Photobac derived from bacteriochlorophyll-a shows potential for treating brain tumor in animal models by photodynamic therapy with desired pharmacokinetics and limited toxicity in rats and dogs.

Photobac is a near infrared photosensitizer (PS) derived from naturally occurring bacteriochlorophyll- a, with a potential for treating a variety of cancer types (U87, F98 and C6 tumor cells in vitro). The main objective of the studies presented herein was to evaluate the efficacy, toxicity and pharmacokinetic profile of Photobac in animals (mice, rats and dogs) and submit these results to the United States Food and Drug Administration (US FDA) for its approval to initiate Phase I human clinical trials of glioblastoma, a deadly cancer disease with no long term cure. The photodynamic therapy (PDT) efficacy of Photobac was evaluated in mice subcutaneously implanted with U87 tumors, and in rats bearing C6 tumors implanted in brain. In both tumor types, the Photobac-PDT was quite effective. The long-term cure in rats was monitored by magnetic resonance imaging (MRI) and histopathology analysis. A detailed pharmacology, pharmacokinetics and toxicokinetic study of Photobac was investigated in both non-GLP and GLP facilities at variable doses following the US FDA parameters. Safety Pharmacology studies suggest that there is no phototoxicity, cerebral or retinal toxicity with Photobac. No metabolites of Photobac were observed following incubation in rat, dog, mini-pig and human hepatocytes. Based on current biological data, Photobac-IND received the approval for Phase-I human clinical trials to treat Glioblastoma (brain cancer), which is currently underway at our institute. Photobac has also received an orphan drug status from the US FDA, because of its potential for treating Glioblastoma as no effective treatment is currently available for this deadly disease.

Excitation of a Single Compound by Light and Ultrasound Enhanced the Long-Term Cure of Mice Bearing Prostate Tumors.

Current treatment for prostate cancer is dependent on the stages of the cancer, recurrence, and genetic factors. Treatment varies from active surveillance or watchful waiting to prostatectomy, chemotherapy, and radiation therapy in combination or alone. Although radical prostate cancer therapy reduces the advancement of the disease and its mortality, the increased disease treatment associated morbidity, erectile dysfunction, and incontinence affect the quality of life of cancer survivors. To overcome these problems, photodynamic therapy (PDT) has previously been investigated using PhotofrinTM as a photosensitizer (PS). However, Photofrin-PDT has shown limitations in treating prostate cancer due to its limited tumor-specificity and the depth of light penetration at 630 nm (the longest wavelength absorption of PhotofrinTM). The results presented herein show that this limitation can be solved by using a near infrared (NIR) compound as a photosensitizer (PS) for PDT and the same agent also acts as a sonosensitizer for SDT (using ultrasound to activate the compound). Compared to light, ultrasound has a stronger penetration ability in biological tissues. Exposing the PS (or sonosensitizer) to ultrasound (US) initiates an electron-transfer process with a biological substrate to form radicals and radical ions (type I reaction). In contrast, exposure of the PS to light (PDT) generates singlet oxygen (type II reaction). Therefore, the reactive oxygen species (ROS) produced by SDT and PDT follow two distinct pathways, i.e., type I (oxygen independent) and type II (oxygen dependent), respectively, and results in significantly enhanced destruction of tumor cells. The preliminary in vitro and in vivo results in a PC3 cell line and tumor model indicate that the tumor specificality of the therapeutic agent(s) can be increased by targeting galectin-1 and galectin-3, known for their overexpression in prostate cancer.

A Remarkable Difference in Pharmacokinetics of Fluorinated Versus Iodinated Photosensitizers Derived from Chlorophyll-a and a Direct Correlation between the Tumor Uptake and Anti-Cancer Activity.

