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Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-β-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.Issue Section: Growth Hormone-Somatostatin-GRH
Human GH (hGH) is a member of a family of closely related hormones that include prolactin and placental lactogen. GH regulates a wide variety of physiological processes, including growth and differentiation of muscle, bone, and cartilage cells. GH is secreted by the somatotrophs of the anterior pituitary gland and acts on various tissues to promote growth and influence metabolism (1–3).
GH is a 22-kDa protein that contains 191 amino acids with two disulfide bonds and four alfa-helixes (4, 5). Signal transduction begins with GH binding to a GH receptor (GHR) on the plasma membrane (6). Structure function studies have determined specific regions of the molecule to be important for GHR binding (4, 7). These studies indicated that GH interacts with a preformed GHR dimmer, which is critical for GH-induced intracellular signal transduction (4). GH has two distinct domains that bind to two identical GHRs at the cell surface in which binding at site 1 followed by binding at site II produces functional receptor dimerization. This resulted in the activation or inactivation of genes that are responsible for GH effects (8).
The use of GH for the treatment of children with impaired linear growth has been accepted as an important therapeutic modality for many years (9–12). In addition, beneficial effects of GH replacement therapy in adults were established. GH substitution in adults increases muscle mass by 5–10%, but part of the effect is attributed to rehydration rather than protein accretion. In addition, GH regulates substrate metabolism in muscle and in particular antagonizes insulin-stimulated glucose disposal. This effect is linked to increased free fatty acid flux (13). One major issue regarding the clinical use of GH is its short half-life due to its rapid clearance (∼12 min) from the circulation (14). Under normal physiological conditions, GH in human plasma is complexes with GH binding protein. One function of the GH binding protein is the relative confinement of hGH to the vascular compartment, thereby protecting it from degradation and prolonging its biological half-life (15, 16).
Previous studies were carried out to stabilize hGH and extend its half-life. It was shown that complexation of hGH with heparin did not cause a major distribution in the tertiary structure of hGH but decreased the hydrophilic environment and stabilize the hormone (17). Other studies demonstrated that crystals of hGH coated with positively charged poly(arginine) allowed delivery of hGH over a period of several days (18). Therefore, GH initiated daily and the dose is based on individual requirement and responsiveness. Previous studies indicated that fusing the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) β-subunit to human follitropin (FSHβ) (19, 20), hCGα subunit (21), TSHβ (22), or to erythropoietin (23) did not affect assembly, secretion, receptor binding affinity, or in vitro bioactivity. However, the in vivo potency and circulatory half-lives of the proteins containing CTP were substantially increased.
In the present study, we designed a long-acting GH by fusing the CTP of hCGβ subunit that contains four O-linked oligosaccharide recognition sites to the coding sequence of the hormone. Four variants of hGH were prepared by fusing one, two, or three CTP sequences to the coding sequence of hGH. Our results indicated that ligation of CTP to the coding sequence of GH did not affect secretion of the chimeric protein into the medium. In vivo studies in hypophysectomized rats indicated that both bioactivity and pharmacokinetic parameters, mean residence time (MRT), area under the curve (AUC), time of maximal plasma concentration (Tmax), maximal plasma concentration (Cmax), and half-life, of GH bearing the CTP were dramatically enhanced.
Materials and Methods
Enzymes used in the construction of DNA vectors and constructs were purchased from New England BioLabs (Beverly, MA). Cell culture media and reagents were obtained from Biological Industries (Beit Hemeek, Israel). Rabbit antisera against GH were purchased from Fitzgerald (Concord, MA). The eukaryotic expression vector [pCI-DHFR (dihydrofolate reductase)] into which the cDNA encoding for the corresponding hGH variants were inserted was purchased from Promega (San Luis Obispo, CA). Commercial recombinant hGH, Biotropin, was obtained from Biotechnology General Ltd. (Kiryat Malachi, Israel).
