The Diagnostic and Therapeutic Importance of Human Growth Hormone Isoforms

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55 Recent Patents on DNA & Gene Sequences 2011, 5, 55-67 The Diagnostic and Therapeutic Importance of Human Growth Hormone Isoforms

Marina María de Jesús Romero-Prado1,* and Noa Beatriz Martín-Cófreces2

1Cardiovascular Unit Research, Department of Physiology, CUCS-Universidad de Guadalajara, 2Department of Inter- cellular Communications in Inflammatory Response, Centro Internacional de Investigaciones Cardiovasculares. Ma- drid, España

Received: November 27, 2010; Accepted: December 20, 2010; Revised: February 9, 2011

Abstract: The study of human growth hormone isoforms has conduced to the elaboration of patents related to very important items: codifying and regulatory sequences, production of the protein at large-scale, modifications to prolong half-life as monomer, dimer and fusion protein for treatments directed to growth-associated diseases. The designed methodologies directed to the identification and quantification of hGH are beside the formers establishing very important basis of patented sources that can be used for a specific and opportune diagnosis and treatment of biological abnormalities or undesirable effects when these growth hormones are involved. Keywords: Growth hormone isoforms, modified growth hormone, growth hormone-related peptides, growth hormone detection.

INTRODUCTION of gene expression and the necessity of other regulatory regions that ameliorate the level of expression (i. e. HSs III The pituitary and placental growth hormones (hGH-N/- and V) [2,3]. Furthermore, the tissue specificity and central V) belong to the superfamily of cytokines which includes location of HS-I,II-GH for the acetylable chromatin 32kb- interleukins, growth factors, cytokines, leukemic inhibitory domain suggested a model in which determinants in the HS factor, and neurotrophic factors. The two human GH genes I,II target the initial binding of histone acetyltransferase share high homology in both coding and proximal promoter (HAT) coactivator complexes that would spread bidirection- regions (>90%) with each other and with placental lactogen ally, encompassing the downstream GH-N promoter (see genes (hPLs). The hGH/PL gene family has been evolution- Fig. 1, Acetyl H3-H4 and double-headed arrow) [1,4]. In the ary expanded by repeated duplications into a 48 kb cluster proposed model for pituitary, the high specificity is attrib- located in the chromosomal region 17q22-q24. The expres- uted to the transcription factor Pit-1 that binds to its binding sion of human hGH-N is principally limited to the somatot- sites in both HS-I,II and proximal promoter regions, interact- rope and somatolactotrope cells of the anterior pituitary and ing with itself and with other transcriptional machinery pro- accounts for 3% of the total pituitary mRNA, whereas hGH- th teins. In this way Pit-1 is considered as the major transcrip- V gene is expressed in placenta from the 9 week of the tional factor that regulate the hGH-N expression in a long- pregnancy until the birth [1]. range manner [3,5,6]. The HSs III to V are common and The Locus Control Region (LCR) for hGH/PL is formed necessary to the placental genes expression. Furthermore, the by five Hypersensitive sites (HS) which control the expres- P-elements, present in each distal promoter of placental sion of hGH-N/-V. Experimental data have revealed two hGH/PL locus gene, have been found to be acetylated re- partially overlapping sets of HSs located between 15 and 32 gions (see Fig. 1, light gray ovals), although a specific inter- kb 5’ to the transcription initiation site of the hGH-N gene action between HSs III to V and P-elements has not been corresponding to two of the five HSs, HS I and II (HS-I,II), demonstrated; both pituitary and placenta genes share the that are strictly necessary for the pituitary-specific expres- HSs III and V for its correct tissue specific expression [4]. sion of hGH-N gene. The HS-I,II acts by driving a genome The structure and regulatory regions of human hGH/PL insertion-independent expression and as a potent enhancer- locus are depicted in Fig. (1). The expression of hGH-N, like sequence as indicated by the proper developmental in- hGH-V and hPL-2 genes has also been detected in other duction of the hGH transgene driven in vivo by these se- tissues such as in peripheral blood monocyte cells (PBMCs) quences in somatotrope cells [2]. However, gigantism was [7] and in IM-9 human lymphocytes [8]. observed in HS-I,II-GH transgenic mice, suggesting an es- The hGH-N polypeptides are single domain proteins cape of the transgene from the normal physiological control composed of a helical bundle composed of four alpha-helices

(A, B, C and D), and a mini-domain (E) which are connected *Address correspondence to this author at the Unidad de Investigación by four interhelical loops (AB, BC, CD and DE); two disul- Cardiovascular, Departamento de Fisiología, Centro Universitario de Cien- fide bridges (cysteines 53-165 and 182-189 of the mature cias de la Salud, Universidad de Guadalajara, C.P. 44340, Jalisco. México; chain) can further stabilize GH structure (Fig. 2A). The pres- Tel: +52.33.1058.5200; Ext: 3657; Fax: +52.33.3617.3499; E-mail: [email protected] ence of disulfide bridges and the amino acids codified by

Pit-1 HS V IV III II I GH-N PL-1 PL-2 GH-V PL-3 SCN4A CD79B

-54 kb 0 +48 kb

PITUITARY Acetyl H3-H4 P-elements

PLACENTA

Fig. (1). The hGH/PL locus. Diagram showing the five genes and its regulatory regions on proximal promoters, as well as long distance regulators (LCR). For more information see the text (Adapted from [1]. A

-S-S- N-ter C-ter

A B C D E

Codifying B Domains -26 -1 +1 a MATGSRTSLLLAFGLLCLPWLQEGSA  +32 +46 +71 b FLQ NPQTSLCFSESIPTPSNREETQQKS

c NLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMG

d RLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSV EGSCGF

+32 +46 Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln GAA GAA GCC TAT ATC CCA AAG GAA CAG AAG TAT TCA TTC CTG CAG Fig. (2). Linear structure and complete amino acids sequence of hGH-N (22K-GH isoform). A, Linear structure of hGH depicting alpha- helix domains (A-E), inter-loop domains and Cysteines (gray circles) as well as disulfide bonds (positions 53-165 and 182-189). B, Amino acid sequence showing codifying domains, corresponding to 22 kDa-GHN immature isoform (217 amino acids): (upper) Codifying domains: (a) Signal peptide (in light gray) -26 to -1 include exons 1 and 2; (b) Exon 3 (in gray): arrow heads () appoint to skipping sites for exon 3 (+32 to +71) (17.5K-GH isoform); inside this sequence is appointed the alternative splicing site ( ) that produces the deletion of +32 to +46 amino acids (20K-GH isoform); (c) and (d) represent exons 4 and 5 respectively. The high-bold amino acids (+1 to +43) correspond to peptide amino acids (+1 to +43) corresponds to peptide described by Bevec (2010) as therapeutic besides natriuretic peptide for different kind of diseases (PAT. US2010/0184680A1); (bottom) Detail of exon 3 showing the consensus nucleotides (underlined) that participate forming the lariat-shape in alternative splicing that produces 20K-GH isoform (based on [20 &21]). exon 3 of hGH-N appear to contribute to increased stability plex activates a signaling cascade that is shown in Fig. (3) and protein half-life in the bloodstream, and favoring a high- [11]. The proteolytic product of hGHR, the soluble receptor affinity binding with the GH receptor (hGHR) (GH-BP), acts as a mediator for somatogenesis [12a-12b]. (WO2006048777A2; EP2241574A1) [9,10]. The complete Proliferation defects as well as growth deficiency form sequence is shown in Fig. (2B). One hGH-N ligand molecule part of a very wide set of genetic or acquired diseases. In the binds to two bound-membrane receptor molecules; this com- case of syndromes that course with growth defects, many 57 Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 Romero-Prado and Martín-Cófreces

pY pY pY JAK2 pY pY JAK2 pY pY pY

pYpY

pY pYpY pY

Transcription of target genes (IGF-I, IGFBP-3, ALS, SOCS) Fig. (3). hGH signalling pathway. The hGH has two receptor binding sites: Site I (S-I includes the C-ter of helix A and parts of helix A and the A-B loop) and Site II (S-II includes amino terminal region of helix A and a portion of helix C). Binding of GH to its receptor (GHR) is sequential: Site I binds to one GHR and then to a second GHR by Site II [89]. The dimerization (1 GH:2 GHR molecules) and activation of the intracellular signalling pathways leads to cellular responses to the hormone (see figure 2). Once the signal is initiated, JAK2 phosphorylation occurs as well as several cytosolic proteins being the STAT5b the most important in gene regulation expression of GH-dependent genes (based on [11]). IGF-I, Insulin-like growth factor-1; IGFBP-3, Insulin-like factor binding protein-3; ALS, acid-labile subunit; SOCS, Sup- pressor of Cytokine signalling. research groups have been looking for symptomatic treatments or for compensating some maturation systemic defects 2. hGH-N AND hGH-V GENE EXPRESSION and, principally, in CNS. In the same sense, the search for Since Li and Evans (1944) discovered the growth hor- hGH therapeutics without secondary or undesirable effects as mone from bovine pituitary gland, many works had been well as their frequency and/or mode of administration have done depicting the biochemical properties, physiology, ge- been the subject of study for several years. This review de- netic and metabolic regulation as well as its therapeutic im- picts an approach to some of the research lines directed to portance for the treatment of diseases that are companied by the use of hGH for therapeutic and diagnostic objectives. growth-derivate problems [15,16]. However, the difficulty in understanding GH regulation is not only based on endoge- 1. HUMAN GROWTH HORMONES: PITUITARY nous factors. It is of relevance that the nature of hormonal AND PLACENTA and physiological responses has been shown to be important for hGH-N in different in vitro and systemic experimental The human Growth Hormone / Placental Lactogen gene systems. The complex nature of the pulsatile secretion of the cluster (hGH/PL), located in chromosome 17q22-q24, is hormone, the time duration of the event, the interference or formed by five highly homologous genes that have evolved antagonism of free fatty acids, glucose or hormones such as through a series of gene duplication events from an ancestral GH gene. The pituitary-expressed growth hormone (hGH-N) glucocorticoids, or the thyroid hormone, age and the physical activity practiced as well as other endocrine systems that is the first of the five genes, close to the regulatory regions regulate the GH secretion (see Table 1), have been made (see above), containing 48 ALU repeats and ALU-like se- using in vitro, in vivo systems and in wild type and trans- quences along the locus. Despite their structural similarity, genic animals that have been well described by others [12b, placental genes are specifically expressed in the syncytiotro- 16-18, 90]. Besides these factors, a possible crosstalk be- phoblast (SCT) on the villous layer of the placenta. During the time, two principal isoforms, 22-kDa and 20-kDa, de- tween the GHs, insulin and Insulin-like growth factor (IGF-I) receptors may be relevant for their physiological functional- tected for each hGH-N and hGH-V hormones, were the rea- ity and for brain development [11, 16, 18]. son for the study of the different expression patterns that they have shown, whose origin was specific splicing variants from intronic regions, or proteolytic degradation as occurs 3. BIOLOGICAL RELEVANCE OF HUMAN for 16kDa-GH isoforms (see below). The 22-kDa growth GROWTH HORMONE ISOFORMS hormone protein differs in 13 amino acids in its mature form 3.1. Pituitary Growth Hormone Patents [13,14]. Although these isoforms have been the focused of several studies, there are actually more isoforms that are The GH regulates growth through hypertrophy, hyperpla- depicted in the Fig. (3). sia, or both, as a result of tissue differentiation, cell prolifera The Diagnostic and Therapeutic Importance of Human Growth Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 58

Table 1. Proteins that Regulate Secretion and Biological Effects of GH*

Abbreviation Effects on GH

Growth Hormone- GH-RF (GH- The mature GH-RF is constituted by 108 amino acids that undergoes proteolytic processing and posttranslational Releasing Factor RH) modification to two forms, one of 40 amino acids -GH-RF(1-40)-, and the second, of 44 amino acids with an ami- dated N-ter -GH-RF(1-44Nter)-. The GH-RF is synthesized in basal hypothalamus and periventricular zone; following secretion into the portal blood, GH-RF reacts with receptors on the membrane of pituitary somatotrophs to stimulate GH secretion and transcription of new GH mRNA.

Somatostatin (Soma- SRIF The SRIF is produced in hypothalamus and suppresses GH release by two ways: 1, by inhibiting the GH-RF pro- totropin Release- duction and release of hypothalamic cells; 2, by suppressing GH release from the pituitary following binding to Inhibiting Factor) receptors on somatotrophs in the anterior pituitary.

Estrogens & Estrogen E, ER The GH promoter region has binding sites for ER. It has been demonstrated the role of Estrogens and to a lesser receptors extent androgens in the stimulation of GH secretion from the pituitary; Estrogens also modify end-organ respon- siveness to GH in a dose-dependent way. Hormone replacement in patients with Turner's syndrome stimulates growth synergistically when estrogens and GH are used in cotreatment. In adult women, endogeouns opioids may also mediate the elevation of serum GH levels in response to estradiol.

Insulin Ins Intravenous infusion of Ins (0.1 units/kg) to induce hypoglycemia as well as high glucose concentrations (5mM) that produce an increase of Ins in a brief period of time, suppress GH secretion for 3-5 hours post glucose infusion.

