Ionic Levothyroxine Formulations: Synthesis, Bioavailability, and Cytotoxicity Studies.

Thyroid diseases affect a considerable portion of the population, with hypothyroidism being one of the most commonly reported thyroid diseases. Levothyroxine (T4) is clinically used to treat hypothyroidism and suppress thyroid stimulating hormone secretion in other thyroid diseases. In this work, an attempt to improve T4 solubility is made through the synthesis of ionic liquids (ILs) based on this drug. In this context, [Na][T4] was combined with choline [Ch]+ and 1-(2-hydroxyethyl)-3-methylimidazolium [C2OHMiM] + cations in order to prepare the desired T4-ILs. All compounds were characterized by NMR, ATR-FTIR, elemental analysis, and DSC, aiming to check their chemical structure, purities, and thermal properties. The serum, water, and PBS solubilities of the T4-ILs were compared to [Na][T4], as well as the permeability assays. It is important to note an improved adsorption capacity, in which no significant cytotoxicity was observed against L929 cells. [C2OHMiM][T4] seems to be a good alternative to the commercial levothyroxine sodium salt with promising bioavailability.

Synthesis and characterization of 64Cu- and Cy5.5-labeled tetraiodothyroacetic acid derivatives for tumor angiogenesis imaging.

It was previously reported that tetraiodothyroacetic acid (tetrac) inhibits angiogenesis by binding to the cell surface receptor for thyroid hormone on integrin αVß3. Therefore, we synthesized and evaluated two 64Cu-labeled tetrac derivatives and a Cy5.5-labeled tetrac derivative for tumor angiogenesis imaging. Tetrac was structurally modified to conjugate with 1,4,7,10-tetraazacyclododecane-N,N',N″,N″'-tetraacetic acid (DOTA) via its hydroxy or carboxylic acid end, and the resulting DOTA-conjugated tetrac derivatives were then labeled with 64Cu. Tetrac was also conjugated with Cy5.5 via its carboxylic acid end. All three tetrac derivatives (1-3) exhibited greater inhibitory activity than tetrac against endothelial cell tube formation. The U87MG cell binding of [64Cu]2 showed a time-dependent increase over 24 h and it was inhibited by 38% at 4 h in the presence of tetrac, indicating specificity of [64Cu]2 to the thyroid hormone receptor site on integrin αVß3. Positron emission tomography (PET) images of U87MG tumor-bearing mice injected with [64Cu]1 and [64Cu]2 revealed that high radioactivity accumulated in the tumors, and that the tumor uptake and tumor-to-nontarget uptake ratio were higher in small tumors than in large tumors. In addition, the Cy5.5-labeled tetrac derivative (3) displayed a strong near-infrared (NIR) signal in the tumors. Taken together, these results suggest that these ligands hold promise as imaging agents for visualization of tumor angiogenesis.

Synthesis of new analogs of tetraiodothyroacetic acid (tetrac) as novel angiogenesis inhibitors for treatment of cancer.

In the angiogenesis process, integrins, which are members of a family of cell surface transmembrane receptors, play a critical role particularly in blood vessel formation and the local release of vascular growth factors. Thyroid hormones such as l-thyroxine (T4) and 3,5,3'-triiodo-l-thyronine (T3) promote angiogenesis and tumor cell proliferation via integrin αvß3 receptor. At or near an arginine-glycine-aspartate (RGD) recognition site on the binding pocket of integrin αvß3, tetraiodothyroacetic acid (tetrac, a deaminated derivative of T4) is a thyrointegrin receptor antagonist and blocks the actions of T3 and T4 as well as different growth factors-mediated angiogenesis. In this study, we synthesized novel tetrac analogs by modifying the phenolic moiety of tetrac and tested them for their anti-angiogenesis activity using a Matrigel plug model for angiogenesis in mice. Pharmacological activity results showed that tetrac can accommodate numerous modifications and maintain its anti-angiogenesis activity.

Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis.

The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality control of newly developed and well-established active pharmaceutical ingredients (APIs). The United States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS absorption detection to determine organic impurities in the drug substance. The impurity profile of the first USP method ("Procedure 1") has already been extensively studied, however for the second method ("Procedure 2"), which exhibits a significantly different impurity profile, no wholistic structural elucidation of impurities has been performed yet. Applying minor modifications to the chromatographic parameters of USP "Procedure 2" and using various comprehensive structural elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted in the characterization of 24 compounds previously unknown to literature and the introduction of two new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified major impurities as well as neutral loss fragmentation patterns of many characterized impurities were discussed.

Synthesis of MR-49, a deiodinated analog of tetraiodothyroacetic acid (tetrac), as a novel pro-angiogenesis modulator.

