Adrenocorticotropic hormone (ACTH) (4-10) analog; adrenocorticotropic hormone-like peptide

Semax (100 nM) has been shown to increase the survival of cholinergic basal forebrain neurons [3], decrease Abeta oligomer (100 μM) levels [2], stimulate choline acetyltransferase activity in isolated basal forebrain tissue cultures [3], and stimulate the synthesis of brain-derived neurotrophic factor (BDNF) in astrocytes cultured in rat basal forebrain [4]. Semax (25 μM and 100 μM, 48 hours) exhibits these effects [2], [3], and [4].

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Here we tested the anti-aggregating properties of a heptapeptide, Semax, an ACTH-like peptide, which is known to form a stable complex with Cu2+ ions and has been proven to have neuroprotective and nootropic effects. We demonstrated through a combination of spectrofluorometric, calorimetric, and MTT assays that Semax not only is able to prevent the formation of Aβ:Cu2+ complexes but also has anti-aggregating and protective properties especially in the presence of Cu2+. The results suggest that Semax inhibits fiber formation by interfering with the fibrillogenesis of Aβ:Cu2+ complexes. [2]

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We studied the effect of Semax (Met-Glu-His-Phe-Pro-Gly-Pro), a behaviorally active adrenocorticotropic hormone (4-10) analogue, on survival of cholinergic basal forebrain neurons in vitro. Semax is known to stimulate learning and memory and can be successfully used for treatment of ischemic stroke.
Methods: Primary cultures of neuronal and glial cells from basal forebrain of rats were used in all experiments. The stability of Semax in cell cultures was tested by HPLC analysis. Cell survival in neuronal cultures was quantitated using immocytochemical and cytochemical analyses as well as detection of choline acetyltransferase activity.
Results: We have shown that Semax may approximately 1.5-1.7 fold increase survival of cholinergic basal forebrain neurons in vitro. Moreover, Semax (100 nM) stimulated activity of choline acetyltransferase in dissociated basal forebrain tissue cultures. However, the numbers of GABA-ergic neurons, total neuron specific enolase neurons were not affected. In concentration from 1 nM to 10 microM, Semax did not affect proliferation of glial cells in primary cultures.
Conclusion: Implications of these findings with respect to Alzheimer's disease remain to be clarified.[3]

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In a previous study, we observed the effects of 'Semax' (Met-Glu-His-Phe-Pro-Gly-Pro), the physiologically active analogue of adrenocorticotropic hormone(4--10), on neuronal cell survival in vitro. We hypothesized that these effects may be mediated by the regulation of expression of some neurotrophic factors. To test this hypothesis we analyzed NGF and BDNF gene expression in glial cells obtained from the basal forebrain of newborn rats, following in vitro treatment with 'Semax'. We observed changes in mRNA levels for both the NGF and BDNF genes. The greatest increase in expression was found after 30 min of 'Semax' administration. At this time, BDNF mRNA level was increased eight-fold in comparison with control, and NGF mRNA level was increased five-fold. [4]

Semax (100 μg/kg, intraperitoneal injection) increases the development and function of the vasculature in ischemic (pMCAO) rats [1]. Semax (50 µg and 250 µg, 100 µL/kg, intranasal inhalation) elevates b BDNF protein levels in the basal forebrain of rats [5].

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Background: The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex tissues to the action of Semax in vivo.
Results: The gene-expression alteration caused by the action of the peptide Semax was compared with the gene expression of the "ischemia" group animals at 3 and 24 h after permanent middle cerebral artery occlusion (pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and, 24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression. Among the immune-response genes, the expression of which was modulated by Semax, genes that encode immunoglobulins and chemokines formed the most notable groups. In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and vasculogenesis.
Conclusions: Semax affects several biological processes involved in the function of various systems. The immune response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote the formation and functioning of the vascular system.The immunomodulating effect of the peptide discovered in our research and its impact on the vascular system during ischemia are likely to be the key mechanisms underlying the neuroprotective effects of the peptide. [1]

