2-Naphthol Moiety of Neocarzinostatin Chromophore as a Novel Protein-Photodegrading Agent and Its Application as a H2 O2 -Activatable Photosensitizer.

A 2-naphthol derivative 2 corresponding to the aromatic ring moiety of neocarzinostatin chromophore was found to degrade proteins under photo-irradiation with long-wavelength UV light without any additives under neutral conditions. Structure-activity relationship studies of the derivative revealed that methylation of the hydroxyl group at the C2 position of 2 significantly suppressed its photodegradation ability. Furthermore, a purpose-designed synthetic tumor-related biomarker, a H2 O2 -activatable photosensitizer 8 possessing a H2 O2 -responsive arylboronic ester moiety conjugated to the hydroxyl group at the C2 position of 2, showed significantly lower photodegradation ability compared to 2. However, release of the 2 from 8 by reaction with H2 O2 regenerated the photodegradation ability. Compound 8 exhibited selective photo-cytotoxicity against high H2 O2 -expressing cancer cells upon irradiation with long-wavelength UV light.

EPR: Evidence and fallacy.

The enhanced permeability and retention (EPR) of nanoparticles in tumors has long stood as one of the fundamental principles of cancer drug delivery, holding the promise of safe, simple and effective therapy. By allowing particles preferential access to tumors by virtue of size and longevity in circulation, EPR provided a neat rationale for the trend toward nano-sized drug carriers. Following the discovery of the phenomenon by Maeda in the mid-1980s, this rationale appeared to be well justified by the flood of evidence from preclinical studies and by the clinical success of Doxil. Clinical outcomes from nano-sized drug delivery systems, however, have indicated that EPR is not as reliable as previously thought. Drug carriers generally fail to provide superior efficacy to free drug systems when tested in clinical trials. A closer look reveals that EPR-dependent drug delivery is complicated by high tumor interstitial fluid pressure (IFP), irregular vascular distribution, and poor blood flow inside tumors. Furthermore, the animal tumor models used to study EPR differ from clinical tumors in several key aspects that seem to make EPR more pronounced than in human patients. On the basis of this evidence, we believe that EPR should only be invoked on a case-by-case basis, when clinical evidence suggests the tumor type is susceptible.

Neocarzinostatin as a probe for DNA protection activity--molecular interaction with caffeine.

Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role.

[Hepatocellular carcinoma with multiple lung metastasis resulting in long-term disease-free survival by transcatheter arterial infusion chemotherapy of SMANCS].

The patient was a 61-year-old female with alcoholic liver cirrhosis, who was admitted to our hospital due to elevation of AFP.During the evaluation, both abdominal ultrasound and enhanced abdominal CT revealed a hepatocellular carcinoma measuring 4 cm in the S6-7 region, complicated with an arteriovenous shunt.Additionally, the lung CT examination showed 20 isolated bilateral lung tumors, all of which were less than 1.4 cm in diameter. Following the diagnosis, we performed a transcatheter arterial infusion chemotherapy of SMANCS at 3 mg through the right heptic artery. Thereafter, the AFP level returned to normal. Additionally, the tumors previously observed in both liver and lung, and exhibited by both lung CT and enhanced abdominal MRI, had disappeared.The patient has been in clinical remission more than 10 years to date.

[Clinical experience of treatment of liver metastasis of renal cell carcinoma treated with SMANCS/Lipiodol therapy].

The treatment for metastatic renal cell carcinoma (RCC) has changed dramatically after the beginning of molecular-targeted therapies. However,the treatment for liver metastasis is still difficult in patients with metastatic RCC. We treated liver metastases (8 target lesions) of RCC with stylene-maleic acid neocarzinostatin (SMANCS)/Lipiodol therapy. At the treatment procedure,a catheter was inserted at the femoral artery (Seldinger's method),a microcatheter was selectively inserted into the branch of hepatic artery which fed the liver metastasis,and then SMANCS/Lipodol was infused. We treated 1,2 and 1 patient 4,2, and 1 time,respectively. One lesion treated with SMANCS/Lipodol was further treated by radiofrequency ablation 13 days later. Of 6 metastatic lesions which could be followed up for more than 6 months after the treatment,one had partial response for 4 months and 4 had stable disease for more than 6 months. Four of the 6 lesions shrunk after SMANCS/Lipiodol treatment. Two of 4 patients survived more than 18 months after the first SMANCS/Lipiodol therapy. In all 9 SMANCS/Lipiodol treatments,grade 1 liver dysfunction (44.4%),ascites (11.1%) and fatigue (11.1%) occurred after the treatments. These adverse events were all improved by conservative treatments. SMANCS/Lipiodol therapy can be a treatment option as local treatment for liver metastasis of RCC.

