Inhibition of dipeptidyl peptidase IV activity by oral
`metformin in Type 2 diabetes
`
`J. R. Lindsay*, N. A. Duffyt, A.M. McKillopt, J. Ardill+, F. P.M. O'Hartet, P.R. Flattt and
`P.M. Bell*
`
`*Regional Centre for Endocrinology and Diabetes,
`Royal Victoria Hospital, tSchool of Biomedical
`Sciences, University of Ulster, Coleraine, and
`tWellcome Laboratory, The Queen's University
`of Belfast, Belfast, Northern Ireland, UK
`
`Accepted 1 June 2004
`
`Abstract
`
`Aims Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP)
`are important insulinotropic hormones that enhance the insulin secretory response
`to feeding. Their potential for treating Type 2 diabetes is limited by short biological
`half-life owing to degradation by dipeptidyl peptidase IV (DPP IV). We investigated
`the acute effects of metformin on DPP IV activity in Type 2 diabetes to elucidate
`inhibition of DPP IV as a possible mechanism of action.
`
`Methods Eight fasting subjects with Type2 diabetes (5M/3F, age 53.1 ± 4.2 years,
`BMI 36.8 ± 1.8 kg/m2, glucose 8.9 ± 1.2 mmol/1, HbA1c 7.8 ± 0.6%) received
`placebo or metformin 1 g orally 1 week apart in a random, crossover design.
`
`Results Following metformin, DPP IV activity was suppressed compared with
`placebo (AUC0_ 6 h 3230 ± 373 vs. 5764 ± 504 nmol ml/1, respectively, P = 0.001).
`Circulating glucose, insulin and total GLP-1 were unchanged. Metformin also
`concentration-dependently inhibited endogenous DPP IV activity in vitro in
`plasma from Type 2 diabetic subjects.
`
`Conclusion Oral metformin effectively inhibits DPP IV activity in Type 2 diabetic
`patients, suggesting that the drug may have potential for future combination
`therapy with incretin hormones.
`
`Diabet. Med. 22, 654-657 (2005)
`
`Keywords dipeptidyl peptidase IV, metformin, GLP-1, Type 2 diabetes
`
`Introduction
`
`In cretin hormones glucagon-like peptide-1 ( GLP-1) and gastric
`inhibitory polypeptide (GIP) are currently under investigation
`as possible agents for treatment ofT ype 2 diabetes [ 1 ,2]. Potential
`therapeutic effects include augmentation of insulin secretion as
`well as inhibition of glucagon secretion and gastric emptying
`[1]. However, the in cretins are metabolized rapidly by dipeptidyl
`peptidase IV (DPP IV), an enzyme widely distributed in plasma
`and most tissues [3]. Dipeptidyl peptidase IV is responsible for
`N-terminal degradation of GLP-1 and GIP, by cleavage of
`N-terminal di-peptide residues yielding biologically inactive
`peptide fragments [3]. Attempts to produce suitable ther(cid:173)
`apies for use in Type 2 diabetes have focused on development
`
`Correspondence to: Prof. P. M. Bell, 1st Floor, East Wing, Royal Victoria
`Hospital, Belfast, BT12 6BA, Northern Ireland, UK
`E-mail: patrick. bell@royalhospitals. n-i. nhs. uk
`
`of degradation-resistant incretin analogues or use of novel
`DPP IV inhibitors [1,2,4].
`Currently there is much interest in the possible effects of
`metformin on DPP IV activity, circulating GLP-1 and the
`enteroinsular axis [5-7]. Mannucci et al. reported increased
`active GLP-1 concentrations following an oral glucose load in
`Type 2 diabetic patients treated with metformin [5]. Although
`enhanced GLP-1 secretion cannot be ruled out, increased GLP-
`1 concentrations were linked to inhibition of GLP-1 degradation
`by metformin, as demonstrated in vitro using pooled human
`serum or buffer containing purified DPP IV [5]. Another study
`described additive glucose-lowering effects of GLP-1 and met(cid:173)
`formin in Type 2 diabetic patients [ 6], but no significant differ(cid:173)
`ences in circulating active or total GLP-1 were observed [ 6]. In
`contrast, observations in DPP IV-deficient rats indicated raised
`levels of active GLP-1 following administration of metformin
`in the fasted state [7]. These authors also questioned signific(cid:173)
`ant inhibition of DPP IV by metformin, at least in vitro [7].
