Tarenflurbil (R-flurbiprofen) is the R-enantiomer of the racemate NSAID flurbiprofen. This study identifies that its effect on amyloid-β (Aβ) generation is mediated through the retinoid X receptor α (RXRα).

- Interferes with the binding of 9-cis-retinoic acid (9-cis-RA) to RXRα, with an IC₅₀ of approximately 75 μM in a competitive ligand binding assay using the RXRα ligand-binding domain (LBD). [1]
- Does not affect cell surface levels of APP, total APP, BACE1, or PS1 levels. [1]

(R)-Flurbiprofen, or tartenflurbil, has the ability to dramatically lower Aβ release while simultaneously raising intracellular Aβ levels. Both 9-cis-RA and unlabeled (R)-flurbiprofen competitively block the interaction of [3H]9-cis-RA with RXRα. (9-cis-retinoid acid, or 9-cis-RA) inhibits Tarenflurbil ((R)-Flurbiprofen)'s lowering of Aβ secretion, and (R)-Flurbiprofen can interfere with the interaction between RXRα and 9-cis-RA). The intracellular Aβ species levels are markedly increased upon treatment with tartenflurbil ((R)-Flurbiprofen)[1]. It is evident that second generation GSMs and nonsteroidal anti-inflammatory drug-based GSMs have different mechanisms of action with regard to Notch processing because Tarenflurbil ((R) )-Flurbiprofen, a well-known nonsteroidal anti-inflammatory drug, affects only Aβ and does not affect Notch β formation[2].

---

Tarenflurbil demonstrates a dual effect on Aβ levels in N2a695 cells (mouse neuroblastoma cells stably expressing human APP695), decreasing extracellular Aβ while increasing intracellular Aβ. This effect is dependent on RXRα.

1. Differential Regulation of Extracellular and Intracellular Aβ: Treatment of N2a695 cells with Tarenflurbil (150 μM, 24 h) significantly reduced the levels of secreted Aβ40, Aβ42, and Aβ38 in the conditioned media, as measured by ELISA. Conversely, it significantly increased the levels of these Aβ species within cell lysates. These findings were confirmed by immunoprecipitation and immunoblotting. Immunocytochemistry also showed stronger intracellular Aβ40 staining in treated cells. Cell surface APP levels, assessed by biotinylation, were unaffected. [1]
2. Requirement of RXRα for Aβ Modulation: The effects of Tarenflurbil on both reducing extracellular Aβ and increasing intracellular Aβ were abolished in cells where RXRα was knocked down by specific siRNA. Overexpression of RXRα did not prevent these effects. As a control, Tarenflurbil remained effective in reducing Aβ secretion in cells treated with TNFα/IFNγ, which also increase Aβ secretion. [1]
3. Interaction with RXRα: In a competitive ligand-binding assay, Tarenflurbil inhibited the binding of [³H]9-cis-RA to the RXRα LBD with an IC₅₀ of approximately 75 μM. Unlabeled 9-cis-RA served as a positive control. [1]
4. Interference by 9-cis-RA: Co-treatment of N2a695 cells with a low concentration of 9-cis-RA (100 nM) significantly attenuated the reduction of Aβ40 and Aβ42 secretion caused by Tarenflurbil (150 μM). At this concentration, 9-cis-RA alone slightly increased Aβ secretion. [1]
5. No Effect on APP Processing Machinery: Modulation of RXRα levels or treatment with Tarenflurbil did not alter total APP, BACE1, or PS1 protein levels, nor did it affect BACE1 enzymatic activity or Notch cleavage (a γ-secretase substrate). RXRα also did not affect the degradation of extracellular Aβ. [1]

