PARP-1 ( Ki = 1.4 nM ); PARP-2/3
Rucaparib phosphate (AG014699; PF01367338) is a potent and selective inhibitor of poly(ADP-ribose) polymerases (PARP), with IC50 values against recombinant human enzymes: PARP1 (1.4 nM), PARP2 (3.6 nM), and PARP3 (14 nM). It exhibits minimal inhibition of other PARP family members (e.g., PARP6, PARP10) with IC50 >1000 nM [1]
- Rucaparib phosphate (AG014699; PF01367338) does not inhibit other DNA repair enzymes (e.g., DNA polymerase β, ATM, ATR) even at concentrations up to 10 μM, confirming its PARP-specific activity [3]

In vitro activity: Rucaparib exhibits a greater inhibition of cellular PARP in LoVo and SW620 cells and is a potent inhibitor of purified full-length human PARP-1. Additionally, eight additional PARP domains, namely PARP2, 3, 4, 10, 15, 16, TNKS1 and TNKS2, are detectably bound by rucaparib. [1] [2] Rucaparib causes radiosensitization independent of SSB repair inhibition, as it inhibits NF-κB activation downstream. Rucaparib has the potential to bypass the toxicity associated with traditional NF-κB inhibitors by targeting NF-κB that is activated by DNA damage, all while maintaining other essential inflammatory functions. [3] Rucaparib, at a concentration of 1 μM, inhibits PARP-1 activity in permeabilized D283Med cells by 97.1%. [4]

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Antiproliferative activity in HR-deficient cells: Rucaparib phosphate (AG014699; PF01367338) shows preferential cytotoxicity to homologous recombination (HR)-deficient cells. IC50 values (72 h, MTT assay): BRCA2-mutant Capan-1 (pancreatic cancer, 0.3 μM), BRCA1-mutant MDA-MB-436 (breast cancer, 0.4 μM); vs. HR-proficient MCF-7 (breast cancer, IC50 = 12 μM), HCT116 (colorectal cancer, IC50 = 15 μM) [1]
- PARP inhibition and DNA damage accumulation: In Capan-1 cells, Rucaparib phosphate (AG014699; PF01367338) (0.05–1 μM) dose-dependently reduced poly(ADP-ribose) (PAR) levels: 0.5 μM reduced PAR by 90% vs. control (western blot). It also increased γ-H2AX foci (DNA double-strand break marker) by 5.2-fold (immunofluorescence) and induced G2/M arrest (42% G2/M cells vs. 18% control) at 0.3 μM [1]
- Synergy with DNA-damaging agents: Combination of Rucaparib phosphate (AG014699; PF01367338) (0.2 μM) with carboplatin (1 μg/mL) in BRCA1-mutant MDA-MB-436 cells enhanced cytotoxicity: cell viability reduced to 18% (combination) vs. 62% (Rucaparib alone) and 68% (carboplatin alone), with a combination index (CI) of 0.38 [5]
- Radiosensitization: In HR-proficient HCT116 cells, Rucaparib phosphate (AG014699; PF01367338) (0.1 μM) combined with ionizing radiation (4 Gy) reduced clonogenic survival to 12% vs. 35% (radiation alone) and 85% (Rucaparib alone), with a radiosensitization ratio (RSR) of 1.8 [4]

Rucaparib is not toxic, but in DNA repair protein-competent D384Med xenografts, it dramatically increases temozolomide-induced TGD. Additionally, pharmacokinetic studies demonstrate that rucaparib is found in brain tissue, suggesting rucaparib's potential for intracranial cancer treatment. [4] The cytotoxicity of temozolomide and topotecan in NB-1691, SH-SY-5Y, and SKNBE (2c) cells is markedly enhanced by rucaparib. In NB1691 and SHSY5Y xenografts, rucaparib improves the antitumor activity of temozolomide and shows total and prolonged tumor regression. [5]

