α-lipoic acid derivative; glucose transport

None of the PEG-derivatives (AN-5, AN-6 and AN-11) or the ester derivatives of hexane (AN-30), octane (AN-31) and decane (AN-32) improved the rate of hexose transport in L6 myotubes (data not shown). However, AN-7 and AN-8 were active in vitro. These two compound as well as LA increased the rate of hexose transport in L6 myotubes in a dose- and a time-dependent manner, as shown in Figure 1, Figure 2, Figure 3, Figure 4. In these experiments the myotubes were pre-conditioned at 5.0 mM glucose or 23.0 mM glucose for 24 h. Pre-incubation of the myotubes at 23.0 mM glucose induced down-regulation of the hexose transport system, as was shown before.20 Maximal stimulatory effects of LA (2.5 mM) on the rate of hexose transport were 39±9% (mean±SEM, n=3) and 51±6% in L6 myotubes maintained at 5.0 and 23.0 mM glucose, respectively (Fig. 1). Higher concentrations of LA were toxic to the myotubes. AN-7 and AN-8 increased the rate of hexose transport by 92±11% and 82±6.8% in L6 myotubes at 5.0 mM glucose and by 120±8.9% and 93±11.2% at 23 mM. These maximal effects were obtained with 200 μM of each compound (Fig. 2). Maximal stimulatory effect of LA on myotubes exposed to 5.0 and 23.0 mM glucose was observed 2 h after its addition and thereafter it rapidly declined (Fig. 3 and 4). In contrast, half maximal and maximal effects of AN-7 and AN-8 were observed at 27 and 36 h, respectively, after their addition to the myotubes cultures exposed to 5.0 mM (Fig. 3). The corresponding values in myotubes exposed to 23.0 mM glucose were 22 and 36 h (Fig. 4). These data suggest a slow release of active LA from the prodrug molecules following accumulation in myotubes. The combined stimulatory effects of insulin and LA, AN-7 or AN-8 were additive both in L6 myotubes under the normo- or hyperglycemic conditions (Fig. 5). AN-7 and AN-8 were not toxic to the myotubes cultures [1].

Table 1 shows the effects of a 2-week treatment with LA (50 mg/kg BW/day), AN-7 or AN-8 (10 mg/kg BW/day) on blood glucose levels in streptozotocin-diabetic male C57/Black mice. AN-7 and LA reduced blood glucose levels in mildly diabetic mice by 30.2±5 (mean±SEM, n=8) and 40±13%, respectively. Lower doses of AN-7 (1 or 5 mg/kg BW/day) had no noticeable effect on blood glucose level of mildly or severly diabetic mice. A higher dose of AN-7 (20 mg/kg BW/day) produced similar glucose lowering effect in mildly diabetic mice as 10 mg/kg/day (data not shown). AN-8 failed to change blood glucose levels in these mice. Neither LA nor AN-7 or AN-8 had a significant effect on blood glucose levels in severely diabetic (Table 1) or in non-diabetic control mice (data not shown). One week after discontinuation of LA and AN-7 treatments, blood glucose levels returned to the same levels of control mice.[1]

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alpha-Lipoic acid [5-[1,2]-dithiolan-3-yl-pentanoic acid (LA)] is a natural antioxidant and cofactor of several enzymes. It increases the glucose transport activity in skeletal muscles and adipocytes in a non-insulin dependent manner. Therefore, LA is widely used in Type 2 diabetic patients as an oral auxiliary drug. However, large doses of LA (0.8-1.8 gr/day p.o.) are required due to its unfavorable pharmacokinetic parameters. In order to improve these parameters, we synthesized ester and amide LA derivates. Two of these newly synthesized compounds, 5-[1,2]-dithiolan-3-yl-pentanoic acid 3-(5-[1,2]dithiolan-3yl-pentanoylamino)-propyl]-amide (AN-7) and 5-[1,2]-dithiolan-3-yl-pentanoic acid 3-(5-[1,2]-dithiolan-3yl-pentanoyloxy)-propyl ester (AN-8) augmented the rate glucose transport in myotubes in culture in the absence or presence of insulin. Their potency was 12-fold higher than that of the parent compound; their maximal stimulatory effect was 1.5-fold higher than that of LA. When tested in vivo in streptozotocin-diabetic C57/Black mice, AN-7 (10 mg/kg/day for 2 weeks, s.c.) reduced blood glucose level by 39% while a higher dose of LA (50 mg/kg/day for 2 weeks, s.c.) lowered it by 30%. These results indicate that AN-7 is more potent than LA in augmenting glucose transport in skeletal muscles and reducing blood glucose in diabetic animals [1].