The tyrosine-based hormones 3,3',5-triiodo-l-thyronine (l-T3) and l-thyroxine (l-T4) that are produced by the thyroid gland control metabolic functions. Iodothyronine deiodinase enzymes convert l-T4 to l-T3, the form of thyroid hormone critical to genomic actions within cells and regulation of metabolism, and to reverse-l-T3, a hormone isoform that is largely inactive. We used tertiary amines in a study of deiodination based on derivatives of tetraiodothyroacetic acid (tetrac)-a naturally occurring derivative of l-T4-to mimic the action of the iodothyronine deiodinases and deiodination of the outer ring iodines. Deiodinated tetrac, MR-49, was found to be pro-angiogenic, with this activity exceeding that of l-T3 and l-T4 in a hemoglobin Matrigel® plug assay of angiogenesis. Tetrac is anti-angiogenic via several nongenomic pathways, and the present studies of MR-49 reveal the critical contribution of outer ring iodines to the angiogenic properties of thyroid hormone analogues, which may have utility as pro-angiogenic pharmaceuticals.

Halogen bonding controls the regioselectivity of the deiodination of thyroid hormones and their sulfate analogues.

The type 1 iodothyronine deiodinase (1D-1) in liver and kidney converts the L-thyroxine (T4), a prohormone, by outer-ring (5') deiodination to biologically active 3,3',5-triiodothyronine (T3) or by inner-ring (5) deiodination to inactive 3,3',5'-triiodothronine (rT3). Sulfate conjugation is an important step in the irreversible inactivation of thyroid hormones. While sulfate conjugation of the phenolic hydroxyl group stimulates the 5-deiodination of T4 and T3, it blocks the 5'-deiodination of T4. We show that thyroxine sulfate (T4S) undergoes faster deiodination as compared to the parent thyroid hormone T4 by synthetic selenium compounds. It is also shown that ID-3 mimics, which are remarkably selective to the inner-ring deiodination of T4 and T3, changes the selectivity completely when T4S is used as a substrate. From the theoretical investigations, it is observed that the strength of halogen bonding increases upon sulfate conjugation, which leads to a change in the regioselectivity of ID-3 mimics towards the deiodination of T4S. It has been shown that these mimics perform both the 5'- and 5-ring deiodinations by an identical mechanism.

Semisynthesis and pharmacological activities of thyroxine analogs: Development of new angiogenesis modulators.

Novel thyroxine analogs with hindered phenol, amino and carboxylic acid groups have been synthesized and the effects of the synthesized compounds on angiogenesis using the chick chorioallantoic membrane and mouse matrigel models have been tested. Pharmacological profiles revealed that thyroxine tolerates numerous modifications on the amino group and remains active. These results provide the rationale for the selection of a novel thyroxine nanoparticle precursor.

Synthesis and physicochemical characterization of Gd-C4-thyroxin-DTPA, a potential MRI contrast agent. Evaluation of its affinity for human serum albumin by proton relaxometry, NMR diffusometry, and electrospray mass spectrometry.

Gd-C(4)-thyroxin-DTPA, a potential MRI contrast agent, was synthesized from Gd-DTPA and thyroxine, which interacts strongly with human serum albumin (HSA). It was characterized in water by its relaxometric properties and its stability versus zinc transmetalation. The affinity of the complex for HSA was studied by using three different methods: proton relaxometry, NMR diffusometry, and electrospray mass spectrometry. From the results, it appears that Gd-C(4)-thyroxin-DTPA exhibits a relatively high relaxivity (r(1) = 9.01 s(-1) mM(-1) at 1.5 T and 310 K), a good stability versus zinc transmetalation, and a strong interaction with HSA (K(a) approximately 10,000 M(-1) with two binding sites). The kinetics of the exchange between the bound and the free form of the complex was evaluated by the NMR diffusometry technique. Competition experiments have allowed the assignment of the chelate's binding site on HSA.

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Semisynthesis and pharmacological activities of Tetrac analogs: angiogenesis modulators.

Novel Tetrac analogs were synthesized and then tested. Anti-angiogenesis efficacy was carried out using the Chick Chorioallantoic Membrane (CAM) model and the mouse matrigel model for angiogenesis. Pharmacological activities showed Tetrac can accommodate numerous modifications and maintain anti-angiogenesis activity.

Thyroid receptor ligands. Part 5: novel bicyclic agonist ligands selective for the thyroid hormone receptor beta.

Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.

Increase in plasma thyrotropin levels in hypothyroid patients during treatment due to a defect in the commercial preparation.

