Jun Ichiro Kuroda

Potent antitumor effect of SN‐38‐incorporating polymeric micelle, NK012, against malignant glioma

Recent published reports on clinical trials of CPT‐11 indicate the effectiveness of this compound, a prodrug of SN‐38, against malignant glioma in combination with anti‐vascular endothelial growth factor antibody. Here, we determined if NK012, and SN‐38 incorporating micelle, can be an appropriate formulation for glioblastoma treatment compared with CPT‐11. In vitro cytotoxicity was evaluated against several glioma lines with NK012, CPT‐11, SN‐38, ACNU, CDDP and etoposide. For the in vivo test, a human glioma line (U87MG) transfected with the luciferase gene was inoculated into nude mice brain for pharmacokinetic analysis by fluorescence microscopy and high‐performance liquid chromatography after intravenous injection of NK012 and CPT‐11. In vivo antitumor activity of NK012 and CPT‐11 was evaluated by bioluminescence image and Kaplan‐Meier analyses. The growth‐inhibitory effects of NK012 were 34‐ to 444‐fold more potent than those of CPT‐11. Markedly enhanced and prolonged distribution of free SN‐38 in the xenografts was observed after NK012 injection compared with CPT‐11. NK012 showed significantly potent antitumor activity against an orthotopic glioblastoma multiforme xenograft and significantly longer survival rate than CPT‐11 (p = 0.0014). This implies that NK012 can pass through the blood brain tumor barrier effectively. NK012, which combines enhanced distribution with prolonged sustained release, may be ideal for glioma treatment. Currently, a phase I study of NK012 is almost complete in Japan and the US. The present translational study warrants the clinical phase II study of NK012 in patients with malignant glioma.

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin‐incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC‐6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid–labile hydrazone bond. The conjugate forms a micellar structure of 40–80 nm in diameter in an aqueous milieu. NC‐6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC‐6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC‐6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC‐6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin‐treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC‐6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC‐6300 in patients with hepatocellular carcinoma or other cancers.

Antitumor Activity of NK012 Combined with Cisplatin against Small Cell Lung Cancer and Intestinal Mucosal Changes in Tumor-Bearing Mouse after Treatment

Purpose: To investigate the advantages of treatment with the SN-38–incorporating polymeric micelles NK012 over CPT-11 in combination with cisplatin [cis-dichlorodiammineplatinum (II) (CDDP)] in mice bearing a small cell lung cancer xenograft in terms of antitumor activity and toxicity, particularly intestinal toxicity. Experimental Design: Cytotoxic effects were evaluated in human small cell lung cancer cell lines [H69, H82, and vascular endothelial growth factor (VEGF)–secreting cells (SBC-3/VEGF and its mock transfectant SBC-3/Neo)]. In vivo antitumor effects were evaluated in SBC-3/Neo–bearing and SBC-3/VEGF–bearing mice after NK012/CDDP or CPT-11/CDDP administration on days 0, 7, and 14. Drug distribution was analyzed by high-performance liquid chromatography or fluorescence microscopy, and the small intestine was pathologically examined. Results: The in vitro growth-inhibitory effects of NK012 were 198- to 532-fold more potent than those of CPT-11. A significant difference in the relative tumor volume on day 30 was found between NK012/CDDP and CPT-11/CDDP treatments (P = 0.0058). Inflammatory changes in the small intestinal mucosa were rare in all NK012-treated mice but were commonly observed in CPT-11–treated mice. Moreover, a large amount of CPT-11 was excreted into the feces and high CPT-11 concentration was detected in the small intestinal epithelium. On the other hand, a small amount of NK012 was found in the feces and NK012 was weakly and uniformly distributed in the mucosal interstitium. Conclusions: NK012/CDDP combination may be a promising candidate regimen against lung cancer without severe diarrhea toxicity and therefore warrants further clinical evaluation.

