Potentiation of the antitumor effect of Merocyanine 540-mediated photodynamic therapy by amifostine and amphotericin B.

Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit-granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540's appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.

Anti-tumor effect of Merocyanine 540-mediated photochemotherapy combined with Edelfosine: potential implications for the ex vivo purging of hematopoietic stem cell grafts from breast cancer patients.

High-dose chemotherapy combined with autologous stem cell support has improved response rates in high-risk and metastatic breast cancer, but has failed to improve long-term survival. Breast cancer has a tendency to metastasize to the bone marrow, and live tumor cells are known to circulate in the peripheral blood of breast cancer patients. Sensitive immunohistochemical, culture-based, and reverse transcriptase polymerase chain reaction (RT-PCR)-based methods have shown that about 50% of histologically normal stem cell grafts from breast cancer patients are contaminated with occult tumor cells, which may cause or contribute to tumor recurrences. Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Unfortunately, most solid tumor cells (including breast cancer cells) are only moderately sensitive or refractory to MC540-PDT. We report here that if MC540-PDT is followed by a 1-h incubation with the alkyl-lysophospholipid, Edelfosine (ET-18-OCH(3)), the depletion of murine and human breast cancer cells is greatly enhanced whereas the recovery of normal hematopoietic stem and progenitor cells is only minimally degraded. When used under conditions that reduce CD34-positive human bone marrow cells only 5.1-fold, and murine and human granulocyte/macrophage progenitors 6.8- and 3-fold, respectively, combination purging with MC540-PDT and Edelfosine depletes murine (Mm5MT) and human (MDA-MB-435S) breast cancer cells >17,000- and >125,000-fold, respectively. These data suggest that combination purging with MC540-PDT and Edelfosine may offer a simple, safe and effective method for the ex vivo purging of autologous stem cell grafts from breast cancer patients.

Preferential inactivation of paediatric solid tumour cells by sequential exposure to Merocyanine 540-mediated photodynamic therapy and Edelfosine: implications for the ex vivo purging of autologous haematopoietic stem cell grafts.

Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.

Crystal violet combined with Merocyanine 540 for the ex vivo purging of hematopoietic stem cell grafts.

The purpose of this study was to determine in a preclinical purging model, how effective crystal violet-mediated photodynamic therapy (CV-PDT) is against solid tumor and drug-resistant mutant tumor cells, and if certain limitations of CV-PDT can be overcome by using crystal violet (CV) in combination with the membrane-active photosensitizer, Merocyanine 540 (MC540). When used under conditions that preserved an adequate fraction of normal human granulocyte/macrophage progenitors (CFU-GM), CV-PDT failed to achieve meaningful reductions of DU145 prostate, H69 small cell lung cancer, and MDA-MB-435S breast cancer cells. Melphalan-resistant L1210/L-PAM1, adriamycin-resistant P388/ADR, and adriamycin-resistant HL-60/ADR leukemia cells were markedly less sensitive to CV-PDT than their wild-type counterparts, whereas cisplatin-resistant H69/CDDP cells were more sensitive than wild-type H69 cells. Sequential exposure to MC540- and CV-PDT under conditions that preserved an adequate fraction (73% and 29%, respectively) of normal CD34-positive hematopoietic stem cells and granulocyte/macrophage progenitors was highly effective against H69 (99.997% reduction) and H69/CDDP (99.999% reduction) cells, but ineffective against HL-60/ADR, MDA-MB-435S, and DU145 cells. CV thus shows only limited promise as a single-modality purging agent. However, in certain situations, clinically meaningful tumor cell depletions can be obtained by using CV in combination with a second photosensitizer such as MC540.

Photochemically generated elemental selenium forms conjugates with serum proteins that are preferentially cytotoxic to leukemia and selected solid tumor cells.

The objective of this study was to determine if and how photoproducts contribute to the antitumor effect of merocyanine-mediated PDT. A panel of barbituric, thiobarbituric and selenobarbituric acid analogues of Merocyanine 540 was photobleached, and the resulting photoproducts were characterized by absorption, fluorescence emission, mass, energy dispersive X-ray, and X-ray photoelectron spectroscopy and tested for cytotoxic activity against tumor cell lines and freshly explanted bone marrow cells. While all dyes were readily photobleached, only photoproducts of selone dyes showed cytotoxic activity. One-hour incubations with micromolar concentrations of selone-derived photoproducts were sufficient to reduce leukemia/lymphoma cells ≥10 000 fold, whereas preserving virtually all normal CD34-positive bone marrow cells. Of six multidrug-resistant tumor cell lines tested, five were as sensitive or more sensitive to photoproducts than the corresponding wild-type lines. Physicochemical characterizations of the cytotoxic activity indicated that it consisted of conjugates of subnano particles of elemental selenium and (lipo)proteins. The discovery of cytotoxic Se-protein conjugates provides a rare example of photoproducts contributing substantially to the antitumor effect of PDT and challenges the long-held view that Se in oxidation state zero is biologically inert. Agents modeled after our Se-protein conjugates may prove useful for the treatment of leukemia.

ELEMENTAL SELENIUM GENERATED BY THE PHOTOBLEACHING OF SELENOMEROCYANINE PHOTOSENSITIZERS FORMS CONJUGATES WITH SERUM MACROMOLECULES THAT ARE TOXIC TO TUMOR CELLS.

Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.