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Non-Clinical Pharmacology and Safety Evaluation of TH9507, a Human Growth Hormone-Releasing Facto


? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

Blackwell Publishing, Ltd.

Non-Clinical Pharmacology and Safety Evaluation of TH9507, a Human Growth Hormone-Releasing Factor Analogue
Eckhardt S. Ferdinandi1, Paul Brazeau2, Kim High1, Bryan Procter3, Stephen Fennell1 and Pascal Dubreuil4

Theratechnologies Inc., 2310 Boulevard Alfred Nobel, Montréal, QC, Canada, 2Neuroendocrinology Laboratory, Notre-Dame Hospital Research Centre, Montréal, QC, Canada, 3ITR Laboratories Canada Inc., 19601 Clark Graham, Baie d’Urfé (Montréal), QC, Canada, and 4 University of Montreal, Faculty of Veterinary Medicine, Department of Clinical Science, 3200, rue Sicotte, Saint-Hyacinthe, QC, Canada (Received March 23, 2006; Accepted September 12, 2006) Abstract: TH9507, an analogue of human growth hormone-releasing factor (hGRF(1– 44)NH 2) minimally modi?ed by addition of a trans-3-hexenoyl moiety to Tyr 1 of the amino acid sequence, was found to be resistant to dipeptidyl aminopeptidase-IV deactivation. Compared to natural hGRF(1– 44)NH 2, the modi?cation slowed the in vitro degradation of the peptide in rat, dog and human plasma and prolonged the in vivo plasma elimination kinetics of immunoreactive TH9507. Plasma growth hormone and insulin-like growth factor-1 (IGF-1) markedly increased in pigs, rats and dogs after daily repeat intravenous or subcutaneous injections of TH9507 at doses up to 600 ?g /kg. Subchronic toxicity studies in rats and dogs with TH9507 treatment for up to 4 months showed a signi?cant, but not dose-related, increase in body weight gain associated with increased biomarker response. Although TH9507 was well tolerated by both rats and dogs, a more pronounced anabolic effect and more evident (reversible) adverse effects (liver and kidney ?ndings, anaemia, clinical chemistry changes, organ weight effects) were observed in dogs after repeat daily subcutaneous injections, which were attributed to prolonged exposure to supraphysiological levels of growth hormone and/or IGF-1. In both rats and dogs, toxicokinetic evaluations indicated that exposure to immunoreactive TH9507 was dose related after both routes of administration. The apparent elimination t1/2 in dogs ranged from 21 to 45 min. In conclusion, TH9507 is a modi?ed hGRF peptide having enhanced potency and duration of action. The adverse treatment-related effects in dogs appear to be associated with sustained exposure to supraphysiological levels of growth hormone and IGF-1 induced by prolonged TH9507 treatment.

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Growth hormone-releasing factor (GRF) is a 44-amino acid peptide that is released from the hypothalamus and acts by binding to somatotropes through a speci?c receptor (GRFR) in the pituitary gland to stimulate the release of growth hormone through a signal transduction cascade [1]. The peptide, initially isolated from a pancreatic tumour in an acromegalic patient and characterized in 1982 [2,3], was later shown by Guillemin to be completely homologous to the native human GRF [4,5]. Subsequently, GRF peptides from other species were isolated and found to share amino acid sequence homology, mainly in the 1–29 amino acid sequence of hGRF(1– 44)NH2, while the remaining 30–44 amino acid sequence was largely species speci?c [6–10]. While the active portion of these molecules is minimally in the ?rst 1– 29 amino acid fragment [11,12], the in vivo deactivation of different GRFs occurs mainly by dipeptidyl aminopeptidase-IV (DPP-IV) catalysed cleavage of the ?rst two N-terminal amino acids in the sequence [13,14]. Structure-activity studies have been carried out on GRF, particularly the 1–29 fragment, in attempts to produce a longacting, therapeutic peptide [11,15,16]. TH9507, a minimally modi?ed analogue of hGRF(1– 44)NH2 having a trans-3Author for correspondence: Eckhardt S. Ferdinandi, Theratechnologies Inc., 2310 Boulevard Alfred Nobel, Ville St-Laurent, Québec, Canada H4S 2A4 (fax +1 514 331 7321, e-mail eferdinandi@theratech.com).

hexenoyl adduct on the terminal nitrogen of Tyr1, was designed to resist DDP-IV deactivation while retaining the physiological characteristics of the natural peptide [16]. This report presents in vitro and in vivo data showing that the trans-3-hexenoyl adduct on TH9507 enhanced the stability and potency of native hGRF with demonstrated functional effects in animal models, and that TH9507 has an acceptable non-clinical safety pro?le for use in human clinical trials. Materials and Methods
Peptides. TH9507 or N-(trans-3-hexenoyl)-[Tyr1]hGRF(1–44)NH2 acetate (CH3CH2CH = CHCH2CO-Tyr1-Ala-Asp-Ala-Ile-Phe-ThrAsn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-LeuLeu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-GlnGlu-Arg-Gly-Ala-Arg-Ala-Arg-Leu44-CONH2) was obtained from Theratechnologies Inc. (Montréal, QC, Canada; non-GMP Lot nos CVAL990524 and CVAL990729) or Bachem Inc. (Torrance, CA, USA; Lot no. FHEXHGRF002B) by standard Fmoc solid phase peptide synthesis using Fmoc-PAL-PEG-PS resin methodology and puri?ed by high pressure liquid chromatography (HPLC). The peptide, having a purity of >95% based on HPLC analysis, was fully characterized by MALDI-TOF mass spectral analysis, amino acid analysis and sequence analysis. Peptide reference standards were obtained as follows: human GRF(1– 44)NH2 (Lot no. 36H49531), purchased from Sigma-Aldrich (St. Louis, MO, USA); H-Tyr-AlaOH (Lot no. 519189), purchased from Chem-Impex International (Wood Dale, IL, USA); and trans-3-hexenoyl-Tyr-Ala-OH, prepared at Theratechnologies Inc. by solid phase synthesis.

