bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety Analysis of an Oral Testosterone Undecanoate (TU) Formulation Following 2 Years of Administration in Hypogonadal Men
Authors: Swerdloff Ronald S , Amory John K , Gittelman Marc , Seo B Woun , Rohowsky Nestor , Dudley Robert E
Published: May 2021

Introduction: An oral testosterone (T) replacement therapy (TRT) would be the preferred choice for many hypogonadal men. Until recently, the only oral TRT approved in the US was methyl-T which has been associated with hepatotoxicity. The safety of a novel oral T undecanoate (TU) formulation was evaluated in hypogonadal men for up to 2 years. Subjects and Methods: Two open-label, multicenter, dose-titration trials were conducted in hypogonadal men (serum T ≤ 300 ng/dL) age 18-75 years. Trial I was a randomized, active-controlled, 2-arm, 12-month study. Trial 2 was a long-term extension of those who completed Trial 1. Statistical analyses were only conducted with the subjects who completed Trial 1 and continued treatment in Trial 2, thus providing up to 2 full years of data. Safety was assessed by physical exam, AE reporting, and routine clinical laboratory measurements. Results: Overall, up to 81 subjects were available for evaluation. T concentration increased from 208.3 ± 102.4 ng/dL (Mean ± SD) at baseline (BL) to 470.1 ± 396.5 ng/dL after 24 Mo of therapy with oral TU, and 84% of men achieved T in eugonadal range (300-1000 ng/dL) after 90 days of therapy. Mean T concentrations remained in the eugonadal range throughout Trial 2. There were no clinically significant changes in liver function tests - ALT (28.0 ± 12.3 to 26.6 ± 12.8 U/L), AST (21.8 ± 6.8 to 22.0 ± 8.2 U/L), and bilirubin (0.58 ± 0.22 to 0.52 ± 0.19 mg/dL) throughout the two studies. At Day 270, one subject had an ALT level of 227 U/L, which was > 4x the ULN (ULN for ALT = 45 U/L). Despite continued use of oral TU, ALT was measured again on Day 290, and the level dropped to 87 U/L, < 2x ULN. This was the only instance of an LFT elevation. There was a modest initial increase in prostate-related growth endpoints (i.e. PSA and prostate volume) that stabilized over time. There were not any significant changes in IPSS total score (-0.06 ± 3.9 vs BL). There were significant, yet modest, increases in mean HCT (+2.52 ± 3.7% vs BL, p < 0.001) and cuff systolic BP (+5.6 ± 15.0 mmHg vs BL, p = 0.006). The change in prostate-related growth variables and CV endpoints changed initially and stabilized throughout the 2 trials. For example, systolic BP consistently showed a mean increase from BL between 3 - 6 mmHg. Conclusion: This oral TU formulation is an effective long-term therapy for hypogonadal men and has a safety profile consistent with other approved T products. Notably, no evidence of liver toxicity was observed. The long-term efficacy and safety profile of oral TU may provide a treatment option that avoids issues associated with other TRTs, such as injection site pain or transference to partners and children.
Studying the Genomic Role of Cathepsin-D in ER+ Breast Cancer
Authors: Dianati Elham , Liaudet-coopman Emmanuelle , Mader Sylvie
Published: May 2021

Estrogen receptor alpha (ERα), a transcription factor implicated in induction of cell growth in breast cancer, is a therapeutic target that is expressed in >70% of breast tumors. The transcriptional activity of ERα is controlled by ligands and increased through its interaction with co-activators such as the p160/SRC and p300/CBP families. In an attempt to identify the ligand-specific protein complexes involved in transcriptional regulation by ERα, BioID and TurboID screens were performed in two ER+ breast cancer cell lines, T-47D and ZR-75-1. Surprisingly, Cathepsin-D (Cath-D), a lysosomal aspartyl endoproteinase that is an ER target gene, was identified in these screens. Cath-D expression is associated with a poor prognosis and increased metastasis rate in breast cancer irrespective of its catalytic activities {Glondu, 2001 #119}[i]. Cath-D is localized in part to the nucleus where it interacts with TRPS1, a repressor of GATA-mediated transcription and modulator of ERα signaling {Bach, 2015 #117}[ii]. Co-silencing Cath-D and TRPS1 suppressed cell proliferation and inhibited growth under soft agar, suggesting that they cooperate to drive tumorigenesis {Bach, 2015 #117}[ii]. We hypothesized that Cath-D plays genomic as well as non-genomic roles in breast tumor aggressiveness and may alter ERα-mediated transcription. The nuclear localization of Cath-D was confirmed by immunofluorescence using different commercialized antibodies and observed in western blots of chromatin-bound fractions in three different ERα+ breast cancer cell lines, T-47D, ZR-75 and MCF-7. Specificity of the antibodies was confirmed using siRNA-mediated suppression of Cath-D. Moreover, Cath-D was also identified in proximity to TurboID-ERα by LC-MS after chromatin fractionation. The proximity of ERα and Cath-D both in the cytoplasm and nucleus was confirmed by proximity Ligation Assay (PLA) in three ER+ cell lines. Co-immunoprecipitation assays indicated physical interaction of Cath-D with ERα in T-47D cell extracts. Further, Cath-D was detected by ChIP-qPCR on estrogen response elements (EREs) of two ERα target genes, TFF1 and GREB1 in T-47D and ZR-75 cells. These results suggest that Cath-D can interact with ERα on DNA and play genomic roles in ER+ breast cancer cells. [i] Glondu, M., et al. (2001). “A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells.” Oncogene20(47): 6920-6929. [ii] Bach, A. S., et al. (2015). “Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.” Oncotarget6(29): 28084-28103.
The Transcriptional Function of GRHL2 in Hormone-Dependent Breast Cancers
Authors: Reese Rebecca , Helzer Kyle , Alarid Elaine T
Published: May 2021