The inhibition of pancreatic cancer invasion-metastasis cascade in both cellular signal and blood coagulation cascade of tissue factor by its neutralisation antibody

Tissue factor (TF), the initiating cell surface receptor for the blood coagulation cascade, plays an important role in malignant transformation of the pancreas, although the precise mechanism remains unresolved. Here, we report that the TF - factor VIIa complex in human pancreatic cancer cells produced a significant amount of MMP-9 and promoted invasion ability in vitro and invasion and metastasis in vivo. For treatment, we successfully developed an anti-human TF monoclonal antibody that inhibits both cellular signalling and blood coagulation cascade via TF. Invasive capability and MMP-9 expression were significantly reduced by the antibody. The antibody inhibited not only tumour invasion in the orthotopic model, but also haematogenous metastasis in the portal-injection liver metastasis model. In conclusion, the TF-VIIa complex plays an important role in invasion-metastasis by enhancing tumour cell infiltration ability and forming microthrombi. The newly established anti-human TF neutralisation antibody may be useful for the treatment of pancreatic and other invasive cancers.

Discovery of an uncovered region in fibrin clots and its clinical significance

Despite the pathological importance of fibrin clot formation, little is known about the structure of these clots because X-ray and nuclear magnetic resonance (NMR) analyses are not applicable to insoluble proteins. In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102–10) recognises an uncovered region that is exposed only when a fibrin clot forms. The epitope of the 102–10 mAb was mapped to a hydrophobic region on the Bβ chain that interacted closely with a counterpart region on the γ chain in a soluble state. New anti-Bβ and anti-γ mAbs specific to peptides lining the discovered region appeared to bind exclusively to fibrin clots. Furthermore, the radiolabelled 102–10 mAb selectively accumulated in mouse spontaneous tumours, and immunohistochemistry using this mAb revealed greater fibrin deposition in World Health Organization (WHO) grade 4 glioma than in lower-grade gliomas. Because erosive tumours are apt to cause micro-haemorrhages, even early asymptomatic tumours detected with a radiolabelled 102-10 mAb may be aggressively malignant.

Antitumour activity of NK012, SN-38-incorporating polymeric micelles, in hypovascular orthotopic pancreatic tumour

Human pancreatic cancer is refractory to chemotherapy partly because of blockage to penetration of anticancer agents. This issue must be taken into account particularly for the drug delivery system (DDS). The aim of the present study is to investigate how NK012 (SN-38-incorporating polymeric micelles) categorised as DDS exerts its antitumour effect in an orthotopic pancreatic tumour model compared with gemcitabine and irinotecan hydrochloride (CPT-11), a low-molecular-weight prodrug of a 7-ethyl-10-hydroxy-camptothecin (SN-38). The maximum tolerated doses (MTDs) of NK012 (30 mg/kg/d), CPT-11 (66.7 mg/kg/d) and gemcitabine (16.5mg/kg/d) were administered to mice bearing human pancreatic cancer cell (SUIT-2) xenografts implanted orthotopically. Antitumour effects of these compounds were evaluated. Drug distribution within the tumour was examined by fluorescence microscopy and high performance liquid chromatography (HPLC). NK012 exerted potent antitumour effects compared with CPT-11 and gemcitabine. A high concentration of NK012 and SN-38 released from NK012 had been observed until 192h. On the other hand, SN-38 converted from CPT-11 was detected only 1h postinjection. Fluorescence from NK012 was detected up to 48h, whereas that from CPT-11 almost disappeared by 24h postinjection. NK012 appeared to exert potent antitumour activity against intractable stroma-rich orthotopic pancreatic tumour xenografts due to its sufficient accumulation followed by the effective sustained release of SN-38 from NK012.

