The Antifungal Action Mode of N-Phenacyldibromobenzimidazoles.

Our study aimed to characterise the action mode of N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure-activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e-f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action (5e with 2,4-Cl2 or 5f with 3,4-Cl2). The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC50 = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160-4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-ß-d-glucosaminide and 4-nitrophenyl-ß-d-N,N',N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing ß-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives.

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h-i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c-d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

Subclinical thyroid dysfunction in the first trimester of pregnancy: 'Disease' versus physiological (pulsatile) variation in TSH concentrations.

BACKGROUND: There is no universal consensus regarding cut-off points for TSH in pregnancy, so concentrations of 2.5 or 4.0 mIU/L were suggested for first trimester (Endocrine Society [2012] and ATA [2017] guidelines, respectively). Yet, the impact of physiological variation in TSH secretion has not been assessed. SUBJECTS AND METHODS: We assessed baseline concentrations of free T4, free T3 and TSH at 30-minute intervals (between 7.00 and 9.00 hours) in 110 healthy pregnant women, age 30.2 ± 6.0 years, 9.9 ± 2.4 weeks of gestation, and in 19 female controls, age 28.9 ± 10.7. RESULTS: Mean TSH concentrations in pregnant women were 1.62 ± 1.26 mIU/L and on average varied by 39.5% (dispersion between the highest and the lowest TSH), with no difference in TSH variation between pregnant women and controls. Taking into account the highest TSH out of five consecutive measurements, TSH >2.5 mIU/L and TSH above 4.0 mIU/L were found in 23 (20.9%) and 10 (9.1%) pregnant women, respectively. In contrast, when the lowest TSH value was considered, then concentrations of TSH >2.5 mIU/L and >4.0 mIU/L were found in 14 (12.7%) and 4 (3.6%) women, respectively. This discrepancy was even more pronounced in aTPO-negative subjects (21 [21.2%] vs 8 [8.1%] women, for TSH >2.5 mIU/L, and six [6.06%] vs one [1.01%], for TSH >4.0 mIU/L). Furthermore, either six (5.4%) or 10 (9.1%) women had TSH concentrations below 0.1 mIU/L. CONCLUSIONS: In a significant number of patients, diagnosis of subclinical thyroid dysfunction could be erroneously made not as a result of 'disease', but as a result of physiological variation in TSH concentrations.

Determination of glucose cut-off points for optimal performance of glucagon stimulation test.

Introduction: The glucagon stimulation test (GST) is widely used to assess growth hormone (GH) and cortisol secretion, nevertheless the precise mechanisms underpinning these hormonal responses remain unclear. We have endeavoured to explore the relationship between glucose and insulin fluctuations during GST and their impact on GH and cortisol secretion. Subjects and methods: We retrospectively studied 139 subjects (mean age 35.5 ± 15.1 years, BMI 26.6 ± 6.61 kg/m²), including 62 individuals with a history of pituitary disease (27 with an intact adrenal axis) and 77 healthy controls. Standard dose intramuscular GST was performed in all subjects. Results: Once BMI and age were excluded from multivariate model, the nadir of glucose concentration during GST was the sole variable associated with maximal GH secretion (ΔGH, p<0.0003), while neither glucose/insulin peak, nor Δglucose/Δinsulin concentrations contributed to ΔGH. 100% pass rate for GH secretion above 3 ng/ml or 1.07 ng/ml cut-offs was observed for glucose concentrations at, or below 60 mg/dl (3.33 mmol/l) (for Controls), or 62 mg/dl (3.44 mmol/l) (for Controls and patients with an intact adrenocortical axis). Such low glucose concentrations were obtained, however, only in about 30% of studied individuals. Conversely, cortisol secretion did not correlate with glucose or insulin fluctuations, suggesting alternative regulatory mechanisms. Conclusions: This study reveals that glucose nadir below 3.33 mmol/l is the only biochemical biovariable linked with optimal GH secretion during GST, whereas mechanisms responsible for cortisol secretion remain unclear. We emphasize the importance of glucose monitoring during GST to validate GH stimulation and support clinical decisions in GH deficiency management.

