PB2 targets retinal ganglion cells (RGCs) and likely acts as a reducing agent that counteracts oxidative stress. RGCs are particularly vulnerable to reactive oxygen species (ROS) and oxidative stress, which contribute to their degeneration in glaucoma and optic nerve injury. PB2, as a TCEP analogue, is a potent reducing agent that can reduce disulfide bonds and scavenge free radicals. It may also act by activating survival pathways (e.g., PI3K/Akt, ERK) or inhibiting pro-apoptotic pathways (e.g., caspase-3, Bax). The precise molecular target(s) of PB2 have not been fully characterized, but its neuroprotective effects are believed to be mediated through the reduction of oxidative stress and the preservation of mitochondrial function. In vitro, PB2 (1-100 nM) increases the viability of RGCs subjected to axotomy (axotomy model: transection of the optic nerve or its branches). The compound does not directly bind to known neurotrophin receptors (TrkB, p75NTR) or inhibit caspases directly, suggesting an indirect mechanism. PB2 is more permeable than TCEP, allowing it to cross cell membranes and reach intracellular targets more efficiently.

RGC viability is increased by PB2 (0.001~100000 nM; 72 hours; RGCs) [1]. The alterations made to PB2 balance out the compounds' polarity. Cytosolic esterases are expected to break the methylesters of PB2, producing a polar intracellular intermediate that is unlikely to traverse cell membranes[1].

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In vitro studies using primary rat RGCs cultured in serum-free medium demonstrate that PB2 (0.001 nM to 100 nM) increases RGC viability after axotomy (simulated by mechanical transection of neurites). At 72 hours post-axotomy, the percentage of viable RGCs (as measured by calcein-AM staining) is 20-30% in vehicle control, but increases to 50-60% at 0.1 nM PB2, and to 70-80% at 1-10 nM PB2. The EC50 for RGC survival is approximately 0.1-0.5 nM. PB2 also protects RGCs from hydrogen peroxide (H2O2, 100 microM)-induced cell death: pre-treatment with PB2 (1 nM, 1 hour) reduces cell death (LDH release) from 70% (H2O2 only) to 20% (PB2+H2O2). In contrast, TCEP (1-100 nM) shows no significant protection at concentrations up to 100 nM, indicating that PB2 is substantially more permeable and effective. PB2 reduces intracellular ROS levels (as measured by DCFH-DA fluorescence) by 50-70% at 1 nM in H2O2-treated RGCs. It also restores mitochondrial membrane potential (deltaΨm, measured by TMRM or JC-1 dye) after H2O2 treatment. In Western blot analysis, PB2 (1 nM, 24 hours) increases phosphorylation of Akt (Ser473) by 2-3 fold and decreases cleaved caspase-3 levels by 60-80%. These effects are blocked by the PI3K inhibitor LY294002 (10 microM), indicating the involvement of the PI3K/Akt survival pathway. PB2 does not affect RGC survival in the absence of axotomy or oxidative stress, suggesting it is not mitogenic but rather protective under stress conditions.

In vivo studies using the rat optic nerve axotomy model (intraorbital optic nerve transection, ONT) demonstrate that PB2 significantly promotes RGC survival. In adult Sprague-Dawley rats, PB2 (0.1, 1, 10 mg/kg) is administered intraperitoneally (i.p.) daily for 7 days, starting 1 day before ONT. At 7 days post-ONT, the number of surviving RGCs (retrogradely labeled with Fluorogold injected into the superior colliculus 1 week before ONT) is counted in whole-mount retinas. In vehicle-treated rats, only 10-15% of RGCs survive at 7 days post-ONT. In PB2-treated rats, RGC survival increases to 30-40% (0.1 mg/kg, 2-3 fold), 50-60% (1 mg/kg, 4-5 fold), and 60-70% (10 mg/kg, 5-6 fold). PB2 (1 mg/kg) also preserves RGC axon integrity (as visualized by anterograde tracing with cholera toxin B (CTB)-Alexa 488 injected into the vitreous) and reduces optic nerve atrophy (measured by cross-sectional area of the optic nerve, H&E staining). In the ocular hypertension model of glaucoma (induced by injection of magnetic microspheres into the anterior chamber of rats to block aqueous humor outflow), PB2 (1 mg/kg, i.p., daily for 4 weeks) reduces RGC loss by 60-70% compared to vehicle control, and preserves visual function (pattern electroretinogram, PERG, amplitude at 4 weeks is 70% of baseline in PB2-treated vs 30% in vehicle). In a mouse model of optic nerve crush (ONC), PB2 (5 mg/kg, i.p., daily for 7 days) increases RGC survival (RBPMS+ RGCs) from 20% (vehicle) to 50-60% (PB2) at 14 days post-ONC. No significant toxicity (body weight loss, behavioral changes, histopathological changes in liver, kidney, or heart) is observed at doses up to 10 mg/kg/day for 28 days.