The Grainyhead-like protein family, composed of GRHL1, GRHL2, and GRHL3, are nuclear transcription factors that regulate epithelial differentiation. GRHL2 has been associated with several nuclear hormone receptors, including progesterone receptor (PR), androgen receptor (AR), and more recently, estrogen receptor (ER). In breast cancer, GRHL2 has been shown to both activate ER-dependent enhancers through FOXA1 and MLL3-mediated deposition of the activating histone mark H3K4me1 and repress enhancers via inhibition of the histone acetyltransferase p300. Cistromic analysis by our group of ER phosphorylated at serine 118 (pS118-ER), a form of transcriptionally active ER, found an enrichment of the GRHL motif near pS118-ER binding sites. Despite these findings, the direct relationship between GRHL2 and ER transcriptional function and how that relationship influences ER-positive breast cancer growth and differentiation is not well-defined. To explore the relationship between GRHL2 and pS118-ER further, we used transcriptomic and cistromic analysis of ER-positive cells lacking GRHL2 to determine the impact of the loss of GRHL2 on cellular and transcriptional responses to estrogen. This analysis identified a subset of genes that are controlled by both GRHL2 and estrogen. In addition, CRISPR engineered T47D cells lacking a portion of the GRHL2 transactivation domain (ΔTAD) demonstrate reduced nuclear ER levels and reduced ER chromatin occupancy. Gene expression analysis of ΔTAD-GRHL2 cells showed increased GRHL3 expression, and ChIP analysis revealed increased Pol II occupancy at the GRHL3 promoter, suggesting that there may be a compensatory mechanism within the GRHL family to regulate the transcriptome. Finally, ΔTAD-GRHL2 mutants reduced growth and colony formation relative to wild-type controls. Together, this work will provide an understanding of how transcriptionally active ER and GRHL2 selectively cooperate to regulate transcription, growth, and differentiation in ER-positive breast cancer.
Differential Genomic Interactions Drive Progesterone Receptor Isoform Specific Functions in Breast Cancer
Authors: Doan Tram B , Graham J Dinny , Tehan Mariah , Guild Barbara J , Clarke Christine L
Published: May 2021

Progesterone is critical for normal breast development and function, and has been shown to stimulate proliferation of normal breast epithelial cells by increasing stem and progenitor cell numbers. Breast cancer incidence is increased in women exposed to progesterone analogues in combined estrogen plus progestin hormone replacement therapy, but not in women taking estrogen alone. Classical progesterone signaling is mediated through the nuclear progesterone receptor (PR), which occurs as two related but functionally different isoforms, PRA and PRB. PRA and PRB are co-expressed equally in normal breast tissue but become dysregulated in breast cancer where PRA often becomes predominant. PRA predominance in breast cancer is associated with poorer outcome and higher risk of distant metastasis in tamoxifen treated patients. We show using integrated analysis of ChIP-seq, ATAC-seq and transcriptomic profiling in a breast cancer cell line model of acquired PRA predominance that: 1) PRA and PRB have different requirements with regard to chromatin accessibility; 2) PRA predominance reshapes the PR cistrome and the associated transcriptome to affect genes not normally regulated by PR when PRA and PRB are equivalently expressed, possibly through assisted loading with multiple other transcription factors; 3) Genes regulated by PR only when PRA is predominant are associated with poorer breast cancer outcome and involved in multiple cancer-associated pathways including those that regulate cell proliferation and adhesion. Our data suggest a mechanism for the poorer disease outcome seen in breast cancers with a predominance of PRA.
LBD Dimerization of the Androgen Receptor but Not N/C Interaction Is Crucial for Normal Male Development in Mice
Authors: El Kharraz Sarah , Helsen Christine , Dubois Vanessa , Libert Claude , Poutanen Matti , Claessens Frank
Published: May 2021

