Background: A novel immunological approach to colon cancer therapy is the antibody targeting of the fibroblast activation protein (FAP), which is highly expressed by stroma cells of this tumour. Unconjugated sibrotuzumab (BIBH 1), which is a humanised version of the murine anti-FAP mAb F19, was investigated for its anti-tumour activity, safety and pharmacokinetics. Patients and Methods: Patients with metastatic colorectal cancer received weekly intravenous infusions of unconjugated sibrotuzumab at a dose of 100 mg over 12 scheduled weeks. The study was implemented as an open-label, uncontrolled, multicentre trial. Results: 25 patients were enrolled. Patients had one or more measurable lesions, predominantly liver lesions, at baseline. At least 8 repeated weekly infusions of sibrotuzumab in 17 evaluable patients did not result in complete or partial remission. Rather, ongoing tumour progression was noted in all patients except for 2 patients with stable disease. However, progressive disease was also observed post-study in these 2 patients who received 1 and 6 additional infusions, respectively, of sibrotuzumab. Sibrotuzumab exhibited 2-compartment pharmacokinetics with a dominant terminal phase and t1/2 ? = 5.3 ± 2.3 days. Adverse drug reactions (rigors/chills, nausea, flushing and one incidence of bronchospasm) were observed in 5 patients. Of the 24 patients given 2 or more infusions of sibrotuzumab, antibodies against sibrotuzumab were found in 3 patients (12.5%) after 4–12 infusions. Conclusions: Sibrotuzumab was well tolerated and safe. The minimal requirement for the continuation of this exploratory trial, of at least one complete or partial remission, or equivalently, of 4 patients with stable disease, was not met.

The human CD44 gene encodes type 1 transmembrane glycoproteins involved in cell-cell and cell-matrix interactions. The structural heterogeneity of the gene products is caused primarily by alternative splicing of at least 10 out of 20 exons. Certain CD44 variant isoforms, in particular those containing CD44 variant domain 6 (CD44v6), have been implicated in tumourigenesis, tumour cell invasion and metastasis. Here we will give an overview of immunohistochemically determined CD44v6 expression in human malignancies (primary epithelial and nonepithelial tumours as well as metastases) and normal tissues, and review several examples of the clinical use of CD44v6-specific antibodies. In nonmalignant tissues, CD44v6 expression is essentially restricted to a subset of epithelia. Intense and homogeneous expression of CD44v6 was reported for the majority of squamous cell carcinomas and a proportion of adenocarcinomas of differing origin, but was rarely seen in nonepithelial tumours. This expression pattern has made CD44v6 an attractive target for antibody-guided therapy of various types of epithelium-derived cancers.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of 0; radicals by microsomes, a stoichiometry of 0; to Hz02 close to 2 : 1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2Oz formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of HzO2.The rates of 0; as well as HzO2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H202 formation dropped to 55 "/,.It was concluded that most of the 0; originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H202 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H202 formation was only slightly decreased at 20 pM dioxygen suggesting that H202 formation via cytochrome P450 should also occur in vivo.After the early observation by Gillette et al. [I] it was confirmed repeatedly that liver microsomes generate hydrogen peroxide during the oxidation of NADPH [2-41. The reaction seemed to be linked to the monooxygenation system for xenobiotics, since pretreatment of the animals with phenobarbital or pregnenolone-l6a-carbonitrile increased the rate of H202 formation [3], and inhibitors of cytochrome P450 affected the production of H202 and the N-demethylation of ethylmorphine in a similar fashion [4]. However, the underlying mechanisms of hydrogen peroxide formation are not well understood [5]. It was found that some substrates of the monooxygenase system stimulate Hz02 formation whereas others are either not effective or are effective only in animals pretreated with phenobarbital but not with pregnenolone16a-carbonitrile [3,4]. A correlation of H202 formation with the spectral binding and hydroxylation of hexobarbital was observed by Heinemeyer et al. [6] from which a hypothetical peroxide complex of cytochrome P450 was postulated as a common intermediate for Hz02 formation and substrate hydroxylation.If this hypothesis is correct, an increased H202 formation should be observable upon addition of pseudo-substrates [7,8] of the microsomal cytochrome P450 monooxygenase system. This was reinvestigated in the present study.As a second possible pathway of NADPH-dependent formation of H202 in liver microsomes the release of superoxide anion radicals by autoxidation of microsomal redox systems, and the subsequent disproportionation of these Saarland, Homburg-Saar, Federal Republic of German...

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.