Insulin-like growth IGF-I, IGF-II Based on animal models, the mechanisms that have been implicated in IGF-I and IGF-II inhibition of GH secretion factor & binding & IGFBPs include processes as secretion and gene transcription of GH. In the same way, GH is necessary for stimulation of proteins IGFs gene expression and longitudinal growth. IGF-I, IGF-binding protein -3 (IGFBP-3) and acid-labile subunits (ALS) comprise the major circulating IGF-I complex. Newborn mice homozygous for a targeted disruption on the gene encoding for IGF-I and IGF-II exhibit a severe growth deficiency. IGF receptor-deficient mice are severely growth-retarded, exhibit multiple growth-and differentiation-dependent abnormalities, and die shortly after birth.IGFBP-2, -5, and -6 have been described as present in microglia and astrocytes of CNS with possible role in development and brain repair.

GH Receptor GH-R The cDNA encodes a mature 620-amino acid polypeptide that is expressed in lymphocytes, liver, kidney, brain and other organs. As few as 54 amino acids, of the 350 amino acid cytoplasmic domain, are capable of transmitting a growth hormone proliferative signal in a promyeloid cell line through a phosphorylation signalling cascade, or through Protein kinase C activation that triggers a rapid induction of early response genes c-fos and c-jun.

GH-Binding Protein GH-BP GH-BP participates transporting GH through the circulation. There are two GH.BPs: the 61 kDa GH-BP1 (high- affinity) that bears structural/immunological resemblance to the extracelluar domain of the GH-R; and the 100 kDa GH-BP2 that exhibits a higher specificity for the 20K-GHV.

Transcription Factors Pit-1, Prop-1 These factors, Pit-1 and Prophet of Pit-1 (Prop-1) are the major regulators of the tissue-specific expression of GH in (TFs) Pituitary- pituitary. Other TFs involved belong to POU homeobox factors (Oct-1, Unc, etc). specific *Based on references 11, 15-18 tion, and protein synthesis. GH requires their membrane system (US4601980A [19]). The second isoforms has 176 bound receptor (GH-R) to activate signal transduction (see amino acids due to a deletion of internal residues 32-46 and Fig. 3) that produces systemic biological effects by regulat- its molecular weight is 20.28 kDa (20K-GHN) [20]. The ing the expression of a variety of different kind of proteins, expression of the 20K-GHN isoform is 5-10% of the level of lipoproteins, lipoprotein receptors as well as hormones. The 22K-GHN isoform in the anterior pituitary gland, and repre- target tissues for GH are cartilage and bone (growth plate), sents 2-7% of the total circulating hGH. A third isoforms has adipose tissue (differentiation from pre-adipocyte to adipo- been detected as the principal hGH expressed in pathological cyte), brain (growth, development and myelination), muscle conditions (genetic GH deficiency type II, GHD-II), which and liver. The relevance of GH in other systems has been has missed exon 3 –residues 32 to 71- and produces a protein depicted before (see Table 1) [15-18]. About the GH gene with a molecular mass of 17.48 kDa (17.5K-GHN) [17]. All and protein product, hGH-N has two major mature isoforms: the isoforms share a signal peptide of 26 amino acids, which the main product of 191 amino acids with a molecular was considered initially as an artifact, in the first complete weight of 22.13 kDa (22K-GHN) that forms a single chain sequence published [21]. N-glycosilation of hGH-N has been with two internal disulfide bridges that link positions 53 and used to prolong its half-life. In this case, recombinantly ex- 165, and positions 182 and 189 (see Fig. 2). The sequence pressed human growth hormone variants carrying additional corresponding to mature 22K-GHN isoform includes amino N-glycosylations, said variant comprising an amino acid acids 24 to 191 which has been produced in a microbial sequence comprising one or more N-glycosylation motifs 59 Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 Romero-Prado and Martín-Cófreces

