Karl-Heinz Glüsenkamp

New indene-derivatives with anti-proliferative properties.

Metabolites of the non-steroidal anti-inflammatory drug Sulindac inhibit cell proliferation by affecting several intracellular signaling pathways including the tumorigenic Ras/Raf/MAPK pathway. Here, we report the synthesis of eight new indene derivatives derived from the Sulindac structure, and present data on their anti-proliferative properties and their effects on the p21ras protein.

Nigericin enhances mafosfamide cytotoxicity at low extracellular pH

SummaryThe cytotoxicity of many alkylating anticancer drugs is increased at reduced intracellular pH (pHi). The therapeutic index of such agents could therefore be improved by lowering pHi in the target cells prior to their application. We have previously demonstrated that the formation of lactic acid can be selectively enhanced in malignant tissues via glucose-mediated stimulation of tumor cell glycolysis. However, the resulting reduction in pHi is partly compensated by the extrusion of H+ equivalents into the extracellular space, with pHi remaining closer to the physiological value than extracellular pH (pHe). For full exploitation of the proton-mediated increase in the cytotoxicity of alkylating agents, pHi should therefore be equilibrated with pHe in lactic acid-producing cells. In the present study we investigated the question as to whether nigericin, an H+/K+ antiporter enabling the entry into cells of H+ ions at low pHe, can be used to enhance the cytotoxic effect of mafosfamide (MAFO; a precursor of “activated” cyclophosphamide) on cultured M1R rat mammary carcinoma cells. At pHe 7.4, the cytotoxic effect of combined treatment with MAFO and nigericin was not superior to treatment with MAFO alone. At acidic pHe, however, MAFO cytotoxicity was potentiated by nigericin as indicated by the colony-forming capacity of M1R cells. For example, at pHe 6.2 (corresponding to the approximate mean “aggregated pH” in actively glycolyzing tumors), the colonyforming fraction of cells treated with a combination of MAFO and nigericin was 3×10−5 that of controls, as compared with a value of 5×10−2 found for cells exposed to MAFO alone. These results suggest that agents counteracting cellular mechanisms that control pHi may be candidate compounds for investigations aimed at the enhancement of alkylating drug cytotoxicity following glucosemediated pH reduction in malignant tumors in vivo.

Diels-alder reactions of malondialdehyde derivatives with reversed electron demand; an easy approach to structurally unique carbohydrates and compounds of the thromboxane type

Abstract 2-Formyl-malondialdehyde ( 1 ) reacts at 22°C in a type of Diels-Alder reaction with reversed electron demand with the enol-ethers ( 18 ) – ( 21 ) and ( 23 ) – ( 25 ) within a frew hours to give the dihydropyrans ( 2 ) – ( 9 ) and ( 12 ) – ( 17 ). Thio-enol-ethers may also be employed in this cycloaddition.

Metabolic response of AH13r rat tumours to cyclophosphamide as monitored by pO2 and pH semi-microelectrodes

The composition of the microenvironment has an important influence on the cellular response to cytotoxic agents. Using pH and pO2 semi-microelectrodes, we have monitored metabolic changes in AH13r rat tumours as a function of time after subcurative chemotherapy. Prior to therapy, tumours contained large areas considered hypoxic (mean pO2 approximately 4 mmHg) and are characterised by a marked accumulation of acidic metabolites (mean pH 6.65). Administration of cyclophosphamide (40 mg/kg body weight) resulted in tumour regression to 15% of pretreatment volumes and a growth delay of 12 days. Concomitant with volume reduction, tumours became reoxygenated (mean pO2 approximately 7 mmHg), with maximum values being reached within 2-4 days, paralleled by a shift of pH to more alkaline values (0.17 U on average). These changes coincided with the development of subtotal necrosis. During early tumour regrowth, the pH and pO2 histograms returned to control values. These data corroborate and extend the results of previous studies in which noninvasive techniques had been applied for the monitoring of treatment-induced metabolic changes in malignant tumours in vivo. In addition, these results support the notion that the effectiveness of anticancer therapy might be improved by selecting and scheduling therapeutic agents in consideration of physiological changes caused by preceding courses of treatment.