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Corticotrophin (ACTH) and its analogues, particularly Semax (Met-Glu-His-Phe-Pro-Gly-Pro), demonstrate nootropic activity. Close functional and anatomical links have been established between melanocortinergic and monoaminergic brain systems. The aim of present work was to investigate the effects of Semax on neurochemical parameters of dopaminergic- and serotonergic systems in rodents. The tissue content of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum was significantly increased (+25%) 2 h after Semax administration. The extracellular striatal level of 5-HIAA gradually increased up to 180% within 1-4 h after Semax (0.15 mg/kg, ip) administration. This peptide alone failed to alter the tissue and extracellular concentrations of dopamine and its metabolites. Semax injected 20 min prior D: -amphetamine dramatically enhanced the effects of the latter on the extracellular level of dopamine and on the locomotor activity of animals. Our results reveal the positive modulatory effect of Semax on the striatal serotonergic system and the ability of Semax to enhance both the striatal release of dopamine and locomotor behavior elicited by D-amphetamine. [6]

ThT Measurements [2]
The kinetics of aβ fiber formation were measured using the Thioflavin T (ThT) assay. Samples were prepared by diluting, in MOPS buffer or in a solution containing bTLE LUVs, the aβ stock solution to reach the final concentration. Semax and/or copper were added from a stock solution to the indicated concentration. Thioflavin T was then added to a final concentration of 40 μM. Experiments were carried out in Corning 96-well non-binding surface plates. Time traces were recorded using a Varioskan plate reader using a λex of 440 nm and a λem of 485 nm at 37 °C, shaking the samples for 10 s before each read. All ThT curves represent the average of three independent experiments.

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Anti-oligomerization Assay [2]
Aβ1–42 oligomers were prepared as previously described from synthetic Aβ1–42 following the protocol of monomerization. Aβ1–42 (0.3 mg) was firstly dissolved in 5 mM DMSO. A solution of 100 μM Aβ1–42 in ice-cold DMEM F-12 without phenol red was prepared and allowed to oligomerize for 48 h at 4 °C according to the Lambert protocol with some modifications as previously described. To evaluate the ability of Semax and/or copper to interfere with Aβ oligomer formation, samples of Aβ1–42 were incubated in the presence or absence of each compound (Aβ/ligand ratios of 1:5 and 1:1 respectively). After 48 h incubation, Aβ/ligand compounds were analyzed for their content of Aβ oligomers.

MTT Assay [2]
Aβ1–42 peptide oligomers were prepared as described in ″Anti-oligomerization Assay″ with/without Semax and copper. d-SH-SY5Y cell viability was tested by incubation with 5 μM Aβ1–42 samples for 48 h in DMEM supplemented with 0.5% of FBS without RA. The viable cells were quantified by the reaction with MTT. After 90 min, the reaction was stopped by adding DMSO, and absorbance was measured at 570 nm; the results were expressed as % of viable cells. The experiments were repeated with n = 8, and results were expressed as mean ± SD.

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Primary cultures of glial cells were derived, with some modifications. Basal forebrains were dissected and mechanically dissociated. Cells were plated on plastic flasks at a density of 15×106 cells in 15 ml of culture medium, and grown in an incubator at 37°C under 5% CO2 in air. Culture medium (MEM) was supplemented with 15% FBS and 100 μg/ml gentamicin. The medium was changed every 3 days. When cell confluence was reached, cells were replated at a dilution of 1:3. At the next passage, cells were plated into four-well plates and confluent cultures were assayed. Medium was changed to MEM with 1% FBS and the cultures were incubated for a further 48 h. Then, aliquots of 100 μl of ‘Semax’ (10 μM final concentration) were injected into the culture medium and the plates were incubated for different times at 37°C in a CO2-incubator. A complete list of the cultures and treatment conditions used in this work is shown in Table 1. [4]

Animal/Disease Models: ischemia (pMCAO) rat [1].
Doses: 100 μg/kg
Route of Administration: intraperitoneal (ip) injection at 15 minutes, 1, 4 and 8 hrs (hrs (hours)) after permanent middle cerebral artery occlusion (pMCAO)
Experimental Results: Enhanced immune response (transcript upregulation).