In vivo characterization of NcsB3 to establish the complete biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore.

Neocarzinostatin (NCS) is an enediyne antibiotic produced by Streptomyces carzinostaticus. The NCS chromophore consists of an enediyne core, a sugar moiety, and a naphthoic acid (NA) moiety. The latter plays a key role in binding the NCS chromophore to its apoprotein to protect and stabilize the bioactive NCS chromophore. In this study, we expressed three genes: ncsB (naphthoic acid synthase), ncsB3 (P450 hydroxylase), and ncsB1 (O-methyltransferase), in Streptomyces lividans TK24. The three genes were sufficient to produce 2-hydroxy-7-methoxy-5-methyl-1-naphthoic acid. Production was analyzed and confirmed by LC-MS and nuclear magnetic resonance. Here, we report the functional characterization of ncsB3 and thereby elucidate the complete biosynthetic pathway of NA moiety of the NCS chromophore.

Importance of loop dynamics in the neocarzinostatin chromophore binding and release mechanisms.

Neocarzinostatin (NCS) is an antitumor chromophore carrier protein with many therapeutic applications. To characterize its binding and release mechanism, we have carried out molecular dynamics (MD) simulations for apo-NCS and holo-NCS. Although the beta-sheet regions of the protein exhibit restricted motion, large atomic fluctuations were observed in the loop regions, especially loop 99-104. This loop undergoes a "door-opening" motion that can facilitate chromophore binding and release. Calculated NMR order parameters confirm the simulated loop flexibility. We also provide a proposed explanation for the release rate difference for two mutants F78L and F78A through our simulation. The binding site structures of holo-NCS were also validated by chemical shift perturbations. Based on these results, a new binding and release mechanism for the NCS chromophore is proposed.

[Successful treatment of multiple pulmonary and peritoneal recurrence of hepatocellular carcinoma with bronchial artery infusion therapy and PEIT followed by surgery--a case report].

A case of hepatocellular carcinoma, successfully treated with multimodal loco-regional treatments, is reported. An 80-year-old male presented with multiple pulmonary and peritoneal metastases 4 months after right heimihepatectomy for ruptured HCC. Bronchial artery infusion of mitomycin C induced pulmonary tumor regression and stabilization. Peritoneal tumor was treated by arterial infusion of SMANCS, followed by percutaneous injection of absolute ethanol, which ended in surgical removal in 28-postoperative month due to abscess formation. He had been well until right adrenal and left pulmonary metastases appeared. Resection of both metastases was carried out in 39-post hepatectomy month. Recurrent left pulmonary metastasis was treated with two sessions of bronchial artery infusion with no effect this time. Video-assisted partial resection of the left lung was performed in 54 post-hepatectomy month. But his AFP level kept rising. Eventually pulmonary metastasis recurred and tumor thrombus reached the left atrium 58 months after hepatectomy. He wanted no more treatment. He died of cerebral infarction caused by tumor thrombus. He enjoyed a good QOL for five years through multimodal loco-regional treatments.

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein.

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein+chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.

Transarterial chemotherapy alone versus transarterial chemoembolization for hepatocellular carcinoma: a randomized phase III trial.

BACKGROUND/AIMS: Transcatheter arterial chemoembolization (TACE) is a combination of transarterial infusion chemotherapy (TAI) and embolization, and has been widely used to treat patients with hepatocellular carcinoma (HCC). However, since the impact of adding embolization on the survival of patients treated with TAI had never been evaluated in a phase III study, we conducted a multi-center, open-label trial comparing TACE and TAI to assess the effect of adding embolization on survival. METHODS: Patients with newly diagnosed unresectable HCC were randomly assigned to either a TACE group or a TAI group. Zinostatin stimalamer was injected into the hepatic artery, together with gelatin sponge in the TACE group and without gelatin sponge in the TAI group. Treatment was repeated when follow-up computed tomography showed the appearance of new lesions in the liver or re-growth of previously treated tumors. RESULTS: Seventy-nine patients were assigned to the TACE group, and 82 were assigned to the TAI group. The two groups were comparable with respect to their baseline characteristics. At the time of the analysis, 51 patients in the TACE group and 58 in the TAI group had died. The median overall survival time was 646 days in the TACE group and 679days in the TAI group (p=0.383). CONCLUSIONS: The results of this study suggest that treatment intensification by adding embolization did not increase survival over TAI with zinostatin stimalamer alone in patients with HCC.

Elevating blood pressure as a strategy to increase tumor-targeted delivery of macromolecular drug SMANCS: cases of advanced solid tumors.

The purpose of this study is to evaluate the improved method of arterial infusion therapy of SMANCS (SX) with lipiodol under the angiotensin-induced hypertensive state for various difficult-to-treat solid tumors. Most patients were unresectable with no other therapeutic options, recurrence after resection, or patients do not respond to common treatments. The new method utilizes angiotensin II (AT) to induce hypertension (e.g. approximately 15-30 mmHg above norm) for 15-20 min. This method was successfully applied to metastatic liver cancer, cholangiocarcinoma, massive renal cell carcinoma, pancreatic and other abdominal solid cancers. This AT-induced hypertension resulted in remarkably enhanced tumor delivery accompanied by improved therapeutic response, and a shorter time to achieve 50% regression of tumor size with least toxicity. We demonstrated clinically herein improved therapy for various advanced solid tumors with SX by elevating the tumor blood flow selectively. This is the first clinical proof that modulations of vascular pathophysiology can uniquely accomplish enhanced tumor selective delivery of polymeric drugs and thus yielded better clinical outcome.

Congeners of the enediyne neocarzinostatin chromophore: designed agents for bulged nucleic acid targets.

Of the commonly recognized structural elements within nucleic acids, bulges are among the least developed as targets for small molecules. Bulges in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these targets would be of considerable utility to chemical biologists and medicinal chemists. Despite such opportunity, there is a dearth of small molecules available with affinity for bulges, which has hampered exploitation of these key targets. We have used guided chemical synthesis to prepare small molecules capable of binding to DNA and RNA bulges. Our design is based on a template which mimics a metabolite of the enediyne neocarzinostatin. The key spirocylic building block was formed through an intramolecular aldol process and the parent template shows pronounced affinity for 2 base bulges. Functionalization with specific aminosugar moieties confers nanomolar binding affinity for selected bulged DNA targets, and installation of reactive functional groups allows covalent modification of bulges. These rationally designed agents can now be used to study the stereochemistry and architecture of bulge-drug complexes and investigate the molecular biology of bulge induced processes. Members of this class have been shown to induce slipped synthesis of DNA, suggesting the agents, in addition to recognizing and binding to pre-formed bulges, can also induce bulge formation on demand.

Synthetic strategies to epoxydiynes and a key synthon of the neocarzinostatin chromophore.

We present herein our recent efforts towards the synthesis of epoxydiynes which represent an unusual structural feature of the neocarzinostatin chromophore. A number of different routes to these epoxydiynes have been explored with varying success. Ultimately a concise and convergent approach was developed, which involved the addition of an allenyl zinc bromide to propargylic ketones/aldehydes followed by epoxide formation. This new protocol enabled us to synthesise a fully elaborated epoxydiyne which will find application for our studies towards the total synthesis of the NCS chromophore.

Solution structure of a designed spirocyclic helical ligand binding at a two-base bulge site in DNA.

The solution structure of the complex formed between an oligodeoxynucleotide containing a two-base bulge (5'-CCATCGTCTACCTTTGGTAGGATGG) and SCA-alpha2, a designed spirocyclic helical molecule, has been elucidated. SCA-alpha2, a close mimic of the metabolite, NCSi-gb, of the DNA bulge-specific enediyne antibiotic neocarzinostatin, differs in possessing a more stable spirocyclic ring system and in lacking certain bulky groupings that compromise bulged DNA binding. This study provides a detailed comparison of the binding modes of the two complexes and provides new insights into the importance of shape and space, as opposed to simple nucleotide sequence, in complex formation at the bulge site. The two rigidly held aromatic rings of SCA-alpha2 form a right-handed helical molecular wedge that specifically penetrates the bulge-binding pocket and immobilizes the two bulge residues (GT), which point toward the minor groove, rather than the major groove as in the NCSi-gb.bulged DNA complex. The ligand aromatic ring systems stack on the DNA bulge-flanking base pairs that define the long sides of the triangular prism binding pocket. Like NCSi-gb, SCA-alpha2 possesses the natural N-methylfuranose moiety, alpha-linked to the benzindanol (BI) moiety. The amino sugar anchors in the major groove of the DNA and points toward the 3'-bulge-flanking base pair. Lacking the bulky cyclocarbonate of NCSi-gb, the SCA-alpha2.bulged DNA complex has a much less twisted and buckled 3'-bulge-flanking base pair (dG20.dC8), and the G20 residue stacks directly above the BI ring platform. Also, the absence of the methyl group and the free rotation of the methoxy group on the dihydronaphthanone (NA) moiety of SCA-alpha2 allow better stacking geometry of the NA ring above the 5'-bulge-flanking dG21.dC5 base pair. These and other considerations help to explain why NCSi-gb binds very poorly to bulged RNA and are consistent with the recent observation of good binding with SCA-alpha2. Thus, although the two complexes resemble each other closely, they differ in important local environmental details. SCA-alpha2 has a better hand-in-glove fit at the bulge site, making it an ideal platform for the placement of moieties that can react covalently with the DNA and for generating congeners specific for bulges in RNA.

An efficient synthesis of epoxydiynes and a key fragment of neocarzinostatin chromophore.

[reaction: see text] A key structural feature of the Neocarzinostatin chromophore is a reactive epoxydiyne. We present here a new method for the preparation of epoxydiynes by the addition of an allenyl zinc bromide to a propargylic ketone.

Formal total synthesis of neocarzinostatin chromophore.

[reaction: see text] An efficient route to the neocarzinostatin chromophore aglycon has been developed. The present strategy involves a stereoselective intramolecular acetylide-aldehyde cyclization to form the C5-C6 bond, followed by efficient installation of alpha-epoxide, naphthoate, and carbonate functionalities. The C8-C9-olefin was introduced by using the Martin sulfurane dehydration reaction to furnish the highly reactive aglycon.

[Case of gastric perforation after TAI (trancatheter arterial infusion) of SMANCS with special reference to accessory left gastric artery].

In 1993, a 55-year-old-man was diagnosed with chronic active hepatitis (HCV). In January 1999, a solitary hepatocellular carcinoma (HCC) was discovered in his liver S8, and a sub-segmental hepatectomy was performed. In July 1999, multiple recurrences in the liver were noticed, and on August 6, 1999, the first SMANCS-TAE was performed. After that, PEIT was added, and then on July 18, 2000 and November 9, 2000, a second and third SMANCS-TAE were carried out, respectively. This time multiple HCCs in the bilateral lobes were discovered, and the 4 th SMANCS-TAE was undergone on April 12, 2001. On a celiac angiogram, the right hepatic artery was shown to have been obliterated by the last TAE. In addition, accessory left gastric artery (accessory LGA) originating in the left hepatic artery (LHA) proximal to the umbilical point (UP) could be seen. So we advanced a microcatheter to the LHA distal to the accessory LGA and injected SMANCS (0.8 mg) into the left hepatic artery. On April 24, he was admitted to hospital by ambulance due to severe upper abdominal pain. The muscular defense was noticed, and an air pocket under the diaphragm was indicated on an X-ray. An emergency total gastrectomy and R-Y re-construction were performed under the diagnosis of gastric perforation. A hole of approximately 10 cm in diameter was found in the anterior wall between the cardia and the upper body, and the accessory left gastric artery (LGA) was obliterated. The principal known side effects of SMANCS are fever, nausea and vomiting. However, as far as this writer has investigated, gastric perforation has never been reported. SMANCS presumably can flow into the stomach wall through the accessory LGA, triggering necrosis of the gastric wall due to circulatory damage. Although arterial infusion of SMANCS is an effective treatment, it causes considerable vascular damage, so intensive follow-up treatment is necessary.

Solution NMR structure investigation for releasing mechanism of neocarzinostatin chromophore from the holoprotein.

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.

Stereochemical control of small molecule binding to bulged DNA: comparison of structures of spirocyclic enantiomer-bulged DNA complexes.

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and ent-DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. Ent-DDI is a left-handed wedge-shaped spirocyclic molecule whose aglycone portion is an enantiomer of DDI, which mimics the spirocyclic geometry of the natural product, NCSi-gb, formed by base-catalyzed activation of the enediyne antibiotic neocarzinostatin. The benzindanone moiety of ent-DDI intercalates between the A6.T21 and the T9.A20 base pairs, overlapping with portions of the purine bases; the dihydronaphthalenone moiety is positioned in the minor groove along the G7-T8-T9 bulge sequence; and the aminoglycoside is in the middle of the minor groove, approaching A20 of the nonbulged strand. This alignment of ent-DDI along the DNA helical duplex is in the reverse direction to that of DDI. The aminoglycoside moiety of ent-DDI is positioned in the 3' direction from the bulge region, whereas that of the DDI is positioned in the 5' direction from the same site. This reverse binding orientation within the bulge site is the natural consequence of the opposite handedness imposed by the spirocyclic ring junction and permits the aromatic ring systems of the two spirocyclic enantiomers access to the bulge region. NMR and CD data indicate that the DNA in the DDI-bulged DNA complex undergoes a larger conformational change upon complex formation in comparison to the ent-DDI-bulged DNA, explaining the different binding affinities of the two drugs to the bulged DNA. In addition, there are different placements of the bulge bases in the helical duplex in the two complexes. One bulge base (G7) stacks inside the helix, and the other one (T8) is extrahelical in the DDI-bulged DNA complex, whereas both bulge bases in the ent-DDI-bulged DNA complex prefer extrahelical positions for drug binding. Elucidation of the detailed binding characteristics of the synthetic spirocyclic enantiomers provides a rational basis for the design of stereochemically controlled drugs for bulge binding sites.