Cell cultures [1]
A subclone of rat skeletal muscle L6 cells, selected for high fusion potential was used. Cells were grown in α-MEM supplemented with 10% (v/v) FCS, 100 U/mL penicillin G and 100 μg/mL streptomycin, in a 95%:5% air/CO2 humidified incubator at 37 °C. Differentiation of myocytes into myotubes was induced with 2% (v/v) FCS, as described.28 The culture medium was changed at least every 48 h.

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Hexose uptake assay [1]
The [3H] dGlc uptake assay was performed as described. Carrier-mediated dGlc uptake was calculated on the basis of protein quantity, determined by Bradford's method.30 Test compounds were added to cell cultures from stocks solution in DMSO by a 1000-dilution (final concentration 1% v/v). DMSO reduced the rate of hexose transport by less than 5%.

Male C57 black mice were housed (eight mice per cage) at the animal facility with a 12-h light–dark cycle at 23 °C. Diabetes was induced in 6-week-old mice by a single ip injection of streptozotocin (150 mg/kg body weight) as described.31 The diabetic mice were divided into two groups: The mildly diabetic group that had non-fasting blood glucose less than 250 mg/dL and a severe diabetic group in which blood glucose ranged between 250 and 600 mg/dL. Two weeks following the induction of diabetes both diabetic and control animals received daily sc injections of LA (50 mg/kg body weight), AN-7 or AN-8 (10 mg/kg body weight) or the vehicle, DMSO, daily, for 2 weeks. The volume of each injection was 20 μL.[1]
Glucose determination: Glucose concentration in culture medium samples and in blood (taken from the tip of tails) was determined with Glucometer Elite™ and blood glucose test strips.

[1]. Synthesis and characterization of new and potent alpha-lipoic acid derivatives. Bioorg Med Chem. 2004 Mar 1;12(5):1183-90.

1. In cultured L6 myotube cells, AN-7 and AN-8 enhanced hexose transport more effectively than LA. Their effects were additive with the stimulatory effect of insulin, suggesting that they act through an intracellular mechanism different from insulin. 2. AN-7 and AN-8 took longer to reach their maximum effect in myotube cells, which appears to depend on the intracellular release rate of LA from its precursor molecules. 3. AN-7 was more effective than its parent compound LA in vivo; its hypoglycemic effect was similar to LA, but at only one-fifth the dose required. 4. AN-7 and similar amide derivatives could serve as prototype drugs for the development of highly effective LA prodrugs as adjunctive therapies for diabetes. [1]

C19H34N2O2S4

450.75

691410-93-2

10433927

Typically exists as solids at room temperature

1.2±0.1 g/cm3

702.1±43.0 °C at 760 mmHg

378.4±28.2 °C

0.0±2.2 mmHg at 25°C

1.572

3.1

2

6

14

27

402

0

O=C(CCCCC1CCSS1)NCCCNC(CCCCC2CCSS2)=O

691410-93-2; 5-(dithiolan-3-yl)-N-[3-[5-(dithiolan-3-yl)pentanoylamino]propyl]pentanamide; N,N'-(Propane-1,3-diyl)bis(5-(1,2-dithiolan-3-yl)pentanamide); A-lipoic acid derivative 1; 3-(5-[1,2]dithiolan-3yl-pentanoylamino)-propyl]-amide; SCHEMBL13834757;

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.)