The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

Cell killing by lysosomotropic detergents.

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen related to Lewis Y that is abundantly expressed at the surface of cells from many human carcinomas; it has a high degree of tumor selectivity and is internalized after binding. BR96-DOX induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured 94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats, like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.

Effect of linker variation on the stability, potency, and efficacy of carcinoma-reactive BR64-doxorubicin immunoconjugates.

The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

Reversal of doxorubicin resistance and catalytic neutralization of lysosomes by a lipophilic imidazole.

A number of lipophilic nitrogenous bases, designed to act as membrane-active, catalytic proton transfer agents, were tested for their ability to neutralize the acidity of lysosomes, a model for other acidic intracellular vesicles involved in drug sorting. The most successful of these, an imidazole 1, caused a 1.7 unit rise in lysosomal pH of RAW cells at 100 microM, compared to a 0.2 and 1.4 unit rise for ammonium chloride at 100 microM and 10 mM, respectively. Compound 1 also exhibited potent reversal of doxorubicin (DOX) resistance in the HCT116-VM46 cell line by a factor of 14 over the sensitive strain, and superior to that of widely used verapamil (VRP) by a factor of 1.75 at 20 microM. It also has antiviral properties, and potential applications in other lysosome-related areas such as immunotoxin potentiation and the control of bacterial toxins, immune response, prion replication, malaria and intralysosomal microorganisms.

Induction of cell death by the lysosomotropic detergent MSDH.

Controlled lysosomal rupture was initiated in lysosome-rich, macrophage-like cells by the synthetic lysosomotropic detergent, O-methyl-serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro-caspase-3-like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro-caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro-apoptotic factors.

Lysosomotropic agents. 1. Synthesis and cytotoxic action of lysosomotropic detergents.

Amines whose pK values lie between about 5 and 9 are lysosomotropic because lysosomes are acidic intracellular compartments. If such amines bear long hydrophobic chains, they become detergents upon protonation inside the lysosomes, rupturing the lysosomal membrane and killing the cell. Six types of lysosomotropic amines have been prepared that all behave in the expected manner. They are cytotoxic to all lysosome-bearing cells but not red blood cells, which lack lysosomes. Their mode of action, the effect of alkyl chain length on activity, and the fact that their cytotoxic action appears only above a threshhold intracellular concentration support the belief that they behave as lysosomotropic detergents. Among the potential applications is cancer chemotherapy.

3-Cyanocephems, and carbon-3 heterocyclic-substituted cephems via 1,3-dipolar cycloadditions.

The transformation is described of 3-formylcephem 1 into its oxime, substituted oximes, and substituted hydrazones and, thence, into the 3-cyano, 3-diazomethyl, and 3-oxonitrilomethyl derivatives. These reactive 1,3-dipoles undergo 1,3-dipolar cycloadditions with various dipolarophiles to give C-3 heterocyclic-substituted cephems.

Total syntheses of (+/-)-1-carbacefoxitin and -cefamandole and (+/-)-1-oxacefamandole.

The total syntheses of the (+/-)-1-carba analogues of cefoxitin (11), 7 alpha-methoxydeacetylcephalothin (5) and cefamandole (31) and the (+/-)-1-oxa analogue of cefamandole (43) are described. Their bioactivity spectra against 14 typical organisms are similar to those of their natural 1-thia counterparts, with the 1-carba compounds somewhat less active and the 1-oxa compound more active than the natural ones.

Lysosomotropic agents. 7. Broad-spectrum antifungal activity of lysosomotropic detergents.

Lysosomotropic detergents, which kill mammalian cells by disrupting lysosomal membranes, have now been found to be antifungals also. All strains in our assay are susceptible. The mode of action is as yet undetermined, but intracellular vacuoles may be the primary targets.

Selective delivery of cytotoxic compounds to cells by the LDL pathway.

Cancer cells need cholesterol to make new membrane. They get it either by de novo synthesis or from low-density lipoprotein (LDL), or both. Some types of cancer have very high LDL requirements. LDL particles, which circulate in the blood, contain a cholesteryl ester core surrounded by a phospholipid coat containing apoproteins that are recognized by LDL receptors on cell surfaces. After attachment to cells, LDL is endocytosed into lysosomes, where the core is exposed and hydrolyzed. A technique is known whereby LDL can be isolated, its core removed and replaced by a compatible lipophilic substance, and then reconstituted into intact LDL particles that are recognized and internalized by cells in the normal manner. A series of cytotoxic compounds has been synthesized, designed to be compatible with reconstituted LDL, and directed against cancers that copiously internalize LDL. They were evaluated by measuring the toxicity of reconstituted LDL toward test cells bearing LDL receptors. Selectivity was determined by comparison, either with mutant cells with few LDL receptors or with reconstituted methylated LDL (which is not recognized by LDL receptors) on normal cells. Two compounds, 19 and 25, were found that reconstitute well, kill or arrest the test cells at reasonably low concentrations, and are completely selective, suggesting that they are delivered to cells exclusively via the LDL pathway.

Lysosomotropic agents. 4. Carbobenzoxyglycylphenylalanyl, a new protease-sensitive masking group for introduction into cells.