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Animal/Disease Models: Rat[5]
Doses: 50 µg, 250 µg, 100 µL/kg
Route of Administration: Intranasal inhalation
Experimental Results: Brain-derived neurotrophic factor (BDNF) protein levels increased in the basal forebrain of rats.

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Animals were divided into two groups: (1) “ischemia” and (2) “ischemia + Semax” groups. pMCAO was performed in all animals. During the experiment, ischemia + Semax animals were given intraperitoneal injections of Semax (100 μg/kg), whereas ischemia animals were injected with saline. The injections of Semax or saline were performed 15 min, 1, 4 and 8 h after pMCAO. The rats were decapitated under anesthesia with ethyl ether 3 and 24 h after the operation. According to data from the literature, significant events in the formation of a stroke area, such as excitotoxicity, mitochondrial damage, emergence of reactive oxygen species, and apoptosis, occur within the first 3 h after occlusion of an artery, and the expression of genes at the early stage of ischemia can be studied at this time point. At the 24 h time point the infarction area reaches its maximal dimensions and the formation of the penumbra is completed[1].

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Drugs. Semax (Met-Glu-His-Phe-Pro-Gly-Pro, 0.15 and 0.6 mg/kg) was dissolved in saline (0.9% NaCl) and administered intraperitoneally (i.p.). [6]
Determination of the Tissue Content of Monoamines and Their Metabolites in the Brain Structures of C57/bl Mice. [6]
C57/bl mice were sacrificed 0.5 or 2 h after Semax (0.15 mg/kg, i.p.) administration (8–10 animals per group). The hypothalamus and striatum were isolated on ice (+4C) and stored in liquid nitrogen. The brain structures were homogenized in 0.1 M perchloric acid with 0.5 lM 3,4-dihydroxybenzoic acid as an internal standard and centrifuged (10,000
g, 10 min, 4C; K70D). The supernatants were analyzed with high performance liquid chromatography with electrochemical detection (HPLC/ED). Dopamine (DA), its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) as well as serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were detected with a glassy carbon electrode set at +0.8 V. The mobile phase contained 0.1 M citrate-phosphate buffer (pH 2.9), 1.85 mM 1- octanesulfonic acid, 0.27 mM ethylenediaminetetra-acetate (EDTA) and 8% acetonitrile. Monoamines and their metabolites were separated by the analytic reverse-phase column PhenomemexC18, 4 lm, 150
4.6 mm. The flow rate was 1.0 ml/min.

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Determination of the Extracellular Levels of Monoamines and Their Metabolites in Freely Moving Sprague–Dawley Rats. [6]
The animals were anesthetized with chloral hydrate (400 mg/kg, i.p.). The custom-made concentric dialysis probes (3 mm dialysis membrane, 0.5 mm outer diameter, 10 kDa cutoff, Hospal, Italy) were implanted into the right striatum (coordinates: AP +0.5, L+3, DV +6.5). At 20–24 h after surgery, the dialysis probes were connected to a microperfusion system and perfused at 2 ll/min with artificial cerebrospinal fluid containing (in mM): Na+ 155.0, K+ 2.9, Ca2+ 1.1, Mg2+ 0.8, and Cl) 133.0; pH 7.4. After an equilibration period of 1.5 h, dialysate fractions were subsequently collected every 20 min. The location of the microdialysis probes was verified post-mortem with cryostat microtome sections of the brain. At least four basal samples were taken before the first drug administration. Semax (0.15 or 0.6 mg/kg, i.p.) was administered either alone or in combination with the dopamine agonist D-amphetamine (D-AMPH) at the dose of 5 mg/kg (free base), i.p., 20 min after the Semax injection. Each group consisted of 6–7 rats. The dialysate fractions were analyzed with HPLC/ED. The potentials of electrochemical detection electrodes were set at )175 mV and +200 mV. The analytic column and the mobile phase were the same as described above.

