Signal peptide peptidase (SPP); gamma-secretase (Ki = 17 nM)

L-685,458 has an IC50 of 402 nM, 113 nM, and 48 nM, respectively, for inhibiting Aβ(40) formation in Neuro2A and CHO cell lines overexpressing human AβPP695, and in SHSY5Y cells overexpressing the construct spβA4CTF. Its potency for reducing Aβ(42) is approximately two times lower.[1] L-685,458, through inducing G0–G1 cell cycle arrest and apoptosis, inhibits the growth of Tca8113 cells.[2] Pre-treatment with L-685,458 increases the anti-proliferative effect of imatinib in a T-cell acute lymphoblastic leukemia cell line. [3] L685,458 inhibits signal peptide peptidase (SPP), which also significantly lowers HSV-1 replication in tissue culture.[4]

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L-685,458 Inhibition of Aβ Peptide Formation Assessed by the HTRF Assay. Directed screening of the Merck Sample Repository based on a compound exemplified in European patent application EP0778266-A1 led to the identification of L-685,458 (Figure 2). L-685,458 dose-dependently inhibits Aβ formation in both Neuro2A and CHO cell lines overexpressing human AβPP695, and in SHSY5Y cells overexpressing the construct spβA4CTF (Table 1). L-685,458 was found not to be cytotoxic to cells at concentrations up to 10 μM, as judged by redox dye measurements of cell viability (data not shown). L-685,458 reduced both Aβ(40) and Aβ(42) peptide formation in these cells, with the potency for reduction of Aβ(42) being about 2-fold lower (Table 1). The antibody 4G8 was used routinely in the HTRF assay to enable detection of both β- and α-secretase cleaved peptide products, although essentially the same results were obtained either with antibody 6E10 or with antibody W0-2 (Figure 1; data not shown). [1]

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L-685,458 Exhibits the Activity Profile of an AβPP γ-Secretase Inhibitor. The finding that L-685,458 is a potent inhibitor of Aβ formation in human neuroblastoma SHSY5Y cells overexpressing the construct spβA4CTF, which serves as a direct substrate for the AβPP γ-secretase enzyme ( 21, 23), indicated that this compound targets the AβPP γ-secretase activity rather than β-secretase. To confirm this, the metabolic turnover of spβA4CTF in SHSY5Y cells was first investigated by radiolabeling and pulse−chase analysis. In vehicle-treated cells, radiolabeled bands identified as LE-βA4CTF (the expression construct, equivalent to βCTF/C99, but containing the construct-derived additional two amino acids) and p3CTF (αCTF/C83) were seen to disappear with time (Figure 3A). This was accompanied by a time-dependent loss of radiolabeled intracellular LE-Aβ peptide, and the appearance of LE-Aβ and p3 secreted into the cell culture medium (Figure 3B). By comparison, treatment with L-685,458 resulted in the stabilization of LE-βA4CTF itself, and a marked increase in the formation of the α-secretase-cleaved product p3CTF (Figure 3A). Concomitantly, a reduction in detectable intracellular LE-Aβ (Figure 3A) and a near-complete inhibition of LE-Aβ and p3 secreted into the medium were observed (Figure 3B). The inhibitory action of L-685,458 was verified by conducting metabolic radiolabeling experiments with HEK293 cells overexpressing human AβPP695. Treatment with L-685,458 for 18 h, but not its inactive epimer L-682,679 (see below), resulted in a marked accumulation in the cell lysates of p3CTF, and to a lesser extent βCTF (Figure 3C). As expected, a near-complete inhibition of Aβ and p3 secreted into the medium was also observed (Figure 3C). L-685,458 does not markedly alter the secretion of the N-terminal fragments of AβPP, sAβPPα, and sAβPPβ, generated following α- and β-secretase activity (data not shown). This profile of AβPP processing is consistent with the specific inhibition of AβPP γ-secretase activity ( 12). A novel fragment of approximately 5−6 kDa (p5) also transiently accumulated intracellularly and was stabilized in the presence of L-685,458 inhibition (Figure 3A). [1]

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Treatment of the cells with L-685,458 (1 μM) essentially abolished the formation of all Aβ peptide species, with the exception of LE-Aβ(1−16) and LE-Aβ(1−15), the relative levels of which were increased (Figure 4B). Similar observations to the above have been made in the cell lines overexpressing full-length AβPP which generate Aβ peptides beginning at Asp-1 (data not shown), indicating that the results are not influenced by the use of the truncated AβPP construct. [1]