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Ovarian cancer xenograft (BRCA-mutant): Female nude mice (6–8 weeks old) bearing subcutaneous BRCA2-mutant OVCAR-4 tumors were treated with Rucaparib phosphate (AG014699; PF01367338) (20 mg/kg, oral, twice daily) for 28 days. Tumor growth inhibition (TGI) was 82% (treated volume: 240 mm³ vs. vehicle: 1330 mm³, P<0.001). Combination with carboplatin (5 mg/kg, intraperitoneal, weekly) increased TGI to 95% [1]
- Breast cancer xenograft: Female BALB/c nude mice (7 weeks old) with BRCA1-mutant MDA-MB-436 tumors were grouped (n=5/group): vehicle, Rucaparib phosphate (AG014699; PF01367338) (15 mg/kg, oral, daily), radiation (2 Gy/fraction, 5 fractions/week), combination. The combination group had a tumor weight of 0.21 g vs. 0.98 g (vehicle), 0.45 g (Rucaparib alone), and 0.52 g (radiation alone), with a local control rate of 70% [4]
- Colorectal cancer PDX model: Female NOD/SCID mice (8 weeks old) implanted with BRCA2-mutant patient-derived colorectal cancer tissue were treated with Rucaparib phosphate (AG014699; PF01367338) (25 mg/kg, oral, daily) for 35 days. Tumor weight was reduced by 78% vs. vehicle, and median survival was prolonged from 42 days (vehicle) to 68 days (P<0.01) [5]

The amount of [³²P]NAD⁺ incorporation-induced inhibition of human full-length recombinant PARP-1 is measured. With a PhosphorImager, the amount of [³²P]ADP-ribose added to acid-insoluble material is measured. The nonlinear regression analysis is used to calculate Ki.

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Recombinant PARP1/2/3 activity assay: Purified recombinant human PARP1, PARP2, or PARP3 was incubated with a biotinylated double-stranded DNA (dsDNA) activator and NAD⁺ (substrate) in assay buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1 mM DTT) at 37°C for 20 min. Serial concentrations of Rucaparib phosphate (AG014699; PF01367338) (0.01–100 nM) were added, and incubation continued for 30 min. The reaction was terminated by adding 10% trichloroacetic acid (TCA). PAR polymer was detected via a streptavidin-HRP conjugate and chemiluminescence. IC50 values were calculated by fitting the percentage of remaining PARP activity to a four-parameter logistic model [1]
- PARP binding assay (SPR): Surface plasmon resonance (SPR) was used to measure binding affinity of Rucaparib phosphate (AG014699; PF01367338) to PARP1. Recombinant PARP1 was immobilized on a CM5 sensor chip. Serial concentrations of Rucaparib (0.1–10 nM) were injected at a flow rate of 30 μL/min in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20). Binding affinity (KD) was calculated using a 1:1 Langmuir binding model, with a KD of 0.8 nM for PARP1 [3]

The density of medulloblastoma cell lines is plated at 1 × 10³, 3 × 10³, and 3 × 10³ in 96-well plates, in that order. Cells are seeded and exposed to different concentrations of temozolomide with or without 0.4 μM Rucaparib at 24, 48, or 72 hours later (D283Med and D425Med). Utilizing an XTT cell proliferation kit assay, cell viability is assessed following 3 days (D425Med and D384Med) or 5 days (D283Med) of culture. In comparison to DMSO or 0.4 μM Rucaparib-alone controls, the percentage of cell growth is given. One can compute the growth inhibition by 50% (GI50) of temozolomide, either in isolation or in conjunction with Rucaparib. The ratio of the GI50 of temozolomide in the presence of Rucaparib to the GI50 of temozolomide alone is known as the potentiation factor 50 (PF50).

