Mitochondrial electron transport chain (ETC); soluble biguanide

IM176OUT05 (0.1-10 μM; 24 hours) has an IC50 of 3.2 μM and inhibits mitochondrial electron transport chain (ETC) activity by reducing oxygen consumption rate (OCR)[1]. In mouse and human induced pluripotent stem cells (iPSCs), IM176OUT05 (10 μM; 6 days) enhances the acquisition and maintenance of stem cell pluripotency [1]. IM176OUT05 (10 and 100 nM) stimulates the expression of genes linked to glycolysis and ETC complex enzymes, thereby promoting the shift to glycolytic metabolism [1].

IM176OUT05 (200 μL, 1%; given once daily to depilated areas) stimulates the hair follicle cycle and increases the number of hair follicles in mice, promoting hair regeneration[1].

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IM176OUT05 (IM) promotes hair regrowth in mice[1]
Preliminarily, we tested whether IM could enhance hair regrowth in mice without toxicity or other side effects (Supplementary Figure S6). The hair cycle was synchronized by the depilation of telogen phase hairs from 7-week-old C57BL/6 mice1, and various concentrations of IM were topically applied daily to the dorsal skin of the mice (Supplementary Figure S6). On day 9, dramatic changes were observed in the area treated with 1% IM, and black pigmentation and hair growth were robustly detected. Hair regrowth was not observed in the control areas or areas treated with 0%, 0.1%, or 0.5% IM, although pigmentation developed on day 11. Next, we compared the abilities of IM and minoxidil, which is an approved drug to treat hair loss, to promote hair regrowth in both male and female mice. IM treatment had a strong promoting effect on hair regrowth, especially in female mice (Fig. 3). By day 8, the skin color was clearly distinguished with darkening in IM-treated mice compared with that in either the control or minoxidil-treated mice. By day 10, hair regrowth was distinctly promoted by IM treatment (Fig. 3) and was reproducibly observed in an independent group of female mice (Supplementary Figure S7). These phenomena were similarly observed in the male mice, but the effect of IM was comparable to that of minoxidil in the male mice (Supplementary Figure S8). Rashes and scars were detected in a few mice after depilation (Supplementary Figure S8b, D0); however, adverse effects, such as skin problems induced by IM treatment, were not observed in any of the animals in the in vivo experiments.

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IM176OUT05 (IM) facilitates the cycle of hair follicle regeneration in mice[1]
The tissues were histomorphometrically analyzed based on the classification by Chase1,30 to show that IM stimulated the progression of the hair follicle cycle (Fig. 4a). On day 20, most hair follicles in the control mice were quantitatively in the anagen III stage, but most hair follicles in the IM-treated mice were at a later stage of anagen, mainly anagen stages V and VI, in longitudinal sections (Fig. 4b). Additionally, the number of hair follicles was obviously increased in the IM-treated mice compared with that in the control mice in transverse sections on day 7 (Fig. 4c), and the number in the IM-treated or minoxidil-treated mice was greater than that in the control mice on day 20 (Fig. 4d). Moreover, keratin 15 (K15), which is a marker of hair follicle stem cells31, was strongly expressed in the hair follicle bulge area of the IM-treated mice compared with that in either the control or minoxidil-treated mice on day 7 (Fig. 5a). The expression of β-catenin, which mediates hair follicle regeneration32, was also increased in the IM-treated mice on day 7. At this time point, Ki67-positive proliferating cells were already apparent in the IM-treated mice, indicating that hair follicle cycling and further expansion of proliferating progenitors were augmented by IM (Fig. 5a). The K15+/β-catenin+ populations were quantified by FACS analysis, and these populations occupied 3.2%, 11.7%, and 6.0% of single cells in the skin of the control and IM-treated, and minoxidil-treated mice, respectively (Fig. 5b). A 3.7-fold increase was observed in the IM-treated mice over the control mice (Fig. 5b). By day 20, K15 and β-catenin were strongly detected in all groups of mice (Fig. 5c), and the K15+/β-catenin+ populations were represented at over 24% in all groups (Fig. 5d). Shh, which is another essential factor for hair follicle development33, was clearly detected in the IM-treated or minoxidil-treated mice (Fig. 5c). The Ki67+/Shh+ populations accounted for <5% in all groups of mice on day 7 (Fig. 5b), but this percentage was significantly increased on day 20 by 13.8%, 36.7%, and 34% in the single cells in the skin of the control, IM-treated, or minoxidil-treated mice, respectively (Fig. 5d). On day 20, the Ki67+/Shh+ populations were increased by 2.7-fold in the IM-treated mice over those in the control mice (Fig. 5d).