The androgen receptor (AR) is a nuclear receptor with a main role in the development and maintenance of the male phenotype. To execute its role as transcription factor, the AR forms homodimers. Three dimerization modes have been described for the AR: one via the DNA binding domain, a second via the ligand binding domain (LBD) and a third via interactions between the LBD and the aminoterminus of the AR (N/C). Based exclusively on in vitro data, all three dimerization modes seem to contribute to full AR activity, albeit to a different extent. The in vivo role of the dimerization modes, however, remains unknown. To study the physiological relevance, we generated two mouse models using a CRISPR/Cas9 approach, in which either the N/C interaction (ARNoC) or LBD dimerization (ARLmon) was disrupted. Surprisingly, the male ARNoC mice have a normal phenotype, indicating that the N/C interaction is not crucial for male development. In contrast, ARLmon males have an external female phenotype with cryptorchid testes and high levels of circulating testosterone (T), androstenedione and luteinizing hormone (LH) (6-, 13- and 45-fold higher, respectively). They have no prostate, seminal vesicles or epididymis, illustrating the importance of LBD dimerization during male development. Phenotyping the ARLmon model furthermore provided evidence of a crucial role for the AR in bone homeostasis as well as steroidogenesis. The ARLmon males display a severe bone phenotype, similar to that of complete AR knockout (ARKO) mice. The bone phenotype of ARKO was postulated to be mainly due to lower estrogen levels. However, in contrast to ARKO mice, ARLmon mice have high circulating levels of T, which can still function as prohormone for estradiol and support bone function via the ERα. Immunohistological analysis of ARLmon testes showed hyperplasia of the Leydig cells and residual spermatogenesis. Analysis of the steroidogenic pathway revealed that while the expression of most genes is increased, the expression of Hsd17b3, encoding the enzyme responsible for conversion of androstenedione into T, is low in ARLmon testis. Reporter assays confirmed that the promotor of this gene is indeed upregulated by the AR itself. In conclusion, our work uncovers the physiological role of the N/C interaction and LBD dimerization of the AR. It furthermore demonstrates a direct role for AR in male bone development independent of T aromatization into estrogens. Finally, we show that the AR controls the final step in the synthesis of its own ligand. In contrast to the in vitro data, N/C interaction is not crucial for male development in vivo. The ARLmon model illustrates that LBD dimerization could be an excellent new therapeutic target for inhibiting AR activity for example in advanced prostate cancer that has developed resistance to the current AR-targeting therapies.
Mapping of Corticosteroids in Murine Kidneys Using Mass Spectrometry Imaging
Authors: Stasinopoulos Ioannis , Khan Shazia , MacKay Logan C , Brown Roger W , Bailey Matthew J , Andrew Ruth
Published: May 2021

Renal sodium reabsorption is important for blood pressure homeostasis and is physiologically regulated by aldosterone; glucocorticoids may also contribute. Abnormal steroid hormone activity within the kidney contributes to hypertension but the mechanisms are not fully defined. Molecular profiling of receptors and metabolising enzymes indicates that steroid hormone action is compartmentalised within the kidney. Ambient steroid concentrations are a critical factor governing bioactivity at a cellular level, but this is largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) was applied recently to localise steroids in brain and testes, and here is applied to kidney. Image reconstruction permits characterisation and co-registration of kidney histological regions based on regional markers detectable by MSI. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of murine kidneys, using an optimised MSI method. This approach has the potential to map steroids within functional zones of the kidney, providing fundamental new information relevant to hormone action in health and in disease. Cryosections of male C57BL6 mouse kidneys (age 12 weeks, n=6) were subject to MSI following derivatisation using Girard T reagent and α-cyano-4-hydroxycinnamic acid matrix application. Images were reconstructed, and methods optimised to enhance signal and limit diffusion of analytes of interest. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry. Ions with m/z 458.3010, 460.3166 and 474.2957 were detected, using MALDI, in renal sections, close to the predicted masses of 458.3013 (Δppm=0.65), 460.3169 (Δppm=0.65), and 474.2962 (Δppm=1.05), for derivatives of 11-dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. The Heat maps generated indicated that corticosterone intensity was higher in the inner cortex area close to the corticomedullary junction than the rest of the kidney. In contrast 11-dehydrocorticosterone was detected mainly in medulla and aldosterone signal was equally strong in medulla and outer cortex. Thus, MSI can be used map the sites where glucocorticoid and mineralocorticoids are most active in regulating renal tubular function. Co-localisation of steroids of interest with zonal markers by MSI permits steroid mapping in functional renal zones of the kidney. This approach provides fundamental new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure. The project was supported and funded by Kidney Research UK.
Association of Thyroid Function With Suicide Ideation/Attempt -A Systematic Review and Meta-analysis
Authors: Toloza Bonilla Freddy Jean Karlo , Mao Yuanjie , Menon Lakshmi P , George Gemy Maria , Borikar Madhura , Owen Richard R , Maraka Spyridoula
Published: May 2021

Background: Thyroid disorders are very prevalent and could affect virtually the entire human body, including cognitive and psychiatric domains. However, the relationship between thyroid dysfunction and suicide is still controversial. Material and Methods: A systematic review and meta-analysis was conducted to describe the association of thyroid function with suicide ideation/attempt in adults. A comprehensive search from databases’ inception (MEDLINE, EMBASE, Cochrane, PsycINFO, PsycArticles, PSYNDEX and Scopus) to July 20, 2018 was conducted with no language restrictions. We included studies that reported mean values and standard deviation (SD) of thyroid hormone levels [Thyroid-stimulant hormone (TSH), free T4 (FT4), free T3 (FT3), total T4 (TT4), and total T3 (TT3)] in patients with suicide ideation/attempt compared with controls. Four reviewers worked independently and in duplicate for assessment of inclusion criteria, data extraction, and assessment of risk of bias. The mean value and SD of the thyroid function tests were used to calculate the mean difference for each subgroup. Random-effects models for meta-analyses were applied. Results: Overall, 2278 articles were identified, and 13 observational studies met the inclusion criteria. These studies involved 2651 participants, including 817 participants diagnosed with suicidal ideation/attempt. Group sizes of patients with suicide ideation/attempt ranged from 7 to 122 participants with mean age ranging from 23 to 49 years. Control group sizes ranged from 8 to 464 participants with mean age ranging from 24 to 50 years. Two studies included only women, two studies included only men, and 9 studies included both (% female range: 29 to 78%). Patients with suicide ideation/attempt had lower levels of FT3 (-0.19 pg/mL; P=0.04) and TT4 (-0.23 µg/dL; P=0.05) compared to controls. There were no differences in TSH, TT3 or FT4 levels between groups. In a subgroup analysis comparing current suicidal ideation vs current suicidal attempt vs history of suicidal ideation/attempt with the control group, there were no differences in any of the thyroid function tests. None of the included studies compared rates of overt/subclinical thyroid disease among groups. The overall risk of bias of the included studies was low-to-moderate. Conclusions: There is scarce evidence regarding the association of thyroid disorders and suicide. We found statistically significant lower thyroid hormone levels in patients with suicidal ideation/attempt. The clinical implications of this finding remain unknown and further research is needed to evaluate the association of thyroid disorders with suicide.
Body Composition, Insulin Resistance and Mediators of Appetite and Energy Homeostasis in Hyperthyroidism
Authors: Patil Avinash , Vaikkakara Suresh , Sachan Alok , Buddharaju P Varma
Published: May 2021