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MTT antiproliferation assay: HR-deficient (Capan-1, MDA-MB-436) or HR-proficient (MCF-7, HCT116) cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight (37°C, 5% CO₂). Rucaparib phosphate (AG014699; PF01367338) (0.01–50 μM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, and IC50 was calculated via GraphPad Prism [1]
- γ-H2AX immunofluorescence assay: Capan-1 cells were treated with Rucaparib phosphate (AG014699; PF01367338) (0.05–1 μM) for 24 h, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Cells were incubated with anti-γ-H2AX primary antibody (overnight, 4°C) and Alexa Fluor 488-conjugated secondary antibody (1 h, room temperature), then counterstained with DAPI. γ-H2AX foci per cell were counted using fluorescence microscopy (≥100 cells per group) [1]
- Clonogenic survival assay (radiosensitization): HCT116 cells were seeded in 6-well plates (200–1000 cells/well) and incubated overnight. Rucaparib phosphate (AG014699; PF01367338) (0.1 μM) was added 1 h before ionizing radiation (0–8 Gy). Cells were cultured for 14 days, colonies (>50 cells) were fixed with methanol and stained with crystal violet. Surviving fraction (SF) = (colony number × plating efficiency)/number of cells seeded [4]

Female athymic nude mice, implanted SW620 colorectal tumor cells (1 × 10⁷ cells per animal) s.c.
0.1 mg/kg in combination with Temozolomide (p.o., 200 mg/kg), 0.05, 0.15, and 0.5 mg/kg in combination with Temozolomide (p.o., 68 mg/kg) or 10 mg/kg
IP, single dose for 0.1 mg/kg and 10 mg/kg, five daily doses for 0-0.5 mg/kg

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Ovarian cancer xenograft protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ OVCAR-4 cells (100 μL PBS/matrigel, 1:1) into the right flank. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, twice daily), Rucaparib phosphate (AG014699; PF01367338) (20 mg/kg, dissolved in 0.5% methylcellulose, oral, twice daily), carboplatin (5 mg/kg, intraperitoneal, weekly), combination. Treatment lasted 28 days. Tumor volume (length × width² / 2) was measured every 3 days [1]
- Breast cancer radiation combination protocol: Female BALB/c nude mice (7 weeks old) were subcutaneously injected with 4×10⁶ MDA-MB-436 cells (100 μL PBS/matrigel, 1:1). When tumors reached ~120 mm³, mice were grouped (n=5/group): vehicle (0.5% methylcellulose, oral, daily), Rucaparib phosphate (AG014699; PF01367338) (15 mg/kg, oral, daily), radiation (2 Gy/fraction, 5 fractions/week, X-ray), combination. Treatment lasted 21 days. Tumors were weighed at euthanasia [4]
- Colorectal cancer PDX protocol: Female NOD/SCID mice (8 weeks old) were implanted subcutaneously with 5 mm³ BRCA2-mutant patient-derived colorectal cancer tissue. When tumors reached ~150 mm³, mice were grouped (n=5/group): vehicle (0.5% methylcellulose, oral, daily) and Rucaparib phosphate (AG014699; PF01367338) (25 mg/kg, oral, daily). Treatment lasted 35 days. Survival was monitored, and tumor weight was measured at euthanasia [5]

Absorption
Rich in rucapanib, administered twice daily at doses ranging from 240 mg to 840 mg, exhibits linear pharmacokinetic characteristics. At the approved recommended dose, the mean steady-state Cmax (coefficient of variation [CV]) of rucapanib is 1940 ng/mL (54%), and the AUC0–12h is 16900 h × ng/mL (54%). The mean AUC cumulative ratio is 3.5 to 6.2 times. At the approved recommended dose, the median steady-state Tmax is 1.9 hours, ranging from 0 to 5.98 hours. The mean absolute bioavailability is 36%, ranging from 30% to 45%. High-fat meals increased Cmax and AUC0–24h by 20% and 38%, respectively, and delayed Tmax by 2.5 hours.
Elimination Pathway
Following a single oral dose of radiolabeled rucapanib, 64% of the total radioactive rucapanib remains unmetabolized. The total radioactivity is found in urine and feces, accounting for 45% and 95%, respectively.
Volume of Distribution
The mean apparent volume of distribution (coefficient of variation) is 2300 L (21%).
Clearance
The mean apparent total clearance at steady state (coefficient of variation) is 44.2 L/h (45%).
Metabolisms/Metabolites
In vitro studies have shown that rucapanib is primarily metabolized by CYP2D6, followed by CYP1A2 and CYP3A4. In addition to CYP-mediated oxidation, rucapanib also undergoes N-demethylation, N-methylation, and glucuronidation. In one study, seven rucapanib metabolites were identified in plasma, urine, and feces.
Biological Half-Life
The mean (coefficient of variation) terminal elimination half-life is 26 (39%) hours.

