Rationale and design of the CRAFT (Continuous ReAssessment with Flexible ExTension in Rare Malignancies) multicenter phase II trial.

BACKGROUND: Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. PATIENTS AND METHODS: Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K-AKT pathway activity; (vi) aberrations predicting increased RAF-MEK-ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon's optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. CONCLUSIONS: CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. TRIAL REGISTRATION NUMBERS: EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521.

Assessment of UV spectrophotometry for determination of plasmid DNA concentration in vector preparations for human gene therapy products.

The European Pharmacopoeia (Ph. Eur.) general chapter 5.14. Gene transfer medicinal products for human use suggests the use of absorbance measurements at 260 nm to determine the DNA concentration of plasmid vectors used for the preparation of gene therapy products for human use. An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to confirm the suitability of UV spectrophotometry for the quantification of plasmid vectors used in gene therapy (GT). Three Official Medicine Control Laboratories (OMCLs of the European OMCL Network) and members of the OMCL Working Group for GT products took part in the study, in which various types of spectrophotometers were assessed using common test samples. Results of the study demonstrated that UV spectrophotometry can be considered suitable for the quantification of plasmid DNA in GT products regardless of the instrument used.

Alpha subunit-dependent glycine receptor clustering and regulation of synaptic receptor numbers.

Accumulation of glycine receptors at synapses requires the interaction between the beta subunit of the receptor and the scaffold protein gephyrin. Here, we questioned whether different alpha subunits could modulate the receptors' diffusion and propensity to cluster at spinal cord synapses. Using quantitative photoactivated localisation microscopy we found that alpha-1 and alpha-3 containing glycine receptors display the same α3:ß2 stoichiometry and gephyrin binding. Despite these similarities, alpha-3 containing receptors are less mobile and cluster at higher density compared to alpha-1, with 1500 versus 1100 complexes µm-2, respectively. Furthermore, we identified a subunit-specific regulation of glycine receptor copy numbers at synapses: when challenged with interleukin 1ß, the synaptic occupancy of alpha-1 but not alpha-3 receptors was reduced. This mechanism may play a role in the cell-type dependent regulation of glycinergic currents in response to interleukin 1ß and highlights the capacity of the alpha subunits to affect receptor-gephyrin binding at synapses.

Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

BACKGROUND: The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. METHODS: Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). RESULTS: As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. CONCLUSION: Curcumin and VAE can inhibit the proliferation and viability of STS cells.

Protein aggregation and prionopathies.

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays.

SS18-SSX fusion protein-induced Wnt/ß-catenin signaling is a therapeutic target in synovial sarcoma.

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/ß-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/ß-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear ß-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/ß-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/ß-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/ß-catenin protein-protein interaction significantly blocked the canonical Wnt/ß-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/ß-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/ß-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer.

BACKGROUND: Biliary tract cancers (BTC) are relatively rare malignant tumours with poor prognosis. It is known from other solid neoplasms that antitumour inflammatory response has an impact on tumour behaviour and patient outcome. The aim of this study was to provide a comprehensive characterisation of antitumour inflammatory response in human BTC. METHODS: Tumour-infiltrating T lymphocytes (CD4+, CD8+, and Foxp3+), natural killer cells (perforin+), B lymphocytes (CD20+), macrophages (CD68+) as well as mast cells (CD117+) were assessed by immunohistochemistry in 375 BTC including extrahepatic (ECC; n=157), intrahepatic (ICC; n=149), and gallbladder (GBAC; n=69) adenocarcinomas. Overall and intraepithelial quantity of tumour-infiltrating immune cells was analysed. Data were correlated with clinicopathological variables and patient survival. RESULTS: The most prevalent inflammatory cell type in BTC was the T lymphocyte. Components of the adaptive immune response decreased, whereas innate immune response components increased significantly in the biliary intraepithelial neoplasia - primary carcinoma - metastasis sequence. BTC patients with intraepithelial tumour-infiltrating CD4+, CD8+, and Foxp3+ T lymphocytes showed a significantly longer overall survival. Number of total intraepithelial tumour-infiltrating Foxp3+ regulatory T lymphocytes (HR: 0.492, P=0.002) and CD4+ T lymphocytes (HR: 0.595, P=0.008) were tumour grade- and UICC-stage-independent prognosticators. The subtype-specific evaluation revealed that the tumour-infiltrating lymphocytic infiltrate is a positive outcome predictor in ECC and GBAC but not in ICC. CONCLUSION: Our findings characterise the immune response in cholangiocarcinogenesis and identify inflammatory cell types that influence the outcome of BTC patients. Further, we show that BTC subtypes show relevant differences with respect to density, quality of inflammation, and impact on patient survival.

