Long-Term Treatment With the Dipeptidyl Peptidase IV
`Inhibitor P32/98 Causes Sustained Improvements in
`Glucose Tolerance, Insulin Sensitivity,
`Hyperinsulinemia, and ␤-Cell Glucose Responsiveness in
`VDF (fa/fa) Zucker Rats
`
`J.A. Pospisilik,1 S.G. Stafford,1 H-U. Demuth,2 R. Brownsey,3 W. Parkhouse,4 D.T. Finegood,4
`C.H.S. McIntosh,1 and R.A. Pederson1
`
`The incretins, glucose-dependent insulinotropic poly-
`peptide (GIP) and glucagon-like peptide 1 (GLP-1) are
`responsible for >50% of nutrient-stimulated insulin
`secretion. After being released into the circulation, GIP
`and GLP-1 are rapidly inactivated by the circulating
`enzyme dipeptidyl peptidase IV (DP IV). The use of DP
`IV inhibitors to enhance these insulinotropic hormonal
`axes has proven effective on an acute scale in both
`animals and humans; however, the long-term effects of
`these compounds have yet to be determined. Therefore,
`we carried out the following study: two groups of fa/fa
`Zucker rats (n ⴝ 6 each) were treated twice daily for 3
`months with the DP IV inhibitor P32/98 (20 mg 䡠 kgⴚ1 䡠
`dayⴚ1, p.o.). Monthly oral glucose tolerance tests
`(OGTTs), performed after drug washout, revealed a
`progressive and sustained improvement in glucose tol-
`erance in the treated animals. After 12 weeks of treat-
`ment, peak OGTT blood glucose values in the treated
`animals averaged 8.5 mmol/l less than in the controls
`(12.0 ⴞ 0.7 vs. 20.5 ⴞ 1.3 mmol/l, respectively). Concom-
`itant insulin determinations showed an increased early-
`phase insulin response in the treated group (43%
`increase). Furthermore, in response to an 8.8 mmol/l
`glucose perfusion, pancreata from controls showed no
`increase in insulin secretion, whereas pancreata from
`treated animals exhibited a 3.2-fold rise in insulin se-
`cretion, indicating enhanced ␤-cell glucose responsive-
`ness. Also, both basal and insulin-stimulated glucose
`
`From the 1Department of Physiology, University of British Columbia, Vancou-
`ver, Canada; 2Probiodrug GmbH, Halle (Saale), Germany; the 3Department of
`Biochemistry, University of British Columbia, Vancouver, Canada; and the
`4School of Kinesiology, Simon Fraser University, Burnaby, Canada.
`Address correspondence and reprint requests to Dr. R.A. Pederson, Depart-
`ment of Physiology, University of British Columbia, 2146 Health Sciences Mall,
`Vancouver, BC, Canada V6T 1Z3. E-Mail: pederson@interchange.ubc.ca.
`Received for publication 28 August 2001 and accepted in revised form 9
`January 2002.
`ACC, acetyl-CoA carboxylase; DP IV, dipeptidyl peptidase IV; GIP, glucose-
`dependent insulinotropic polypeptide-(1– 42); GLP-1, glucagon-like peptide
`1-(7-36)amide; GLP-1a, active GLP-17–36; GS, glycogen synthase; OGTT, oral
`glucose tolerance test.
`H.-U.D. is the Chief Executive Officer and Chief Scientific Officer of and a
`shareholder in Probiodrug GmbH, a pharmaceutical company in the process
`of developing a DP IV inhibitor treatment for diabetes and its complications.
`R.A.P. and C.H.S.M. are both members of a scientific advisory panel to
`Probiodrug and receive consulting fees for their participation. R.A.P. and
`C.H.S.M. also receive grant/research support from Probiodrug to support
`studies on the drug candidate P32/98 and its utility in treating diabetes and its
`complications.