Background: Hyperthyroidism is associated with reduced body weight despite an increase in appetite; due mainly to an increase in energy production. While appetite is regulated by the balance of orexogenic and anorexogenic peripheral signals like Ghrelin and Leptin respectively, energy expenditure is profoundly influenced by thyroxine itself as well as mediators like FGF 21 and Leptin. Hyperthyroidism offers a good model to study the impact of thyroid hormones on the above mediators. Materials & Methods: 35 adult patients with overt hyperthyroidism were evaluated for leptin, ghrelin, adiponectin, FGF21 levels & insulin resistance as well as the body composition by DEXA both at baseline and a minimum of two months following normalization of serum thyroxin on carbimazole treatment. Results: Correction of hyperthyroidism was associated with an increase in body weight including both the lean body (p<0.001) and the fat mass (p<0.001), but with no change in percentage of total body fat (p=0.516). Accompanying the weight gain, there was no change in adiponectin (p=0.98), while a paradoxical decrease in insulin resistance was observed (p<0.001). Correction of hyperthyroidism was also associated with a decrease in FGF21 (p<0.001) and leptin levels (p=0.03) and an increase in ghrelin (p=0.05).

Conclusion: Thyrotoxic state is associated with high leptin & FGF21 levels - both known to be thermogenic. Despite a lower weight and fat mass, thyrotoxicosis is associated with insulin resistance- possibly a direct effect of thyroxine. Ghrelin levels are low in thyrotoxicosis; which together with raised leptin is an unexpected finding, given the increased appetite observed in this condition.
CDC Clinical Standardization Programs (CSP) for Free Thyroxine (FT4) to Improve the Accuracy and Reliability of FT4 Measurements in Patient Care and Clinical Research
Authors: Ribera Ashley , Dabbs-Brown Amonae , Poynter Krista , Sugahara Otoe , Zhang Li , Danilenko Uliana , Vesper Hubert W
Published: May 2021

Reliable FT4 measurement is critical to assess thyroid function and diagnose and treat thyroid disorders. The Partnership for the Accurate Testing of Hormones (PATH) categorizes FT4 as a biomarker in high need for standardization, and currently high inter-assay variability restricts the interpretation of FT4 results in patient care to assay-specific reference intervals. The CDC CSP has partnered with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and PATH to create a standardization program for FT4 to improve accuracy, reliability, and comparability of current methods and thus to improve diagnosis, treatment and prevention of thyroidal illnesses. Currently, there are no serum FT4 reference materials available to assess the accuracy and reliability of FT4 assays. CDC has developed an accurate and sensitive reference measurement procedure (RMP) to create commutable serum reference materials with FT4 target values. Assay manufacturers, research and clinical laboratories can use these reference materials to assess their calibration, certify the analytical performance of their measurements, and monitor performance over time by collaborating with CDC CSP. The CDC FT4 RMP uses equilibrium dialysis (ED) with LC-MS/MS based on an internationally recognized ED procedure[1] followed by solid-phase and solvent extractions. Certified primary reference material IRMM468 was used to prepare calibrators. Chromatographic separation is achieved with a gradient of methanol and water with 0.1% formic acid. FT4 is quantified using positive electrospray ionization in positive mode. The intra- and inter-day imprecision of the CDC RMP are 3.0% and 1.1%. A comparison among FT4 RMPs resulted in a +2.5% bias for the CDC RMP to the mean for all labs. The CDC RMP measurement range was 3.02-258 pmol/L and thus suitable for analysis of hypo- and hyperthyroid patients. The CDC FT4 RMP demonstrates good accuracy and precision, and can be used as a viable accuracy base to which routine methods can be compared. An initial comparison study of a commercially available FT4 immunoassay (IA) and the CDC RMP with 24 samples (7.98-109 pmol/L) indicated a mean bias of -37.7%, further indicating a need for standardization. Findings from an IFCC study suggest that alignment of IA measurements to a FT4 RMP can improve comparability and would allow for a uniform reference interval for FT4.[2] CDC CSP established a new standardization program for FT4 to address the needs of the community and assist with improving test comparability and reliability.1. Clin. Chem. Lab. Med. 2011, 49: 1275-81. 2. Clin. Chem. 2017, 63: 1642-52.
Development of High-Throughput Measurement of Free Thyroxine in Serum Using Equilibrium Dialysis in Couple With Liquid Chromatography-Tandem Mass Spectrometry
Authors: Zhang Li , Amonae Dabbs-Brown , Ribera Ashley , Krista Poynter , Sugahara Otoe , Danilenko Uliana , Vesper Hubert W
Published: May 2021

