iron chelator; α-synuclein aggregation
Iron and alpha-synuclein. PBT434 (ATH434) is a novel, brain-penetrant small molecule that acts as a modulator of iron metabolism. It reduces iron accumulation and iron-mediated redox activity, which in turn inhibits the aggregation of alpha-synuclein protein, lowers its tissue levels, and prevents its associated neurotoxicity in preclinical models. This dual mechanism is designed to halt the progression of diseases like Multiple System Atrophy (MSA).

Iron-generated H2O2 is greatly inhibited and the rate of Fe-mediated α-synuclein aggregation is significantly reduced by PBT434 methanesulfonate (0–20 µM; 3 hours) [1]. Brain microvascular endothelial cells are not cytotoxically affected by PBT434 methanesulfonate (0-100 µM; 24 h) [2]. The expression of total TfR and Cp protein levels in hBMVEC is increased by PBT434 methanesulfonate (20 µM; 24 h) [2].

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In this study, researchers determined that the iron chelator PBT434, which is currently being developed for treatment of Parkinson's disease and multiple system atrophy, modulates the uptake of iron by human brain microvascular endothelial cells (hBMVEC) by chelation of extracellular Fe2+. Treatment of hBMVEC with PBT434 results in an increase in the abundance of the transcripts for transferrin receptor (TfR) and ceruloplasmin (Cp). Western blot and ELISA analyses reveal a corresponding increase in the proteins as well. Within the cell, PBT434 increases the detectable level of chelatable, labile Fe2+; data indicate that this Fe2+ is released from ferritin. In addition, PBT434 potentiates iron efflux likely due to the increase in cytosolic ferrous iron, the substrate for the iron exporter, ferroportin. PBT434 equilibrates rapidly and bi-directionally across an hBMVEC blood-brain barrier. These results indicate that the PBT434-iron complex is not substrate for hBMVEC uptake and thus support a model in which PBT434 would chelate interstitial iron and inhibit re-uptake of iron by endothelial cells of the blood-brain barrier, as well as inhibit its uptake by the other cells of the neurovascular unit. Overall, this presents a novel and promising mechanism for therapeutic iron chelation.[2]

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In vitro, PBT434 (ATH434) has been shown to prevent alpha-synuclein toxicity in cellular models. By chelating and redistributing iron, it reduces oxidative stress and the formation of toxic alpha-synuclein aggregates. This neuroprotective effect is central to its development for synucleinopathies. It is a brain-penetrant and small molecule inhibitor of alpha-syn aggregation.

PBT434 methanesulfonate (30 mg/kg; oral; once daily for 21 days) demonstrated considerably less rotation in the L-DOPA paradigm, greatly reduced SNpc neuron loss in the MPTP model, and significantly conserved neuronal number in the 6-OHDA toxicity model[1].
In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. Researchers conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.[1]

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In a Phase 2 clinical trial for Multiple System Atrophy (MSA), ATH434 was found to be safe and well-tolerated. The trial showed robust clinical efficacy, with treated participants showing significant reductions in disease progression. Specifically, in a 12-month placebo-controlled study, ATH434 treatment led to a nearly 50% slowing of disease progression in patients with moderate-stage MSA, with 30% of patients showing stable symptoms. These positive results indicate strong disease-modifying potential.

α-synuclein aggregation assay[1]
Each batch of recombinant α synuclein that was synthesised underwent protein sequencing and mass spectrometry to ensure purity. The lyophilised purified WT recombinant α synuclein was reconstituted with Tris Buffer Saline (TBS) pH 7.4. Pooled aliquots were spun at 100,000 g for 30 mins at 4° to remove pre-formed aggregates/seeds. The supernant containing the monomeric form was collected and used in the assay. The protein concentration was determined using BCA method Iron Nitrate was weighed and dissolved in TBS solution. PBT434  was dissolved in 100% DMSO, then diluted to stock solution using milliQ water. To each tube, TBS, Fe, Compound/Veh then α synuclein was added in sequence with equal concentrations. The final concentration of α synuclein, Fe and compound was 186.6 μM.

Once all solutions were in the tubes, samples were vortex for 2 s before plating up. Samples were assayed in the presence of ThT (20 μM). The assay was read in a Perkin-Elmer Enspire multi-mode plate reader set at 37°, reading every 30 mins (1800 s), shaking at 800 rpm (1800 Seconds) between each read up to 42 h. ThT fluorescence intensity was measured over time at wavelengths 450 emission and 485 nm excitation. The RFU values were normalised to TBS ThT blank wells and were plotted over time. The lag-time and the maximal relative fluorescent units (RFU) were reported as a measure of kinetic profiling of compounds.