`
`uptake were increased in soleus muscle strips from the
`treated group (by 20 and 50%, respectively), providing
`direct evidence for an improvement in peripheral insu-
`lin sensitivity. In summary, long-term DP IV inhibitor
`treatment was shown to cause sustained improvements
`in glucose tolerance, insulinemia, ␤-cell glucose respon-
`siveness, and peripheral insulin sensitivity, novel ef-
`fects that provide further support for the use of DP IV
`inhibitors in the treatment of diabetes. Diabetes 51:
`943–950, 2002
`
`In 1995, Kieffer et al. (1) showed glucose-dependent
`
`insulinotropic polypeptide-(1– 42) (GIP) and gluca-
`gon-like peptide 1-(-36)amide (GLP-1) to be sub-
`strates of
`the
`circulating
`enzyme dipeptidyl
`peptidase IV (DP IV) in vivo. DP IV is an ubiquitous
`ectopeptidase that preferentially cleaves oligopeptides
`with a penultimate prolyl, alanyl, or seryl residue at the
`NH2-terminus, a substrate specificity that encompasses a
`number of bioactive peptides including GIP, GLP-1, and
`the counterregulatory hormone glucagon (2,3). DP
`IV⫺mediated cleavage of GIP and GLP-1 is a rapid pro-
`cess, yielding a circulating half-life of 1–2 min for the
`parent peptides. The resultant NH2-terminally truncated
`products GIP3– 42 and GLP-19 –36amide have been shown in a
`number of studies to be inactive at the receptor level and
`thus noninsulinotropic (4,5). Subsequent studies have
`clearly established DP IV⫺mediated NH2-terminal trunca-
`tion as the primary mechanism for GIP and GLP-1 inacti-
`vation (1,6 – 8).
`Also known as the incretins, GIP and GLP-1 make up the
`endocrine component of the entero-insular (gut-pancreas)
`axis—a concept describing the neural, endocrine, and
`substrate signaling pathways between the small intestine
`and the islets of Langerhans (9). Together, the incretins
`are responsible for ⬎50% of nutrient-stimulated insulin
`release. In addition, the incretins share a number of
`non-insulin-mediated effects that contribute to effective
`glucose homeostasis. GIP and GLP-1 have both been
`shown to inhibit gastric motility and secretion (10,11),
`promote ␤-cell glucose competence (12), and stimulate
`insulin gene transcription and biosynthesis (13,14). In
`addition, GIP has been reported to play a role in the
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`DP IV INHIBITOR P32/98 IMPROVES ␤-CELL FUNCTION
`
`regulation of fat metabolism (15), and GLP-1 has been
`shown to stimulate ␤-cell differentiation and growth (16),
`as well as to restore islet-cell glucose responsiveness (17).
`We have previously shown that acute administration of
`the specific DP IV inhibitor P32/98 (isoleucyl-thiazolidine)
`in Zucker rats enhances insulin secretion and glucose
`tolerance (18), improvements that were much more pro-
`found in the diabetic, fatty animals than in their lean
`littermates (19). Balkan et al. (20) confirmed these findings
`using the DP IV inhibitor NVP-DPP728 and went on to
`provide evidence for the previously postulated stabiliza-
`tion of, and rise in, plasma active GLP-17–36 (GLP-1a) after
`inhibitor treatment. However, despite its efficacy, the use
`of DP IV inhibitors on an acute scale is unlikely to exploit
`the longer term incretin actions involving altered intracel-
`lular protein function and gene expression. It was there-
`fore hypothesized that chronic DP IV inhibitor treatment
`of diabetic animals,
`in addition to improving glucose
`tolerance, would enhance ␤-cell glucose responsiveness,
`replication, and turnover, and thus result in sustained
`improvements in ␤-cell function.
`In the present study, two groups of Vancouver diabetic
`fatty (VDF) rats were treated for 3 months with the DP IV
`inhibitor P32/98. VDF rats are a substrain of the fatty
`(fa/fa) Zucker rat, which display abnormalities character-
`istic of type 2 diabetes, including mild hyperglycemia,
`hyperinsulinemia, glucose intolerance, hyperlipidemia, im-
`paired insulin secretion, and peripheral and hepatic insulin
`resistance (21). Parameters including body weight, food
`and water intake, and oral glucose tolerance were regu-
`larly examined to track the progression of the disease and
`study the possible therapeutic effects of the inhibitor. At
`the end of the treatment period, ex vivo fat and muscle
`insulin sensitivity were assessed, and pancreas perfusion
`was performed to measure ␤-cell glucose responsiveness.
`The results provided the first demonstration that long-term
`DP IV inhibitor treatment causes progressive and sus-
`tained improvements in glucose tolerance, insulin sensi-
`tivity, and ␤-cell glucose responsiveness.