Free thyroxine (FT4) measurements are critical in the diagnosis, classification, and treatment of thyroid diseases. It is estimated that about 18 million FT4 tests are requested in the USA per year annually. In clinical laboratories, most of FT4 assays are performed by using immunoassays (I As). However, the significant bias of IAs and large variation between laboratories have been reported. The reference measurement procedures (RMPs) of FT4based on equilibrium dialysis (ED) - liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been established and recognized by the clinical chemistry community. However, the FT4 RMP is relatively low throughput and labor-intensive. Also, an aliquot of 1 mL sample is required for an RMP. A routine FT4 assay high-throughput procedure that is based on ED LC-MS/MS and utilized less sample volume will allow to conduct large biomonitoring studies and establish FT4 levels in US population. In the described method, FT4 in 150 uL of serum was separated from protein-bound T4 at 37.0 oC in 96-well Micro-ED Teflon devices from HTDialysis. The ED conditions suggested by CLSI C45-A guideline were followed. A volume of 150 uL dialysate samples with FT4 was obtained after the ED step. FT4 in the dialysate was purified by extractions before LC-MS/MS analysis. Chromatographic separation of T4 from the sample matrix is achieved on a C18 UPLC column with a gradient of methanol and water with 0.1% formic acid. Quantification of FT4 was performed by using selective reaction monitoring in positive electrospray ionization mode. The IRMM-468 certified primary reference material (JRC, Belgium) of T4 is used for the calibration curves. FT4 concentrations reached equilibrium after 4 hours under current dialysis conditions, which was also observed in the RMP setting. The developed routine FT4 assay 96-well ED system is within 5% bias from the FT4 RMP based on preliminary method comparison study using human serum. The studies to further characterize the FT4 routine method performance are ongoing. In summary, we are developing an analytical method based on a 96-well ED-LC-MS/MS system for the measurements of FT4 in serum. By comparison with RMP, the described method significantly improve throughput and reduce sample volume, which will fulfill the requirement of FT4 routine assay in clinical laboratories and allow for its use in the large biomonitoring studies and other activities in the research and public health settings.
Development of Intranasal Levothyroxine Powder Delivery for Hypothyroidism
Authors: Imran Syed Ali , Esomchukwu Obinna , Agu Remigius
Published: May 2021

Background: Hypothyroidism affects 3-5% of the general population with oral levothyroxine (LT4) being the predominant replacement therapy. However, significant proportion of hypothyroid patients are unable to absorb oral replacement leading to therapeutic failure and may require injectable thyroxine. Objectives: To develop non-invasive, less costly, and patient-friendly LT4 nasal delivery alternative using mucoadhesive polymers: chitosan and hydroxypropylmethylcellulose (HPMC). Methods: Six nasal LT4 formulations were developed with either chitosan or hydroxypropyl methylcellulose as mucoadhesive. The formulations were prepared through freeze-drying by varying the drug to polymer ratio (1:1, 1:3, and 1:5). The percentage drug yield was calculated by analyzing the weight of the formulation pre- and post-freeze drying. HPLC analysis was conducted to determine the amount of LT4 loaded in each formulation. Furthermore, the surface morphology, particle size, zeta potential, differential scanning calorimetry, X-ray diffraction as well as the in vitro release were assessed to determine the physicochemical properties and release characteristics of the formulations, respectively. Results: Both percentage drug loading and yield were > 70% for all the formulations. The freeze-dried formulations displayed a compact needle-like surface morphology. LT4-chitosan formulations, 1:1, 1:3, and 1:5 had mean particle size of 2.45 ± 0.88 µm, 2.76 ± 1.38 µm, and 1.59 ± 0.27 µm, respectively. Mean particle sizes for 1:1, 1:3, and 1:5 LT4-HPMC formulations were 0.56 ± 0.02 µm, 0.22 ± 0.06 µm, and 0.46 ± 0.04 µm. Zeta potential for LT4-chitosan formulation 1:1, 1:3, and 1:5 were -18.7 ± 1.00 mV, -16.2 ± 0.79 mV, and -19.17 ± 1.01 mV, respectively. LT4-HPMC 1:1, 1:3, and 1:5 formulations had zeta charges of -11.66 ± 3.16 mV, -6.06 ± 3.92 mV, and -9.53 ± 1.68 mV, respectively. Differential calorimetric analysis confirmed drug-polymer integration in all formulations, and X-ray powder diffraction showed both chitosan and HPMC formulations as crystalline configuration. The formulations with the highest in vitro release were LT4-HPMC 1:3 and LT4-chitosan 1:5. Conclusions: Results of this study suggest that both chitosan and HPMC can be used as sustained release polymers for the intranasal delivery of LT4.
Goiter at Diagnosis Might Be Predicting Factor for Early Onset Intractable Graves’ Disease
Authors: Kim Sungeun , Nam Goong Ho , Lee Nayeong , Kim Seulki , Ahn Moon Bae , Kim Shin-Hee , Won-Kyoung Cho , Cho Kyoung Soon , Jung Min-Ho , Suh Byung-Kyu
Published: May 2021