Synergistic antitumor activity of the SN-38-incorporating polymeric micelles NK012 with S-1 in a mouse model of non-small cell lung cancer

The combination therapy of CPT‐11, a prodrug of SN‐38, with S‐1, a dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine, shows a high clinical response rate in non‐small cell lung cancer (NSCLC). However, this combination causes severe toxicities such as diarrhea. Here, we investigated the advantages of treatment with the SN‐38‐incorporating polymeric micelles NK012 over CPT‐11 in combination with S‐1 in mice bearing a NSCLC xenograft in terms of antitumor activity and toxic effects, particularly intestinal toxicity. In vitro cytotoxic effects were examined in human NSCLC cell lines (A549, PC‐9, PC‐14, EBC‐1 and H520). In vivo antitumor effects were evaluated in PC‐14‐ and EBC‐1‐bearing mice after NK012 or CPT‐11 administration on Days 0 and 7 and S‐1 administration on Days 0–13. Pathological changes in the small intestine were also investigated. The in vitro growth inhibitory effects of NK012 were 56.8‐ to 622‐fold more potent than those of CPT‐11. NK012/S‐1 treatment showed significantly higher antitumor activity both in PC‐14‐bearing (p = 0.0007) and EBC‐1‐bearing mice (p < 0.0001) than CPT‐11/S‐1 treatment. The deformity and decrease in the density of intestinal villi were more severe in CPT‐11/S‐1‐treated mice than in NK012/S‐1‐treated mice. NK012/S‐1 combination is a promising candidate regimen against NSCLC without inducing toxicities such as severe diarrhea and therefore warrants clinical evaluation.

The antitumor activity of NK012, an SN-38-incorporating micelle, in combination with bevacizumab against lung cancer xenografts.

It has been demonstrated that NK012, a novel 7‐ethyl‐10‐hydroxycamptothecin (SN‐38)‐incorporating polymeric micelle, exerts significantly more potent antitumor activity against various human tumor xenografts than irinotecan (CPT‐11) (a water‐soluble prodrug of SN‐38). Combination therapy of anticancer agents with bevacizumab (Bv), an anti‐vascualr endothelial growth factor humanized monoclonal antibody, has more potently inhibited tumor growth than either agent alone. In the current study, the authors examined the antitumor effect of NK012 in combination with Bv against human lung cancer.

Multiple peripheral middle cerebral artery aneurysms associated with Behcet's disease.

SummaryWe report a 19-year-old woman with Behcet’s disease who suffered a subarachnoid hemorrhage and had bilateral peripheral middle cerebral artery aneurysms. After steroid therapy for 3 days, the smaller aneurysm disappeared. The larger aneurysm was excised and the artery reconstructed using a superficial temporary artery graft. Histological examination showed vasculitis restricted to the wall of the aneurysm. This is the first report of arterial reconstruction for an aneurysm associated with Behcet’s disease. Steroid therapy before the operation may facilitate repair of the arterial wall.

Higher incidence of epilepsy in meningiomas located on the premotor cortex: a voxel-wise statistical analysis

BackgroundA substantial number of patients with brain tumors develop recurrent seizures, known as tumor-associated epilepsy. It is important to identify specific subgroups of brain tumor patients with higher incidences of epilepsy because a meta-analysis failed to certify the effectiveness of prophylactic anti-epileptic drugs (AEDs) to abort tumor-associated epilepsy as a whole.MethodsTo investigate the relationship between tumor location and incidence of epilepsy, we performed voxel-wise comparison between 3D MRI scans obtained from patients with meningioma-associated epilepsy and those from control patients using spatial normalization techniques on neuroimaging data. Variables such as age, tumor size, the degree of edema, and pathological diagnosis were also compared between the two groups.ResultsOur results showed the highest incidence of epilepsy when the tumor was located on the premotor cortex in the frontal lobe (Z-scores >2.0, Liebermeister’s quasi-exact test). The stepwise multiple regression analysis on the clinical data revealed that the tumor diameter (p < 0.001) and the patient’s age (p = 0.024) were positive and negative predictors, respectively, for the onset of epilepsy.ConclusionsThe incidence of epilepsy was higher in meningiomas located on the premotor cortex than on the other cortex. Larger volume also contributed to the onset of epilepsy. We suggest that variations of epilepsy incidence dependent on tumor characteristics can be considered when treating tumor-associated epilepsy.