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Experimental Biology and Medicine 231:1373-1378 (2006)
© 2006 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Inhibition of Dipeptidyl-Peptidase IV Does Not Increase Circulating IGF-1 Concentrations in Growing Pigs

T. D. Faidley*,1, B. Leiting{dagger},2, K. D. Pryor{dagger}, K. Lyons{ddagger}, G. J. Hickey* and D. R. Thompson*

* Department of Pharmacology; {dagger} Department of Metabolic Disorders; and {ddagger} Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065

1To whom requests for reprints should be addressed at Merck Research Laboratories, Branchburg Farm, 203 River Road, Somerville, NJ 08876. E-mail: terry_faidley{at}merck.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The enzyme dipeptidyl peptidase-IV (DPP-IV) inactivates a variety of bioactive peptides, including glucagon-like peptide-1 (GLP-1) and growth hormone releasing hormone (GHRH). Inhibiting DPP-IV in order to increase circulating GLP-1 is of interest as a treatment for Type II diabetes. Inactivation of DPP-IV may also increase circulating GHRH, potentially enhancing growth in domestic animals. To test the hypothesis that inhibition of DPP-IV activity will influence the growth hormone/ IGF-1 axis, growing pigs (Sus scrofa domesticus, 78 kg) were treated with a DPP-IV inhibitor (Compound 1, the 2,5-difluor-ophenyl analog of the triazolopiperazine MK0431, sitagliptin), and plasma concentrations of IGF-1 were monitored. Pigs were administered either sterile saline (0.11 ml/kg followed by a continuous infusion at 2 ml/hr for 72 hrs, controls, n = 2), Compound 1 (2.78 mg/kg followed by a continuous infusion at 0.327 mg/kg·hr for 72 hrs, n = 4) or GHRH (0.11 ml/kg sterile saline, followed by a continuous infusion of GHRH at 2.5 µg/ kg·hr for 48 hrs, n = 4). Plasma concentrations of Compound 1 were maintained at 1 µM, which resulted in a 90% inhibition of circulating DPP-IV activity. Relative to the predose 24-hr period, area under the IGF-1 concentration curve (AUC) tended to be lower (P = 0.062) with Compound 1 (·79 ± 130 ng/ml·hr) than controls (543 ± 330 ng/ml·hr). GHRH treatment increased the IGF-1 AUC (1210 ± 160 ng/ml·hr, P = 0.049 vs. controls and P = 0.001 vs. Compound 1). We conclude that inhibition of DPP-IV does not alter the circulating levels of IGF-1 in the growing pig.

Key Words: growth hormone releasing hormone • DPP-IV • sitagliptin analog


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growth hormone (GH) secretion from the pituitary gland is regulated by the stimulatory activity of growth hormone releasing hormone (GHRH) and the inhibitory activity of somatostatin (1, 2). Circulating GH concentrations in turn stimulate secretion of insulin-like growth factor 1 (IGF-1) (3). Administration of either exogenous GHRH or GH leads to an increase in IGF-1, enhanced growth performance, and decreased fat deposition in swine (4–6).

GHRH belongs to a protein superfamily that includes glucago, glucagon-like peptides (GLPs), secretin, and other bioactive peptides (7). GHRH shares an {alpha}-helical structure (7) and susceptibility (8) to inactivation by dipeptidyl peptidase IV (DPP-IV) with many other family members (e.g., GLP-1, gastric-inhibiting peptides [GIPs], and peptide histidine methionine). Indeed, inhibition of DPP-IV increases the circulating half-life of the incretin hormones GLP-1 and GIP, improving glucose tolerance in Type II diabetics (9). Complete inhibition of DPP-IV does not appear to be necessary: 2- to 3-fold increases in plasma concentrations of GLP-1 were achieved in mice with inactivation of 84% to 96% of plasma DPP-IV (17). Thus, there has been much interest in the pharmaceutical industry in developing DPP-IV inhibitors for the treatment of Type II diabetes (10, 11).

