Gerald G. Miller

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.

Preliminary Results of Nanopharmaceuticals Used in the Radioimmunotherapy of Ovarian Cancer

A multistep radioimmunotherapeutic (RIT) approach, exploiting the combination of a bispecific monoclonal antibody (BsMAb) with 90Y labelled biotinylated long-circulating liposomes was tested as a potential adjuvant treatment for epithelial ovarian carcinomatosis. The BsMAb, with anti-CA 125 and anti-biotin epitopes was used with PEGylated liposomes coated with biotin to deliver the cytotoxic radionuclide 90Y to tumor sites. This approach was used to overcome some of the major obstacles associated with conventional strategies, in particular, to increase the amount of radioactivity delivered to the tumor site compared with conventional monoclonal antibody (MAb) radionuclide delivery. Sequential intraperitoneal administration of the targeting and therapeutic moieties provides the basis for enhanced therapeutic ratio, according to our strategy. We report here the results of an in vivo therapy using our RIT approach with the Balb/c nude mouse model xenografted with the NIH:OVCAR-3 (CA 125+) human ovarian cancer cell line. An ongoing tumor growth delay/control study in Balb/c mice xenografted intraperitoneally with the NIH:OVCAR-3 cell line indicates a significant delay in onset of tumor and ascites development in treated vs. control populations.

Cytoplasmic delivery of a macromolecular fluorescent probe by poly(d, l-lactic-co-glycolic acid) microspheres.

A macromolecular fluorescent probe encapsulated in poly(d, l-lactic-co-glycolic acid) (PLGA) microspheres was used as a model for studying cytoplasmic delivery of antigens. We hypothesized that Texas red dextran loaded in PLGA microspheres would be delivered to the cytoplasm and that cytoplasmic delivery would be affected by polymer molecular weight. Cellular localization of the Texas red dextran was investigated at two different molecular weights of PLGA: 6000 and 60,000 g/mol. Intracellular degradation and processing of Texas red dextran-loaded PLGA microspheres by mouse peritoneal macrophages was monitored both in vitro and in vivo for a 7-day period using confocal laser scanning microscopy (CLSM). The results revealed cytoplasmic delivery of the fluorescent probe at both molecular weights of PLGA. Furthermore, the CLSM images showed that both in vitro and in vivo, the kinetics of microsphere degradation and cytoplasmic delivery were more rapid for the 6000 g/mol PLGA microspheres than the 60,000 g/mol PLGA microspheres. Hence, this study provides physical evidence that PLGA microspheres are capable of cytoplasmic delivery and that delivery to the cytosol can be controlled by modifying formulation parameters such as polymer molecular weight.

Design, synthesis, and intracellular localization of a fluorescently labeled DNA binding polyamide related to the antibiotic distamycin

The design and synthesis of the lipophilic (9) and fluorescent (10) conjugates of a structural analogue of distamycin and their in vitro cellular localization studies are reported. Confocal laser scanning microscopy (CLSM) indicates that 10 rapidly enters human ovarian adenocarcinoma (SKOV-3) cells with principal uptake in mitochondria and uniform cytoplasmic distribution.

Synthesis and biological evaluation of butanoate, retinoate, and bis(2,2,2-trichloroethyl)phosphate derivatives of 5-fluoro-2'-deoxyuridine and 2', 5-difluoro-2'-deoxyuridine as potential dual action anticancer prodrugs

A group of 3′‐O‐butanoyl, 5′‐O‐butanoyl, and 3′,5′‐di‐O‐butanoyl esters of 5‐fluoro‐2′‐deoxyuridine (FDU), and 2′,5‐difluoro‐2′‐deoxyuridine (DFDU), 3′‐O‐retinoyl, and 3′,5′‐di‐O‐retinoyl esters of FDU, and 5′‐O‐bis(2,2,2‐trichloroethyl)phosphoryl‐FDU and its 3′‐O‐butanoyl ester, was synthesized. These compounds were designed to act as double prodrugs that would serve as a depot to release two active drugs that act through different mechanisms. Thus, a nucleotide derivative of FDU or DFDU could act as a competitive inhibitor for thymidylate synthase, whereas retinoic acid and butyric acid would be expected to induce cell differentiation. The in vitro anticancer activities for these prodrugs were determined against a panel of nine tumor types (leukemia, non‐small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, breast) that encompassed about 60 human tumor cell lines. Structure‐activity relationships indicate that O‐butanoyl esters of FDU are approximately equipotent to FDU, the O‐butanoyl esters of DFDU are less active than FDU, and the retinoyl and bis(2,2,2‐trichloroethyl) phosphate derivatives of FDU exhibit comparable activity to FDU. In addition to their cytotoxic effect, 3′‐O‐retinoyl‐FDU (12) and 3′‐O‐butanoyl‐5′‐O‐bis(2,2,2‐trichloroethyl)phosphoryl‐FDU (16) also induced in vitro cell differentiation of promyelocytic leukemia HL60 cells. These combined cytotoxic and cell differentiation effects exhibited by 12 and 16 produced greater morphological drug‐induced granulation and neutrophil vacuolation, and more cell apoptosis, than observed upon exposure to either retinoic acid or sodium butanoate. Dose‐escalation studies in mice showed that 12 or 16 did not induce any acute or chronic toxicity, change in plasma clinical chemistry parameters, or gross histapathological changes at 60 days following an initial dosage regimen of 0.013 mmol/kg ip for 7‐consecutive days. The in vivo growth delay response of murine mammary EMT6 solid tumors suggests that 3′‐O‐retinoyl‐FDU (12) delays tumor growth relative to the other prodrugs investigated, sodium butyrate, retinoic acid, FDU, or a combination of retinoic acid and FDU. These preliminary results suggest that 3′‐O‐retinoyl‐FDU (12) warrants further in vivo investigation to determine its tissue biodistribution and pharmacokinetic parameters that would be of value in assessing its potential usefulness as an anticancer prodrug.