Construction of chimeric genes and expression vectors
A cassette gene containing the CTP of hCGβ was fused in tandem to the coding sequence of human GH constructing four chimeric genes, in which one, two, or three CTPs were ligated to the N-terminal or C-terminal ends of hGH coding sequence. The variants that were prepared are: GH-wild type (WT; a recombinant GH that is produced in DG44 cells), GH-CTP (CTP was ligated to the C terminal), CTP-GH (CTP was ligated to the N terminal), CTP-GH-CTP, and CTP-GH-CTP-CTP (GH-LA) (Fig. 1). DNA fragments containing sequences of hGH cDNA-WT and hGH cDNA-CTPs were synthesized by GeneArt (Regensburg, Germany). The DNA fragments contain the recognition sites of the restriction enzymes: XbaI (in the N terminal) and NotI (in the C terminal). Fragments containing hGH and CTP sequences of the different variants were completely sequenced to ensure that no errors were introduced during synthesis and ligated into the XbaI-NotI sites at the cloning site of the eukaryotic expression vector, pCI-DHFR.FIG. 1.
Construction of hGH chimeric genes. The chimeric genes containing the cDNA of hGH and the hCGβ carboxyl-terminal peptide were synthesized. The DNA fragments contain the recognition sites of the restriction enzymes: XbaI (in the N terminal) and NotI (in the C terminal). Fragments containing hGH and CTP sequences of the different variants were ligated into the XbaI-NotI sites at the cloning site of the eukaryotic expression vector, pCI-DHFR.
Cell culture and DNA transfection
Chinese hamster ovary-DG44 cells, which are DHFR negative, were used. Cells were cultured in MEM-α medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM), and 10% heat-inactivated fetal bovine serum at 37 C in humidified incubator containing 5% CO2. These cells were transfected with 2 μg DNA of plasmid by using FuGENE6 (Roche, Mannheim, Germany) according to manufacturer protocol.
Cells were selected for insertion of the plasmid DNA by growth in culture medium of CD DG44 without hypoxanthine and thymidine (Life Technologies) supplemented with 8 mML-glutamine (Biological Industries) and 18 ml/liter of 10% Pluronic F-68 solution (Life Technologies).
For hormone collection, cells secreting GH variants were plated and grown to confluency in T-75 flasks. Cells were washed twice with serum-free medium, and 20 ml of medium without fetal calf serum (FCS) were added. Medium was collected every 24 h, clarified by centrifugation, and concentrated using centriprep concentrators (Amicon Corp., Danvers, MA). Concentrations of GH were determined by GH immunoradiometric assay and a double-antibody RIA (Diagnostic Products Corp., Los Angeles, CA).
Samples of condition medium that were collected from stable clones were electrophoresed on denaturing 15% sodium dodecyl sulfate-polyacrylamide gels as described before (24). Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol. Proteins were transferred to a 0.2 μm pore size nitrocellulose membrane (Sigma, St. Louis, MO) at 250 mA for 3 h using a Mini Trans-Blot electrophoresis cell (Bio-Rad Laboratories, Richmond, CA) according to the method described in the manual accompanying the unit. The nitrocellulose membrane was incubated in 5% nonfat dry milk for 2 h at room temperature. The membrane was incubated with GH antiserum (1:1000 titers) overnight at 4 C followed by three consecutive washes in PBS containing 0.1% Tween 20 (10 min/wash). Then the membrane was incubated with secondary antibody conjugated to horseradish peroxidase (Zymed, San Francisco, CA) for 2 h at room temperature followed by three washes. Finally, the nitrocellulose paper was reacted with enhanced chemiluminescent substrate (Pierce, Rockford, IL) for 5 min, dried with a Whatman sheet (Whatman, Kent, UK), and exposed to x-ray film.
Hypophysectomized Sprague Dawley-derived male rats were obtained from Charles River Laboratories (Jerusalem, Israel) and housed in air-conditioned quarters with a 12-h light, 12-h dark schedule. Standard food and water were available ad libitum. Institute ethical committee approved the in vivo protocols. Animals were treated with condition medium containing GH analogs as specified.
In vivo bioassay
Efficacy of GH-CTPs in vivo was assessed by weight gain and IGF-I levels in hypophysectomized rats. To select the most active variant and avoid possible antibody formation, short protocols were chosen. Eleven days of incremental weight gain was measured in hypophysectomized male rats after two injections sc of (0.6 mg/kg) once every 5 d (total dose of 1.2 mg/kg). This was compared with a standard protocol of once-daily injections of 0.12 mg/kg (total dose of 1.2 mg/kg) of commercial hGH (Biotropin). Weight gain was measured in all animals before treatment, 24 h after first injection, and then twice weekly until the end of the study on d 11.
