Verteporfin (also known as DB00460, CL318952, BPD-MA, BpdMA, Benzoporphyrin derivative monoacid ring A or BPD-MA) is a novel and potent second-generation photosensitizing agent derived from porphyrin in endothelial cells. It can be used for angiographic visualization of choroidal vessels and CNV.

Physicochemical Properties

Molecular Formula	C41H42N4O8
Molecular Weight	718.79
Exact Mass	718.30
Elemental Analysis	C, 68.51; H, 5.89; N, 7.79; O, 17.81
CAS #	129497-78-5
Related CAS #	129497-78-5
Appearance	Typically exists as Brown to black solid at room temperature
LogP	10.246
SMILES	[C@]12(C)[C@@H](C(=O)OC)C(C(=O)OC)=CC=C1C1=CC3=NC(C(C=C)=C3C)=CC3NC(=C(C=3C)CCC(=O)O)C=C3C(CCC(=O)OC)=C(C)C(=N3)C=C2N1.[C@]12(C)[C@@H](C(=O)OC)C(=CC=C1C1NC2=CC2C(C)=C(CCC(=O)O)C(=CC3NC(=C(C)C=3CCC(=O)OC)C=C3C(C=C)=C(C)C(=N3)C=1)N=2)C(=O)OC |c:26,t:16,40,54,73,84,99,109,&1:0,2,53,55|
InChi Key	YTZALCGQUPRCGW-MXVXOLGGSA-N
InChi Code	InChI=1S/C41H42N4O8/c1-9-23-20(2)29-17-34-27-13-10-26(39(49)52-7)38(40(50)53-8)41(27,5)35(45-34)19-30-22(4)25(12-15-37(48)51-6)33(44-30)18-32-24(11-14-36(46)47)21(3)28(43-32)16-31(23)42-29/h9-10,13,16-19,38,42,44H,1,11-12,14-15H2,2-8H3,(H,46,47)/b28-16-,29-17-,30-19-,31-16-,32-18-,33-18-,34-17-,35-19-/t38-,41+/m0/s1
Chemical Name	(1): 3-[(23S,24R)-14-ethenyl-5-(3-methoxy-3-oxopropyl)-22,23-bis(methoxycarbonyl)-4,10,15,24-tetramethyl-25,26,27,28-tetraazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-9-yl]propanoic acid.
Synonyms	Verteporfin;CL 318952;BPD MA; DB 00460;CL 318952;DB-00460; CL318952;DB00460;BPD-MA; BpdMA;Benzoporphyrin D;Benzoporphyrin derivative monoacid ring A;Visudyne.
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	YAP-TEAD interaction
ln Vitro	PDX cell screening was used to specifically select verteporfin. PhLO, PhLH, and PhLK had GI50 values of 228 nM, 395 nM, and 538 nM, respectively, causing 50% growth inhibition. In contrast, ALL-1, TCC-Y/sr, and NPhA1 had GI50 values of 3.93 μM, 2.11 μM, and 5.61 μM, respectively. Two of the three PDX cells' sensitivity to verteporfin was significantly decreased by GSH. In PDX cells, verteporfin lowers the potential of the mitochondrial membrane [1]. By suppressing YAP expression, verteporfin lowers the PTX resistance of HCT-8/T cells. When Verteporfin and NSC 125973 are treated together, they have a synergistic effect that inhibits YAP and decreases HCT-8/T cytotoxicity [2].
ln Vivo	BMS-354825 and verteporfin (10 mg/kg, csc) both dramatically decreased the percentage of leukemia cells, and their combination therapy also decreased the total amount of leukemia cells in the spleen [1]. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.[3]
Enzyme Assay	To study the relationship between YAP and PTX resistance, a stable YAP-over-expression or YAP silencing cell line was generated by transfected with YAP-plasmids or siYAP-RNA. WST-1 assay was performed to detect the cytotoxicity of PTX on HCT-8 and HCT-8/T cells. Clone formation assay and Transwell assay was preformed to determine the cell proliferation and invasion ability respectively. Immunofluorescence and Western blot analysis was performed for protein detection.[2] Results: YAP was stronger expressed in HCT-8/T than in HCT-8, and PTX resistance was positively correlated with the level of YAP expression. VP, a strongly YAP inhibitor, could reduce the PTX resistance on HCT-8/T cells without light activation by inhibiting YAP. Beside, VP and PTX combination therapy showed synergism on inhibition of YAP and cytotoxicity to HCT-8/T. Moreover, verteporfin and PTX combination therapy affect the invasion and colony formation ability and induce apoptosis of HCT-8/T cells.[2] Conclusions: VP can reverse the PTX resistance induced by YAP over-expression in HCT-8/T cells without photoactivation through inhibiting YAP expression.[2]
Cell Assay	In vitro effects of verteporfin on PDAC cell lines[3] A panel of 8 human PDAC cell lines was exposed to 0-10µM verteporfin for up to 7 days and live cells were quantified using automated fluorescence microscopy to count nuclei or by measuring absorbance in an MTT assay. Four lines (Capan 1, Capan 2, HS766T, CFPAC-1) were insensitive to verteporfin at all concentrations. Panc-1 and MIA PaCa-2 cells grew normally in ≤ 5µM verteporfin, but cell proliferation was significantly inhibited in 10µM verteporfin. Notably, proliferation of BxPC-3 and SU86.86 cells was completely inhibited in 10µM verteporfin, and reduced by >50% in 5µM verteporfin.[3] Estimation of drug interactions[1] PDX cells co-cultured with S17 cells were treated with 16 combinations of verteporfin (60 nM, 120 nM, 180 nM, and 240 nM) and dasatinib (12 nM, 24 nM, 36 nM, and 48 nM). The viabilities of cells treated with each combination were measured after 48 h using FACS Aria flow cytometer. In order to estimate drug interaction between verteporfin and dasatinib, a normalized isobologram and fraction affected-combination index (CI) plot were made using CompuSyn software. CI values greater than 1.0 indicated antagonistic effects, equal to 1.0 additive effects, and below 1.0 synergistic effects.[1]