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The Assessment of Locomotor Activity of C57/bl Mice. [6]
The effects of Semax (0.6 mg/kg, i.p.), D-AMPH (2.0 mg/kg, i.p.) and their combination on the locomotor activity of C57/bl mice were also studied. The dose of D-AMPH was chosen to elicit a clear enhancement in the locomotor activity. Each group consisted of 10–12 mice. The locomotor activity was recorded in every mouse 3 times for 10-min periods using the Opto-Varimex apparatus. The first session (intact mice) was 0–10 min, followed by an injection of Semax or saline. The second session was 20–30 min. The animals received an injection of D-AMPH or saline at 30 min. The third 1494 Eremin et al. session 50–60 min began 20 min after D-AMPH administration. The scheme of drug administration in the microdialisys and behavioral experiments was the same.

[1]. The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis. BMC Genomics. 2014 Mar 24;15:228.

[2]. Semax, a Synthetic Regulatory Peptide, Affects Copper-Induced Abeta Aggregation and Amyloid Formation in Artificial Membrane Models. ACS Chem Neurosci. 2022 Feb 16;13(4):486-496.

[3]. Effects of behaviorally active ACTH (4-10) analogue - Semax on rat basal forebrain cholinergic neurons. Restor Neurol Neurosci. 2008;26(1):35-43.

[4]. Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analog. Neurosci Lett. 2001 Aug 3;308(2):115-8.

[5]. Semax, an analogue of adrenocorticotropin (4-10), binds specifically and increases levels of brain-derived neurotrophic factor protein in rat basal forebrain. J Neurochem. 2006 Apr;97 Suppl 1:82-6.

[6]. Semax, an ACTH(4-10) analogue with nootropic properties, activates dopaminergic and serotoninergic brain systems in rodents. Neurochem Res. 2005 Dec;30(12):1493-500.

In this study, we analyzed the action of the neuroprotective peptide Semax on the transcriptome of rat brain cortical cells in conditions of experimental focal ischemia. Although Semax has been shown to be effective in brain stroke therapy, the molecular mechanisms underlying its neuroprotective action remain unknown. As shown here, Semax influenced various biological processes that contribute to the functioning of the different systems of the organism. The immune response was most markedly affected by the action of Semax. The peptide increased the amount and mobility of immune cells and enhanced the expression of chemokine and immunoglobulin genes. Our data showed that Semax is likely to influence processes that accompany the formation of new blood vessels during early ischemia cascade stages, as well as their stabilization at later stages. The expression of genes responsible for the intracellular level of Ca2+ was sensitive to Semax administration against the background of the unfolding pMCAO-induced neurodegenerative processes. Our results showed that Semax enhanced the expression of genes encoding protein products that promote intracellular Ca2+ accumulation. Possibly, the neuroprotective effect of Semax on ischemia-damaged nervous tissues includes an impact on processes involved in the incorporation of Ca2+ into cells. Thus, the immunomodulating effects of Semax described here, as well as its influence on the vascular system in conditions of ischemia, are likely to be key factors in the neuroprotective effects of the peptide. It cannot be ruled out that the large amount of genes that exhibited changed levels of expression, the functions of which remain unknown or not well studied, will help disclose other, hitherto unknown pathways of Semax action on damaged brain tissues. We must state at the same time that the baffling complexity of the multicomponent nature of cerebral ischemia and the ability of Semax to affect a large number of biological processes require future research to uncover the full scope of the mechanisms of action of this peptide. [1]

