Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model.

INTRODUCTION: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. METHODS: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. RESULTS: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. CONCLUSIONS: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Useful Parameters in Dynamic Contrast-enhanced Ultrasonography for Identifying Early Response to Chemotherapy in a Rat Liver Tumor Model.

OBJECTIVES: The objective of the study is to determine a parameter on the time-intensity curve (TIC) of dynamic contrast-enhanced ultrasonography (DCE-US) that best correlates with tumor growth and to evaluate whether the parameter could correlate with the early response to irinotecan in a rat liver tumor model. MATERIAL AND METHODS: Twenty rats with tumors were evaluated (control: Saline, n = 6; treatment: Irinotecan, n = 14) regarding four parameters from TIC: Peak intensity (PI), k value, slope (PI × k), and time to peak (TTP). Relative changes in maximum tumor diameter between day 0 and 10, and parameters in the first 3 days were evaluated. The Mann-Whitney U-test was used to compare differences in tumor size and other parameters. Pearson's correlation coefficients (r) between tumor size and parameters in the control group were calculated. In the treatment group, relative changes of parameters in the first 3 days were compared between responder and non-responder (<20% and ≥20% increase in size on day 10, respectively). RESULTS: PI, k value, PI × k, and TTP significantly correlated with tumor growth (r = 0.513, 0.911, 0.665, and 0.741, respectively). The mean RC in k value among responders (n = 6) was significantly lower than non-responders (n = 8) (mean k value, 4.96 vs. 72.5; P = 0.003). CONCLUSION: Parameters of DCE-US could be a useful parameter for identifying early response to irinotecan.

Comparison of embolic effect between water-in-oil emulsion and microspheres in transarterial embolization for rat hepatocellular carcinoma model.

AIM: To compare two different embolic materials, water-in-oil (W/O) emulsion followed by gelatin particles and microspheres in transarterial embolization (TAE), using a rat hepatocellular carcinoma model. METHODS: Twenty rats bearing N1S1 cells were divided into the W/O emulsion group and Microsphere group. Water-in-oil emulsion was created by a glass membrane emulsification device. The tumor vascularity was measured by contrast-enhanced ultrasonography 24 h before and 10 min and 48 h after TAE. Tumor necrosis, hepatic infarction ratio surrounding the tumor, and locations of the embolic materials 48 h after TAE were assessed. The changes of serum liver enzymes were also evaluated. Statistical significance was determined by using either the Mann-Whitney U-test or Fisher's exact test. RESULTS: The tumor vascularity 48 h after TAE was significantly higher in the Microsphere group (20.1 vs. 3.76%, P = 0.016). The overflow of Lipiodol into the portal veins surrounding the tumor was seen, whereas microspheres were seen only in the artery. The percentage of necrotic area and complete response ratio in the W/O emulsion group was significantly higher (99.9 vs. 87.6%, P = 0.029 and 87.5 vs. 28.6%, P = 0.041, respectively). Serum aspartate aminotransferase and serum alanine aminotransferase levels 48 h after TAE were significantly higher in the W/O emulsion group (P < 0.01). CONCLUSIONS: The embolization using W/O emulsion followed by gelatin particles showed stronger antitumor effects with the occlusion of both the tumor feeding artery and the portal vein compared with microspheres, which occluded only the arteries.

Drug Release Property of Lipiodol Emulsion Formed by Glass Membrane Emulsification Device for Transarterial Chemoembolization.

PURPOSE: To evaluate physiochemical characteristics of emulsions formed by a modified emulsification device and to compare in vitro drug release properties of ethiodized oil (Lipiodol)-drug solution emulsion formed by the device and a 3-way-stopcock for conventional transarterial chemoembolization. MATERIALS AND METHODS: A V-shaped pumping emulsification device with a 100-µm-micropore glass membrane was developed to reduce the resistance of pumping. Epirubicin solution was mixed with Lipiodol (ratio 1:2) with pumping exchanges through the device. The percentage of water-in-oil (W/O) and droplet size distribution and viscosity were evaluated. The in vitro drug release properties were compared between using the device and a 3-way-stopcock. RESULTS: Percentage of W/O was 98.45 ± 0.03%. The median droplet size was 22.58 ± 1.70 µm, and the viscosity was 143.70 ± 12.36 cP. The released epirubicin at 0 min was 1.73 ± 1.05% in the device, whereas 41.02 ± 7.27% in a 3-way-stopcock (P < 0.001). The half-life of release (t50%) of the device was significantly longer than that of a 3-way-stopcock (175 ± 25 vs. 8 ± 6 min, P < 0.001). CONCLUSION: The V-shaped emulsification device with a 100-µm-micropore glass membrane can form nearly 100% W/O emulsion with homogenous droplet sizes. Emulsion formed by the device showed a slower epirubicin release property compared with that of a 3-way-stopcock.

