Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen's clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class' therapeutic potential. Finally, the structure-activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen's mechanism.

X-ray psoralen activated cancer therapy (X-PACT) is a new therapeutic approach that has been shown to induce tumor cell apoptosis and cytotoxicity in vitro, and slow tumor growth in BALB/c mice with syngeneic 4T1 tumors. X-PACT is accomplished by injection of co-incubated psoralen and phosphors; the phosphors emit UV to activate psoralen, and are activated by an external kV source. Here we describe application of a kV x-ray source mounted on board a medical linear accelerator for X-PACT in preparation for a phase I clinical trial of X-PACT for spontaneous tumors in pet dogs. We commissioned a 80 kVp beam at 50-80 cm from the source with varied blade settings to achieve rectangular collimated beams. Commissioning included dosimetry measurements, developing a formalism for absolute dose calculation in water, FLUKA Monte Carlo based planning and evaluation, and verification measurements. Dosimetry measurements included AAPMTG-61 absolute dose calibration, depth dose curves, backscatter and collimator scatter factors, heel effect, and leakage. Reasonable agreement was achieved between measurement and Monte Carlo, and between calculated dose and verification measurements. Finally, we demonstrate the X-PACT treatment process for an example dog with a 3-5 cc left hip sarcoma located at a 2 cm depth. The absolute dose formalism indicated 21 pulses of 160 mAs were required to deliver the prescription dose of 0.6 Gy to 2.8 cm depth. The dose distribution was calculated with the Monte Carlo planning tool and visualized at 1 x 1 x 2 mm(3) spatial resolution. A dose enhancement in the hip bone of up to 4.5 Gy was observed. This work demonstrates that X-PACT is feasible utilizing diagnostic kV sources such as those mounted on a clinical linear accelerator, and reports commissioning and treatment planning data and formalism respectively.

Purpose: This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with immunogenic anti-cancer potential. Psoralen therapies have been limited due to the requirement for psoralen activation by UVA light. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and reradiate (phosphoresce) at UV wavelengths. Methods: The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry to investigate treatment induced apoptosis. Methylene blue staining, and WST assays were also used. X-PACT was then evaluated in an in-vivo pilot study on BALBc mice with syngeneic 4T1 tumors, including control arms for X-PACT components. Analysis focused on tumor growth delay. Results: A multivariable regression analysis of 36 independent in-vitro irradiation experiments demonstrated that X-PACT induces significant tumor cell apoptosis and cytotoxicity on all three tumor cell lines in-vitro (p<0.0001). Neither psoralen nor phosphor alone had a strongly significant effect. The in-vivo studies show a pronounced tumor growth delay when compared to controls (42% reduction at 25 days, p=0.0002). Conclusions: These studies demonstrate for the first time a therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation generate UVA light in-situ (including deep seated lesions) which in-turn photo-activates powerful anticancer therapeutics which may lead to short and long term therapeutic effect. This work was supported by Immunolight Llc

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of solid cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with current application to proliferative disease and extracorporeal photopheresis (ECP) of cutaneous T Cell Lymphoma. An immunogenic role for light-activated psoralen has been reported, contributing to long-term clinical responses. Psoralen therapies have to-date been limited to superficial or extracorporeal scenarios due to the requirement for psoralen activation by UVA light, which has limited penetration in tissue. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and re-radiate (phosphoresce) at UV wavelengths. The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry in combination with complimentary assays, and the in-vivo mouse study. In an in-vitro study, we show that X-PACT induces significant tumor cell apoptosis and cytotoxicity, unlike psoralen or phosphor alone (p<0.0001). We also show that apoptosis increases as doses of phosphor, psoralen, or radiation increase. Finally, in an in-vivo pilot study of BALBc mice with syngeneic 4T1 tumors, we show that the rate of tumor growth is slower with X-PACT than with saline or AMT + X-ray (p<0.0001). Overall these studies demonstrate a potential therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. In summary, X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation are delivered to a specific tumor site to generate UVA light which in-turn unleashes both short- and potentially long-term antitumor activity of photo-active therapeutics like psoralen.

Purpose:Psoralen is a UV‐light activated anti‐cancer biotherapeutic used for treating skin lesions (PUVA) and advanced cutaneous T‐cell lymphoma (ECP). To date psoralen has not been used to treat deep seated tumors due to difficulty in generating UV‐light at depth. We recently demonstrated psoralen activation at depth by introducing energy converting particles that absorb kV x‐ray radiation and re‐emit UV‐light. Our in‐vitro work found that 0.2–1Gy using 40–100kVp x‐rays combined with psoralen and particles can induce a substantial apoptotic response beyond that expected from the sum of individual components. In preparation for a phase I clinical trial of canine companion animals, we address the physics and dosimetry considerations for applying this new teletherapy paradigm to an in‐vivo setting.Methods:The kV on‐board imaging (OBI) system mounted on a medical linear accelerator (Varian) was commissioned to deliver the prescribed dose (0.6Gy) using 80 and 100kVp. Dosimetric measurements included kVp, HVL, depth dose, backscatter factors, collimator and phantom scatter factors, field size factors, and blade leakage. Absolute dosimetry was performed following AAPM TG61 recommendations and verified with an independent kV dose meter. We also investigated collimated rotational delivery to minimize skin dose using simple dose calculations on homogeneous cylindrical phantoms.Results:Single beam delivery is feasible for shallow targets (<5cm) without exceeding skin tolerance, while a rotational delivery may be utilized for deeper targets; skin dose is ∼75% of target dose for 80kVp collimated rotational delivery to a 3cm target within a 20cm phantom. Heat loading was tolerable; 0.6Gy to 5cm can be delivered before the anode reaches 75% capacity.Conclusion:KV teletherapy for Psoralen activation in deep seated tissue was successfully commissioned for a Varian OBI machine for use in a phase I clinical trial in canines. Future work will use Monte Carlo dosimetry to investigate dose in presence of bone.Research funded by Immunolight LLC. H. Walder, Z. Fathi, & W. Beyer are employees of Immunolight LLC which holds a patent on the technology. Drs. Adamson and Oldham are consultants to Immunolight LLC.

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

An ex vivo trial utilizing photopheresis with Benzoporphyrin Derivative as the photoactive compound, identified the minimum energy levels of light and concentrations of BPD that eradicated both cell-free and cell-associated HIV-1 infectivity without destroying the virus particles or infected leukocytes. Leukocytes remained viable with altered chemokine/cytokine expression. Apoptosis was induced in a minority of CD4 but not CD8 positive cells with a statistically significant increase in cytolytic T-cell activity. In the 24 week clinical trial in 7 HIV-1 infected patients, three who had rapidly rising viral loads prior to initiating therapy stabilized. Two had a sustained greater than 0.5 log decrement and 5 had stable plasma viral loads (less than a 0.5 log increment or decrement) with varied effects on absolute CD4 and CD8 positive lymphocytes counts. One achieved a greater than 1 log decrement in HIV-1 plasma viral load and undetectable in vivo cell-free and cell-associated HIV-1 infectivity with an increased in vitro lymphocyte mitogen stimulation index. Under amended protocol, 5 additional 12 month courses were administered to three additional patients and two of the previous enrollees. Area under the curve for viral load showed a significant decrease from pre- to post-therapy (p 0.007). No associated toxicities were observed.