BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 7 ppt

inactivated, thus promoting glycogen breakdown. The rate of gluconeogenesis is also stimulated by the inactivation of pyruvate kinase and simultaneous activation of fructose 1,6 bisphos- phatase. Hypoglycaemia remains the most frequent complication of insulin administration to diabetics. It usually occurs due to (a) administration of an excessive amount of insulin; (b) administration of insulin prior to a mealtime, but with subsequent omis sion of the meal; or (c) due to increased physical activity. In severe cases, this can lead to loss of consciousness and even death. Although it may be treated by oral or i.v. administration of glucose, insulin-induced hypoglycaemia is sometimes treated by administration of glucagon. Glucagon is also used medically as a diagnost ic aid during certain radiological examinations of the stomach, small and large intestine where decreased intestinal motility is advantage ous (the hormone has an inhibitory effect on the motility of the smooth muscle lining the walls of the gastrointestinal tract). 322 BIOPHARMACEUTICALS Figure 8.9. Overview of the extraction of pancreatic cells from a human donor pancreas. Typically, a pancreas will house approximately 1 million such cells, of which around 40% are recovered by this procedure Traditionally, glucagon preparations utilized therapeutically are chromatographically purified from bovine or porcine pancreatic tissue (the structure of bovine, porcine and human glucagon is identical, thus eliminating the possibility of direct immunological complications). Such commercial preparations are generally formulated with lactose and sodium chloride and sold in freeze-dried form; 0.5–1.0 units of glucagon (approximately 0.5– 1.0 mg freeze-dried hormone) are administered to the patient by s.c. or i.m. injection. More recently, glucagon preparations produced via recombinant means have also become available. ‘GlucaGen’ is the trade name given to one such product, produced by Novo Nordisk using an engineered Saccharomyces cerevisiae strain. Upstream processing (aerobic batch-fed fermentation) is followed by an upward adjustment of media pH in order to dissolve precipitated product (glucagon is insoluble in aqueous-based media between pH 3–9.5) . This facilitates subsequent removal of the yeast by centrifugation. Glucagon is then recover ed and purified from the media by a series of further precipitation as well as high-resolution HORMONES OF THERAPEUTIC INTEREST 323 Figure 8.10. Initiation of a metabolic response to the binding of glucagon to its receptor. (1)¼ glucagon cell surface receptor; (2)¼G protein; (3)¼ adenylate cyclase (see text for further detail) chromatographic steps. Eli Lilly also produces a recombinant glucagon product using an engineered E. coli strain. HUMAN GROWTH HORMONE (hGH) Human growth hormone (hGH , somatotrophin; Figure 8.11) is a polypeptide hormone synthesized in the anterior pituitary. It promotes normal body growth and lactation and influences various aspects of cellular metabolism. Mature hGH contains 191 amino acid residues and displays a molecular mass of 22 kDa. It also contains two characteristic intra-chain disulphide linkages. hGH mRNA can also undergo alternate splicing, yielding a shortened GH molecule (20 kDa), which appears to display biological activities indistinguishable from the 22 kDa species. hGH displays significant, although not absolute, species specificity. GHs isolated from other primates are the only preparations biologically active in humans (this precluded the earlier use of bovine/porcine preparations for medical use in humans). Growth hor mone synthesis and release from the pituitary is regulated by two hypothalamic factors: growth hormone releasing hormone (GHRH, also known as growth hormone releasing factor, GHRF, or somatorelin) and grow th hormone release inhibiting hormone (GHRIH) or 324 BIOPHARMACEUTICALS Figure 8.11. 3-D structure of human growth hormone. Photo from Chantalat et al. (1995), by courtesy of the Protein Data Bank: http://www.rcsb.org/pdb/ somatostatin (Table 8.5). Furthermore, while GH directly mediates some of its biological actions, its major influence on body growth is mediated indirectly via IGF-1, as discussed below. GHRH, GHR IH, GH and IGF-1 thus form a hormonal axis, as depicted in Figure 8.12. Growth hormone releasing factor (GHRF) and inhibitory factor (GHRIF) GHRF and GHRIF are peptides secreted by hypothalamic neurons termed ‘neuroendocrine transducers’ (the name is apt, as these interface between the nervous and endocrine systems). The factors that regulate their secretion are poorly understood but probably involve both nerve impulses originating from within the brain and feedback mechanisms, possibly involving pituitary hormo nes. 37, 40 and 44 amino acid GHRF variants have been identified. All can promote GH release from the pituitary, an activity which apparently resi des in the first 