Enhancement of the radiation response of EMT-6 tumours by a copper octabromotetracarboranylphenylporphyrin.

OBJECTIVE: The carborane-containing porphyrin, copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPBr), was investigated as a potential radiation enhancing agent for X-ray radiotherapy (XRT) in a subcutaneously implanted EMT-6 murine carcinoma. METHOD: The biodistribution and toxicological profile of this porphyrin has been shown to be favourable for another bimodal radiotherapy technique, boron neutron-capture therapy. For the XRT studies, CuTCPBr was formulated in either 9% Cremophor (BASF Corporation, Ludwigschafen, Germany) EL and 18% propylene glycol (9% CRM) or a revised formulation comprising 1% Cremophor ELP, 2% Tween 80 (JT Baker, Mansfield, MA), 5% ethanol and 2.2% PEG 400 (CTEP formulation), which would be more clinically acceptable than the original 9% CRM formulation. Using the 9% CRM formulation of CuTCPBr, doses of 100, 210 or 400 mg kg(-1) of body weight were used in combination with single doses of 25-35 Gy 100 kVp X-rays. RESULTS: While doses of 100 mg kg(-1) and 210 mg kg(-1) did not result in any significant enhancement of tumour response, the 400 mg kg(-1) dose did. A dose modification factor of 1.20±0.10 was obtained based on the comparison of doses that produced a 50% local tumour control probability. With the CTEP formulation of CuTCPBr, doses of 83 and 170 mg kg(-1) produced significant radiation enhancement, with dose modification factors based on the TCP(50) of 1.29±0.15 and 1.84±0.24, respectively. CONCLUSION: CuTCPBr significantly enhanced the efficacy of XRT in the treatment of EMT-6 carcinomas in mice. The CTEP formulation showed a marked improvement, with over 9% CRM being associated with higher dose modification factors. Moreover, the radiation response in the skin was not enhanced.

[Pleomorphic high-grade soft tissue sarcomas: is the subclassification up to date?]. / Pleomorphe High-grade-Sarkome der Weichgewebe : Ist die Subklassifikation zeitgemäß?

The conceptual evolution in the classification of pleomorphic high-grade sarcomas is a paradigm of how the integrative morphological, immunohistochemical and molecular genetic analysis has contributed to a clinical, prognostic and therapy-oriented characterization of this complex group of tumors. The clinical and prognostic relevance of a refined subtyping of pleomorphic high-grade sarcomas, which until recently was considered a mere academic exercise, is now undisputed. It is imperative to unequivocally differentiate sarcomas from non-sarcomatous, clearly defined malignancies to start adequate therapy. Furthermore, pleomorphic sarcomas which are particularly aggressive and prone to poor prognosis, have to be separated from sarcomas which, in contrast to the pleomorphic phenotype, are characterized by a less aggressive behavior. Also, morphologically pleomorphic but benign mesenchymal tumors must be recognized. Finally, it is important to promote the promising, array-based identification of diagnostic, prognostic and clinically relevant gene signatures on larger collections of pathomorphologically and clinically precisely defined subtypes of pleomorphic high-grade sarcomas.

[Molecular pathology of sarcomas. Update on the research group "Molecular Diagnosis of Sarcomas"]. / Molekularpathologie von Sarkomen. Erste Ergebnisse des Sarkomforschungsverbundes KoSar.

To establish precise diagnostic algorithms and standardised treatment of sarcomas in specialized centers, the interdisciplinary research group KoSar (sarcoma competence network) has been funded by German Cancer Aid. A sarcoma tissue repository and a diagnostic reference center have been set up, presently containing about 1000 accurately diagnosed sarcomas of different entities. Significant gene expression profiles for synovial sarcomas, leiomyosarcomas, myxoid liposarcomas and a small profile for myxofibrosarcomas as well as a new classification of angiosarcomas were defined. We systematically searched for activated signal transduction pathways in sarcoma cell lines and xenograft transplant models and candidate targets for molecular therapies were identified. Based on these results first clinical studies have been initiated by the German Interdisciplinary Sarcoma Study Group (GISG).