In C57BL6/J mice that develop a non-remitting form of the disease and in SJL mice that develop a relapsing-remitting (RR)-EAE, the effects of both an early and late start of treatment with Tarenflurbil ((R)-Flurbiprofen) are evaluated. When administered within three days of immunization, tartenflurbil ((R)-Flurbiprofen) totally prevents the development of clinical EAE scores in C57BL6/J mice. We call this regimen "preventive treatment." Since the effect is dose-dependent, 5 mg/kg/day is the minimal daily dose required for full prevention. The positive control, Fingolimod (FTY720, 0.5 mg/kg/day), has effects similar to those of Tarenflurbil ((R)-Flurbiprofen). In C57BL6/J mice, tareflurbil ((R)-Flurbiprofen) also significantly lowers clinical EAE scores when treatment is started just prior to the onset of clinical manifestations, a strategy known as semi-therapeutic (10 mg/kg/day), and when treatment is started on day 13 (5 mg/g/day), after the disease has fully developed[3].

---

Tarenflurbil demonstrates potent therapeutic effects in two mouse models of multiple sclerosis (EAE), preventing and attenuating disease progression, reducing inflammation, and alleviating pain.

1. Prevention and Attenuation of Clinical EAE (C57BL6/J mice - Primary Progressive EAE): Oral Tarenflurbil (2.5, 5, 10 mg/kg/day in drinking water) dose-dependently prevented the development of clinical EAE scores when started early (3 days post-immunization). At 5 mg/kg/day, it completely prevented disease. It also significantly reduced scores with semi-therapeutic (started 7-8 days post-immunization, 10 mg/kg/day) and late-therapeutic (started 13 days post-immunization, 5 mg/kg/day) regimens. Its efficacy was comparable to fingolimod (FTY720, 0.5 mg/kg/day). [3]
2. Attenuation of Relapsing-Remitting EAE (SJL mice): Preventive treatment with Tarenflurbil (5 mg/kg/day from day 3) almost completely prevented clinical scores. Late-therapeutic treatment (started at first remission, day 19) significantly reduced the relapse rate and severity of subsequent scores. It also improved rotarod performance during remission. [3]
3. Reduction of EAE-Evoked Pain: In C57BL6/J mice, Tarenflurbil (10 mg/kg/day from day 3) significantly reduced heat hyperalgesia (hot plate), mechanical hyperalgesia (dynamic plantar test), and cold allodynia (acetone test). In SJL mice with late-therapeutic treatment, it significantly reduced heat hyperalgesia (Hargreaves test and tail flick test) during the second remission. [3]
4. Reduction of Immune Cell Infiltration and Microglial Activation (Immunofluorescence and FACS): In C57BL6/J mice, Tarenflurbil prevented the infiltration of CD11b+ myeloid cells, CXCR3+ cells, and CD3+ T cells into the spinal cord, and preserved a resting, branching phenotype of resident microglia. Bone marrow transplant studies with β-actin-EGFP donors confirmed a strong reduction of invading CD11b+ and CD4+ cells. In SJL mice with late treatment, Tarenflurbil reduced CD11b+ and F4/80+ macrophage/microglia infiltrates and prevented myelin destruction. [3]
5. Increase in Regulatory T Cells and IL-10 (FACS of Splenocytes): Tarenflurbil treatment increased the frequency of CD4+CD25+FoxP3+ regulatory T cells (Tregs) and CTLA4+ inhibitory T cells in the spleen. It also strongly increased the levels of the anti-inflammatory cytokine IL-10. [3]
6. Reduction of Pro-inflammatory Gene Expression (Microarray): Microarray analysis of the spinal cord showed that Tarenflurbil strongly reduced the EAE-evoked upregulation of 523 pro-inflammatory genes, particularly those involved in response to stimulus, immune system processes, and signal transduction, while genes related to metabolic processes were unaffected. [3]
7. Reduction of Optic Neuritis and Brain Inflammation (In Vivo Imaging): In SJL mice, Tarenflurbil significantly reduced optic neuritis (measured with a bioluminescent inflammation probe) and brain/spinal cord inflammation (measured with MMPsense-680 near-infrared probe) during the first disease flare. [3]
8. Reduction of Blood-Brain Barrier Leakage and Myelin Damage: Late-therapeutic Tarenflurbil treatment reduced blood-brain barrier leakage (BSA-Cy5.5 imaging) in SJL mice and reduced myelin inflammation/destruction (DBT imaging and MBP ELISA) in C57BL6/J mice. Immunofluorescence of the optic nerve showed preserved myelin basic protein (MBP) and neurofilament (NF200) staining. [3]

The study did not employ direct enzyme activity assays on purified enzymes for Tarenflurbil. The primary molecular interaction assay was a competitive ligand-binding assay.