To investigate and compare the pharmacokinetic profile and anti-cancer activity of fluorinated and iodinated photosensitizers (PSs), the 3-(1'-(o-fluorobenzyloxy)ethyl pyropheophorbide and the corresponding meta-(m-) and para (p-) fluorinated analogs (methyl esters and carboxylic acids) were synthesized. Replacing iodine with fluorine in PSs did not make any significant difference in fluorescence and singlet oxygen (a key cytotoxic agent) production. The nature of the delivery vehicle and tumor types showed a significant difference in uptake and long-term cure by photodynamic therapy (PDT), especially in the iodinated PS. An unexpected difference in the pharmacokinetic profiles of fluorinated vs. iodinated PSs was observed. At the same imaging parameters, the fluorinated PSs showed maximal tumor uptake at 2 h post injection of the PS, whereas the iodinated PS gave the highest uptake at 24 h post injection. Among all isomers, the m-fluoro PS showed the best in vivo anti-cancer activity in mice bearing U87 (brain) or bladder (UMUC3) tumors. A direct correlation between the tumor uptake and PDT efficacy was observed. The higher tumor uptake of m-fluoro PS at two hours post injection provides a solid rationale for developing the corresponding 18F-agent (half-life 110 min only) for positron imaging tomography (PET) of those cancers (e.g., bladder, prostate, kidney, pancreas, and brain) where 18F-FDG-PET shows limitations.

Impact of mono- and di-ß-galactose moieties in in vitro / in vivo anticancer efficacy of pyropheophorbide-carbohydrate conjugates by photodynamic therapy.

To investigate the impact of mono- and di-ß-galactose moieties in tumor uptake and photodynamic therapy (PDT) efficacy, HPPH [3-(1'-hexyloxy)ethyl-3-devinylpyropheophorobide-a], the meso pyropheophorbide-a [3-ethyl-3-devinyl-pyropheophorbide-a], and the corresponding 20-benzoic acid analogs were used as starting materials. Reaction of the intermediates containing one or two carboxylic acid functionalities with 1-aminogalactose afforded the desired 172- or 20(4')- mono- and 172, 20(4')-di galactose conjugated photosensitizers (PSs) with and without a carboxylic acid group. The overall lipophilicity caused by the presence of galactose in combination with either an ethyl or (1'-hexyloxy)ethyl side chain at position-3 of the macrocycle made a significant difference in in vitro uptake by tumor cells and photoreaction upon light exposure. Interestingly, among the PSs investigated, compared to HPPH 1 the carbohydrate conjugates 2 and 11 in which ß-galactose moieties are conjugated at positions 172 and 20(4') of meso-pyro pheophorbide-a showed similar in vitro efficacy in FaDu cell lines, but in SCID mice bearing FaDu tumors (head & neck) Ps 11 gave significantly improved long-term tumor cure.

Tumor-Avid 3-(1'-Hexyloxy)ethyl-3-devinylpyrpyropheophorbide-a (HPPH)-3Gd(III)tetraxetan (DOTA) Conjugate Defines Primary Tumors and Metastases.

3-(1'-Hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH or Photochlor), a tumor-avid chlorophyll a derivative currently undergoing human clinical trials, was conjugated with mono-, di-, and tri-Gd(III)tetraxetan (DOTA) moieties. The T1/T2 relaxivity and in vitro PDT efficacy of these conjugates were determined. The tumor specificity of the most promising conjugate was also investigated at various time points in mice and rats bearing colon tumors, as well as rabbits bearing widespread metastases from VX2 systemic arterial disseminated metastases. All the conjugates showed significant T1 and T2 relaxivities. However, the conjugate containing 3-Gd(III)-aminoethylamido-DOTA at position 17 of HPPH demonstrated great potential for tumor imaging by both MR and fluorescence while maintaining its PDT efficacy. At an MR imaging dose (10 µmol/kg), HPPH-3Gd(III)DOTA did not cause any significant organ toxicity in mice, indicating its potential as a cancer imaging (MR and fluorescence) agent with an option to treat cancer by photodynamic therapy (PDT).

Photodynamic Therapy in Combination with Doxorubicin Is Superior to Monotherapy for the Treatment of Lung Cancer.

