment analysis for kinetics of gene silencing by RNA interference.

RNA interference (RNAi) was quantitatively evaluated from a kinetic viewpoint. A simple kinetic evaluation based on moment analysis was proposed, assuming suppression and recovery phases of gene expression. We defined the area under the curve of the inhibitory effect (AUC(IE)) as an index of the total intensity of RNAi and the mean response time of the inhibitory effect (MRT(IE)) as an index of its duration. The proposed kinetic analysis helps to understand the RNAi effect in a quantitative and time-dependent manner, which will be beneficial for designing RNAi-based gene silencing for both experimental and therapeutic purposes.

Histogram analysis of pharmacokinetic parameters by bootstrap resampling from one-point sampling data in animal experiments.

A bootstrap method is proposed for assessing statistical histograms of pharmacokinetic parameters (AUC, MRT, CL and V(ss)) from one-point sampling data in animal experiments. A computer program, MOMENT(BS), written in Visual Basic on Microsoft Excel, was developed for the bootstrap calculation and the construction of histograms. MOMENT(BS) was applied to one-point sampling data of the blood concentration of three physiologically active proteins ((111)In labeled Hsp70, Suc(20)-BSA and Suc(40)-BSA) administered in different doses to mice. The histograms of AUC, MRT, CL and V(ss) were close to a normal (Gaussian) distribution with the bootstrap resampling number (200), or more, considering the skewness and kurtosis of the histograms. A good agreement of means and SD was obtained between the bootstrap and Bailer's approaches. The hypothesis test based on the normal distribution clearly demonstrated that the disposition of (111)In-Hsp70 and Suc(20)-BSA was almost independent of dose, whereas that of (111)In-Suc(40)-BSA was definitely dose-dependent. In conclusion, the bootstrap method was found to be an efficient method for assessing the histogram of pharmacokinetic parameters of blood or tissue disposition data by one-point sampling.

An evaluation method for nonlinear local disposition in rat liver and kidney.

A two-sampling sites method was developed to separately estimate the nonlinear local disposition in the liver and kidney by sampling blood simultaneously from the hepatic vein and an artery after intravenous administration. Using this method, it was attempted to predict the renal elimination from the systemic and hepatic elimination. Etoposide, a substrate of both P-glycoprotein and CYP3A, was used as a model drug. The blood samples from the hepatic vein and an artery were simultaneously taken from a rat after intravenous administration of etoposide at a dose of 20 or 80 mg/kg. At a dose of 20 mg/kg, the total clearance (CL), hepatic clearance (CLH), and renal clearance (CLR=CL-CLH), which were almost constant, were 2.82 +/- 0.24, 0.742 +/- 0.214, and 2.09 +/- 0.34 l/h/kg, respectively. At a dose of 80 mg/kg, CL and CLR considerably decreased with an increase in plasma concentration, whereas CLH slightly decreased. By means of the two-sampling sites method, we estimated the local drug disposition in the liver and kidney. The present local pharmacokinetic method would be applicable to assess the local disposition of other drugs that are mainly metabolized in these organs.

Analysis methods and recent advances in nonlinear pharmacokinetics from in vitro through in loci to in vivo.

An attempt has been made to review the nonlinearities in the disposition in vitro, in situ, in loci and in vivo mainly from a theoretical point of view. Parallel Michaelis-Menten and linear (first-order) eliminations are often observed in the cellular uptake, metabolism and efflux of drugs. The well-stirred and parallel-tube models are mainly adopted under steady-state conditions in perfusion experiments, whereas distribution, tank-in-series and dispersion models are often used under nonsteady-state conditions with a pulse input. The analysis of the nonlinear local disposition in loci is reviewed from two points of view, namely an indirect method involving physiologically based pharmacokinetics (PBPK) and a direct (two or three samplings) method using live animals. The nonlinear global pharmacokinetics in vivo is reviewed with regard to absorption, elimination (metabolism and excretion) and distribution.

Efficient scavenger receptor-mediated hepatic targeting of proteins by introduction of negative charges on the proteins by aconitylation: the influence of charge density and size of the proteins molecules.

The in vivo disposition characteristics of some aconitylated proteins in mice were studied after intravenous injection in relation to their molecular properties such as overall negative charge and size of the molecules. Superoxide dismutase (SOD, M(w)=32000) and bovine serum albumin (BSA, M(w)=67000) were used to produce aconitylated derivatives with a different extent of modification. Aconitylated SOD (Aco-SOD) was only moderately taken up by the liver in spite of its negative charge density, whereas aconitylated BSA (Aco-BSA) with a smaller charge density was taken up by the liver very efficiently. Aco-BSA was more rapidly cleared by the liver than succinylated BSA due to the introduction of more anionic groups, especially when the degree of modification was low. Interestingly, highly aconitylated BSAs exhibited significant accumulation in the kidney at higher doses, especially when the hepatic uptake was saturated. Further analysis that was based on a physiological pharmacokinetic model including a saturable hepatic uptake process revealed that the higher the number of negative charges on the proteins, the higher was the apparent affinity of aconitylated proteins for the hepatic SRs. In general, the affinity of aconitylated proteins was higher than that of succinylated proteins when the degree of acylation was the same. Thus, the present study indicates that apart from charge density on the proteins the molecular size of the proteins is important for SR-mediated uptake in the liver. Aconitylation of proteins seems more suitable than succinylation for targeting of proteins to the liver nonparenchymal cells, in particular, at a low degree of acylation of the proteins at which the enzymatic activity is better retained for sufficient negative charges introduced.