1. RXRα Ligand-Binding Assay: The human RXRα ligand-binding domain (LBD, amino acids 223-462), prepared as a polyhistidine-tagged fusion protein, was incubated with 7.5 nM [³H]9-cis-RA in the presence of increasing concentrations of unlabeled 9-cis-RA or Tarenflurbil for 14 hours at 4°C. The RXRα LBD was then captured using nickel-coated beads, and bound radioactivity was measured with a scintillation counter to determine competitive inhibition. [1]
2. BACE1 Activity Assay: Cells with modulated RXRα levels were lysed, and the lysates were assayed for β-secretase (BACE1) activity using a commercial kit, following the manufacturer's protocol. This was done to rule out an effect of RXRα on BACE1. [1]

Multiple cell-based assays were used to characterize the effects of Tarenflurbil and RXRα.

1. Cell Line and Culture: N2a mouse neuroblastoma cells stably transfected with human APP695 (N2a695 cells) were used. They were cultured in standard N2a media supplemented with 400 μg/mL G418. [1]
2. Aβ Quantification (ELISA): After treatments, conditioned media and cell lysates were collected. Levels of Aβ38, Aβ40, and Aβ42 were measured using commercial MSD ELISA kits according to the manufacturer's protocols. [1]
3. Aβ Immunoprecipitation and Immunoblotting: Aβ from conditioned media and cell lysates was immunoprecipitated and then immunoblotted with the 6E10 antibody. Densitometry was used to quantify Aβ levels. [1]
4. Immunocytochemistry: N2a695 cells treated with or without Tarenflurbil were immunostained with a rabbit anti-Aβ40 antibody, followed by a fluorescently labeled secondary antibody and DAPI nuclear stain. Images were collected using a fluorescence microscope. [1]
5. Cell Surface Biotinylation: Cells were incubated with 0.5 mg/mL Sulfo-NHS-LC-Biotin at 4°C. Excess biotin was quenched with glycine. Cell lysates were precipitated with streptavidin beads, and samples were immunoblotted with an anti-APP antibody (22C11) to detect cell surface APP. [1]
6. RXRα Modulation (Overexpression and Knockdown): To overexpress RXRα, cells were transfected with an RXRα expression vector using Lipofectamine 2000. To knock down RXRα, cells were transfected with a SMARTpool of RXRα-specific siRNAs or control GFP siRNA using Lipofectamine RNAi MAX. [1]
7. Notch Cleavage Assay: Cells were transfected with a Myc-tagged Notch construct (NAE). After modulation of RXRα levels, cell lysates were immunoblotted with an anti-Myc antibody (9E10) to detect total Notch and an antibody specific for the cleaved Notch intracellular domain (NICD). [1]
8. Aβ Degradation Assay: Naïve N2a cells with modulated RXRα levels were treated with Aβ-containing media collected from N2a695 cells. Conditioned media were collected at various time points and residual Aβ40 levels were measured by ELISA. [1]

Multiple detailed in vivo protocols were used to evaluate the effects of Tarenflurbil in EAE models.