We have previously shown that a radioactive (123I)-analog of methyl 3-(1'-(iodobexyloxy) ethyl-3-devinylpyropheophorbide-a (PET-ONCO), derived from chlorophyll-a can be used for positron emission tomography (PET) imaging of a variety of tumors, including those where 18F-FDG shows limitations. In this study, the photodynamic therapy (PDT) efficacy of the corresponding non-radioactive photosensitizer (PS) was investigated in a variety of tumor types (NSCLC, SCC, adenocarcinoma) derived from lung cancer patients in mice tumor models. The in vitro and in vivo efficacy was also investigated in combination with doxorubicin, and a significantly enhanced long-term tumor response was observed. The toxicity and toxicokinetic profile of the iodinated PS was also evaluated in male and female Sprague-Dawley rats and Beagle dog at variable doses (single intravenous injections) to assess reversibility or latency of any effects over a 28-day dose free period. The no-observed-adverse-effect (NOAEL) of the PS was considered to be 6.5 mg/kg for male and female rats, and for dogs, 3.45 mg/kg, the highest dose levels evaluated, respectively. The corresponding plasma Cmax and AYClast for male and female rats were 214,000 and 229,000 ng/mL and 3,680,000 and 3,810,000 h * ng/mL, respectively. For male and female dogs, the corresponding plasma Cmax and AYClast were 76,000 and 92,400 ng/mL and 976,000 and 1,200,000 h * ng/mL, respectively.

Charged groups on pyropheophorbide-based photosensitizers dictate uptake by tumor cells and photodynamic therapy efficacy.

This study investigated the impact of anionic and cationic substituents of the pyropheophorbide-based photosensitizers (PS) on uptake and retention by tumor epithelial cells and photodynamic therapy (PDT). A series of PSs were generated that bear carboxylic acid functionalities, alkyl amines with variable length of carbon units or as a quaternary ammonium salt introduced at position 172 of 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH). The nature of the functionalities in the macrocycle made a significant difference in overall lipophilicity (log D values at pH 7.4), and in binding to and retention by human and murine tumor cells. Depending on the presence of functional groups, the PSs showed a change in cellular uptake from diffusion to endocytosis and in the preference for subcellular localization to mitochondria/ER or lysosomes. Two and more carboxylic groups drastically reduced uptake by all cell types. In contrast, PSs with amine and quaternary amine salt showed higher cellular binding, uptake and in vitro PDT efficacy than HPPH. The enhanced cellular uptake of the cationic PSs was accompanied by a loss of tumor cell specificity and contributed to severe systemic toxicity in tumor-bearing mice intravenously injected with the PS and subjected to investigate their therapeutic potential.

Synthesis, Tumor Specificity, and Photosensitizing Efficacy of Erlotinib-Conjugated Chlorins and Bacteriochlorins: Identification of a Highly Effective Candidate for Photodynamic Therapy of Cancer.

Erlotinib was covalently linked to 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) and structurally related chlorins and bacteriochlorins at different positions of the tetrapyrrole ring. The functional consequence of each modification was determined by quantifying the uptake and subcellular deposition of the erlotinib conjugates, cellular response to therapeutic light treatment in tissue cultures, and in eliminating of corresponding tumors grown as a xenograft in SCID mice. The experimental human cancer models the established cell lines UMUC3 (bladder), FaDu (hypopharynx), and primary cultures of head and neck tumor cells. The effectiveness of the compounds was compared to that of HPPH. Furthermore, specific functional contribution of the carboxylic acid side group at position 172 and the chiral methyl group at 3(1') to the overall activity of the chimeric compounds was assessed. Among the conjugates investigated, the PS 10 was identified as the most effective candidate for achieving tumor cell-specific accumulation and yielding improved long-term tumor control.

Sonodynamic therapy in combination with photodynamic therapy shows enhanced long-term cure of brain tumor.

This article presents the construction of a multimodality platform that can be used for efficient destruction of brain tumor by a combination of photodynamic and sonodynamic therapy. For in vivo studies, U87 patient-derived xenograft tumors were implanted subcutaneously in SCID mice. For the first time, it has been shown that the cell-death mechanism by both treatment modalities follows two different pathways. For example, exposing the U87 cells after 24 h incubation with HPPH [3-(1'-hexyloxy)ethyl-3-devinyl-pyropheophorbide-a) by ultrasound participate in an electron-transfer process with the surrounding biological substrates to form radicals and radical ions (Type I reaction); whereas in photodynamic therapy, the tumor destruction is mainly caused by highly reactive singlet oxygen (Type II reaction). The combination of photodynamic therapy and sonodynamic therapy both in vitro and in vivo have shown an improved cell kill/tumor response, that could be attributed to an additive and/or synergetic effect(s). Our results also indicate that the delivery of the HPPH to tumors can further be enhanced by using cationic polyacrylamide nanoparticles as a delivery vehicle. Exposing the nano-formulation with ultrasound also triggered the release of photosensitizer. The combination of photodynamic therapy and sonodynamic therapy strongly affects tumor vasculature as determined by dynamic contrast enhanced imaging using HSA-Gd(III)DTPA.