The Utility of Intravenous Methylprednisolone as an Adjunct Treatment for Drug-Resistant Amiodarone-Induced Thyrotoxicosis.

BACKGROUND: Amiodarone-induced thyrotoxicosis (AIT) may pose treatment challenges. We present a series of patients in which we achieved the normalisation of free T3 (FT3) using intravenous methylprednisolone (ivMP) in AIT refractory to thiamazole and oral prednisone. Namely, in three males (aged 56, 50 and 64, all with a history of AF and/or a low ejection fraction), an addition of ivMP resulted in the normalisation of FT3, which allowed successful thyroidectomy. In another case of a 65-year-old man, we initially succeeded in the normalisation of FT3 using ivMP from FT4 > 7.77 ng/dL (0.93-1.7) to 2.41 ng/dL and in that of FT3 from 14.95 pg/mL (2-4.4) to 2.05 pg/mL), but four weeks after stopping ivMP, despite the continuation of thiamazole and prednisone, there was rebound thyrotoxicosis: FT4 > 7.77 ng/dL and FT3-5.46 pg/mL. Intravenous MP was restated leading to a decline in FT4 to 2.51 ng/dL and in FT3 to 1.92 pg/mL, thus allowing a successful thyroidectomy. Finally, in a 78-year-old man with AF, goitre, and AIT resistant to thiamazole, prednisone and lithium carbonate, we obtained a reduction in FT4 to 1.51 ng/dL and in FT3 to 3.17 pg/mL after seven pulses of ivMP. Oral prednisone was gradually reduced and successfully stopped about six months later. He remained on low-dose thiamazole (5 mg od). CONCLUSIONS: Pulse ivMP in addition to oral steroids may be a useful adjunct therapy either for the preparation of a thyroidectomy or as a treatment modality in drug-resistant AIT. Though a total cure is possible, there is a danger of a rebound worsening of thyrotoxicosis after premature discontinuation of ivMP.

Metformin paradoxically worsens insulin resistance in SHORT syndrome.

BACKGROUND: SHORT syndrome is an autosomal dominant condition associated severe insulin resistance (IR) and lipoatrophy due to post-receptor defect in insulin signaling involving phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), where no clear treatment guidelines are available. METHODS: We attempted to test the efficacy metformin in a female patient with SHORT syndrome by measuring glucose and insulin during an extended Oral Glucose Tolerance Test (OGTT) in a 21-year old patient (BMI 17.5 kg/m2), who presented for endocrine assessment with a history of amenorrhoea. RESULTS: She had lipid concentrations within the reference range, normal thyroid function tests, prolactin, gonadotropins, estradiol and androgens with Free Androgen Index 4.52. Extended Oral Glucose Tolerance Test was performed and showed severe IR. She was then started on metformin 850 mg twice a day, and had repeated OGTT. This showed dramatic worsening of glucose tolerance (e.g. glucose 96 mg/dl versus 187 mg/dl and 68 mg/dl versus 204 mg/dl at 120 and 150 min of OGTT, respectively). This was accompanied by a massive increase of already high insulin concentrations (e.g. from 488.6 to > 1000 µIU/ml, and from 246.8 to > 1000 µIU/ml at 120 and 150 min of OGTT, respectively). Insulin concentrations remained above upper assay detection limit also at 180 min of OGTT on metformin treatment (> 1000 µIU/ml versus 100.6 µIU/ml without metformin). CONCLUSIONS: Metformin treatment may paradoxically lead to deterioration of insulin resistance and to development of glucose intolerance in SHORT syndrome. Hence, metformin treatment might be potentially harmful in these patients. Though, the precise cause of such profound and paradoxical worsening of glucose tolerance post metformin remains unknown, SHORT syndrome might prove to be an interesting model to study the mechanism(s) of metformin action.