Development and In Vivo Evaluation of Sustained Release Microparticles Loaded with Levothyroxine for Hypothyroidism Treatment.

Hypothyroidism is a chronic condition combated by a daily oral supplementation of levothyroxine. In addition to the need for frequent dosing, oral administration may result in variable absorption of the drug leading to a failure in achieving normal thyroid function. Therefore, the development of a long-acting injectable system capable of delivering the drug is necessary. This work was aimed at developing sustained release microparticles loaded with levothyroxine. The microparticles were produced through the emulsification-solvent evaporation method using 2 grades of biocompatible and biodegradable polyesters: poly(ᴅ,ʟ-lactide-co-glycolide) (PLGA) and poly(ᴅ,ʟ-lactide) (PLA). Both polymers produced microparticles with very similar sizes (1.9 µm) and zeta potential values (around -22.0 mV). However, PLA microparticles had a significantly higher drug loading (6.1% vs. 4.4%, respectively) and encapsulation efficiency (36.8%, vs. 26.1%, respectively) when compared to PLGA counterparts. While both types of microparticles displayed a biphasic release pattern in vitro, a slower rate of release was observed with PLA microparticles. Moreover, a similar biphasic release pattern was found in vivo, with an initial phase of rapid release followed by a slower phase in the subsequent 10 days. These results indicate the possibility of developing levothyroxine loaded polyester microparticles as a potential long-acting thyroid hormone replacement therapy.

A Simple Substitution on Thyroid Hormones Remarkably Alters the Regioselectivity of Deiodination by a Deiodinase Mimic.

The regioselective deiodinations of L-thyroxine (T4) play key roles in the thyroid hormone homeostasis. These reactions are catalyzed by three isoforms of the selenoenzymes, iodothyronine deiodinases (Dio1, Dio2 and Dio3), which are highly homologous in nature. Dio1 mediates 5'- or 5-deiodinations of T4 to produce T3 and rT3, respectively. In contrast, Dio2 and Dio3 are selective to 5'- or 5-deiodination to produce T3 and rT3, respectively. Understanding of the regioselectivity of deiodination at the molecular level is important as abnormal levels of thyroid hormone have been implicated in various clinical conditions, such as hypoxia, myocardial infarction, neuronal ischemia and cancer. In this paper, we report that the electronic properties of the iodine atoms in thyroxine (T4) can be modulated through a simple substitution in the 4'-phenolic moiety. This leads to the change in the regioselectivity of deiodination by different small molecule mimics of Dio enzymes. By using this chemical approach, we also show that the substitution of a strong electron withdrawing group facilitates the removal of all four iodine atoms in the T4 derivative. Theoretical investigations on the hydrogen bonded adducts of T4 with imidazole indicate that the charge on the iodine atoms depend on the nature of hydrogen bond between the -OH group of T4 and the imidazole moiety. While the imidazole can act as either hydrogen bond acceptor (HBA) or hydrogen bond donor (HBD), the protonated imidazole acts exclusively as HBD in T4-imidazole complex. These studies support the earlier observations that the histidine residue at the active sites of the deiodinases play an important role not only in the substrate binding, but also in altering the regioselectivity of the deiodination reactions.

Modeling Type-1 Iodothyronine Deiodinase with Peptide-Based Aliphatic Diselenides: Potential Role of Highly Conserved His and Cys Residues as a General Acid Catalyst.

Type-1 iodothyronine deiodinase (ID-1) catalyzes the reductive elimination of 5'-I and 5-I on the phenolic and tyrosyl rings of thyroxine (T4), respectively. Chemically verifying whether I atoms with different chemical properties undergo deiodination through a common mechanism is challenging. Herein, we report the modeling of ID-1 using aliphatic diselenide (Se-Se) and selenenylsulfide (Se-S) compounds. Mechanistic investigations of deiodination using the ID-1-like reagents suggested that the 5'-I and 5-I deiodinations proceed via the same mechanism through an unstable intermediate containing a Se⋅⋅⋅I halogen bond between a selenolate anion, reductively produced from Se-Se (or Se-S) in the compound, and an I atom in T4. Moreover, imidazolium and thiol groups, which may act as general acid catalysts, promoted the heterolytic cleavage of the C-I bond in the Se⋅⋅⋅I intermediate, which is the rate-determining step, by donating a proton to the C atom.

[Treatment of hypothyroidism in childbearing age]. / Il trattamento dell'ipotiroidismo in età fertile.