Weight gain was also detected after three injections and different dosing patterns of GH-LA variant. Animals were treated with a single injection of different doses (0.6 or 1.8 mg/kg), of GH-LA variant every 4 d for 2 wk, and it was compared with once-daily injection of 0.12 mg/kg of Biotropin. Each point represents the group average of weight gain (grams) ± SE.
To test the effect of GH on IGF-I level, rats were administered sc with a single dose (0.6 or 1.8 mg/kg) of Biotropin or with GH-LA variant. After injection, plasma samples for IGF-I analyses were obtained at different times, 2–96 h. Samples were analyzed for IGF-I concentration using a commercial ELISA kit (R&D Systems, Minneapolis, MN).
Metabolic clearance rate
The metabolic clearance of commercial hGH (Biotropin) and GH-LA variant was determined after iv or sc injection of 50 μg/kg into hypophysectomized Sprague Dawley male rats. At selected intervals after injection, blood samples were collected and the concentrations of GH were determined by a GH ELISA kit according to the method described in the manual accompanying the kit (Diagnostic Products).
Mean serum concentrations were determined for each group at each time point, and composite serum concentration vs. time curves were generated. Concentration values below the limit of quantitation of the assay (1400 pg/ml) were set to zero for the calculation of group means. Noncompartmental analysis was performed with WinNonlin professional version 5.2.1 (Pharsight Corp., Mountain View, CA).
The AUC from time zero to the last measurable time point (AUC0-t) was estimated using the trapezoid method. Linear regression over the last three or more time points was used to estimate the elimination rate constant (λ), which was used to estimate terminal half-life (t½) and AUC from zero to infinity (AUC0-∞) from the following equations: t½ = ln (2)/λ; AUC0-∞ = AUC0-t + Ct/λ, where Ct is the last measurable concentration as predicted by the linear regression. Serum clearance was calculated from the dose divided by the AUC0-∞. The Cmax and Tmax were determined directly from the data.
Data were expressed as the mean ± SEM. Statistical analysis of the data were performed using Student’s t test and one-way multivariate ANOVA to calculate Pvalue. P < 0.05 was considered statistically significant.
Secretion of hGH variants from transfected cells
hGH cDNAs, WT, and chimeras were inserted into the pCI-DHFR plasmid, a eukaryotic expression vector, and transfected into Chinese hamster ovary-DG44 cells. Stable clones that secreted hGH variants were selected. Concentrations of hGH variants in the condition media were measured using a GH ELISA kit.
Using Western blot analysis and hGH antiserum after denaturing SDS-PAGE allowed detecting the secretion of GH variants into the media. The hGH-WT and the commercial hGH, Biotropin, migrated faster than hGH-CTPs and exhibited molecular mass of approximately 22 kDa (Fig. 2). GH variants containing one, two, or three CTPs exhibited higher molecular mass of 30, 39, and 47.5 kDa, respectively. The increase in molecular mass of hGH-CTPs due to the addition of 28 amino acids (molecular mass of ∼2800 Da) and probably to the attached O-linked oligosaccharide chains linked to CTP. These data may indicate that the O-linked glycosylation recognition site of the CTP is preserved, even though the sequence is fused to different proteins.FIG. 2.
Expression of GH-WT and GH-CTP variants from transfected DG44 cells. Conditioned media from transfected cells were prepared for SDS-PAGE and proteins were detected by Western blot using GH antiserum.
In vivo bioactivity
For further pharmacological evaluation of hGH variants, comparative bioactivity studies of GH-CTP variants, recombinant GH-WT (produced in DG44 cells), and commercial hGH, Biotropin, were performed in hypophysectomized Sprague Dawley derived male rats (n = 10/group) using different frequencies and wide dose range as described in Materials and Methods. Efficacy of GH-CTPs was assessed by weight gain in a hypophysectomized rat model. Weight gain was measured in all animals before treatment, 24 h after the first injection, and then twice weekly until the end of the study on d 11. Subcutaneous injections of GH-WT, Biotropin, GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d, for 11 d, increased the weight gain by 4, 4.9, 5.1, and 7 g, respectively, compared with untreated controls that have a weight gain of 2 g (Fig. 3). Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once a week for 2 wk or with Biotropin (0.12 mg/kg) daily for 10 d resulted in a dramatic increase (P < 0.001) in weight gain (Fig. 3).FIG. 3.