Effect of interferons on P-glycoprotein-mediated rhodamine-123 efflux in cultured rat hepatocytes.

The effect of interferon (IFN)-beta and IFN-gamma on P-glycoprotein (P-gp)-mediated efflux of rhodamin-123(Rho-123), a typical substrate of P-gp, was studied in rat hepatocytes in primary culture. After treatment with IFN-beta, IFN-gamma, or both for 3 days, steady-state levels of Rho-123, incorporated into the hepatocytes, were measured to evaluate the P-gp activity. Whereas IFN-beta did not affect the intracellular level of Rho-123, IFN-gamma treatment caused a significant increase of the level, suggesting that IFN-gamma treatment suppresses the expression of P-gp or its activity. A combination of the two types of IFN exhibited a similar effect to that of IFN-gamma alone. The effect of IFN-gamma was still observed in the presence of H(2)O(2), which enhances the expression and activity of P-gp. Immunoblot analysis using a monoclonal antibody C219 revealed, however, that P-gp expression was increased after treatment with IFN-gamma, but only slightly by IFN-beta treatment. These results suggest that the enhanced Rho-123 uptake of rat primary hepatocytes induced by IFN-gamma does not result from reduced expression of P-gp but, rather, from impaired maturation or dysfunction of the efflux transporter.

Michaelis-Menten elimination kinetics of acetaldehyde during ethanol oxidation.

BACKGROUND: Acetaldehyde (AcH) is a toxic metabolite of ethanol (EtOH). The pharmacokinetics of blood AcH during EtOH oxidation was studied with or without the administration of aldehyde dehydrogenase 2 inhibitor (cyanamide) in rabbits. METHODS: An bolus of EtOH saline solution (0.25, 0.5, 1.0, 1.5, and 2.0 g/kg) was injected intravenously. Cyanamide was administered intraperitoneally (25 mg/kg body weight) to the cyanamide-treated group. Blood EtOH and AcH concentrations were measured by using head-space gas chromatography. RESULTS: In the control group, the first peak of the blood AcH appeared immediately and the second elevation appeared 1 to 4 hr after administration at a high EtOH dose. The blood AcH levels other than the second elevation part were significantly correlated to the blood EtOH levels. In the cyanamide-treated group, a peak and a plateau formed at the time corresponding to the second peak in the control group. The peak and plateau concentration of AcH increased markedly. We attempted simultaneous curve fitting, using the five blood EtOH and AcH concentration-time curves, to determine the pharmacokinetic model. Consequently, the AcH elimination was best described by a Michaelis-Menten kinetic model in both groups. CONCLUSIONS: The blood AcH profile was suggested to consist of the first and second components that are related to the blood EtOH concentration itself and the metabolic formation of AcH, respectively. With higher EtOH doses or aldehyde dehydrogenase 2 inhibition, the second component becomes prominent as a result of the capacity-limited property of the metabolism of AcH, which is described by Michaelis-Menten elimination kinetics.

Evaluation of capacity-limited first-pass effect through liver by three-points sampling in portal and hepatic veins and systemic artery.

PURPOSE: The three-points method was newly developed by sampling the portal and hepatic veins and systemic artery. A model of hepatic local disposition with the Michaelis-Menten elimination was proposed to explain the concentration dependency of the hepatic recovery ratio (F(H)). METHODS: 5-fluorouracil (5-FU) was selected as a model drug. 5-FU was administered orally 90 min after its intraarterial dose. Blood specimens in both femoral artery and hepatic vein were sampled after intraarterial dose, and blood specimens in both femoral artery and portal vein were taken after oral administration. RESULTS: It was shown that F(H) increased with an increase in the input drug concentration into the liver. The mean absorption time (MAT) estimated by nonlinear analysis agreed with the mean local absorption time (t(n)) whereas MAT by linear analysis was significantly smaller than t(a). CONCLUSIONS: The three-points method was newly developed, and the proposed nonlinear model explained well the capacity-limited elimination of 5-FU through the liver. MAT by the nonlinear analysis was in good agreement with t(a).

Pharmacokinetic analysis of in vivo disposition of succinylated proteins targeted to liver nonparenchymal cells via scavenger receptors: importance of molecular size and negative charge density for in vivo recognition by receptors.

In vivo disposition characteristics of succinylated (Suc-) proteins were studied after intravenous injection in mice in relation to their molecular characteristics as negatively charged macromolecules. Recombinant superoxide dismutase (SOD; molecular mass, 32 kDa), bovine serum albumin (BSA; molecular mass, 67 kDa), and bovine IgG (molecular mass, 150 kDa) were used to produce succinylated derivatives with different degrees of modification. (111)In-labeled Suc-SODs were rapidly excreted into the urine with no significant hepatic uptake. In contrast, (111)In-Suc-BSA and Suc-IgG were significantly taken up by liver nonparenchymal cells via scavenger receptors (SRs) according to the degree of succinylation and the dose injected. Interestingly, highly succinylated BSAs exhibited significant accumulation in the kidney at higher doses when the hepatic uptake was saturated. Pharmacokinetic analysis demonstrated that the hepatic uptake of succinylated proteins depended on the molecular size and the estimated surface density of succinylated amino residues. Further analysis based on a physiological pharmacokinetic model, involving a saturable process with Michaelis-Menten kinetics, revealed that the surface density of negative charges was correlated with the affinity of larger succinylated proteins for the hepatic SRs. Thus, the present study has provided useful basic information for a therapeutic strategy and the molecular design of succinylated proteins for use as drug carriers and therapeutic agents per se for SR-mediated targeting in vivo.