(N-X-S/T), which are not present in the wild type hGH quencing the growth hormone genes as well as its large-scale (WO2009/156511A2 [22]). Indeed, specific glycosylations purification (see above) (US004670393 [13], US4446235 patterns for hGH-V have been established (see below). [32]). In 1997, Uchida and coworkers reported the production of authentic 20K-GH in Escherichia coli from which Different isoforms have been described as consequence highly purified recombinant 20K-GH was obtained at Mitsui of proteolytic partial degradation (5 and 12 kDa). The di- Chemicals Inc. A study group of 20K-GH isoform compris- meric form referred as MER-45-kDa hGH (45K-GHN), ing basic and clinical investigators was formed; they under- described for first time by Lewis in 1977, has been detected took a number of novel studies of this GH isoform in man. in pituitary extract as 1% of total serum circulating hGH [13a] and represents the 2%-5% of the hGH-N in human The summary of this work includes some of the main findings on recombinant 20K-GH, especially the data about its pituitary adenomas [12b,17]. The 45K-GHN biochemical biological effect, measurement, regulation of secretion and properties, as result of 22K-GHN isoform dimerization, had its application in the detection of GH doping abuse [33]. The been analyzed and the model proposed for the extraordinary large-scale production of 20K-GHN was proposed to im- stability is attributed to a special topology of disulfide link- prove body composition, to stimulate lipolysis and/or to ages that are dissymmetric or that result in formation of catenane structures [23]. The biological potential of the very increase the serum IGF-I concentration, as a source of hGH agent for replacement therapies in adults, particularly for stable dimer MER-45K-hGHN (also called “big big GH”) growth hormone-deficient adult patients (see below) has been analyzed and found to be a poor competitor for (US6399565B1 [34]). other GH receptor species monomer in microsomes from liver (rat) and mammary gland (rabbit), compared to monomeric hGH. Furthermore the hGH-N dimer does not promote 3.2. Placental Growth Hormone Patents proliferation of T47-D human breast cancer cells but instead The placental protein hGH-V is produced during preg- partially blocks their proliferation. The authors of these stud- nancy by the 9th gestational week and totally substitutes ies conclude that the dimerization of hGH through unusually plasmatic levels of hGH-N at about the 20th week [35], being stable disulfide bonds forms a quaternary arrangement that able to bind either to soluble or plasma membrane receptors attenuates hGH’s receptor-binding and growth- promoting [16, 17]. The established placental choriocarcinoma cell lines activities [24]. Finally, a new isoforms that is processed by have been a very useful tool for the study of the metabolism, alternative splicing involving 3’ and 5’ of the exons 2 and 3 gene expression regulation and physiological phenomena respectively, has been claimed by Fagan and coworkers both ex vivo or in vivo due to the systems used to analyze the (2005) and called INSP101 (PT1569961E [25]) and hGH/PL locus. The knowledge about the somatotrope prop- INSP105 (US007605129B2 [26]) (see below). erties of 22K-GHV come from studies made by Selden and In humans, hGH is secreted in a pulsatile manner into the coworkers [36]. They designed a fusion plasmid for the blood. By infusion of recombinant human GH (rhGH-N) in complete codifying region of hGH-V (2.1 kb) linked to the healthy volunteers (n=24) for three periods of 180 min each, metallothionein 1 promoter (Mt1p). The hGH-V construct the half-life of GH was estimated as 13.0±3.8, 18± 5.3 and was compared with hGH-N one in transiently transfected L1 20.5±4.5 min for bolus of 60, 180 and 310 mg/m2 [27]. An mouse cell line, and using a transgenic model. hGH-V interesting aspect is the biochemical dimorphism shown by showed significant differences from hGH-N respectively in hGH-N: women show higher GH-N levels than men both in the following characteristics: i) an isoelectric point (pI) of controls and acromegalic patients (P<0.001) [28]. The pat- 8.8 for hGH-V deamidated products vs. a values ranging terns of secretion for 20-kDa and 22-kDa hGH are sexually from 8.35 to 8.5 for hGH-N; ii) differential extent of gene differentiated at the basal levels in acromegalic patients vs expression under identical cell culture conditions (hGH-N controls because in part of estradiol, but not testosterone 10-times more than hGH-V); and iii) the ability to cross- concentrations [29]. Some of the principal biological proper- react with three different antibodies. Indeed, the principal ties and patented use of hGH for diagnostics and therapeutics molecules recovered from supernatant of L1 cell cultures, directed to growth-associated diseases are enumerated in isolated with exclusion chromatography, were monomeric Table 2. Wang and coworkers [30] demonstrated the equipo- hGH-N (70-75%) and hGH-V (25%), and the reminder as a tent osteo-anabolic effects of 20K- and 22K-hGH on several dimer of 44 kDa. Finally, they showed the expression of functions such as the induction of cell-growth promoting, the hGH-V in tissues different from pituitary or placenta in a synthesis of Osteocalcin, Al-P activity and IL-6 release from transgenic mouse model. When expressed in the liver and primary cultures of Human Osteoblast-like cells (HOB), other locations, hGH-V caused a 1.4 to 1.9 fold increase in suggesting that this phenomenon could be happening in animal size. The variants hGH-V2(88) and hGH-V3(53) are human subjects that are treated with the 20K-GHN. These claimed by Rosen et al. (US5597709 [37]) as generated by effects of 20K-GH on human bone metabolism as well as an alternative splicing on a common pre-mRNA where exon-3 increase in IGF-I and IGFBP-3, were corroborated in human may use two alternate sites to fuse with the donor site of beings. Besides a significant decrease in the percentage of exon-2. The deleted regions by these alternative splicings body fat (the body fat mass, BFM), there were very impor- corresponds to amino acids 33 to 51 for hGHV-2(88) and 33 tant changes in abdominal subcutaneous and visceral fat to 72 for hGHV-3(53) (see Fig. 4). Rosen et al. areas, without affecting lipid profile in a significant manner (US5962411A1 [38]) claimed for these discoveries as well [31]. The development of molecular tools based on recombi- as for the bacterial expression, purification and use of these nant technologies established the basis for cloning and se- isoforms for diagnostic preventative and therapeutic

The Diagnostic and Therapeutic Importance of Human Growth Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 60

Table 2. Patent Registration of GH Products: Isoforms and Variants, Modified Proteins, Therapeutic and Diagnostic Uses

Concept Publication Number Title Inventor(s) Publication Date

hGH-N variants: identifi- US2003153003 (A1); [53] Growth hormone variants; Method WELLS JAMES A [US]; CUNNINGHAM 8/14/03 cation of active domains, for identifying active domains and amino BRIAN C [US] amino acid residues, acid residues in polypeptides and hor- derivatives and related mone variants; hormones

AU2008203051 (A1) [54] Derivatives of Growth Hormone COX III GEORGE N [US] 31/07/2008 and Related Proteins

US2010029563 (A1), [55a-55b] Derivatives of growth hor- COX III GEORGE N [US]; DOHERTY 04/02/2010, US2010266529 (A1) mone and related proteins, and methods DANIEL J [US] 20/10/2010; of use thereof.

PT1568772 (E) [56] Human Growth Hormone variants CLARK ROSS G [US]; LOWMAN HENRY 4/14/10 B [US]; WELLS JAMES A [US]; OLSON KENNETH [US]; FUH GERMAINE G [US]; CUNNINGHAM BRIAN C [US]

Novel antiangiogenic WO9851323 (A1), [57-60] Novel antiangiogenic peptide WEINER RICHARD I [US]; MARTIAL 22/04/2004, peptide agents: therapeu- US2003186382 (A1), agents and their therapeutic and diagnos- JOSEPH A [BE]; STRUMAN INGRID [BE]; 24/04/2008, tic and diagnostic use US2004077054 (A1), tic use TAYLOR ROBERT [US]; BENTZIEN 02/10/2003, US2008096795 (A1). FRAUKE [US] 19/11/1998

Therapeutic uses of hGH- WO2005018659 (A2), [61-63] Somatogenic and non- GLUCKMAN PETER [NZ]; GILMOUR 02/02/2006, N and hGH-V isoforms WO2006012525 (A2), diabetogenic therapy using a 20kda ROBERT S [GB]; VICKERS MARK H 14/12/2006, US2006281675 (A1) placental variant of growth hormone [NZ]; BREIER BERNHARD H H [NZ] 03/03/2005

WO2009/033680 A2, [64-65] Use of a peptide as therapeutic BEVEC DORIAN [DE]; CAVALLI FABIO 19/03/2009, US 2010/0184680 A1 agent. Therapeutic uses of B-type Natri- [CH]; CAVALLI VERA [CH]; BACHER 22/07/2010 uretic peptide and human Growth Hor- GERALD [DE] mone 1-43

US2008249020 (A1) [66] Human Growth Hormone Treatment BRIGHT GEORGE [US]; AN BOB ROB- 10/9/08 Methods ERT [US]

US2010137200 (A1) [67] Methods for treatment of Growth CLARK ROSS G [NZ]; CLARK GILLIAN 6/3/10 disorders [NZ]

PT1140149 (E) [68] Use of human Growth Hormone to GIANNI ALESSANDRO MASSIMO [IT] 1/31/07 increase the number of circulating CD34+ cells, intended for reconstitution of the hematopoietic and immune system following myeloablative or antiblastic therapies, by transplantation, re-infusion or engraftment.

