Summary Tumour-selective acidification is of potential interest for enhanced therapeutic gain of pH sensitive drugs. In this study, we investigated the feasibility of a tumour-selective reduction of the extracellular and intracellular pH and their effect on the tumour response of selected anti-cancer drugs. In an in vitro L1210 leukaemic cell model, we confirmed enhanced cytotoxicity of chlorambucil at low extracellular pH conditions. In contrast, the alkylating drugs melphalan and cisplatin, and bioreductive agents mitomycin C and its derivative EO9, required low intracellular pH conditions for enhanced activation. Furthermore, a strong and pH-independent synergism was observed between the pHequilibrating drug nigericin and melphalan, of which the mechanism is unclear. In radiation-induced fibrosarcoma (RIF-1) tumour-bearing mice, the extracellular pH was reduced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) or its analogue benzylguanidine (BG) plus glucose. To simultaneously reduce the intracellular pH, MIBG plus glucose were combined with the ionophore nigericin or the Na + /H + exchanger inhibitor amiloride and the Na + -dependent HCO 3 -/Cl -exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS). Biochemical studies confirmed an effective reduction of the extracellular pH to approximately 6.2, and anti-tumour responses to the interventions indicated a simultaneous reduction of the intracellular pH below 6.6 for at least 3 h. Combined reduction of extra-and intracellular tumour pH with melphalan increased the tumour regrowth time to 200% of the pretreatment volume from 5.7 ± 0.6 days for melphalan alone to 8.1 ± 0.7 days with pH manipulation (P < 0.05). Mitomycin C related tumour growth delay was enhanced by the combined interventions from 3.8 ± 0.5 to 5.2 ± 0.5 days (P < 0.05), but only in tumours of relatively large sizes. The interventions were non-toxic alone or in combination with the anti-cancer drugs and did not affect melphalan biodistribution. In conclusion, we have developed non-toxic interventions for sustained and selective reduction of extra-and intracellular tumour pH which potentiated the tumour responses to selected anti-cancer drugs.

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meta-Iodobenzylguanidine (MIBG) is a multipotent drug used in its radiolabeled form as a tumor-seeking radiopharmaceutical in the diagnosis and treatment of pheochromocytoma and neuroblastoma. Nonradiolabeled MIBG has also proved to be effective in the palliation of carcinoid syndromes and, on a predosing schedule, in enhancing the relative tumor uptake of a subsequent [131I]-MIBG dose in tumors of neuroadrenergic origin. In addition, MIBG is under investigation as an inhibitor of mitochondrial respiration and, as such, for its use in tumor-specific acidification. In this report we describe the side effects of nonradiolabeled MIBG on kidney function in mice. High doses of MIBG (40 mg/kg) reduced renal blood perfusion as measured by 86Rb distribution by 50%, which could be antagonized by the bioamine receptor blockers prazosin and cyproheptadine. MIBG also induced reversible renal damage as evidenced from a decrease in [51Cr]-ethylenediaminetetraacetic acid (EDTA) clearance and from histological damage, which was most pronounced in the distal tubuli. These effects were unrelated to reduced perfusion, however, and could not be antagonized by bioamine receptor blockers, Ca2+-channel blockers, or diuretics. Clearance effects of MIBG were mimicked by N-nitro-L-arginine methyl ester (L-NAME), a known inhibitor of nitric oxide synthase (NOS), and MIBG itself (100 microM) also inhibited NOS in vitro, suggesting that NOS inhibition by MIBG may have contributed to the observed reduction in renal clearance. The MIBG analog benzylguanidine (BG), which is equipotent in terms of mitochondrial inhibition, did not affect renal clearance, thus excluding mitochondrial inhibition as the main mechanism of MIBG-induced damage. MIBG, however, was much more cytotoxic than BG to kidney tubular cells in primary cultures. Although the renal effects of high-dose MIBG were reversible, alterations in the pharmacokinetics of concomitant medications by a temporary reduction in renal function should be taken into account in its clinical application.

Abstract-Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Key Words: neointima formation Ⅲ matrix metalloproteinases Ⅲ plasmin(ogen) Ⅲ urokinase-type plasminogen activator Ⅲ adenovirus I njury to the vessel wall resulting from vascular intervention triggers neointima formation and vascular remodeling, which together have been implicated as an important cause of (re)stenosis and graft failure after balloon angioplasty. 1 Various studies have demonstrated that neointimal thickening involves smooth muscle cell (SMC) migration and proliferation as well as extracellular matrix (ECM) deposition. 2 SMCs synthesize many of the major components of the vessel wall ECM and have the ability to digest all of these components through production and secretion of degradative enzymes. 3,4 This feature enables SMCs to invade and migrate, and to remodel the vessel wall, thereby contributing to the pathological process of (re)stenosis. Among these degradative enzymes, the matrix metalloproteinases (MMPs) and the plasminogen activation (PA) system play key roles in vascular remodeling. 5 The MMPs are a family of enzymes that can be divided into four subclasses: collagenases, gelatinases, stromelysins, and membrane-type MMPs. Increased MMP activity after vascular injury was demonstrated in various model systems, both in vitro and in vivo. 6 -14 MMP activity is controlled by specific inhibitors: tissue inhibitors of metalloproteinases (TIMPs). To date, four members of the tissue inhibitor family have been identified: TIMP-1, -2, -3, and -4. They are expressed by a variety of cells and are present in most tissues and body fluids. TIMP-1 and TIMP-2 have the broadest substrate specificity, acting against all members of the MMP family. 15 Studies using the rat balloon injury model have demonstrated that TIMP-1 and TIMP-2 overexpression reduces or delays, respectively, neointimal development. 16 -18 Furthermore, in 14-day organ cultures of saphenous vein TIMP-1, -2, and -3 were found to be potent inhibitors of neointima formation, whereas only TIMP-3 overexpression inhibited stenosis in a porcine vein graft model in vivo. 19 -21 The second degradative enzyme system, the plasminogen activation system, revolves around the serine protease plas-