Effect of glucose-mediated pH reduction and cyclophosphamide on oxygenation of transplanted rat tumors☆

PURPOSE Glucose-mediated reduction of tumor pH is under investigation as a means to improve the therapeutic index of anticancer agents. An improvement of glucose supply to tumors is, however, likely to influence various metabolic and pathophysiological parameters apart from pH which, in turn, could modulate H+ ion-mediated effects. As a first step to identify changes in these parameter, we have investigated the effect of glucose-mediated pH reduction on oxygenation of malignant tissues either per se or in combination with a pH-sensitive drug, cyclophosphamide. METHODS AND MATERIALS H+ ion and oxygen-sensitive semi-microelectrodes were used to measure pH and pO2 in transplanted TV1A and AH13r rat tumors at normoglycemia and following high-dose intravenous glucose infusion. RESULTS In both tumors analyzed, pH reduction was accompanied by a decrease in pO2. In TV1A tumors, for example, the mean (median) pO2 decreased from 8.2 mm Hg (3.7 mm Hg) to 3.9 mm Hg (1.7 mm Hg) at 2 hr and 2.9 mm Hg (1.9 mm Hg) at 24 hr, respectively, after raising the plasma glucose concentration to 25 +/- 2 mmol/l. At the same time points, the mean pH had declined from 6.89 to 6.29 and 6.24, respectively. The class of pO2 readings < 5 mm Hg increased from a pretreatment value of 65% to approximately 90% at 24 hr. In contrast, cyclophosphamide treatment resulted in improved oxygenation of AH13r tumors, an effect first observed at 24 hr after drug administration. When both modalities were combined, cyclophosphamide partly prevented the acidosis-associated decrease in oxygen partial pressure. CONCLUSION The results of this study indicate that, within the context of the therapeutic approach used, cytotoxic agents should be employed which are more active against hypoxic than against well-oxygenated cells. In particular, glucose-mediated reduction of oxygen partial pressure in malignant tissues may be exploited to increase the fraction of hypoxic cells prior to administration of drugs activated in hypoxic cells by reductive metabolism (bioreductive agents).

Rapid hydrolysis of amides under physiological conditions: Influence of the microenvironment on the stability of the amide bond

A new class of bicyclic carboxyamides 1a-9a differing with respect to substitution patterns and exo-endo geometry has been synthesized. These amides are characterized by a structure-dependent unusual rapid hydrolysis rate at physiological conditions. The corresponding bicyclic anhydrides might be used as tools for masking and modifying therapeutic agents containing amine functionalities.

New modular delivery system for diagnostic and therapeutic pre‐targeting using tautomer‐specific monoclonal antibody EM‐6‐47 and 3‐substituted adenines

We have developed a new modular affinity system for the 2‐step delivery of functional molecules to target cells. The system is based on the tautomer‐specific monoclonal antibody (MAb) EM‐6–47, which binds to 3‐ and 3,8‐substituted adenines with high affinity (Ka > 109 l/mol) without cross‐reacting with naturally occurring purine derivatives. This MAb serves as the hapten‐specific fusion partner to produce bispecific MAbs (bs‐MAbs) recognizing a target cell antigen and a low‐m.w. hapten as carrier molecule for, e.g., radionuclides. Either the C‐8 or the N‐3 position of adenines can be used for conjugation with effector molecules; the remaining position may be substituted with different moieties to modulate the pharmacokinetics of the haptens. Different 3‐ and 3,8‐substituted adenines conjugated to the chelates DOTA and DTPA or to the drug daunomycin were synthesized. Adenine‐chelate derivatives were efficiently labeled with 111In and 90Y, while high‐affinity binding of 3‐substituted adenines to MAb EM‐6–47 remained almost unaffected by the conjugation to radiochelates. To confirm the validity of the delivery system, a prototype bs‐MAb, EM‐168–47, was generated by somatic cell fusion of MAb EM‐6–47 and MAb EM‐168–2, the latter recognizing a surface antigen on canine hematopoietic cells. Two‐step targeting assays in vitro verified the bs‐MAb‐mediated, dose‐dependent delivery of 111In‐labeled adenine‐chelate derivatives to myeloid cells. This system represents a powerful tool for new pre‐targeting approaches relying on bs‐MAbs and low‐m.w. haptens. Suitable cellular antigens can be targeted by fusing the appropriate MAbs with hapten‐specific MAb EM‐6–47, and tailor‐made 3‐substituted adenines may be labeled with diagnostic or therapeutic radionuclides, cytotoxic drugs or other functional molecules. Int. J. Cancer 77:610–619, 1998.