Additional recombinant hormones now approved

Three additional recombinant hormones have recently gained marketing approval: thyroid-stimulating hormone, parathyroid hormone and calcitonin.

Structurally, thyroid-stimulating hormone (TSH or thyrotrophin) is classified as a member of the gonadotrophin family, although functionally it targets the thyroid gland as opposed to the gonads. As with other gonadotrophins, it is a heterodimeric glycoprotein displaying a common a-subunit and a unique P-subunit. The P-subunit shows less homology to that of other members of the group. It consists of 118 amino acids, is particularly rich in cysteine residues and contains one N-linked glycosylation site (Asn 23).

TSH is synthesized by a distinct pituitary cell type: the thyrotroph. Its synthesis and release are promoted by thyrotrophin-releasing hormone (a hypothalamic tripeptide hormone). TSH exerts its characteristic effects by binding specific receptors found primarily, but not exclusively, on the surface of thyroid gland cells. Binding to the receptor activates adenylate cyclase, leading to increased intracellular cAMP levels. Ultimately, this triggers TSH's characteristic effects on thyroid function, including promoting iodine uptake from the blood, synthesis of the iodine-containing thyroid hormones thyroxine (T4) and triiodothyronine (T3) and the release of these hormones into the blood, from where they regulate many aspects of general tissue metabolic activity. Elevated plasma levels of T4 and T3 also promote decreased TSH synthesis and release by a negative feedback mechanism.

TSH is approved for medical use as a diagnostic aid in the detection of thyroid cancer/thyroid remnants in post-thyroidectomy patients. Thyroid cancer is relatively rare, exhibiting highest incidence in adults, particularly females. First-line treatment is surgical removal of all/most of the thyroid gland (thyroidectomy). This is followed by thyroid hormone suppression therapy, which entails administration of T3 or T4 at levels sufficient to maintain low serum TSH levels through the negative feedback mechanism mentioned earlier. TSH suppression is required in order to prevent TSH-mediated stimulation of remnant thyroid cancer cells. The reoccurrence of active thyroid cancer can be detected by administration of TSH along with radioactive iodine. TSH promotes uptake of radioactivity, which can then be detected by appropriate radioimaging techniques.

The commercial recombinant TSH product (tradename, thyrogen; international non-proprietary name; thyrotrophin alfa) is produced in a CHO cell line co-transfected with plasmids harbouring the DNA sequences coding for the a- and P-TSH subunits. The cells are grown in batch harvest animal cell culture bioreactors. Following recovery and concentration (ultrafiltration), the TSH is chromatographically purified and formulated to a concentration of 0.9 mg ml-1 in phosphate buffer containing mannitol and sodium chloride as excipients. After sterile filtration and aseptic filling into glass vials, the product is freeze-dried. Finished product has been assigned a shelf life of 3 years when stored at 2-8 °C.

Human parathyroid hormone (hPTH) is an 84 amino acid polypeptide that functions as a primary regulator of calcium and phosphate metabolism in bones. It stimulates bone formation by osteoblasts, which display high-affinity cell surface receptors for the hormone. PTH also increases intestinal absorption of calcium.

A truncated version of PTH (tradenames Forsteo and Forteo) has been approved for the treatment of osteoporosis in postmenopausal women. This 4 kDa polypeptide is identical in sequence to the N-terminal residues 1-34 of endogenous hPTH, it binds to the native PTH receptor and triggers the same effects. The product is produced in E. coli, purified and presented as a sterile solution containing 250 ^g active substance per millilitre.

Osteoporosis affects some 75 million people in Europe, Japan and the USA combined. The condition is characterized by progressive thinning of the bones, leading to bone fragility and increased risk of fracture. Treatment with Forsteo increases bone mineral density and generally entails daily s.c. injection for several months at dosage levels of 20 ^g active/dose.

Calcitonin is a polypeptide hormone that (along with PTH and the vitamin D derivative, 1,25-dihydroxycholecalciferol) plays a central role in regulating serum ionized calcium (Ca2+) and inorganic phosphate (Pi) levels. The adult human body contains up to 2 kg of calcium, of which 98 per cent is present in the skeleton (i.e. bone). Up to 85 per cent of the 1 kg of phosphorus present in the body is also found in the skeleton (the so-called mineral fraction of bone is largely composed of Ca3(PO4)2, which acts as a body reservoir for both calcium and phosphorus). Calcium concentrations in human serum approximate to 0.1 mg ml-1 and are regulated very tightly (serum phosphate levels are more variable).

Calcitonin lowers serum Ca2+ and Pi levels, primarily by inhibiting the process of bone resorption, but also by decreasing resorption of Pi and Ca2+ in the kidney. Calcitonin receptors are predictably found primarily on bone cells (osteoclasts) and renal cells, and generation of cAMP via adenylate cyclase activation plays a prominent role in hormone signal transduction.

Calcitonin is used clinically to treat hypercalcaemia associated with some forms of malignancy and Paget's disease. The latter condition is a chronic disorder of the skeleton in which bone grows abnormally in some regions. It is characterized by substantially increased bone turnover rates, which reflects overstimulation of both osteoclasts (promote bone resorption, i.e. degradation of old bone) and osteoblasts (promotes synthesis of new bone).

In most mammals, calcitonin is synthesized by specialized parafollicular cells in the thyroid. In sub-mammalian species, it is synthesized by specialized anatomical structures known as ulti-mobranchial bodies.

Calcitonin produced by virtually all species is a single-chain 32 amino acid residue polypeptide, displaying a molecular mass in the region of 3500 Da. Salmon calcitonin differs in sequence from the human hormone by nine amino acid residues. It is noteworthy, however, as it is approximately 100-fold more potent than the native hormone in humans. The higher potency appears due to both a greater affinity for the receptor and a greater resistance to degradation in vivo. As such, salmon, as opposed to human calcitonin, is used clinically. Traditional clinical preparations were manufactured by direct chemical synthesis, although a recombinant form of the molecule has now gained marketing approval. The recombinant calcitonin is produced in an engineered E. coli strain. Structurally, salmon calcitonin displays C-terminal amidation. A C-terminal amide group (—CONH2) replacing the usual carboxyl group is a characteristic feature of many polypeptide hormones. If present, it is usually required for full biological activity/stability. As E. coli cannot carry out post-translational modifications, the amidation of the recombinant calcitonin is carried out in vitro using an a-amidating enzyme, which is itself produced by recombinant means in an engineered CHO cell line. The purified, amidated finished product is formulated in an acetate buffer and filled into glass ampoules. The (liquid) product exhibits a shelf life of 2 years when stored at 2-8 °C.

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