`
`RESEARCH DESIGN AND METHODS
`Materials. The DP IV inhibitor P32/98 (di-[2S,3S]-2-amino-3-methyl-pen-
`tanoic-1,3-thiazolidine fumarate) was synthesized, as previously described
`(22).
`Animals. We randomly assigned six pairs of male fatty (fa/fa) VDF Zucker rat
`littermates to a control or treatment (P32/98) group at 440 g body wt (age 11 ⫾
`0.5 weeks). Animals were housed on a 12-h light/dark cycle (lights on at 0600)
`and allowed access to standard rat diet and water ad libitum. The techniques
`used in this study were in compliance with the guidelines of the Canadian
`Council on Animal Care and were approved by the University of British
`Columbia Council on Animal Care, Certificate # A99-006.
`Protocol for daily monitoring and drug administration. The treatment
`group received P32/98 (10 mg/kg) by oral gavage twice daily (0800 and 1700)
`for 100 days, and the control animals received concurrent doses of vehicle
`consisting of a 1% cellulose solution. Every 2 days, body weight, morning and
`evening blood glucose, and food and water intake were assessed. Blood
`samples were acquired from the tail, and glucose was measured using a
`SureStep analyzer (Lifescan Canada, Burnaby, Canada). Food and water
`intake were measured by subtraction.
`Protocol for monthly assessment of glucose tolerance. Every 4 weeks
`from the start of the experiment, an oral glucose tolerance test (OGTT; 1 g/kg)
`was performed after an 18-h fast and complete drug washout (⬃12 circulating
`half-lives for P32/93). No 0800 dose was administered in this case. Blood
`samples (250 ␮l) were collected from the tail using heparinized capillary
`tubes, centrifuged, and stored at –20°C. In the case of the 12-week OGTT,
`blood was collected directly into tubes containing the DP IV inhibitor P32/98
`(final concentration 10 ␮mol/l) for analysis of active GLP-1 (EGLP-35K; Linco
`
`Research, St. Charles, MO). Plasma insulin was measured by radioimmuno-
`assay using a guinea pig anti-insulin antibody (GP-01), as previously described
`(23), and blood glucose was measured as described above. Plasma DP IV
`activity was determined using a colorimetric assay measuring the liberation of
`p-nitroanilide (A405 nm) from the DP IV substrate H-gly-pro-pNA (Sigma;
`Parkville, Ontario, Canada). It is important to note that the assay involves a
`20-fold sample dilution and therefore underestimates the actual degree of
`inhibition occurring in the undiluted sample when using rapidly reversible
`inhibitors such as P32/98.
`Estimations of insulin sensitivity made from OGTT data were performed
`using the composite insulin sensitivity index proposed by Matsuda and
`DeFronzo (24). Calculation of the index was made according to the following
`equation:
`
`CISI ⫽
`
`10,000
`冑(FPG 䡠 FPI)(MG 䡠 MI)
`
`where FPG and FPI are fasting plasma glucose and insulin concentrations,
`respectively, and MG and MI are the mean glucose and insulin concentrations,
`respectively, over the course of the OGTT.
`Protocol for 24-h glucose, insulin, and DP IV profile. To determine the
`effects of DP IV inhibition over a 24-h period, blood glucose, insulin, and DP
`IV activity levels were measured as described above, every 3 h for 24 h, 6
`weeks into the study. Drug dosing was continued at the appropriate times
`during the profile.
`Skeletal muscle insulin sensitivity. Uptake of 14C-labeled glucose in soleus
`muscle strips was measured as an indicator of skeletal muscle insulin
`sensitivity. In brief, after an overnight fast and 18 h after the last dose of
`P32/98, the animals were anesthetized with pentobarbital sodium (Somnotol;
`⬃50 mg/kg). The soleus muscles of both hindlimbs were exposed and isolated.
`After freeing the muscle by severing the proximal and distal tendons, strips of
`⬃25–35 mg were pulled from the muscle (the two outer thirds of each muscle
`were used). After being weighed, the strips were fixed onto stainless steel
`clips at their resting length and allowed to stabilize for 30 min in a
`Krebs-Ringer bicarbonate buffer supplemented with 3 mmol/l pyruvate, con-
`tinuously gassed with 95% O2:5% CO2 and held at 37°C in a shaking water bath.