Use of a Glass Membrane Pumping Emulsification Device Improves Systemic and Tumor Pharmacokinetics in Rabbit VX2 Liver Tumor in Transarterial Chemoembolization.

PURPOSE: To evaluate the phamacokinetics of epirubicin in conventional transarterial chemoembolization using a developed pumping emulsification device with a microporous glass membrane in VX2 rabbits. MATERIALS AND METHODS: Epirubicin solution (10 mg/mL) was mixed with ethiodized oil (1:2 ratio) using the device or 3-way stopcock. Forty-eight rabbits with VX2 liver tumor implanted 2 weeks prior to transarterial chemoembolization were divided into 2 groups: a device group (n = 24) and a 3-way-stopcock group (n = 24). Next, 0.5 mL of emulsion was injected into the hepatic artery, followed by embolization using 100-300-µm microspheres. The serum epirubicin concentrations (immediately after, 5 minutes after, and 10 minutes after) and the tumor epirubicin concentrations (20 minutes after and 48 hours after) were measured after transarterial chemoembolization. Histopathologic evaluation was performed with a fluorescence microscope. RESULTS: The area under the curve and maximum concentrations of epirubicin in plasma were 0.45 ± 0.18 µg min/mL and 0.13 ± 0.06 µg/mL, respectively, in the device group and 0.71 ± 0.45 µg min/mL and 0.22 ± 0.17 µg/mL, respectively, in the 3-way-stopcock group (P = .013 and P = .021, respectively). The mean epirubicin concentrations in VX2 tumors at 48 hours in the device group and the 3-way-stopcock group were 13.7 ± 6.71 and 7.72 ± 3.26 µg/g tissue, respectively (P = .013). The tumor necrosis ratios at 48 hours were 62 ± 11% in the device group and 51 ± 13% in the 3-way-stopcock group (P = .039). CONCLUSIONS: Conventional transarterial chemoembolization using the pumping emulsification device significantly improved the pharmacokinetics of epirubicin compared to the current standard technique using a 3-way stopcock.

Development of Repeatable Microcatheter Access Port for Intra-arterial Therapy of Liver Cancer.

PURPOSE: To develop an implantable port in which a microcatheter can be inserted for a combination therapy of repeated transarterial chemoembolization (TACE) and hepatic arterial infusion chemotherapy (HAIC) for advanced liver cancer. MATERIALS AND METHODS: The design of a currently used implantable port was modified. A funnel part was constructed in the port. The septum was punctured by a 20-gauge indwelling needle, and 2.0-Fr non-tapered microcatheter was inserted into the port. In the in vitro studies, the advance of a microcatheter out of the funnel part was evaluated via seven different septum puncture sites. A 5-Fr indwelling catheter connected to the port was placed in a vascular model, and a microcatheter catheterization was evaluated. In an in vivo study, the port-catheter system was implanted in the hepatic artery in a pig. A microcatheter was percutaneously inserted through the port into the hepatic arterial branches, and embolization was performed. RESULTS: In the in vitro studies, the microcatheter was smoothly advanced out of the port and catheterizations into the hepatic arteries were successful via all septum puncture sites. In the in vivo study, repeated selective embolization through the port was successfully conducted on 7, 14 and 21 days after the implantation. CONCLUSION: The developed implantable port can be used for repeated catheter insertion into the hepatic artery. The combination of repeated TACE and HAIC could be possible using this device.

Superabsorbent Polymer Microspheres Prepared with Hypertonic Saline to Reduce Microsphere Expansion.