29 amino acid residues of these molecules. Administration of GHRH to GH-deficient individuals generally promotes modest increases in GH secretion, thereby increasing growth rate. Hypothalamic GHRIF is a cyclic 14 amino-acid peptide, although a 28 amino acid form is also found in some other tissues. GHRIF inhibits the release not only of GH but also of tyrotrophin and corticotrophin from the pituitary, and insulin and glucagon from the pancreas. It can also regulate the level of duodenal secretions. The GH receptor GH induces its characteristic biological effects by binding to a specific cell surface receptor. The human receptor is a single chain 620 amino acid transmembrane polypeptide. Sequence analysis indicates it is a member of the haemopoietic receptor superfamily (which includes receptors for several ILs, GM-CSF and EPO). X-ray crystallographic analysis shows that GH binds simultaneously to the extracellular domains of two receptor molecules, effectively promoting receptor dimerization (Figure 8.13). The exact molecular detail of the subsequent signal transduction events remain to be determined. However, ligand binding does induce receptor autophosphorylation, as well as phosphorylation of additional cellular substances and protein kinase C activation. HORMONES OF THERAPEUTIC INTEREST 325 Table 8.5. Some factors known to affect the rate of secretion of GH. Most of these factors influence GH release indirectly by affecting the rate and level of secretion of GHRH and/or GHRIH Factors promoting increased GH secretion Factors promoting decreased GH secretion Starvation Obesity Sleep Elevated blood glucose Stress b-Adrenergic antagonists Exercise b-Adrenergic agonists Low blood glucose Several amino acids Glucagon Vasopressin a-Adrenergic agonists b-Adrenergic antagonists Soluble GH-binding proteins (GH BPs) are also found in the circulation. In humans, these GHBPs are generated by enzymatic cleavage of the integral membrane receptor, releasing the GH-binding extracellular domain. In rodents, however, the GHBPs are derived from alternatively cleaved GH-receptor mRNA. The exact physiological role of these binding proteins remains to be elucidated. In serum, GH binds to two such GHBPs, an action which prolongs the hormone’s plasma half-life. 326 BIOPHARMACEUTICALS Figure 8.12. Overview of the mechanisms by which GH induces its biological effects and how its secretion from the pituitary is regulated Biological effects of GH GH primarily displays an anabolic activity. It partially stimulates the growth of bone, muscle and cartilage cells directly. Binding of GH to its hepatic receptor results in the synthesis and release of insulin-like growth factor (IGF-1), which mediates most of GH’s growth-promoting activity on, for example, bone and skeletal muscle (Chapter 7). The major effects mediated by hGH are summarized in Table 8.6. A deficiency in the secretion of hGH during the years of active body grow th results in pituitary dwarfism (a condition responsive to exogenous hGH administration). On the other hand, overproduction of hGH during active body growth results in gigantism. hGH overproduction after primary body growth has occurred results in acromegaly, a condition characterized by enlarged hands and feet, as well as coarse features. HORMONES OF THERAPEUTIC INTEREST 327 Figure 8.13. Growth hormone found in the circulation is generally bound to GH-binding proteins. Binding to the cell surface receptor promotes receptor dimerization and phosphorylation and hence activation. This leads to the phosphorylation of various cystolic protein substrates, which mediate intracellular effects of the hormone Table 8.6. Some of the major biological effects promoted by growth hormone. While many of these are direct, other effects are mediated via IGF-1 (Chapter 7) Increased body growth (particularly bone and skeletal muscle) Stimulation of protein synthesis in many tissues Mobilization of depot lipids from adipose tissue (lipolytic effect) Elevation of blood glucose levels (anti-insulin effect) Increase of muscle and cardiac glycogen stores Increased kidney size and enhanced renal function Reticulocytosis (increased reticulocyte production in the bone marrow) Therapeutic uses of GH GH has a potentially wide range of therapeutic uses (Table 8.7). To date, however, its major application has been for the treatment of short stature. hGH extracted from human pituitary glands was first used to treat pituitary dwarfism (i.e. caused by sub-optimal pituitary GH secretion), in 1958. It has subsequently proved effective in the treatment of short stature caused by a variety of other conditions, including: . Turner’s syn drome; . idiopathic short stature; . chronic renal failure. The use of hGH extracted from the pituitaries of deceased human donors came to an abrupt end in 1985, when a link between treatment and Creutzfeldt–Jakob disease (CJD, a rare, but fatal, neurological disorder) was discovered. In this year, a young