`These conditions were maintained for the duration of the experiment, unless
`otherwise stated.
`To assess glucose uptake in response to insulin, muscle strips underwent
`two preincubations (30 and 60 min) followed by a 30-min test incubation. Both
`the second preincubation and the test incubation contained 0 or 800 ␮U/ml
`insulin. The test incubation was performed in media supplemented with
`[3H]inulin (0.1 ␮Ci/ml) as a measure of extracellular space and the nonme-
`tabolizable glucose analogue [14C]-3-O-methylglucose (0.05 ␮Ci/ml) for mea-
`surement of glucose uptake. After incubation, each strip was blotted dry and
`digested with proteinase K (0.25 ␮g/ml), and the radioactivity of the muscle
`digests was measured with a liquid scintillation⫺counting dual-isotopic
`program.
`Adipose tissue insulin sensitivity. To estimate insulin sensitivity in adipose
`tissue, glycogen synthase (GS) and acetyl-CoA carboxylase (ACC) levels were
`measured, as previously described (25,26). In brief, 3-cm3 samples of ependy-
`mal adipose tissue were obtained from anesthetized animals and subjected to
`a 16-min collagenase digestion (0.5 mg/ml). Recovered adipocytes were
`washed three times and allowed to stabilize for 1 h in 37°C Krebs buffer
`repetitively gassed with 95% O2:5% CO2. Then 2-ml aliquots of the adipocyte
`suspension containing 0, 100, 250, 800, and 1,500 ␮U/ml insulin were incu-
`bated for 30 min and immediately flash frozen on liquid nitrogen and stored at
`–70°C. Before ACC and GS assessment, stored samples were thawed, homog-
`enized in buffer (pH 7.2) containing 20 mmol/l MOPS, 250 mmol/l sucrose, 2
`mmol/l EDTA, 2 mmol/l EGTA, 2.5 mmol/l benzamidine, and centrifuged (15
`min at 15,000g).
`For measurement of ACC activity, 50-␮l aliquots of supernatant, preincu-
`bated in the presence or absence of 20 mmol/l citrate, were added to 450 ␮l of
`[14C]HCO3 containing assay buffer (pH 7.4; 50 mmol/l HEPES, 10 mmol/l
`MgSO4, 5 mmol/l EDTA, 5.9 mmol/l ATP, 7.8 mmol/l glutathione, 2 mg/ml BSA,
`15 mmol/l KHCO3, 150 ␮mol/l acetyl CoA). After 3 min, the reaction was
`arrested by the addition of 200 ␮l of 5 mol/l HCl. Samples were dried for 6 h,
`resuspended in 400 ␮l of distilled water, combined with 3 ml scintillation
`cocktail, and counted on a Beckman LS 6001C ␤-counter.
`GS activity was measured using a modification of a filter paper method
`(26): 25 ␮l of the cell extracts, prepared as indicated above, were added to
`assay buffer (pH 7.0; 75 mmol/l MOPS, 75 mmol/l NaF, 10 mg/ml glycogen, 2
`mmol/l UDP-[14C]glucose) held at 30°C in the presence or absence of 15
`mmol/l glucose-6-phosphate. Each reaction was stopped by spotting 50 ␮l of
`the reaction mixture onto Whatmann 3MM filter paper and immersing the
`paper in 66% ethanol. After three ethanol washes, the samples were air dried
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`and the [14C] activity (UDP-[14C]glucose incorporation into glycogen) was
`determined.
`Protocol for pancreas perfusion. After excision of soleus and ependymal
`adipose tissue samples, the pancreas was isolated and perfused with a
`low-to-high glucose (4.4 to 8.8 mmol/l) perfusion protocol, as previously
`described (27). After exposure through a mid-line incision on the ventral
`aspect, the pancreas was isolated, all minor vessels were ligated, and a
`glucose perfusate was introduced through the celiac artery. Perfusion effluent
`was collected at 1-min intervals via the portal vein, with a perfusion rate of 4
`ml/min. Samples were stored at –20°C until analysis.
`Immunohistochemistry and ␤-cell mass determination. Pancreata were
`removed from anesthetized animals (50 mg/kg sodium pentobarbital) and
`placed directly into fixative for 48 h (44% formaldehyde, 47% distilled H2O, 9%
`glacial acetic acid). After being embedded in paraffin, 5-␮m tissue sections
`were cut, mounted onto slides, and dried ready for staining. To assess ␤-cell
`area, sections were stained first with a guinea pig anti-insulin primary
`antibody and then with peroxidase-conjugated goat anti-guinea pig secondary.