IM176OUT05 (IM) penetration assay[1]
The SK-OV-3, MDA-MB-435, 786-O, MDA-MB-231, and MCF-7 cells were seeded at 500,000 cells per well in 12-well plates. The cells were treated with 10 μM of biguanides for 30 min. The cells in each well were washed with cold PBS, and the amount of biguanides were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS), and the data were analyzed using MassHunter B 01.03. For the AMP-activated protein kinase (AMPK) activation assay, 500,000 MCF-7 cells/well were plated in six-well plates. The cells were treated with concentrations ranging from 3 μM to 10 mM of biguanides and were incubated for 12 h. The cells were treated with 1% Triton X-100 cell lysis buffer, and the supernatant was collected after centrifugation at 12,000 rpm for 20 min. The protein samples (25 μg each) were added to the p-AMPK ELISA plates, and the absorbances of samples were measured at 450 nm.

Oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) measurement[1]
A549 cells were treated in triplicate with serially diluted IM176OUT05 (IM) for 24 h and were washed prior to the OCR measurements. In the reprogramming experiments, OSKM-transduced MEFs were reseeded in triplicate at a density of 3 × 103 cells per well in poly-l-lysine-coated 96-well XF plates 4 days after reprogramming. On the following day (day 5), the medium was replaced with mESC medium with or without the test chemicals. On day 7, OCR/ECAR was measured using a Seahorse XFe96 Flux analyzer according to the manufacturer’s instructions. The probe cartridge was calibrated without CO2 for 1 h, and then basal OCR/ECAR measurement was performed. The following ETC-targeting compounds were sequentially added at each indicated time point: 1.5 μM oligomycin (ATP synthase, complex V, inhibitor), 5 μM FCCP (uncoupler), and 0.5 μM rotenone (complex I inhibitor) + antimycin A (complex III inhibitor). The value was normalized against the cell number.

Animal/Disease Models: 7weeks old C57BL/6 mice[1]
Doses: 200 μL, 1%
Route of Administration: Apply to the depilated area; 200 μL, 1%, one time/day
Experimental Results: Strongly promoted the hair regrowth, especially in female mice.

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Hair regeneration model[1]
Dorsal skin hairs in the telogen phase from 7-week-old C57BL/6 mice1 were depilated with an animal clipper and wax. The following day, 200 μl of placebo control, 1% IM176OUT05 (IM), 1% minoxidil, or 1% metformin were applied daily to the area with a sterilized cotton swab. Images of each animal were captured daily, and the level of pigmentation was quantified by the intensity of the darkness of the back color in the same area (1.6 × 3 cm) using ImageJ software. The mice were sacrificed, and skin tissues were obtained on days 0, 7, 14, and 20. Half of the tissue was used for RNA isolation, and the other half of the tissue was fixed with 4% paraformaldehyde overnight for histochemistry.[1]

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Ear hole punch assay For pinnal tissue repair assays, three holes of 2 mm in diameter in each ear were punched with ear punches. The indicated concentration of IM176OUT05 (IM) was applied with a sterilized cotton swab every day. The hole area was measured with digital calipers

[1]. A novel and safe small molecule enhances hair follicle regeneration by facilitating metabolic reprogramming. Exp Mol Med. 2018 Dec 6;50(12):1-15.

Targeting hair follicle regeneration has been investigated for the treatment of hair loss, and fundamental studies investigating stem cells and their niche have been described. However, knowledge of stem cell metabolism and the specific regulation of bioenergetics during the hair regeneration process is currently insufficient. Here, we report the hair regrowth-promoting effect of a newly synthesized novel small molecule, IM176OUT05 (IM), which activates stem cell metabolism. IM facilitated stemness induction and maintenance during an induced pluripotent stem cell generation process. IM treatment mildly inhibited mitochondrial oxidative phosphorylation and concurrently increased glycolysis, which accelerated stemness induction during the early phase of reprogramming. More importantly, the topical application of IM accelerated hair follicle regeneration by stimulating the progression of the hair follicle cycle to the anagen phase and increased the hair follicle number in mice. Furthermore, the stem cell population with a glycolytic metabotype appeared slightly earlier in the IM-treated mice. Stem cell and niche signaling involved in the hair regeneration process was also activated by the IM treatment during the early phase of hair follicle regeneration. Overall, these results show that the novel small molecule IM promotes tissue regeneration, specifically in hair regrowth, by restructuring the metabolic configuration of stem cells.[1]

C11H18CLN5

255.75

255.125

C, 51.66; H, 7.09; Cl, 13.86; N, 27.38

1643659-96-4

1544871-16-0; 1643659-96-4 (HCl)

137796975

White to off-white solid powder

4

1

4

17

268

0

C1C=CC(CCNC(NC(N)=N)=N)=C(C)C=1.Cl

HUMHPPQJBIWIPM-UHFFFAOYSA-N

InChI=1S/C11H17N5.ClH/c1-8-4-2-3-5-9(8)6-7-15-11(14)16-10(12)13;/h2-5H,6-7H2,1H3,(H6,12,13,14,15,16);1H

1-(diaminomethylidene)-2-[2-(2-methylphenyl)ethyl]guanidine;hydrochloride

IM176OUT05; 1643659-96-4; IM-176OUT05

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: 125 mg/mL (488.76 mM)

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.)