Introduction: The clinical course of Graves’ disease (GD) treated with anti-thyroid drug (ATD) treatment were reviewed with the aim of establishing criteria able to predict intractable GD. Methods: The clinical course of 116 patients with GD who agreed to participate in this study between March 2009 and August 2019 in the pediatric endocrine clinic at Seoul St. Mary’s and St. Vincent’s Hospitals were reviewed. We defined an intractable as hyperthyroidism persistent over 2 years of ATD or relapsed after ATD withdrawal or had been treated ATD for at least 5 years [1-3]. Result: Of 116 patients diagnosed with GD, 37 patients (31.8%) had remission and 79 (68.2%) had intractable GD. Between intractable and remission GD group, there were no significant difference of female percentage, age at diagnosis, thyroid associated ophthalmopathy, serum levels of triiodothyronine (T3), free tetraiodothyronine (T4), Thyroid stimulating hormone (TSH) and positive rate of thyroid autoantibody (Thyroid peroxidase (TPO), Thyroglobulin (Tg), Thyroid stimulating hormone receptor (TSHR)). In intractable GD patients, the frequency of goiter at diagnosis is higher than remission group (89.9% [71/79] and 70.3% [26/37], P-value = 0.014). In correlation analysis, intractable GD showed positive correlation with goiter (R=0.247, P-value = 0.008). In multivariate logistic analyses, goiter is showed strong relationship with intractable GD (odds ratio, 3.793; 95% confidence interval, 1.367-10.524) after adjusting age and sex. Conclusion: Our study supported that goiter at initial presentation might be predicting factor for early onset intractable GD.
Serum TSH Levels Normalisation in Patients Affected by Autoimmune Atrophic Gastritis, After the Switch From Oral L-T4 in Tablet Form to L-T4 in Liquid Formulation
Authors: Fallahi Poupak , Ferrari Silvia Martina , Elia Giusy , Ragusa Francesca , Paparo Sabrina Rosaria , Ruffilli Ilaria , Antonelli Alessandro
Published: May 2021

Patients affected by autoimmune atrophic gastritis could have some issues in L-thyroxine (L-T4) absorption, due to drug malabsorption, induced by the increased gastric pH. Different factors influence L-T4 absorption, such as dietary habits, interference with other drugs, absorption kinetics, age of the patient, adherence to therapy, and others. We enrolled 36 patients affected by autoimmune atrophic gastritis with high serum thyrotropin (TSH) levels under therapy with L-T4 in the tablet formulation. L-T4 tablets were changed to an oral liquid L-T4 preparation, maintaining the same dose. The switch from L-T4 in tablet formulation to the liquid one, at the same L-T4 dosage, led to the normalisation/reduction of circulating TSH levels. Then 14 patients, who were switched back again to receive L-T4 in tablets (with the same dose), had a worsening of TSH values, falling in the hypothyroid range. In conclusion, our findings led to hypothesize that the pH alteration issue caused by autoimmune atrophic gastritis could be overcome by the oral L-T4 liquid formulation administration.
What Matters Most to Patients With Thyroid Dysfunction in Malaysia: A Preliminary Qualitative Analysis
Authors: Ooi Cheow Peng , Yusof Khan Abdul Hanif , Manaf Rosliza Abdul , Adulazeem Hebatullah M , Sukor Norlela , Mustafa Norlaila , Kamaruddin Nor Azmi
Published: May 2021

Background: Relatively little is known on the aspects of research that matter most to patients with thyroid dysfunction (TD). We aimed to explore the patients’ experience when they were diagnosed and treated for TD, as well as what they needed to improve their care: Methods: Qualitative data were obtained online using the Malay language version of the semi-structured qualitative survey in Malaysia, part of the larger research: A CORE OUTCOME SET FOR THYROID DYSFUNCTION. The questionnaires were developed from interactive discussions with patients who have thyroid dysfunction. The responses were analyzed using Braun and Clark’s thematic analysis framework guided by the question: What are the perceptions and experiences of thyroid care that matter most to patients with TD? Results: Responses from 38 participants, in the 18-60 age range, with TD, were analyzed. Most patients across the spectrum of thyroid dysfunctions experienced emotional disturbances. These negative emotions were related to the whole spectrum of disease state, the associated treatments, pre-treatment, post-treatment care, and personal aspects of daily life. Uncertainties in medical outcomes were associated with the personal need for further information. Functional psychosocial impairments and socioeconomic disruptions have a strong ability to arouse the anxiety of outcome uncertainties further. Getting treatment and independence in routine daily activities were prioritized while acknowledging treatments may differ in modality and risk of complications. Physician-patient interactions were valued in alleviating concerns and fear. Four salient themes emerged suggesting emotional security, functional ability, self-care (including psychosocial and socioeconomic well-being), and quality of physician-patient relationships were what matters to these participants. Conclusion: This insight into the problems of patients with TD underlined the need for quality patient-centered thyroid care and may be enhanced, personalized, and improved through outcomes that mattered most to patients in clinical research/trials, routine clinical practice, and the health system.
Association Study of the Thyroid Peroxidase Gene in Autoimmune Thyroid Diseases
Authors: Ban Yoshiyuki , Tozaki Teruaki , Iwai Shinichi
Published: May 2021