`Slides were developed using diaminobenzidine and counterstained with
`hematoxylin. Analyses were performed using Northern Eclipse Software
`(Empix Imaging, Mississauga, Ontario, Canada), as previously described (28).
`Statistical Analysis. Student’s t test and ANOVA were used, where appro-
`priate, to test statistical significance of the data (P ⬍ 0.05). Analyses were
`performed using Prism 3.0 data analysis software (GraphPad Software, San
`Diego, CA).
`
`RESULTS
`Effects of P32/98 treatment on body weight, daily
`blood glucose, and food and water intake. VDF rats
`treated with P32/98 displayed a 12.5% (25 g) reduction in
`weight gain over the 3-month treatment period (control:
`211 ⫾ 8 g; treated: 176 ⫾ 6 g) (Fig. 1A). Measurements of
`food and water intake revealed a minor decrease in water
`intake (Fig. 1B) in the treated animals concomitant with
`unaltered food intake. Food intake over the course of the
`experiment averaged 30.0 ⫾ 0.4 and 30.4 ⫾ 0.3 g 䡠 rat⫺1 䡠
`day⫺1 in the treated and control groups, respectively. Food
`and water intake decreased over the course of the exper-
`iment, paralleling the decrease in the rate of weight gain as
`the growth of the animals began to plateau at around
`600 – 650 g (data not shown). Twice-daily monitoring of
`blood glucose revealed no differences in morning or
`evening blood glucose values between the experimental
`groups, although neither group displayed notably hyper-
`glycemic values (data not shown). Morning blood glucose
`levels over the course of the experiment averaged 5.0 ⫾
`0.1 and 5.3 ⫾ 0.1 mmol/l in the treated and control animals,
`respectively. Evening blood glucose values averaged 6.7 ⫾
`0.1 and 7.0 ⫾ 0.2 mmol/l, respectively. Hematocrit, mea-
`sured at 4-week intervals, indicated no adverse effects of
`the blood-sampling protocol used, averaging 43.4 – 45.3% in
`both groups.
`Effects of P32/98 treatment on blood glucose, insulin,
`and DP IV levels over 24 h. After 6 weeks of treatment,
`a 24-h profile of blood glucose, insulin, and DP IV activity
`levels was obtained by taking blood samples at 3-h inter-
`vals, interrupting neither treatment administration nor the
`light/dark cycle. The profile confirmed that administration
`of P32/98 caused significant inhibition of DP IV activity
`over the majority of the 24-h cycle, with at least 65%
`inhibition during the feeding cycle (Fig. 2A). The inte-
`grated blood glucose excursion in the treated animals was
`75% that of the controls, peaking at 7.7 ⫾ 0.3 mmol/l, as
`compared to 9.8 ⫾ 0.6 mmol/l for the untreated animals
`(Fig. 2B). The corresponding plasma insulin profile
`showed a decrease not only in peak insulin values, but also
`in “basal,” nonfeeding values (⬃0800 to 1800) in the
`treated animals (Fig. 2C).
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`intake measured in DP IV
`FIG. 1. Body weight and water
`inhibitorⴚtreated (E) or control (f) VDF rats (n ⴝ 6 in each group).
`Body weight (A), and water intake (B) were measured along with
`morning and evening blood glucose levels and food intake (not shown)
`every 2 days. *P < 0.05 vs. other group.
`
`Effects of P32/98 treatment on oral glucose toler-
`ance. The three OGTTs, performed in the absence of
`circulating P32/98 and at 1-month intervals, were used to
`monitor the progression of the disease state in the control
`animals and to document any improvements displayed in
`the treated group. The initial OGTT, administered after 4
`weeks of treatment, showed significant decreases (⬃2
`mmol/l) in basal and 45-, 60-, and 90-min blood glucose
`values in the treated group, despite overlapping plasma
`insulin excursions (Fig. 3A). Data from the second OGTT
`were very similar, with the exception that the 120-min
`blood glucose value was also significantly lowered in the
`treated group than in the control group (10.8 ⫾ 0.8 vs.