man who had received hGH therapy some 15 years previously died from CJD, which, investigato rs concluded, he had contracted from infected pituitary extract (CJD appears to be caused by a prion). At least an additional 12 CJD cases suspected of being caused in the same way have subsequently been documented. Fortunately, several recombinant hGH (rhGH) preparations were coming on- stream at that time (Table 8.8), and now all hGH preparations used clinically are derived from recombinant sources. Currently, in excess of 20 000 people are in receipt of rhGH therapy. rhGH was first produ ced in E. coli in the early 1980s. The initial recombinant preparations differed from the native human hormone only in that they contai ned an extra methionine residue (due to the AUG start codon inserted at the beginning of the gene). Subsequently, a different cloning strategy allowed production in E. coli of products devoid of this terminal methionine. In vitro analysis, including tryptic peptide mapping, amino acid analysis and comparative immunoassays, show the native and recombinant form of the molecule to be identical. Clinical trials in humans have also confirmed that the recombinant version promotes identical biological responses to the native hormone. rhGH was first purified (on a lab scale) by Genentech scientists using the strategy outlined in Figure 8.14. A somewhat similar strategy is likely used in its process-scale purification. Recombinant hGH (rhGH) and pituitary dwarfism Various studies have confirmed that rhGH promotes increased linear growth rates in children suffering from pituitary dwarfism (classical growth hormone deficiency). Dosages are generally 328 BIOPHARMACEUTICALS Table 8.7. Some actual or likely therapeutic uses for hGH. Refer to text for details Treatment of short stature caused by GH deficiency Treatment of defective growth caused by various diseases/medical conditions Induction of lactation Counteracting ageing Treatment of obesity Body building Induction of ovulation administered on a weekly basis by i.m. or s.c. injection. Duration of treatment typic ally varies from 6 months to 2 years, although on occasion administration has continued for up to 4 years. Increased growth rates are generally observed, although the extent varies with, for example, the recipient’s age at onset of treatment, sex and baseline growth rates. rhGH-induced growth HORMONES OF THERAPEUTIC INTEREST 329 Table 8.8. Recombinant human growth hormone (rhGH) preparations approved for general medical use Product (trade name) Company Indication Humatrope Eli Lilly hGH deficiency in children Nutropin Genentech hGH deficiency in children Nutropin AQ Schwartz Pharma AG Growth failure, Turner’s syndrome BioTropin Biotechnology General hGH deficiency in children Genotropin Pharmacia and Upjohn hGH deficiency in children Saizen Serono Laboratories hGH deficiency in children Serostim Serono Laboratories Treatment of AIDS-associated catabolism/wasting Norditropin Novo Nordisk Treatment of growth failure in children due to inadequate growth hormone secretion Figure 8.14. Production of recombinant human growth hormone (rhGH) in E. coli (as an intracellular protein). Subsequent to fermentation, the cells are collected by centrifugation or filtration. After homogenization, nucleic acids and some membrane constituents are precipitated by the addition of polyethyleneimine. Ammonium sulphate precipitation of the supernatant concentrates the crude rhGH preparation. Chromatographic purification follows, as illustrated acceleration is most notable during the initial stages of treatment, with relative effect decreasing with time. In most cases, growth hormone treatment ensures a final body height several centimetres greater than would otherwise be attained in recipients. Idiopathic short stature and Turner’s syndrome In many cases, a root cause for slower than normal growth rate in children of short stature is not immediately obvious (idiopathic short stature). Endogenous GH levels are often considered to be within a normal range (although there may be changes in its pusatile secretion patterns). A host of clinical trials have shown, however, that rhGH administration can increase the growth rate of many children with idiopathic short stature. Several trials lasting up to 3 years showed that, although the response was most dramatic during the first year, even during year three, mean growth rates were over 3 cm/year greater than expected. Turner’s syndrome is a genetic defect that affects females (sufferers carry only one of the usual two X chromosomes). These individuals are infertile, often show developmental defects, mental retardation and short statu re. Virtually all clinical trials involving Turner’s syndrome patients confirm that administration of GH significantly increases growth velocity, indicating its therapeutic usefulness in these cases. Metabolic effects of hGH The twin metabolic effects of hGH in pro moting increased