Sakae Unezaki

Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy

Abstract The extravasation of liposomes of different sizes into solid tumors after i.v. injection was visualized by in vivo fluorescence microscopy in mouse neuroblastoma C-1300-bearing mice. Liposomes composed of distearoylphosphatidylcholine/cholesterol (1/1 molar ratio) and 6 mol% distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared. The PEG-coated liposomes were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a liposome marker or with doxorubicin (DXR) as an aqueous-phase marker. Liposomes with an average diameter of 100–200 nm showed the greatest tumor accumulation. With time after injection of DiI-labeled liposomes, the tumor interstitial fluorescence intensity increased. Most fluorescent spots were located outside and around the vessel wall, indicating extravasation of intact liposomes. The perivascular distribution was heterogeneous. We also obtained the same fluorescence localization pattern with DXR released from extravasated liposomes after injection of DXR-encapsulated liposomes. No fluorescence from extravasated liposomes was detected in normal s.c. tissue; the fluorescent spots were observed only in the vessel wall. Our results indicate that small-size long-circulating liposomes are able to traverse the endothelium of blood vessels in tumors and extravasate into interstitial spaces. Moreover, encapsulated drug was released from extravasated liposomes in the tumor.

Enhanced delivery of doxorubicin to tumor by long-circulating thermosensitive liposomes and local hyperthermia.

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (180-200 nm), prepared from dipalmitoylphosphatidylcholine (DPPC)/distearoylphosphatidylcholine (DSPC) (9:1 (m/m)) and 6 mol% of ganglioside GM1 (GM1), with 95-98% entrapping efficiency by the pH-gradient method. 45% of the entrapped DXR was released from these GM1/DPPC/DSPC liposomes by incubation at 42 degrees C for 5 min in 20% serum or saline (this degree of release was lower than that of hydrophilic drugs such as cisplatin, due to the basic and amphiphilic nature of DXR). Inclusion of GM1 (6 mol%) endowed DPPC/DSPC liposomes with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system uptake over 6 h after injection. Concomitantly, DXR levels in blood remained high for long time. Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR loaded, long-circulating, thermosensitive (DXR-GM1/DPPC/DSPC) liposomes was 2.5-times or 6-times higher than that after treatment with DXR-DPPC/DSPC liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the treatment with DXR-GM1/DPPC/DSPC liposomes and hyperthermia resulted in effective tumor-growth retardation and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.

Enhanced tumor targeting and improved antitumor activity of doxorubicin by long-circulating liposomes containing amphipathic poly(ethylene glycol)

Abstract Enhanced delivery of doxorubicin (DXR) to colon 26 in mice was studied by the long-circulating liposomes (LCL) composed of distearoylphosphatidylcholine cholesterol (DSPC CH) (1 1. m m) and 6 mol° distearoyl phosphatidyl ethanolamine (DSPE) derivatives of poly(ethylene glycol) (PEG) with an average molecular weight of 1000. LCL was approximately 100 nm in mean diameter and encapsulated DXR with > 98% entrapping efficiency by the transmembrane pH gradient method. The control liposomes (LP) which had the same lipid composition, similar size and DXR entrapment as LCL. but did not have amphipathic PEG. were used for comparison. Liposomal DXR and free DXR were injected intravenously at a dose of 5 mg DXR kg to Balb c mice implanted subcutaneously with colon 26 carcinoma. DXR encapsulated in LCL showed high blood levels up to 24 h after injection, compared with the corresponding DXR-LP and free DXR. The value of the area under the curve (AUC) of blood was approximately 2.4-or 810-fold higher than that of DXR-LP or free DXR. respectively. LCL decreased the uptake amount of DXR by the reticuloendothelial system (RES) of liver and spleen. Both liposomal formulations effectively reduced the DXR concentrations in heart compared with free DXR. Compared with DXR-LP or free drug. DXR-LCL produced an approximately 3.6-or 10.5-fold increased DXR level in tumor. respectively, at 6 h after injection. The AUC value of tumor tissue for DXR-LCL was 3.4-or 9.4-fold higher than that of DXR-LP or free DXR. respectively. These high tumor levels of DXR by LCL corresponded to the prolonged residence feature of liposomes. Therapeutic experiments were performed with three different formulations of DXR. Administration of DXR-LCL at a dose of 10 mg DXR kg resulted in effective retardation of tumor growth and 2-fold prolongation of survival times compared with DXR-LP. free drug and saline. Our results indicate that DXR encapsulated in long-circulating liposomes is significantly more active against colon 26 carcinoma than the conventional liposome (DSPC CH. 1:1. m m) formulation of DXR and free drug. Thus long-circulating liposomes should be useful carriers of chemotherapeutic agents for the treatment of solid tumor.