`12.3 ⫾ 0.8, respectively); once again, the insulin profiles
`were superimposable (data not shown). The final OGTT,
`performed after 12 weeks of treatment, showed a marked
`difference in glucose tolerance between the two groups,
`with significantly decreased blood glucose values ob-
`served at all time points. Peak blood glucose values in the
`treated group averaged 12.0 ⫾ 0.7 mmol/l, 8.5 mmol/l less
`than that of control animals (Fig. 3B), whereas 2-h values
`in the treated group had returned to 9.2 ⫾ 0.5 mmol/l, a
`40% reduction compared to control values. GLP-1a levels,
`measured during the final OGTT using an NH2-terminally
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`DP IV INHIBITOR P32/98 IMPROVES ␤-CELL FUNCTION
`
`FIG. 2. A 24-h profile of plasma DP IV activity (A), blood glucose (B),
`and plasma insulin (C) levels in VDF rats after 6 weeks of treatment,
`either with (E) or without (f) the DP IV inhibitor P32/98 (n ⴝ 6 in each
`group). Treated animals were administered 10 mg/kg P32/98 twice
`daily, as indicated by the arrows, and whereas the control group
`received only the 1% cellulose injection vehicle. *P < 0.05 vs. other
`group.
`
`directed enzyme-linked immunosorbent assay, were found
`to be unchanged (Fig. 3B). Despite this lack of altered
`GLP-1a levels, the early-phase insulin response measured
`in the treated group exceeded that of the control animals
`by 43%. However, the integrated insulin responses be-
`tween the two groups showed no significant differences.
`Analysis of the OGTT data using the composite insulin
`sensitivity index of Matsuda and DeFronzo (24) revealed a
`progressive increase in estimated insulin sensitivity of the
`treated animals relative to controls (Fig. 3C).
`Comparison of the OGTTs over the course of the
`experiment revealed a progressive decrease in both fasting
`and peak blood glucose values in animals treated with
`P32/98, improvements that were not observed in control
`animals (Fig. 4A and B). Peak insulin values did not differ
`significantly between the two experimental groups until
`the final, 12-week, OGTT, at which time the peak insulin
`levels in the treated animals exceeded those of the control
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`animals by an average of 43% (Fig. 4C). Plasma DP IV
`activity, measured at the start of each OGTT, was signifi-
`cantly increased in the treated group by week 8 of the
`study, and the elevation was maintained at week 12 (Fig.
`4D).
`Effects of chronic DP IV inhibitor treatment on pan-
`creatic glucose responsiveness. A low-to-high step glu-
`cose perfusion protocol was performed on the pancreata
`of half of each group of animals. The shift from 4.4 to 8.8
`mmol/l glucose perfusate caused a 3.2-fold increase in
`insulin secretory rate in the pancreata from the treated
`animals (Fig. 5). The insulin secretory rate shifted from a
`basal 570 ⫾ 170 to over 2,100 ␮U/min within 2 min of high
`glucose perfusion. The same glucose step procedure failed
`to elicit any significant response in the control pancreata
`until well over 20 min of high glucose perfusion (Fig. 5).
`Effects of chronic DP IV inhibitor treatment on mus-
`cle and fat insulin sensitivity. To further define the
`apparent improvements in insulin sensitivity observed in
`the OGTT data, assays of muscle and fat insulin sensitivity
`were performed. GS and ACC activity were measured in
`isolated adipocytes along with uptake of 14C-labeled glu-
`cose into soleus muscle strips. ACC levels in adipose from
`both experimental groups were minimal (approaching
`limits of detection), lacked insulin responsiveness, and
`showed no differences between the two groups (data not
`shown). GS activity also appeared insensitive to insulin,
`although the activity of the enzyme at all measured insulin
`concentrations was higher in the treated animals than in
`their control littermates (Fig. 6A). Soleus muscle strips
`taken from the treated animals exhibited significantly
`higher rates of glucose uptake, in both the basal and the
`insulin-stimulated states. Glucose uptake in the nonstimu-
`lated state was 22% higher in the treated rats (Fig. 6B). The
`insulin-stimulated rise in glucose uptake was enhanced in
`the treated compared to in the control group (87.5 ⫾ 10.4
`vs. 58.5 ⫾ 3.5 cpm/mg tissue at 800 ␮U/ml insulin, respec-
`tively).
