Supplemental Data Steroidomics LC-MS/MS

Supplementary Data Steroidomics LC-MS/M

A Micellar Mitotane Formulation with High Drug-Loading and Solubility: Physico-Chemical Characterization and Cytotoxicity Studies in 2D and 3D In Vitro Tumor Models

Adrenocortical carcinoma (ACC) is a rare tumor and prognosis is overall poor but heterogeneous. Mitotane (MT) has been used for treatment of ACC for decades, either alone or in combination with cytotoxic chemotherapy. Even at doses up to 6 g per day, more than half of the patients do not achieve targeted plasma concentration (14–20 mg L$^{-1}$) even after many months of treatment due to low water solubility, bioavailability, and unfavorable pharmacokinetic profile. Here a novel MT nanoformulation with very high MT concentrations in physiological aqueous media is reported. The MT‐loaded nanoformulations are characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, and powder X‐ray diffraction which confirms the amorphous nature of the drug. The polymer itself does not show any cytotoxicity in adrenal and liver cell lines. By using the ACC model cell line NCI‐H295 both in monolayers and tumor cell spheroids, micellar MT is demonstrated to exhibit comparable efficacy to its ethanol solution. It is postulated that this formulation will be suitable for i.v. application and rapid attainment of therapeutic plasma concentrations. In conclusion, the micellar formulation is considered a promising tool to alleviate major drawbacks of current MT treatment while retaining bioactivity toward ACC in vitro

A Micellar Mitotane Formulation with High Drug Loading and Solubility: Physico-Chemical Characterization and Cytotoxicity Studies in 2D and 3D in Vitro Tumor Models.

Adrenocortical carcinoma (ACC) is a rare tumor and prognosis is overall poor but heterogeneous. Mitotane (MT) has been used for treatment of ACC for decades, either alone or in combination with cytotoxic chemotherapy. Even at doses up to 6 g per day, more than half of the patients do not achieve targeted plasma concentration (14-20 mg/L) even after many months of treatment which is caused by low water solubility and unfavorable pharmacokinetic properties such as poor bioavailability and high volume of distribution of MT. The clinical need and previously reported extraordinary high drug loading of poly(2-methyl-2-oxazoline)-block-poly(2-butyl-2-oxazoline)-block-poly(2-methyl-2-oxazoline) (A-pBuOx-A) based micelles for paclitaxel (PTX), led us to develop MT loaded micelles which may enable an injectable formulation. We successfully solubilized up to 6 g/L of MT in an aqueous formulation. The MT loaded nanoformulations were characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD), confirmed the amorphous nature of drug in the formulations. The polymer itself did not show cytotoxicity in adrenal and liver cell lines. By using the ACC model cell line NCI-H295 both in monolayers and tumor cell spheroids, we demonstrated micellar MT to exhibit comparable efficacy to its ethanol solution. We postulate that this formulation would be suitable for i.v. application and more rapid attainment of therapeutic plasma concentrations. In conclusion, we consider our micellar formulation a promising tool to alleviate major drawbacks of current MT treatment while retaining bioactivity towards ACC in vitro.</p

Experimental adaptation of wild-type canine distemper virus (CDV) to the human entry receptor CD150.

Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red) adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2) pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5) pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs

Abiraterone Acetate for Cushing's Syndrome : Study in a Canine Primary Adrenocortical Cell Culture Model

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer, but could also be an interesting treatment of Cushing's syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM - 10 μM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In conclusion, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model

Abiraterone Acetate for Cushing's Syndrome : Study in a Canine Primary Adrenocortical Cell Culture Model

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer, but could also be an interesting treatment of Cushing's syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM - 10 μM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In conclusion, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model

Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA▿

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA

Infection of Vero-hSLAM cells with CDV-A75/17^red.

<p>Human CD150-expressing Vero cells (Vero-hSLAM) were infected with recombinant wild-type CDV-A75/17^red at a MOI of 1.0 (<b>A</b>), incubated for the indicated times and pictures of the red autofluorescence taken under UV light. At 4 days post-infection (dpi) virus was prepared and titrated, and Vero-hSLAM cells infected at a MOI of 0.01 (<b>B</b>). After 4 dpi virus was again prepared and titrated, and Vero-hSLAM cells were infected with this virus at a MOI of 0.01 for indicated times (<b>C</b>). Titres of the virus preparations were determined using Vero-cSLAM cells and are given in (<b>D</b>). Photomicrographs of the autofluorescent virus-encoded tD tomato red were taken under UV light (bar = 100 µm).</p

Structural model of the CDV-H-CD150 interaction.

<p>Side (A) and top (B) view of the globular head of CDV-H (amino acids 188 to 602) presented in magenta with highlighted amino acid 540 G (green), and of the interacting first V-like domain of human CD150 (amino acids 32 to 140) shown in blue with highlighted amino acid 71 E (yellow). The structures were modelled based on the crystal structure of MV-H bound to CD150 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057488#pone.0057488-Hashiguchi2" target="_blank">[33]</a>.</p

Comparison of single step growth curves of parental and adapted CDV-A75/17^red using canine and human CD150-expressing target cells.

<p>Vero-cSLAM cells (open circles) and Vero-hSLAM cells (closed circles) were infected with parental CDV-A75/17^red, and Vero-cSLAM cells (blue triangles) and Vero-hSLAM cells (red squares) were infected with human CD150-adapted CDV-A75/17^red (CDV-A75-ad) at a MOI of 0.01 and incubated for indicated times before harvesting cell bound plus released virus. The viruses were then titrated using the optimal target cells for each virus (n = 3, using 3 independently adapted viruses).</p