PURPOSE: To analyze size changes of superabsorbent polymer (SAP) microspheres with the reduced expansion technique, and to evaluate pharmacological advantages of transarterial chemoembolization using cisplatin-loaded SAP microspheres with the reduced expansion technique. MATERIALS AND METHODS: In an in vitro study, diluted contrast materials containing different concentrations of sodium ions were examined to expand SAP microspheres and determined the reduced expansion technique. Size distributions of cisplatin-loaded SAP microspheres were analyzed. In an in vivo study, TACE was performed using cisplatin-loaded SAP microspheres with the reduced expansion and control techniques in 18 VX2 rabbits. RESULTS: The degree of expansion was reduced to the greatest extent by using a mixture of non-ionic contrast material and 10% NaCl at a 4:1 ratio. The mean diameter of the reduced expansion of cisplatin-loaded SAP microspheres was 188.4 µm, while that of the control expansion was 404.9 µm. The plasma platinum concentrations of the reduced expansion group at 5 min after TACE were significantly higher than those of the control expansion group (2.19 ± 0.77 vs. 0.75 ± 0.08 µg/mL, P = .01). The tumor platinum concentrations of the reduced expansion group at 1 h were significantly higher than those of the control expansion group (10.76 ± 2.57 vs. 1.57 ± 0.14 µg/g, P = .044). CONCLUSION: The expanding level of SAP microspheres can be reduced by using hypertonic saline. Cisplatin-loaded SAP microspheres with the reduced expansion technique have the advantages of achieving higher cisplatin tissue concentration in TACE for liver tumors.

Development of pumping emulsification device with glass membrane to form ideal lipiodol emulsion in transarterial chemoembolization.

PURPOSE: To evaluate a pumping emulsification device that can improve the physiochemical properties and stability of lipiodol emulsion for conventional transarterial chemoembolization. MATERIALS AND METHODS: A pumping emulsification device constructed of a glass membrane with a hydrophobic surface with pore size of 50 µm in diameter was placed between two syringe adaptors. Epirubicin solutions were mixed with lipiodol with pumping exchanges using the emulsification device or a three-way cock. The ratios of epirubicin solution to lipiodol were 1:2 or 1:1. A total of 120 emulsions were created. RESULTS: The emulsification device showed significantly higher percentages of water-in-oil when compared with the three-way cock (97.9 % vs. 68.9 % in 1:2 ratio, and 82.1 % vs. 17.8 % in 1:1 ratio, p < .001). Droplet sizes in the emulsification device were more homogenous. Mean droplet sizes and viscosities in the emulsification device did not show any significant changes for 30 min after pumping, whereas in the three-way cock, the droplet sizes significantly enlarged and viscosities significantly decreased (p=.023 and p=.002). CONCLUSION: The emulsification device can form a high percentage of water-in-oil emulsion with stable droplets sizes and viscosities. This developed device is promising to increase therapeutic effects in conventional transarterial chemoembolization. KEY POINTS: • We developed new device for transarterial chemoembolization for liver cancer. • The device can improve the physiochemical properties of lipiodol emulsion. • The device can increase the therapeutic effects in conventional transarterial chemoembolization.

Techniques to Form a Suitable Lipiodol-Epirubicin Emulsion by Using 3-Way Stopcock Methods in Transarterial Chemoembolization for Liver Tumor.

PURPOSE: To compare physicochemical properties of emulsions of ethiodized oil (Lipiodol; Guerbet, Villepinte, France) and epirubicin prepared using different techniques for conventional transarterial chemoembolization. MATERIALS AND METHODS: Lipiodol was mixed with epirubicin solution (8.33 mg/mL) by using a 3-way stopcock. The following technical parameters were compared: ratio of epirubicin solution to Lipiodol (1:2 vs 1:1), number of pumping exchanges through the stopcock (20 exchanges vs 10 exchanges), pumping speed (1 s/push vs 2 s/push), and first push syringe (epirubicin solution vs Lipiodol). RESULTS: The mean percentage of water-in-oil was 70.45 ± 1.51 in the 1:2 epirubicin-Lipiodol ratio and 16.03 ± 2.95 in the 1:1 ratio (P < .001). The first push syringe did not influence emulsion type. Median droplet sizes were significantly larger in the slower pumping speed (52.0 µm in 2 s vs 33.7 µm in 1 s; P < .001), whereas there was no significant difference in number of pumping exchanges. Droplet sizes enlarged during 30 minutes after pumping. Viscosity was lower in the 1:1 ratio and the slower pumping speed. Viscosity decreased during 30 minutes after pumping. CONCLUSIONS: The ratio of epirubicin to Lipiodol is a significant factor to form water-in-oil emulsions with higher viscosity. The percentage of water-in-oil is limited to 70% using current pumping techniques. The pumping speed strongly influences droplet size and viscosity.

Intraarterial Therapy Using Micellar Nanoparticles Incorporating SN-38 in a Rabbit Liver Tumor Model.