1. **Animals:** Female C57BL6/J mice (for primary progressive EAE) and female SJL mice (for relapsing-remitting EAE), aged 10-12 weeks at immunization, were used. [3]
2. **Drug Formulation and Administration:** Tarenflurbil was administered orally via the drinking water at doses of 2.5, 5, or 10 mg/kg/day. For late-therapeutic treatment in severely affected C57BL6/J mice, it was administered via drug-soaked sweet cornflakes to ensure intake. FTY720 (fingolimod, 0.5 mg/kg/day) was used as a positive control. [3]
3. **EAE Induction:**
- **C57BL6/J:** Mice were immunized subcutaneously with 200 μg MOG35-55 peptide emulsified in Complete Freund's Adjuvant (CFA), followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) at 1-2 h and 24 h post-immunization. [3]
- **SJL:** Mice were immunized subcutaneously with 200 μg PLP139-151 peptide in CFA, followed by two i.p. injections of 200 ng PTX. [3]
4. **Treatment Regimens:**
- **Preventive:** Started 3 days after immunization. [3]
- **Semi-therapeutic:** Started 7-8 days after immunization (before symptom onset). [3]
- **Late-therapeutic:** Started after full disease development (day 13 for C57BL6/J; day 19 during first remission for SJL). For late treatment, mice were allocated as score-matched pairs. [3]
5. **Bone Marrow Transplantation (BMX):** Recipient C57BL6/J mice were irradiated (9.5 Gy) and received an i.v. injection of 6 × 10⁶ bone marrow cells from β-actin-EGFP donor mice. EAE was induced 3 weeks after transplantation. [3]
6. **Clinical Scoring and Behavioral Tests:** EAE scores were assessed daily. Rotarod performance and nociceptive behavior (hot plate, Hargreaves, dynamic plantar, acetone, tail flick tests) were evaluated at various time points. [3]
7. **In Vivo Imaging:** Mice were anesthetized with isoflurane and imaged using an IVIS Lumina Spectrum. Probes used: Xenolight RediJect Inflammation Probe (i.p., for MPO activity), MMPsense-680 (i.v., for metalloproteinase activity), BSA-Cy5.5 (i.v., for blood-brain barrier leakage), and DBT (i.v., for myelin imaging). [3]
8. **Tissue Collection and Ex Vivo Analysis:** At defined time points, mice were perfused, and tissues (spinal cord, brain, spleen, optic nerve) were collected for immunofluorescence, FACS analysis, LC-MS/MS (endocannabinoids, prostaglandins, flurbiprofen enantiomers), ELISA (MBP), and microarray gene expression analysis. [3]

---

Multiple detailed in vivo protocols were used to evaluate the effects of Tarenflurbil in EAE models.

1. Animals: Female C57BL6/J mice (for primary progressive EAE) and female SJL mice (for relapsing-remitting EAE), aged 10-12 weeks at immunization, were used. [3]
2. Drug Formulation and Administration: Tarenflurbil was administered orally via the drinking water at doses of 2.5, 5, or 10 mg/kg/day. For late-therapeutic treatment in severely affected C57BL6/J mice, it was administered via drug-soaked sweet cornflakes to ensure intake. FTY720 (fingolimod, 0.5 mg/kg/day) was used as a positive control. [3]
3. EAE Induction:
- C57BL6/J: Mice were immunized subcutaneously with 200 μg MOG35-55 peptide emulsified in Complete Freund's Adjuvant (CFA), followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) at 1-2 h and 24 h post-immunization. [3]
- SJL: Mice were immunized subcutaneously with 200 μg PLP139-151 peptide in CFA, followed by two i.p. injections of 200 ng PTX. [3]
4. Treatment Regimens:
- Preventive: Started 3 days after immunization. [3]
- Semi-therapeutic: Started 7-8 days after immunization (before symptom onset). [3]
- Late-therapeutic: Started after full disease development (day 13 for C57BL6/J; day 19 during first remission for SJL). For late treatment, mice were allocated as score-matched pairs. [3]
5. Bone Marrow Transplantation (BMX): Recipient C57BL6/J mice were irradiated (9.5 Gy) and received an i.v. injection of 6 × 10⁶ bone marrow cells from β-actin-EGFP donor mice. EAE was induced 3 weeks after transplantation. [3]
6. Clinical Scoring and Behavioral Tests: EAE scores were assessed daily. Rotarod performance and nociceptive behavior (hot plate, Hargreaves, dynamic plantar, acetone, tail flick tests) were evaluated at various time points. [3]
7. In Vivo Imaging: Mice were anesthetized with isoflurane and imaged using an IVIS Lumina Spectrum. Probes used: Xenolight RediJect Inflammation Probe (i.p., for MPO activity), MMPsense-680 (i.v., for metalloproteinase activity), BSA-Cy5.5 (i.v., for blood-brain barrier leakage), and DBT (i.v., for myelin imaging). [3]
8. Tissue Collection and Ex Vivo Analysis: At defined time points, mice were perfused, and tissues (spinal cord, brain, spleen, optic nerve) were collected for immunofluorescence, FACS analysis, LC-MS/MS (endocannabinoids, prostaglandins, flurbiprofen enantiomers), ELISA (MBP), and microarray gene expression analysis. [3]