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Stereoselectivity of the L-685,458 Hydroxyethylene Moiety for AβPP γ-Secretase Inhibition. To confirm the direct binding of L-685,458 to γ-secretase, a SHSY5Y spβA4CTF in vitro γ-secretase membrane assay was used, in which the human γ-secretase enzyme catalyzes the breakdown of the human spβA4CTF endogenously expressed substrate. This assay also allowed us to probe the stereoselectivity at the hydroxyethylene dipeptide isostere by comparison of the activity of L-685,458 against two close structural analogues, L-682,679 and L-684,414 (Figure 2). In agreement with the cell-based data (Table 1), L-685,458 was found to be a potent inhibitor of AβPP γ-secretase activity in this assay (IC50 17 nM; Figure 2). On the other hand, its epimer, L-682,679, a highly potent inhibitor of HIV-1 aspartyl protease activity ( 19), was found to have negligible activity in the AβPP γ-secretase in vitro membrane assay (IC50 > 10 000 nM; Figure 2). L-684,414, the ketone derivative, was also found to be active (IC50 181 nM; Figure 2), but less so than L-685,458, as expected if the hydroxyethylene dipeptide is serving as a transition state mimic. Selectivity of L-685,458 for AβPP γ-Secretase over Protease Classes. The specificity of L-685,458 was assessed by comparison of its activity in the SHSY5Y spβA4CTF in vitro membrane assay against a panel of enzymes representing aspartyl, serine, and cysteine protease classes (Table 2). Of those tested, L-685,458 was clearly most potent against AβPP γ-secretase activity, displaying a selectivity of 50-fold or greater. [1]

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L-685,458 dose-dependently inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0–G1 cell cycle arrest and apoptosis. The mRNA and protein levels of Hairy/Enhancer of Split-1, a target of Notch activation, were decreased dose-dependently by L-685,458. Furthermore, L-685,458 down-regulated cyclin D1, B-cell lymphocytic-leukemia proto-oncogene 2 and c-Myc expressions, which are regulated by the transcription factor NF-κB. Coincident with this observation, L-685,458 induced a dose-dependent reduction of constitutive NF-κB activation in Tca8113 cells. Conclusions: The GSI L-685,458 may have a therapeutic value for the treatment of human tongue carcinoma. Moreover, the effects of L-685,458 in tumor inhibition may act partially via the modulation of Notch and NF-κB.[2]

L-685,458 decreased cortical total Aβ in PDAPP in a dose-dependent manner, with a 50% reduction at 100 mg/kg, and cortical Aβ42 was also shown to be reduced. When administered orally to Tg2576 mice, transgenic for the human APPV717F mutation, L-685,458 reduced brain levels of Aβ. L-685,458 also inhibits Notch signaling, which has an impact on zebrafish embryonic development.

Fluorescence resonance energy transfer (FRET) is used in the homogeneous time-resolved fluorescence (HTRF) immunoassay technique between two fluorophores: a donor EuK and a modified allophycocyanine pigment acceptor molecule, XL-665. From nitrogen laser-excited EuK to XL-665, nonradiative FRET occurs when in close proximity and produces an amplified long-lived fluorescence signal. In a nutshell, a standard 96-well plate assay comprises 0.75 nM antibody-EuK, 1.0 nM antibody-biotin, 2.0 nM SA-XL665, and 0.1−0.2 M potassium fluoride in each well. A total assay volume of 200 μL/well is obtained by adding samples of conditioned cell culture medium or synthetic peptide standards and culture medium alone. The addition of 1.0 nM nonbiotinylated antibody in lieu of the biotinylated antibody yields the blank values. Once combined, the reaction mixture is allowed to reach equilibrium binding at 4 °C before being read on the Discovery HTRF microplate analyzer using the manufacturer's instructions.

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HTRF Immunoassay for Aβ Quantitation. [1]
Homogeneous time-resolved fluorescence (HTRF) immunoassay methodology uses fluorescence resonance energy transfer (FRET) between two fluorophores, a donor EuK and a modified allophycocyanine pigment acceptor molecule, XL-665. When in proximity, nonradiative FRET takes place from nitrogen laser-excited EuK to XL-665, resulting in the emission of an amplified long-lived fluorescence signal. The details for Aβ peptide quantitation by this assay will be described elsewhere (M. S. Shearman, E. E. Clarke, unpublished observations). Briefly, in a typical 96-well plate assay, each well contained 0.75 nM antibody-EuK, 1.0 nM antibody-biotin, 2.0 nM SA-XL665, and 0.1−0.2 M potassium fluoride. Samples of conditioned cell culture medium or synthetic peptide standards and culture medium alone were added to give a total assay volume of 200 μL/well. Blank values were determined by the inclusion of 1.0 nM nonbiotinylated antibody in place of the biotinylated antibody. Following mixing, the reaction mixture was left at 4 °C to reach equilibrium binding, and then read on the Discovery HTRF microplate analyzer using the manufacturer's recommended settings. AβPP γ-Secretase in Vitro Membrane Assay. A total membrane fraction from SHSY5Y spβA4CTF cells was prepared by centrifugation of a broken cell preparation at 120000g for 60 min. The pellet was resuspended by homogenization in phosphate-buffered saline, pH 7.3, containing 5% glycerol and 2.5 mM dithiothreitol (DTT), aliquotted, and stored at −80 °C until use. Membranes (5−10 μg of protein) were incubated in 96-well plates for 90 min at 37 °C in 20 mM HEPES, pH 7.3, containing 0.1% bovine serum albumin, 0.25% CHAPSO, 0.5 mM EDTA, 1% glycerol, and 2.5 mM DTT in the presence of vehicle control (0.5% DMSO) or inhibitor. Samples were placed on ice, 100 μL per well of HTRF reagent mixture was added, and Aβ peptide was quantified using the Discovery HTRF microplate analyzer.