`
`654
`
`© 2005 Diabetes UK. Diabetic Medicine, 22, 654-657
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01566
`Page 1
`
`

`
`~~~ ~------------------------------ Short report 655
`enzyme (10 J..L]) was incubated for 60 min at 37 oc with 25 J.l]
`of 50 mmol HEPES buffer (pH 7.4) containing the Gly-Pro-AMC
`substate (final substrate concentration 1 mmol/1). Where appro(cid:173)
`priate, metformin was incorporated into the 25 J.l] of HEPES
`buffer at the concentrations stated in Fig. 2. The reaction was
`stopped by addition of 70 J.l] of 3 mol/! acetic acid. AMC was
`measured by comparison with a standard curve (range 3.9-
`500 nmol/1) by fluorimetric assay using excitation and emis(cid:173)
`sion wavelengths of 370 nm and 440 nm, respectively, using a
`Flexstation (Molecular Devices, Crawley, West Sussex, UK).
`Results of DPP IV activity are displayed as nmol!ml!min. The
`intra and interassay coefficients of variation of the assay were
`2.1% and 6.9%, respectively. Glucose concentrations were deter(cid:173)
`mined using a glucose-oxidase method. HbA1 c was meas(cid:173)
`ured in whole blood by ion-exchange HPLC. Serum insulin was
`determined using a microparticulate enzyme immunoassay
`(Abbott Laboratories Ltd, Berkshire, UK). Glucagon-like
`peptide-1 was measured by a competitive C-terminally directed
`radioimmunoassay, using rabbit antibody R600-8 (Bachem;
`Department of Medicine, Queen's University, Belfast, UK) with
`charcoal separation of bound and free moieties.
`
`This study was designed to investigate the effects of an acute
`dose of metformin on DPP N activity and total GLP-1 concentra(cid:173)
`tions compared with placebo in Type 2 diabetes. In addition,
`we have evaluated the in vitro effects of metformin on DPP IV
`activity using plasma from patients with Type 2 diabetes.
`
`Methods
`
`Subjects and protocol
`
`Eight patients with Type 2 diabetes (five males and three females,
`age 53.1 ± 4.2 years, duration of diabetes 2.5 ± 1.3 years, BMI
`36.8 ± 1.8 kg/m2 and HbA1c, 7.8 ± 0.6%) were recruited from
`clinics at the Royal Victoria Hospital, Belfast. All were treated
`as outpatients with dietary therapy. The Queen's University of
`Belfast Ethics Committee approved the study and all subjects
`gave written informed consent. Patients fasted from 22:00 h on
`the evening preceding the investigation. An intravenous cannula
`was sited at the antecubital fossa for blood sampling at the times
`shown in Fig. 1. Each patient was studied on two occasions in
`a randomized, crossover design, receiving either placebo or 1 g
`of metformin (Merck Pharmaceuticals, West Drayton, UK) on
`separate study mornings 1 week apart. A final sample was taken
`after non-fasting 24 h from the start of the protocol.
`
`Biochemical assays
`
`Dipeptidyl peptidase N activity was determined [8] in triplicate
`using a fluorometric method for measurement of free AMC
`(7-amino-4-methyl-coumarin) liberated from the DPP IV sub(cid:173)
`strate, Gly-Pro-AMC. An aliquot of plasma or purified DPP IV
`
`Statistical analysis
`
`Significant differences between groups of data were assessed
`using repeated measures ANOVA with Greenhouse Geisser(cid:173)
`corrected F-tests. Area under the curve (AUC) was calculated
`using the trapezoidal rule between t = 0 to t = 6 h and compared
`using a Student's paired t-test. Observations at each time point
`were similarly compared in the event of a statistically significant
`interaction between treatment and time arising in the ANOVA.