`Effects of chronic DP IV inhibitor treatment on ␤-cell
`area and islet morphology. The 3-month oral DP IV
`inhibitor regimen yielded no significant differences in
`␤-cell area or islet morphology. Islets from control and
`treated animals comprised 1.51 ⫾ 0.04 and 1.50 ⫾ 0.03% of
`the total pancreatic area, respectively. Large, irregularly
`shaped islets with significant ␤-cell hyperplasia were ob-
`served in both groups, a morphology characteristic of the
`fa/fa Zucker rat.
`
`DISCUSSION
`The use of DP IV inhibitors to enhance the entero-insular
`axis has attracted much recent interest as a potential
`therapeutic strategy in the treatment of diabetes. Several
`recent studies have established the efficacy of these com-
`pounds on an acute scale (18 –20,29). However, investiga-
`tions performed on an acute scale do not exploit the
`potential benefits of long-term incretin effects, such as the
`enhancement of ␤-cell glucose sensitivity and the stimula-
`tion of ␤-cell mitogenesis, differentiation, and insulin bio-
`synthesis. Here we reported the results of the first study of
`long-term DP IV inhibitor treatment in a model of type 2
`diabetes, the VDF fa/fa rat. The results demonstrated that
`long-term DP IV inhibition arrested the progression of the
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`FIG. 3. OGTT administered to both DP IV inhibitorⴚtreated (E) and control (f) VDF rats (n ⴝ 6 in each group) after 4 (A) and 12 (B) weeks
`of treatment. Blood glucose and plasma insulin measurements were performed in both series of tests, while the active fraction of plasma GLP-1
`was also measured at 12 weeks. The inset in B shows the integrated plasma insulin responses for the 12-week OGTT. C: Relative insulin
`sensitivity, control versus treated, corresponding to the 4- and 12-week OGTTs shown in A and B. *P < 0.05 vs. other group.
`
`fa/fa Zucker diabetic syndrome and caused a progressive
`improvement in glucose tolerance, insulin sensitivity, and
`␤-cell glucose responsiveness.
`
`Daily monitoring revealed a 12.5% decrease in body
`weight gain (4% reduction in final body weight) in the
`treated animals compared to untreated controls (Fig. 1A).
`
`FIG. 4. Comparison of fasting (A) and peak (B) blood glucose, peak plasma insulin (C), and fasting plasma DP IV activity (D) measured during
`OGTTs performed at 4-week intervals in control (f) or DP IV inhibitorⴚtreated (E) VDF rats (n ⴝ 6 in each group). *P < 0.05 vs. other group.
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`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
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`DP IV INHIBITOR P32/98 IMPROVES ␤-CELL FUNCTION
`
`FIG. 5. Insulin release measured during perfusion of pancreata from
`VDF rats after 3 months of treatment with (E) or without (f) the DP
`IV inhibitor P32/98 (n ⴝ 3 in each group).
`
`Although not statistically significant, mean food intake in
`the treated animals averaged 0.4 g 䡠 day⫺1 䡠 rat⫺1 (41 g/rat
`over the course of the study) less than those in the control
`
`FIG. 6. Adipose tissue glycogen synthase activity (A) and uptake of
`3-O-[14C] methyl-D-glucose into soleus muscle strips (B) isolated from
`VDF rats after 12 weeks of P32/98 treatment (䡺) or a control 1%
`cellulose solution (f) (n ⴝ 6 in each group). *P < 0.05 vs.
`corresponding control value; †P < 0.01 vs. corresponding control value;
`‡statistically significant difference from basal ([insulin] ⴝ 0 ␮U/ml).
`
`948
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`group. The cumulative 41 g/rat nonsignificant difference in
`food intake over the course of the experiment might
`partially account for the decreased weight gain in the
`treated animals. These findings rule out neither the possi-
`bility that the gastric inhibitory actions of the incretins nor
`the reported satiety effects of GLP-1 played a role in the
`decrease in weight gain.