Modified growth hor- WO2009/133137A2 [70] Pegylated recombinant human HERSEL ULRICH; RAU HARALD; 11/5/09 mones:amino acid or growth hormone compounds WEGGE THOMAS; LESSMANN TOR- polypeptide modifications, BEN; SPROGOE KENNETT; KINDER- pegylation, N-glycosilated MANN SUSANNE; RASMUSSEN GRE- GH; fusion proteins THE NORSKOV

WO2006069220(A2) [71] Modified human Growth Hormone SIM BEE-CHENG; LITZINGER D; CHO 4/29/10 HO SUNG; DANIEL THOMAS O; DI- MARCHI RICHARD; HAYS ANNA- MARIA; WILSON TROY

IL133974 (A) [73] Cysteine variant of Growth Hor- SHLOMO COHEN & CO. (BOLDER BIO- 5/17/10 mone TECHNOLOGY) UAE

AU2008201889 (A1) [74] Growth hormone fusion protein ARTYMIUK PETER; ROSS RICHARD; 5/22/08 SAYERS JON 61 Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 Romero-Prado and Martín-Cófreces

(Table 2) Contd….

Concept Publication Number Title Inventor(s) Publication Date

US2010197582 (A1) [75] Growth Hormone fusion proteins ROSS RICHARD [GB]; ARTYMIUK PE- 8/5/10 TER [GB]; SAYERS JON [GB]

WO2010029107 (A1) [76] Growth Hormone conjugate with JOHANSEN NILS LANGELAND [DK] 3/18/10 increased stability

US2007/0299006 [77] Recombinant fusion proteins to BALLANCE DAVID JAMES [GB] 12/27/07 Growth Hormone and serum albumin

Secretagogues or inhibi- US2009048340 (A1) [79] Growth Hormone secretion regula- ONO KAORI [JP]; YONEZAWA TOMO- 2/19/09 tors of GH tor HIRO [JP]; MORI MASATO [JP]

PT1077941 (E) [80] Compounds with Growth Hormone ANKERSEN MICHAEL [DK]; HANSEN 7/1/10 releasing properties THOMAS KRUSE [DK]

US2010010467 (A1) [81] Growth Hormone releasing hor- DRAGHIA-AKLI RUXANDRA [US]; 1/14/10 mone treatment to decrease cholesterol KHAN AMIR S [US]; FIORROTO MARTA levels L [US]

MX2009008561 (A) [82] Method of treating cell proliferative POLVINO WILLIAMS J [US] 1/15/10 disorders using growth hormone secretagogues.

US2010203060 (A1) [83] Inhibitors for growth hormone and LOBIE PETER E [NZ] 8/12/10 related hormones, and methods of use thereof

hGH detection systems WO9736929 (A1); [84-85] Monoclonal antibodies binding HANSSON YNGVE ELOF [SE], BARON 10/9/97 for diagnose and/or CA2250047 (A1) human growth hormone (hGH) LEONOR KREMER [ES], ALONSO CAR- research LOS MARTINEZ [ES]

US5945296 (A) [86] Monoclonal antibody HANSSON YNGVE ELOF [SE], BARON 8/31/99 LEONOR KREMER [ES], ALONSO CAR- LOS MARTINEZ [ES]

AU712583 (B1) [87]A method of measurement of human HASHIMOTO YOSHIHIDE, KONO 11/11/99 growth hormone NAOKO, MAKINO TADASHI, IRIE MI- NORU

US2009305300 (A1) [88] Methods and kits to diagnose LARSEN FINN [GB] 12/10/09 Growth Hormone deficiency purposes in certain human diseases and for the design of and evidenced that this last isoform was unable to activate specific antibodies against the described isoforms. lactogenic response in Nb2-11C, Baf/3 and Baf 3 LP cells stably transfected with either the rat or the human prolactin The 22K-hGH-V/GHV-1 protein shows differences in receptor (r/hPRLR). There are more biochemical properties 13-15 amino acids compared to 22K-hGH-N [35]. The other hGH-V known isoforms are 26K-GHV/GHV-2, 24K- of hGH-N isoforms and methods of GH isoforms cloning, isolation and characterization that have been patented [52- GHV/GHV-3, and 20K-GHV/GHV-4 [39,40]. The 26K- 56] (see Table 2). hGHV/hGHV-2 is a 230-amino acid membrane-bound protein that includes the amino acids codified by the D intron showing a complete divergence in the carboxy-terminus 4. PATENTS RELATED TO CLINICAL AND THERA- when compared to hGH-N. The hGH-V3 isoform lacks of PEUTIC RELEVANCE OF GROWTH HORMONES four bp forming a 219-amino acid protein with a molecular 4.1. Clinical Relevance of Growth Hormones weight of 24 kDa [41]. Two more isoforms have been described: hGH-V4, that is equivalent in molecular weight to The high homology between both growth hormone genes 20K-GH pituitary isoforms [42] and the recently patented, have allowed the design of genetic chimaeras by removing newly described isoform that shows a new exon 2 and a and replacing the exon 3 in a reciprocal way, forming hGH- truncated exon 3 which results in a A-B loop modified, re- NV3 and hGH-VN3 constructs. In this case, the alternative ferred as INSP105 (US007605129B2 [26]). Additionally, splicing pattern disappears upon exon 3 replacement by the Solomon and coworkers [43] produced at large-scale the corresponding region from the hGH-V gene (hGH-NV3) (see isoforms 22K-GHN, 22K-GHV, 20K-GHN and 20K-GHV Fig. 5). These data suggest that sequences in the 3’ end of The Diagnostic and Therapeutic Importance of Human Growth Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 62

hGH-N/-V

Relative Relative Abundance* Abundance** (%) (%) 22K-GHN(1) 22K-GHV(1)

90 53.5 24-26K-GHV(2) 20K-GHN(2) 26.7 5-10

24K-GHV(3) 17K-GHN(3) 19.2

0.5-1 20K-GHV(4)

0.6

hGH-N isoforms hGH-V isoforms Fig. (4). The hGH-N and GH-V isoforms. Diagram depicting gene sequence as common for hGH-N and hGH-V; this last differs 13 amino acids from hGH-N [35]. The principal isoforms are shown (in parenthesis the number of isoforms), pointing to translated regions that are involved in each one. The relative abundance is based on different authors as protein (hGH-N) or mRNA (hGH-V). Note that 20K-GHN(2) is the equivalent to 20K-GHV(4) but with notorious differences in its relative abundance. * The relative abundance of serum protein detectable by adequate polyclonal and monoclonal antibodies; ** the relative abundance of each gene alternative mRNA transcripts (adapted from [42]). hGH-N hGH-V

