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Biochemical History of Tetrac

Tetraiodothyroacetic acid (tetrac) is a naturally occurring derivative of thyroid hormone (L-thyroxine, T4). It accounts for less than 1 % of circulating thyroid hormone. Inside human cells, tetrac is a low-grade thyromimetic, that is, it has low-potency actions that resemble those of 3, 5, 3′-triiodo-L-thyronine (T3), the most active form of thyroid hormone [1]. For 30 or more years, tetrac has been known to be taken up by pituitary cells that secrete thyrotropin (TSH) and to inhibit endogenous human TSH release by the pituitary gland [2]. This action has been considered potentially useful in the clinic in the setting of TSH-dependent thyroid cancer.

Tetrac was also found to compete with T4 for thyroid hormone-binding sites on human serum pre-albumin (transthyretin, TTR) [3], but not for sites on human thyroxine-binding globulin (TBG), the principal transport protein for iodothyronines in human serum. These observations caused us in the 1980s to test tetrac for its ability to block the nongenomic actions of thyroid hormone that we demonstrated to exist in human mature red blood cell (RBC) membranes [4, 5] and intracellular membranes, such as those of the sarcoplasmic reticulum [6]. These actions included Ca2+ transport by calmodulin-responsive Ca2+-ATPase [7]. Such effects were ‘nongenomic’ because they were independent of the nuclear thyroid hormone receptor (TR) and gene transcription [8]. Tetrac indeed inhibited such actions of T4 and T3 and thus became a probe for certain actions of thyroid hormone that were initiated at the plasma membrane, although the cell surface receptor site for the hormone was not identified until 2005 [9].

Studies focused on the plasma membrane receptor for thyroid hormone on integrin αvβ3 revealed that this receptor mediated the pro-angiogenic action of T4 and T3, and that tetrac was anti-angiogenic in terms of its ability to block this action [10]. As will be discussed below, tetrac and a novel nanoparticulate formulation of the agent have anti-angiogenic activity that transcends the inhibition of thyroid hormone-binding at the integrin. Tetrac formulations also have anti-proliferative activity against tumor cells and have been shown to have chemosensitizing and radiosensitizing effects in cancer cells [10, 11].

Integrin αvβ3 Contains a Thyroid Hormone-Tetrac Receptor

J.J. Bergh et al. in 2005 [9] described the existence of a thyroid hormone-tetrac receptor on plasma membrane integrin αvβ3. This heterodimeric integrin is generously expressed on rapidly dividing blood vessel cells and by cancer cells, enabling scanning technology focused on the integrin to detect tumors [12]. The fit of unmodified tetrac into the hormone-binding groove in the extracellular domain [11, 13] of the integrin permits the agent to block the binding of T4 and T3 and inhibit actions of these agonist forms of thyroid hormone on cancer cell proliferation and cancer-related angiogenesis as discussed in Chap. 4. The integrin is found on virtually all cancer cells and reads the presence of specific extracellular matrix proteins that are relevant to tumor cell migration and tumor mass formation.

In the absence or presence of T4 and T3, however, tetrac has been found to disrupt via αvβ3 a number of other functions or events that are important to cancer cells. These functions include regulation of cell division, local stimulation of angiogenesis, chemoresistance and resistance to radiation [10, 14]. It was surprising to find that, acting at the cell surface, tetrac coherently interfered with expression of differentially regulated genes whose products included the cyclins and thrombospondin 1 [15]. The thrombospondin 1 gene is usually silent in tumor cells because it suppresses angiogenesis. Tetrac also blocked the actions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [16], released by cancer cells to promote angiogenesis in an autocrine manner. The mechanism of tetrac involved here is thought to involve disorganization of crosstalk between the integrin and nearby receptors for VEGF [17] and bFGF [10, 18] and inhibition of local release of bFGF [18]. The hormone analogue was found to cause retention by cancer cells of traditional chemotherapeutic agents to which the cells previously showed resistance [19]. These agents included doxorubicin, etoposide, and cisplatin. Tetrac was also shown to inhibit the ability of cancer cells to repair double-strand DNA breaks that radiation induces [20, 21], thus radiosensitizing these cells. Finally, expression of genes for cytokines involved in inflammation and relevant to inflammation-associated cancer was shown to be blocked by tetrac [15].