Pluronic F-127: An Efficient Delivery Vehicle for 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH or Photochlor).

To determine the impact of delivery vehicles in photosensitizing efficacy of HPPH, a hydrophobic photosensitizer was dissolved in various formulations: 1% Tween 80/5% dextrose, Pluronic P-123 and Pluronic F-127 in 0.5%, 1% and 2% phosphate buffer solutions (PBS). HPPH was also conjugated to Pluronic F-127, and the resulting conjugate (PL-20) was formulated in PBS. Among the different delivery vehicles, only Pluronic P-123 displayed significant vehicle cytotoxicity, whereas Pluronic F127 was nontoxic. Compared to PL-20, HPPH formulated in Tween80 and Pluronic F-127 showed higher cell-uptake, but lower long-term retention in Colon26 cell compared to PL-20. The higher retention of PL-20 was similarly observed during in vivo uptake with BALB/c mice baring Ct26 tumors. In contrast to the in vitro uptake experiments, PL-20 showed slightly higher uptake compared to HPPH formulated in Tween or Pluronic-F127. A significant difference in pharmacokinetic profile was also observed between the HPPH-Pluronic formulation and PL-20. Under similar in vivo treatment parameters (drug dose 0.47 µmol kg-1 , light dose: 135 J cm-2 at 24 h post-injection of PS), HPPH formulated either in Tween or Pluronic F-127 formulation showed similar in vivo PDT efficacy (20-30% tumor cure on day 60), whereas PL-20 showed 40% tumor cure (day 60).

Epidermal Growth Factor Receptor-Targeted Multifunctional Photosensitizers for Bladder Cancer Imaging and Photodynamic Therapy.

The in vitro and in vivo anticancer activity of iodinated photosensitizers (PSs) with and without an erlotinib moiety was investigated in UMUC3 [epidermal growth factor (EGFR)-positive] and T24 (EGFR-low) cell lines and tumored mice. Both the erlotinib-conjugated PSs 3 and 5 showed EGFR target specificity, but the position-3 erlotinib-PS conjugate 3 demonstrated lower photodynamic therapy efficacy than the corresponding non-erlotinib analogue 1, whereas the conjugate 5 containing an erlotinib moiety at position-17 of the PS showed higher tumor uptake and long-term tumor cure (severe combined immunodeficient mice bearing UMUC3 tumors). PS-erlotinib conjugates in the absence of light were ineffective in vitro and in vivo, but robust apoptotic and necrotic cell death was observed in bladder cancer cells after exposing them to a laser light at 665 nm. In contrast to 18F-fluorodeoxyglucose, a positron emission tomography agent, the position-17 erlotinib conjugate (124I-analogue 6) showed enhanced UMUC3 tumor contrast even at a low imaging dose of 15 µCi/mouse.

Phototriggered Release of Tumor-Imaging and Therapy Agents from Lyophilized Multifunctional Polyacrylamide Nanoparticles.

The success of polyacrylamide nanoparticles in drug delivery spurred the creation of variations in surface functional groups. We report herein a simple, reproducible, and efficient approach for the creation of modifiable nanoparticles that are characterized by their long-term stability and high loading efficiency. In our experiments, a hydrophobic photosensitizer, such as 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), was able to be postloaded at more than 90% efficiency across all types of nanoparticles (NPs). Moreover, the NPs have tunable release kinetics, ranging from 9% to 23%, released by 96 h in 1% serum albumin, depending on the surface modification used. Additionally, it was observed that the NPs had a photorelease mechanism where >60% of the payload was released when exposed to at least 2 J of light. This held true with the photosensitizer and hydrophobic chemotherapeutics like curcumin. To test the impact these modifications have in vitro, two different bladder cancer cell lines were chosen (UMUC3 and T24). These nanoparticles increase the efficacy of the photosensitizer by 4-fold in UMUC3, with the cationic and amino-functionalized particles having the highest efficacy. This increase in efficacy, high uptake, and favorable subcellular localization makes the cationic modification of the nanoparticle extremely attractive for future studies.