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ECKHARDT S. FERDINANDI ET AL. ad libitum access to certi?ed diet (Teklad Certi?ed Rodent Diet no. 8728C, Teklad Inc., Madison, WI, USA). Dogs were given access to 400 g certi?ed diet for 2 hr daily (Teklad Certi?ed 25% Laboratory Dog Diet no. 8727C). Both rats and dogs had ad libitum access to treated municipal tap water. Protocols for all animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of each testing facility. Pharmacology studies in pigs. Three studies were carried out to determine the growth hormone (GH) and IGF-1 release in pigs after single injections of TH9507 and hGRF(1–44)NH2 at escalating doses. In each of the ?rst two studies (growth hormone release), 72 animals (body weight, 42.1 ± 2.1 kg and 41.5 ± 1.9 kg) were surgically implanted with an indwelling jugular catheter 5 days prior to the beginning of the experiment [17] and randomly assigned to nine groups of eight animals/group comprising one control (isotonic saline, 3.0 ml/injection) and seven treatment groups: hGRF(1–44)NH2 at 0.11, 0.33, 1.00 and 3.00 ?g/kg (intravenous injection) and 3.0, 9.0, 27.0 and 81.0 ?g/kg (subcutaneous injection); TH9507 at 0.11, 0.33, 1.00 and 3.00 ?g/kg (intravenous and subcutaneous injection). Serial blood samples were collected from each animal through a jugular catheter every 20 min. from 1 hr prior to and up to 8 hr after the intravenous and subcutaneous injections, and additionally at 10 and 30 min. after each intravenous dose injection for determination of serum growth hormone concentrations. In the third study, 40 male pigs (42.2 ± 2.2 kg body weight) were randomly assigned (eight animals/group) to one control (isotonic saline, 3.0 ml/injection) and four treatment groups: hGRF(1– 44)NH2, 30 ?g/kg; TH9507, 7.5, 15.0 and 30 ?g/kg. The doses were administered by subcutaneous injections at 12-hr intervals for 5 consecutive days. Blood samples were collected from each animal through venipuncture for determination of serum IGF-1 concentrations just prior to the ?rst dose on Day 1 then daily at 24-hr intervals (prior to peptide injection) up to Day 8 (60 hr after the last injection). Blood samples were allowed to clot at room temperature for 3 hr, centrifuged (1,500 ×g at 4°C), and the separated sera were stored at ?20°C pending measurement of GH or IGF-1 concentrations. Toxicology studies in rats and dogs. In subchronic toxicity studies, Sprague-Dawley rats and Beagle dogs were treated daily with escalating doses of TH9507 administered by intravenous and subcutaneous injection for up to 4 months’ duration as indicated in table 1. In each study, in-life observations included the following: daily evaluation of the clinical condition of the animals; body weight measurement at pretreatment, Day 1 and weekly thereafter, and prior to termination; and weekly food intake. Haematology, coagulation, and serum and urine chemistry parameters were determined in samples collected prior to termination (following an overnight food deprivation). At the end of the treatment period, the animals were euthanized (rats by exsanguination during methoxy?uorane anaesthesia; dogs by intravenous overdose of sodium pentobarbital followed by exsanguination) and

Reagents. DPP-IV (Lot no. 102K3781) was obtained from SigmaAldrich. Blank plasma was purchased from the following sources: human plasma, Biological Specialty Corp. (Colmar, PA, USA, Lot no. 22-70154A); rat and dog plasma, Lampire Biological Laboratories (Pipersville, PA, USA, Lot nos 102945084 and 091944295, respectively). The porcine growth hormone reference standard, RASG14276-A-1, was supplied by Hoffman-La Roche (Nutley, NJ, USA). Anti-IGF-1 primary antibody (Lot UBK487), raised in rabbit against human IGF-1, was provided by the National Institute of Diabetes, Digestive and Kidney Diseases programme. Polyethylene glycol 6000 and bovine gammaglobulin were purchased from Sigma-Aldrich. In vitro biodegradation studies. Aliquots of TH9507 and hGRF(1– 44)NH2 stock solutions (10 ?l; 1 mg peptide/ml in 0.2% aqueous acetic acid) were added to 90 ?l of rat, dog or human plasma (?nal concentration, 100 ?g peptide/ml). Plasma samples were incubated at 37°C and removed at the following times for electrospray ionizationliquid chromatography-mass spectrometry (ESI-LC-MS) analysis: 0, 1, 2, 3, 4 and 5 hr; samples containing hGRF(1– 44)NH2 (in human plasma only) were also removed at 10, 20, 30 and 40 min. Blank plasma controls were incubated for 0 and 5 hr. TH9507, trans-3-hexenoyl-Tyr-Ala-OH and H-Tyr-Ala-OH were also spiked into quenched human, dog and rat plasma at 100 ?g/ml (?nal concentration) to evaluate extraction recovery. For assessment of enzyme (DPP-IV) stability, 10 ?l of TH9507 and hGRF stock solutions were added to 90 ?l aliquots of solutions containing 1.1 unit/ml DPP-IV in 0.1 M Tris pH 8.0 (100 ?g peptide/ml ?nal) and incubated for 0 and 15 min., and 3 hr (TH9507 only) at 37°C. After incubation, samples were quenched with 200 ?l of 0.5% formic acid in acetonitrile, vortexed, centrifuged (9,600 ×g for 5 min.) and analysed by ESI-LC-MS. Animal treatment. Pathogen-free male Yorkshire-Landrace barrows, obtained from a local supplier (F. Ménard, Ange-Gardien, QC, Canada), were individually housed in 1 × 2-m pens under controlled conditions (room temperature ranged from 18°C to 20°C with a photoperiod of 12 hr light : 12 hr dark) and were given free access to municipal water and a commercial diet containing 18.0% crude protein with a lysine content of 0.7%. For the toxicity studies, Sprague-Dawley rats were obtained from Charles River Canada Inc. (St. Constant, QC, Canada), and beagle dogs were obtained from Covance Research Products Inc. (Kalamazoo, MI, USA). Upon arrival and prior to study group assignments, the animals were examined by veterinary staff for assessment of general health and acceptance into the study. Animals were housed under controlled conditions with room temperature set at 21 ± 3°C (rat) and 22 ± 2°C (dog), relative humidity 50 ± 20% (both species), and a photoperiod of 12 hr light : 12 hr dark. A minimum acclimation period of 2 and 4 weeks for rats and dogs, respectively, was allowed prior to initiation of dosing. Except for an overnight fast prior to termination or collection of blood and urine for laboratory investigations, rats had