`Monitored every 2 days, morning and evening blood
`glucose values showed no significant response to the
`inhibitor treatment, a likely reflection of two points. First,
`the blood-sampling times (0800 and 1700) corresponded to
`postabsorptive and early feeding states, respectively, with
`blood glucose values in the ranges of 4.5–5.5 and 6.0 – 8.0
`mmol/l, respectively. In light of the hypothesized, glucose-
`dependent mechanism of action of the treatment, large
`decreases in glucose values would not be anticipated at
`these glycemic levels. Second, both morning and evening
`blood samples were collected immediately before drug
`dosing, at times of minimum DP IV inhibition, when the
`potential for any acute therapeutic effects of the treatment
`were at a minimum. Both points are supported by the 24-h
`profile shown in Fig. 2.
`The unaltered postabsorptive blood glucose values not-
`withstanding, DP IV inhibitor treatment effectively re-
`duced both prandial blood glucose and blood glucose
`responses to an OGTT (Figs. 2 and 3). During the 24-h
`profile, the control animals exhibited a 105% rise in plasma
`insulin in response to a 5.2-mmol/l
`increase in blood
`glucose, whereas the treated animals displayed a 160%
`insulin response to a much smaller glucose excursion (3.0
`mmol/l). Although these differences were likely attribut-
`able, at least in part, to an acute increase in circulating
`incretin levels induced by P32/98, the pronounced early-
`phase insulin peak exhibited during the OGTT was not
`(the OGTT took place after complete drug washout). The
`latter data suggest not only an increased insulin sensitiv-
`ity, but also an enhanced ␤-cell glucose responsiveness, in
`treated animals. Ultimately, an increase in ␤-cell glucose
`responsiveness was clearly demonstrated through pan-
`creas perfusion. After exposure to an elevated (8.8 mmol/l)
`glucose perfusate, pancreata from the control animals
`showed an absence of first-phase insulin release, whereas
`those from the treated group exhibited an immediate
`3.2-fold insulin response (Fig. 5). The absence of an
`early-phase insulin release seen in the control group is
`characteristic of the VDF rat and is a hallmark of type 2
`diabetes (21). Considering the lack of altered ␤-cell area or
`islet morphology, these data suggest that long-term treat-
`ment with P32/98 causes an improvement in the ability of
`the existing ␤-cell population to sense and respond to
`increases in glucose concentration. These findings are
`consistent with the reported effects of GLP-1 on ␤-cell
`differentiation, as well as numerous reports showing the
`glucose-sensitizing effects of GIP and GLP-1 in both islets
`and immortalized ␤-cell models (30,31).
`Elevated fasting blood glucose in the face of hyperinsu-
`linemia and poor clearance of an oral glucose load are
`consistent with the hepatic and muscle insulin resistance,
`respectively, described in the fa/fa Zucker rat. Findings in
`the present study showed that DP IV inhibitor treatment at
`least partially corrected both of these metabolic devia-
`tions, suggesting improvements in both sites of insulin
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`DIABETES, VOL. 51, APRIL 2002
`
`Merck Exhibit 2113, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
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`

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`resistance. An increased glucose-to-insulin ratio evident
`during the postabsorptive state of the 24-h profile (Fig. 2),
`as well as fasting values of the 12-week OGTT (Figs. 3 and
`4), were consistent with a decrease in insulin resistance in
`the treated animals. The latter increase in insulin sensitiv-
`ity was shown to be significant at both 4 and 12 weeks
`using the composite insulin sensitivity index of Matsuda
`and DeFronzo (24). This mathematical analysis was previ-
`ously validated (with high correlation) against the hyper-
`insulinemic-euglycemic clamp technique in 153 subjects
`with varying degrees of insulin resistance. The relative
`insulin sensitivity of the treated animals improved with
`each successive OGTT, ultimately reaching a relative
`index score 1.56 ⫾ 0.26 times that of the control animals.
`The results of the 24-h glucose/insulin/DP IV profile and
`the OGTT were corroborated by direct measurements of
`glucose uptake in soleus muscle strips, which clearly
`demonstrated improved glucose uptake in both the non-
`stimulated and the insulin-stimulated states (Fig. 6).
`Though somewhat controversial, both GIP and GLP-1 (and
`exendin-4) have been reported to increase muscle insulin
`sensitivity through the stimulation of glycogen synthesis
`and glucose uptake (32–35). In addition, a number of
`whole animal studies using GLP-1 or related GLP-1 recep-
`tor agonists have observed similar improvements in
`glucose tolerance and insulin sensitivity. Young et al. (36)
`showed that long-term administration of the GLP-1 agonist
`exendin-4 causes glucose-lowering an