Regulatory requirements for clinical trial and marketing authorisation application for gene therapy medicinal products.

Over the last two decades, clinical trials using gene therapy medicinal products (GTMPs) have been carried out for a large number of rare, inherited monogeneic disorders as well as common multigeneic diseases such as cancer, cardiovascular and infectious diseases including AIDS. Despite some early difficulties and setbacks, the gene therapy field has slowly progressed and, nowadays, offers the promise of novel treatments for a growing number of diseases. On the other hand, gene therapy approaches are often associated with additional risks due to limited clinical experience with a given gene transfer system, long-lasting effects of the therapeutic gene, and/or a complex mode of action. As a result, specific regulations and guidelines have been introduced within the EU to help address these uncertainties. This article summarises the legislative framework and will provide an overview on the regulatory requirements for clinical trials and marketing authorisation applications.

Respiratory Deleted in Malignant Brain Tumours 1 (DMBT1) levels increase during lung maturation and infection.

Deleted in Malignant Brain Tumours 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein that binds and aggregates various bacteria and viruses in vitro. Studies in adults have shown that DMBT1 is expressed mainly by mucosal epithelia and glands, in particular within the respiratory tract, and plays a role in innate immune defence. We hypothesized that respiratory DMBT1 levels may be influenced by various developmental and clinical factors such as maturity, age and bacterial infection. DMBT1 levels were studied in 205 tracheal aspirate samples of 82 ventilated preterm and full-term infants by enzyme-linked immunosorbent assay. Possible effects of various clinical parameters were tested by multiple regression analysis. DMBT1 levels increased significantly with lung maturity (P < 0.0001 for both gestational and postnatal age) and in small-for-gestational-age infants (P = 0.0179). An increase of respiratory DMBT1 levels was detected in neonatal infections (P < 0.0001). These results were supported by Western blotting. Immunohistochemical analyses of archived newborn lung sections (n = 17) demonstrated high concentrations of DMBT1 in lungs of neonates with bacterial infections. Our data show that preterm infants are able to up-regulate DMBT1 in infection as an unspecific immune reaction.

DMBT1 as an archetypal link between infection, inflammation, and cancer

Los estudios epidemiológicos y moleculares indican vínculosentre infección, inflamación y cáncer, que parece que convergena nivel molecular en mecanismos asociados con la inmunidadinnata. Aquí, presentamos un resumen del conocimientosobre la proteína secretada "scavenger receptor cysteine-rich(SRCR)" Deleted in Malignant Brain Tumors 1 (DMBT1), tambiénconocida como glicoproteína-340 o aglutinina de la saliva. DMBT1se expresa diferencialmente en varios tipos de cáncer, en muchoscasos disminuyendo su regulación. Como proteína secretada allumen, tiene funciones en la defensa innata contra los patógenos,y la regulación de la inflamación. En contraste, podría inducir ladiferenciación epitelial y de células madre, como proteína de lamatriz extracelular. Su amplia respuesta a estímulos patofisiológicossugiere un papel general en la protección celular y tisular,probablemente uniendo la defensa contra patógenos y la regulaciónde la respuesta inflamatoria a procesos regenerativos. Existensimilitudes muy interesantes con las funciones de otras proteínasSRCR presentes en metazoos primitivos, como las esponjasy los erizos de mar. Esto sugiere que sus diferentes funcionespodrían basarse en un principio antiguo y simple, que seríala mediación diferencial de adhesión y anti-adhesión. De manerasimilar a las vías de señalización de NF-κB, que también estánreguladas indirectamente por DMBT1, el conocimiento actualindica que DMBT1 no sólo podría tener funciones de prevenciónde enfermedad, sino probablemente también funciones generadorasde enfermedad. En resumen, DMBT1 podría representarun paradigma del vínculo arquetípico entre infección, inflamación,y cáncer. La comprensión de su complejo modo de acciónpromete nuevos puntos de vista sobre el origen y las bases molecularesde las grandes enfermedades humanas