Table 1. Treatment groups, number of animals/group, body weight ranges and ages of the rats and dogs used in repeat dose toxicity studies. For both routes of administration, the animals in each respective dose group were injected once daily with TH9507 at doses of 100, 300 and 600 ?g/kg per day. Control animals received vehicle only in the same manner (dose volume, 1 ml/kg). Additional six rats/sex/treatment group was used to collect blood for toxicokinetic and biomarker evaluations, and in the 3- to 4-month studies, additional animals (rats, 10/sex/group; dogs, 2/sex/group) were used in the control and high dose treatment groups for recovery assessment. 28-day studies Intravenous injection Species Rat Dog Sex Male Female Male Female Dose groups 4 4 4 4 Animals/ group 10 10 3 3 Body weight range 242 – 297 g 155 –198 g 7.2–9.0 kg 7.1–8.7 kg Age 9 weeks 9 weeks 7 months 7 months Dose groups 4 4 4 4 3- to 4-month studies Subcutaneous injection Animals/ group 15 15 4 4 Body weight range 242 – 297 g 155 –198 g 8.9 –10.8 kg 7.5–10.0 kg Age 9 weeks 9 weeks 7–8 months 7–8 months

? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

PHARMACOLOGY AND TOXICOLOGY OF HUMAN GROWTH HORMONE-RELEASING FACTOR ANALOGUE subjected to full external and internal examinations. All major organs were weighed and samples from each major organ and tissue were retained in ?xative, embedded in paraf?n wax, sectioned, stained with haematoxylin and eosin, and examined histopathologically. Additional sections of liver in the 3-month study were stained with Oil-Red-O for neutral lipids. For the determination of TH9507 toxicokinetics and biomarker (rat GH and rat IGF-1) concentrations in rats, blood was collected from the retro-orbital sinus (for serial samples) or via the abdominal aorta (for terminal samples) on Day 1 and Day last (Day 28 or Day 91) before and at 5, 60 and 120 min. after administration. In dogs, serial blood samples were withdrawn via a jugular vein from each animal on Day 1 and Day last for measurement of TH9507 and canine GH (28-day study only) concentrations before and at 5, 15, 30, 60, 90, 120 and 240 min. after intravenous injection; and before at 15, 30, 60, 90 and 120 min., and 12 and 24 hr after subcutaneous injection. Blood samples were also withdrawn from each dog for measurement of canine IGF-1 concentrations at the initiation (Day 1 or 2) and end (Day last) of both subchronic studies. Dose preparation. For intravenous and subcutaneous injections to pigs, dose solutions of hGRF(1– 44)NH2 or TH9507 were prepared 10–15 min. prior to injection by dissolving the peptide in isotonic saline at a concentration to deliver the target dose in a ?nal volume of 3.0 ml/injection. In the rat and dog toxicity studies, the TH9507 doses were prepared daily by dilution of aqueous stock solutions of the peptide (600 ?g/ml; prepared weekly in sterile water) to concentrations of 300 ?g/ml and 100 ?g/ml to deliver the target daily dose (600 ?g/kg, 300 ?g/kg and 100 ?g/kg) by intravenous (or subcutaneous) injection of 1 ml/kg dose volume. Mannitol was added to each dose solution to achieve a ?nal concentration of 5% mannitol (w/v). The dose solution concentrations, monitored throughout the studies by HPLC analysis, were within acceptable limits (±10%) of nominal values. LC-MS. ESI-LC-MS analysis, conducted on a Quantum Ultra triple quadrupole mass spectrometer (Thermo Electron Corp., San Jose, CA, USA) ?tted with a Surveyor autosampler/pump system and a SpectraSYSTEM P4000 quaternary gradient pump, was carried out under the following conditions: LC ?ow, split 1 : 4 before the ESI source; source voltage, 5.0 kV; capillary offset, 35.0 V; capillary temperature, 350°C; nebulizing gas, 30 units; auxiliary gas, 10 units. Analytical conditions including validation of LC-MS identi?cations were established by analysis of trans-3-hexenoyl-Tyr-Ala-OH and H-Tyr-Ala-OH in stock solutions and in water/acetonitrile 50 : 50 (v/v) and water, respectively. Aliquots (25 ?l) of supernatants from the processed plasma incubations were injected and analysed by ESI-LC-MS in positive ion and centroid mode with a mass range scan of m/z 200 to 1500 every second. MS data were acquired using Finnigan Xcalibur version 1.3 software and mass chromatograms and spectra were prepared using Qual Browser (contained in Xcalibur).

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Spectra from initial (t = 0 hr) and blank (t = 5 hr) samples were subtracted from TH9507 and hGRF(1– 44)NH2 incubate spectra to minimize the assignment of an endogenous peak as a metabolite. TH9507 radioimmunoassay. Plasma concentrations of immunoreactive TH9507 in rat and dog plasma were determined by a double antibody competitive binding radioimmunoassay (RIA) method [18,19]. In brief, the method comprised the use of a polyclonal rabbit anti-hGRF (Peninsula Laboratories, San Carlos, CA, USA) as the primary antibody reagent, 125I-Tyr10-hGRF(1– 44) amide (Amersham Biosciences, Piscataway, NJ, USA) as the competitive tracer, TH9507 as the standard, and a second goat anti-rabbit antibody (Cederlane Laboratories, Hornby, ON, Canada) to precipitate the bound fraction prior to gamma radioactivity measurement of the pellet. Biomarkers. Growth hormone in pig and dog sera was measured by a double antibody procedure using porcine growth hormone and canine GH antibodies, both raised in monkey and prepared to a ?nal dilution of 1 : 100,000 for porcine GH [20] and 1 : 100 for canine growth hormone. In each case, the secondary antibody was anti-monkey immunoglobulin G obtained from Linco (Lot no. 5040–10). Tracer reagents (125I-porcine growth hormone or 125Icanine GH) were prepared by standard iodination procedures and stored at 4°C. Rat growth hormone concentrations were measured by Cirion Inc. (Montreal, QC, Canada) using a commercial kit (ELISA method) obtained from BIO-PRI (Massy, France). IGF-1 concentrations in pig and dog sera were determined by a standard RIA method following an acid extraction [21]. Pharmacokinetic and statistical analyses. Non-compartmental pharmacokinetic analyses were carried out by application of WinNonlin software (version 1.5A) on the available immunoreactive-TH9507 plasma concentrations measured by RIA. The tmax and Cmax parameters were obtained by data inspection. Areas under the plasma (and serum) concentration versus time curve (AUC) were generated by the WinNonlin on the basis of the trapezoidal rule from 0 to the last sampling time having a quanti?able value (tlast). Elimination rate constant (kel) for the estimation of the apparent terminal elimination half-life (t1/2 = ln2/kel) was determined by linear regression analysis on selected time points in the apparent terminal phase of each log plasma concentration versus time curve. Total plasma clearance (CL) and apparent volume of distribution (Vd) were calculated as follows: CL = Dose/AUCinf; Vd = CL/kel. Numerical data were subjected to calculation of group means and standard deviations. Body weight data were analysed for homogeneity of variance (?????) using Levene’s test. For homogeneous data, a one-way ????? was performed and the signi?cance of any inter-group differences in mean values was determined by Dunnett’s test. For heterogeneous data, the Kruskal–Wallis test was used to compare all considered groups and the signi?cance of any differences between the control and treated groups was assessed using Dunn’s test. Biomarker (GH,