Around mid-1995, the Molecular Endocrinology Laboratory of the Regional Hospital (Malaga, Spain) began detecting an increase in TSH levels in the serum of patients under study to control the treatment of hypothyroidism with levothyroxine. Over a period of 5 months, of a total of 467 hypothyroid patients treated with Levothyroid, 53% had TSH levels higher than 6 microU/mL. The reliability of the biochemical results was verified by duplicating 56 randomly chosen samples from all those with high TSH levels and by an external control performed in four different laboratories. The amount of levothyroxine in the tablets was analyzed by RIA, high performance liquid chromatography, and their iodine contents. The lowest levels of levothyroxine found in the 50 micrograms Levothyroid tablets were those determined by RIA, with a mean value of 32.3 micrograms, resulting in a 35.3% loss of activity. The mean value of levothyroxine found in these same tablets by high performance liquid chromatography was 39.3 micrograms, amounting to a 21.3% loss in activity. The iodine showed no significant loss in these tablets, with a mean experimental value of 48 micrograms. The commercial laboratory withdrew lot J from the market, the one in which these deficiencies were found.

Avaliação do Tratamento Supressivo com Levotiroxina na Doença Nodular de Tireóide: Resultados de um Estudo Clínico Prospectivo, Randomizado, Duplo-Cegoe Controlado com Placebo

O objetivo do presente estudo foi avaliar o efeito do tratamento supressivo comlevotiroxina em reduzir o tamanho de nódulos tireoideanos clinicamente solitários. Os critérios para inclusão dos pacientes no estudo foram: nódulo frio ou morno à cintilografia, neste último caso suprimível com o uso de hormônio tireoideano; nódulos sólidos oupredominantemente sólidos à ecografia; e resultado citológico da PAAF de lesão negativa para malignidade. Critérios de exclusão foram: mais do que um nódulo palpável, nódulos quentes ou mornos não suprimíveis pelo uso do hormônio tireoideano; císticos ou predominantemente císticos à ecografia, achados sugestivos ou positivos para malignidade na PAAF; gestação e qualquer contra-indicação para terapia supressiva de tireóide. Um totalde 48 pacientes foram randomizados para receber levotiroxina (n = 25) ou placebo (n = 23) por um período de 1 ano. A dose de levotiroxina (LT) foi de 200 (ig/dia para pacientes com menos de 70 kg e 250 [ig/dia para aqueles pesando mais do que 70 kg. Foi realizada ecografia de alta resolução antes e após 6 e 12 meses de tratamento, para avaliar as mudanças notamanho dos nódulos. Dosagens de TSH-sensível (TSH-S) e prova do TRH foram realizadas para confirmar a supressão do eixo hipofisário-tireoideano, correlacionando-se os níveis basaisde TSH-S com suas respostas na prova do TRH. A tireoglobulina (TG) foi dosada com intuito de verificar sua possível utilidade como um "marcador bioquímico" da eficácia do tratamento supressivo. Após 1 ano, ocorreu diminuição do volume nodular em 44% dos pacientes dogrupo levotiroxina (GT) contra 35% do grupo placebo (GP). A diferença entre os dois grupos não foi significativa. Aos 6 meses, ocorreu uma significativa diminuição do volume nodularmédio no GT (p < 0,05; teste t de Student), mas aos 12 meses a diminuição no volume nodular não foi significativa...

The purpose of the present study was to evaluate the effect of thyroid suppressiontherapy on clinically solitary thyroid nodules. Criteria for inclusion were the following features of the nodule: "cold" or "warm" on thyroid scintigraphic scanning (in the latter case, suppressible with thyroid hormone); solid or predominantly solid on ultrasonography; and negative for malignancy by fine-needle aspiration biopsy. Exclusion criteria were: more than one palpable nodule; "hot" or "non-suppressible-warm" nodule on scintigraphic scanning;cytologic findings suggestive of a neoplasy by fine-needle aspiration biopsy; cystic or predominantly cystic nodule on ultrasonography, and pregnancy or any other contraindication for thyroid suppression therapy. A total of 48 patients were randomly assigned to receive either levothyroxine (n = 25) or placebo (n = 23), for a one-year period. The dose of levothyroxine (LT) given was 200 (ig/day for patients weighing less and 250 ug/day for those weighing more than 70 kg. High-frequency ultrasonography was performed before, at six months, and after one year of treatment, to evaluate changes in nodule size. TSH-sensitive (TSH-S) levels and TRH test were performed to confirm the suppression of thyrotropin. TheTSH-S basal level and its variation with TRH test were correlated. Thyroglobulin (TG) was measured to evaluate its possible usefulness as a "biochemical marker" of thyroid suppression.After one year, nodule volume was reduced in 44% of LT-treated patients, and in 35% of patients given placebo. The difference between the two groups was not significant. At six months, there was a significant reduction in mean nodule volume in the LT-treated group(p < 0,05; Student's t test). At one year, however, this volume reduction was no longer found to be significant. In the placebo group, there was a significant increase in the mean nodule volume at both six months and one year (p < 0,05; Student's t test)

Synthesis and properties of N-bromoacetyl-L-thyroxine.