In vivo bioactivity of recombinant GH derivatives. Hypophysectomized Sprague Dawley derived male rats (n = 10/group) received a single sc injection per week (50 μg/kg) for 2 wk of GH-WT, GH-CTP, CTP-GH, CTP-GH-CTP, CTP-GH-CTP-CTP (GH-LA), or commercial Biotropin. In addition, animals were treated with daily injections of 10 μg/kg Biotropin for 2 wk. Control animals were injected sc with PBS. Weight gain was measured in all animals before treatment, 24 h after first injection, and then twice weekly until the end of the study. Each point represents the group average of weight gain (grams) ± SE. *, P < 0.0.5; **, P < 0.01; ***, P < 0.001.
Weight gain was also detected after repeated injections with different doses (0.6 or 1.8 mg/kg,) of GH-LA every 4 d, and it was compared with once-daily injection of Biotropin (0.12 mg/kg). Injection of 0.6 mg/kg significantly (P < 0.001) increased the weight gain compared with controls by 16 ± 2 g, whereas a similar effect was achieved by daily administration of 0.12 mg/kg Biotropin that increased the weight gain by 18 ± 3 g. Injection of a higher amount (1.8 mg/kg) of GH-LA, once every 4 d dramatically increased the weight gain up to 28 ± 4 g after 2 wk (Fig. 4).FIG. 4.
Weight gain profile after repeated dosing of hGH variants. Hypophysectomized Sprague Dawley derived male rats (n = 10/group) received a single sc injection once every 4 days (0.6 or 1.8 mg/kg) for 2 wk of GH-LA variant. In addition, animals were treated with daily injection of 0.1 mg/kg commercial Biotropin for 2 wk. Control animals were injected sc with PBS. Weight gain was measured in all animals before treatment, 24 h after first injection, and then twice weekly until the end of the study. Each point represents the group average of weight gain (grams) ± SE.
The effect of GH on IGF-I serum levels in hypophysectomized rats after a sc single injection was detected. The cumulative serum levels of IGF-I after injection of CTP-LA was significantly higher than that detected after injection of Biotropin (Fig. 5). The maximal effect of GH-LA was generally proportional to dose, and it was 3–4 times higher than that of Biotropin. The maximal effect reached by GH-LA was 36–48 h as compared with 20–24 h of Biotropin.FIG. 5.
IGF-I serum levels in hypophysectomized rats after sc injection of CTP-GH-CTP-CTP (GH-LA) or Biotropin. Single doses of GH-LA or Biotropin (0.6 or 1.8 mg/kg) were injected sc to hypophysectomized rats. Serum IGF-I after injection was measured using a specific ELISA. Each pointrepresents the group average of seven rats ± SE.
The circulatory half-lives of GH variants were determined after iv or sc injections into hypophysectomized Sprague Dawley male rats. At selected intervals after injection of 50 μg/kg, blood samples were collected and the concentrations of GH were determined. The results indicated that after iv or sc injections, a higher level of the chimera is still detectable in serum after 50 and 72 h, respectively, in which the levels of Biotropin after 24 h (iv injection) and 48 h (sc injection) were undetectable (Fig. 6). The estimated plasma half-lives for Biotropin or GH-LA after iv or sc injections were 0.5 and 1.7 vs. 6.9 and 9 h, respectively (Table 1).FIG. 6.
Metabolic clearance of commercial Biotropin and CTP-GH-CTP-CTP (GH-LA) in vivo. Hypophysectomized Sprague Dawley derived male rats (n = 7) were injected iv (A) or sc (B) with 50 μg/kg of Biotropin or GH, and blood samples were drawn at the indicated time. Serum GH levels were determined by RIA. Each point represents the group average of seven rats ± SE.