EcoRI SacI SmaI SmaI EcoRI EcoRI SacI SmaI SmaI EcoRI

1 2 3 4 5 1 2 3 4 5

XhoI XhoI SacI SmaI

1 2 3 4 5 1 2 3 4 5

hGH-NV3 hGH-VN3 Fig. (5). Chimeric hGHN-V3 & hGHV-N3. Diagram of constructs made to test recognition of the recombinant proteins by GH-BP. Restric- tion sites used to interchange the exon 3 and for cloning the gene into the vector are shown; ER, Eco RI; Sc, Sac I; Sm, Sma I; Xh, Xho I;. (Adapted from [12a]). intron B, or in exon III outside the AG region and its imme- tities using other promoter or regulatory regions as insert in a diate consensus region contribute to the usage of the alterna- Transposon-based vector (Tn-MCS). tive splice-site. Furthermore the chimerical proteins pro- In nature, the 17.5K-hGH-N isoform has shown to arise duced from the constructs showed different biological prop- from skipping of exon 3 which therefore causes the lack of erties [39] and binding capacities [12a]. At the present, residues 32-71 being the principal isoform that acts as nega- Cooper et al. (US2010093036A1 [44]), had claimed for the tive dominant protein in the isolated autosomal dominant development of more vectors to produce hGH in large quan- growth hormone deficiency type II (IGHD type II, OMIM 63 Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 Romero-Prado and Martín-Cófreces

