Abstract
Hematologic malignancies constitute a diverse group of diseases, each characterized by distinct pathophysiological features. These include, in part, acute and chronic forms of lymphoblastic leukemia (ALL and CLL), acute and chronic forms of myeloid leukemia (AML and CML) multiple myeloma (MM), and myelodysplastic syndrome (MDS). Although many forms of therapy exist which may produce initial disease remission, recurrence and/or progression of these diseases present formidable challenges in treatment efficacy. We propose targeting the heme pathway via sonodynamic therapy (SDT) as a tailored drug-device combination treatment modality to optimize patient outcomes avoiding the debilitating side effects of chemotherapy, immunotherapy, radiation, and bone marrow transplantation, all which comprise current standard of care. The drug, 5-aminolevulinic acid (5-ALA, SONALA-001), is the first committed intermediate in the heme biosynthetic pathway while the device provides low-intensity focused ultrasound. When 5-ALA is provided in excess amounts to rapidly growing cancer cells, it is taken up by those cells in great quantities and converted to protoporphyrin IX (PpIX), a photoactive molecule which accumulates in cancer cells. PpIX fluorescence detection is an FDA-approved use as a visual aid for neurosurgeons and urologists to identify brain and bladder cancers, respectively. When activated by light of an energy higher than that which results in fluorescence, PpIX transfers its energy to molecular oxygen in the cell, creating a highly reactive, short-lived oxygen species, singlet oxygen. Photodynamic therapy (PDT) specifically kills tumor cells through a combination of necrosis (caused by immediate tumor cell death via lipoperoxidation of the cell membrane) and programmed cell death (apoptosis) caused by lipoperoxidation of the mitochondrial membrane and release of cytochrome C. The lower level of PpIX accumulation in normal healthy cells should translate to a significant therapeutic index favoring death of the malignant cells. Topical 5-ALA PDT is FDA-approved for the treatment of precancerous skin lesions (actinic keratoses) and has treated over 5 million subjects since approval. To determine the potential for 5-ALA PDT to treat hematologic malignancies, we sought to determine the 5-ALA incubation time and dose for maximal PpIX accumulation across a panel of hematologic cancers using commercially available cell lines, and isolated PBMCs and bone marrow (BMMCs) samples from both normal controls and cancer patients. In some cases, PBMCs and BMMCs from the same patient were evaluated. To determine peak PpIX accumulation, we exposed cells to various 5-ALA concentrations (0.3-1mM) and measured PpIX fluorescence over time (2-24hrs) using either a plate reader or flow cytometer. For our initial studies we used a fluorescent plate reader with multiple cell lines (e.g. Jurkat, RPMI-8226, K562). For healthy and patient-derived PBMC and BMMCs (e.g. AML, CML, MDS), we assessed PpIX accumulation using flow cytometry. For experiments conducted using hematologic cancer patient samples (PBMCs), we first sought to determine whether results from samples with high proportion of blast (high blasts) cells differed from those with a low proportion of blast cells (mid/low blasts). We observed a bias toward greater PpIX accumulation in the CD45 mid/low population (high proportion of blast cells) as compared to the CD45 high (low proportion blast cell population). In vitro PDT studies showed that cell death was dependent on PpIX accumulation in models of leukemia and myeloma (e.g. T-ALL, MM, and CML), whereas cells receiving 5-ALA, but no UV light exposure appeared unaffected. We propose that these results demonstrate the potential 5-ALA/PDT as treatment for leukemic cell populations in vivo. While the use of ALA PDT would restrict our treatment to a form of extracorporeal photopheresis, we propose to develop ultrasound as a source of photons (via sonoluminescence). This would allow us to treat circulating blood in situ noninvasively and would also have the potential to treat bone marrow infiltrated with malignant cells.This research was, in part, funded by the Advanced Research Projects Agency for Health (ARPA-H). The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the United States Government.