Mean pharmacokinetic parameters after iv or sc administration of a single dose (50 μg/kg) of Biotropin or GH-LA in Sprague Dawley rats
|AUC (h/μg · liter)||162||1568||41||680|
|t½ (α) (h)||ND||0.74||ND||1.58|
|t½ (β) (h)||0.5||6.9||1.7||9|
Parameters were generated for individual rats and the mean data are presented here. ND, Not detected.Open in new tab
Estimations of the MRT, the AUC, the Cmax, and the Tmax of GH-LA variant are dramatically higher than that of Biotropin after iv or sc injections (Table 1). MRT values of GH-LA after sc or iv administrations were 9.9 and 12.9 h, respectively, whereas the estimated values of Biotropin were 0.5 and 2.5 h, respectively. AUC values of GH-LA after sc or iv administrations were 680 and 1568 h/μg · liter, respectively, whereas the estimated values of Biotropin were 41 and 162 h/μg · liter, respectively. The Cmax and Tmax values of GH-LA were higher than that of Biotropin after sc administration (37 vs. 13 μg/liter and 8 vs. 0.5 h, respectively). These data suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP.
Results of the present study demonstrated that the addition of hCG-CTP extension to the coding region of hGH cDNA significantly increases the bioactivity and prolonged its circulating half-life in vivo. Moreover, the results indicated that a single injection of the GH variant is sufficient for a significant increase of weight gain compared with a daily injection of GH-WT.
These results are consistent with earlier studies that indicated that fusing the CTP sequence to hFSHβ (19, 20), hCGα subunit (21), human TSHβ subunits (22), or the C terminal of erythropoietin (23) resulted in a significant increase in the in vivobioactivity and half-lives of the hormones in the circulation. On the other hand, deletion of the CTP from hCGβ subunit caused a significant reduction in the in vivobioactivity of hCG without effect on receptor binding affinity and in vitro biological activity (25).
The CTP extension contains 28 amino acids with several proline and serine residues and four O-linked oligosaccharides sites. Previous studies indicated that the CTP sequence can be shuttled into different proteins and still be an acceptor for the O-linked oligosaccharides (19–23). It was postulated that the O-linked oligosaccharides add flexibility and hydrophilicity to the protein (26). This may explain the disinterference of CTP on the protein conformation and thus on receptor binding and bioactivity in vitro. On the other hand, it was suggested that the four O-linked oligosaccharides play an important role in preventing plasma clearance and thus increasing the half-life of the protein in the circulation (27, 28).
These roles have been postulated because the O-linked oligosaccharides are ended with sialic acid, which is negatively charged. It is known that negatively charged forms of the hormones are less cleared through the glomerular filtration (29). Thus, addition of four O-linked oligosaccharide chains to the backbone of the protein decreased the renal clearance, in which the kidney is the main site of clearance for glycoprotein hormones (30) and thus prolonged its half-life in the circulation.
Recombinant hGH became a novel therapeutic option for adults with acquired GH deficiency (GHD). Recent studies indicated that many of the metabolic and psychological abnormalities associated with GHD can be reversed with GH replacement. This treatment is an effective and rational therapy for adult men and women with known pituitary disease or risk factors for hypopituitarism. The impact of childhood GHD on bone health has attracted numerous publications in the scientific literature over the past 20 yr. There is a perception that GHD in a child may lead to abnormal bone density and fractures, and GHD is frequently listed as a cause of secondary osteoporosis in children (31). At the transition from childhood to adulthood, when bone elongation is completed, GH therapy is currently offered to all patients with confirmed severe GHD.
One major issue regarding the clinical use of GH is its relative short half-life due to rapid clearance mechanisms. The significant interest in extended release therapeutic proteins is well justified. Extended the half-life of the protein can provide greater safety and efficacy and improve patient compliance because of less frequent administration. Several second-generation therapeutic proteins with extended half-lives and improved potencies in vivo have been created by modifying the proteins with polymers such as polyethylene glycol (PEG) or using crystalline biopharmaceuticals (18, 32–34). Covalent attachment of PEG to a protein increases the protein’s effective size and reduces its rate of clearance from the body. Previous studies indicated that hGH derivatives conjugated with PEG polymers have longer circulating half-lives than that of hGH WT after sc or iv administration in rats. Moreover, the potency of GH-PEG derivatives was significantly higher by about 10-fold compared with unmodified hGH (33, 34). Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor. Therefore, in vitro bioactivity of GH-PEG derivatives was reduced 100- to 1000-fold (33).