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Potentiometry[1]
Potentiometric titrations of the peptides were performed on a MettlerTitrando 907/Dosino 800 titration system, using InLab 422 combined glass-Ag/AgCl electrodes, which were calibrated daily by nitric acid titrations. 0.1 M NaOH (carbon dioxide free) was used as titrant. Sample volumes of 1.2–1.5 ml were used. The samples contained typically 0.8 mM PBT434 , dissolved in 4 mM HNO3/96 mM KNO3. The Fe (II) and Fe (III) complex formation was studied using a 2.5–4-fold excess of the compound over the metal ion, added as nitrate. All experiments were performed under argon at 25 °C, in the pH range of 2.3 to 12.2. The collected data were analyzed using the HYPERQUAD program [1]. Three to five titrations were included simultaneously into calculations, separately for protonation, Fe (II) and Fe (III) complexation.

The UV-visible spectra were recorded at 25 °C on a Cary 50 or a Perkin Elmer spectrophotometer, over the spectral range of 230–800 nm. The optical path for all experiments was 1 cm. The samples containing PBT434  alone or with Fe (II), Fe (III), Cu (II) or Zn (II) ions were titrated with NaOH in the pH range of 2.0–12.0, by careful manual additions of very small amounts of the concentrated base solution. For Fe (III) and Fe (II) the PBT434  concentration used was 0.1 mM, and the ligand-to-metal ratio was 4:1, to keep in line with conditions that delivered good potentiometic titrations. For Cu (II) the PBT434  concentration used was 0.1 mM, and the ligand-to-metal ratios used varied between 1:1 and 4:1. For Zn (II) spectroscopic titrations were performed at a lower concentration of 0.04 mM PBT434  and 0.02 mM Zn (II) to avoid precipitation. The Fe (II) samples were prepared under nitrogen, in a Coy glove box, and transferred to the spectrophotometer

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Cell‑free assays for PBT434 (ATH434) focus on its metal‑binding properties. To assess its ability to bind iron, a colorimetric or fluorescent assay can be used. A solution of ferric iron (Fe3+) (10 uM) is incubated with varying concentrations of ATH434 (1-100 uM) in a 50 mM HEPES buffer (pH 7.0). The mixture is allowed to reach equilibrium. The amount of free or iron-bound ATH434 can be measured using spectrophotometry or a metal-sensitive fluorescent probe such as calcein, which is quenched upon binding iron. The binding affinity (Kd) and stoichiometry of the iron-ATH434 complex can be determined. Additionally, an alpha-synuclein aggregation assay can be performed to measure its anti-aggregation activity in a cell‑free system.

Cell Cytotoxicity Assay[2]
Cell Types: hBMVEC
Tested Concentrations: 1, 10, 20, 50, 100 µM
Incubation Duration: 24 h
Experimental Results: demonstrated no cytotoxic effects on brain microvascular endothelial cells.

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Western Blot Analysis[2]
Cell Types: hBMVEC
Tested Concentrations: 20 μM
Incubation Duration: 24 h
Experimental Results: Increased the expression of total TfR, Cp protein level.

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MTT assay[2]
hBMVEC were grown to confluency in a 24-well plate, then treated with PBT434  at the indicated concentrations in cell culture media for 24h at 37°C. The next day, media was removed, and cells were incubated with RPMI+serum media containing MTT at 0.5mg/ml for 2h at 37°C, followed by incubation with 10% SDS/0.01N HCl for an additional 16h at 37°C to solubilize the MTT formazan crystals. Once solubilized, the solution was transferred to a 96-well plate in triplicates and the absorbance was read at 570nm on a plate reader. Values were blank corrected and normalized to the untreated control. Cells treated with 0.1% Triton X-100 were used as a positive control for cell death.

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14C-PBT434 accumulation and efflux assays[2]
For 14C-PBT434 uptake, hBMVEC monolayers were loaded with 20 μM 14C-PBT434  in RPMI1640 plus serum growth media for up to 3h at 37°C. Reactions were quenched with ice-cold quench buffer, as previously described, and lysed in lysis buffer. The lysates were assayed for 14C counts (Beckman LS6500 Scintillation Counter) and normalized to protein content determined by BCA assay.

For 14C-PBT434  efflux, hBMVEC monolayers were loaded with 20 μM 14C-PBT434  in RPMI plus serum growth media for 30min at 37°C, then washed twice with pre-warmed RPMI plus citrate and incubated in RPMI plus serum efflux media for an additional 2.5h. Every 30min, cells were quenched with ice-cold quench buffer, lysed, and processed as above. Cell-associated 14C counts were normalized to protein content.

For 14C-PBT434  trajectory assays, hBMVEC were grown in the apical chamber of transwell inserts were loaded with 20 μM 14C-PBT434 in either the apical chamber (RPMI+serum) or the basal chamber (RPMI-serum). Media samples were collected from both the apical and basal chamber at the indicated timepoints, and after 3h cells were quenched with ice-cold quench buffer, lysed, and processed the same as above. For a rough approximation of the intracellular PBT434  concentration at the endpoint of the uptake and efflux assays, the concentration was calculated from the pmol of 14C-PBT434 remaining in the cells using an estimation of 200K – 250K cells at confluence based on initial seeding density, and an approximated endothelial cell volume of 10,000 μm3.