Thyroid diseases play a crucial role in conditions of infertility and recurrent miscarriages. We here reported a case of a 34 years old woman, affected by autoimmune thyroiditis with subclinical hypothyroidism. The patient previously had a miscarriage and she was planning another pregnancy. Therefore, she started levothyroxine (LT4) therapy. However, despite optimal TSH levels, she did not get pregnant and she underwent medically assisted procreation. During stimulation, TSH levels slightly increased and LT4 therapy has been increased to reach optimal TSH. After the conception, a monthly biochemical evaluation of the thyroid function test has been performed, with further adjustments of LT4 therapy, until the 30th week of pregnancy. In the post-partum period, LT4 therapy has been reduced and periodical biochemical evaluation has been performed. The case confirms the importance of the thyroid function evaluation during fertile age to obtain optimal TSH levels, with the aims to favorite the conception and to assure a favorable pregnancy outcome.

Levothyroxine Sodium Pentahydrate Tablets - Formulation Considerations.

Even though levothyroxine sodium pentahydrate tablets have been in the market since 1955, there continue to be recalls due to sub potency. We have comprehensively reviewed the factors affecting its stability in solid oral dosage forms. A compilation of marketed formulation compositions enabled the identification of the potential 'problem excipients'. Two excipient properties, hygroscopicity and microenvironmental acidity, appeared to be responsible for inducing drug instability. In drug products, depending on the formulation composition and storage conditions, the pentahydrate can dehydrate to highly reactive levothyroxine sodium monohydrate, or undergo salt disproportionation to the free acid form of the drug. The USP assay method (HPLC based) is insensitive to these different physical forms of the drug. The influence of physical form of levothyroxine on its chemical stability is incompletely understood. The USP has five product-specific dissolution tests reflecting the complexity in its evaluation.

The role of transthyretin in cell biology: impact on human pathophysiology.

Transthyretin (TTR) is an extracellular protein mainly produced in the liver and choroid plexus, with a well-stablished role in the transport of thyroxin and retinol throughout the body and brain. TTR is prone to aggregation, as both wild-type and mutated forms of the protein can lead to the accumulation of amyloid deposits, resulting in a disease called TTR amyloidosis. Recently, novel activities for TTR in cell biology have emerged, ranging from neuronal health preservation in both central and peripheral nervous systems, to cellular fate determination, regulation of proliferation and metabolism. Here, we review the novel literature regarding TTR new cellular effects. We pinpoint TTR as major player on brain health and nerve biology, activities that might impact on nervous systems pathologies, and assign a new link between TTR and angiogenesis and cancer. We also explore the molecular mechanisms underlying TTR activities at the cellular level, and suggest that these might go beyond its most acknowledged carrier functions and include interaction with receptors and activation of intracellular signaling pathways.

Design, synthesis, and biological evaluation of novel bifunctional thyrointegrin antagonists for neuroblastoma.

Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma membrane integrin αvß3. Dual targeting of the NET and integrin αvß3 receptors using a Drug-Drug Conjugate (DDC) might provide effective treatment strategy in the fight against neuroblastoma and other neuroendocrine tumors. In this work, we synthesized three dual-targeting BG-P400-TAT derivatives, dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400-PAT containing di-iodobenzene, di-methoxybenzene, and piperazine groups, respectively. These derivatives utilize to norepinephrine transporter (NET) and the integrin αvß3 receptor to simultaneously modulate both targets based on evaluation in a neuroblastoma animal model using the neuroblastoma SK-N-F1 cell line. Among the three synthesized agents, the piperazine substituted BG-P400-PAT exhibited potent integrin αvß3 antagonism and reduced neuroblastoma tumor growth and cancer cell viability by >90%. In conclusion, BG-P400-PAT and derivatives represent a potential therapeutic approach in the management of neuroblastoma.

Investigating the Influence of Excipients on the Stability of Levothyroxine Sodium Pentahydrate.