`Statistical significance was assumed if P < 0.05.
`
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`
`Figure 1 Effectsofmetformin 1 gorallycompared
`with placebo on circulating dipeptidyl peptidase
`IV (DPP IV) activity, glucose, insulin and total
`glucagon-like peptide-1 (GLP-1) responses in
`Type 2 diabetic patients. Values are mean± SEM
`for eight subjects (**P < 0.01 metformin
`compared with placebo using AUC t= 0_6 h).
`
`© 2005 Diabetes UK. Diabetic Medicine, 22, 654-657
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01566
`Page 2
`
`

`
`656 Metformin inhibition of DPP IV • ]. R. Lindsay et al.
`
`_________ __, ~~~'~I
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`(Metformin l!mol/1)
`
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`
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`
`Figure 2 In vitro effects of metformin on dipeptidyl peptidase IV (DPP IV)
`activity in (a) plasma from Type 2 diabetic subjects and (b) purified DPP
`IV enzyme. Values aremean±SEM for three subjects. *P < 0.05, **P < 0.01,
`* * * P < 0.001 compared with activity in the absence of added metformin.
`
`Results
`
`Circulating DPP IV, glucose and hormone profiles following
`metformin and placebo
`
`Dipeptidyl peptidase IV activity at baseline was similar for the
`metformin and placebo days (Fig. 1). Following metformin there
`was rapid suppression compared with the placebo across 6 has
`measured by AUC,=0--<5 h (3230 ± 373 and 5764 ± 504 nmol!ml,
`respectively, P = 0.001 ). Dipeptidyl peptidase IV activity follow(cid:173)
`ing metformin fell from baseline to a nadir of 4 ± 1 nmol!ml!
`min at 5 h, which was lower than the nadir of 11 ± 2 following
`the placebo (P < 0.0001 ). Dipeptidyl peptidase IV activity 24 h
`following metformin had returned to 17 ± 6 nmol!ml!min,
`which was comparable to basal levels and those 24 h after the
`placebo (17 ± 4 nmol!ml!min). Fasting glucose, insulin and
`GLP-1levels were comparable for the placebo and metformin
`days. ANOVA showed no differences in these parameters in the
`6 h following metformin compared with placebo (Fig. 1).
`
`Metformin effects on endogenous DPP IV activity in Type 2
`diabetic plasma in vitro and on purified DPP IV activity
`
`Metformin concentration-dependently inhibited endogenous
`DPP IV activity (IC50 56 1-!mol!l) in plasma from subjects with
`
`Type 2 diabetes (Fig. 2A). The effect was significant by
`50 1-1mol!l of metformin and increased to an observed maxi(cid:173)
`mal84% inhibition at the highest concentration of metformin
`tested (500 1-1mol!l). We can report that metformin also causes
`a dose-dependent decrease in DPP IV activity in plasma from
`healthy individuals (which starts with higher basal DPP IV
`activity compared with diabetic subjects) with an approximate
`IC50 value of 80 1-!mol!l (data not shown). Similarly, met(cid:173)
`formin caused a dose-dependent inhibition of purified DPP IV
`enzyme activity (IC50 98 1-!mol!l, Fig. 2B), which was signi(cid:173)
`ficantly decreased (by 28%, P < 0.05) at a concentration of
`25 1-1mol!l and with a maximal inhibition (80%) occurring at
`500 /-!mol!!.
`
`Discussion
`
`Therapeutic strategies proposed to enhance the incretin effect
`by administration of GLP-1 or GIP are under consideration for
`Type 2 diabetes [1,2]. However, this approach is frustrated by
`the short half-lives of these peptides owing to rapid degrada(cid:173)
`tion by DPP IV [3,4]. One way of circumventing this difficulty
`is to develop stable, long-acting DPP IV-resistant analogues.
`N-terminal analogues of both GIP and GLP-1 have been
`shown to exhibit resistance to DPP IV in vitro and prolong
`insulinotropic activity in vivo in Type 2 diabetes [1,2]. N(cid:173)
`acetyl and N-pyroglutamyl analogues of GIP plus the GLP-1
`analogue NN2211 appear to be particularly effective in vivo,
`raising the possibility of their use in therapy of Type 2 diabetes
`mellitus [1,2].