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For cellular assays, human neuroblastoma cells (e.g., SH-SY5Y) are treated with ferric ammonium citrate to induce iron overload and oxidative stress, mimicking conditions in MSA. Cells are pre-treated with varying concentrations of PBT434 (0.1-10 uM) for 24 hours, followed by exposure to the iron challenge. The levels of alpha-synuclein protein are measured by Western blot, and markers of oxidative stress (e.g., reactive oxygen species (ROS) levels) are assessed using fluorescent dyes (e.g., DCFH-DA). The aggregation of alpha-synuclein can be visualized by immunocytochemistry. The compound's ability to prevent iron-induced neurotoxicity is measured by an MTT cell viability assay.

Animal/Disease Models: 12 weeks, 25 g, Male C57BL/6 J mice (6-OHDA intoxication model)[1]
Doses: 30 mg/kg
Route of Administration: Po; daily for 21 days (commencing 3 days following induction of lesion)
Experimental Results: Prevented neuronal loss following 6-OHDA, preserving up to 75% of the SNpc neurons remaining (both Nissl and tyrosine hydroxylase (TH) positive neurons) after the initial phase of cell death.

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Animal/Disease Models: 12 weeks, 25 g, Male C57BL/ 6 J mice (MPTP model)[1]
Doses: 1, 3, 10, 30, 80 mg/kg
Route of Administration: Po; daily for 21 days (commenced 24 h after induction of lesion)
Experimental Results: Increased the proportion of SNpc cells rescued , increased there was a trend to improved turning behavior, Dramatically increased varicosity abundance, prevented the decline in levels of the presynaptic marker synaptophysin (SYNP) in a dose-dependent manner.

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6-OHDA intoxication model[1]
Mice anesthetized with 2.5–3% isoflurane were placed into a stereotaxic apparatus and 3.0 μg of 6-OHDA was injected into the right SNpc, as described before. Amphetamine induced (5 mg/kg) rotational behavior was measured three days after 6-OHDA lesion using an automated Rotacounter system. Robust rotational behavior has been observed as early as one-day post-lesion. Only mice that exhibited rotations at day 3 between 200 and 450 times per hour were included in the trial. Mice were then randomly assigned to the PBT434  treatment group or sham-vehicle (VEH) treatment group. The PBT434  treatment group was gavaged at 30 mg/kg/day, commencing 3 days following induction of lesion. Experimenters were blinded to the assignment of treatments for each of the groups. Mice were retested and then culled twenty-one days post 6-OHDA lesion.

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MPTP model[1]
Mice were administered an acute dosing regimen of four injections of MPTP two hours apart. Each experimental trial contained MPTP lesioned animals that were randomly subdivided into a sham treated group (vehicle alone) and drug treatment group (30 mg/kg/day PBT434 , commencing 24 h after MPTP until culled at day 21). Experimenters were blinded to the assignment of treatments for each of the groups. In one group of animals the mice were treated with analog of PBT434  (PBT434-met 30 mg/kg/day) which does not have the ability to bind metals as a control.

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Cerebrospinal fluid collection from dogs[1]
The collection of cerebrospinal fluid (CSF) was performed at the conclusion of a 28 day toxicology study in 10 month old Beagle dogs. PBT434  was administered by oral gavage once a day for 28 days at the following doses: vehicle control (0 mg/kg/day), 10 mg/kg/day, 30 mg/kg/day and 50 mg/kg/day. Each treatment arm included 3 male and 3 female dogs. The CSF was extracted at necropsy into collection tubes containing 10uL of butylated hydroxytoluene, frozen on dry ice and stored at −80 °C until analysis. Any samples showing signs of hemolysis were excluded due to the potential for contamination of α-synuclein from blood [52].

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Cerebrospinal fluid collection from rats[1]
Cannulas were inserted into the lateral cerebral ventricles of wild-type rats by stereotactic surgery. CSF sampling was performed using a rodent microdialysis bowl (BASi, n = 8). Baseline CSF was sampled after which the animals were gavaged with PBT434  at 30 mg/kg. CSF samples were collected at one and four hours after gavage with PBT434 . Samples were analysed by Western blot for the presence of α-synuclein as described.