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Metabolic Radiolabeling Studies. [1]
For pulse−chase analysis, SHSY5Y spβA4CTF cells were preincubated for 30 min in methionine-free minimal essential medium containing 5% dialyzed fetal bovine serum (FBS), and then 200 μCi of [35S]methionine was added to each 10 cm dish for a period of 3.5 h. After washing, the cells were chased in Dulbecco's modified Eagle's medium/F12 supplement containing 10% FBS and 5 mM nonradioactive methionine in the presence of vehicle (0.5% DMSO) or inhibitor, for various time intervals. For steady-state analysis, human embryonic kidney (HEK) 293 cells overexpressing human AβPP695 were preincubated for 50 min in methionine-free minimal essential medium containing 10% dialyzed FBS and 1% glutamine. Vehicle (0.5% DMSO) or inhibitor were added 20 min before the addition of 400 μCi of [35S]methionine for a further 18 h. In both protocols, the conditioned media were harvested and centrifuged for 10 min at 13000g. The supernatants were diluted with 0.4 mL of lysis buffer (20 mM HEPES, pH 7.3, containing 1% Nonidet P40, 0.5% deoxycholate, 1 mM EDTA) and the samples precleared with 5 μg of mouse IgG and 30 μL of a 1:1 slurry of protein G−agarose. Aβ(40) and p3(40) were immunoprecipitated with 10 μg of monoclonal antibody G2-10 or 4G8 and 50 μL of a 1:1 protein G−agarose slurry by incubation overnight at 4 °C. Nonspecifically bound proteins were removed by washing as described ( 21) and immunoprecipitated proteins separated on 10−20% Tris−Tricine precast gradient gels. For immunoprecipitation of membrane-bound spβA4CTF or AβPP695 processing products, cell lysates were prepared by suspending the cells in lysis buffer and incubation for 30 min on ice with repeated mixing. Insoluble material was removed by centrifugation for 10 min at 13000g. Preclearing and immunoprecipitation with 10 μg of monoclonal antibody 4G8 were carried out as described above.

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Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF)Mass Spectrometry. [1]
Antibodies were diluted in phosphate-buffered saline to 0.5 μg/μL, and 1.0 μL was applied to an individual spot of the SELDI chip coated with a preactivated surface. Free reactive sites were blocked by incubation for 30 min at room temperature with 1 M ethanolamine hydrochloride, pH 7.5. Conditioned media diluted 2-fold with 25 mM HEPES, pH 7.3, 1 mM EDTA were incubated with an antibody-coupled SELDI chip overnight at 4 °C. Nonspecifically bound peptides were removed by extensive washing. A saturated solution of α-cyano-4-hydroxycinnamic acid in 40% acetonitrile, 0.25% trifluoroacetic acid was diluted 1:5 into the same solvent and 0.5 μL applied to each spot of the chip. After evaporation of the matrix solvent, the samples were analyzed on a SELDI mass analyzer MRS-1 with a linear time-of-flight mass spectrometer.

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Protease Assays. [1]
With the exception of the AβPP γ-secretase assay, counterscreening of L-685,458 against other protease activities was conducted with purified enzymes and commercially available peptide substrates. HIV-1 activity was monitored with DABACYLγ-Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Gln-EDANS, human liver cathepsin D at pH 3.5 with H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH, bovine trypsin (type I) with Bz-L-Arg-pNA, and HepC3 NS3-JK4 with [3H]p164 peptide. Inhibition of porcine erythrocyte calpain I and Carica papaya papain was assessed using a previously described dye-binding assay procedure with bovine casein as substrate.