PURPOSE: To evaluate the pharmacokinetics of intraarterial (IA) administration of micellar nanoparticles incorporating SN-38 injection compared with intravenous (IV) administration in a rabbit liver tumor model. MATERIALS AND METHODS: In this animal care committee-approved study, 18 rabbits (mean weight, 3.89 kg; range, 3.20-4.70 kg) with VX2 liver tumors were divided into two groups (IA and IV). Micellar nanoparticles incorporating SN-38 (30 mg/kg) were injected through the left hepatic artery in the IA group and the right femoral vein in the IV group. NK012 and free SN-38 in the plasma, liver parenchyma, and tumors were measured within 24 hours. Histologic examinations were conducted at 2 and 24 hours. RESULTS: There were no significant differences in the serum area under the concentration-time curve (0-24 h) for free SN-38, at 1,500 and 1,310 µg∙min/mL in the IA and IV groups, respectively (P = .152). The IA group showed significantly higher free SN-38 concentrations in tumor tissues at all time points compared with the IV group (P = .002 at 3 min, P = .011 at 2 h, and P = .047 at 24 h). Histologic findings showed that significantly higher tumor necrosis ratios were observed in the IA group compared with the IV group at 24 hours (P = .028). CONCLUSIONS: Micellar nanoparticles could be a promising IA drug delivery system to achieve high tumor tissue concentrations of SN-38.

Pharmacokinetics and Histopathological Findings of Chemoembolization Using Cisplatin Powder Mixed with Degradable Starch Microspheres in a Rabbit Liver Tumor Model.

PURPOSE: The purpose of this study is to evaluate the pharmacokinetics and histopathological findings of transarterial chemoembolization (TACE) using cisplatin powder mixed with degradable starch microspheres (DSM) (Cis/DSM-TACE) compared with cisplatin arterial infusion (Cis-AI). MATERIALS AND METHODS: Eighteen rabbits with VX2 liver tumors were divided into two groups: Cis/DSM-TACE (n = 9) and Cis-AI (n = 9) groups. In the Cis/DSM-TACE group, a mixture of cisplatin powder and DSM was injected until stasis of hepatic arterial flow was achieved. In the Cis-AI group, cisplatin solution was infused. RESULTS: The platinum concentrations in VX2 tumors in the Cis/DSM-TACE group at 24 and 72 h were significantly elevated compared with those in the Cis-AI group (P = .016 and .019, respectively). There were no significant differences in the platinum concentrations in plasma. Histopathological examination revealed the presence of several microspheres inside the tumors at 1 h, which completely disappeared at 24 h. Tumor cell apoptosis at 1 h in the Cis/DSM-TACE group was more frequently observed compared with that in the Cis-AI group (P = .006). CONCLUSIONS: TACE using cisplatin powder mixed with DSM provides a higher drug concentration in tumors, thereby achieving stronger antitumor effects compared with arterial infusion of cisplatin solution.

Compatibility of an Ultraselective Microcatheter and Epirubicin Loaded 300-500-µm DC Bead in Ex Vivo Study.

PURPOSE: The purpose of this study is to examine whether epirubicin loaded DC Bead 300-500 µm in size can pass through a 1.8-Fr ultraselective microcatheter in ex vivo study. METHODS: Epirubicin (25 mg/1 mL) loaded 100-300 and 300-500 µm DC Bead were tested. Both sizes were diluted 5, 10, and 30 times using contrast material. Ultraselective microcatheter with the outer diameter of 1.8 Fr and the inner diameter of .017 inch (431.8 µm) was used. The diluted DC Bead was injected at a speed of 1 mL/min, and the pressure was continuously measured. The microspheres' shapes after ejection were observed by a stereomicroscope. RESULTS: The maximum pressure of contrast material alone was 8.40 ± 0.21 psi. The maximum pressure in 5, 10, and 30 times dilution groups of 100-300 µm were 9.67 ± 1.18, 9.25 ± 0.25, and 9.71 ± 0.28 psi, respectively, whereas 21.10 ± 10.2, 10.48 ± 2.14, 10.09 ± 0.37 psi, respectively in 300-500 µm groups. The maximum pressure in 5 times dilution group of 300-500 µm was significantly higher than the other groups (P < 0.05). In 300-500 µm, 4 of 10 measurements showed high pressure over 24 psi (the maximum value was 43.5 psi) in 5 times dilution group, whereas in 10 times and 30 times dilution groups, all measurements showed less than 12 psi. No damages of microspheres were found. CONCLUSIONS: Epirubicin loaded DC Bead 300-500 µm in size can pass through a 1.8-Fr ultraselective microcatheter. To avoid high resistance due to microspheres' aggregation, dilution more than 10 times is needed.

Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues.

Dihydropyrimidine dehydrogenase (DPD) is important to the antitumor effect of 5-fluorouracil (5-FU). DPD gene (DPYD) expression in tumors is correlated with sensitivity to 5-FU. Because the 5-FU accumulated in cancer cells is also rapidly converted into inactivated metabolites through catabolic pathways mediated by DPD, high DPD activity in cancer cells is an important determinant of the response to 5-FU. DPD activity is highly variable and reduced activity causes a high risk of 5-FU toxicity. Genetic variation in DPYD has been proposed as the main factor responsible for the variation in DPD activity. However, only a small proportion of the activity of DPD can be explained by DPYD mutations. In this study, we found that DPYD is a target of the following microRNAs (miRNA): miR-27a, miR-27b, miR-134, and miR-582-5p. In luciferase assays with HepG2 cells, the overexpression of these miRNAs was associated with significantly decreased reporter activity in a plasmid containing the 3'-UTR of DYPD mRNA. The level of DPD protein in MIAPaca-2 cells was also significantly decreased by the overexpression of these four miRNAs. The results suggest that miR-27a, miR-27b, miR-134, and miR-582-5p post-transcriptionally regulate DPD protein expression. The levels of miRNAs in normal lung tissue and lung tumors were compared; miR-27b and miR-134 levels were significantly lower in the tumors than normal tissue (3.64 ± 4.02 versus 9.75 ± 6.58 and 0.64 ± 0.75 versus 1.48 ± 1.39). DPD protein levels were significantly higher in the tumors. Thus, the decreased expression of miR-27b would be responsible for the high levels of DPD protein. This study is the first to show that miRNAs regulate the DPD protein, and provides new insight into 5-FU-based chemotherapy.

[Clinical evaluation of an oxygen concentrator and humidifier that does not require additional reservoir water].

A conventional humidifier with a reservoir of water for humidification can produce micro-aerosols contaminated with bacteria. The present study was undertaken to determine the clinical efficiency of a membrane humidifier that does not require additional reservoir water. We analyzed relative room air humidity and oxygen levels obtained from 2 pressure-swing adsorption (PSA)-type oxygen concentrators with membrane humidifiers. A significant correlation was found between relative room air humidity and that of oxygen moistened by a membrane humidifier. Several patients with chronic respiratory failure experienced improvements in subjectively reported nasal dryness using an oxygen concentrator with a membrane humidifier. This device avoids the need to change reservoir water, and may improve patient quality of life in the home.

Influence of intermittent hypoxia on the signal transduction pathways to inflammatory response and circadian clock regulation.

AIMS: Obstructive sleep apnea syndrome (OSAS), characterized by intermittent hypoxia/reoxygenation (IHR), is often associated with changing levels of circulating inflammatory cytokines and causes excessive daytime sleepiness, mood disturbances, and cardiovascular disease. An abnormal rhythm in the expression of circadian clock genes is observed in OSAS patients, and is also implicated in OSAS-related clinical symptoms. IHR-induced signal transduction is thought to underlie OSAS-associated complications. The aim of this study is to elucidate the influence of IHR on signal transduction pathways to inflammatory response and circadian clock regulation. MAIN METHODS: To evaluate the direct action of IHR on intracellular signaling, we used a cell culture model to explore the underlying transcriptional events initiated by IHR. KEY FINDINGS: Treatment of cultured human lung adenocarcinoma epithelial cells (A549) with IHR resulted in the elevation of mRNA levels of an inflammation cytokine interleukin-6 (IL-6), due to activation of the signaling pathway of nuclear factor-kappaB, a potent transcriptional activator of IL-6. On the other hand, the treatment of cells with IHR had little effect on clock gene response element-driven transcription. As a consequence, there was no significant change in mRNA levels of clock genes in IHR-treated cells. SIGNIFICANCE: These results suggest that IHR can activate signal transduction to an inflammatory response, but not to circadian clock regulation. The abnormal rhythm in the expression of clock genes in OSAS patients is attributable to the changed levels of circulating factors that have the ability to modulate clock gene expression.

Day-night variations of serum interleukin-6 in patients with severe obstructive sleep apnea syndrome before and after continuous positive airway pressure (CPAP).