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In vivo efficacy is studied in a transgenic mouse model of Multiple System Atrophy (MSA) that overexpresses alpha-synuclein. PBT434 (ATH434) is formulated in a 0.5% methylcellulose suspension and administered by oral gavage at doses of 10-30 mg/kg once daily for 3 months. Control animals receive vehicle. Motor function is assessed using the Rotarod test (measuring latency to fall) and the pole test (assessing bradykinesia). At the study endpoint, the animals are perfused, and their brains are collected. The levels of aggregated alpha-synuclein in the brainstem and striatum are quantified by immunohistochemistry and Western blot. The levels of iron in specific brain regions are measured by inductively coupled plasma mass spectrometry (ICP-MS).

PBT434 mesylate (ATH434 mesylate) is an orally bioavailable, small molecule. It is designed to be brain-penetrant, allowing it to reach its targets in the central nervous system. In preclinical models, it is well-absorbed after oral administration, achieving sufficient brain concentrations to modulate iron levels. Detailed human pharmacokinetic data from its Phase 1 study have been completed, supporting its progression into Phase 2 trials.

Preclinical toxicology studies have shown that PBT434 (ATH434) is safe and well-tolerated. In the 12-month Phase 2 clinical trial for MSA, the drug was reported to be safe, with positive clinical efficacy results. No severe adverse events have been reported in published trial results. The compound has demonstrated a clean safety profile, with no noticeable in vivo toxicity in animal models.

[1]. The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease. Acta Neuropathol Commun. 2017 Jun 28;5(1):53.

[2]. Bailey DK, Clark W, Kosman DJ. The iron chelator, PBT434, modulates transcellular iron trafficking in brain microvascular endothelial cells. PLoS One. 2021 Jul 26;16(7):e0254794.

Genetic and experimental evidence strongly suggests that α-synuclein is closely associated with the etiology of Parkinson's disease (PD), and therefore this protein is considered a potential target for disease-modifying therapy. As our understanding of the role of iron in the pathogenesis of PD deepens, more and more evidence suggests that selectively targeting this ubiquitous biometal can modulate the level of α-synuclein. The development of PBT434 is precisely to take advantage of this therapeutic opportunity. In addition to its potential clinical application value, it will also become an important tool for studying the role of metals in regulating the level of α-synuclein, the role of oxidative stress as an initiation and persistence factor of substantia nigra lesions, and the involvement of other components of the neuronal iron transport mechanism. Current therapies for PD and atypical Parkinson's syndrome can at best provide limited symptom relief and cannot change the progression of the disease. In three different animal models of Parkinson's disease, the beneficial effects of PBT434 on motor function, neuropathology, and biochemical markers of disease state suggest its potential for disease modification. [1] In summary, we provide in vitro evidence that PBT434 can cross the blood-brain barrier and enter the interstitial space, which is consistent with the results of early clinical trials. Furthermore, we found that while PBT434 had a moderate effect on the regulation of LIP and downstream iron-dependent protein expression, unlike high-affinity iron chelators, it did not significantly interfere with normal cellular physiological functions. In addition, PBT434 was able to bind and redistribute extracellular ions Fe2+, thereby limiting downstream oxidative stress associated with this pro-oxidant and its role in the aggregation of cytotoxic proteins. This novel mechanism of action provides a compelling reason for the continued development of PBT434 as a treatment for neurodegenerative diseases associated with metal accumulation. [2]

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PBT434 mesylate (ATH434 mesylate) is an oral, brain-penetrant metal chaperone in clinical development for Multiple System Atrophy (MSA). It has been granted Fast Track designation by the U.S. FDA. Positive Phase 2 clinical trial results have been reported, showing a significant slowing of disease progression in MSA patients. The compound works by modulating iron homeostasis, reducing alpha-synuclein aggregation, and providing neuroprotection. It is not yet FDA-approved and is for research use. CAS: 2387898-69-1.

C13H17CL2N3O5S

398.262180089951

397.026

C, 39.21; H, 4.30; Cl, 17.80; N, 10.55; O, 20.09; S, 8.05

2387898-69-1

1232840-87-7; 2387898-69-1 (mesylate); 1232841-78-9 (HBr)

139593496

White to off-white solid powder

3

7

3

24

484

0

S(O)(=O)(=O)C.O=C1N(C(CNCC)=NC2=C(C(Cl)=CC(Cl)=C12)O)C

UBTJWJNTOFSHON-UHFFFAOYSA-N

InChI=1S/C12H13Cl2N3O2.CH4O3S/c1-3-15-5-8-16-10-9(12(19)17(8)2)6(13)4-7(14)11(10)18;1-5(2,3)4/h4,15,18H,3,5H2,1-2H3;1H3,(H,2,3,4)

5,7-dichloro-2-(ethylaminomethyl)-8-hydroxy-3-methylquinazolin-4-one;methanesulfonic acid

PBT434 MESYLATE; PBT434 (methanesulfonate); 2387898-69-1; ATH434 MESYLATE; ATH-434 MESYLATE; ATH434; ATH-434; ATH 434; PBT-434 MESYLATE; 826P1VAG3U; EX-A8324;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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