DPP-IV exists as both a membrane-spanning form present in cells throughout the body and a soluble circulating form. Both forms of DPP-IV have identical enzymatic activity (9) and cleave a wide range of bioactive peptides in vitro, including hormones, neuropeptides, and chemokines (12). One potential regulatory role of DPP-IV is the inactivation of GHRH through cleavage of the active form, GHRH(1–44)-NH2, to the N-terminally shortened inactive form, GHRH(3–44)-NH2, (13). While trypsin-like degradation of GHRH also occurs, in vitro studies using GHRH analogs designed to resist cleavage at the N-terminus have demonstrated that the primary degradation of GHRH is via DPP-IV (13, 14). Substitution of Ala2 with DAla prevents DPP-IV proteolysis (13, 15), and administration of this analog increases GH release in swine up to 2-fold (7). The His1, Val2 analog of GHRH is also not degraded by DPP-IV in vitro, and it demonstrates increased plasma stability over native GHRH (16). GHRH analogs containing the His1, Val2 substitutions were 5.4- to 12.5-fold more potent than native GHRH in release of GH in swine (16). Thus, inhibition of DPP-IV in vivo may increase endogenous concentrations of GHRH and enhance GH secretion.

One of the concerns surrounding DPP-IV inhibition therapies for Type II diabetes in humans is, that in addition to stabilizing the target incretins GLP-1 and GIP, there is potential for stabilization of multiple bioactive peptide substrates such as GHRH. Increasing circulating active GHRH would stimulate the GH/IGF-1 axis, resulting in increased IGF-1 concentration. While studies have shown beneficial effects of exogenous administration of GHRH (17, 18) and GH (19) in humans, elevation of IGF-1 in diabetics would not be desired, given its potential to exacerbate insulin resistance (20).

The present study was designed using IGF-1 plasma concentrations in pigs as a biomarker for the physiological relevance of DPP-IV activity in GHRH degradation. Domestic swine approaching market weight were dosed with a DPP-IV inhibitor to evaluate the potential for growth enhancement and/or adverse effects as a result of stabilization of endogenous GHRH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, Housing, and Surgical Procedures.
Ten male castrate, crossbred swine (Stonehurst Farms, Strasburg, PA) with a pretrial mean weight of 78 ± 1.1 kg were used. Pigs were fed an 18% crude protein, 5% fat corn-soybean–based meal (F.M. Brown’s Sons Inc., Birdsboro, PA) and housed individually in 1.4 x 1.4-m pens in an environmentally controlled facility. Water was available at all times via a nipple drinker system. All procedures were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.

Pigs were surgically fitted with double-lumen jugular catheters (Micro-Renathane, 0.2-cm o.d. x 0.1-cm i.d.; Braintree Scientific, Braintree, MA) approximately 14 days before the study. Anesthesia was induced with a cocktail containing Telazol (2 mg/kg, tiletamine HCl and zolazepam; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (5 mg/kg; Phoenix Scientific, St. Joseph, MO). Pigs were then endotracheally intubated, and anesthesia was maintained with 2% to 3% isoflurane (Anaquest, Liberty Corner, NJ) in oxygen with a flow rate of approximately 1 liter per min. Catheters were tunneled subcutaneously to a 5 x 5-cm subcutaneous pocket formed approximately 10 cm ventral to the dorsal midline and 10 cm caudal to the ear. Catheters were then connected to vascular access ports (Access Technologies, Skokie, IL). Patency of ports and catheters was maintained with a saline flush solution containing 50 U/ ml heparin (Elkins-Sinn, Cherry Hill, NJ) and 1000 U/ml penicillin G (Pfizer Roerig Division, New York, NY). Pigs were treated with a prophylactic antibiotic (oxytetracycline, 22 mg/kg; Butler Co., Columbus, OH) and an analgesic (flunixin meglumine, 0.5 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA).

Test Compounds.
Compound 1 (7-[(3R)-3-amino-1-oxo-4-(2,5-difluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine L-tartaric acid salt; Process Research, Merck Research Laboratories, Rahway, NJ), a small molecule inhibitor of DPP-IV, is the 2,5-difluorophenyl analog of the triazolopiperazine MK0431 (sitagliptin) (21). Compound 1 was dissolved in sterile saline (25 mg/ml). Growth hormone releasing factor amide (GHRH, fragment 1–29; Sigma-Aldrich, St. Louis, MO) was first dissolved in 0.1 N HCl and then normalized with 0.1 N NaOH for a final concentration of 5 mg/ml.

