Antileishmanial

The results showed that dihydrocyclosporin A (DHCsA-d) was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 μM and 12.14 μM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 μM and 4.84 μM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 μM (24 h) and 6.65 μM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. Conclusions: The findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.[1]

In vitro inhibitory assays[1]
Promastigotes, in logarithmic phase, were grown at a cell density of 1.0 × 106 cells/ml, CsA, DHCsA-d, or SSG was added at different concentrations prepared from concentrated stock solutions: 5, 10, 15, 20 and 25 μM for CsA [11]; 5, 10, 15, 20 and 25 μM for DHCsA-d; and 5, 10, 25, 50 and 90 μM for SSG. Parasite inhibition rates were evaluated at 24 h and 48 h using flow cytometry, employing an FITC Annexin V Apoptosis Detection Kit I, according to the manufacturer’s instructions. Inhibition rate (%) was calculated as follows: Inhibition rate (%) = 100% − [(No. of live parasites in treated sample/No. of live parasites in untreated control) × 100%].
To evaluate the inhibitory effects of CsA, DHCsA-d, or SSG on intracellular amastigotes, we infected macrophages of a murine macrophage stable cell line (RAW 264.7 with logarithmic phase promastigotes. RAW 264.7 cells (5.0 × 105 cells/per well) were plated on round glass coverslips in 24-well plates and allowed to adhere to the slides for 24 h at 37 °C, 5% CO2, in PRMI 1640 medium supplemented with 10% FBS. Adherent macrophages were infected with L. donovani promastigotes, at a macrophages-to-parasite ratio of 1:20 for 6 h at 37 °C, 5% CO2. Next, the non-infected parasites were removed by washing three times with PBS, and the infected macrophages were incubated in 37 °C in 5% CO2 with PRMI 1640 medium and 10% FBS without drugs for 24 h. The medium was then removed and different concentrations of CsA (3, 6, 10, 15, 20 μM), DHCsA-d (3, 6, 10, 15, 20 μM) or SSG (5, 10, 25, 50, 90 μM) dissolved in fresh medium were added, and the coverslips were incubated for 2 days. At 24 and 48 h, the glass coverslips were fixed in methanol and stained with Wright’s stain. The numbers of parasites were determined using light microscopy by counting at least 200 cells per slide. The results are expressed as means of three independent experiments. The 50% inhibitory concentration (IC50) was calculated for promastigotes and intracellular amastigotes by fitting the values to a non-linear curve analysis.

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Electron microscopy[1]
CsA, DHCsA-d, or SSG-treated control promastigotes and intracellular amastigotes within the macrophages of RAW 264.7 cell line were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and post-fixed in a solution containing 1% OsO4, 1.25% potassium ferrocyanide and 0.1 M cacodylate buffer, pH 7.2. Next, the cells were dehydrated in acetone and embedded in epoxy resin. Stained with lead citrate and observed under a Hitachi HT7700 transmission electron microscope.

In this study, researchers evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control.[1]

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Cytotoxicity assessment: Cytotoxicity effects of CsA, DHCsA-d, or SSG against murine macrophages (RAW 264.7) were assessed by using the Cell Counting Kit-8. Murine macrophages were cultivated in a 96-well plate using PRIM 1640 medium containing 10% FBS and maintained at 37 °C, 5% CO2, for 24 h. Next, different concentrations of the drugs (7, 10, 20, 50 and 100 μM for CsA and DHCsA-d; 60, 75, 90, 105 and 120 μM for SSG) were added. The cytotoxicity was measured at 24 and 48 h following a standard protocol.[1]

[1]. Evaluation of in vitro antileishmanial efficacy of cyclosporin A and its non-immunosuppressive derivative, dihydrocyclosporin A. Parasit Vectors. 2020 Feb 21;13(1):94.
[2]. Isolation of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, the characteristic structural element of cyclosporins, from a blocked mutant of Tolypocladium inflatum. J Antibiot (Tokyo). 1990 Jun;43(6):707-14.

See also: Dihydrocyclosporin A (annotation moved to).

C62H113N11O12

1204.651

1203.857

C, 61.82; H, 9.46; N, 12.79; O, 15.94

59865-15-5

59865-15-5;

25023708

Typically exists as solid at room temperature

1.0±0.1 g/cm3

1285.3±65.0 °C at 760 mmHg

120-125ºC

731.1±34.3 °C

0.0±0.6 mmHg at 25°C

1.465

3.67

5

12

16

85

2280

12

O[C@H]([C@H](C)CCCC)[C@H]1C(N[C@H](C(N(C)CC(N(C)[C@H](C(N[C@H](C(N(C)[C@H](C(N[C@@H](C)C(N[C@H](C)C(N(C)[C@@H](CC(C)C)C(N(C)[C@H](C(N(C)[C@H](C(N1C)=O)C(C)C)=O)CC(C)C)=O)=O)=O)=O)CC(C)C)=O)C(C)C)=O)CC(C)C)=O)=O)CC)=O

TYFOVYYNQGNDKH-HHPJSCBPSA-N

InChI=1S/C62H113N11O12/c1-25-27-28-40(15)52(75)51-56(79)65-43(26-2)58(81)67(18)33-48(74)68(19)44(29-34(3)4)55(78)66-49(38(11)12)61(84)69(20)45(30-35(5)6)54(77)63-41(16)53(76)64-42(17)57(80)70(21)46(31-36(7)8)59(82)71(22)47(32-37(9)10)60(83)72(23)50(39(13)14)62(85)73(51)24/h34-47,49-52,75H,25-33H2,1-24H3,(H,63,77)(H,64,76)(H,65,79)(H,66,78)/t40-,41+,42-,43+,44+,45+,46+,47+,49+,50+,51+,52-/m1/s1

(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-[(1R,2R)-1-hydroxy-2-methylhexyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21-di(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone

Dihydrocyclosporin A; Dihydrocyclosporin-A; Dihydrocyclosporin A; 59865-15-5; Dihydro Cyclosporin A; (3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-[(1R,2R)-1-hydroxy-2-methylhexyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21-di(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone; Dihydrocyclosporine A; 3eov; Dihydrociclosporin A; 6-[(2S,3R,4R)-3-Hydroxy-4-methyl-2-(methylamino)octanoic acid]cyclosporin A; 6-[(3R,4R)-3-Hydroxy-N,4-dimethyl-L-2-aminooctanoic acid]cyclosporin A; 1,4,7,10,13,16,19,22,25,28,31-Undecaazacyclotritriacontane, cyclic peptide deriv.; Cyclosporin A, dihydro-; Dihydrocyclosporin A; 2x2c; Dihydrocyclosporin A.

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.)