Metabolism / Metabolites
The known human metabolites of flurbiprofen include (2S,3S,4S,5R)-6-[(2R)-2-(3-fluoro-4-phenylphenyl)propionyl]oxy-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

---

Detailed pharmacokinetic data for Tarenflurbil were provided.

- Plasma Concentration-Time Course (Single Oral Dose): After a single oral dose of 10 mg/kg in C57BL6/J mice, R-flurbiprofen plasma concentrations were measured over time. Based on AUCs from 0-8 h, the percentage of R-to-S inversion was approximately 23%. [3]
- Continuous Oral Administration (Drinking Water): Plasma concentrations of R- and S-flurbiprofen in C57BL6/J and SJL mice receiving continuous R-flurbiprofen (10 mg/kg/day) showed equivalent bioavailability and inversion rates between strains, with circadian rhythms reflecting drinking behavior. [3]
- Tissue Distribution: Seven hours after a single oral dose of 10 mg/kg R-flurbiprofen in C57BL6/J mice, tissue concentrations in the liver, skin, and brain regions (prefrontal cortex, hippocampus) were about 5,000- to 10,000-fold lower than plasma concentrations, consistent with high plasma protein binding. [3]

The study provides information on the safety profile of Tarenflurbil.

- Low Toxicity Profile: The introduction states that R-flurbiprofen is "almost free of the side-effects typical of classical NSAIDs, such as gastrointestinal or renal toxicity." It is noted to have low toxicity even on high daily doses in humans. [3]
- Gastrointestinal Toxicity: The study explicitly contrasts R-flurbiprofen with S-flurbiprofen, stating that "only administration of S-flurbiprofen causes gastrointestinal toxicity." [3]
- In Vivo Safety in Mice: In the EAE experiments, mice treated with Tarenflurbil did not show obvious adverse effects. Body weight was monitored in one late-treatment experiment, and the R-flurbiprofen group had a significantly higher mean body weight than the vehicle group, suggesting better overall health. [3]
- No Formal Toxicity Assessment: The paper does not provide formal toxicological endpoints such as LD₅₀ or detailed organ histopathology for toxicity, as the primary focus was on efficacy. [3]

[1]. Retinoid X receptor-alpha mediates (R )-flurbiprofen's effect on the levels of Alzheimer's beta-amyloid. J Neurochem. 2009 Oct;111(1):142-9.

[2]. Second generation γ-secretase modulators exhibit different modulation of Notch β and Aβ production. J Biol Chem. 2012 Sep 21;287(39):32640-50.

[3]. R-flurbiprofen attenuates experimental autoimmune encephalomyelitis in mice. EMBO Mol Med. 2014 Sep 30;6(11):1398-422.