A range of tablet excipients were evaluated for their influence on the physical form and chemical stability of levothyroxine sodium pentahydrate (LSP; C15H10I4NNaO4·5H2O). LSP-excipient binary powder blends were stored under two conditions: (a) in hermetically sealed containers at 40 °C and (b) at 40 °C/75% RH. By use of synchrotron X-ray diffractometry, the disappearance of LSP could be quantified and the appearance of crystalline levothyroxine (free acid) could be identified. Under hermetically sealed conditions (40 °C) hygroscopic excipients such as povidone induced partial dehydration of LSP to form levothyroxine sodium monohydrate. When stored at 40 °C/75% RH, acidic excipients induced measurable disproportionation of LSP resulting in the formation of levothyroxine (free acid). HPLC analyses of drug-excipient mixtures revealed that lactose monohydrate, microcrystalline cellulose, and croscarmellose sodium caused pronounced chemical decomposition of LSP. On the other hand, magnesium stearate, sodium stearyl fumarate, and alkaline pH modifiers did not affect the physical and chemical stability of the API following storage at 40 °C/75% RH. HPLC, being a solution based technique, revealed chemical decomposition of the API, but the technique was insensitive to physical transformations. Excipient properties such as hygroscopicity and microenvironmental acidity were identified to be critical determinants of both physical and chemical stability of LSP in a drug product. For drugs exhibiting both physical and chemical transformations, simultaneous solid-state and solution based analyses will enable comprehensive stability evaluation.

New Formulations of Levothyroxine in the Treatment of Hypothyroidism: Trick or Treat?

Background: Levothyroxine (LT4) as a medication is used by up to 5.3% of the adult population. For optimal efficacy, the traditional tablet formulation (LT4tab) requires that patients avoid concomitant ingestion with food, drinks, and certain medications, as well as excellent patient compliance. Some comorbidities influence bioavailability of LT4 and may mandate repeated dose adjustments. Summary: New LT4 formulations (soft gel [LT4soft] and liquid [LT4liq]) containing predissolved LT4 are claimed to improve bioavailability, presumably by facilitating absorption. Thus, these formulations may well be more suitable than LT4tab for patients whose daily requirements are subjected to variations in bioavailability. Here, we review the evidence and indications for use of new LT4 formulations and highlight areas of uncertainty that are worthy of further investigation. While bioequivalence is established for LT4soft and LT4liq administered to healthy volunteers compared with LT4tab in pharmacokinetic (PK) studies, therapeutic equivalence of the new formulations seems to be different in several clinical settings. Some evidence suggests that new formulations of LT4 may mitigate against the strict requirements relating to concomitant ingestion with food, drinks, and certain medications, which apply to traditional LT4 tablets. The principal indication is in selected patients with disease fluctuations and intermittent therapies with interfering medications, where the need for frequent dose adjustments and office visits may be diminished. Whether the use of LT4soft or LT4liq in patients with impaired gastric acid secretion results in better control of hypothyroidism than LT4tab remains unclear. Conclusions: The evidence in favor of using LT4soft and LT4liq in clinical practice over LT4tab is weak, and the underlying putative PK mechanisms unclear. Additional studies to investigate these potential benefits, define the cost-effectiveness, and understand the PK mechanisms involved with new LT4 formulations are needed.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

Thyroxine binding to type III iodothyronine deiodinase.

Iodothyronine deiodinases (Dios) are important selenoproteins that control the concentration of the active thyroid hormone (TH) triiodothyronine through regioselective deiodination. The X-ray structure of a truncated monomer of Type III Dio (Dio3), which deiodinates TH inner rings through a selenocysteine (Sec) residue, revealed a thioredoxin-fold catalytic domain supplemented with an unstructured Ω-loop. Loop dynamics are driven by interactions of the conserved Trp207 with solvent in multi-microsecond molecular dynamics simulations of the Dio3 thioredoxin(Trx)-fold domain. Hydrogen bonding interactions of Glu200 with residues conserved across the Dio family anchor the loop's N-terminus to the active site Ser-Cys-Thr-Sec sequence. A key long-lived loop conformation coincides with the opening of a cryptic pocket that accommodates thyroxine (T4) through an I⋯Se halogen bond to Sec170 and the amino acid group with a polar cleft. The Dio3-T4 complex is stabilized by an I⋯O halogen bond between an outer ring iodine and Asp211, consistent with Dio3 selectivity for inner ring deiodination. Non-conservation of residues, such as Asp211, in other Dio types in the flexible portion of the loop sequence suggests a mechanism for regioselectivity through Dio type-specific loop conformations. Cys168 is proposed to attack the selenenyl iodide intermediate to regenerate Dio3 based upon structural comparison with related Trx-fold proteins.

Partial Dehydration of Levothyroxine Sodium Pentahydrate in a Drug Product Environment: Structural Insights into Stability.