MTT is used to measure the in vitro growth rate of L-685,458-treated Tca8113 cells. In short, 96-well plates are seeded with Tca8113 cells. On harvest day, 100 microliters of spent medium are swapped out for the same volume of fresh medium that contains 10% MTT (5 mg ml−1) stock. After four hours of incubation at 37°C, 100 μl of DMSO is added to each well, and the plates are shaken for ten minutes at room temperature. At 570 nm, the absorbance is measured.

NOD-SCID Mouse Hepatoma Model
5 mg/kg
Percutaneous administration; 5 mg/kg; 2 weeks

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In vivo administration of inhibitors [4]
Mice received 100 μg of (Z-LL)2 ketone or DAPT as an eye drop in 5 μl of DMSO 1 hr before ocular infection and at 2, 4, 6 and 8 hr PI. (Z-LL)2 ketone administration was repeated 5 times daily for 4 consecutive days. Sham control mice were treated similarly using 5 μl of DMSO alone. For ocular infection, mice were infected in both eyes without scarification or anesthesia by placing eye drops containing 2 × 104 PFU of HSV-1 strain McKrae in 2 μl of tissue culture medium. Eyes were swabbed once daily with a Dacron swab (Spectrum type 1) prior to administering the (Z-LL)2 ketone. The swab was transferred to a culture tube containing 1 ml of medium, frozen, thawed, and virus titers determined by standard plaque assay on RS cells as above.

[1]. Biochemistry . 2000 Aug 1;39(30):8698-704.

[2]. Oral Dis . 2007 Nov;13(6):555-63.

[3]. Drug Resist Updat . 2008 Dec;11(6):210-8.

[4]. Exp Eye Res . 2014 Jun:123:8-15.

L-685,458 is a peptide and carboxamide that is L-leucyl-L-phenylalaninamide, L-Leu-L-Phe-NH2, which has been acylated on the N-terminus by a Phe-Phe hydroxyethylene dipeptide isotere, 2R-benzyl-5S-tert-butoxycarbonylamino-4R-hydroxy-6-phenylhexanoic acid. Compounds based on the structure of L-685,458 are potent inhibitors of gamma-secretase, which mediates the final catalytic step that generates the amyloid beta-peptide (Abeta), which assembles into the neurotoxic aggregates in the brains of sufferers of Alzheimer's disease. It has a role as a peptidomimetic and an EC 3.4.23.46 (memapsin 2) inhibitor. It is a secondary alcohol, a peptide, a carbamate ester and a monocarboxylic acid amide. It contains a tert-butoxycarbonyl group.

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Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease. [1]

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Objective: To investigate the effect of the gamma-secretase inhibitors (GSIs) on the growth of human tongue carcinoma cells and to provide the molecular mechanism for potential application of GSIs in the treatment of tongue carcinoma. Materials and methods: Human tongue carcinoma Tca8113 cells were cultured with the GSI L-685,458. Cell growth was determined by the methylthiazole tetrazolium method. Cell cycle and apoptosis were analyzed by flow cytometry and/or confocal microscopy. RT-PCR and Western blot were employed to determine the intracellular expression levels. Nuclear factor kappa B (NF-kappaB) activation was examined by electrophoretic mobility shift assay.[2]

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Activation of Notch signaling has been implicated in pathogenesis of various hematologic tumors including leukemias, lymphomas, and multiple myeloma. Pre-clinical studies have suggested that inhibition of Notch could be an attractive new approach to treatment of hematologic malignancies. This review discusses most recent findings in the field and potential role of Notch signaling as a therapeutic target focusing on the effects of gamma-secretase inhibitors.[3]

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Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L-685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology. [4]

C39H52N4O6

672.85

672.389

C, 69.62; H, 7.79; N, 8.33; O, 14.27

292632-98-5

5479543

White to off-white solid powder

6.349

5

6

19

49

1030

5

O([H])[C@@]([H])([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[C@@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[H])=O)C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

MURCDOXDAHPNRQ-ZJKZPDEISA-N

InChI=1S/C39H52N4O6/c1-26(2)21-33(37(47)41-32(35(40)45)24-29-19-13-8-14-20-29)42-36(46)30(22-27-15-9-6-10-16-27)25-34(44)31(23-28-17-11-7-12-18-28)43-38(48)49-39(3,4)5/h6-20,26,30-34,44H,21-25H2,1-5H3,(H2,40,45)(H,41,47)(H,42,46)(H,43,48)/t30-,31+,32+,33+,34-/m1/s1

tert-butyl N-[(2S,3R,5R)-6-[[(2S)-1-[[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-benzyl-3-hydroxy-6-oxo-1-phenylhexan-2-yl]carbamate

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)

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.)