A one-step bromoacetylation of L-thyroxine (T4) produces N-bromoacetyl-L-thyroxine (BrAcT4) in good yield. The reaction product is best purified by high-speed countercurrent chromatography. While HPLC is satisfactory only for purification of microgram and submicrogram quantities, amounts ranging from about 1 ng to 1 g of BrAcT4 can be processed by high-speed countercurrent chromatography (HSCCC), a method which we have previously used for the purification of N-bromoacetyl-3,3',5-triiodo-L-thyronine (BrAcT3). Operating conditions for the one-step synthesis of BrAcT4 and BrAcT3 differ due to differences in solubility and reactivity of the two hormones. BrAcT4 purified by HSCCC and shown to be pure by analytical HPLC has been characterized by alpha max and epsilon max in the near and far uv in several solvents, mass spectrum, 1H NMR spectrum, TLC in three solvent systems, retention time in reverse-phase HPLC (C18) in relation to the retention times of two internal standards, 3,3',5-triiodo-L-thyronine and T4, and melting point. Corresponding data for BrAcT3, not previously reported, have also been determined. The described procedure can provide not only substantial amounts of highly purified BrAcT4 for competition studies, but also 125I-labeled BrAcT4 of high specific activity for affinity labeling. Since solutions of BrAcT4 and of BrAcT3 undergo partial decomposition on evaporation to dryness, suitable procedures for the preparation of these hormones in solid form and for storage in solutions have been devised.

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

Preparation and separation of the glucuronide and sulfate conjugates of thyroxine and triiodothyronine.

An enzymatic method for synthesis of labelled thyroxine glucuronide (T4G) and triiodothyronine glucuronide (T3G) from labelled thyroxine (T4) and triiodothyronine (T3) is presented. The synthetic glucuronides are completely digested by beta-glucuronidase, with recovery of the parent T4 or T3. They have distinctive elution patterns on HPLC and on Sephadex G25 chromatography, and can be clearly separated from T4 and T3 as well as from synthetic T4 sulfate (T4S) and T3 sulfate (T3S). On LH 20 chromatography, elution of T4G and T3G is intermediate between that of T4 and T3 and that of T4S and T3S. T3G can be well separated from other thyronines by HPLC alone, but T4G coelutes with rT3 on HPLC; these are then separated by adding a Sephadex G25 chromatography step. Biosynthetic 131I-T3G and 125I-T4G from the bile of a cat given 131I-T3 and 125I-T4 had similar HPLC chromatographic patterns to those of synthetic T3G and T4G. That the identified peaks from analysis of the bile were indeed T3G and T4G was confirmed by recovery of the parent T3 and T4 after beta-glucuronidase digestion.

Preparation, purification, and stability of high specific activity 125I-labeled thyronines.

A modified chloramine-T method is described for the preparation of several radioiodothyronines by an exchange reaction, which results in low specific activity preparations, or by an addition reaction, which yields radioiodothyronines with specific activities up to 7 mCi/microgram. Purification by paper chromatography is shown to be more convenient than by LH-20 chromatography and provides better resolution among the various thyronines. Radioiodothyronines with only a single iodine atom in the outer (3,5,3'-triiodothyronine and 3,3'-diiodothyronine) are stable for several months when stored in organic solvents. The least stable radioiodothyronines are those with two 125I atoms in the outer ring (3,3',5'-triiodothyronine (rT3) produced from 3-iodothyronine and thyroxine (T4) from 3,5-diiodothyronine). The stability of rT3 and T4 stored in human plasma at 4 C is much greater than when stored in buffer at the same pH. The use of high specific activity [125I]rT3 has permitted the development of a radioimmunoassay with a sensitivity of 1 pg rT3/ml incubation volume.

[Investigation on the synthesis of 125I labelled triiodothyronine and thyroxine of high specific radioactivity (author's transl)]. / Untersuchungen zur Herstellung von 125J-markiertem Trijodthyronin und Thyroxin mit hoher spezifischer Radioaktivität

The reaction conditions of the monoiodination of L-diiodothyronine and L-triiodothyronine by nonradioactive iodide have been investigated by separation of the reaction mixture on sephadex G-25 with sodium hydroxide 10 mmol/l as eluent and quantitative iodine estimation in the eluate. When labelling with 125I was performed under optimal conditions, a good yield of chromatographically pure L-triiodothyronine or L-thyroxine was obtained with a specific radioactivity between 2 and 3 Ci/mg. The synthesized labelled hormones were tested by radioimmunoassay. They enable a detection of less than 2 pg T3 and less than 5 pg T4.