Enhanced Tumor Targeting of Doxorubicin by Ganglioside GM1-bearing Long-circulating Liposomes

Doxorubicin (DXR) was encapsulated in long-circulating liposomes, composed of ganglioside GM1 (GM1)/distearoylphosphatidylcholine (DSPC)/cholesterol (CH) (0.13:1:1 in molar ratio) and sized to approximately 100 nm in mean diameter, with 98% entrapping efficiency by the transmembrane pH gradient method. Free DXR, DXR-DSPC/CH and DXR-GM1/DSPC/CH liposomes were injected intravenously into Colon 26 tumor-bearing Balb/c mice via the tail vein at a dose of 5.0 mg DXR/kg. DXR-GM1/DSPC/CH liposomes gave a higher blood level of the drug than did DXR-DSPC/CH liposomes or free DXR up to 24 hours after injection, and the area under the blood concentration-time curve (AUC) for DXR-GM1/DSPC/CH liposomes was 1.5 or 526 times higher than that for DXR-DSPC/CH liposomes or free DXR, respectively. DXR-GM1/DSPC/CH liposomes gave a decreased DXR concentration in the reticuloendothelial system (RES) of the liver and the spleen. Both liposomal formulations effectively reduced the DXR concentration in the heart as compared with that in the case of free DXR. At 6 hours after i.v. injection, DXR-GM1/DSPC/CH liposomes provided an approximately 3.3- or 9-fold higher peak DXR level in the tumor as compared with DXR-DSPC/CH liposomes or the free drug, respectively. These high tumor levels of DXR appear to reflect the prolonged residence time of the liposomes. The results suggest that encapsulation of DXR in GM1-bearing long-circulating liposomes will be useful for cancer chemotherapy.

Comparative study of the cellular pharmacodynamics of calcineurin inhibitors between patients with chronic renal failure awaiting renal transplantation and cirrhosis patients awaiting liver transplantation.

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1–7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC50s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10–100 times stronger than those of cyclosporine. The median IC50 value for cyclosporine against the CRF PBMCs was not significantly different from the median IC50 value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC50 value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs (p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.

Assessment of Cancer-Related Fatigue, Pain, and Quality of Life in Cancer Patients at Palliative Care Team Referral: A Multicenter Observational Study (JORTC PAL-09)

Introduction Cancer-related fatigue greatly influences quality of life in cancer patients; however, no specific treatments have been established for cancer-related fatigue, and at present, no medication has been approved in Japan. Systematic research using patient-reported outcome to examine symptoms, particularly fatigue, has not been conducted in palliative care settings in Japan. The objective was to evaluate fatigue, pain, and quality of life in cancer patients at the point of intervention by palliative care teams. Materials and Methods Patients who were referred to palliative care teams at three institutions and met the inclusion criteria were invited to complete the Brief Fatigue Inventory, Brief Pain Inventory, and European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 15-Palliative. Results Of 183 patients recruited, the majority (85.8%) were diagnosed with recurrence or metastasis. The largest group (42.6%) comprised lung cancer patients, of whom 67.2% had an Eastern Cooperative Oncology Group Performance Status of 0–1. The mean value for global health status/quality of life was 41.4, and the highest mean European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 15-Palliative symptom item score was for pain (51.0). The mean global fatigue score was 4.1, and 9.8%, 30.6%, 38.7%, and 20.8% of patients’ fatigue severity was classified as none (score 0), mild (1–3), moderate (4–6), and severe (7–10), respectively. Discussion Cancer-related fatigue, considered to occur more frequently in cancer patients, was successfully assessed using patient-reported outcomes with the Brief Fatigue Inventory for the first time in Japan. Results suggested that fatigue is potentially as problematic as pain, which is the main reason for palliative care.

Falsely Abnormally Elevated Blood Trough Concentration of Tacrolimus Measured by Antibody-Conjugated Magnetic Immunoassay in a Renal Transplant Recipient: A Case Report

This report presents a falsely abnormally elevated blood trough concentration (C(t)) of tacrolimus measured by antibody-conjugated magnetic immunoassay (ACMIA) methods in a renal transplant recipient. Because the C(t) of tacrolimus was 78.5 ng/mL at day 2 after a 52-year-old man underwent renal transplantation, we stopped the tacrolimus extended-release formulation. However, because the abnormally elevated blood C(t) continued in the range of 41.1-59.1 ng/mL, we then measured the tacrolimus concentration in a stored blood sample before renal transplantation, it was 43 ng/mL. Consequently, the day-7 blood sample was measured with both ACMIA and enzyme-linked immunoassay, showing C(t) values of 42.8 ng/mL and 0.89 ng/mL, respectively. Because the abnormally elevated C(t) was falsely measured by the ACMIA method, we restarted tacrolimus However, the calcineurin inhibitor was subsequently converted to cyclosporine at day 21 after renal transplantation. Although cyclosporine was also measured by ACMIA, there was not an abnormally elevated C(t). Subsequently, the tacrolimus concentration ratio in plasma and whole blood (P/B-tacrolimus concentration ratio) was measured by ACMIA in a posttacrolimus blood sample. The P/B-tacrolimus concentration ratio was 100%. In contrast, the P/B-tacrolimus concentration ratio was <30% in 2 control patients administered tacrolimus. It has been reported recently that there were cases showing falsely slightly elevated C(t) of tacrolimus within the therapeutic range of concentrations. Therefore, we must be careful not to reduce the tacrolimus dose falsely. We consider confirmatory methods for a falsely abnormally elevated C(t) of tacrolimus measured by ACMIA to (1) measure P/B-tacrolimus concentration ratio, (2) compare ACMIA with another measurement, and (3) evaluate a blood sample stored before tacrolimus administration.

Liposome Drug Delivery System for Murine Neuroblastoma

PURPOSE The effects of liposome-infused doxorubicin on C-1300 murine neuroblastoma were studied. The liposome surface was covered with polyethylene glycol to avoid migration toward the reticuloendothelial system and to prolong its presence in the bloodstream. Liposome-infused doxorubicin hydrochloride (DXR), an anthracycline was used as an anticancer antibiotic substance. METHODS Each A/J mouse was transplanted with 1 x 10(5) C-1300 murine neuroblastoma cells subcutaneously in the thigh. The experiment was conducted when the maximum tumor dimension was 1 cm. The control group was given only physiological saline solutions, the second group was given DXR alone, and the third group received liposome-infused DXR (Lip-DXR). The survival and doubling times were measured. One, 12, and 24 hours after the injection, the DXR concentration in the cardiac tissues was measured for statistical comparison. RESULTS The survival time of the mice was found to be 27+/-5.10 days in the control group, 31.40+/-3.15 days in the DXR group, and 43.86+/-2.13 days in the Lip-DXR group. The Lip-DXR group showed the longest survival time. The tumor-doubling time was found to be 9.07+/-2.30, 10.75+/-3.49, and 19.80+/-3.26 days, for each group, respectively. When comparing the DXR concentration in the heart tissues, the Lip-DXR-administered mice showed significantly lower DXR accumulation in the cardiac tissues after 1 and 12 hours than the DXR-administered mice. CONCLUSION This study proved that liposome-infused DXR could be used effectively on murine neuroblastoma (C-1300 tumor cell model) and may reduce the incidence of cardiac toxicity as compared with DXR alone.

Prevalence of Autoantibodies and the Efficacy of Immunotherapy for Autoimmune Cerebellar Ataxia.

OBJECTIVE Autoimmune cerebellar ataxias were recently reported to be treatable. However, the proportion of patients with cortical cerebellar atrophy of unknown etiology with autoimmune-associated cerebellar ataxia and the actual effectiveness of immunotherapy in these diseases remain unknown. METHODS We measured the level of autoantibodies (including anti-gliadin antibody, anti-glutamic acid decarboxylase (GAD) antibody, and anti-thyroid antibody) in 58 Japanese patients with cerebellar ataxia, excluding those with multiple system atrophy, hereditary spinocerebellar ataxia, cancer, or those who were receiving phenytoin, and the efficacy of immunotherapy was assessed. RESULTS Thirty-one of 58 (53%) patients were positive for anti-GAD antibody, anti-gliadin antibody, or anti-thyroid antibody. Seven of the 12 anti-gliadin antibody-positive patients, three of the four anti-GAD antibody-positive patients, and three of the six anti-thyroid antibody-positive patients responded well to immunotherapy, indicating that 59% of patients with ataxia-associated antibody-positive cerebellar ataxia undergoing immunotherapy responded well. CONCLUSION Some patients with cerebellar ataxia have autoimmune conditions and diagnosing autoimmune cerebellar ataxia is therefore an important component in the care of patients with this disease entity.