Obstructive sleep apnea syndrome (OSAS) causes intermittent hypoxia and increases in sympathetic activity and contributes to cardiovascular disorders. Interleukin-6 (IL-6) is one of the important proinflammatory cytokines. We examined the levels of serum IL-6 concentrations in nine patients with severe OSAS at four different clock times during the 24 h before and after three months of continuous positive airway pressure (CPAP) therapy. Serum IL-6 levels were significantly reduced after CPAP therapy by 46% (6.2+/-1.0 vs. 3.3+/-0.4 pg/ml, p<0.005). No significant 24 h variation of serum IL-6 in severe OSAS patients was found before CPAP; however, a significant 24 h variation of serum IL-6 was found after CPAP. Intermittent hypoxia during sleep may contribute to systemic inflammation and result in an elevation of serum IL-6 in severe OSAS patients.

Severe toxicities after irinotecan-based chemotherapy in a patient with lung cancer: a homozygote for the SLCO1B1*15 allele.

Irinotecan is used widely in the treatment of several malignancies, but unpredictable severe toxicities such as myelosuppression and delayed-type diarrhea are sometimes experienced. Polymorphism of the UGT1A1 gene is one of the likely reasons for interindividual differences in irinotecan pharmacokinetics and severe toxicity. Also, polymorphic organic anion-transporting polypeptide 1B1 (OATP1B1, SLCO1B1) is reported to be involved in the hepatocellular uptake of SN-38. A 61-year-old man with lung cancer developed severe toxicities, including grade 3 diarrhea, grade 4 leukopenia, and grade 4 neutropenia, after the first cycle of irinotecan (60 mg/m) plus cisplatin chemotherapy. The irinotecan and SN-38 areas under the concentration-time curve from time zero to infinity in this patient were 43% and 87% higher than the corresponding mean values for 10 other patients with lung cancer treated with irinotecan (60-100 mg/m) normalized for the dose of irinotecan. Analysis of genetic variants in genes encoding the drug-metabolizing enzyme (UGT1A1) and transporter (SLCO1B1) involving irinotecan disposition revealed that this patient was homozygous for the SLCO1B1*15 allele, which may result in severe toxicities attributable to the extensive accumulation of SN-38. Screening of SLCO1B1*15 is suggested to be useful in irinotecan chemotherapy to avoid unpredicted severe toxicity, although the homozygous genotype is rare among the Japanese.

[A case of small-cell lung cancer effectively treated by bi-weekly administration of amrubicin].

The patient was a 62-year-old man with small-cell lung cancer (limited disease). He was treated with cisplatin (CDDP) plus etoposide(ETP) and concurrent radiotherapy as first-line treatment. Although a complete response was achieved, his pro-GRP elevated 10 months later. He was treated with several anticancer agents including, CDDP plus irinotecan (CPT-11), CPT-11, paclitaxel and amrubicin (AMR). Although, as a monotherapy, the administration of AMR (30 mg/m(2)) for 3 consecutive days (3-day schedule) seemed to be most effective, severe myelosuppression was observed, and this treatment was discontinued. We changed the treatment schedule to biweekly administration of AMR (30 mg/m(2)). The level of pro-GRP decreased, and it was maintained at a similar level for 7 months. No severe toxicity was observed during this period. This case suggests that the bi-weekly administration of AMR may be a useful option for the treatment of small-cell lung cancer when a 3-day AMR schedule is highly myelosuppressive.

Beta2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro.

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the beta2-adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of beta2-adrenoceptor agonists in BEAS-2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS-2B cells as verified by immunoblot analysis. beta2-adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP-CREB in BEAS-2B cells.

Cellular immune profile in patients with non-small cell lung cancer after weekly paclitaxel therapy.

Paclitaxel is a new agent for advanced non-small cell lung cancer (NSCLC). Weekly doses may enhance antitumor activity while minimizing toxicity, but little is known about immune recovery. Paclitaxel (80 mg/m2) was administered to 10 patients with NSCLC, weekly during 3-week cycles. Natural killer (NK) activity, CD3-CD16+CD56+ NK cells, and differential counts were monitored. NK activity appeared in all patients after treatment with paclitaxel therapy NK activity showed a 27 +/- 9% decrease (mean +/- SE) on protocol day 8 and a 37 +/- 7% decrease on day 15 (p < 0.05) recovering to 89 +/- 5% of baseline on day 29. With weekly paclitaxel, a decrease in NK cell function persisted through the first cycle but then recovered. Weekly paclitaxel may be less immunosuppressive than agents such as cisplatin.