Table 2. Sequence of the major TH9507 biodegradation products detected by liquid chromatography-mass spectrometry (LC-MS) analysis in human, dog or rat plasma incubations of the peptide. The peptide fragments X-Tyr[1–2]Ala, Tyr[1–2]Ala and Asp[3–44]Leu were identi?ed by comparison to authentic analytical standards. For all the other fragments, only MS analysis was used to provide putative sequence identi?cation. Other minor degradation products, although not listed in this table, were also identi?ed by MS. Plasma matrix Human Human Dog Rat Total no. of products 14 10 15 31 Major peptide fragments (abundance >5% of total mixture) Hexenoyl-adductsa 0 X-Tyr[1– 42]Ala X-Tyr[1– 43]Arg, X-Tyr[1–20]Arg X-Tyr[1– 20]Arg X-Tyr[1–12]Lys, X-Tyr[1–11]Arg Other fragments Tyr[1–2]Ala, Asp[3 – 44]Leu 0 Ala[42 – 44]Leu Lys[21– 44]Leu, Lys[12 – 44]Leu

Peptide hGRF(1–44)NH2 TH9507 TH9507 TH9507
a

X, trans-3-hexenoyl group on Tyr1 of TH9507 and related peptide fragments.
? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

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ECKHARDT S. FERDINANDI ET AL. Table 3. Mean (±S.D.) porcine GH AUC0?480 min. (?g/min./ml) in pigs administered single, escalating doses of TH9507 by intravenous and subcutaneous injections and hGRF(1– 44)NH2 by subcutaneous injection. Dose (?g/kg) 0 (control) 0.11 0.33 1 3 9 27 81 TH9507 Intravenous 4.91 ± 0.57 4.41 ± 0.48 4.95 ± 0.39 7.34 ± 0.62* 9.59 ± 0.61* ND ND ND Subcutaneous 2.20 ± 0.40 2.64 ± 0.40 3.48 ± 0.30 5.69 ± 0.77* 6.09 ± 0.96* ND ND ND hGRF(1– 44)NH2 Subcutaneous 2.20 ± 0.40 ND ND ND 2.74 ± 0.24 3.17 ± 0.46 3.42 ± 0.33* 3.66 ± 0.52*

ND, not determined. Indicated values (*) were signi?cantly different versus control (P < 0.05) based on a one-way ????? test between treatment pairs.

Fig. 1. Ex vivo disappearance of TH9507 in human, rat and dog plasma (A), and disappearance of human growth hormone-releasing factor (hGRF(1–44)NH2) with concomitant increase of H-Tyr-AlaOH and hGRF(3–44)NH2 peptide fragments in human plasma (B) determined by liquid chromatography-mass spectrometry (LC-MS) analysis of aliquots withdrawn at selected sampling times up to 5 hr from incubations (37°C) of the respective plasma matrices containing TH9507 and hGRF(1– 44)NH2 at concentrations of 100 ?g/ml.

of >8 hr was estimated for TH9507 compared to approximately 33 min. for hGRF. LC-MS analyses of incubates con?rmed the presence of various TH9507-related peptide fragments in each plasma matrix (31 in rat plasma; compared to 10 and 15 fragments in human and dog plasma, respectively) of which none corresponded to trans-3-hexenoyl-Tyr1Ala2 or GRF(3–44) amide (table 2). Also, the trans-3-hexenoylTyr1Ala2 dipeptide was not observed when TH9507 was incubated directly with DPP-IV enzyme con?rming the enzyme resistance imparted by the trans-3-hexenoyl adduct (data not shown). Pharmacology and toxicology. TH9507 administered by both intravenous and subcutaneous injections resulted in an increase in growth hormone response in pigs (table 3), rats (table 4) and dogs (table 5). In pigs, the TH9507-induced increase in porcine growth hormone was dose-related after both routes of administration and markedly higher than after subcutaneous injection of
Table 4. Mean percent increase (?%) in GH concentrations in rats (n = 6 animals/sex/group) at 5 min. after TH9507 administration on Day 1 (single dose) and the last day (Day 28 or Day 91) of the daily repeat dose regimen. In both studies, the overall mean rat GH concentrations at 5 min. after administration were signi?cantly different (P < 0.05) from pre-dose values both on Day 1 and Day 28 or 91 based on a t-test using the ????? error term. Intravenous injection Dose (?g/kg) 100 300 600 Sex Male Female Male Female Male Female Day 1 (?%) 333 272 224 436 122 573 Day 28 (?%) 339 687 398 1060 389 502 Subcutaneous injection Day 1 (?%) 201 180 206 245 395 187 Day 91 (?%) 271 175 230 295 305 312

IGF-1) data were analysed for homogeneity using Bartlett’s test. A two-way ????? was used to determine if there were any signi?cant group, sex or group*sex differences for each comparison of interest. Depending on the results obtained, further statistical analyses were performed as indicated in the legends to the tables.