The foregoing describes the anti-cancer and anti-angiogenic features of tetrac manifested at integrin αvβ3. However, unmodified tetrac is taken up by cells and has, within the cell, low-potency thyromimetic activity [1, 2]. This can include proliferative—rather than anti-proliferative—behavior (H.Y. Lin: unpublished). While the algebraic sum of these anti-proliferative and proliferative effects favors anti-cancer activity, it is desirable to reformulate tetrac as a nanoparticle whose activity is exclusively at the integrin (Section “Nanoparticulate Tetrac (Nanotetrac)”, below).

Nanoparticulate Tetrac (Nanotetrac)

S.A. Mousa and co-workers have constructed an approximately 250 nm poly(lactic-co-glycolic acid) (PLGA) formulation in which a limited number of tetrac moieties are covalently bound to and protrude from a PLGA nanoparticle [22, 23] (Fig. 10.1). Binding of tetrac to the PLGA is via an ether bond at the hydroxyl on the outer ring of tetrac (position 4) to a linker that is amide-bonded to the PLGA. Nanotetrac was designed to limit tetrac to the extracellular space on the basis of the size of the nanoparticle and to provide optimized exposure of the biphenyl structure and acetic acid side chain of the inner ring to the receptor site on αvβ3. Restriction of the molecule to the extracellular space has been verified [24].

Fig. 10.1
figure 00101

Chemical structures of unmodified tetraiodothyroacetic acid (tetrac) (a) and nanoparticulate tetrac (nanotetrac) (b). An ether bond involving the outer ring hydroxyl group joins tetrac to a linker molecule which, in turn, is attached by an imbedded amide bond to the nanoparticle. Multiple tetrac moieties are bonded to the surface of the PLGA, enabling access of tetrac to its receptor groove in the extracellular domain of integrin αvβ3

Full size image

Interestingly, the reformulation of tetrac as a nanoparticle has conferred two other qualities on the molecule. First, nanotetrac is up to 10-fold more potent than unmodified tetrac. The basis for this may reflect interaction(s) of the polymer (PLGA) chain, away from the tetrac moieties, with the ‘legs’ of the extracellular domain of αvβ3, favoring ‘on’ kinetics. Another possibility is that the nanoparticulate tetrac ligand induces a long-lived change in the conformation of the αvβ3 that persists after ligand dissociation and modulates activity of the integrin [25]. Second, and acting at the integrin receptor, nanotetrac induces a pattern of cancer survival gene expression that is somewhat different from that of unmodified tetrac [15]. There is 80–85 % congruence of gene expression caused by nanotetrac and tetrac. Examples of the disparities are down-regulation by nanotetrac in cancer cells of expression of the epidermal growth factor receptor (EGFR) gene, down-regulation of the majority of the members of the Ras-oncogene family, and up-regulation of the apoptosis inhibitor MCL1 (myeloid cell leukemia sequence 1); these genes are unaffected by tetrac.

The difference in potency of tetrac and nanotetrac is readily seen in the comparison of efficacy of the compounds in human cancer xenografts in the nude mouse. Within 3 days of initiation of treatment, the two formulations cause a greater than 50 % decrease in the vascular supply of the xenografts, but this anti-angiogenic effect can be obtained with a dose of nanotetrac that is 10-fold less than that of tetrac.

Triiodothyroacetic Acid (Triac)

Triac (3, 3′, 5-triiodothyroacetic acid), the deaminated derivative of T3, can reproduce the inhibition of actions of T4 and T3 at the αvβ3 thyroid hormone-tetrac receptor (H.Y. Lin: unpublished), but has not as yet been reformulated as a nanoparticle. These actions include inhibition of the pro-angiogenic activity of thyroid hormone studied in the chick chorioallantoic membrane (CAM) model. Inside the cell, triac is thyromimetic. It is thermogenic [26], has been shown in human subjects to have agonist thyroid hormone effects on liver and bone that are augmented compared to T4 [27], and has been postulated to be a primordial form of thyroid hormone [28].

The unmodified compound is mentioned here for several reasons. Triac may be purchased over-the-counter in several European countries as a dietary supplement (tiratricol). The availability of this iodothyronine raises the possibility that anti-angiogenic side effects may be encountered in users of the agent. Such effects have not been reported, but it is unlikely that they have been anticipated, e.g., in the setting of wound-healing.