`An alternative strategy to enhance the action of GLP-1 and
`GIP is the use of DPP IV inhibitors, which decrease peptide
`degradation and increase the physiological incretin effect [ 4].
`Inhibitors tested both in vitro and in vivo include isoleucine
`thiazolidide (P32/98), valine pyrrolidine, FE99901 and NVP(cid:173)
`DPP728, which result in improved insulin secretion and glu(cid:173)
`cose tolerance in animals and humans. Although, many other
`bioactive peptides are substrates for DPP IV [3], this approach
`might be useful in promoting endogenous incretin hormone
`effects or for prolongation of biologically active GLP-1 or GIP
`given by infusion [4].
`In contrast to these novel approaches, metformin has been
`available for treating diabetes since the 1950s [9]. Metformin
`improves insulin sensitivity and reduces hepatic glucose output
`and gluconeogenesis. The current study demonstrated signific(cid:173)
`ant inhibitory effects on circulating DPP IV activity in Type 2
`diabetic patients between 1.5 and 6 h after administration,
`consistent with the T max for metformin. Following a 1-g dose
`the metformin concentration and effective dose in plasma of
`diabetic subjects would be expected to reach approximately
`20 1-1mol!l [10]. The in vitro study that examined the dose(cid:173)
`dependent effect of metformin on DPP IV activity (Fig. 2B)
`demonstrated that a comparable concentration of 25 1-1mol!l
`metformin (18 1-1mol!l in final reaction mixture) is effective at
`inhibiting DPP IV activity. Return of DPP IV activity to basal
`levels was observed within 24 h, suggesting an escape from the
`
`© 2005 Diabetes UK. Diabetic Medicine, 22, 654-657
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01566
`Page 3
`
`

`
`inhibitory effects of metformin from 6 h after drug administra(cid:173)
`tion. To achieve more sustained inhibition of DPP IV activity,
`metformin dosing regimens of two or three times daily may be
`required.
`It is not certain if the acute effects of metformin on DPP IV
`activity demonstrated in the present study will be reproducible
`in chronic dosing regimens in Type 2 diabetes [5]. Nevertheless
`our data support a new mechanism of metformin action as
`suggested previously [5]. The inability of others to reproduce
`such effects [7, 11] is difficult to explain but may relate to the
`use of alternative assay methodologies. Interestingly, there is
`evidence also that chronic inhibition of DPP IV activity may
`delay the onset of diabetes in animal models of impaired glu(cid:173)
`cose tolerance [12]. Together with the present observations,
`therapeutic inhibition of DPP IV activity may partly explain
`the mechanism of metformin action in diabetes prevention in
`man [13].
`As expected, metformin given in the fasting state did not
`acutely lower circulating glucose or increase insulin concentra(cid:173)
`tions [9]. However, our study design did allow analysis of
`responses of DPP IV activity to metformin without the influ(cid:173)
`ence of other variables. An additional limitation to the current
`study was the lack of an available N-terminally directed assay
`to detect biologically active GLP-1. Glucagon-like peptide-1
`levels determined by a C-terminally directed assay measuring
`active and cleaved inactive fragments remained unchanged
`following metformin. This would not support a direct stimu(cid:173)
`latory effect of metformin on GLP-1 secretion as suggested by
`observations in DPP IV-deficient rats [7]. However, further
`studies testing metformin under conditions of entero-endocrine
`nutrient stimulation are required to clarify this proposal [11].
`In conclusion, this study has demonstrated that oral met(cid:173)
`formin inhibits DPP IV activity acutely in patients with Type 2
`diabetes. Although there was no antihyperglycaemic or insuli(cid:173)
`notropic effects in the fasting state, metformin may contribute
`to glycaemic control by reducing the degradation of entero(cid:173)
`insular hormones secreted following feeding. Knowing that
`metformin inhibits DPP IV activity supports the possible use of
`metformin in combination with incretin hormones for therapy
`in Type 2 diabetes.