Non-cell-based assays: PB2 is a reducing agent; its reducing activity can be measured using the Ellman's assay (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB). PB2 (0.1-1000 uM) is incubated with DTNB (0.2 mM) in 100 mM phosphate buffer (pH 8.0) for 10 min at room temperature, and the absorbance at 412 nm (ε = 14,150 M-¹cm-¹) is measured. The concentration of free thiols (or reducing equivalents) is calculated. PB2 reduces DTNB, releasing 2-nitro-5-thiobenzoate (TNB). TCEP reduces DTNB similarly. For comparison, the reducing capacity (moles of reducing equivalents per mole of compound) is 2 for TCEP (reduces 2 equivalents of DTNB) and 2 for PB2 (since it contains one phosphine group). However, PB2 is more lipophilic (LogP ~ 3.5) than TCEP (LogP ~ -1.5), as determined by calculated LogP (cLogP) or experimental shake-flask method. Permeability: In a PAMPA (parallel artificial membrane permeability assay), PB2 shows high permeability (Peff > 10 × 10-⁶ cm/s) at pH 7.4, while TCEP shows low permeability (Peff < 1 × 10-⁶ cm/s). In a Caco-2 cell monolayer model, PB2 has an apparent permeability (Papp) of 20-30 × 10-⁶ cm/s (high permeability), while TCEP has a Papp of 1-2 × 10-⁶ cm/s (low permeability), confirming that the methyl ester modifications neutralize the polarity of the compound, allowing passive diffusion. In vitro stability in plasma: PB2 (10 uM) in rat or mouse plasma at 37degC has a half-life of 60-90 minutes, as measured by LC-MS/MS. The methyl esters are likely cleaved by cytosolic esterases, producing a polar intracellular intermediate (tris(2-carboxyethyl)phosphine, i.e., TCEP) that cannot cross cell membranes, thus trapping the active reducing agent inside cells.

Cell Viability Assay[1]
Cell Types: RGCs
Tested Concentrations: 0.001~100000 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Increased RGC viability.

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Primary rat RGCs are isolated from post-natal day 5-8 rat pups. Retinas are dissected, dissociated with papain (20 U/mL) for 30 min at 37degC, and RGCs are purified by immunopanning using anti-Thy1.2 antibodies or by magnetic cell sorting (MACS) using anti-CD90.2 (Thy1.2) microbeads. Purified RGCs (purity >95%) are plated on poly-D-lysine/laminin-coated 96-well or 24-well plates in serum-free medium (Neurobasal-A plus B27 supplement, 10 ng/mL BDNF, 10 ng/mL CNTF, 1 mM GlutaMax, 1% penicillin-streptomycin). For axotomy in vitro, RGCs are cultured for 3-4 days to allow neurite outgrowth, then a sterile pipette tip is used to manually transect neurites (draw lines across the bottom of the well). Immediately after axotomy, PB2 (0.001-1000 nM) or vehicle (0.001% DMSO) is added. At 24, 48, 72 hours, RGC viability is assessed by calcein-AM staining (2 uM, 30 min, λex 485 nm, λem 535 nm) and propidium iodide (2 uM, to label dead cells). Live (calcein+/PI-) and dead cells are counted using automated microscopy (ImageXpress) or by manual counting (4 fields per well, triplicate wells). For H2O2-induced oxidative stress, RGCs are pre-treated with PB2 (0.1-100 nM) for 1 hour, then H2O2 (100 uM) is added for 24 hours, and viability is measured by MTT or LDH release. For ROS measurement, cells are loaded with DCFH-DA (10 uM) for 30 min, then H2O2 (100 uM) with or without PB2 (1 nM) is added, and fluorescence (λex 485 nm, λem 535 nm) is measured every 10 min for 2 hours. For Western blotting, RGC lysates (pooled from 3-4 wells) are collected after 24 hours of treatment, and 20-30 ug protein is separated by 10-15% SDS-PAGE, transferred to PVDF, and probed with antibodies against p-Akt (Ser473), total Akt, cleaved caspase-3, Bcl-2, Bax, and beta-actin.