Results In vitro biodegradation. The disappearance of TH9507 in rat, dog and human plasma incubations, determined by LC-MS analysis, is shown in ?g. 1A. The results show that the fraction of TH9507 remaining in rat plasma declined faster than in human and dog plasma. In contrast, hGRF(1– 44)NH2 biodegradation in human plasma was much faster than TH9507 (?g. 1B). In addition, the disappearance of the parent peptide resulted in the concomitant appearance of the dipeptide H-Tyr1-Ala2OH and hGRF(3–44)NH2, the expected fragments resulting from DPP-IV cleavage of the parent peptide. In a similar study (data not shown), the ex vivo disappearances of TH9507 and hGRF(1–44)NH2 were determined in human plasma up to 54-hr and, based on these data, an in vitro half-life (t1/2)

? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

PHARMACOLOGY AND TOXICOLOGY OF HUMAN GROWTH HORMONE-RELEASING FACTOR ANALOGUE Table 5.

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Mean (±S.D.) AUC0-tlast (?g.min./ml) canine GH and IGF-1 concentrations in dogs (n = 3 or 4 animals/sex/group) administered repeat daily doses of TH9507 by intravenous (28 days) or subcutaneous injections (16 weeks). IGF-1 concentrations were measured prior to dosing (predose), and at 24 hr after the ?rst dose (Day 1) and last doses (Day 28 or Week 16). In the 28-day study, a t-test using the ????? error term indicated that overall IGF-1 values for treated animals at 24 hr after administration on Day 1 were signi?cantly different ( P < 0.001) from values before treatment and that overall GH AUC values for treated animals on Days 1 and 28 were signi?cantly different ( P < 0.001) from Day 28 controls. A t-test on each group least-squares mean indicated that Day 28 IGF-1 values were signi?cantly different ( P < 0.001) from values before treatment. In the 16-week study, Dunnett’s test indicated that Week 16 IGF-1 values for both males and females of each treated group were signi?cantly different (P < 0.0001) from Day 1 controls. Intravenous injection Dose (?g/kg) 0 100 300 600 cGH AUC0-tlast (?g.min./ml) Sex Male Female Male Female Male Female Male Female Day 1 ND ND 4.72 ± 1.19 5.54 ± 0.56 9.83 ± 3.02 8.40 ± 1.97 6.93 ± 2.68 7.22 ± 5.89 Day 28 2.59 ± 0.45 3.46 ± 1.59 11.9 ± 2.23 15.4 ± 3.64 19.9 ± 8.26 24.6 ± 10.7 24.1 ± 13.1 8.97 ± 1.62 Pre-dose ND ND 323 ± 123 314 ± 150 384 ± 103 337 ± 13 283 ± 85 341 ± 129 IGF-1 (ng /ml) Day 1 ND ND 370 ± 103 334 ± 122 523 ± 154 394 ± 51 330 ± 113 416 ± 140 Day 28 ND ND 658 ± 122 549 ± 195 871 ± 62 831 ± 121 754 ± 159 759 ± 162 Subcutaneous injection IGF-1 (ng /ml) Day 1 218 ± 44 167 ± 50 221 ± 80 187 ± 84 159 ± 58 210 ± 44 165 ± 32 138 ± 39 Week 16 156 ± 52 128 ± 55 854 ± 178 854 ± 219 873 ± 21 1199 ± 313 881 ± 30 814 ± 496

ND, not determined.

hGRF(1– 44)NH2 (table 3). Also, TH9507 induced a potent, pulsatile porcine GH response with two or three distinct peaks over an 8-hr interval, whereas with hGRF(1– 44)NH2 a multi-peak response was obtained for an interval of <3 hr at the high intravenous dose (?g. 2). Figure 3 shows the increase in serum IGF-1 concentrations in pigs administered repeat subcutaneous injections of TH9507 at 12-hr intervals for 5 consecutive days at escalating doses up to 30 ?g / kg compared to a similar repeat-dose regimen of hGRF at a dose of 30 ?g / kg. The results indicated that, similar to the effect on the porcine GH biomarker, TH9507 induced a signi?cantly larger increase in IGF-1 at each dose level compared to hGRF(1– 44)NH2 at 30 ?g/kg. The changes (?) in IGF-1 levels were not signi?cantly different between TH9507 dose groups, but all the changes in the TH9507 treatment groups were signi?cantly larger than

those in the control (saline) and hGRF(1– 44)NH2 treatment groups. For example, on Day 6 at 24 hr after the last dose, the ? IGF-1 in the TH9507 groups ranged from 89 ± 21 ng/ml to 166 ± 42 ng/ml compared to ?12 ± 24 ng/ml in the control group and 26 ± 23 ng/ml in the hGRF(1–44)NH2 group (P < 0.05). In rats, serum concentrations of rat GH increased multifold relative to pre-dose levels after single (Day 1) and repeat daily TH9507 doses (table 4). The results indicate that changes in rat growth hormone concentrations, albeit highly variable in rats due to the pulsatility and sex differences in response

Fig. 2. Mean (n = 8 animals/group) plasma concentrations of porcine GH in Yorkshire-Landrace pigs after single intravenous injections of TH9507 or hGRF(1–44)NH2 at a dose of 3 ?g/kg.

Fig. 3. Mean (±S.D.) concentrations of serum of pigs (8 animals/ group) during and after repeat subcutaneous injections of escalating TH9507 doses (7.5 to 30 ?g /kg) and of hGRF(1– 44)NH2 at one dose level (30 ?g /kg). The peptides were admininstered at 12-hr intervals starting on Day 1 to Day 5. Blood sampling for IGF-1 analysis occurred just prior to the ?rst dose and subsequently at 24-hr intervals to Day 8 (3 days after the last dose).

? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

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ECKHARDT S. FERDINANDI ET AL. Table 6.