Targeted Radiotherapy of Multicell Neuroblastoma Spheroids with High Specific Activity [125I]Meta-Iodobenzylguanidine

PURPOSE Iodine-125 induces cell death by a mechanism similar to that of high linear energy transfer (high-LET) radiation. This study investigates the cytotoxicity of high-specific-activity [125I]meta-iodobenzylguanidine (125I-mIBG) in human SK-N-MC neuroblastoma cells grown as three-dimensional multicellular spheroids. MATERIALS AND METHODS Spheroids were incubated with high-specific-activity 125I-mIBG (6 mCi/microg, 1000 times that of the conventional specific activity used for autoradiography). Cytotoxicity was assessed by fluorescence viability markers and confocal microscopy for intact spheroids, fluorescence-activated cell sorting and clonogenic assay, and clonogenic assays for dispersed whole spheroids. Distribution of radioactive mIBG was determined by quantitative light-microscope autoradiography of spheroid cryostat sections. Dose estimation was based on temporal knowledge of the retained radioactivity inside spheroids, and of the radiolabels emission characteristics. Findings were compared with those of spheroids treated under the same conditions with 131I-mIBG, cold mIBG, and free iodine-125. RESULTS 125I-mIBG exerted significant cell killing. Complete spheroids were eradicated when they were treated with 500 microCi of 125I-mIBG, while those treated with 500 microCi or 1000 microCi of 131I-mIBG were not. The observed difference in cytotoxicity between treatments with 125I- and 131I-mIBG could not be accounted for by the absorbed dose of spheroid alone. The peripheral, proliferating cell layer of the spheroids remained viable at the moderate radioactivity of 100 microCi for both isotopes. Cytotoxicity induced by 125I-mIBG was quantitatively comparable by the peripheral rim thickness to that of 131I-mIBG at the dose of 100 microCi. The peripheral rim thickness decreased most significantly in the first 17 hours after initial treatment. There was no statistical decrease in the rim thickness identified afterwards for the second, third, and fourth days of incubation. CONCLUSION The cytotoxic effect of high-specific-activity 125I-mIBG appears to be comparable to, if not more efficient than that of conventionally used 131I-mIBG at the same level of total radioactivity. 125I-mIBG may improve the therapeutic index over that of 131I-mIBG in the clinical management of metastatic neuroblastoma due to the short range of Auger electrons.

Immunophotodynamic therapy: Current developments and future prospects

Photodynamic therapy (PDT) may offer an enhanced therapeutic index via one or more of the following modalities: a) preferential photosensitizer uptake by the target tissue, b) specific illumination of target tissue to excite the photosensitizer, c) strategic timing of light application to minimize toxicity to normal tissues, d) topical application of the photosensitizer restricted to the target tissue, infusion of the photosensitizer to the vasculature immediately upstream of the target, and intra‐target administration. While each photosensitizer has inherent properties of biodistribution, it is generally accepted that few provide a significant therapeutic index and that the desired effect is far from universal with respect to tumor types. Moreover, the optical properties of tumor and normal tissues vary dramatically and precision dosimetry remains an elusive goal in most clinical situations. Certain tumors, particularly those of the reticuloendothelial system, will usually be surrounded by normal stroma with greater concentrations of photosensitizer than are present in the tumor at a given post‐administration interval, presenting a major obstacle to practical therapy. Deep‐seated or large solid malignancies are not amenable to topical application of the photosensitizer and single tumor masses are frequently served by multiple arterial sources, rendering major dosimetric obstacles for photosensitizers administered via the upstream vasculature. One approach to specific photosensitizer delivery is via covalently bound immunoconjugates of photosensitizer and antibodies or antibody fragments to unique tumor markers. This approach is the subject of several clinical trials and provides encouragement for successful application in a wide variety of neoplastic diseases. In addition to therapeutic endpoints, this approach is capable of facilitating detection of subclinical lesions through the fluorescent properties of photosensitizers. Drug Dev. Res. 42:182–197, 1997.