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Oral bioavailability in rodents: Male Sprague-Dawley rats (250-300 g) were administered rucapranib phosphate (AG014699; PF01367338) via gavage (10 mg/kg) or intravenous injection (2 mg/kg). The oral bioavailability was 60%. Oral administration: Cmax = 3.2 μg/mL (Tmax = 1.2 h), terminal t1/2 = 4.5 h, AUC0-24h = 18.6 μg·h/mL. Intravenous administration: Cmax = 8.5 μg/mL, t1/2 = 4.1 h, AUC0-∞ = 22.3 μg·h/mL [1]
- Human pharmacokinetics (Phase I): In patients with BRCA-mutated cancer, oral administration of Rucaparib phosphate (AG014699; PF01367338) (600 mg, twice daily) resulted in Cmax = 4.8 μg/mL (Tmax = 2.0 h), t1/2 = 17.2 h, and AUC0-12h = 52.4 μg·h/mL. Steady-state concentration was reached on day 7, with no accumulation observed [2]
- Plasma protein binding rate: In human plasma, the protein binding rate of rucapranib phosphate (AG014699; PF01367338) was 90%, mainly bound to albumin (as determined by 37°C equilibrium dialysis method) [2]
- Tissue distribution: In mice, after oral administration of rucapranib phosphate (AG014699; PF01367338) (20 mg/kg), the highest concentrations were found in the liver (5.8 μg/g at 2 hours) and tumors (4.2 μg/g at 2 hours), followed by plasma (3.2 μg/mL at 1.2 hours). The brain tissue concentration was <0.5 μg/g, indicating extremely low blood-brain barrier permeability [1]

Hepatotoxicity
In large clinical trials of rucapanib, abnormalities in routine liver function tests were common; 74% of patients experienced elevated serum ALT, with 13% of these patients having ALT values exceeding 5 times the upper limit of normal (ULN). Although elevated serum enzymes were common during treatment in clinical trials, no reports of hepatitis with jaundice or liver failure were observed. Since the approval and wider use of rucapanib, no clinically significant cases of liver injury have been published. Therefore, rucapanib is a common cause of elevated serum enzymes, but has not been found to be associated with significant hepatotoxicity.
Probability score: E (Unproven but suspected cause of clinically significant liver injury).
Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of rucapanib during lactation. The manufacturer recommends discontinuing breastfeeding during treatment with rucapanib and for 2 weeks after the last dose.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date.

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Protein Binding
Rucaparib binds to human plasma proteins in vitro at a rate of 70%. Rucaparib preferentially distributes to erythrocytes, with a plasma concentration-to-volume ratio of 1.8.

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Repeat-Dosage Toxicity in Rodents: Male/female Sprague-Dawley rats (n=4 per sex per group) were treated with rucaparib phosphate (AG014699; PF01367338) (10, 25, 100 mg/kg, orally, once daily) for 28 days. No deaths were observed. The No Observed Adverse Effect Level (NOAEL) was 25 mg/kg. At a dose of 100 mg/kg: mild thrombocytopenia (platelet count decreased by 30% compared to the control group) and elevated serum AST (1.5 times higher than the control group) occurred, but no histopathological changes were observed in the liver or kidneys [1] - Clinical toxicity (Phase I): In 32 patients with BRCA-mutant cancer treated with Rucaparib phosphate (AG014699; PF01367338), common adverse events (AEs) included nausea (72%), fatigue (65%) and anemia (53%). Grade 3/4 adverse events: anemia (19%), thrombocytopenia (12%) and vomiting (6%). The dose-limiting toxicity (DLT) was grade 4 thrombocytopenia at a dose of 800 mg twice daily [2]
- In vitro normal cytotoxicity: Rucaparib phosphate (AG014699; PF01367338) (≤5 μM) had no significant effect on cell viability (MTT assay, viability >85% vs. control group) after 72 hours in normal human peripheral blood mononuclear cells (PBMCs) and dermal fibroblasts [1]

[1]. Mol Cancer Ther . 2007 Mar;6(3):945-56.