body protein synthesis and increased lipolytic activity suggest a role for the hormone in influencing body lean mass/fat composition. Attention in this regard has focused upon treating obesity and burns, as well as counteracting some of the effects of old age. Clinical studies in dieting obese people suggest that GH treatment (typically for 3–12 weeks) did not promote reduction of body fat levels any faster than in persons’ dieting, but without treatment. A lipolytic effect was, however, observed in obese people who were not subject to caloric restriction during rhGH treatment. A future role for this hormone in treating obesity is, therefore, far from certain. Clinical trials have also revealed a role for rhGH in the treatment of severe burns, particularly in children. The fear and emotional trauma (as well as physical damage) associated with receiving a severe burn triggers a neurological and immune-mediated response termed the stress response. This is characterized by: . protein catabolism; . loss of lean body mass; . increased metabolic rate; . futile substrate cycling and lipolysis; . elevated body temperature. rhGH treatment is aimed largely at slowing/preventing elevated protein catabolism. Initial trials in burn patients showed that GH administration reduced protein loss by 50% compared to (untreated) controls. Subsequent GH studies in children with massive burning (over 50% of total body surface), revealed accelerated wound healing, particularly at the skin graft donor site. This, in turn, facilitated further skin grafting within shorter time periods, thus reducing the time to close the burn wound. On the basis of such results, GH may well play a future expanding role in burn care management. 330 BIOPHARMACEUTICALS The production of GH is age-modulated. The highest production levels are recorded immediately after birth, with a second increase noted at puberty. GH secretion decreases steadily after age 40, and this decline is likely linked to age-associated decreased muscle, bone and skin mass, all of which contribute to age-associated frailty. In recent years, several pilot clinical trials, assessing the effects of GH administration to ageing adults, have been carried out. Typically, trial duration is 4–6 months. A 7% increase in lean body mass and skin thickness, along with a 14% drop in body fat, was observed in one trial, although results recorded in other trials were less striking. More detailed clinical trials and cost:benefit analysis must be carried out in order to fully assess the potential of GH to counteract some of the effects of ageing in the elderly population. GH, lactation and ovulation Recombinant bovine GH (bovine somatotrophin, BST), has been used for a number of years to boost milk yield in dairy cattle (by up to 20%). More recent studies in monkeys showed that GH administration increased milk yields as well as promoting a slight increase in milk fat levels. This suggested that GH might be beneficial in the treatment of human lactation failure. Treatment of a small group of breast-feeding women with rhGH for 1 week has been shown to increase milk production (although it had no effect on the milk’s nutritional composition, including fat levels). GH also appears to impact upon ovarian physiology, mainly although not exclusively through IGF-1. IGF-1 stimulates replication of cultured mammalian granulosa cells (see gonadotrophin section) and potentiates FSH action on the ovary. While GH appears not to play an essential role in ovulation or fertility, it seems to act synergistically with gonadotrophins and other reproductive hormones. One clinical trial, involving women subfertile due to the lack of endogenous FSH secretion, showed that co-treatment with rhGH decreased the quantity of exogenous FSH required to induce ovulation. The range of potential applications of rhGH in clinical medicine continues to grow. The use of GH is also facilitated by the absence of any serious side effects in most instances. Although its efficacy in promoting growth in persons of short stature is beyond doubt, more convincing clinical evidence is required before its approved clinical applications are expanded further. THE GONADOTROPHINS The gonadotrophins are a family of hormones for which the gonads represent their primary target (Table 8.9). They directly and indirectly regulate reproductive function and, in some cases, the development of secondary sexual characteristics. Insufficient endogenous production of any member of this family will adversely affect reproductive function, which can be treated by administration of an exogenous preparation of the hormone in question. Most gonadotrophins are synthesized by the pituitary, although some are made by reproductive and associated tissues. Follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) Follicle stimulating hormone (FSH) and luteinizing hormone (LH) play critical roles in the development and maintenance of male and particularly female reproductive function (Box 8.4). Human chorionic gonadotrophin (hCG), produced by pregnant women, plays a central role in maintaining support systems for the developing embryo during early pregnancy. All three are HORMONES OF THERAPEUTIC INTEREST 331 [...]