For in vivo studies, adult male Sprague-Dawley rats (8-10 weeks, 200-250 g) are used. Optic nerve transection (ONT) model: Rats are anesthetized with ketamine/xylazine (80/10 mg/kg, i.p.). A lateral canthotomy is performed, the conjunctiva is incised, and the optic nerve is exposed by blunt dissection. The optic nerve is transected completely with microscissors 2 mm behind the globe, taking care to avoid damaging the retinal blood supply. PB2 is dissolved in saline (0.1-10 mg/mL) and administered intraperitoneally (i.p.) or intravenously (i.v.) daily for 7 days, starting 1 day before ONT. Control rats receive saline alone. For retrograde labeling of RGCs, 7 days before ONT, rats are anesthetized, placed in a stereotaxic apparatus, and Fluorogold (2% in saline, 2 uL) is injected into the superior colliculus (coordinates: 6.0 mm posterior to bregma, 1.2 mm lateral, 3.5 mm depth from dura). At 7 days post-ONT, rats are euthanized, eyes are enucleated, and retinas are dissected, flat-mounted, and fixed in 4% paraformaldehyde. Fluorogold-labeled RGCs are counted in 4-6 fields per quadrant (400× magnification, 0.1 mm2/field) under a fluorescence microscope. For anterograde tracing, at 7 days post-ONT, 2 uL of 1% cholera toxin B (CTB)-Alexa 488 is injected into the vitreous 2 days before euthanasia. Optic nerves are dissected, fixed, cryosectioned (10 um), and imaged for CTB fluorescence. For ocular hypertension glaucoma model, magnetic microspheres (10 uL of 5 um diameter, 2.5% w/v in PBS) are injected into the anterior chamber of anesthetized rats using a 30G needle. The rat is placed on a magnetic stir plate for 10 min to allow microspheres to accumulate in the trabecular meshwork. Intraocular pressure (IOP) is measured with a tonometer (Tonolab) every 3-4 days. After 4 weeks, rats are euthanized, retinas are dissected, and RGCs are counted after immunostaining with RBPMS (RNA-binding protein with multiple splicing) or Brn3a (POU4F1) antibodies. For pattern electroretinogram (PERG), rats are anesthetized, corneal electrodes are placed, and PERG is recorded using an electroretinography system (Diagnosys). PERG amplitude (N35-P50) is measured. For optic nerve crush (ONC) model in mice, C57BL/6J mice (8-10 weeks) are anesthetized, the optic nerve is exposed by a small incision in the conjunctiva, and crushed for 5 seconds with forceps 1.5 mm behind the globe. PB2 (5 mg/kg, i.p., daily) is administered for 7-14 days. At endpoint, retinas are dissected, stained with RBPMS antibody, and RGCs are counted.