Levothyroxine sodium pentahydrate (LSP; C15H10I4NNaO4·5H2O) gradually loses one molecule of water of crystallization as the water vapor pressure is decreased from 90% to 15% RH (40 °C), a behavior characteristic of nonstoichiometric hydrates. LSP loses four molecules of water of crystallization to form levothyroxine sodium monohydrate (LSM; C15H10I4NNaO4·H2O) under realistic storage conditions (40 °C/0% RH for 3 h). The crystal structure of LSP was determined following which the specimen was partially dehydrated in situ to form LSM. The crystal structure of LSM provided insight into its potential for high reactivity. Thus, its presence in a drug product is undesirable. In LSP-oxalic acid mixtures stored in a hermetic container at 40 °C, there was moisture transfer from drug to excipient. Synchrotron X-ray diffractometry revealed dehydration of LSP resulting in LSM, while anhydrous oxalic acid transformed to its dihydrate. In formulations of LSP, chemical degradation of levothyroxine sodium may be preceded by its partial dehydration.

Thyroxine impregnated chitosan-based dressings stimulate angiogenesis and support fast wounds healing in rats: Potential clinical candidates.

This research paper demonstrates efficacy of chitosan and thyroxine loaded chitosan (CS) dressings for their angiogenic and wound healing potential. The dressings were prepared by freeze gelation method. Thyroxine was loaded by physical adsorption into chitosan membranes. The porosity was analyzed by scanning electron microscopy (SEM) and chemical structures were investigated by Fourier transform infra-red spectroscopy (FTIR). Cell culture studies showed materials were non-toxic and chorioallantoic membranes assay (CAM) confirmed that the thyroxine loaded chitosan stimulated angiogenesis much higher than simple chitosan dressings. In addition, thyroxine loaded dressings showed excellent wound healing potential when tested on full thickness rats wounds. A good epithelialization was obtained along with robust wound closure. Overall, as compared to chitosan, thyroxine containing membranes showed high level of angiogenesis and fast wound healing.

Cyclodextrin-based superparamagnetic host vesicles as ultrasensitive nanobiocarriers for electrosensing.

A carbohydrate-based nanohybrid of superparamagnetic nanoparticles embedded in unilamellar bilayer vesicles of amphiphilic ß-cyclodextrins (magnetic cyclodextrin vesicles, mCDVs) has been engineered as a novel magnetic biorecognition probe for electrosensing. As a proof-of-concept, the synergistic properties of these mCDVs on a magneto nanocomposite carbon-paste electrode (mNC-CPE) have been used for the picomolar determination of thyroxine (T4) as a model analyte (taking advantage of the host-guest chemistry of ß-cyclodextrin and T4), resulting in the most sensitive electrochemical T4 system reported in the literature. Accordingly, a first demonstration of mCDVs as alternative water-soluble magnetic nanobiocarriers has been devised foreseeing their successful use as alternative electrochemical biosensing platforms for the supramolecular trace determination of alternative targets.

Why Were More Than 200 Subjects Required to Demonstrate the Bioequivalence of a New Formulation of Levothyroxine with an Old One?

At the request of French Regulatory Authorities, a new formulation of Levothyrox® was licensed in France in 2017, with the objective of avoiding the stability deficiencies of an existing licensed formulation. Before launching the new formulation, an average bioequivalence (ABE) trial was conducted, having enrolled 204 subjects and selected for interpretation a narrow a priori bioequivalence range of 0.90-1.11. Bioequivalence was concluded. In a previous publication, we questioned the ability of an ABE trial to guarantee the switchability within patients of the new and old levothyroxine formulations. It was suggested that the two formulations should be compared using the conceptual framework of individual bioequivalence. The present paper is a response to those claiming that, despite the fact that ABE analysis does not formally address the switchability of the two formulations, future patients will nevertheless be fully protected. The basis for this claim is that the ABE study was established in a large trial and analyzed using a stringent a priori acceptance interval of equivalence. These claims are questionable, because the use of a very large number of subjects nullifies the implicit precautionary intention of the European guideline when, for a Narrow Therapeutic Index drug, it recommends shortening the a priori acceptance interval from 0.80-1.25 to 0.90-1.11.

Halogen Bonding in Biomimetic Deiodination of Thyroid Hormones and their Metabolites and Dehalogenation of Halogenated Nucleosides.