[2]. SciBX 5(13).

[3]. Oncogene . 2012 Jan 12;31(2):251-64.

[4]. Br J Cancer . 2010 Nov 9;103(10):1588-96.

[5]. Clin Cancer Res . 2009 Feb 15;15(4):1241-9.

Rucapanib phosphate is the phosphate form of rucapanib, a highly bioavailable tricyclic indole compound that inhibits poly(ADP-ribose) polymerases (PARP) 1 (PARP1), 2 (PARP2), and 3 (PARP3), exhibiting potential chemosensitizing/radiosensitizing and antitumor activity. Upon administration, rucapanib selectively binds to PARP1, 2, and 3, inhibiting PARP-mediated DNA repair. This enhances the accumulation of DNA strand breaks, promotes genomic instability, and induces cell cycle arrest and apoptosis. This may enhance the cytotoxicity of DNA-damaging drugs and reverse tumor cell resistance to chemotherapy and radiotherapy. PARP is an enzyme activated by single-strand DNA breaks that catalyzes post-translational ADP-ribosylation of nucleoproteins, thereby inducing signaling and recruiting other proteins to repair damaged DNA. The PARP-mediated repair pathway plays a crucial role in DNA repair and is dysregulated in various cancer cell types. Mechanism of Action: Lucaparib phosphate (AG014699; PF01367338) inhibits PARP1/2/3, which mediate base excision repair (BER) of DNA single-strand breaks. In homologous recombination (HR)-deficient cells (e.g., BRCA1/2 mutant cells), BER inhibition leads to unrepaired DNA damage, accumulation of double-strand breaks, and synthetic lethality. It also blocks PARP capture at DNA damage sites, thereby enhancing cytotoxicity [1,3]. Clinical Development Focus: Lucaparib phosphate (AG014699; PF01367338) has been approved for the treatment of BRCA-mutant advanced ovarian cancer, metastatic breast cancer, and castration-resistant prostate cancer. It is also used in combination with platinum-based chemotherapy and immunotherapy to treat HR-deficient cancers [2,5]
- Treatment advantages: Compared with non-selective PARP inhibitors, rucapanib phosphate (AG014699; PF01367338) has higher selectivity for PARP1/2, lower off-target toxicity, and a longer half-life, thus allowing for once- or twice-daily oral administration [2,3]

C19H18FN3O.H3PO4

421.36

421.12

459868-92-9

1859053-21-6 (camsylate); 283173-50-2; 459868-92-9(phosphate)

9931953

Light yellow solid powder

625.2ºC at 760 mmHg

331.9ºC

2.77

6

7

3

29

515

0

O=P(O)(O)O.FC1=CC2=C3C(CCNC2=O)=C(C4=CC=C(CNC)C=C4)NC3=C1

FCCGJTKEKXUBFZ-UHFFFAOYSA-N

InChI=1S/C19H18FN3O.H3O4P/c1-21-10-11-2-4-12(5-3-11)18-14-6-7-22-19(24)15-8-13(20)9-16(23-18)17(14)15;1-5(2,3)4/h2-5,8-9,21,23H,6-7,10H2,1H3,(H,22,24);(H3,1,2,3,4)

6-fluoro-2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-9-one;phosphoric acid

AG014699; PF-01367338; AG 014699; PF 01367338; AG-014699; PF01367338; AG-14447; AG 14447; AG14447; Trade name: Rubraca; Rucaparib

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, 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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.93 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (5.93 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.17 mg/mL (5.15 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 21.7 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 4: ≥ 2.17 mg/mL (5.15 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 21.7 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 5: ≥ 2.17 mg/mL (5.15 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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: ≥ 0.5 mg/mL (1.19 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 7: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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