#173100) [45-47]. A misbalance in synthesis and secretion Turner Syndrome, Prader Willi Syndrome, Intrauterine of GH has been found in prostate and lung cancer. The predi- growth retardation, idiopathic short stature, renal failure, lection for one of two principal isoforms (22- and 20-kDa) catabolic states (for example during chemotherapy treat- suggests that the possibility of predicting the evolution of ment), in cachexia related to AIDS and short bowel syn- patients in therapeutic anti-cancer treatments will be highly drome. For example, the therapy for hGH-N-deficient pa- dependent on the development of very specific diagnostic tients is principally based in the recombinant 20K-GHN tools or by monitoring the treatment with drug ocreotide for protein. The administration of 20K-GH at three different acromegalic patients [48]. In contrast, the recombinant hGH- dose (0.006, 0.012 and 0.024 mg/kg) during 16 weeks had N protein has been used to obtain liver reparation by repopu- effects dose-dependent on increase of IGF-I, IGFBP-3, os- lation and substitution of the damaged tissue. To date, the teocalcin and urinary deoxypyridinoline (U-DPD); the ab- patents about different hormone replacement therapies based dominal and visceral fat was also reduced significantly [51]. on hGH-N as recombinant protein, modified polypeptide and GH Deficiency (GHD) includes a group or different patholo- /or stably and functional protein with longer half-life, have gies all with a failure or reduction of GH secretion. Turner been elaborated and are depicted in Table 2. Syndrome is caused by the total or partial absence of one X In a very interesting approach, Männik et al. [42] de- chromosome; the TS females (95%) had a notorious short stature and reduced final height. The invention claimed by scribed the expression of hGH-V in placentae from mothers Tuefferd et al. [52] stems for the pharmacogenomics analysis whose children had appropriated (AGA), small (SGA) or evaluating gene expression and gene variations in a group of large (LGA) size for gestational age. In accordance with 310 Growth Hormone Disease (GHD) and Turner Syndrome previous studies, 22K-GHV was the major transcript (53.5%) (TS) patients. The results of the studies according to the in AGA placentas; hGH-V2, hGH-V3 and hGH-V4 repre- sented the 26.7%, 19.2% and 0.6% respectively. The hGH- invention show in particular for (1). The GHD population: (a) one SNP that may be used to identify low responders to V4 is equivalent in molecular weight to 20K-GH pituitary treatment with GH: rs941798 in the PTPN1 gene as a ge- isoforms but with unknown relevance during gestational nomic marker; (b) two SNPs that may be used to identify development. In the case of SGA, the hGH-V2 isoform high responders to treatment with GH: rs2270777 in the showed a significantly lowered expression: compared with CDK4 gene as a genomic marker, rs 970467 in the LEPR normal-size babies, the expression of 22K-GHV/GH2-1 was about 1.1-fold lower in the SGA (p=0.05) and about 1.3-fold gene as an allelic marker predictive of a high response based on IGF-I response to GH treatment in the GDH population. higher in the LGA group (p=0.006). Similar differences were (2). The TS population: (a) One SNP that may be used to observed among the groups studied also for 24K-GHV/GH2- identifying high responders to treatment with GH: rs2276310 3 (p<0.04). During normal or pathological pregnancies the in the ARRB1 gene as an allelic marker; (b) five SNPs that levels of hGH-V may be altered, which can be raised as well may be used to identify low responders to treatment with by the maternal IGF-I. In this sense, Chellakooty et al. [49] found that (i) the gestational age-length of pregnancy- was GH: rs2069502 in the CDK4 gene, rs4456611 in the BCL2 gene, rs2270777 in the CDK4 gene as genomic marker, coincident when maximal peak concentrations of hGH-V rs2906713 in the SH2B2 gene and rs10513055 in the were reached; (ii) there is a highly significant positive asso- PIK3CB gene as allelic markers. Patients with markers pre- ciation between increase in hGH-V and IGF-I during preg- dictive of a low response would be given an optimized dose nancy. The authors hypothetically suggested that hGH-V of GH that may be increased dose compared to that is con- influences fetal growth, whereas IGF-I may stimulate fetal growth indirectly by affecting placental size or function. The sidered as standard dose. Low responders can also be candi- date patients for therapies with long-acting GH analogues. serum levels of both hGH-V and IGF-I had been reported as Other methods described for cloning, isolation and bio- low in women with intrauterine growth retardation (IUGR) chemical characterization of GH variants have been also pregnancies and to correlate with birth weight and placental described [53-56]. weight in pregnant women with Type I Diabetes Mellitus [50]. A) Anti-Angiogenic Peptide Agents: Therapeutic and Di- agnostic Use (WO 9851323A1 [57], US 2003186382A [58], 4.2. hGH Isoforms, Modified hGH and/or hGH-Related US 2004/0077054A1 [59], US 2008096795A1 [60] Peptides for Therapeutic Use The invention by Weiner et al. [57-60] consists on an During the last years different molecules and strategies anti-angiogenic peptide of 16kDa substantially identical to have been designed to produce or modify biochemical char- about 10 to about 150 consecutive amino acids selected from acteristics of the hGH, such as more stability, longer half-life or thorough the production of fusion proteins, and others that the N-terminal end of human placental lactogen (16K hPL), human GH-V (16K GH-N and 16K hGH-V) or human can maintain the most important lipolytic and growth- prolactin (16K hPRL), with the following biological activi- stimulating properties, avoiding the development of insulin- ties: (i) the peptide inhibits the endothelial cell proliferation resistance. In this sense, the molecules or the modifications and organization in capillars; (ii) it inhibits angiogenesis in depicted below are the prototype of improved models. The chick chorioallantoic membrane; and (iii) binds to at least principal uses of hGH-N isoforms have been directed to the compensation of low levels of hormone in the case of hGH- one specific receptor which can not bind an intact full length growth hormone, placental lactogen, prolactin, or hGH-V. N deficiencies (as IGHD II, see above), or other kind of The invention is supported by evidence coming from the diseases that are companied with low or retarded somatic analysis of the effects of some of these anti-angiogenic pep- growth. The therapeutic use of growth hormone is for its tides on a tumour mass in a human patient; and by the design own deficiency and includes for example the treatment of of a method for diagnosing a probable abnormality in placen- The Diagnostic and Therapeutic Importance of Human Growth Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 64 tal vascularization during pregnancy comprising the assay of ylene glycol (PEG) by a process known as “pegylation” that the level of at least one of endogenous N-terminal fragments has several beneficial effects. Firstly, the PEG coat increases above mentioned. The inventors suggest that the peptides of the effective molecular weigh from 22kDa-GH to approxi- the invention can also be used as contraceptive agents. mately 40kDa-GH. The effect obtained is the decrease in glomerular filtration of GH thereby increasing the half-life of B) Therapeutic Use of 20-kDa hGH-V Isoform GH in vivo which reduces the administered dose needed to (WO2005018659 [61], US2006281675A1 [62], produce the desired effect. Other groups have developed WO2006012525 [63] modified hGH including non-naturally encoded amino acids The invention by Gluckman et al. [61-63] refers to the that are linked to water soluble polymer with a linker or is biological properties of the 20-kDa hGH-V isoform that are bonded to the water soluble polymer (as PEG) shared with the 22- and 20-kDa hGH-N isoforms for thera- (US2006069220) [71] However, the PEGylation has the peutic use, and to those modifications in amino acids that disadvantage that it reduces affinity of hormone for receptor improve the molecule to favour somatogenic but no diabeto- [72], and chemical modification with subsequent purification genic effects, stability and half-life. This variant provides the is expensive. beneficial effects of the conventional therapy such as growth E) Cystein Variant of hGH-N ( IL133974-[73]) promotion, stimulation of secretion of IGF-I and lipolysis but avoids unwanted properties, including diabetogenic ef- The hGH-N variants included in this patent [73] are the fects which are reduced. result of substitutions of a cysteine residue for an amino acid at a specific position within the protein. Preferred sites for C) hGH-N Peptide (Amino Acids 1-to-43) and Type-B Na- substitution are the N- and O-linked glycosylation sites. The triuretic Peptide (BNP) (WO 2009/033680 [64], KR20100059866 [65]) methodological strategy used for cysteine residue substitutions is based on mutagenic oligonucleotide primers. The The peptides hGH-N (amino acids 1-to-43, see Fig. 2) patent describes the production of GH mutants (called and type-B Natriuretic Peptide (BNP) used in a peptide com- “muteins”) as the following: T135C, S132C, T148C, S144C. bination, enclosed in this invention by Bevec et al. [49] was The GH muteins were tested for biological activity and ca- tested using different kinds of assays to challenge their effect pacity to bind receptors in an in vitro assay; the GH that as active therapeutic agents in the prophylaxis and/or treat- retains in vitro activity can be PEGylated by standard proce- ment of cancer, proliferative diseases, tumours and their dures (see above). metastases. Peptides of the invention and the peptide combi- F) Fusion Protein LR (AU2008201889 [74], nation showed no inhibitory or irregular effects on (i) the cell US2010197582A1 [75] ) cycle of tested human lung cells, (ii) on proliferation of specifically stimulated human T-cells or B-cells, (iii) induction The LR-fusion protein, by Ross et al. [75] is the result of apoptosis on human lung cells. For this last case, the pre- from the fusion of GH to the extracellular domain of GHR – ventive properties of the peptides on apoptosis were tested. exGHR- in a head-to-tail