Mean (±S.D.) body weights (g) of rats (n = 10 rats/sex/group, 28-day study; n = 15 rats/sex/group, 91-day study) and mean ( ±S.D.) percent increase in body weight relative to Day 1 (?%) after daily administration of TH9507 by intravenous and subcutaneous injections at doses of 100, 300 and 600 ?g/kg per day. Dose (?g/kg) 0 100 300 600 Intravenous injection Sex Male Female Male Female Male Female Male Female Day 1 266 ± 9.6 172 ± 10.6 267 ± 13.7 173 ± 13.1 263 ± 12.2 174 ± 11.0 263 ± 11.0 179 ± 11.5 Day 29 347 ± 16.5 194 ± 13.8 377 ± 26.5* 213 ± 18.1* 357 ± 24.2 217 ± 16.4* 358 ± 18.5 226 ± 16.4* ?% 30.4 ± 4.7 13.2 ± 5.5 41.2 ± 7.2 22.8 ± 4.0 35.6 ± 7.6 24.5 ± 5.6 36.4 ± 5.5 26.6 ± 5.5 Day 1 308 ± 13.4 220 ± 9.1 312 ± 19.3 215 ± 9.8 307 ± 14.6 217 ± 11.5 307 ± 17.5 217 ± 6.7 Subcutaneous injection Day 91 542 ± 50.2 310 ± 31.2 564 ± 60.4 334 ± 47.0 572 ± 44.4 340 ± 28.5* 555 ± 58.4 365 ± 32.1* ?% 76.2 ± 13.9 40.5 ± 9.2 80.2 ± 11.2 54.9 ± 15.5 86.0 ± 9.7 56.3 ± 9.8 80.7 ± 12.8 68.2 ± 11.5

The indicated (*) body weights were signi?cantly different from control values ( P < 0.05) based on Dunnett’s test (28-day study) and Dunn’s test (91-day study).

pro?les [22], were independent of the route of administration and dose level. Although IGF-1 levels were also measured in the 28-day rat study, the increases in that biomarker response were not statistically signi?cant (P < 0.05) (data not shown). By contrast, canine GH AUC increased signi?cantly in dogs after repeat daily TH9507 dose administrations (Day 1 vs. Day 28, P < 0.001) and IGF-1 concentrations were signi?cantly higher after repeat daily injections of TH9507 regardless of the route of administration (Day 1 vs. Day 28 and Day 1 vs. Day 116; P < 0.001). Similar to the other two species, the increases in the growth hormone and IGF-1 biomarkers were not dose related in the dog (table 5). Treatment of rats and dogs by daily intravenous and subcutaneous injections of TH9507 for up to 4 months at doses up to 600 ?g/kg per day resulted in modest increases in body weight gain with no mortality or observed systemic toxicity in either species. Tables 6 and 7 show that in both species, the increase (?%) in body weight gain of treated versus control animals was larger in female compared to male animals, and more pronounced after the prolonged subcutaneous regimen. The magnitude of the body weight increase was generally larger in dogs than rats especially

after subcutaneous injection. In addition to reversible injection site reactions (both species), clinical laboratory ?ndings in dogs (but not rats) after daily subcutaneous injections included the development of a reversible, mild regenerative anaemia at all dose levels. Reticulocytosis and reversible increases in cholesterol, triglycerides, protein and globulin were also observed in dogs at all dose levels as well as some electrolyte changes (e.g. increased serum phosphorus), although for most electrolytes (serum sodium, chloride, calcium, potassium) the changes were small, remaining within normal ranges, and were inconsistent in relation to dose level. In dogs (but not rats), weights of liver and pituitary glands were increased, and spleen weights were decreased in absolute terms and relative to terminal body weight in all treatment groups, although there was no clear evidence of dose-dependency in the magnitude of these effects. Histopathology investigations showed no abnormalities in the pituitary or spleen. Reversible centrilobular hepatocellular vacuolation and tubular basophilia were noted in the liver and kidneys, respectively, of dogs in each dose group. Because the appearance of the cytoplasm of the hepatocytes was consistent with glycogen accumulation, the vacuolar

Table 7. Mean (±S.D.) body weights (kg) of dogs (n = 3 dogs/sex/group, 28-day study; n = 4 or 6 dogs/sex/group, 16-week study) and mean ( ±S.D.) percentage increase in body weight relative to Day 1 (?%) after daily administration of TH9507 by intravenous and subcutaneous injections at doses of 100, 300 and 600 ?g/kg per day. Dose (?g/kg) 0 100 300 600 Intravenous injection Sex Male Female Male Female Male Female Male Female Day 1 7.8 ± 0.53 7.7 ± 0.55 8.2 ± 0.72 8.0 ± 0.59 8.1 ± 0.49 8.0 ± 0.59 8.1 ± 0.72 8.0 ± 0.56 Day 28 8.4 ± 0.52 8.1 ± 0.46 9.5 ± 0.78 8.8 ± 0.53 9.3 ± 0.55 9.3 ± 0.21 9.3 ± 0.64 9.1 ± 0.75 ?% 7.7 ± 1.4 5.7 ± 2.4 15.9 ± 2.4 9.6 ± 1.4 15.7 ± 0.3 17.5 ± 6.3 14.6 ± 4.7 13.7 ± 2.7 Day 1 9.6 ± 0.70 8.5 ± 0.94 9.6 ± 0.51 8.7 ± 0.96 9.5 ± 0.50 8.6 ± 0.75 9.7 ± 0.62 8.5 ± 0.98 Subcutaneous injection Day 113 10.5 ± 0.72 9.3 ± 1.28 12.0 ± 0.97* 12.8 ± 1.39* 11.7 ± 0.82 12.4 ± 0.60* 12.0 ± 0.80* 11.3 ± 2.18* ?% 9.9 ± 6.9 8.2 ± 4.2 25.8 ± 13.1 46.7 ± 0.6 23.5 ± 12.5 43.7 ± 9.8 25.0 ± 13.4 32.7 ± 13.1

On Day 113 of the 16-week study, the indicated (*) body weights were signi?cantly different from control values ( P < 0.05) based on Dunnett’s test.
? 2007 Theratechnologies Journal compilation ? 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 100, 49–58