Experimental Design.
Pigs were randomly assigned to one of three different treatment groups. All treatments were administered iv with a primed, continuous infusion delivered using Baxter APII Ambulatory Pumps (Baxter Healthcare Corp., Round Lake, IL). Controls (n = 2) were administered sterile saline consisting of a bolus infusion of 0.11 ml/kg followed by a continuous infusion at 2 ml/hr for 72 hrs. Compound 1 (n = 4) was administered in a bolus infusion at a dosage of 2.78 mg/kg followed by a continuous infusion at a dosage of 0.327 mg/kg·hr for 72 hrs. Animals dosed with GHRH (n = 4) were first administered a bolus infusion of 0.11 ml/kg sterile saline followed by a continuous infusion of GHRH at a dosage of 2.5 µg/kg·hr for 48 hrs and then a continuous infusion of sterile saline at 2 ml/hr for an additional 24 hrs.

Blood Sampling
Samples of blood (5 ml) were collected into tubes containing EDTA every 6 hrs beginning 24 hrs prior to the initiation of dosing. Blood collection and drug infusion were accomplished simultaneously using the dual-lumen catheters attached to individual subcutaneous access ports. The infusion side of the catheter was advanced approximately 2 cm further downstream in the jugular than the blood collection side. Harvested plasma was stored at –70°C until assayed.

Determination of Plasma Concentrations of Compound 1.
Concentrations of Compound 1 were determined by high performance liquid chromatography tandem mass spectrometry using an ABI Sciex API 3000 mass spectrometer (Applied Biosystems, Foster City, CA) operated in positive ion atmospheric pressure chemical ionization mode with multiple-reaction monitoring. The high-performance liquid chromatography system interfaced to the mass spectrometer consisted of 2 Perkin-Elmer Series 200 micro pumps and a Perkin-Elmer Series 200 autosampler (Perkin-Elmer, Norwalk, CT). A volume of 0.05 ml plasma was spiked with 50 ng internal standard. Plasma was prepared for LC-MS/MS analysis by solid phase extraction using OASIS HLB extraction plates (30 mg; Waters Corp., Milford, MA). Plasma samples (diluted with 200 µl water) were added to plates that had been preconditioned with methanol and then water (1 ml each), washed with water (1 ml), and eluted with methanol (1 ml). The organic eluent was concentrated to dryness under nitrogen at 350 °C and reconstituted with 0.2 ml mobile phase. The reconstituted extracts were chromatographed using a Fluophase PFP column ((Thermo Hypersil-Keystone, Bellefont, PA) 50 x 2 mm, 5 micron) and eluted at 0.2 ml/min under isocratic conditions with acetonitrile–water (9:1) containing 5 mM ammonium formate/0.1% formic acid. Under these conditions Compound 1 eluted at 2.3 min. A standard curve was generated from the mean of two replicates that were made by spiking an equal volume of plasma from untreated animals with increasing amounts (0.05–4000 ng; 12 concentrations). The lower limit of quantification was 1 ng/ml.

Determination of Plasma DPP-IV Activity.
The in vitro assay for measuring inhibition of plasma DPP-IV has been previously described (21). Briefly, plasma DPP-IV activity was measured using a continuous fluorometric assay with the substrate Gly-Pro-AMC, which is cleaved by DPP-IV to release the fluorescent AMC leaving group. The data are reported as percentage inhibition calculated as follows: %Inhibition = 100 (1 – (Vt/Vc)), where Vt is the rate of reaction of treated sample and Vc is the rate of reaction of control sample.

Determination of Plasma IGF-1.
Analyses of plasma samples for IGF-1 concentrations were performed by the Endocrinology Laboratory at the Cornell University Animal Health Diagnostic Center (Ithaca, NY) using an immunoradiometric assay (DSL-5600 Active IRMA kit; Diagnostic Systems Laboratories, Webster, TX). The intra-and interassay coefficients of variation were 5.7% and 10.0%, respectively.

Data and Statistical Analysis.
Area under the IGF-1 concentration curve (AUC) was calculated as increase over the predose 24 hrs and is reported as mean ± SE. Statistical analyses were performed using the General Linear Model of SAS (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preliminary studies demonstrated that in pigs Compound 1 has an in vivo half-life of 2.7 hrs and that plasma concentrations of 0.6 µM result in DPP-IV inhibition of greater than 94% (data not shown). Plasma concentrations of pigs infused with Compound 1 in the current study are shown in Figure 1Go. The primed, continuous infusion resulted in Compound 1 concentrations at or above 1 µM throughout the 72 hrs of infusion.


