Pafolacianine (OTL-0038; OTL-38; Cytalux), is a folate receptor-directing/optical imaging agent (fluorescent marker) approved in 2021 as an adjunct imaging agent for intraoperative identification of malignant lesions in adult patients with ovarian cancer. It is a folate analogue conjugated with a fluorescent dye that absorbs light in the near-infrared (NIR) spectrum within a range of 760 nm to 785 nm, with a peak absorption of 776 nm. It emits fluorescence within a range of 790 nm to 815 nm with a peak emission of 796 nm. The longer wavelength allows for deeper penetration of the fluorescent light through tissues for better imaging and detection of tumours. Pafolacianine targets the folate receptor alpha (FRα) which is upregulated in numerous epithelial malignancies. It is currently under investigation for use in FRα-positive pituitary adenoma, lung cancer, and renal-cell carcinoma, which is thought to be the second-highest folate receptor-expressing cancer.

Physicochemical Properties

Molecular Formula	C61H67N9O17S4
Molecular Weight	1326.4948
Exact Mass	1325.353
CAS #	1628423-76-6
Related CAS #	1628858-03-6 (sodium);1628423-76-6 (free acid);
PubChem CID	135565623
Appearance	Green to dark green solid powder
LogP	4
Hydrogen Bond Donor Count	8
Hydrogen Bond Acceptor Count	22
Rotatable Bond Count	24
Heavy Atom Count	91
Complexity	3330
Defined Atom Stereocenter Count	1
SMILES	S(C1C([H])=C([H])C2=C(C=1[H])C(C([H])([H])[H])(C([H])([H])[H])/C(=C(/[H])\C(\[H])=C1\C(=C(C([H])([H])C([H])([H])C\1([H])[H])/C(/[H])=C(\[H])/C1C(C([H])([H])[H])(C([H])([H])[H])C3C([H])=C(C([H])=C([H])C=3[N+]=1C([H])([H])C([H])([H])C([H])([H])C([H])([H])S(=O)(=O)O[H])S(=O)(=O)[O-])OC1C([H])=C([H])C(C([H])([H])[C@@]([H])(C(=O)O[H])N([H])C(C3C([H])=C([H])C(=C([H])C=3[H])N([H])C([H])([H])C3=C([H])N=C4C(C(N([H])C(N([H])[H])=N4)=O)=N3)=O)=C([H])C=1[H])/N2C([H])([H])C([H])([H])C([H])([H])C([H])([H])S(=O)(=O)O[H])(=O)(=O)O[H]
InChi Key	PDXNSXLPXJFETD-DYVQZXGMSA-N
InChi Code	InChI=1S/C61H67N9O17S4/c1-60(2)46-33-44(90(81,82)83)22-24-49(46)69(28-5-7-30-88(75,76)77)51(60)26-16-38-10-9-11-39(17-27-52-61(3,4)47-34-45(91(84,85)86)23-25-50(47)70(52)29-6-8-31-89(78,79)80)54(38)87-43-20-12-37(13-21-43)32-48(58(73)74)66-56(71)40-14-18-41(19-15-40)63-35-42-36-64-55-53(65-42)57(72)68-59(62)67-55/h12-27,33-34,36,48H,5-11,28-32,35H2,1-4H3,(H9-,62,63,64,66,67,68,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86)/t48-/m0/s1
Chemical Name	hydrogen (S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-3-(4-(((E)-2-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)-3H-indol-1-ium-2-yl)vinyl)-6-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)oxy)phenyl)propanoate
Synonyms	OTL0038 OTL-38OTL 0038 OTL 38OTL-0038 OTL38 Cytalux
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