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55

Mean (±S.D.) toxicokinetic parameters for immunoreactive TH9507: C5 min. (after intravenous injection) in rats; and tmax, Cmax and AUC0-tlast (after subcutaneous injection), in rats and dogs after daily administration of TH9507 at doses of 100, 300 and 600 ?g /kg per day. Because there was no apparent sex effect on the toxicokinetic parameters, the means ( ±S.D.) were calculated on combined male and female values (n = 12 (rats) or n = 6–8 (dogs) animals/group). Rats TH9507 Dose (?g/kg) 100 300 600 28-day, intravenous injection Occasion Day 1 Day last Day 1 Day last Day 1 Day last C5 min., ng/ml 26.0 ± 23.3 35.4 ± 14.3 74.1 ± 42.7 106 ± 54.8 210 ± 81.5 244 ± 140 13-week, subcutaneous injections tmax, min. 26 ± 42 19 ± 25 5±0 5±0 12 ± 19 12 ± 19 Cmax, ng/ml 5.5 ± 3.3 10.3 ± 7.5 9.0 ± 4.6 17.7 ± 9.3 14.4 ± 4.5 35.7 ± 17.0 AUC0-tlast, ng.min./ml 248 ± 267 455 ± 640 367 ± 273 543 ± 713 447 ± 274 1860 ± 1170 Dogs 16-week, subcutaneous injections tmax, min. 32 ± 19 41 ± 34 45 ± 35 47 ± 25 36 ± 27 41 ± 21 Cmax, ng/ml 6.7 ± 2.0 37.8 ± 30.7 16.4 ± 4.3 84.5 ± 40.4 35.0 ± 20.5 220 ± 152 AUC0-tlast, ?g.min./ml 1.11 ± 0.62 9.36 ± 8.87 1.70 ± 1.08 17.6 ± 13.1 6.84 ± 4.19 44.4 ± 29.7

change in the liver cells was likely related to a pharmacological effect of TH9507 on carbohydrate metabolism, rather than a toxic effect of TH9507-treatment. Taken together, the results from the toxicity studies showed that while the no observed adverse effect level (NOAEL) was determined to be the high dose (600 ?g/kg) in both species after 28 days of intravenous injection, a NOAEL (600 ?g/kg) was only determined in rats after daily subcutaneous injections. In dogs, a NOAEL was not found after subcutaneous injection indicating that dogs were more sensitive to prolonged TH9507 treatment than rats.

Toxicokinetics. Toxicokinetics of immunoreactive TH9507 in both rats and dogs are presented in table 8 and ?g. 4 as mean (±S.D.) of the combined male and female values in each dose group because there was no apparent sex effect in the measured concentrations of the peptide. After intravenous injection, immunoreactive TH9507 was detected up to 4 hr after administration in dogs (?g. 4), while in rats the concentrations were below the limit of detection at 1 hr after administration thereby precluding calculation of the pharmacokinetic parameters. Non-compartmental pharmacokinetic analysis on the measured plasma concentrations

Fig. 4. Mean (±S.D.) plasma concentrations of immunoreactive TH9507 in beagle dogs following a single intravenous injection (Day 1) (A) and after 28 daily intravenous injections of TH9507 (Day 28) (B) at doses of 100, 300 and 600 ?g/kg/day. The inserted tables present mean (±S.D.) values for the calculated pharmacokinetic parameters: AUC, half-life (t 1/2), apparent volume of distribution (Vd) and total body clearance (CL).
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ECKHARDT S. FERDINANDI ET AL.

in dogs indicated that immunoreactive TH9507 had a volume of distribution of 658 to 788 ml/kg and was eliminated from plasma at a t1/2 of approximately 44 min. with a plasma clearance of 670 to 900 ml/hr/ kg. The somewhat shorter t1/2 (20.6 and 26.6. min.) and higher CL in the low dose dogs was likely due to insuf?cient characterization of the elimination phase. In both species, the exposure to immunoreactive TH9507 after intravenous and subcutaneous injection increased in a dose-related manner based on Cmax and AUCtlast values (?g. 4 and table 8). After subcutaneous injection, the uptake was somewhat slower in dogs (tmax, 32– 47 min.) than rats (mean tmax, 5–26 min.). Although highly variable, the Cmax and AUC values in both species were higher after subcutaneous injection on Day last, compared to Day 1, whereas after intravenous injection the concentrations of immunoreactive TH9507 were similar on Day 1 and Day 28 of treatment. Furthermore, the magnitude of the increase was somewhat larger in dogs (about 10 times) than rats (about two to three times). These data suggested that repeat daily subcutaneous injections of TH9507 resulted in an accumulation of immunoreactive TH9507 in both species, albeit to a somewhat lower extent in the rat compared to the dog. The reason for this apparent increase in exposure, which remains to be established, may be related to the presence of anti-TH9507 antibody detected in both species after repeated subcutaneous injections (Theratechnologies Inc., unpublished results). Discussion These studies have shown that the addition of a trans-3hexenoyl group to Tyr1 of the N-terminal human GRF sequence, a minimal change to the peptide structure, resulted in a retention of the physiological characteristics of the natural peptide with enhanced stability and in vivo potency. The in vitro studies indicated that the modi?ed peptide was resistant to DPP-IV catalysed cleavage resulting in a slower biodegradation of TH9507, likely by other, perhaps species-speci?c peptidases (?g. 1 and table 2). Indeed, the main TH9507 biodegradation product found in each plasma matrix corresponded to large peptide fragments retaining the hexenoyl group, indicating that cleavage occurred mainly from the C-terminal end of the molecule (table 2). In addition, the trypsin-like endopeptidase cleavage between Arg11 and Lys12, identi?ed as an hGRF(1– 44)NH2 deactivation site secondary to DPP-IV [23], was also found to occur in each matrix. The enhanced in vivo stability of TH9507 was demonstrated in a Phase I clinical trial in which the t1/2 of immunoreactive peptide was estimated to be approximately 1 hr in normal healthy human subjects (Theratechnologies Inc., unpublished results) compared to t1/2 of 6.8 min. for immunoreactive hGRF(1–44)NH2 [13]. Furthermore, the in vitro plasma biodegradation of TH9507 appeared to be most rapid in rats and similar in dog and human (?g. 1). This apparent rank order of in vitro stability was re?ected in vivo where the t1/2 of immunoreactive TH9507 in dogs was estimated to be 42.3 to 45.3 min. (?g. 4), approximately the