Figure 1
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  		Figure 1. Plasma concentrations of Compound 1 from individual pigs dosed at Time 0 with a bolus of 2.78 mg/kg followed by a continuous infusion of 0.327 mg/kg·hr for 72 hrs.

 
Selected reaction curves for Pig 1, typical of all assays, are shown in Figure 2Go. The rates of reaction at baseline and 72 hrs, calculated from the rates at the end of the 180-sec reactions, were 0.327 and 0.031 nmol/min, respectively. Inhibition at 72 hrs for Pig 1 was 91%. For all of the pigs, inhibition of DPP-IV by Compound 1 averaged 91% ± 1% over the 72-hr period (Fig. 3Go).


Figure 2
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  		Figure 2. Typical DPP-IV reaction curves in plasma obtained from Pig 1, which was dosed with a bolus of Compound 1 (2.78 mg/kg), followed by a continuous infusion of 0.327 mg/kg·hr for 72 hrs.

 

Figure 3
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  		Figure 3. Inhibition of DPP-IV (mean ± SE) in plasma obtained from pigs (n = 4) dosed with a bolus of Compound 1 (2.78 mg/kg), followed by a continuous infusion of 0.327 mg/kg·hr for 72 hrs. Baseline DPP-IV activity for Pig 1 was 59 nmol/min. (The percentage of inhibition as determined using the in vitro assay underestimates the percentage of inhibition achieved in vivo, as Compound 1 is a competitive, rapidly reversible inhibitor, and assay of plasma DPP-IV activity requires: (1) dilution of plasma, which results in a dilution of the total inhibitor; and (2) presence of substrate that competes with inhibitor for binding to the enzyme.)

 
Inhibition of circulating DPP-IV did not result in a significant increase in circulating IGF-1 (Fig. 4Go). Relative to the predose 24-hr period, the AUC of IGF-1 following treatment with Compound 1 was –79 ± 130 ng/ml·hr, and the IGF-1 AUC from saline controls was 543 ± 330 ng/ ml·hr (P = 0.062). In contrast, pigs treated with GHRH had a robust increase in IGF-1, with the AUC increased over the predose 24 hrs by 1210 ± 160 ng/ml·hr (P = 0.049 compared with saline controls and P = 0.001 compared with Compound 1–dosed pigs).


Figure 4
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  		Figure 4. IGF-1 plasma concentrations (mean ± SE) in pigs infused with saline (bolus of 0.11 ml/kg, continuous infusion of 2 ml/hr; n = 2; {blacktriangledown}), growth hormone releasing factor (bolus of 0.11 ml/kg saline, continuous infusion of GHRH at 2.5 µg/kg·hr for 48 hrs, then saline at 2 ml/hr; n = 4; •), or Compound 1 (bolus of 2.78 mg/kg, continuous infusion of 0.327 mg/kg·hr for 72 hrs; n= 4; •). Dosing began at Time 0.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, inhibition of DPP-IV with a potent, selective (21) inhibitor did not result in increased circulating IGF-1 in pigs. This result indicates that DPP-IV inhibition does not significantly modulate the GH/IGF-1 axis in these animals, and, hence, precludes DPP-IV inhibition as a growth-enhancing strategy in production animals. Conversely, these data ameliorate concerns that inhibition of DPP-IV as a therapy for Type II diabetes may elevate circulating IGF-1 levels and adversely affect the insulin sensitivity of the individual.

There are several possible explanations for why there is no change in circulating IGF-1 levels following DPP-IV inhibition. While enzymatic degradation of GHRH by DPP-IV is efficient in vitro, it is not the only pathway of GHRH metabolism in vivo. For example, GHRH can be inactivated by enzymatic cleavage at Arg11, Lys12, Arg20, and Lys21 (22). Furthermore, inhibition of DPP-IV would not prevent inactivation of GHRH by oxidation of methionine (Met27 conversion to Met(0)27) (16). In addition, the rate of GHRH degradation has been reported to differ across tissues. GHRH(1–29)NH2 is degraded more rapidly in liver homogenates (4) but at a slower rate in pituitary and hypothalamus preparations (23); when compared to serum, however, the major cleavage in theses tissues is not DPP-IV dependant.