design. The LR-fusion protein was The model used was based on C2 ceramide as apoptosis more potent in vivo compared to GH but not in vitro where inductor. Peptide 1 and Peptide 2 prevented by 14% and the bioactivity of LR-fusion was 10 times less. This effect is 18.4% respectively the ceramide-induced apoptosis on hu- probably attributable to the formation of dimers by the LR- man lung cells. The peptide combination in two different fusion protein. Indeed, LR-fusion showed a 300-fold reduc- mole ratios prevented by 22.7% and 25.9% the ceramide- tion in clearance compared to GH and a 10 to 30-fold reduc- induced apoptosis on human lung cells. Furthermore, the tion compared to that previously reported for a GH/GHBP peptides were able to induce the overproduction of Th1/Th2 complex [74] or conjugate upon intravenous administration cytokines IL-4 and IL-2 versus controls. Also, these peptides in rats. The therapeutic use of growth hormone has been induced a minor production of INF versus controls. Other based on its basic sequence plus modifications made to give systems used to test the two peptides properties were endo- it stability or as fusion protein as the case of the 75K-LR- thelial cell migration and endothelial tube formation. In the fusion protein (US2010197582A1) [75], and it is considered former, the peptide 1 inhibited by 21% and the peptide 2 the pathway of administration (oral, intravenous or subcuta- induced by 14% the migration of human endothelial cells, neously). Other conjugates as hGH-GHBP have also been respectively. The combination of both peptides did not in- developed and patented [76]. hibit or induce the tube formation endothelial cells. Finally, G) Fusion Proteins: hSA-hGH for Therapeutic Objectives peptide 1 inhibited by 3%, the peptide 2% inhibited by 19% (US 2010261650A1/ US 2007/0299006 A1 [77]). and the combination of both peptides inhibited by 17% the tube formation of endothelial cells, respectively. In Table 2 The hGH has been shown to be secreted into the blood in are mentioned other therapeutic uses of modified hGH to pulses in vivo. By infusion of recombinant human GH repopulate specific tissues or to treat myeloproliferative (rhGH-N) in healthy volunteers (n=24) during three periods syndromes as well as compositions and methods for treating of 180 min each, the half-life of GH was estimated as GH deficiency (see above) [66-69]. 13.0±3.8, 18± 5.3 and 20.5±4.5 min for bolus of 60, 180 and 2 D) Pegylated Recombinant hGH (PEG-HGH-52) 310 mg/m [27]. Human serum albumin (hSA) is a protein of 585 amino acids that is responsible of a significant propor- (WO2009/133137A2 [70]) tion of the osmotic pressure of serum and that functions as a The invention developed by Hersel et al. [70] and related carrier for endogenous and exogenous ligands. The use of herein refers to a modified hGH protein that shows a longer albumin as a component of a fusion protein for the stabiliza- half-life. Furthermore, the modified GH is coated in polyeth- tion of other proteins has been described [77]. Poznansky et 65 Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 Romero-Prado and Martín-Cófreces al. [78] increased the half-life of porcine GH (pGH) with A) Antibodies Development to hGH Detection either porcine or human serum albumin (HSA:HGH fusion (WO9736929A1 [84]; CA2250047A1 [85], US5945296 [86]) protein); and the resulting 180KD conjugate was found to have and extended half-life in the blood stream of rats after 2 In general, has been demonstrated that the assay systems that involve polyclonal antibodies yield similar results but to 3-hours upon administration, compared to the 5 min de- very notorious discrepancies are detected when monoclonal tected for unconjugated growth hormone. In both cases the antibodies are used [17,33]. It is well established that GH in polypeptides are bridged by linker peptide to maximise the the pituitary and in circulation consists of a variety of mo- availability of the hGH portion for the binding to its hGH lecular isoforms including monomers such as 27-, 20-, 17- receptor. The HAS-HGH fusion protein has another properties as being highly reproducible in large-scale production by and 5-kDa, grouped as non-22kDa GH [32] and oligomers as big as >45 kDa. [20], which have different cross-reactivity in yeast (S. cerevisiae) and to be very stable for a relative long- various GH immunoassays. Indeed, GH-binding protein time (> 1 month at 4°C). (GHBP) is considered as a discrete source of interference in 4.3. Growth Hormone Secretagogues and Inhibitors GH assays because binds to circulating hGH [69]. Hansson et al. [84] claim for the invention of monoclonal antibodies A very interesting approach is based on regulating pulsatile secretion of GH by administrating amino acids aspartic capable of specific binding to 20K-hGHN isoform for its identification and measurement in body fluids, especially in acid and valine (US2009048340). This patent, claimed by serum. Ono et al. [79], is considered as anti-aging product that, when is administered as the authors propose, could mimic B) Methods for Detecting Human Growth Hormone the pulsated secretion of a GH, being considered as provider (AU712583 [87], US2009305300A1 [88]) of an excellent effect of suppressing induction of insulin resistance compared with a continuous secretion. Other in- Hashimoto et al. [87] claim for this invention designed to detect and distinguish two types of human GH (22K-GH ventions have been developed as peptide-based secretagogue from 20K-GH) in blood or urine of human subjects to whom formulations to obtain GH secretion by pituitary cells [80]. one of them is externally administered at least. Therefore, the The axis GH-GHRF includes the elements depicted in method serves to confirm or judge whether or not a hGH has Table 1, that are considered as necessary to be taken in ac- been externally administered. The immunodetection is made count to design a strategy to diminish abnormal cholesterol using monoclonal antibodies specific for each isoform. A levels (US2010010467A1). Daghia-Akli et al. [81] claims very ingenious design to detect growth hormone deficiency for the design of plasmids and constructs containing GH-RH, is by the measurement of hGH in serum after the infusion or regulatory sequences and methods of delivering into the injection of a growth hormone secretagogue, that can be an tissue of a subject the nucleic acid expression construct hormone (GHRH) or a peptide (GHRP). The invention by (electroporation, transporter systems –spermine, histone, Larsen [88] refers to the administration of GH secretagogues cationic peptides and/or polylysine-, etc.). Other organic (GHSs) EP 1572 (Formula I) or EP 1573 (Formula II), ob- compounds have been considered as GH secretagogues, taining a post-administration sample to determining the level depicted as structural formulas (I to XVI) that were tested to of growth hormone in the sample. This determination is treat subjects with proliferative diseases (MX200900856) indicative or normal or abnormal growth hormone levels [82]. Finally, there are methods designed to inhibit hGH and indicating hGH deficiency. Preferably, the amount of EP related hormones expression by the use of antisense polynu- 1572 or EP 1573 administered to a subject is suggested to be cleotide iRNAs or siRNAs [83]. between 18 and 75 mg, considering a dose of 0.5mg/kg 4.4. Diagnostic Relevance of Human Growth Hormones (ranges of 0.1 to 1.0 mg/kg). and Related Patents CURRENT & FUTURE DEVELOPMENTS In a very complete review, Irie et al. [33] describe a new method of measurement of 22K-hGH and 20K-GH for dop- A sample of the existing systems to design, synthesize, ing test, remarking the importance in establishing stable produce, identify and quantify wild type and modified hGH sources of critical regents and materials, and in the increase proteins as well as its biological effects both in vitro and in of their stability and the reproducibility of the test. An aspect vivo have been shown. Additionally, other study forms exist that is very important is the pulsatile secretion of hGH-N that that imply transient transfection to test epigenome effects on varies with age, in contrast with hGH-V that is secreted hGH-V expression [90] and hGH-N [91]. An important focus continuously. The hGH-N pulsatile secretion allows single of research is to identify those transcription factors that are measurements to estimate its levels. The hGH-V hormone modulated under metabolic, endocrine and/or nutritional has been considered as an importantly protein involved in conditions dictating developmental abnormalities in tro- regulation of the nutrients availability to the placenta, influ- phoblastic invasiveness and how can these influence on encing the fetal substrate supply either via autocrine or human GHs expression and function. paracrine mechanisms. Additionally, the role of IGF-I and – The complexity and possible functions of all isoforms II as well as IGFBPs (IGFBP-3) in growth regulation during variety is under analysis yet. To prolong half-life of a potent neurological development in mouse, in growth and placental somatogenic protein avoiding its undesirable effects contin- differentiation has been studied (see [11] and references ues to be a very important objective. The methods designed therein).

The Diagnostic and Therapeutic Importance of Human Growth Recent Patents on DNA & Gene Sequences 2011, Vol. 5, No. 1 66 to quantify the isoforms different from the considered as the [15] Tuggle CK, Trenkle A. Control of growth hormone synthesis. major isoforms has recovered relevance in light of the poten- Domest Anim Endocrinol 1996; 13: 1-33. [16] Strobl JS, Thomas MJ. Human growth hormone. Pharmacol Rev tial biologic effects in diseases as cancer, neovascularization, 1994; 46: 1-34. immunomodulation, and metabolic processes where the [17] Baumann GP. Growth hormone isoforms. Growth Horm IGF Res growth hormones are coupled to the IGF system. Indeed, the 2009; 19: 333-40. perspectives looking for the patents derivated from all these [18] Aberg ND, Brywe KG, Isgaard J. Aspects of growth hormone and findings establish objectives that will be done in the next insulin-like growth factor-I related to neuroprotection, regeneration, and functional plasticity in the adult brain. 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