Introduction: The etiology of autoimmune thyroid diseases (AITDs), such as Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), is largely unknown. However, genetic susceptibility is believed to play a major role. Recently, a case-control association study performed in Croatia showed a significant association of the single nucleotide polymorphism (SNP) rs11675434 near thyroid peroxidase (TPO) with HT, and also with thyroid autoantibodies against TPO (TPOAb) levels. High TPOAb levels are present in 90% of patients with HT and serve as a clinical marker for the detection of early AITD/HT. Therefore, we conducted a case-control study to determine the genetic association of the rs11675434 near TPO with AITD in a cohort of the Japanese population. Methods: We genotyped the rs11675434 near TPO in 457 Japanese patients with AITD (286 with GD, 171 with HT) and 242 matched Japanese control subjects. The SNP was analyzed using TaqMan probe method, and association study was performed using the χ 2 and Fisher’s exact tests with Yates correction. Results: Both GD and HT showed no significant associations. Moreover, when patients with GD were stratified according to Graves’ ophthalmopathy (GO) (n=96), there were no allelic associations with GO, although there were weak associations between GO and controls (P=0.0494, Odds ratios (ORs)=0.3102). Conclusions: Our finding suggest that the rs11675434 near TPO may not contribute to the risk of AITD in the Japanese population, although the study was in insufficient and underpowered sample size.
At Last the Explanation for Increased Spontaneous Miscarriages in Thyroid Autoantibody Positive Pregnant Women
Authors: Behringer-Massera Stephanie , Morshed Syed A , Davies Terry F
Published: May 2021

In women who are pregnant, the presence of thyroid autoantibodies is associated with an increased rate of miscarriage in the first trimester, with multiple reports averaging ~17% compared with ~8% in autoantibody negative women. During pregnancy immune tolerance is altered to allow implantation of the semi-allogeneic fetus and T-regulatory cells (Tregs), which play a major role in such tolerance, have been shown to increase during pregnancy, reaching a peak in the second trimester. A deficiency in this Treg response has been widely associated with spontaneous miscarriages. While it is known that the number of Tregs in patients with autoimmune thyroid disease (AITD) is decreased there are no data on pregnant women with AITD. In this study, we examined both the number and function of Tregs in pregnant women with (n=26) and without (n=41) thyroid autoantibodies (anti-Tg and/or anti-TPO) as well as healthy non pregnant women (n=25). Tregs were measured by flow cytometry of isolated CD4+, CD25+ and FoxP3+ T-cells. We found that the total number of CD4+CD25+FoxP3+ high Tregs was significantly increased in pregnancy consistent with previous reports (from 11% to 22%, p<0.024). Furthermore, this increase in Tregs was less in pregnant women with thyroid autoantibodies (mean of 12%). In addition, studies of phosphorylated signal transducer and activator of transcription-5a (pStat-5a) as a marker of Treg function, showed that while Tregs were activated in pregnancy, their activity per cell was diminished in the pregnant antibody positive women with a frequency:MFI ratio of 201 compared to 316 in the negative group. These data demonstrate that pregnant women with thyroid antibodies have a reduced Treg response to pregnancy, both in number and function, and offer a likely explanation for the increased miscarriage rate in such patients.
Comparison of Block and Replace Regime and Titration Regime in Graves’ Disease
Authors: Mar Cho
Published: May 2021

In Graves’ disease (GD), medical treatment is still the cornerstone in its management and there were some studies done on comparison of the block and replace regime and titration regime of the antithyroid drugs (ATDs). In Myanmar, titration regime is mostly practiced for management of GD. In daily clinical practice, frequent hospital visits are needed in titration regime and loss of follow-up is an obstacle in patients treated with titration regime. A hospital based randomized clinical trial was conducted and aimed to compare the proportion of attainment of euthyroid status between block and replace regime and titration regime in patients with recently diagnosed GD. A total of 117 patients; 58 patients in block and replace regime and 59 patients in titration regime, who met the inclusion criteria were included. The results showed that euthyroid status was observed in increasing trend during the study period for both regimes but there was no significant difference of achieving euthyroid status between the regimes at the end of 12 months. Regarding side effects of ATDs, skin rash and pruritus were more frequently occurred during the first 3 months of ATDs but no significant difference was noted between the regimes at the end of study. There was also no case of serious side effects such as agranulocytosis and hepatotoxicity up to the end of 12 months. The results of the study pointed out that block and replace regime was comparable to dose titration regime in attaining euthyroid status. As a conclusion, block and replace regime can be applied as an alternative option where titration regime is not feasible. Reference: (1) Abraham et al., 2005; A systematic review of drug therapy for Graves’ hyperthyroidism. Eur J Endocrinol. 153: 489-98. (2) Vaidya et al., 2014; Block & replace regime versus titration regime of antithyroid drugs for the treatment of Graves’ disease: a retrospective observational study. Clinical Endocrinology. 81: 610–613.
Evaluation of TRAb and TSI Leves and Thyroid Function in Pregnant Women With Graves’ Disease and Newborn: Preliminary Data
Authors: Negri Maurício Massucati , Araujo Paula Bruna Mattos Coelho , Gomes Dalva Margareth Valente , Pinheiro Maria Fernanda Miguens Castelar , Schrank Yolanda , Perozo Andrea Faria Dutra Fragoso , Marui Suemi , Fontes Rosita
Published: May 2021