Another study extended the half-life of GH using crystalline biopharmaceuticals (18). Crystals of recombinant hGH were coated with monomolecular layer of positively charged poly(arginine). The efficacy of this crystalline formation injected sc once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using hypophysectomized rats model and in measurement of serum IGF in monkeys (18). Protein crystallization is a difficult step in the structural-crystallographic pipeline. Crystallization image classification and automated scoring is a multistage process. In addition, the low loading of hGH in microspheres and relatively large particle size leads to high viscously of formation and necessitates administration through a large diameter needles. This may result in local irritation and pain and may lead to poor patient compliance.
In the present study, we hypothesized that increasing the sialic acid containing carbohydrate of GH would increase its serum half-life and thereby the in vivobiological activity. Increased sialic acid content will result in producing a more negatively charged form of GH with a longer half-life due to decreased glomerular filtration. This technique was proved by us previously in designing long-acting glycoprotein hormones (19, 23). Interestingly, human exposure to FSH-CTP in phase I studies was safe (35, 36). No antibodies against FSH-CTP were detected, and measurements of local tolerance demonstrated that sc administration of FSH-CTP was well tolerated and no increase in intensity of injection-site responses was observed after repeated exposure to FSH-CTP.
In addition, the hCG subunit is normally secreted in both men and women, and the immune system may not recognize the GH-CTP chimeras as a foreign protein. In addition, other studies have demonstrated that the CTP is weakly immunogenic in human (37). Therefore, we expect that addition of CTP to human GH backbone will not be immunogenic in human. However, the immunogenicity of GH-CTP should be tested in human clinical trials. These studies may emphasize the rationale for using the CTP in designing long-acting recombinant proteins for clinical use.
IGF-I is mainly secreted by the liver as a result of stimulation by GH. The results of the present study indicate that a single dose of GH (0.6 or 1.8 mg/kg) significantly increased the levels of IGF-I in the circulation. The maximal levels of IGF-I in circulation were activated after 48 h of treatment. Later peak of IGF-I may suggest prolonged bioactivity of GH-LA. IGF-I level begins to decline with the drop of GH-LA level. Other studies indicated that the accelerated clearance of IGF-I from serum results in the transfer of IGF-I to the extracellular space and enhances its degradation by proteases or binding to IGF binding proteins, and the rest is free forms with a half-life of 10 min (38). Therefore, the level of IGF-I in circulation is rising after sc treatment in which the Cmax and AUC is higher and begins to decline as soon as GH drops below the therapeutic level.
An interesting observation from the present study was the ability of a single injection of GH-CTP variant to be sufficient for increasing the weight gain in rats, whereas the same total dose of GH-WT administered daily was required to produce an increase as effective as one injection of GH-CTP variant. These results indicated the importance of pulsatile secretion and the pharmacokinetic parameters, MRT, AUC, and Cmax, rather than total dose of GH, in increasing in vivo bioactivity.
In summary, the present study describes a novel long-acting recombinant GH agonist designed by fusing of multi-CTP sequences to the coding sequence of GH. Ligation of three CTP sequences significantly increased the in vivo bioactivity and half-life of GH. The results indicate that administration of a single dose of GH-CTP variant every 4–5 d is sufficient for increasing weight gain in rats. These data establish a rationale for using this chimera as a long-acting GH analog. The therapeutic efficacy of this analog needs to be established in higher animals and in human clinical trials.
We thank the Israel Ministry of Industry and Trade for supporting this research.This work was supported by the Israel Ministry of Industry and Trade.Disclosure Summary: F.F. is a consultant for ModigenTech. R.G., A.B.-I., Y.F. and E.F. are employed by ModigeneTech.First Published Online July 21, 2010
- AUC,Area under the curve;
- Cmax,maximal plasma concentration;
- CTP,carboxyl-terminal peptide;
- FSHβ,β-subunit to human follitropin;
- DHFR,dihydrofolate reductase;
- GHD,GH deficiency;
- GHR,GH receptor;
- hCG,human chorionic gonadotropin;
- hGH,human GH;
- MRT,mean residence time;
- PEG,polyethylene glycol;
- t½,terminal half-life;
- Tmax,maximal time concentration;
- WT,wild type.