Thyroid hormones (THs) are key players in the endocrine system and play pivotal roles in carbohydrate and fat metabolism, protein synthesis, overall growth, and brain development. The thyroid gland predominantly produces thyroxine or 3,5,3',5'-tetraiodothyronine (T4) as a prohormone; three isoforms of a mammalian selenoenzyme-iodothyronine deiodinase (DIO1, DIO2 and DIO3)-catalyze the regioselective deiodination of T4 to produce biologically active and inactive metabolites. Whereas DIO1 catalyzes both 5- and 5'-deiodination of T4, DIO2 and DIO3 selectively mediate 5- and 5'-deiodination, respectively. In this review we discuss the regioselective deiodination of THs in the presence of organochalcogen compounds. Naphthalene-based compounds containing sulfur and/or selenium at the peri positions mediate regioselective 5-deiodination of THs, detailed mechanistic studies having revealed that the heterolytic cleavage of the C-I bond is facilitated by the formation of cooperative Se/S⋅⋅⋅I halogen bonds and Se/S⋅⋅⋅Se chalcogen bonds. We also discuss the biomimetic deiodination of several TH metabolites, including sulfated THs, iodothyronamines, and iodotyrosines. A brief discussion on the dehalogenation of halogenated nucleosides and nucleobases in the presence of organochalcogen compounds is also included.

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.

Thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels enhance angiogenesis in in-ovo experiments.

Angiogenesis is one of the most important processes in repair and regeneration of many tissues and organs. Blood vessel formation also play a major role in repair of dental tissue(s) after ailments like periodontitis. Here we report the preparation of chitosan/carboxymethyl cellulose/hydroxyapatite based hydrogels, loaded with variable concentrations of thyroxin i.e., 0.1 µg/ml, 0.5 µg/ml and 1 µg/ml. Scanning electron microcopy images (SEM) showed all hydrogels were found to be porous and solution absorption study exhibited high swelling potential in aqueous media. FTIR spectra confirmed that the used materials did not change their chemical identity in synthesized hydrogels. The synthesized hydrogels demonstrated good bending, folding, rolling and stretching abilities. The hydrogels were tested in chick chorioallantoic membrane (CAM) assay to investigate their angiogenic potential. Hydrogel containing 0.1 µg/ml of thyroxine showed maximum neovascularization. For cytotoxicity analyses, preosteoblast cells (MC3T3-E1) were seeded on these hydrogels and materials were found to be non-toxic. These hydrogels with pro-angiogenic activity possess great potential to be used for periodontal regeneration.

Triazole Modified Tetraiodothyroacetic Acid Conjugated to Polyethylene Glycol: High Affinity Thyrointegrin αvß3 Antagonist with Potent Anticancer Activities in Glioblastoma Multiforme.

Discovery of bioactive molecules that target integrins has implicated their role in tumor angiogenesis, tumor growth, metastasis, and other pathological angiogenesis processes. Integrins are members of a family of cell surface receptors that play a critical role in the angiogenesis process. Tetraiodothyroacetic acid (tetrac), a deaminated derivative of l-thyroxine (T4), is a "thyrointegrin" antagonist that blocks the actions of l-triiodothyronine (T3) and T4 with an interaction site that is located at or near the RGD recognition site identified on integrin αvß3's binding pocket (thyrointegrin αvß3 receptors). We have enhanced the biological activity of a tetrac-based inhibitor via significantly improving its αvß3 receptor binding affinity by introducing a triazole ring on the outer ring of tetrac and covalently conjugating to polymer to increase the product's hydrophilicity via PEGylation. The product, P-bi-TAT, was restricted from nuclear translocation and demonstrated high blood brain barrier permeability and retention in contrast to the non-PEG conjugated derivative. Results of biological activity indicated that this macromolecule new chemical entity P-bi-TAT has greater than 400-fold potent integrin αvß3 affinity versus the parent compound tetrac and has potent anticancer/anti-angiogenesis efficacy against glioblastoma multiforme (GBM). P-bi-TAT administered subcutaneously once daily for 21 days at 1-10 mg/kg mouse body weight resulted in a dose-dependent suppression of GBM tumor growth and viability as monitored with IVIS imaging (P < 0.001). GBM tumors had >95% volume loss and maximal loss of GBM cell viability during the 21 days ON-treatment experiment as well as in the 21 days ON followed by 21 days OFF-treatment experiment (P < 0.001). In conclusion, P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM.