Cyclosporine EP Impurity B

By mutagenic treatment of a strain of Tolypocladium inflatum, a cyclosporin non-producing mutant was obtained which accumulated the characteristic building unit of cyclosporins, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation Bmt; systematic name: (2S,3R,4R,6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid) in free form. The isolation from a culture filtrate was performed by extraction, chromatographic separation and final crystallization from methanol - water. The structure and stereochemistry of this amino acid was determined by chemical transformation and correlation to dihydro-MeBmt, with known chirality [(2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-octanoic acid], obtained by hydrolysis of dihydrocyclosporin A.

A novel metabolite of cyclosporin A was observed in human blood and urine. An analytical sample of this metabolite was isolated from human urine and the structure was determined to be (8-hydroxy-6,7-dihydro-MeBMT1) cyclosporin based on the 1H-NMR, 13C-NMR, FAB-MS, and HPLC characteristics of the biological sample as well as by comparison with a synthetically derived authentic sample. The significance of this metabolite in terms of the pathway by which cyclosporin A is metabolized is discussed.

Background: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. Methods: In this study, we 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. Results: Our results showed that 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: Our 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.