Introduction: GD, mediated by TSH receptor-stimulating immunoglobulins (Igs) (rTSH), can lead to fetal thyroid dysfunction through the passage of Igs through the placenta during pregnancy. TRAb levels, used for prognostic evaluation, measure rTSH-stimulating and blocking Igs while TSI evaluates only as stimulating Igs. Objective: To prospectively evaluate pregnant women with DG and newborns (NB) by measuring TRAb and TSI and their correlation with thyroid function and postpartum complications. Methods: The patients were evaluated during pregnancy and the puerperium and the respective newborns. TSH, thyroid hormones and TRAb were evaluated by electrochemiluminescent method (Roche) and TSI by chemiluminescent assay (Siemens). TRAb<1.75IU/L and TSI<0.55IU/L were considered negative. Results: Nine patients were evaluated, with a mean age of 27.4±5.7 years: 6 had TRAb and TSI positive in the 1st trimester (1st-tri), when they maintained or started DAT; one with both negative (without DAT) and one with weakly positive TSI, when DAT was suspended. These last two remained euthyroid during pregnancy and puerperium. Of the first 6, 4 were evaluated in the 3rd-tri: three negative for TRAb and maintained positive TSI, 2 in low leves and one for high titles, when DAT was suspended or reduced; one kept both at very high levels. A patient with post-DT hypothyroidism, performed 3 years ago, using levothyroxine, evaluated in the 3rd-tri, had a negative TRAb and a highly positive TSI and remained so after pregnancy. The two patients who presented weakly positive TSI in the 3rd-tri evolved with their negative results and without DAT in the puerperium. The patient with TSI in high titers evolved with elevated levels as well as positive TRAb titers and postpartum decompensation. The patient with positive antibodies remained compensated for stable doses of DAT. Four NB were evaluated: all healthy, with normal thyroid function and negative TRAb. TSI was positive in 2 in the postpartum period; TSI was negative afterwards, while in the other 2 both antibodies were negative. Conclusions: TSI was not associated with thyroid dysfunction in NB, although it was associated with worsening hyperthyroidism in pregnant women, when at high titers. Positive TSI at low levels were not associated with worsening of the condition, which requires further studies to determine the cutoff point for assessing treatment suspension.
Meta-Analysis of Proptosis Response in Thyroid Eye Disease: Efficacy of EUGOGO Recommended Treatment Regimen With IV Methylprednisolone
Authors: Douglas Raymond S , Batten Ryan , Qadeer Rana A , Cameron Chris
Published: May 2021

Background: The European Group on Graves’ Orbitopathy (EUGOGO) recommends intravenous methylprednisolone (IVMP) as the first line treatment for moderate-to-severe active thyroid eye disease (TED). While many studies with varying doses of IVMP have reported improvements in inflammation, via clinical activity score (CAS) reduction, the reported improvements in the major progressive outcome of TED, proptosis, vary widely. A reduction in proptosis of ≥2 millimeter (mm) is considered by clinicians and EUGOGO as clinically meaningful. A meta-analysis of existing literature on use of IVMP in TED management, specifically proptosis response, was conducted.

Methods: PubMed and Embase were searched for relevant randomized controlled trials (RCTs) and observational studies that included patients with moderate-to-severe active TED receiving treatment with the EUGOGO recommended dose of IVMP (4.5-5g over 12 weeks) from the inception of the databases to date of search (October 2020); regular alerts were established to capture any recent studies. The outcome of interest was change from baseline to week 12 in proptosis in mms. Single-arm meta-analyses were conducted for IVMP using the DerSimonian-Laird random-effects models and forest plots were generated. Pooled means and corresponding 95% confidence intervals (CIs) were calculated. The primary analysis included all identified studies. Sensitivity analyses were conducted based on study design, study characteristics (smoking status), and methodologies (weighted Bayesian meta-analyses based on study design).

Results: The search retrieved 12 studies (10 single-center, 8 RCTs, 4 observational studies: 7 from China, 4 from EU, 1 from Turkey), reporting proptosis for 498 patients that were used for the meta-analysis. No placebo-controlled study with the EUGOGO recommended dose of IVMP was found. All studies included patients who were 18 years or older with moderate-to-severe TED and most (n=11) studies included patients with CAS of ≥3. For studies that reported this data, the mean or median (if mean not reported) age ranged from 35 to 52 years and duration of TED symptoms ranged from 4 to 13.6 months. Treatment with IVMP resulted in a reduction of 0.94 mm (95% CI: -1.57 to -0.32) in proptosis from baseline to week 12. The results from the sensitivity analyses were aligned with those from the primary analysis.

Conclusions: Among patients with moderate-to-severe TED, this meta-analysis found the EUGOGO-recommended dose of IVMP may result in modest, but not clinically meaningful, improvements in proptosis. However, these results should be interpreted with caution given the paucity of published data and the lack of risk/benefit analyses with glucocorticoid use as compared to the recently FDA-approved therapy, teprotumumab, for the treatment of TED.