`
`Acknowledgements
`
`We thank Dr Chris Patterson for statistical advice. These studies
`were supported by the R & D Office of the Department of
`
`Short report 657
`
`Health and Personal Social Services (NI) and University of
`Ulster research strategic funding. JRL received a R & D Office
`Research Fellowship and NAD received a R & D Office
`Research Studentship.
`
`References
`
`1 Meier JJ, Gallwitz B, Nauck MA. Glucagon-like peptide 1 and gastric
`polypeptide: potential applications in type 2 diabetes mellitus. Bio(cid:173)
`drugs 2003; 17:93-102.
`2 O'Harte PPM, Gault VA, Parker JC, Harriott P, Mooney MH, Bailey
`CJ, Flatt PR. Improved stability, insulin-releasing activity and anti(cid:173)
`diabetic potential of two novel N-terminal analogues of gastric inhib(cid:173)
`itory polypeptide. N-Acetyl-GIP Pglu-GIP. Diabetologia 2002; 45:
`1281-1291.
`3 Mende in R, Gallwitz B, Schmidt WE. Dipeptidyl-peptidase IV hydro(cid:173)
`lyses gastric inhibitory polypeptide, glucagon-like peptide-1 (7-36)
`amide, peptide histidine methionine and is responsible for their
`degradation in human serum. Eur J Biochem, 1993; 214: 829-835.
`4 Drucker DJ. Therapeutic potential of dipeptidyl peptidase IV inhibi(cid:173)
`tors for the treatment of type 2 diabetes. Expert Opin Invest Drugs
`2003; 12: 87-100.
`5 Mannucci E, Ognibene A, Cremasco F, Bardini G, Mencucci A,
`Pierazzuoli E, Ciani S, Messeri G, Rotella CM. Effect of metformin
`on glucagon-like peptide 1 (GLP-1) and leptin levels in obese non(cid:173)
`diabetic subjects. Diabetes Care 2001; 24: 489-494.
`6 Zander M, Taskiran M, Toft-Nielsen MB, Madsbad S, Holst JJ.
`Additive glucose-lowering effects of glucagon-like peptide-1 and
`metformin in type 2 diabetes. Diabetes Care 2001; 24: 720-725.
`7 Yasuda N, Inoue T, Nagakura T, Yamazaki K, Kira K, Sarki T,
`Tanaka I. Enhanced secretion of glucagon-like peptide 1 by bigua(cid:173)
`nide compounds. Biochem Biophy Res Commun 2002; 298: 779-
`784.
`8 Fujiwara K, Tsuru D. New chromogenic and fluorogenic substrates
`for pyrrolidonyl peptidase. J Biochem 1978; 83: 1145-1149.
`9 Bell PM, Hadden DR. Metformin. Endocrinol Metab Clin North Am
`1997; 26: 523-537.
`10 Zarghi A, Faroutan SM, Shafaati A, Khoddam A. Rapid determina(cid:173)
`tion of metformin in human plasma using ion-pair HPLC. J Pharma(cid:173)
`ceut Biomed Ana/2002; 31: 197-200.
`11 Hinke SA, Kunh-Wache K, Hoffmann T, Pederson RA, Mcintosh
`CH, Demuth H-U. Metformin effects on dipeptidyl peptidase IV deg(cid:173)
`radation of glucagon-like peptide-1. Biochem Biophys Res Commun
`2002; 291: 1302-1308.
`12 Sudre B, Broqua P, White RB, Ashworth D, Evans DM, Haigh R,
`Junien JL, Aubert ML. Chronic inhibition of circulating dipeptidyl
`peptidase IV by FE 999011 delays the occurrence of diabetes in male
`Zucker diabetic fatty rats. Diabetes 2002; 51: 1461-1469.
`13 Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin
`JM, Walker EA, Nathan DM. Reduction in the incidence of type 2
`diabetes with lifestyle intervention or metformin. N Eng! J Med
`2002; 346: 393-403.
`
`© 2005 Diabetes UK. Diabetic Medicine, 22, 654-657
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01566
`Page 4