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In summary, we performed a series of biophysical experiments that show that the heptapeptide ACTH(4–7)-PGP (Semax) is able to inhibit, in the absence of the model membrane, Aβ fiber formation. In particular, we showed that in the presence of Cu2+ ions, Semax is able not only to sequester metal ions, inhibiting Aβ:Cu2+ complex formation, but also to interact, most probably, with Aβ:Cu2+ oligomers, retarding the formation of protofibrillar and fibrillar species. DSC results showed that the heptapeptide modulates the interaction of Aβ:Cu2+ with the hydrophobic core of the model membrane, preventing or retarding the insertion of prefibrillar species in the hydrophobic core of the model membrane. This finding is corroborated by the evidence that in the presence of the model membrane of bTLE, Semax could by itself inhibit fiber formation but exerts the maximum of its anti-aggregating properties in the presence of Cu2+ ions. This suggests that the heptapeptide interacts differently with monomeric/oligomeric species of Aβ and Aβ:Cu2+ complexes. This different behavior is confirmed by dye leakage experiments that, according to the ThT assay, showed a protective role of Semax. Finally, the MTT assay revealed that Semax or the Semax:Cu2+ complex protects cells from the toxicity of Aβ1–42 oligomers. More in-depth experiments should be done especially to investigate the effect of Semax on ROS production, which is known to increase as an effect of Aβ:Cu2+ interaction. [2]

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We were able to detect changes in mRNA levels for both NGF and BDNF genes in cultures treated with ‘Semax’. Moreover, the greatest increase in expression for both genes was found in glial cells that had been incubated with ‘Semax’ for 30 min. Levels of BDNF mRNA were increased eight-fold in comparison with controls at time zero (C, 0 min), and mRNA levels for the NGF gene were increased five-fold. At the same time, we observed constant levels of both gene expressions for control glial cells at times zero and 24 h (C, 0 min, C, 24 h), and a modest three-fold increase for both genes’ expression, during incubation with 15% FBS (60 min and 24 h: see Table 1). Reductions in mRNA levels for these genes in glial cell cultures incubated in the presence of ‘Semax’ over longer times (from 1 to 4 h for BDNF gene expression and from 1 to 3 h for NGF gene expression) were demonstrated. Further, we found a small increase in expression of both genes in glial cells incubated with ‘Semax’ for 8–16 h. The results obtained in this work clearly show that ‘Semax’, a synthetic analogue of ACTH(4–10) can increase NGF and BDNF mRNA expression in primary glial cells obtained from newborn rat brains. Thus, these results support our hypothesis that at least some effects of ‘Semax’ on neuronal cell survival may be mediated through the regulation of the expression of some neurotrophic factors. [4]

C39H55N9O12S

873.97

873.369

2828433-33-4

Semax;80714-61-0

155977617

H-Met-Glu-DL-His-Phe-Pro-Gly-Pro-OH.CH3CO2H; L-methionyl-L-alpha-glutamyl-DL-histidyl-L-phenylalanyl-L-prolyl-glycyl-L-proline acetic acid; Met-Glu-His-Phe-Pro-Gly-Pro

MEHFPGP

White to off-white solid powder

9

15

21

61

1480

5

CC(=O)O.CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)NC(CC1=CN=CN1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N3CCC[C@H]3C(=O)NCC(=O)N4CCC[C@H]4C(=O)O)N

SIUVGMURDLGHSO-MMTLCEBMSA-N

InChI=1S/C37H51N9O10S.C2H4O2/c1-57-16-13-24(38)32(50)42-25(11-12-31(48)49)33(51)43-26(18-23-19-39-21-41-23)34(52)44-27(17-22-7-3-2-4-8-22)36(54)46-15-5-9-28(46)35(53)40-20-30(47)45-14-6-10-29(45)37(55)56;1-2(3)4/h2-4,7-8,19,21,24-29H,5-6,9-18,20,38H2,1H3,(H,39,41)(H,40,53)(H,42,50)(H,43,51)(H,44,52)(H,48,49)(H,55,56);1H3,(H,3,4)/t24-,25-,26?,27-,28-,29-;/m0./s1

acetic acid;(2S)-1-[2-[[(2S)-1-[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-4-carboxybutanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-phenylpropanoyl]pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid

SEMAX ACETATE; 2828433-33-4; Semax acetate(80714-61-0 free base); AKOS040744796; DA-77789; TS-08141; acetic acid;(2S)-1-[2-[[(2S)-1-[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-4-carboxybutanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-phenylpropanoyl]pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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.)