ADME/Pharmacokinetics	Absorption, Distribution and Excretion The mean Cmax of pafolacianine was 59.1 ± 5.94 ng/mL and AUCinf was 63.6 ± 12.6 ng x hr/mL. Following a single intravenous infusion of radiolabeled pafolacianine sodium, approximately 35% of the dose was recovered in urine (19.1%) and in feces (15.8%) after approximately 3-5 weeks. The mean volume of distribution (Vz) is 17.1 ± 5.99 L, indicating distribution into tissues. During intraoperative molecular imaging for surgically resectable pulmonary adenocarcinoma, pafolacianine accumulated in nearly 70% of squamous cell carcinomas. The mean plasma clearance is 28.6 ± 4.97 L/hr. Metabolism / Metabolites The metabolism of pafolacianine is unknown; however, pafolacianine is not metabolized by cytochrome P450 (CYP) enzymes. Biological Half-Life The elimination half-life of pafolacianine is 0.44 ± 0.23 hours.
Toxicity/Toxicokinetics	Protein Binding Plasma protein binding of pafolacianine is 93.7%. No notable partitioning into red blood cells was observed.
References	[1]. Intraoperative near-infrared fluorescence imaging targeting folate receptors identifies lung cancer in a large-animal model. Cancer. 2017 May 15;123(6):1051-1060.
Additional Infomation	Pafolacianine, or OTL38, is a folate analogue conjugated with a fluorescent dye that absorbs light in the near-infrared (NIR) spectrum within a range of 760 nm to 785 nm, with a peak absorption of 776 nm. It emits fluorescence within a range of 790 nm to 815 nm with a peak emission of 796 nm. The longer wavelength allows for deeper penetration of the fluorescent light through tissues for better imaging and detection of tumours. Pafolacianine targets the folate receptor alpha (FRα) which is upregulated in numerous epithelial malignancies. On November 29, 2021, the FDA approved pafolacianine as an adjunct imaging agent for intraoperative identification of malignant lesions in adult patients with ovarian cancer. It is currently under investigation for use in FRα-positive pituitary adenoma, lung cancer, and renal-cell carcinoma, which is thought to be the second-highest folate receptor-expressing cancer. Pafolacianine is a fluorescent imaging agent composed of a folate receptor-alpha (FRa)-targeting ligand conjugated to a fluorescent near infrared (NIR) dye, that can be used for imaging of FRa-expressing tumor cells. Upon administration, the FRa-targeting moiety of pafolacianine specifically binds to FRa expressed on tumor cells thus selectively delivering the fluorescent dye to FRa-expressing tumor cells. Upon NIR imaging, tumor cells fluoresce, which allows for the visualization and identification of FRa-overexpressing tumor cells. FRa, a high-affinity folate-binding protein and a member of the folate receptor family, is overexpressed in various cancer cell types. See also: Pafolacianine Sodium (active moiety of). Drug Indication Pafolacianine is an optical imaging agent indicated in adult patients with ovarian cancer as an adjunct for intraoperative identification of malignant lesions. Mechanism of Action Pafolacianine is consists of folic acid linked by its γ-carboxyl and a short spacer to an indocyanine green-related near-infrared dye called SO456. It is used for intraoperative fluorescence for tumour identification and surgical resection. It targets the folate receptor alpha (FRα), which is often overexpressed in various cancers including ovarian cancer. Pafolacianine binds to FRα-expressing cancer cells with an affinity of ~1 nM and is internalized via receptor-mediated endocytosis to be concentrated in FR-positive cancer tissues. Pafolacianine absorbs light and in the near-infrared (NIR) region within a range of 760 nm to 785 nm with a peak absorption of 776 nm and, upon excitation, emits fluorescence within a range of 790 nm to 815 nm with a peak emission of 796 nm. Pharmacodynamics Tumor to background ratios changed with different mass doses studied. High tumor to background ratio was observed with 0.025 mg/kg dose. Pafolacianine exposure-response relationships and the time course of pharmacodynamic responses are unknown.

Solubility Data

Solubility (In Vitro)

DMSO : ~25 mg/mL (~18.85 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (1.57 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 20.8 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: 2.08 mg/mL (1.57 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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	0.7539 mL	3.7693 mL	7.5386 mL
	5 mM	0.1508 mL	0.7539 mL	1.5077 mL
	10 mM	0.0754 mL	0.3769 mL	0.7539 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.