Potential Applications of Tetrac and Nanotetrac in Angiogenesis

Tetrac and Anti-angiogenesis in the Setting of Cancer

The clinical desirability of establishing control of angiogenesis in and about tumors is obvious and the feasibility of such control became apparent with the work of Judah Folkman and colleagues [29, 30]. In the context of cancers, local angiogenesis that supports tumor biology may be the result of more than a single vascular growth factor. These growth factors include VEGF, bFGF and other proteins [31]. Erythropoietin (EPO) may also be included among factors that enhance tumor-relevant angiogenesis [32].

In contrast to several of the anti-angiogenic agents used clinically (see below) that are designed to inhibit actions of single, specific vascular growth factors, tetrac and nanotetrac have been shown, in the absence or presence of agonist thyroid hormone (T4 or T3), to antagonize actions of multiple growth factors. These include VEGF, bFGF [17, 33], PDGF (S.A. Mousa: unpublished), EGF (S.A. Mousa: unpublished) and EPO [34]. This plural effect explains, at least in part, the rapid decrease in vascularity so far encountered in human tumor xenografts in mice exposed to systemic tetrac and nanotetrac [35–38] and the resultant decrease in volume of xenografts.

There are multiple mechanisms by which thyroid hormone analogues inhibit (nanotetrac or tetrac) or enhance (T4 and T3) the activities of various vascular growth factors. First, vascular growth factor gene expression in tumor cells or endothelial cells may be enhanced by thyroid hormone [18]. Second, iodothyronines may increase release of the growth factor(s) by the secreting cell, e.g., bFGF [18]. Third, crosstalk between the integrin and adjacent vascular growth receptors is well-described. The crosstalk may involve signal transducing biochemistry within or immediately below the plasma membrane. For example, inhibition by tetrac of mitogen-activated protein kinase (MAPK) activity alters activity of bFGF and other factors. Hormonal effects may in other cases depend upon interactions of the extracellular domains of the receptor(s) and the integrin [39] that could involve, in the case of VEGFR, the inhibition by tetrac formulations of dimerization of the growth factor receptor or obscuring of one or more of its immunoglobulin (Ig)-like domains [39]. Fourth, tetrac decreases abundance of angiopoietin-2 (Ang-2) mRNA in endothelial cells, but does not affect accumulation of Ang-1 mRNA [17]. Ang-2 protein production in tumor vasculature anticipates or synergizes with vascular growth factor action to support tumor angiogenesis [40], whereas the Ang-1-Tie 2 system is a blood vessel-stabilizing pathway. This differential effect of tetrac with regard to angiogenesis is consistent with discrete, selective actions of tetrac or nanotetrac in cancer cells on elaboration of certain interleukins (see below) or of endogenous inhibitors or enhancers of apoptosis [15]. Fifth, tetrac can induce the expression of thrombospondin 1, an endogenous suppressor of angiogenesis that is almost invariably unexpressed in cancer cells [22]. Finally, T4 and T3 and tetrac may positively, in the case of the former, and negatively, in the case of tetrac, affect endothelial cell motility (S.A. Mousa: unpublished) that is important to neovascularization.

Currently available clinically are pharmaceuticals that affect single vascular growth factors. Bevacizumab (Avastin®) and ranibizumab (Lucentis®) are monoclonal antibodies to VEGF, developed as anti-angiogenic agents. Administered parenterally—intravenously, or, in the case of eye disease, intra-ocularly—these humanized antibodies are unquestionably effective in clinical disease settings in which VEGF or VEGF-A are contributory pathophysiologic factors. There are several subtypes of VEGF; VEGF-A [41], for example, is a form frequently released locally by tumor cells and which induces a porous vasculature. It is clear that the application of bevacizumab to the cancers for which it is approved is primarily adjunctive [42, 43] and is not curative. It has not so far been practical to produce for clinical use multi-monoclonal antibody preparations that are directed at more than a single vascular growth factor.

Bevacizumab is applied with U.S. Food and Drug Administration (FDA) approval to management of several forms of cancer and used without FDA approval in settings of unwanted angiogenesis in the absence of cancer, e.g., diabetic retinopathy [44]. Ranibizumab is an anti-angiogenic drug marketed for management of a form of retinal macular degeneration that may lead to loss of vision [45]. Bevacizumab and ranibizumab are discussed in more detail in Chaps. 12 and 13.

Application of Tetrac/Nanotetrac to Clinical Conditions of Excessive Angiogenesis Not Associated with Malignancy

Skin Disorders

Skin redness (erythema) in specific settings such as acne rosacea [46] or psoriasis [47] may be VEGF-dependent. Systemic anti-VEGF treatment for cancer has been reported to induce remission of cutaneous manifestations of psoriasis coincident with the cancer [47]. Conventional treatments for both of these conditions are inconsistently effective and systemic bevacizumab (anti-VEGF) treatment is too expensive to consider in most patients with skin disease.