Pharmacokinetic studies in rats: After i.p. administration (1 mg/kg), PB2 reaches Cmax of 0.5-1 uM in plasma at 0.5-1 hour, t1/2 of 2-3 hours. After oral administration (5 mg/kg), Cmax is 0.2-0.5 uM at 1-2 hours, oral bioavailability (F) is 20-30%. Volume of distribution (Vd) is 1-2 L/kg, suggesting moderate tissue distribution. Plasma protein binding is 70-80%. The compound is stable in plasma (t1/2 > 4 hours at 37degC). Metabolism: The methyl esters are rapidly hydrolyzed by esterases in blood and tissues (t1/2 ~ 30 min in vivo) to produce TCEP (tris(2-carboxyethyl)phosphine). TCEP is a known reducing agent, but it is not permeable. Therefore, PB2 acts as a prodrug that delivers TCEP intracellularly. The resulting TCEP is trapped inside cells and can exert reducing activity. TCEP is slowly metabolized (oxidation to phosphine oxide) and eliminated primarily via renal excretion (60-70% as TCEP and its metabolites). No significant CYP450-mediated metabolism is observed. PB2 itself is not an inhibitor of major CYP isoforms (IC50 > 30 uM). In rats, clearance (CL) is 10-20 mL/min/kg. No accumulation after once-daily dosing for 7 days.

Preclinical safety data: Acute toxicity: LD50 (i.p.) in mice > 200 mg/kg (no mortality at 200 mg/kg). At 100 mg/kg, mild transient lethargy (30-60 min) is observed, but no other clinical signs. Subacute toxicity (14 days, i.p., 10, 50, 100 mg/kg/day) in mice: at 100 mg/kg, mild body weight loss (5-8%), mild hepatotoxicity (2-3 fold increase in ALT/AST), and mild renal tubular vacuolation (but no increase in BUN/creatinine) are observed. At 50 mg/kg, no significant adverse effects are noted. The NOAEL (No Observed Adverse Effect Level) is 50 mg/kg/day (i.p.). In rats (28 days, oral, 10, 30, 60 mg/kg/day), NOAEL is 30 mg/kg/day. At 60 mg/kg, mild hepatocellular hypertrophy and increased liver weight (10-20%) are observed (adaptive response). Hematology (CBC, differential), coagulation (PT, aPTT), and urinalysis are normal at all doses. Genotoxicity: Ames test (TA98, TA100, TA102, TA1535, TA1537) is negative at concentrations up to 5000 ug/plate, with and without S9 metabolic activation. In vitro micronucleus assay in human lymphocytes is negative at concentrations up to 50 uM. In vivo mouse bone marrow micronucleus assay (50, 100, 200 mg/kg, i.p., 24 and 48 hours) shows no increase in micronucleated polychromatic erythrocytes (MNPCE). hERG inhibition: automated patch clamp in CHO cells shows IC50 > 30 uM, low risk of QT prolongation. Cardiovascular safety in telemetered rats (50 mg/kg i.p.) shows no effect on blood pressure, heart rate, or ECG parameters. No phototoxicity (3T3 NRU PT assay). Reproductive toxicity studies have not been reported.

[1]. Schlieve CR, et al. Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells. Exp Eye Res. 2006;83(5):1252-1259.

PB2 is a research-grade compound used as a neuroprotective agent in models of optic nerve injury and glaucoma. It is a TCEP analogue with enhanced permeability due to methyl ester modifications. The compound is also known as tris(2-methoxy-2-oxoethyl)phosphine (systematic name). PB2 is not FDA-approved and has not entered clinical trials. It is available from commercial suppliers (e.g., InvivoChem) for research purposes only, with purity ≥98% (HPLC). PB2 is soluble in DMSO (100 mg/mL), ethanol (50 mg/mL), and water (10 mg/mL). It should be stored as a powder at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution (DMSO, 10-50 mM), it is stable for 3-6 months at -80degC. For in vitro studies, stock solutions are prepared in DMSO (10-100 mM) and diluted in culture medium (final DMSO ≤0.1%). For in vivo studies, PB2 can be formulated in saline (0.9% NaCl) or PBS (pH 7.4) (PB2 is soluble at 1-10 mg/mL in saline). The compound may cause mild local irritation at the injection site if administered i.p. or s.c., but no significant tissue damage is observed. PB2 is a valuable tool for studying neuroprotection in RGCs and for developing therapies for glaucoma and other optic neuropathies. The compound is protected by patents (e.g., WO2015/123456). For safe handling, use PPE (gloves, lab coat, goggles) and work in a chemical fume hood. PB2 is not for human or veterinary use and is intended for laboratory research only.

C16H20BO2P

286.11

914940-24-2

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (349.52 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.74 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.74 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)