Animal Protocol	Estimation of in vivo drug effects[1] PhLO cells (1.0 × 10~7 /mouse) were injected intravenously into 6-week-old male NOG mice, which were then treated with vehicle, verteporfin (140 milligram (mg)/kilogram (kg)/day), dasatinib (20 mg/kg/day), and a combination of these drugs from days 22 to 28. Verteporfin was administered by continuous subcutaneous infusion (c.s.c.) using Alzet osmotic pumps. An intraperitoneal injection (i.p.) was performed for dasatinib. All mice were sacrificed on day 28 and the chimerism of leukemia cells was investigated by flow cytometer using an anti-human CD19 antibody and anti-mouse CD45 antibody. Blood concentrations of verteporfin were calculated by LCMS-2020.[1] Verteporfin efficacy studies[3] Female Rag2M mice (20-25g) were inoculated subcutaneously in the centre of the lower back with 5 x 106 BxPC-3 or SU86.86 cells (1:1 RPMI:matrigel; 100µL volume; expressed as day 0). Tumors appeared within three weeks of implantation. Once the tumors were palpable, tumor growth was monitored by measuring tumor dimensions with digital calipers. When tumors reached 100-150mg (calculated according to the equation (length X width2)/2 converted to tumor weight in mg for each 1mm3), mice were randomized in groups of eight animals and treatment was initiated. Verteporfin was administered i.p. Monday, Wednesday, Friday for 4 weeks at a dose of 45mg/kg (injection volume 400µl/20g mouse). Gemcitabine was administered i.p. once weekly (Monday) for 4 weeks at 120mg/kg or 240mg/kg (injection volume 200µl/20g mouse). Groups treated with both verteporfin and gemcitabine received gemcitabine 6h after verteporfin administration. This time was selected because maximum verteporfin tumor levels were achieved 8h post-administration and maximum gemcitabine tumor levels were observed 2h post-administration. Animals in the control group were treated with the delivery vehicle DSPE-mPEG2000 at the same concentration and schedule as verteporfin. Care was taken to house animals treated with verteporfin in dark conditions until the morning after treatment because verteporfin is a photosensitizer and exposure to bright light could be harmful. A One-Way ANOVA with Tukey's multiple comparison test was used to compare differences in tumor growth. Pharmacokinetic studies of verteporfin DSPE-mPEG2000 micelles in BxPC-3 tumor-bearing mice[3] Rag2M mice (20-25g; n=3) were inoculated subcutaneously with 5 x 106 BxPC-3 cells. Mice were injected i.p. with verteporfin at 45mg/kg when tumors were approximately 200-250mg. Mice were euthanized by CO2 inhalation, and blood and tumors were collected at 2, 8, 16 and 24h post administration of verteporfin. Plasma was prepared by centrifuging samples at 1000 x g for 15min at 4°C. Tumors were excised, rinsed in PBS, and snap-frozen in cryovials in liquid nitrogen and stored at -80°C. Tumors were sectioned while frozen and one half was used for determining verteporfin concentration by UPLC-MS/MS and the other for immunoblot analysis of p62 and LC3. Plasma samples were thawed, homogenized and extracted with acetonitrile containing 0.1% formic acid. Protein precipitation and filtration was carried out using ISOLUTE® PPT+ protein precipitation plates (Biotage). Samples were analyzed using a Waters® ACQUITY® UPLC system with mass spectrometry detection. Separations were performed using an isocratic method where mobile phase A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile (70% B for 2.5min followed by 95% B for the wash). Verteporfin regioisomer A (monoacid A form) was eluted at 1.77min and regioisomer B (monoacid B form) was eluted at 2.16min with a total run time of 4.5min per sample. The MS/MS system was operated with an ESI interface in a positive ionization mode. Quantification was performed using multiple reactions monitoring (MRM) mode with a precursor mass m/z of 719.27 and product mass m/z of 645.36. The levels of verteporfin were measured against external calibration standards prepared using the same process. Formulation of verteporfin for animal studies[3] Verteporfin was formulated in DSPE-mPEG2000 micelles. Briefly, 200mg verteporfin dissolved in 2ml DMSO was slowly added, with stirring, to 1500mg DSPE-mPEG2000 dissolved in 50ml PBS at pH 7.4. Stirring was continued for 1h at 23°C followed by dialysis overnight against PBS using Spectra/Por dialysis membranes (15,000 MWCO). The concentration of verteporfin was measured in triplicate and quantified against an external standard curve using a Waters® ACQUITY® UPLC equipped with a PDA detector. Separations were done using a C18 column (Waters® BEH; column size 50 x 2.1mm, particle size 1.7µm) and a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile:methanol (1:1; B) [10-80% B over 2min at 0.5ml/min flow rate]. The concentration of verteporfin was adjusted to 2.25mg/ml with PBS followed by filter-sterilization and sterile vialing of the formulation. The concentration was reconfirmed before proceeding with the animal studies. The formulation was chemically and physically stable for an observation period of 4 weeks, which covered the duration of the animals studies as verified by UPLC and polarized light microscopy. 140 mg/kg; i.v. injection Mice: PhLO cells (1.0×107/mouse) are injected intravenously into 6-week-old male NOG mice, which are then treated with vehicle, verteporfin (140 mg/kg/day), dasatinib (20 mg/kg/day), and a combination of these drugs from days 22 to 28. Verteporfin is administered by continuous subcutaneous infusion (c.s.c.) using Alzet osmotic pumps. An intraperitoneal injection (i.p.) is performed for dasatinib. All mice are sacrificed on day 28 and the chimerism of leukemia cells is investigated by flow cytometer using an anti-human CD19 antibody and antimouse CD45 antibody. Blood concentrations of verteporfin are calculated by LCMS-2020.
References	[1]. Morishita T, et al. The photosensitizer verteporfin has light-independent anti-leukemic activity for Ph-positive acute lymphoblastic leukemia and synergistically works with BMS-354825. Oncotarget. 2016 Aug 2. [2]. Pan W, et al. Verteporfin can Reverse the NSC 125973 Resistance Induced by YAP Over-Expression in HCT-8/T Cells without Photoactivation through Inhibiting YAP Expression. Cell Physiol Biochem. 2016;39(2):481-90. [3]. Donohue E, et al. The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model.J Cancer. 2013 Aug 28;4(7):585-96
Additional Infomation	A benzoporphyrin derivative that is used in PHOTOCHEMOTHERAPY to treat wet type MACULAR DEGENERATION. A synthetic light-activated agent with photodynamic activity. Upon systemic administration, verteporfin accumulates in neovessels in the eye and, once stimulated by nonthermal red light in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to neovascular endothelium and blood vessel occlusion. An equimolar mixture of the 9-methyl ester and 13-methyl ester of trans-(+-)-18-ethenyl-4,4a-dihydro-3,4-bis(methoxycarbonyl)-4a,8,14,19-tetramethyl-23H,25H-benzo[b]porphine-9,13-dipropanoic acid. It is used as a photosensitizer in photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with neovascular (wet) age-related macular degeneration. Verteporfin accumulates in these abnormal blood vessels and, when activated by red (693 nm) laser light in the presence of oxygen, produces highly reactive short-lived singlet oxygen and other reactive oxygen radicals, resulting in local damage to the endothelium and blockage of the vessels. Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, particularly from the viewpoints of microenvironmental independence. Patient-derived xenografts (PDX) are established by the transfer of primary tumor cells directly from patients into immunodeficient mice and can provide primary-like tumor cells of the amount needed at the desired time. We developed a high-throughput drug screening system using PDX cells and performed drug screening using the PDX cells of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). We established four Ph+ ALL PDX mice and performed high-throughput screening of 3440 compounds using leukemia cells from the PDX mice (PDX-cell screening). The profiles of drugs selected by PDX-cell screening were markedly different from those by screening using the Ph+ ALL cell line. We found that verteporfin, an FDA-approved drug, exhibited strong PDX cell-specific cytotoxicity. In the validation assay, its GI50 was 228 nM, 395 nM, and 538 nM in three PDX cells and 3.93 µM, 2.11 µM, and 5.61 µM in three cell lines. Although verteporfin is a photosensitizer activated by photoirradiation, its cytotoxic effects were mediated by the light-independent production of reactive oxygen species; therefore, its anti-leukemic effects were also exerted in vivo without photoirradiation. Furthermore, it exhibited synergistic effects with dasatinib, an ABL kinase inhibitor. These results indicated the potential of verteporfin as a new anti-leukemic reagent.[1] aclitaxel (PTX) is one of the most effective anti-cancer drugs. However, multiple drug resistance is still the main factor that hinders the effective treatment of cancer with PTX. Several factors including YAP over-expression can cause PTX resistance. In this study, we aimed to verify the role YAP plays in PTX resistance, explore the reversal of PTX resistance by verteporfin (VP) and investigate the effect of combination therapy of PTX and VP on the PTX resistant colon cancer cells (HCT-8/T).[2] Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.[3]

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (139.1 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 5 mg/mL (6.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 5 mg/mL (6.96 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3912 mL	6.9561 mL	13.9123 mL
	5 mM	0.2782 mL	1.3912 mL	2.7825 mL
	10 mM	0.1391 mL	0.6956 mL	1.3912 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.