same as in humans, while in rats TH9507 was not detected at 1 hr after administration suggesting a more rapid plasma clearance. These results indicated that the N-[trans-3hexenoyl]-Tyr1 modi?cation of hGRF signi?cantly enhanced the stability of the peptide in rat, dog and human plasma matrices. Structure-activity studies on GRF have shown that receptor binding characteristics are affected by changes in the peptide sequence and structure [24]. In vitro studies on baby hamster kidney cells transfected with the hGRF receptor have shown that the binding characteristics (IC50) of TH9507 and natural hGRF were similar, indicating that the N-[trans-3-hexenoyl]Tyr1 modi?cation had no apparent effect on hGRF receptor binding (P. Gaudreau, unpublished results). The enhanced in vivo potency of TH9507 relative to unmodi?ed hGRF was demonstrated in the marked increase in GH and IGF-1 response obtained in pigs treated with the two peptides (?gs 2 and 3). Although the pig is an appropriate model for potency evaluation [25], a functional effect related to TH9507 treatment was not evaluated in this study. However, the treatment of pigs with [desNH2Tyr1,DAla2,Ala15]hGRF(1–29)NH2, another DPP-IV-resistant analogue of hGRF [24], showed effects on several growth parameters and carcass characteristics similar in magnitude to porcine GH treatment [25]. Further studies showed that, compared to controls, repeated daily intravenous or subcutaneous injections of TH9507 induced a release of GH and IGF-1 in rats and dogs similar to that observed in pigs (tables 4 and 5). Because control animals of both species showed a normal increase in body weight gain, the larger body weight change in the treated animals (tables 6 and 7) was probably a result of the treatment-related biomarker response. Furthermore, rats and dogs treated with growth hormone alone showed a similar elevation of circulating IGF-1 levels and related increase in body weight gain [26,27]. Overall, in both rats and dogs, the level and duration of circulating IGF-1 was more closely associated with the anabolic effect than exposure to TH9507. Based on Cmax and AUC values (table 8 and ?g. 4), exposure to immunoreactive TH9507 was consistently dose-related and higher after intravenous than subcutaneous injection. By contrast, body weight increases were dose-independent after both routes of administration and the magnitude of the effect was larger after the prolonged subcutaneous dosing regimen (tables 6 and 7). However, GH and IGF-1 concentrations increased independently of TH9507 doses in rats, dogs and pigs (?g. 3 and tables 4 and 5) and reached plateau concentrations with duration of treatment, possibly due the IGF-1 feedback mechanism in the somatotropic axis [28,29]. Because IGF-1 is known to be responsible for anabolic effects in both animals and man [30], the observed pharmacodynamics in these studies were consistent with a TH9507-induced elevation and maintenance of high circulating IGF-1 concentrations likely through the somatotropic axis. In contrast to humans treated with growth hormone [29,30], the anabolic effect of the TH9507 treatment tended to be higher in female animals than male animals of both species (tables 6 and 7), an apparent sex effect that was also observed in rats treated

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with human GH [26]. The apparent lower anabolic response in rats (compared to dogs) may be due to a species difference in the pro?le of IGF expression [31,32]. The results of the toxicity studies in rats and dogs indicated that, similar to the effect on body weight gain, the treatment-related effects observed in the animals were likely not to be directly due to TH9507 exposure, but rather to prolonged exposure to elevated, supraphysiological concentrations of biomarkers, particularly IGF-1. Because the endocrine physiology of the rats precluded a signi?cant augmentation of IGF-1 concentrations [31,33], TH9507 treatment-related ?ndings were limited to modest increases in body weight gain and injection site reactions after 91 daily subcutaneous injections, and the NOAEL was found to be the high dose (600 ?g/kg per day) after both intravenous and subcutaneous dosing regimens. By contrast, the NOAEL in dogs, also found to be the high dose (600 ?g/kg) after 28 daily intravenous injections, was not determined after 116 days (4 months) of daily subcutaneous injections due to the observation of adverse effects at the low dose (100 ?g/kg per day). Similar to the increase in body weight gain, many of the other treatment-related effects observed in dogs were attributed to prolonged exposure to high concentrations of GH and IGF-1 induced by TH9507. Effects on haematological parameters (anaemia), increased serum calcium and phosphorus concentrations, and increased total cholesterol and triglycerides changes found in dogs administered TH9507 were also observed in dogs treated with porcine GH [27]. Also, porcine growth hormone treatment-related increases in organ weights such as pituitary, liver and adrenals, attributed to an increased volume of the normal cell population in those tissues [34], were also found in dogs treated with TH9507. However, some differences in growth hormone and TH9507 treatment effects may have occurred in kidneys as TH9507 induced a reversible tubular basophilia, whereas animals treated with porcine GH presented enlarged glomeruli with thicker mesangium. In addition, the TH9507-treated dogs had signi?cantly lower thyroid/parathyroid and spleen weights relative to total body weight in contrast to the porcine GHtreated animals in which no effect was observed in those tissues [27]. Whether the effects on thyroid and spleen weights were speci?cally due to TH9507 treatment, or to elevated GH and/or IGF-1 concentrations, remains to be determined. Nevertheless, none of the tissues showing a TH9507 treatment-related change in weight was found to have abnormal histological ?ndings and the changes in each of the affected tissues were found to be reversible. In conclusion, the addition of a trans-3-hexenoyl moiety to Tyr1 of hGRF(1–44)NH2 gave a novel peptide (TH9507) that was resistant to DPP-IV catalysed deactivation, and was shown to have enhanced stability and potency in stimulating the release of GH and/or IGF-1. Repeat daily intravenous and subcutaneous injections of TH9507 for up to 4 months were well tolerated in both rats and dogs and produced a functional increase in body weight gain. Adverse reactions at all dose levels were found in dogs but not rats after

longer-term (up to 4 months) daily subcutaneous injections. In addition to the anabolic effect, many of the observed adverse reactions in dogs could be attributed to prolonged exposure to supraphysiological concentrations of GH and IGF-1 induced by TH9507 treatment although some effects could not be explained by the action of elevated biomarkers. Based on these data, TH9507 was considered safe for use in human clinical trials, which were initiated for various indications including the treatment of lipodystrophy syndrome in human immunode?ciency virus patients [35]. Acknowledgements The authors thank Aristidis Gritsas of MDS Pharma Services (Montreal, Canada) for carrying out the ex vivo biodegradation studies and Justin Rousselle for the radioimmunoassay analysis of rat and dog plasma samples.

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