... Amino acid sequence Buserelin acetate C2H5-NH-Pro-Arg-Leu-Ser-Tyr-Ser-Trp-His-5oxo pro j butyl Goserelin acetate NH2CO(NH)2-Pro-Arg-Leu-Ser-Tyr-Ser-Trp-His-5oxo pro j butyl Leuprorelin acetate Nafarelin acetate C2H5-NH-Pro-Arg-Leu-D-Leu-Tyr-Ser-Trp-His-5oxo pro NH2 -Gly-Pro-Arg-Leu-D-Ala-Tyr-Ser-Try-His-5oxo pro j naphthyl NH2 -Gly-Pro-Arg-Leu-Gly-Tyr-Ser-Trp-His-5oxo pro GnRH slow clearance rate appears... factor-b (TGF-b) family of proteins The inhibins are heterodimers consisting of a- and b-polypeptide subunits Activins are bb dimers The mature form of the a-subunit is termed ac, and it consists of 134 amino acid residues Two closely related (but structurally distinct) b-subunit forms have been characterized: bA and bB These exhibit in 338 BIOPHARMACEUTICALS excess of 70 % amino acid homology, and differ... hormones, containing an identical a-polypeptide subunit and a unique bpolypeptide subunit which confers biological specificity to each gonadotrophin In each case, both subunits of the mature proteins are glycosylated Human FSH displays four N-linked (asparagine or Asn-linked) glycosylation sites, located at positions Asn 52 and 78 of the asubunit and Asn 7 and 24 of the b-subunit Some 30% of the hormone’s... gestation, and reach maximum size at about day 70 , after which they steadily regress They synthesize high levels of PMSG and secrete it into the blood, where it is detectable between days 40 and 130 of gestation Box 8.6 The androgens and oestrogens The androgens and oestrogens represent the major male and female sex hormones, respectively The testicular Leydig cells represent the primary source of androgens... Human growth hormone and extracellular domain of its receptor; crystal structure of the complex Science 255, 306–312 Gibson, F & Hinds, C (19 97) Growth hormone and insulin-like growth factors in critical illness Intens Care Med 23(4), 369– 378 Hathaway, D (2002) Growth hormone: challenges and opportunities for the biotechnology sector J Anti-aging Med 5(1), 57 62 Neely, E (1994) Use and abuse of human... Caeruloplasmin Haptoglobin Type 1-1 Type 2-1 Type 2-2 Transferrin Haemopexin 0.1–0.6 151 1.0–2.2 1.6–3.0 1.2–2.6 2.0–3.2 0.5–1.0 100 200 400 76 .5 57 b2-microglobulin 0.002 11.8 g-Globulins Transthyretin 7. 0–15.0 0.1–0.4 150 55 Function Osmoregulation transport Retinol transport Binds/transports thyroxine Cortisol and corticosterone transport Copper transport Binds and helps conserve haemoglobin Iron... N-linked glycosylation also takes place, as does intra-chain disulphide bond formation The a- and b-subunits combine non-covalently and appear to be stored in secretory vesicles, separately to those containing LH While free a-subunits are also found HORMONES OF THERAPEUTIC INTEREST Box 8.4 333 An overview of the female reproductive cycle The human female reproductive (ovarian) cycle is initiated and. .. Med 45, 4 07 420 Piwien-Pilipuk, G et al (2002) Growth hormone signal transduction J Pediat Endocrinol Metab 15(6), 77 1 78 6 Sharara, F & Giudice, K (19 97) Role of growth hormone in ovarian physiology and onset of puberty J Soc Gynaecol Invest 4(1), 2 7 Simpson, H et al (2002) Growth hormone replacement therapy for adults: into the new millennium Growth Hormone IGF Res 12(1), 1–33 Gonadotrophins and TSH... BIOPHARMACEUTICALS Matzuk, M et al (1992) a-Inhibin is a tumor-suppressor gene with gonadal specificity in mice Nature 360, 313–319 McCann, S et al (2001) Control of gonadotrophin secretion by follicule stimulating hormone-releasing factor, luteinizing hormone-releasing hormone and leptin Arch Med Res 32(6), 476 –485 Rao, C & Sanfilippo, J (19 97) New understanding in the biochemistry of implantation — potential... direct roles of luteinizing hormone and human chorionic gonadotropin Endocrinologist 7( 2), 1 07 111 Risbridger, G et al (2001) Activins and inhibins in endocrine and other tumors Endocr Rev 22(6), 836–858 Schwartz, N (1995) The 1994 Stevenson Award lecture Follicle-stimulating hormone and luteinizing hormone: a tale of two gonadotrophins Can J Physiol Pharmacol 73 , 675 –684 Siarim, M & Krishnamurthy, H . glycosylated. Human FSH displays four N-linked (asparagine or Asn-linked) glycosylation sites, located at positions Asn 52 and 78 of the a- subunit and Asn 7 and 24 of the b -subunit. Some 30% of the hormone’s. into the endoplasmic recticulum. N-linked glycosylation also takes place, as does intra-chain disulphide bond formation. The a-andb-subunits combine non-covalently and appear to be stored in secretory. species). Furthermore, it displays both FSH-like and LH-like biological activities. This glycoprotein hormone is a heterodimer, composed of an a- and b-subunit, and approximately 45% of its molecular