Although administration of Compound 1 to the animals in this study caused essentially complete inhibition of soluble DPP-IV activity in plasma, that inhibition may not have prevented the membrane-bound enzyme from degrading GHRH in vivo. While the activity of the circulating form of DPP-IV can be successfully blocked in vitro (13), DPP-IV activity in several tissues can be orders of magnitude greater than that in serum (24). For instance, serum DPP-IV activity with Gly-Pro-4-nitroanilide as a substrate was reported to be approximately 10 nmol/min·mg, whereas liver activity (1.6 x 103 nmol/min·mg) and kidney activity (1.5 x 104 nmol/min·mg) were considerably higher (24). In contrast, there is evidence the plasma DPP-IV inhibition is a good surrogate for inhibition of the membrane-bound form in vivo. Inhibition of plasma DPP-IV activity in preclinical species and in man at a similar percentage as we obtained in the pig is correlated with stabilization of GLP-1 and a subsequent increase in circulating levels (25, 26).

In addition, GHRH is an excellent substrate for DPP-IV, with a rate constant at physiologic concentrations that is several times greater than that of GLP-1 or GIP (24). Therefore, it is possible that a small percentage of DPP-IV remains uninhibited and could metabolize sufficient amounts of GHRH to prevent a physiologic shift in the GH axis. GHRH homologous desensitization is another possible mechanism controlling GH secretion and, thus, IGF-1 secretion. An increase in GHRH may act to decrease the number of GHRH receptors in the pituitary. Incubation of primary rat pituitary cells with GHRH, but not GH, decreased the mRNA of GHRH receptors (27). However, GH release in primary rat pituitary cell cultures (27) was significantly stimulated by as little as 0.01 nM (50 pg/ml) GHRH, whereas a 10-fold increase in GHRH (0.1 nM) was necessary to significantly reduce receptor mRNA levels. Furthermore, in the current study, infusion of GHRH in vivo led to an increase in circulating IGF-1. These data suggest that increases in circulating GHRH may not induce downregulation of GHRH receptors; however, receptor mRNA upregulation is present following immunoneutralization of GHRH in rats (28). Therefore, GHRH may contribute to regulation of the GH axis through upregulation, but not downregulation of receptor expression.

To assess the question of whether DPP-IV plays a physiologic role in the metabolism of a particular peptide, four criteria need to be assessed (9). The first criterion, in vitro cleavage by DPP-IV, is met in the cases of GLP-1, GIP, and GHRH, as well as various other endogenous bioactive peptides. The second, successful inhibition of degradation by DPP-IV inhibitors in vitro, is also met for GLP-1, GIP, and GHRH. GHRH degradation is decreased in plasma incubations with DPP-IV inhibitors (14), and the plasma stability of GHRH analogs that are resistant to DPP-IV is greater than that of native GHRH (16). The third criterion, an increase in the relative proportions of intact to degraded endogenous peptide following in vivo treatment with a DPP-IV inhibitor, has been met by GLP-1 and GIP but has not been demonstrated for GHRH and was not directly measured in the current study. The final criterion is demonstration of a physiologic change in animals that lack expression of functional DPP-IV. This criterion has been met for GLP-1 in the Japanese Fischer 344 rat and the CD26–/– C57/B6 mouse, which are DPP-IV negative and have increased circulating GLP-1 and improved glucose tolerance (29, 30). There is no indication of enhanced GH secretion in the DPP-IV–negative rodents, as the Japanese Fischer 344 rats grow at the same rate as DPP-IV–positive Fischer 344 rats (31), and the male CD26–/– mice are slightly smaller than wild-type mice (29, 30).

We conclude that the failure to induce increases in IGF-1 concentrations in pigs with inhibition of DPP-IV activity, as seen in the current study, and the normal (or slightly slower) growth rate of DPP-IV–negative rodents suggest that DPP-IV is not a major regulator of endogenous GHRH activity nor, by extension, the GH/IGF-1 axis.


   Acknowledgments
 
We thank TP Murphy and SE Nicolich for technical support.


   Footnotes
 
2 Deceased Back

Received for publication January 11, 2006. Accepted for publication March 16, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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