Epidemiological and molecular studies have pointed to linksbetween infection, inflammation and cancer, which appear to convergeat the molecular level in mechanisms associated with innateimmunity. Here, the present knowledge about the secreted scavengerreceptor cysteine-rich (SRCR) protein Deleted in MalignantBrain Tumors 1 (DMBT1), also known as glycoprotein-340or salivary agglutinin, is summarized. DMBT1 is differentially expressed in various cancer types with most of these displayinga downregulation. As a lumenally secreted protein, it exerts functionsin innate pathogen defense and the regulation of inflammation.By contrast, it may trigger epithelial and stem cell differentiationas an extracellular matrix protein. Its broad responsivenessto pathophysiological stimuli points to a general role incell and tissue protection, which possibly is best circumscribedby linking pathogen defense and regulation of the inflammatoryresponse to regenerative processes. Compelling similaritiesto the functions of SRCR proteins in primitive metazoa such assponges and sea urchins exist, which support that its various functionsmay rely on an ancient and simple principle, i.e. the differentialmediation of adhesion and anti-adhesion. Similar to NF-κB signaling pathways, which are also indirectly regulated byDMBT1, the present state of the art indicates that DMBT1 not onlycould exert disease-preventing, but probably also disease-promotingfunctions. Taken together, DMBT1 may represent a paradigmfor an archetypal link between infection, inflammation, andcancer. Understanding its complex mode of action promises novelinsights into the origin and the molecular basis of major humandiseases

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain.

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.

Recent progress in the syntheses and biological evaluation of boronated porphyrins for boron neutron-capture therapy.

Boronated porphyrins are an important class of tumor-localizing agents in two bimodal therapies for cancer currently under study experimentally and clinically; boron neutron-capture therapy (BNCT) and photodynamic therapy (PDT). The desirable properties for the boronated porphyrins are that they are easily synthesized, pure and well-characterized drugs, and that in vivo, they are stable, tumor-specific, with high tumor:blood and tumor:normal tissue boron concentration ratios, and cause minimal toxicity. A large number of new porphyrins and their syntheses are presented herein. The focus is primarily on porphyrins published within the past 5 years, but the implications and trends from porphyrins studied in vivo over the past 15 years are also reviewed. Many possess quite unusual, novel structures and others have appended cell-targeting moieties for greater tumor specificity. Besides the commonly used closo- and nido-o-carboranes other boron cages and modes of attachment are presented. These boron cages can selectively alter the lipophilic, hydrophilic and amphiphilic properties of the porphyrins as well as their boron content. New delivery modalities have also greatly improved the targeting potential of compounds previously deemed unsuitable for applications in BNCT.

Oxidative chemistry of nickel porphyrins.

The oxidative chemistry of nickel(II) porphyrins is reviewed. Whether electron abstraction occurs from the metal to yield Ni(III) or from the porphyrin to yield Ni(II) pi cation radicals is discussed in terms of the relative energy levels of the metal and porphyrin orbitals. The effects of axial ligands in further modulating this ordering as well as the orbital occupancy of Ni(III) are also reviewed. Structural considerations, based on existing stereochemical data for Ni(I), high spin Ni(II) and related Ni(III) tetraaza complexes, are used to predict the metrics of Ni(III) porphyrins for which no structural data are available.

Molecular structures and magnetic resonance spectroscopic investigations of highly distorted six-coordinate low-spin iron(III) porphyrinate complexes.

