Solubilization of cyclosporin A

This study investigated the solubilization of cyclosporin A (CsA), a neutral undecapeptide, by cosolvency, micellization, and complexation. Cosolvents (ethanol, propylene glycol, polyethylene glycol, tetrahydrofurfuryl alcohol polyethyleneglycol ether, and glycerin), surfactants (polyoxyethylene sorbitan monooleate [(Tween 80)], polyoxyethylene sorbitan monolaurate [(Tween 20)], and Cremophor EL), and cyclodextrins (α-cyclodextrin [(αCD)] and hydroxypropyl-β-cyclodextrin[(HP\CD)] were used as solubilizing agents in this study. Surfactants had a noticeable effect in increasing CsA solubility. Twenty percent solutions of Tween 20, Tween 80, and Cremophor EL increased the solubility by 60 to 160 fold. Cyclodextrins can increase the CsA solubility, but αCD was more effective than HP\CD. Cosolvents on the other hand did not increase the solubility of CsA as much as expected from the LOGP (logrithm of wateroctanol partition coefficent) value of CsA.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Effects of repeated injection of cyclosporin A on pentylenetetrazol-induced convulsion and cyclophilin mRNA levels in rat brain

To investigate the relationship between the immune system and convulsions in an animal model, we examined the effects of repeated administration with the immunosuppressant cyclosporin A on pentylenetetrazol (PTZ)-induced convulsions and the changes in the mRNA expression of its binding protein cyclophilin in the rat brain. The consecutive administration of cyclosporin A (5 mg/kg s.c., 14 days) significantly aggravated the severity of convulsions induced with PTZ 75 mg/kg i.p. Furthermore, it down-regulated the levels of cyclophilin mRNA in several brain regions and inhibited the PTZ-induced increase of hippocampal cyclophilin mRNA. Compared with the group without PTZ pretreatment or the group treated with chronic vehicle administration after the PTZ-preinjection, chronic cyclosporin A administration after the initial injection of PTZ apparently aggravated convulsions after the second PTZ injection. Interestingly, the increase in hippocampal cyclophilin mRNA observed after a single PTZ injection was not found after the second PTZ injection in the group with PTZ pretreatment. Therefore, these findings suggest that cyclosporin A administered peripherally can affect the central nervous system, and that an immune response associated with the first convulsive episode plays a key role in severity during subsequent attacks.     

NMR studies of [U-C]cyclosporin A bound to human cyclophilin B

NMR data (¹H and ¹³C chemical shifts, NOEs) on [[U-¹³C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-¹³C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574–6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.     

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.     

Oligomycin strengthens the effect of cyclosporin A on mitochondrial permeability transition by inducing phosphate uptake

The purpose of this work was to assess the effect of oligomycin on the mitochondrial membrane permeability transition. The antibiotic was found to strengthen cyclosporin A (CSA)-induced protection of non-specific permeability, which is triggered by a matrix Ca2+ load in the absence of ADP. Oligomycin also reinforced the protective effect of CSA on carboxyatractyloside-induced pore opening in the absence of ADP, but failed to do so in mitochondria incubated under anaerobic conditions or after addition of CCCP. Analyzing the efflux of matrix Ca2+, we found that mitochondrial swelling and the collapse of the transmembrane electric gradient coincided with membrane leakage. The effects of the antibiotic were observed in phosphate-containing media but not in the presence of acetate. Furthermore, N-ethylmaleimide hindered the protective effect of oligomycin-CSA. In addition, the matrix phosphate concentration increased concurrently with a diminution in the matrix-free fraction of Ca2+. We concluded that oligomycin increases phosphate uptake by stimulating the phosphate-/OH- exchange reaction.     

Interactions of a bacterial biosurfactant trehalose lipid with phosphatidylserine membranes

Trehalose lipids are biosurfactants produced by rhodococci that, in addition to their well known potential industrial and environmental uses, are gaining interest in their use as therapeutic agents. The study of the interaction of biosurfactants with membranes is important in order to understand the molecular mechanism of their biological actions. In this work we look into the interactions of a bacterial trehalose lipid produced by Rhodococcus sp. with dimyristoylphosphatidylserine membranes by using differential scanning calorimetry, X-ray diffraction and infrared spectroscopy. Differential scanning calorimetry and X-ray diffraction show that trehalose lipid broadens and shifts the phospholipid gel to liquid-crystalline phase transition to lower temperatures, does not modify the macroscopic bilayer organization and presents good miscibility both in the gel and the liquid-crystalline phases. Infrared experiments show that trehalose lipid increases the fluidity of the phosphatidylserine acyl chains, changed the local environment of the polar head group, and decreased the hydration of the interfacial region of the bilayer. Trehalose lipid was also able to affect the thermotropic transition of dimyristoylphosphatidyserine in the presence of calcium. These results support the idea that trehalose lipid incorporates into the phosphatidylserine bilayers and produces structural perturbations which might affect the function of the membrane.     

GRP78 induction by cyclosporin A in human HeLa cells

Immunosuppressive drugs such as cyclosporin A (CsA) and FK506 are known to have pleiotropic effects on cells. Here we demonstrate that treatment of HeLa cells with low concentrations of CsA (but not of FK506) induces the synthesis of a stress protein, GRP78, located inside the endoplasmic reticulum. High concentrations of CsA lead to a general decrease in protein synthesis. When cells are stressed (heat-shocked) during the CsA treatment, the synthesis of heat shock proteins is reinforced. FK506 has no detectable effects at any concentration. The mechanism of induction of GRP78 by CsA remains presently unknown. Whatever the mechanism involved, GRP78 overexpression might be responsible for some of the physiological effects of CsA.     

Modulation by substrates of the protective effect of cyclosporin A on mitochondrial damage

The influence of substrates on the role of cyclosporin A, to promote the closure of the permeability transition pore, was studied. It was found that in succinate-oxidizing mitochondria, cyclosporin inhibited pore opening as induced by carboxyatractyloside. The opposite occurred when mitochondrial respiration was supported by malate-glutamate, i.e., cyclosporin A was unable to block pore opening promoted by carboxyatractyloside. We propose that the failure of cyclosporin A to induce pore closure could be due to a low NADH matrix content.     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K⁺ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na⁺-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca²⁺ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.     

Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.     

Anti-IL-17A blocking antibody reduces cyclosporin A-induced relapse in experimental autoimmune encephalomyelitis mice

Cyclosporin A (CsA) is effective at reducing pathogenic immune responses, but upon withdrawal of CsA the immune response often “rebounds” resulting in a relapse or exacerbation of disease. The mechanisms, cells and cytokines involved in the relapse or exacerbation after CsA withdrawal are unknown. We hypothesized that CsA withdrawal induces IL-17 production that could be responsible for relapse, and examined the effect of anti-IL-17A antibody on relapse induced after CsA withdrawal in mouse experimental autoimmune encephalomyelitis (EAE). CsA treatment markedly decreased the EAE disease score during the first episode, but augmented disease severity after CsA withdrawal, compared to untreated mice. After discontinuation of CsA the production of IL-17A was increased and the severity of relapse in EAE was reduced by treatment with anti-IL-17A antibody. These results suggest that the resumption of T cell immune responses after CsA withdrawal leads to a burst of IL-17A production that is at least partially responsible for relapse in EAE mice.     

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.     

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O

The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ubo), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light-scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca(2+)-sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ubo, but when the concentration of Ubo exceeded 20 microM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.