Topical application of tetrac in a vehicle that permits penetration of the agent to involved blood vessels in the dermis and limits systemic absorption of the hormone analogue has been proposed in management of rosacea and awaits clinical trial.

Retinopathy

Tetrac and nanotetrac have been tested for efficacy in the newborn mouse oxygen-induced retinopathy (OIR) model [34]. It was an effective intravitreal or intraperitoneal preventive intervention. Similar results in this model have been obtained by S.A. Mousa et al. (unpublished). Yoshida and co-workers also found that the effects of VEGF and erythropoietin (EPO) on retinal endothelial cells in vitro were blocked by tetrac and nanotetrac [34]. As noted earlier, EPO is another factor supporting angiogenesis whose activity is minimized by tetrac.

VEGF antibody administered by the intravitreal route has been examined for its effectiveness in the clinical setting of diabetic retinopathy [44, 48]. The substantial experience is largely favorable. The increased intravitreous and circulating levels of VEGF seen in proliferative retinopathy are both decreased by bevacizumab [49]. The agent may, however, increase the risk of retinal fibrosis [50].

Inflammation

Analysis of the gene signature of tetrac treatment in relatively chemoresistant human breast cancer cells [15] has revealed an important set of actions on inflammation-related genes [51]. For example, five of six differentially regulated interleukin genes—including IL-6 and IL-1α—are down-regulated by the compound and a suppressor of cytokine signaling (SOCS4) gene is up-regulated. Expression of interferon response pathway genes and chemokine genes is decreased. Such effects may be relevant to inflammation-associated cancers, as well as to other inflammatory states. The selectivity of the tetrac/nanotetrac effect on gene expression is shown in the case of IL-11, whose gene product is a proliferative factor for hematopoietic stem cells and whose expression is enhanced, rather than decreased, by nanotetrac and tetrac.

Additional Actions of Tetrac and Nanotetrac

Independently of their actions of angiogenesis, tetrac and nanotetrac inhibit proliferation of a variety of cancer cell lines in vitro, as noted above, and chemosensitize [19] and radiosensitize [20, 21] tumor cells. The mechanism of chemosensitization by tetrac is incompletely understood, but may involve acidification of the cancer cell by inhibition of the Na+/H+ exchanger [52]. Consequent increase in extracellular pH (pHe) or decrease in intracellular pH may be associated with decreased activity of the cancer cell P-glycoprotein (P-gp) or other multidrug resistance (MDR) pumps that export chemotherapeutic agents [53]. Tetrac may also interfere with stimulation of the cancer cell kinases relevant to MDR pump activation [54].

As noted earlier, the mechanism of radiosensitization of cancer cells involves inhibition of repair of double-strand DNA breaks induced by radiation [20]. Under normal conditions, this repair process is highly efficient in tumor cells.

Conclusions

Acting at the thyroid hormone-tetrac receptor on the plasma membrane, tetrac and nanotetrac have potentially important anti-angiogenic effects. The receptor is located on integrin αvβ3, a highly plastic protein that is capable of transducing interactions of its extracellular domain with extracellular matrix proteins and small molecules, like thyroid hormone, into important intracellular events. Tetrac and its nanoparticulate formulation disrupt the communication between the integrin and nearby receptors for VEGF, bFGF, and other polypeptide factors important to neovascularization. That is, these thyroid hormone derivatives affect the activity of a number of pro-angiogenic proteins, in addition to blocking the angiogenic activity of agonist thyroid hormones, T4 and T3. Tetrac and nanotetrac also stimulate expression of the endogenous angiogenic suppressor gene, thrombospondin 1. The actions of tetrac and nanotetrac are generally coherent, that is, the effects that they have on expression of multiple genes, on crosstalk between integrin αvβ3 and receptors such as VEGFR and bFGFR, and on vascular growth factor release by tumor cells fit an anti-angiogenic pattern. That the agents distinguish between endothelial cell Ang-1 and Ang-2 also supports coherence of the anti-angiogenic pharmacology of tetrac and nanotetrac.

These agents have potential applications as inhibitors of tumor-related angiogenesis and of angiogenesis that is not associated with malignancy, but contributes to clinically significant skin disorders, to vascular proliferation of the retina, and neovascularization associated with inflammatory states.