Three bis-axially ligated complexes of iron(III) octaethyltetraphenylporphyrin, (OETPP)Fe(III), have been prepared, which are low-spin complexes, each with two axial nitrogen-donor ligands (N-methylimidazole (N-MeIm), 4-(dimethylamino)pyridine (4-NMe(2)Py), and 2-methylimidazole (2-MeImH)). The crystal and molecular structure of the bis-(2-MeImH) complex shows the macrocycle to be in a saddled conformation, with the ligands in perpendicular planes aligned at 14 degrees to the porphyrin nitrogens so as to relieve the steric interaction between the 2-methyl groups and the porphyrin. The Fe-N(por) bond lengths are typical of nonplanar six-coordinate low-spin Fe(III) complexes, while the axial Fe-N(ax) bond lengths are substantially longer than those of [(TPP)Fe(2-MeImH)(2)](+) (2.09(2) A as compared to 2.015(4) and 2.010(4) A). The crystal and molecular structure of the bis-(4-NMe(2)Py) complex also shows the macrocycle to be in a mainly saddled conformation, but with a significant ruffled component. As a result, the average Fe-N(por) bonds are significantly shorter (1.951 A as compared to 1.974 A) than those of the bis-(2-MeImH) complex. One ligand is aligned at 9 degrees to two trans porphyrin nitrogens, while the other is at 79 degrees to the same porphyrin nitrogens, producing a dihedral angle of 70 degrees between the ligand planes. The EPR spectrum of this complex, like that of the bis-(2-MeImH) complex, is of the "large g(max)" type, with g(max) = 3.29 and 3.26, respectively. However, in frozen CD(2)Cl(2), [(OETPP)Fe(N-MeIm)(2)](+) exhibits both "large g(max)" and normal rhombic signals, suggesting the presence of both "perpendicular" and "parallel" ligand orientations. The 1- and 2D (1)H NMR spectra of each of these complexes, as well as the chloroiron(III) starting material, were investigated as a function of temperature. The COSY and NOESY/EXSY spectra of the chloride complex are consistent with the expected J-coupling and saddle inversion dynamics, respectively. Complete spectral assignments for the bis-(N-MeIm) and -(4-NMe(2)Py) complexes have been made using 2D (1)H NMR techniques. In each case, the number of resonances due to methylene (two) and phenyl protons (one each) is consistent with D(2)(d)() symmetry, and therefore an effective perpendicular orientation of the axial ligands on the time scale of the NMR experiments. The temperature dependences of the (1)H resonances of these complexes show significant deviations from Curie behavior, and also evidence of extensive ligand exchange and rotation. Spectral assignment of the eight methylene resonances of the bis-(2-MeImH) complex to the four ethyl groups was possible through the use of 2D (1)H NMR techniques. The complex is fluxional, even at -90 degrees C, and ROESY data suggest that the predominant process is saddle inversion accompanied by simultaneous rotation of the axial ligands. Saddle inversion becomes slow on the 2D NMR time scale as the temperature is lowered in the ligand order of N-MeIm > 4-NMe(2)Py > 2-MeImH, probably due mainly to progressive destabilization of the ground state rather than progressive stabilization of the transition state of the increasingly "hindered" bis-ligand complexes.

Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma.

Pancreatic cancer can seldom be resected, and chemotherapy has only a limited effect on survival or tumour load. We did a phase I/II trial in 14 patients with pancreatic cancer to assess the safety of local activation of low-dose ifosfamide. We encapsulated genetically modified allogeneic cells, which expressed a cytochrome P450 enzyme, in cellulose sulphate and delivered them by supraselective angiography to the tumour vasculature. These cells locally activated systemically administered ifosfamide. The tumours of four patients regressed after treatment, and those of the other ten individuals who completed the study remained stable. Median survival was doubled in the treatment group by comparison with historic controls, and 1-year survival rate was three times better. Further studies of this cell-therapy-based treatment combined with chemotherapy for inoperable pancreatic cancer are warranted.

Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide.

Feline kidney cells were transfected with a vector overexpressing cytochrome P450 2B1 (CYP2B1). Transfected cells acquired a new specific biochemical activity, which could be demonstrated by a rapid CYP2B1 detection assay and showed selective sensitivity to the antitumorigenic prodrug ifosfamide (IFO). Further, the cell-killing effect was also mediated on nonmodified cells like feline kidney cells, mouse lymphoma, and human pancreatic cells in the vicinity of the CYP2B1-expressing cells due to the diffusible nature of the activated IFO metabolites. One of these, phosphoramide mustard, causes interstrand DNA cross-linking and it has been thought that the inability to repair this damage results in apoptosis. Surprisingly, our results clearly demonstrate a necrotic mechanism of IFO-induced cell death. This may have important implications for the activation of the immune system during CYP2B1/IFO suicide gene therapy of cancer.