Development of a new minimally invasive phototherapy for lung cancer using antibody-toxin conjugate.

BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and laser irradiation. The effectiveness of current PDT using red light for advanced cancers is limited, because red light can only reach depths within a few millimeters. To enhance the antitumor effect for lung cancers, we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin and a PS, mono-L-aspartyl chlorin e6 (NPe6). METHODS: We examined whether cetuximab encapsulated in endosomes was released into the cytosol by PS in PDT under light irradiation. A431 cells were treated with fluorescein isothiocyanate-labeled cetuximab, NPe6, and light irradiation and were observed with fluorescence microscopy. We analyzed the cytotoxicity of saporin-conjugated cetuximab (IT-cetuximab) in A431, A549, and MCF7 cells and the antitumor effect in model A549-bearing mice in vivo using the iTAP method. RESULTS: Fluorescent microscopy analysis showed that the photodynamic effect of NPe6 (20 µM) and light irradiation (37.6 J/cm2 ) caused the release of cetuximab from the endosome into the cytosol. In vitro analysis demonstrated that the iTAP method enhanced the cytotoxicity of IT-cetuximab by the photodynamic effect. In in vivo experiments, compared with IT-cetuximab alone or PDT alone, the iTAP method using a low dose of IT-cetuximab showed the greatest enhancement of the antitumor effect. CONCLUSIONS: Our study is the first report of the iTAP method using NPe6 for lung cancer cells. The iTAP method may become a new, minimally invasive treatment superior to current PDT methods.

Development of drug discovery screening system by molecular interaction kinetics-mass spectrometry.

RATIONALE: Drug discovery studies invariably require qualitative and quantitative analyses of target compounds at every stage of drug discovery. We have developed a system combining molecular interaction analysis and mass spectrometry (LC-MS) using the principle of nanopore optical interferometry (nPOI) called molecular interaction kinetics-mass spectrometry (MIK-MS). Since nPOI has high binding capacity, the bond-dissociated compound can be directly detected using LC-MS. In this study, we use carbonic anhydrase II (CAII) as a ligand and apply six small compounds as analytes and report the affinity analysis using MIK-MS. METHODS: CAII was immobilized onto a COOH sensor chip using standard amine coupling. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions. An amount of 50 µL of mix solution was injected over the reference channel and sample channel for CAII immobilization. The solutions eluting from the sensor chip were collected from the waste-line of the SKi Pro system every 30 s. Reconstructed elution samples were then injected into the LC-MS/MS system. RESULTS: A mixture containing furosemide, acetazolamide, 4-sulfamoylbenzoic acid, 5-(dimethylamino)-1-naphthalene sulfonamide (DNSA), sulfanilamide and sulpiride (15 µM each) was injected into the CAII-immobilized sensor chip, and the fractions eluted from the SKi Pro system were collected and subjected to selected reaction monitoring LC-MS characterization. Specific results were obtained for acetazolamide, DNSA, furosemide and sulpiride. The results suggest that the association-dissociation curve of a mixed sample can be obtained by one-time MIK-MS analysis. CONCLUSIONS: Six small-molecule binders of CAII were analyzed quantitatively using nPOI and MIK-MS, and the results were compared to published surface plasmon resonance (SPR) results. The nPOI and SPR results show good agreement, confirming the reliability of the analysis. Time-dependent binding results may be obtained by our MS sensorgram approach. Drugs that meet medical needs in a short period are required; this nPOI-LC-MS system is considered an important tool for rapid drug discovery.

Primary malignant hepatic glucagonoma: an autopsy case.

A 73-year-old woman was admitted to our department for treatment of diabetes (plasma glucose 289 mg/dl, HbA(1C) 7.1%, and glycated albumin 34.9%). She displayed the signs and symptoms of glucagonoma syndrome, including necrolytic migratory erythema (NME), low aminoacidemia, and a marked increase of the serum glucagon level (4,940 pg/ ml). Thus, we suspected a glucagonoma causing secondary diabetes. However, we could not detect any mass in the pancreas or the gastrointestinal tract, and only found a liver lesion resembling a hemangioma. Her NME improved markedly after intravenous infusion of amino acids, and her plasma glucose was controlled reasonably well by intensive insulin therapy. However, her general condition deteriorated and she died on day 57 after hospitalization. At autopsy, the only tumor detected was the liver mass. This was a large solid tumor (8 x 6 x 5 cm) with a pattern of white and dark brown stripes located in the left lobe, while two white nodules were also found in the right lobe. Based on the histopathological and immunohistochemical findings, the liver lesion was shown to be a malignant glucagonoma with intrahepatic metastases. Since primary malignant hepatic glucagonoma has not been reported before, we present this extremely rare case of primary malignant glucagonoma of the liver.

No ghrelin response to oral glucose in diabetes mellitus with gastroparesis.

To investigate the role of ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, in diabetic gastroparesis, we evaluated the plasma ghrelin profile during the oral glucose tolerance test in 55 patients with diabetes (men/women: 36/19, mean +/- SE of age: 55.1 +/- 1.7 years) with or without gastroparesis (diagnosed by the (13)C-acetate breath test). We also further examined cardiac autonomic neuropathy by assessing 24-hour variation of the R-R interval in randomly selected 32 patients with diabetes (men/women: 23/9, mean +/- SE of age: 54.2 +/- 2.5 years), and evaluated the influence of autonomic neuropathy on ghrelin. The fasting plasma ghrelin level was significantly lower in diabetes mellitus with gastroparesis than in healthy controls (7.9 +/- 0.7 fmol/ml versus 16.6 +/- 5.3 fmol/ml, p = 0.006). Patients with diabetes with gastroparesis showed no decrease of plasma ghrelin after glucose loading, unlike patients without gastroparesis or healthy controls. Diabetes mellitus with autonomic neuropathy, but not those without it, also showed no decrease of plasma ghrelin after glucose loading. Diabetic gastroparesis may be related to ghrelin-associated neurohormonal abnormalities, but the pathophysiological meaning of this abnormal ghrelin response needs further clarification.

Fulminant type 1 diabetes complicated by leukemoid reaction.

A 28-year-old woman with severe ketoacidosis was admitted to our hospital on day 11 after giving birth. However, her HbA(1C) level was normal (5.2%) and both GAD and anti-insulin autoantibody were negative, and the WBC count was extremely high (57,500/ml) with immature leucocytes in the peripheral blood. Her WBC count decreased along with control of ketoacidosis and reduction of the plasma glucose level, and was normalized on day 5 after admission. Bone marrow aspiration subsequently showed no malignant cells, so the final diagnosis was fulminant type 1 diabetes combined with a leukemoid reaction. This is the first report of a patient with both fulminant type 1 diabetes and a leukemoid reaction. The mechanism that triggered the leukemoid reaction could not be clarified, but severe ketoacidosis may have been involved.