Formulation development of a novel targeted theranostic nanoemulsion of docetaxel to overcome multidrug resistance in ovarian cancer.

OBJECTIVE: Ovarian cancer is a highly lethal disease in which the majority of patients eventually demonstrate multidrug resistance. Develop a novel active targeted theranostic nanomedicine designed to overcome drug efflux mechanisms, using a Generally Regarded As Safe (GRAS) grade nanoemulsion (NE) as a clinically relevant platform. MATERIALS AND METHODS: The NEs surface-functionalized with folate and gadolinium, were made using GRAS grade excipients and a high-shear microfluidization process. Efficacy was evaluated in ovarian cancer cells, SKOV3 and SKOV3TR. The NE accumulation in tumors was evaluated in SKOV3 tumor-bearing mice by magnetic resonance imaging (MRI). RESULTS AND DISCUSSION: The NE with particle size < 150 nm were stable in plasma and parenteral fluids for 24 h. Ovarian cancer cells in vitro efficiently took up the non-targeted and folate-targeted NEs; improved cytotoxicity was observed for the folate-targeted NEs showing a 270-fold drop in the IC50 in SKOV3TR cells as compared to docetaxel alone. The addition of gadolinium did not affect cell viability in vitro, but showed relaxation times comparable to Magnevist®. Folate-targeted NEs accumulated in tumors for prolonged period of time compared to Magnevist® and showed enhanced contrast compared to non-targeted NEs with MRI in SKOV3 tumor-bearing mice suggesting active targeting of NEs due to folate modification. CONCLUSIONS: A folate-targeted, theranostic NE delivers docetaxel by receptor mediated endocytosis that shows enhanced cytotoxicity capable of overcoming ABC transporter mediated taxane resistance. The diagnostic capability of the targeted nanomedicine showed enhanced contrast in tumors compared to clinically relevant MRI contrast agent Magnevist®.

Development of EGFR-targeted nanoemulsion for imaging and novel platinum therapy of ovarian cancer.

PURPOSE: Platinum-based chemotherapy is the treatment of choice for malignant epithelial ovarian cancers, but generalized toxicity and platinum resistance limits its use. Theranostic nanoemulsion with a novel platinum prodrug, myrisplatin, and the pro-apoptotic agent, C6-ceramide, were designed to overcome these limitations. METHODS: The nanoemulsions, including ones with an EGFR binding peptide and gadolinium, were made using generally regarded as safe grade excipients and a high shear microfluidization process. Efficacy was evaluated in ovarian cancer cells, SKOV3, A2780 and A2780CP. RESULTS: The nanoemulsion with particle size <150 nm were stable in plasma and parenteral fluids for 24 h. Ovarian cancer cells in vitro efficiently took up the non-targeted and EGFR-targeted nanoemulsions; improved cytotoxicity was observed for the these nanoemulsions with the latter showing a 50-fold drop in the IC50 in SKOV3 cells as compared to cisplatin alone. The addition of gadolinium did not affect cell viability in vitro, but showed relaxation times comparable to Magnevist(®). CONCLUSION: The myrisplatin/C6-ceramide nanoemulsion synergistically enhanced in vitro cytotoxicity. An EGFR binding peptide addition further increased in vitro cytotoxicity in EGFR positive cancer cells. The diagnostic version showed MR imaging similar to the clinically relevant Magnevist® and may be suitable as a theranostic for ovarian cancer.

Pathogen-inspired drug delivery to the central nervous system.

For as long as the human blood-brain barrier (BBB) has been evolving to exclude bloodborne agents from the central nervous system (CNS), pathogens have adopted a multitude of strategies to bypass it. Some pathogens, notably viruses and certain bacteria, enter the CNS in whole form, achieving direct physical passage through endothelial or neuronal cells to infect the brain. Other pathogens, including bacteria and multicellular eukaryotic organisms, secrete toxins that preferentially interact with specific cell types to exert a broad range of biological effects on peripheral and central neurons. In this review, we will discuss the directed mechanisms that viruses, bacteria, and the toxins secreted by higher order organisms use to enter the CNS. Our goal is to identify ligand-mediated strategies that could be used to improve the brain-specific delivery of engineered nanocarriers, including polymers, lipids, biologically sourced materials, and imaging agents.