(R)-Flurbiprofen is a flurbiprofen, and it is the enantiomer of (S)-flurbiprofen. Tarenflurbil is an investigational drug that was previously used to treat patients with mild Alzheimer's disease. It is a selective amyloid depressant (SALA) that reduces the levels of the toxic peptide β-amyloid 42 (Aβ42) in cultured human cells and animal models. Aβ42 is a major initiator of neurotoxicity and amyloid plaque formation in the brains of Alzheimer's patients. Development of this drug for the treatment of Alzheimer's disease was terminated in June 2008. Tarenflurbil was also used in clinical trials for the treatment of prostate cancer. Tarenflurbil is the oral active synthetic enantiomer of flurbiprofen. Tarenflurbil activates c-Jun N-terminal kinase, increases AP-1 binding to DNA, and downregulates the expression of cyclin D1, thereby causing tumor cells to arrest in the G1 phase of the cell cycle and undergo apoptosis. This drug also affects the expression of nuclear factor κB, a fast-response transcription factor that stimulates the body's immune response to tumor cells. R-flurbiprofen does not inhibit cyclooxygenase.

---

Drug Indications
Its application/treatment in Alzheimer's disease and prostate cancer is under investigation.

---

Mechanism of Action
MPC-7869 is not an inhibitor of cyclooxygenases (COX-1 and COX-2). This compound regulates signal transduction and transcriptional activation pathways associated with nuclear factor κB (NF-κB), a major transcription factor involved in the expression of various molecules, including cell growth, cell death, and inflammation. Furthermore, MPC-7869 has recently been shown to regulate γ-secretase activity and selectively reduce Aβ42 peptide levels in vitro and in vivo, thereby reducing amyloid pathology in the brain. MPC-7869 has a good safety profile and has demonstrated high efficacy in animal models of cancer and Alzheimer's disease. In transgenic mouse studies, MPC-7869 reduced amyloid levels in the brain and prevented memory loss.

---

Tarenflurbil (R-flurbiprofen) is the R-enantiomer of the racemic non-steroidal anti-inflammatory drug (NSAID) flurbiprofen. Unlike the S-enantiomer, it has negligible cyclooxygenase (COX) inhibitory activity. It garnered significant interest as a potential Alzheimer's disease (AD) therapeutic based on earlier studies showing that some NSAIDs, including flurbiprofen, could reduce the generation of the amyloidogenic Aβ42 peptide. However, a large phase III clinical trial failed to show clinical benefit. This study provides a potential mechanistic explanation for this failure. It demonstrates that while Tarenflurbil reduces the secretion (extracellular levels) of Aβ, it simultaneously increases the levels of intracellular Aβ, which is believed to be more directly correlated with AD pathogenesis. The study identifies retinoid X receptor α (RXRα) as a key mediator of this effect. Tarenflurbil binds to RXRα, competing with its natural ligand 9-cis-RA, and this interaction is necessary for its modulation of Aβ. The findings suggest that a simple measurement of total or extracellular Aβ may not fully capture the complex effects of such compounds and that an increase in the intracellular pool of Aβ could counteract any potential benefit from lowering extracellular Aβ. [1]

C15H13FO2

244.26

244.089

51543-40-9

Flurbiprofen;5104-49-4

92337

White to off-white solid powder

1.2±0.1 g/cm3

376.2±30.0 °C at 760 mmHg

110-113ºC

181.3±24.6 °C

0.0±0.9 mmHg at 25°C

1.568

4.11

1

3

3

18

286

1

C[C@H](C1=CC(=C(C=C1)C2=CC=CC=C2)F)C(=O)O

SYTBZMRGLBWNTM-SNVBAGLBSA-N

InChI=1S/C15H13FO2/c1-10(15(17)18)12-7-8-13(14(16)9-12)11-5-3-2-4-6-11/h2-10H,1H3,(H,17,18)/t10-/m1/s1

(2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid

(R)-Flurbiprofen; E 7869; MPC 7869; E-7869; MPC7869; E7869; Flurizan; Furbiprofen; MPC-7869;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 50 mg/mL (~204.70 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.23 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (10.23 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (10.23 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.

---

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