Bioactive primary and secondary amines, when acylated with the Z-Gly-Phe group, are transported into pinocytic cells, such as macrophages, P-815 mastocytoma, SV-40 3T3, and leukemia 1210, much faster than the parent compounds. Amines such as lysosomotropic detergents [R. A. Firestone, J. M. Pisano, and R. J. Bonney, J. Med. Chem., 22, 1130 (1979) and nitrogen mustard, which are deactivated by acylation, are unmasked by enzymic action intracellularly, probably in lysosomes because an acidic pH maximum in activity exists which acts only on the L isomer. The added polarity and molecular weight brought about by acylation prevents the amines' normally facile entry into cells by simple diffusion, restricting it to an active-transport mechanism.

Effects of lysosomotropic detergents on the human malarial parasite Plasmodium falciparum in in vitro culture.

Various lysosomotropic detergents were tested in this work on in vitro cultures of Plasmodium falciparum and are shown to be potent antimalarial agents. The order of antimalarial potency was similar to that of cell toxicity on mammalian cells in culture (Miller DK et al., J Cell Biol 97, 1841-51 (1983]. The most efficient agents, N-dodecyl-imidazole (NDI) and N-dodecyl morpholine (NDM) displayed IC50 values of 6.7 +/- 0.7 microM and 23 +/- 5 microM. The mechanism of action of NDI measured at IC50 concentrations displayed the following features: irreversible antimalarial effect after 15 min exposure of cells to drug; alkalinization of the parasite food vacuole; inhibition of protein synthesis; inhibition of host cell protein digestion by the parasite; lack of vacuolar membrane disruption; lack of effect on the rate of constitutive autoproteolysis. No biochemical or ultrastructural indications were found to support a detergent-like action of NDI and its structural congeners on the major acidic compartment of the parasite, the food vacuole. Rather, alkalinization of that compartment by weak-base accumulation properties of the amphiphilic drugs and ensuing protonophoric effect are likely to play a major role in the various parasite-associated properties affected by these drugs.

Cathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicin.

A series of lysosomal protease-sensitive peptides attached to doxorubicin (DOX) was prepared as model substrates for internalizing anticancer immunoconjugates and potential antimetastasis prodrugs. Rates of cathepsin B-mediated release of free drug was measured for each, and human plasma stabilities for representative examples.

Improved cytotoxicity of antitumor compounds deliverable by the LDL pathway.

The concept of LDL-based chemotherapy of cancer is based on the fact that many tumors have high LDL requirements. A series of compounds has been synthesized, some of which meet all criteria for such therapy, i.e., they can be reconstituted with LDL, they do not leak out of the reconstituted LDL (rLDL), and they are potent enough to kill cells exclusively via the LDL receptor pathway. Two of these compounds are significantly superior to the best one from our earlier study [Firestone et al. (1984) J. Med. Chem. 27, 1037-1043], being cytotoxic in rLDL at concentrations reasonably attainable in vivo.

Cathepsin B-sensitive dipeptide prodrugs. 2. Models of anticancer drugs paclitaxel (Taxol), mitomycin C and doxorubicin.

Substrates containing doxorubicin (DOX), paclitaxel (taxol), and mitomycin C (MMC) attached to the cathepsin B-sensitive dipeptide Phe-Lys via a self-immolative spacer were prepared as model compounds for internalizing anticancer immunoconjugates. Cathepsin B-mediated release rates of free drug, rat liver lysosomal susceptibility and human plasma stability were measured for each.

pH dependent cytotoxicity of N-dodecylimidazole: a compound that acquires detergent properties under acidic conditions.

N-dodecylimidazole is a compound which acquires detergent properties under acidic conditions and might be useful in killing selectively cells in those regions of solid tumours which have a reduced extracellular pH (pHe). We have therefore studied the effects of N-dodecylimidazole against malignant cells in tissue culture. N-dodecylimidazole displayed pHe-dependent cytotoxicity against EMT-6 and MGH U1 cells; cell killing was dose dependent and was 100-fold greater at pHe 6.0 than pHe 7.0. Reduced toxicity of N-dodecylimidazole was observed at higher cell concentrations (> 10(6) cells ml-1), and only minor effects were observed against multicellular tumour spheroids. Potential mechanisms of action of N-dodecylimidazole include detergent-mediated lysis of the cell membrane at low pHe, and selective uptake into lysosomes where detergent activity leads to rupture of the lysosomal membrane and release of cytolytic enzymes. Inhibition of activity of cysteine proteases by the inhibitor E-64 did not protect cells against the toxicity of N-dodecylimidazole, suggesting that these lysosomal enzymes do not play a major role in the mechanism of action of this compound. Lysis of erythrocytes (which contain no lysosomes) was observed with low concentrations of N-dodecylimidazole. Dependence of cell lysis on cell concentration was similar to that observed for two other detergents that act on the plasma membrane, Triton X-100 and sodium dodecyl sulfate. We conclude that N-dodecylimidazole causes pHe dependent cell killing in two cultured tumour cell lines, and that its mechanism of action is probably due to acid mediated production of detergent activity which acts primarily on the cell plasma membrane.

(6-Maleimidocaproyl)hydrazone of doxorubicin--a new derivative for the preparation of immunoconjugates of doxorubicin.

The (6-maleimidocaproyl)hydrazone of doxorubicin was synthesized and conjugated to several mAbs, including chimeric BR96, via a Michael addition reaction to thiol-containing mAbs. DTT reduction of disulfides present in the mAb was a reliable and general method for generating a consistent number of reactive SH groups. The conjugates, after purification by Bio-Beads, were free of unreacted linker and/or doxorubicin. All conjugates released doxorubicin under acidic conditions that mimic the lysosomal environment, while they were relatively stable at neutral pH. BR96 conjugates showed antigen-specific cytotoxicity.